Claudio Basilico, M.D.

Sox2 maintains self renewal of tumor-initiating cells in osteosarcomas
Basu-Roy U; Seo E; Ramanathapuram L; Rapp TB; Perry JA; Orkin SH; Mansukhani A; Basilico C
2011 Sep 19;:?-?, Oncogene
Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.Oncogene advance online publication, 19 September 2011; doi:10.1038/onc.2011.405
— id: 137542, year: 2011, vol: , page: ?, stat: Journal Article,

The Sox2 high mobility group transcription factor inhibits mature osteoblast function in transgenic mice
Holmes, Greg; Bromage, Timothy G; Basilico, Claudio
2011 Oct;49(4):653-661, Bone
We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1alpha1 promoter, which extended Sox2 expression into more mature osteoblasts. In long bones, trabecular cartilage remodeling was delayed and the transition from endochondral to cortical bone was disrupted, resulting in porous and undermineralized cortical bone. Collagen deposition was disorganized, and patterns of osteoclast activity were altered. Calvarial bones were thinner and parietal bones failed to develop the diploic space. Microarray analysis showed significant up- or downregulation of a variety of genes coding for non-collagenous extracellular matrix proteins, with a number of genes typical of mature osteoblasts being downregulated. Our results position Sox2 as a negative regulator of osteoblast maturation in vivo
— id: 137066, year: 2011, vol: 49, page: 653, stat: Journal Article,

Regulation of non-classical FGF1 release and FGF-dependent cell transformation by CBF1-mediated notch signaling
Kacer D.; Mcintire C.; Kirov A.; Kany E.; Roth J.; Liaw L.; Small D.; Friesel R.; Basilico C.; Tarantini F.; Verdi J.; Prudovsky I.
2011 ;226(11):3064-3075, Journal of cellular physiology
FGF1, a widely expressed proangiogenic factor involved in tissue repair and carcinogenesis, is released from cells through a non-classical pathway independent of endoplasmic reticulum and Golgi. Although several proteins participating in FGF1 export were identified, genetic mechanisms regulating this process remained obscure. We found that FGF1 export and expression are regulated through Notch signaling mediated by transcription factor CBF1 and its partner MAML. The expression of a dominant negative (dn) form of CBF1 in 3T3 cells induces transcription of FGF1 and sphingosine kinase 1 (SphK1), which is a component of FGF1 export pathway. dnCBF1 expression stimulates the stress-independent release of transduced FGF1 from NIH 3T3 cells and endogenous FGF1 from A375 melanoma cells. NIH 3T3 cells transfected with dnCBF1 form colonies in soft agar and produce rapidly growing highly angiogenic tumors in nude mice. The transformed phenotype of dnCBF1 transfected cells is efficiently blocked by dn forms of FGF receptor 1 and S100A13, which is a component of FGF1 export pathway. FGF1 export and acceleration of cell growth induced by dnCBF1 depend on SphK1. Similar to dnCBF1, dnMAML transfection induces FGF1 expression and release, and accelerates cell proliferation. The latter effect is strongly decreased in FGF1 null cells. We suggest that the regulation of FGF1 expression and release by CBF1-mediated Notch signaling can play an important role in tumor formation. 2011 Wiley-Liss, Inc
— id: 137084, year: 2011, vol: 226, page: 3064, stat: Journal Article,

Distinct functions of Sox2 control self-renewal and differentiation in the osteoblast lineage
Seo E; Basu-Roy U; Zavadil J; Basilico C; Mansukhani A
2011 Nov;31(22):4593-4608, Molecular & cellular biology
The transcription factor Sox2 is a key player in the maintenance of pluripotency and stemness. We have previously shown that Sox2 maintains self-renewal in the osteoblast lineage while inhibiting differentiation. Sox2 also interferes with Wnt signaling by binding beta-catenin, a central mediator of the Wnt pathway. Here we show that these multiple functions of Sox2 are encoded in distinct domains. The self-renewal function of Sox2 is dependent on its transcriptional activity, and requires both its DNA-binding and C-terminal activation regions, while only the third C-terminal-transactivation region is required for binding beta-catenin and interfering with Wnt-induced transcription. Gene expression analysis upon Sox2 deletion strongly supports the notion that Sox2 maintains stemness. We also show that Sox2 suppresses differentiation by attenuating Wnt signaling by posttranscriptional and transcriptional mechanisms and that negative regulators of the Wnt pathway, APC and GSK3beta are direct Sox2 targets in osteoblasts. Several genes associated with stemness such as FoxP1 and BMI-1 are downregulated upon Sox2 inactivation. Constitutive expression of the polycomb complex member, BMI-1, can bypass the Sox2 requirement for self-renewal, but does not affect differentiation. Our results establish a connection between Sox2 and BMI-1 in maintaining self-renewal and identify BMI-1 as a key mediator of Sox2 function
— id: 137541, year: 2011, vol: 31, page: 4593, stat: Journal Article,

The transcription factor Sox2 is required for osteoblast self-renewal
Basu-Roy, U; Ambrosetti, D; Favaro, R; Nicolis, S K; Mansukhani, A; Basilico, C
2010 Aug;17(8):1345-1353, Cell death & differentiation
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2-null cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2-null cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage
— id: 110867, year: 2010, vol: 17, page: 1345, stat: Journal Article,

Sox2 Is Required For The Self-renewal Of The Osteoblast Lineage
Mansukhani, A; Roy, UB; Nicolis, S; Basilico, C
2010 MAR ;46(5):S53-S53, Bone
— id: 109713, year: 2010, vol: 46, page: S53, stat: Journal Article,

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G(2) arrest in RCS chondrocytes
Tran, Tri; Kolupaeva, Victoria; Basilico, Claudio
2010 Nov;9(21):4379-4386, Cell cycle
Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G(1) phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G(2) phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G(1) arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G(2) arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G(2) checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G(2) arrest. Additionally our results indicate that the transient G(2) arrest is induced by FGF in RCS cell through mechanisms that are independent of the G(1) arrest, and that the G(2) block is not strictly required for the sustained G(1) arrest but may provide a pausing mechanism that allows the FGF response to be fully established
— id: 114189, year: 2010, vol: 9, page: 4379, stat: Journal Article,

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology
Holmes, Greg; Rothschild, Gerson; Roy, Upal Basu; Deng, Chu-Xia; Mansukhani, Alka; Basilico, Claudio
2009 Apr 15;328(2):273-284, Developmental biology (Orlando)
Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form
— id: 99591, year: 2009, vol: 328, page: 273, stat: Journal Article,

Fibroblast growth factor signaling uses multiple mechanisms to inhibit Wnt-induced transcription in osteoblasts
Ambrosetti, Davide; Holmes, Greg; Mansukhani, Alka; Basilico, Claudio
2008 Aug;28(15):4759-4771, Molecular & cellular biology
Fibroblast growth factor (FGF) and Wnt signals are both critical for proper bone development. We previously reported that the expression of activating FGF receptor mutations in osteoblasts downregulated the expression of many genes reported as targets of Wnt signaling, suggesting an antagonistic effect between Wnt signaling, which promotes osteoblast differentiation and function, and FGF signaling, which inhibits these processes. To analyze the effect of FGF on Wnt signaling in osteoblasts, we created reporter cell lines where a Wnt-responsive promoter drives luciferase expression and showed that Wnt3a-induced luciferase expression was specifically inhibited by FGF treatment. FGF specifically prevented the formation of a Wnt-induced transcriptional complex of TCF1 and -4 with beta-catenin on DNA. FGF did not significantly affect the activation of beta-catenin, although it reduced both the expression of TCF/LEF factors and their induction by Wnt. Microarray analysis using osteoblasts treated with Wnt3a and FGF alone or in combination showed that about 70% of the genes induced by Wnt3a were downregulated by combined FGF treatment. These included novel and previously identified Wnt target genes and genes involved in osteoblast differentiation. Furthermore, FGF alone could downregulate the expression of four Fzd Wnt receptor genes. Our results show that FGF antagonizes Wnt signaling by inhibiting Wnt-induced transcription and suggest that multiple mechanisms, including downregulation of TCFs and Wnt receptors, contribute to this effect
— id: 80816, year: 2008, vol: 28, page: 4759, stat: Journal Article,

Protein phosphatase PP2A mediates FGF-induced p107 dephosphorylation in chondrocytes
Kolupaeva, V; Laplantine, E; Basilico, C
2008 MAR ;42(5):S56-S56, Bone
— id: 76443, year: 2008, vol: 42, page: S56, stat: Journal Article,

PP2A-mediated dephosphorylation of p107 plays a critical role in chondrocyte cell cycle arrest by FGF
Kolupaeva, Victoria; Laplantine, Emmanuel; Basilico, Claudio
2008 ;3(10):e3447-e3447, PLoS ONE
FGF signaling inhibits chondrocyte proliferation, a cell type-specific response that is the basis for several genetic skeletal disorders caused by activating FGFR mutations. This phenomenon requires the function of the p107 and p130 members of the Rb protein family, and p107 dephosphorylation is one of the earliest distinguishing events in FGF-induced growth arrest. To determine whether p107 dephoshorylation played a critical role in the chondrocyte response to FGF, we sought to counteract this process by overexpressing in RCS chondrocytes the cyclin D1/cdk4 kinase complex. CyclinD/cdk4-expressing RCS cells became resistant to FGF-induced p107 dephosphorylation and growth arrest, and maintained significantly high levels of cyclin E/cdk2 activity and of phosphorylated p130 at later times of FGF treatment. We explored the involvement of a phosphatase in p107 dephosphorylation. Expression of the SV40 small T-Ag, which inhibits the activity of the PP2A phosphatase, or knockdown of the expression of the PP2A catalytic subunit by RNA interference prevented p107 dephosphorylation and FGF-induced growth arrest of RCS cells. Furthermore, an association between p107 and PP2A was induced by FGF treatment. Our data show that p107 dephosphorylation is a key event in FGF-induced cell cycle arrest and indicate that in chondrocytes FGF activates the PP2A phosphatase to promote p107 dephosphorylation
— id: 93562, year: 2008, vol: 3, page: e3447, stat: Journal Article,

Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways
Raucci, Angela; Bellosta, Paola; Grassi, Roberta; Basilico, Claudio; Mansukhani, Alka
2008 May;215(2):442-451, Journal of cellular physiology
Skeletal development requires the correct balance of osteoblast proliferation, survival, and differentiation which is modulated by a network of signaling pathways and transcription factors. We have examined the role of the AKT (PKB), and ERK1/2 signaling pathways in the osteoblast response to FGFs, which inhibit differentiation, and to IGF-1 and Wnt signaling, which promote it. Using osteoblastic cell lines as well as primary calvarial osteoblasts, we show that ERK1/2 and AKT have distinct effects in FGF-induced osteoblast proliferation and differentiation. ERK1/2 is a primary mediator of FGF-induced proliferation, but also contributes to osteoblast differentiation, while AKT is important for osteoblast survival. Signaling by IGF-1, that promotes osteoblast differentiation, strongly activates AKT and weakly ERK1/2, while the opposite results are obtained with FGF, which inhibits differentiation. By introducing a constitutively active form of AKT, we found that increased AKT activity drives osteoblasts to differentiation. Increasing the AKT signal in osteoblasts that harbor FGFR2 activating mutations, found in Crouzon (342Y) and Apert (S22W) syndromes, is also able to drive differentiation in these cells, that normally fail to differentiate. Wnt signals, that promotes differentiation, also induce AKT phosphorylation, and cells expressing active AKT have increased levels of stabilized beta-catenin, a central molecule in Wnt signaling. Our results indicate that the relative strengths of ERK and AKT signaling pathways determine whether osteoblasts are driven into proliferation or differentiation, and that the effects of AKT may be due, in part, to synergy with the Wnt pathway as well as with the Runx2 transcription factor. J. Cell. Physiol. (c) 2007 Wiley-Liss, Inc
— id: 74656, year: 2008, vol: 215, page: 442, stat: Journal Article,

Identification of active transcriptional regulatory modules by the functional assay of DNA from nucleosome-free regions
Yaragatti, Mahesh; Basilico, Claudio; Dailey, Lisa
2008 Jun;18(6):930-938, Genome research
The identification of transcriptional regulatory modules within mammalian genomes is a prerequisite to understanding the mechanisms controlling regulated gene expression. While high-throughput microarray- and sequencing-based approaches have been used to map the genomic locations of sites of nuclease hypersensitivity or target DNA sequences bound by specific protein factors, the identification of regulatory elements using functional assays, which would provide important complementary data, has been relatively rare. Here we present a method that permits the functional identification of active transcriptional regulatory modules using a simple procedure for the isolation and analysis of DNA derived from nucleosome-free regions (NFRs), the 2% of the cellular genome that contains these elements. The more than 100 new active regulatory DNAs identified in this manner from F9 cells correspond to both promoter-proximal and distal elements, and display several features predicted for endogenous transcriptional regulators, including localization within DNase-accessible chromatin and CpG islands, and proximity to expressed genes. Furthermore, comparison with published ChIP-seq data of ES-cell chromatin shows that the functional elements we identified correspond with genomic regions enriched for H3K4me3, a histone modification associated with active transcriptional regulatory elements, and that the correspondence of H3K4me3 with our promoter-distal elements is largely ES-cell specific. The majority of the distal elements exhibit enhancer activity. Importantly, these functional DNA fragments are an average 149 bp in length, greatly facilitating future applications to identify transcription factor binding sites mediating their activity. Thus, this approach provides a tool for the high-resolution identification of the functional components of active promoters and enhancers
— id: 80304, year: 2008, vol: 18, page: 930, stat: Journal Article,

Downregulation of Akt activity contributes to the growth arrest induced by FGF in chondrocytes
Priore, Riccardo; Dailey, Lisa; Basilico, Claudio
2006 Jun;207(3):800-808, Journal of cellular physiology
Unregulated FGF signaling produced by activating FGFR3 mutations causes several forms of dwarfism-associated chondrodysplasias in humans and mice. FGF signaling inhibits chondrocyte proliferation by activating multiple signal transduction pathways that all contribute to chondrocyte growth arrest and induction of some aspects of differentiation. Previous studies had identified the Stat1 pathway, dephosphorylation of the Rb family proteins p107 and p130, induction of p21 expression and sustained activation of MAP kinases as playing a role in the FGF response of chondrocytes. We have examined the role of Akt (PKB) in the response of chondrocytes to FGF signaling. Differently from what is observed in many other cell types, FGF does not activate Akt in chondrocytes, and Akt phosphorylation is actually downregulated after FGF treatment. By expressing a constitutively activated, myristylated form of Akt (myr-Akt) in the RCS chondrosarcoma cell line, we show that Akt activation partially counteracts the inhibitory effect of FGF signaling. The response of myr-Akt expressing cells to FGF is identical to parental RCS in the first few hours after treatment, but then diverges as myr-Akt cells show decreased p130 phosphorylation, increased cyclin E/cdk2 activity and continue to proliferate at a slow rate. Constitutive Akt activation does not affect p21 expression but appears to influence directly cdk/cyclin activity. On the other hand, the induction of differentiation-related genes is unchanged in myr-Akt cells. These results identify Akt downregulation as an important aspect of the response of chondrocytes to FGF that, however, only affects chondrocyte proliferation and not the ability of FGF to induce differentiation genes
— id: 64470, year: 2006, vol: 207, page: 800, stat: Journal Article,

Introduction to the special FGF issue
Basilico C
2005 Apr;16(2):105-106, Cytokine & growth factor reviews
— id: 55972, year: 2005, vol: 16, page: 105, stat: Journal Article,

A promiscuous liaison between IL-15 receptor and Axl receptor tyrosine kinase in cell death control
Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Thon, Lutz; Mamat, Uwe; Bellosta, Paola; Basilico, Claudio; Adam, Dieter; Paus, Ralf; Bulfone-Paus, Silvia
2005 Dec 21;24(24):4260-4270, EMBO journal
Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha
— id: 95115, year: 2005, vol: 24, page: 4260, stat: Journal Article,

Mechanisms underlying differential responses to FGF signaling
Dailey, Lisa; Ambrosetti, Davide; Mansukhani, Alka; Basilico, Claudio
2005 Apr;16(2):233-247, Cytokine & growth factor reviews
Fibroblast growth factors (FGFs) are key regulators of several developmental processes in which cell fate and differentiation to various tissue lineages are determined. The importance of the proper spatial and temporal regulation of FGF signals is evident from human and mouse genetic studies which show that mutations leading to the dysregulation of FGF signals cause a variety of developmental disorders including dominant skeletal diseases and cancer. The FGF ligands signal via a family of receptor tyrosine kinases and, depending on the cell type or stage of maturation, produce diverse biological responses that include proliferation, growth arrest, differentiation or apoptosis. A central issue in FGF biology is to understand how these diverse cellular responses are determined and how similar signaling inputs can generate distinct patterns of gene expression that govern the specificity of the cellular response. In this review we draw upon studies from the past fifteen years and attempt to construct a molecular picture of the different levels of regulation by which such specific cellular responses could be achieved by FGF signals. We discuss whether specificity could lie in the nature of the ligand, the particular receptor, the signal transduction pathways utilized, or the transcriptional regulation of specific genes. Finally, we also discuss how the interplay of FGF signals with other signaling systems could contribute to the cellular response. In particular we focus on the interaction with the Wnt pathway since FGF/Wnt cross-talk is emerging as an important nexus in regulating a variety of biological processes
— id: 55971, year: 2005, vol: 16, page: 233, stat: Journal Article,

Fibroblast growth factors share binding sites in heparan sulphate
Kreuger, Johan; Jemth, Per; Sanders-Lindberg, Emil; Eliahu, Liat; Ron, Dina; Basilico, Claudio; Salmivirta, Markku; Lindahl, Ulf
2005 Jul 1;389(Pt 1):145-150, Biochemical journal
HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs
— id: 95116, year: 2005, vol: 389, page: 145, stat: Journal Article,

Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation
Mansukhani, Alka; Ambrosetti, Davide; Holmes, Greg; Cornivelli, Lizbeth; Basilico, Claudio
2005 Mar 28;168(7):1065-1076, Journal of cell biology
Activating mutations in fibroblast growth factor receptor 2 (FGFR2) cause several craniosynostosis syndromes by affecting the proliferation and differentiation of osteoblasts, which form the calvarial bones. Osteoblasts respond to FGF with increased proliferation and inhibition of differentiation. We analyzed the gene expression profiles of osteoblasts expressing FGFR2 activating mutations (C342Y or S252W) and found a striking down-regulation of the expression of many Wnt target genes and a concomitant induction of the transcription factor Sox2. Most of these changes could be reproduced by treatment of osteoblasts with exogenous FGF. Wnt signals promote osteoblast function and regulate bone mass. Sox2 is expressed in calvarial osteoblasts in vivo and we show that constitutive expression of Sox2 inhibits osteoblast differentiation and causes down-regulation of the expression of numerous Wnt target genes. Sox2 associates with beta-catenin in osteoblasts and can inhibit the activity of a Wnt responsive reporter plasmid through its COOH-terminal domain. Our results indicate that FGF signaling could control many aspects of osteoblast differentiation through induction of Sox2 and regulation of the Wnt-beta-catenin pathway
— id: 55788, year: 2005, vol: 168, page: 1065, stat: Journal Article,

A conserved enhancer element that drives FGF4 gene expression in the embryonic myotomes is synergistically activated by GATA and bHLH proteins
Iwahori, Akiyo; Fraidenraich, Diego; Basilico, Claudio
2004 Jun 15;270(2):525-537, Developmental biology (Orlando)
FGF4 is the earliest member of the fibroblast growth factor (FGF) family expressed during embryogenesis where it plays essential roles in post-implantation development and limb growth and patterning. The expression of the Fgf4 gene in specific developmental stages, including the ICM of the blastocyst, the myotomes, and the limb bud AER, is regulated by distinct enhancer elements (Hom) in the 3' UTR. We previously identified the Hom3a region as the major DNA element responsible for Fgf4 expression in the myotomes and AER, and showed that a conserved E-box is a target for the myogenic bHLH transcription factors MYF5 and MYOD. To further define the cis- and trans-acting elements that determine Hom3a activity, we conducted a mutational analysis of the ability of the Hom3a region to drive lacZ expression in the myotomes of transgenic mice. We identified a minimal enhancer of 226nt that contains four elements, including the E-box, necessary to drive gene expression in the myotomes. One of these elements is a binding site for the GATA family of transcription factors, and we show here that GATA 1-4 and 6 can synergize with MYF5 or MYOD to activate transcription of a reporter plasmid driven by a portion of the Hom3a enhancer including the GATA site and the E-box. In line with this finding, we could show a direct interaction between MYF5/MYOD and GATA-3 or GATA-4, mediated by the N-terminal and bHLH domains of MYF5/MYOD and the C-terminal zing finger domain of GATA-3/4. To further study the role of the Hom3a enhancer in directing Fgf4 expression and the function of FGF4 in limb and muscle development, we generated mutant mice in which the Fgf4 Hom3a region had been deleted (Delta3a). In situ hybridization analysis of sections from Delta3a/ Delta3a embryos at E11.5 showed a drastically reduced expression of Fgf4 mRNA in the myotomes and AER. However, these mice developed normally and show no limb or muscle defects, and the same was true of heterozygous mice in which one Fgf4 allele carried the Hom3a deletion and the other was a null allele (Delta3a/Fgf4(-)). Together, these results show that Hom3a is the major DNA enhancer element directing Fgf4 expression in myotomes and limb bud AER, and that its activity in the myotomes results at least in part from the synergistic action of GATA and bHLH myogenic factors that bind to evolutionary conserved sequences in the Hom3a enhancer. However, expression of Fgf4 in the myotomes or AER of murine embryos does not appear to be essential for muscle or limb development
— id: 44724, year: 2004, vol: 270, page: 525, stat: Journal Article,

Insights into the molecular basis for fibroblast growth factor receptor autoinhibition and ligand-binding promiscuity
Olsen, Shaun K; Ibrahimi, Omar A; Raucci, Angela; Zhang, Fuming; Eliseenkova, Anna V; Yayon, Avner; Basilico, Claudio; Linhardt, Robert J; Schlessinger, Joseph; Mohammadi, Moosa
2004 Jan 27;101(4):935-940, Proceedings of the National Academy of Sciences of the United States of America
The prototypical fibroblast growth factor receptor (FGFR) extracellular domain consists of three Ig domains (D1-D3) of which the two membrane-proximal D2 and D3 domains and the interconnecting D2-D3 linker bear the determinants of ligand binding and specificity. In contrast, D1 and the D1-D2 linker are thought to play autoinhibitory roles in FGFR regulation. Here, we report the crystal structure of the three-Ig form of FGFR3c in complex with FGF1, an FGF that binds promiscuously to each of the seven principal FGFRs. In this structure, D1 and the D1-D2 linker are completely disordered, demonstrating that these regions are dispensable for FGF binding. Real-time binding experiments using surface plasmon resonance show that relative to two-Ig form, the three-Ig form of FGFR3c exhibits lower affinity for both FGF1 and heparin. Importantly, we demonstrate that this autoinhibition is mediated by intramolecular interactions of D1 and the D1-D2 linker with the minimal FGF and heparin-binding D2-D3 region. As in the FGF1-FGFR2c structure, but not the FGF1-FGFR1c structure, the alternatively spliced betaC'-betaE loop is ordered and interacts with FGF1 in the FGF1-FGFR3c structure. However, in contrast to the FGF1-FGFR2c structure in which the betaC'-betaE loop interacts with the beta-trefoil core region of FGF1, in the FGF1-FGFR3c structure, this loop interacts extensively with the N-terminal region of FGF1, underscoring the importance of the FGF1 N terminus in conferring receptor-binding affinity and promiscuity. Importantly, comparison of the three FGF1-FGFR structures shows that the flexibility of the betaC'-betaE loop is a major determinant of ligand-binding specificity and promiscuity
— id: 42613, year: 2004, vol: 101, page: 935, stat: Journal Article,

Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes
Raucci, Angela; Laplantine, Emmanuel; Mansukhani, Alka; Basilico, Claudio
2004 Jan 16;279(3):1747-1756, Journal of biological chemistry
Fibroblast growth factors (FGFs) regulate long bone development by affecting the proliferation and differentiation of chondrocytes. FGF treatment inhibits the proliferation of chondrocytes both in vitro and in vivo, but the signaling pathways involved have not been clearly identified. In this report we show that both the MEK-ERK1/2 and p38 MAPK pathways, but not phospholipase C gamma or phosphatidylinositol 3-kinase, play a role in FGF-mediated growth arrest of chondrocytes. Chemical inhibitors of the MEK1/2 or the p38 MAPK pathways applied to rat chondrosarcoma (RCS) chondrocytes significantly prevented FGF-induced growth arrest. The retinoblastoma family members p107 and p130 were previously shown to be essential effectors of FGF-induced growth arrest in chondrocytes. The dephosphorylation of p107, one of the earliest events in RCS growth arrest, was significantly blocked by MEK1/2 inhibitors but not by the p38 MAPK inhibitors, whereas that of p130, which occurs later, was partially prevented both by the MEK and p38 inhibitors. Furthermore, by expressing the nerve growth factor (NGF) receptor, TrkA, and the epidermal growth factor (EGF) receptor, ErbB1, in RCS cells we show that NGF treatment of the transfected cells caused growth inhibition, whereas EGF did not. FGF- and NGF-induced growth inhibition is accompanied by a strong and sustained activation of ERK1/2 and p38 MAPK and a decrease of AKT phosphorylation, whereas EGF induces a much more transient activation of p38 and ERK1/2 and increases AKT phosphorylation. These results indicate that inhibition of chondrocyte proliferation by FGF requires both ERK1/2 and p38 MAPK signaling and also suggest that sustained activation of these pathways is required to achieve growth inhibition
— id: 48184, year: 2004, vol: 279, page: 1747, stat: Journal Article,

p21(WAF1/CIP1) acts as a brake in osteoblast differentiation
Bellosta, Paola; Masramon, Laia; Mansukhani, Alka; Basilico, Claudio
2003 May;18(5):818-826, Journal of bone & mineral research
Continuous fibroblast growth factor signaling inhibits the differentiation of primary osteoblasts and osteoblastic cell lines. We studied the expression of several cell cycle regulatory molecules in response to fibroblast growth factor, and found that fibroblast growth factor strongly upregulates the expression of p21(WAF1/CIP1), a CDK inhibitor that has also been implicated in the regulation of apoptosis and cell differentiation. To test the hypothesis that p21 mediated the fibroblast growth factor effects on osteoblasts, we studied the differentiation of primary osteoblasts and osteoblastic cell lines derived from p21 null mice in the presence or absence of fibroblast growth factor. While the results obtained indicate that p21 is not the major mediator of the inhibition of osteoblast differentiation by fibroblast growth factor, we found that p21 per se acts as a brake on osteoblast proliferation and differentiation. p21 is strongly downregulated during differentiation and is highly expressed in osteoblastic cell lines expressing activated FGFR2, which do not differentiate. p21 null osteoblasts differentiate faster than wild-type cells, are more susceptible to the differentiation-promoting action of BMP-2, and undergo increased differentiation-related apoptosis. Furthermore, transient overexpression of p21 from an adenovirus vector delayed the onset of differentiation both in wild-type and in p21 null osteoblasts. These results highlight a new function for p21 in osteoblast differentiation
— id: 34520, year: 2003, vol: 18, page: 818, stat: Journal Article,

FGF signaling initiates multiple pathways to induce growth arrest and promote hypertrophic differentiation of chondrocytes
Dailey, L; LaPlantine, E; Priore, R; Basilico, C
2003 MAY ;32(5):S144-S144, Bone
— id: 38552, year: 2003, vol: 32, page: S144, stat: Journal Article,

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation
Dailey, Lisa; Laplantine, Emmanuel; Priore, Riccardo; Basilico, Claudio
2003 Jun 23;161(6):1053-1066, Journal of cell biology
Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E-Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate
— id: 39184, year: 2003, vol: 161, page: 1053, stat: Journal Article,

Fibroblast growth factor (FGF) signaling in immature and mature osteoblasts
Mansukhani, A; Cornivelli, L; Basilico, C
2003 MAY ;32(5):S144-S144, Bone
— id: 38551, year: 2003, vol: 32, page: S144, stat: Journal Article,

Fibroblast growth factor 2 promotes tumor progression in an autochthonous mouse model of prostate cancer
Polnaszek, Nathaniel; Kwabi-Addo, Bernard; Peterson, Leif E; Ozen, Mustafa; Greenberg, Norman M; Ortega, Sagrario; Basilico, Claudio; Ittmann, Michael
2003 Sep 15;63(18):5754-5760, Cancer research
Fibroblast growth factor (FGF) 2 (or basic FGF) is expressed at increased levels in human prostate cancer. FGF2 can promote cell motility and proliferation, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGF2 plays a critical role in prostate cancer progression, we have used the transgenic adenocarcinoma of the mouse prostate (TRAMP) model system. A high percentage of TRAMP mice develop metastatic prostate cancer, and thus the TRAMP model is useful for evaluating cancer progression. TRAMP mice were crossed with FGF2 knockout (FGF2(-/-)) mice, and tumor progression in TRAMP mice that were either hemi- or homozygous for inactivation of the FGF2 allele was compared with progression in wild-type TRAMP mice. Inactivation of even one FGF2 allele resulted in increased survival, a decrease in metastasis, and inhibition of progression to the poorly differentiated phenotype in primary prostatic tumors. When compared with wild-type mice, poorly differentiated tumors arising in FGF(+/-) and FGF(-/-) mice expressed higher levels of vascular endothelial growth factor and, in some cases, increased levels of acidic FGF intracellular binding protein, a nuclear FGF1-binding protein. These findings suggest that both FGF2-mediated angiogenesis and intranuclear FGF2 activities may promote tumor progression and support the hypothesis that FGF2 plays a significant role in prostate cancer progression in vivo
— id: 44725, year: 2003, vol: 63, page: 5754, stat: Journal Article,

Role of fibroblast growth factor type 1 and 2 in carbon tetrachloride-induced hepatic injury and fibrogenesis
Yu, Chundong; Wang, Fen; Jin, Chengliu; Huang, Xinqiang; Miller, David L; Basilico, Claudio; McKeehan, Wallace L
2003 Oct;163(4):1653-1662, American journal of pathology
Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis
— id: 44726, year: 2003, vol: 163, page: 1653, stat: Journal Article,

Fibroblast growth factor 2 is necessary for the growth of glutamate projection neurons in the anterior neocortex
Korada, Sailaja; Zheng, Wei; Basilico, Claudio; Schwartz, Michael L; Vaccarino, Flora M
2002 Feb 1;22(3):863-875, Journal of neuroscience
Basic fibroblast growth factor (Fgf2) is required for the generation of founder cells within the dorsal pseudostratified ventricular epithelium, which will generate the cerebral cortex, but the ganglionic eminences are not affected. We report here that the Fgf2 null mutant mice show an approximately 40% decrease in cortical glutamatergic pyramidal neurons. In contrast, no change in pyramidal or granule cell number is detected in the hippocampus of Fgf2 -/- mice. In addition, the soma of the pyramidal cells in the frontal and parietal cortices are smaller in Fgf2 knock-out mice. The decrease in the number and size of glutamatergic neuronal population affects all cortical layers but is restricted to the frontal and parietal cortices without any change in the occipital cortex, indicating that Fgf2 is necessary to regulate cell number and size in the anterior cerebral cortex. In contrast to pyramidal neurons, cortical GABA interneurons are unaffected by the lack of Fgf2. The resulting imbalance between the excitatory and inhibitory neurotransmission in the cerebral cortex is reflected by an increased duration of sleep when the animals receive a GABA receptor agonist. Thus, Fgf2 signaling may contribute to the regional specification of the cerebral cortex and may play a role in increasing the size of anterior cortical regions during vertebrate evolution
— id: 34523, year: 2002, vol: 22, page: 863, stat: Journal Article,

FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest
Laplantine, Emmanuel; Rossi, Ferdinand; Sahni, Malika; Basilico, Claudio; Cobrinik, David
2002 Aug 19;158(4):741-750, Journal of cell biology
Unregulated FGF signaling affects endochondral ossification and long bone growth, causing several genetic forms of human dwarfism. One major mechanism by which FGFs regulate endochondral bone growth is through their inhibitory effect on chondrocyte proliferation. Because mice with targeted mutations of the retinoblastoma (Rb)-related proteins p107 and p130 present severe endochondral bone defects with excessive chondrocyte proliferation, we have investigated the role of the Rb family of cell cycle regulators in the FGF response. Using a chondrocyte cell line, we found that FGF induced a rapid dephosphorylation of all three proteins of the Rb family (pRb, p107, and p130) and a blockade of the cells in the G1 phase of the cell cycle. This cell cycle block was reversed by inactivation of Rb proteins with viral oncoproteins such as polyoma large T (PyLT) antigen and Adenovirus E1A. Expression of a PyLT mutant that efficiently binds pRb, but not p107 and p130, allowed the cells to be growth inhibited by FGF, suggesting that pRb itself is not involved in the FGF response. To investigate more precisely the role of the individual Rb family proteins in FGF-mediated growth inhibition, we used chondrocyte micromass culture of limb bud cells isolated from mice lacking Rb proteins individually or in combination. Although wild-type as well as Rb-/- chondrocytes were similarly growth inhibited by FGF, chondrocytes null for p107 and p130 did not respond to FGF. Furthermore, FGF treatment of metatarsal bone rudiments obtained from p107-/-;p130-/- embryos failed to inhibit proliferation of growth plate chondrocytes, whereas rudiments from p107-null or p130-null embryos showed only a slight inhibition of growth. Our findings indicate that p107 and p130, but not pRb, are critical effectors of FGF-mediated growth inhibition in chondrocytes
— id: 34521, year: 2002, vol: 158, page: 741, stat: Journal Article,

Fibroblast growth factor induces divergent signals in immature versus mature osteoblasts
Mansukhani, A; Basilico, C
2002 Mar;30(3):C27-, Bone
— id: 27515, year: 2002, vol: 30, page: C27, stat: Journal Article,

Lack of ERK activation and cell migration in FGF-2-deficient endothelial cells
Pintucci, Giuseppe; Moscatelli, David; Saponara, Fiorella; Biernacki, Peter R; Baumann, F Gregory; Bizekis, Costas; Galloway, Aubrey C; Basilico, Claudio; Mignatti, Paolo
2002 Apr;16(6):598-600, FASEB journal
The formation of blood capillaries from preexisting vessels (angiogenesis) and vascular remodeling secondary to atherosclerosis or vessel injury are characterized by endothelial cell migration and proliferation. Numerous growth factors control these cell functions. Basic fibroblast growth factor (FGF-2), a potent angiogenesis inducer, stimulates endothelial cell proliferation, migration, and proteinase production in vitro and in vivo. However, mice genetically deficient in FGF-2 have no apparent vascular defects. We have observed that endothelial cell migration in response to mechanical damage in vitro is accompanied by activation of the extracellular signal-regulated kinase (ERK) pathway, which can be blocked by neutralizing anti-FGF-2 antibodies. Endothelial cells from mice that are genetically deficient in FGF-2 neither migrate nor activate ERK in response to mechanical wounding. Addition of exogenous FGF-2 restores a normal cell response, which shows that impaired migration results from the genetic deficiency of this growth factor. Injury-induced ERK activation in endothelial cells occurs only at the edge of the wound. In addition, FGF-2-induced ERK activation mediates endothelial cell migration in response to wounding without a significant effect on proliferation. These data show that FGF-2 is a key regulator of endothelial cell migration during wound repair
— id: 34522, year: 2002, vol: 16, page: 598, stat: Journal Article,

Identification of receptor and heparin binding sites in fibroblast growth factor 4 by structure-based mutagenesis
Bellosta P; Iwahori A; Plotnikov AN; Eliseenkova AV; Basilico C; Mohammadi M
2001 Sep;21(17):5946-5957, Molecular & cellular biology
Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-A resolution. FGF4 adopts a beta-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the betaC'-betaE loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O- and 6-O-sulfates in heparin to exert its optimal biological activity
— id: 26713, year: 2001, vol: 21, page: 5946, stat: Journal Article,

Polio vaccine samples not linked to AIDS
Blancou P; Vartanian JP; Christopherson C; Chenciner N; Basilico C; Kwok S; Wain-Hobson S
2001 Apr 26;410(6832):1045-1046, Nature
— id: 34526, year: 2001, vol: 410, page: 1045, stat: Journal Article,

Coevolution of HMG domains and homeodomains and the generation of transcriptional regulation by Sox/POU complexes
Dailey L; Basilico C
2001 Mar;186(3):315-328, Journal of cellular physiology
The highly conserved homeodomains and HMG domains are components of a large number of proteins that play a role in the transcriptional regulation of gene expression during embryogenesis. Both the HMG domain and the homeodomain serve as interfaces for factor interactions with DNA, as well as with other proteins, and it is likely that the high degree of structural and sequence conservation within these domains reflects the conservation of basic aspects of these interactions. Classical HMG domain proteins have an ancient origin, being found in all eukaryotes, and are thought to have given rise to the metazoan-specific class of HMG domain proteins called the Sox proteins. Similarly, the metazoan-specific POU domain proteins are thought to have arisen from genes encoding ancestral homeodomain proteins. In this review, we summarize several examples of different HMG-homeodomain interactions that illustrate not only the ancient origin of each of these protein families, but also their relationship to each other, and discuss how coevolution of HMG and homeodomains may have lead to creation of the specialized Sox/POU protein complexes. Using the FGF-4 gene as an example, we also speculate on how coevolution of regulatory Sox/POU target DNA sequences may have occurred, and how the summation of these changes may have lead to the emergence of new developmental pathways
— id: 26799, year: 2001, vol: 186, page: 315, stat: Journal Article,

STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2
Sahni M; Raz R; Coffin JD; Levy D; Basilico C
2001 Jun;128(11):2119-2129, Development
Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. FGF signaling inhibits chondrocyte proliferation and we have previously shown that the transcription factor STAT1 mediates the growth inhibitory effect of FGF in vitro. We provide genetic evidence that STAT1 is a modulator of the negative regulation of bone growth by FGF in vivo. We crossed Stat1(-/-) mice with a transgenic mouse line overexpressing human FGF2 (TgFGF). TgFGF mice exhibit phenotypes characterized by chondrodysplasia and macrocephaly, which affect endochondral and intramembranous ossification. We found that the chondrodysplasic phenotype of these mice results both from reduced proliferation and increased apoptosis of growth plate chondrocytes. Loss of STAT1 function in TgFGF mice led to a significant correction of the chondrodysplasic phenotype, but did not affect the skull malformations. The reduced proliferation of TgFGF growth plate chondrocytes, as well as their excessive apoptosis, were restored to near-normal levels in the absence of STAT1 function. Unregulated FGF signaling in TgFGF mice also induced apoptosis in calvarial osteoblasts that was not, however, corrected by the absence of STAT1. Detailed analysis of Stat1(-/-) growth plates uncovered a transient phenotype, characterized by an expansion of the proliferative zone and by acceleration of longitudinal bone growth, that attenuated as the animals grew older. These results document an essential role for STAT1 in FGF-mediated regulation of cell growth that is specific to the epiphyseal growth plate
— id: 26721, year: 2001, vol: 128, page: 2119, stat: Journal Article,

Serum levels of interleukin-18 in patients with uncomplicated Plasmodium falciparum malaria
Torre D; Giola M; Speranza F; Matteelli A; Basilico C; Biondi G
2001 Apr-Jun;12(2):361-364, European cytokine network
Interleukin (IL)-18, a newly discovered cytokine produced primarily by macrophages, has been shown to induce gamma interferon (IFN-gamma) production by natural killer cells, to induce the T helper type 1 response. To further elucidate the role of this cytokine in uncomplicated malaria caused by Plasmodium falciparum, serum levels of IL-18, and gamma interferon (IFN-gamma), determined by an immunoenzymatic assay, were analyzed in 40 adult patients, and in 15 healthy control subjects. A significant increase in serum levels of IL-18 was observed in patients with uncomplicated P. falciparum malaria on admission, whereas serum levels of IFN-gamma tended to increase although not significantly. Serum levels of IL-18 decreased three days later, but still remained significantly high, whereas IFN-gamma levels returned to normal levels compared to the controls. No significant correlation was found between parasitemia and serum levels of IL-18 and IFN-gamma. The increase of IL-18 levels during acute and recovery phases of uncomplicated P. falciparum malaria may reflect a proinflammatory role of IL-18 in these patients. An early and effective immune response regulated by proinflammatory Th1 cytokines, including tumor necrosis factor (TNF), interleukin (IL)-12, and possibly IFN-gamma may limit the progression from uncomplicated malaria to severe and life-threatening complications
— id: 34525, year: 2001, vol: 12, page: 361, stat: Journal Article,

Evolution of coinfection with human immunodeficiency virus and hepatitis C virus in patients treated with highly active antiretroviral therapy
Torre D; Tambini R; Cadario F; Barbarini G; Moroni M; Basilico C
2001 Nov 1;33(9):1579-1585, Clinical infectious diseases
A retrospective analysis of data from a cohort of patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) who were treated with highly active antiretroviral therapy (HAART) at 3 infectious diseases units in northern Italy was performed. While the patients were receiving HAART, CD4(+) cell counts significantly increased and HIV RNA serum levels decreased. However, no significant overall changes in alanine aminotransferase (ALT) levels and HCV RNA serum levels were observed. Fifteen (4.6%) of 323 patients died within 3 years of follow-up; death was related to cirrhosis in 5 patients (1.6%). No significant difference was observed between cirrhosis-related mortality and mortality related to other causes. Patients with ALT levels >4 times the normal values at initiation of HAART showed a significant decrease in ALT levels, whereas patients with normal ALT levels at initiation of HAART showed a significant increase over time, suggesting that HAART may have long-term beneficial or detrimental effects, depending on patient characteristics
— id: 34524, year: 2001, vol: 33, page: 1579, stat: Journal Article,

Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer
Ambrosetti DC; Scholer HR; Dailey L; Basilico C
2000 Jul 28;275(30):23387-23397, Journal of biological chemistry
Fibroblast growth factor (FGF)-4 gene expression in the inner cell mass of the blastocyst and in EC cells requires the combined activity of two transcriptional regulators, Sox2 and Oct-3, which bind to adjacent sites on the FGF-4 enhancer DNA and synergistically activate transcription. Sox2 and Oct-3 bind cooperatively to the enhancer DNA through their DNA-binding, high mobility group and POU domains, respectively. These two domains, however, are not sufficient to activate transcription. We have analyzed a number of Sox2 and Oct-3 deletion mutants to identify the domains within each protein that contribute to the activity of the Sox2 x Oct-3 complex. Within Oct-3, we have identified two activation domains, the N-terminal AD1 and the C-terminal AD2, that play a role in the activity of the Sox2 x Oct-3 complex. AD1 also displays transcriptional activation functions in the absence of Sox2 while AD2 function was only detected within the Sox2 x Oct-3 complex. In Sox2, we have identified three activation domains within its C terminus: R1, R2, and R3. R1 and R2 can potentiate weak activation by Sox2 in the absence of Oct-3 but their deletion has no effect on the Sox2 x Oct-3 complex. In contrast, R3 function is only observed when Sox2 is complexed with Oct-3. In addition, analysis of Oct-1/Oct-3 chimeras indicates that the Oct-3 homeodomain also plays a critical role in the formation of a functional Sox2 x Oct-3 complex. Our results are consistent with a model in which the synergistic action of Sox2 and Oct-3 results from two major processes. Cooperative binding of the factors to the enhancer DNA, mediated by their binding domains, stably tethers each factor to DNA and increases the activity of intrinsic activation domains within each protein. Protein-protein and protein-DNA interactions then may lead to reciprocal conformational changes that expose latent activation domains within each protein. These findings define a mechanism that may also be utilized by other Sox x POU protein complexes in gene activation
— id: 11713, year: 2000, vol: 275, page: 23387, stat: Journal Article,

Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog [In Process Citation]
Fraidenraich D; Iwahori A; Rudnicki M; Basilico C
2000 Sep 15;225(2):392-406, Developmental biology (Orlando)
The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.
— id: 11501, year: 2000, vol: 225, page: 392, stat: Journal Article,

Different point mutations in the met oncogene elicit distinct biological properties
Giordano S; Maffe A; Williams TA; Artigiani S; Gual P; Bardelli A; Basilico C; Michieli P; Comoglio PM
2000 Feb;14(2):399-406, FASEB journal
The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met(PRC) receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET(PRC) mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.-Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties
— id: 14406, year: 2000, vol: 14, page: 399, stat: Journal Article,

Distal most limb structures develop despite absence of Shh signaling in mouse
Kraus, P; Fraidenraich, D; Basilico, C; Loomis, CA
2000 JUN 1 ;222(1):267-267, Developmental biology (Orlando)
— id: 54562, year: 2000, vol: 222, page: 267, stat: Journal Article,

Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts
Mansukhani A; Bellosta P; Sahni M; Basilico C
2000 Jun 12;149(6):1297-1308, Journal of cell biology
Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts
— id: 11658, year: 2000, vol: 149, page: 1297, stat: Journal Article,

Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice [published erratum appears in Mol Cell Biol 2000 May;20(10):3752]
Miller DL; Ortega S; Bashayan O; Basch R; Basilico C
2000 Mar;20(6):2260-2268, Molecular & cellular biology
Fibroblast growth factor 1 (FGF1) and FGF2, the prototypic members of the FGF family of growth factors, have been implicated in a variety of physiological and pathological processes. Unlike most other FGFs, FGF1 and FGF2 are ubiquitously expressed and are not efficiently secreted. Gene knockouts in mice have previously demonstrated a role for FGF2 in brain development, blood pressure regulation, and wound healing. The relatively mild phenotypic defects associated with FGF2 deletion led to the hypothesis that the continued expression of other FGFs partially compensated for the absence of FGF2 in these mice. We now report our generation of mice lacking FGF1 and their use, in combination with our previously described FGF2 null mice, to produce mice lacking both FGF1 and FGF2. FGF1-FGF2 double-knockout mice are viable and fertile and do not display any gross phenotypic defects. In the double-knockout mice we observed defects that were similar in extent to those previously described for the FGF2 null mice. Differences in the organization of neurons of the frontal motor cortex and in the rates of wound healing were observed. We also observed in FGF2(-/-) mice and in FGF1-FGF2 double-knockout mice novel impairments in hematopoiesis that were similar in severity. Essentially no abnormalities were found in mice lacking only FGF1. Our results suggest that the relatively mild defects in FGF2 knockout animals are not a consequence of compensation by FGF1 and suggest highly restricted roles for both factors under normal developmental and physiological conditions
— id: 11824, year: 2000, vol: 20, page: 2260, stat: Journal Article,

Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice (vol 20, pg 2260, 2000)
Miller, DL; Ortega, S; Bashayan, O; Basch, R; Basilico, C
2000 MAY ;20(10):3752-3752, Molecular & cellular biology
— id: 54696, year: 2000, vol: 20, page: 3752, stat: Journal Article,

Circulating levels of IL-18 in adult and paediatric patients with HIV-1 infection
Torre D; Speranza F; Martegani R; Pugliese A; Castelli F; Basilico C; Biondi G
2000 Sep 29;14(14):2211-2212, AIDS
— id: 14405, year: 2000, vol: 14, page: 2211, stat: Journal Article,

Levels of the bcl-2 protein, fibronectin and alpha(5)beta(1) fibronectin receptor in HIV-1-infected patients with Kaposi's sarcoma
Torre D; Zeroli C; Martegani R; Pugliese A; Basilico C; Speranza F
2000 Dec;2(15):1831-1833, Microbes & infection
Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of neoplastic cells (spindle cells) mixed with endothelial and inflammatory cells. In this study we evaluated the role of the adhesive glycoprotein, fibronectin (FN) and its receptor alpha(5)beta(1) (FNR), and the proto-oncogene bcl-2, an anti-apoptotic protein. Significantly decreased serum levels of FN were noted in HIV-1-infected patients with KS, whereas serum levels of FNR were significantly increased in the same patients. Furthermore, increased FNR expression was observed on CD4 cells from KS patients. Serum levels of bcl-2 protein were significantly decreased in asymptomatic seropositive patients, whereas HIV-1-infected patients with KS showed increased serum levels of bcl-2. These results provide further information about interaction between integrins and the extracellular matrix and bcl-2 protein that can support cell survival either of neoplastic cells or endothelial and inflammatory cells
— id: 34527, year: 2000, vol: 2, page: 1831, stat: Journal Article,

Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signaling promotes gonadotropin-releasing hormone neuronal survival via extracellular signal-regulated kinase (ERK) and Akt
Allen MP; Zeng C; Schneider K; Xiong X; Meintzer MK; Bellosta P; Basilico C; Varnum B; Heidenreich KA; Wierman ME
1999 Feb;13(2):191-201, Molecular endocrinology
We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1-7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Ax1) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1-7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Ax1) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration
— id: 14408, year: 1999, vol: 13, page: 191, stat: Journal Article,

Mutant Met-mediated transformation is ligand-dependent and can be inhibited by HGF antagonists
Michieli P; Basilico C; Pennacchietti S; Maffe A; Tamagnone L; Giordano S; Bardelli A; Comoglio PM
1999 Sep 16;18(37):5221-5231, Oncogene
Mutations in the genes encoding for Met, Ret and Kit receptor tyrosine kinases invariably result in increased kinase activity and in the acquisition of transforming potential. However, the requirement of receptor ligands for the transformation process is still unclear. We have investigated the role of hepatocyte growth factor (HGF), the high-affinity ligand for Met, in mutant Met-mediated cell transformation. We provide evidence that the transforming potential displayed by mutant forms of Met found in human cancer is not only sensitive but entirely dependent on the presence of HGF, by showing that mutant Met transforms NIH3T3 fibroblasts, which produce endogenous HGF, but is not able to transform epithelial cells, unless exogenous HGF is supplied. Accordingly, mutant Met-induced transformation of NIH3T3 cells can be inhibited by HGF antagonists and increased by HGF stimulation. We also show that an engineered Met receptor which contains an oncogenic mutation but is impaired in its ability to bind HGF completely loses its transforming activity, which can be rescued by causing receptor dimerization using a monoclonal antibody. These results indicate that point mutations resulting in Met kinase activation are necessary but not sufficient to cause cell transformation, the latter being dependent on ligand-induced receptor dimerization. They also suggest that mutant Met-driven tumour growth depends on the availability and tissue distribution of active HGF, and provide proof-of-concept for the treatment of mutant-Met related pathologies by HGF-antagonizing drugs
— id: 14407, year: 1999, vol: 18, page: 5221, stat: Journal Article,

FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway
Sahni M; Ambrosetti DC; Mansukhani A; Gertner R; Levy D; Basilico C
1999 Jun 1;13(11):1361-1366, Genes & development
Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis
— id: 12005, year: 1999, vol: 13, page: 1361, stat: Journal Article,

Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth
Bardelli A; Longati P; Gramaglia D; Basilico C; Tamagnone L; Giordano S; Ballinari D; Michieli P; Comoglio PM
1998 Nov 24;95(24):14379-14383, Proceedings of the National Academy of Sciences of the United States of America
The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of RET and KIT. Recently, homologous residues were found mutated in MET, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear. METPRC mutations result in increased kinase activity and-in some instances, i.e., M1250T substitution-in changes in substrate specificity. A direct correlation occurs between the transforming potential of METPRC mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y1349VHVNATY1356VNV) located in the receptor tail. Substitution of these 'docking tyrosines' with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness in vitro. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (YpVNV) inhibits invasive growth induced by METPRC mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated MET and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene
— id: 14409, year: 1998, vol: 95, page: 14379, stat: Journal Article,

Distinct regulatory elements govern Fgf4 gene expression in the mouse blastocyst, myotomes, and developing limb
Fraidenraich D; Lang R; Basilico C
1998 Dec 1;204(1):197-209, Developmental biology (Orlando)
Embryonic development requires a complex program of events which are directed by a number of signaling molecules whose expression must be rigorously regulated. We previously showed that expression of Fgf4, which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in EC cells by the synergistic interaction of Sox2 and Oct-3 with the Fgf4 EC cell-specific enhancer. To verify whether this mechanism was also operating in vivo, and to identify new elements controlling Fgf4 gene expression in distinct developmental stages, we have analyzed the expression of LacZ reporter plasmids containing different fragments of the Fgf4 gene in transgenic mouse embryos. Utilizing these transgenic constructs we have been able to recapitulate, for the most part, Fgf4 gene expression during embryonic development. We show here that most of the cis-acting regulatory elements determining Fgf4 embryonic expression are located in conserved regions within the 3' UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct-proteins binding sites. We were also able to identify specific and distinct enhancer elements that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Finally, we present evidence that the very restricted pattern of expression of Fgf4 transcripts in the AER results from the combined action of positive and negative regulatory elements located 3' of the Fgf4 coding sequences. Thus the Fgf4 gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression.
— id: 7333, year: 1998, vol: 204, page: 197, stat: Journal Article,

FGF signaling activates STAT1 and p21 and inhibits the estrogen response and proliferation of MCF-7 cells
Johnson MR; Valentine C; Basilico C; Mansukhani A
1998 May;16(20):2647-2656, Oncogene
Normal breast tissue as well as most breast tumors are dependent on estrogen for growth. Breast tumors often progress to a hormone-independent state which is associated with poor prognosis. It has been proposed that activation of growth factor signaling pathways in the tumor cells may free them from hormonal control. Certain growth factors can mimic estrogen responses by activating the estrogen receptor via its phosphorylation by mitogen-activated protein (MAP) kinase. In this report, however, we show that fibroblast growth factor (FGF), despite activating MAP kinase, is growth-inhibitory for estrogen-dependent MCF-7 breast cancer cells. MCF-7 cells treated with FGFs exhibit slower growth than controls in both the presence and absence of estrogen, with a concomitant increase in the number of cells in G0/G1. Expression of a constitutively activated FGF receptor in these cells further decreases their growth rate, which is no longer influenced by FGF treatment. Activation of the FGF signaling pathway also reduces the induction of an estrogen-responsive CAT reporter plasmid by estrogen, an effect which appears to be independent of serine 118 in the estrogen receptor, a MAP kinase target site. The inhibitory effects of FGF are probably mediated through the sustained induction of the cyclin kinase inhibitor p21/WAF1/CIP1, which is upregulated at the mRNA and protein level by FGF. FGF treatment also results in the phosphorylation of STAT1. This upregulation of p21 and phosphorylation of STAT1 is not detectable in T47D breast cancer cells upon which FGF has no inhibitory effect
— id: 8389, year: 1998, vol: 16, page: 2647, stat: Journal Article,

Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor 2
Ortega S; Ittmann M; Tsang SH; Ehrlich M; Basilico C
1998 May 12;95(10):5672-5677, Proceedings of the National Academy of Sciences of the United States of America
Basic fibroblast growth factor (FGF2) is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiological and pathological processes, including limb development, angiogenesis, wound healing, and tumor growth, but its physiological role is still unclear. To determine the function of FGF2 in vivo, we have generated FGF2 knockout mice, lacking all three FGF2 isoforms, by homologous recombination in embryonic stem cells. FGF2(-/-) mice are viable, fertile and phenotypically indistinguishable from FGF2(+/+) littermates by gross examination. However, abnormalities in the cytoarchitecture of the neocortex, most pronounced in the frontal motor-sensory area, can be detected by histological and immunohistochemical methods. A significant reduction in neuronal density is observed in most layers of the motor cortex in the FGF2(-/-) mice, with layer V being the most affected. Cell density is normal in other regions of the brain such as the striatum and the hippocampus. In addition, the healing of excisional skin wounds is delayed in mice lacking FGF2. These results indicate that FGF2, although not essential for embryonic development, plays a specific role in cortical neurogenesis and skin wound healing in mice, which, in spite of the apparent redundancy of FGF signaling, cannot be carried out by other FGF family members
— id: 56758, year: 1998, vol: 95, page: 5672, stat: Journal Article,

Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization [see comments]
Ozaki H; Okamoto N; Ortega S; Chang M; Ozaki K; Sadda S; Vinores MA; Derevjanik N; Zack DJ; Basilico C; Campochiaro PA
1998 Sep;153(3):757-765, American journal of pathology
Basic fibroblast growth factor (FGF2) is constitutively expressed in the retina and its expression is increased by a number of insults, but its role in the retina is still uncertain. This study was designed to test the hypothesis that altered expression of FGF2 in the retina affects the development of retinal neovascularization. Mice with targeted disruption of the Fgf2 gene had no detectable expression of FGF2 in the retina by Western blot, but retinal vessels were not different in appearance or total area from wild-type mice. When FGF2-deficient mice were compared with wild-type mice in a murine model of oxygen-induced ischemic retinopathy, they developed the same amount of retinal neovascularization. Transgenic mice with a rhodopsin promoter/Fgf2 gene fusion expressed high levels of FGF2 in retinal photoreceptors but developed no retinal neovascularization or other abnormalities of retinal vessels; in the ischemic retinopathy model, they showed no significant difference in the amount of retinal neovascularization compared with wild-type mice. These data indicate that FGF2 expression is not necessary nor sufficient for the development of retinal neovascularization. This suggests that agents that specifically antagonize FGF2 are not likely to be useful adjuncts in the treatment of retinal neovascularization and therapies designed to increase FGF2 expression are not likely to be complicated by retinal neovascularization
— id: 7727, year: 1998, vol: 153, page: 757, stat: Journal Article,

Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model
Tobe T; Ortega S; Luna JD; Ozaki H; Okamoto N; Derevjanik NL; Vinores SA; Basilico C; Campochiaro PA
1998 Nov;153(5):1641-1646, American journal of pathology
Choroidal neovascularization (CNV) is the major cause of severe visual loss in patients with age-related macular degeneration. Laser treatment is helpful for a minority of patients with CNV, and development of new treatments is hampered by a poor understanding of the molecular signals involved. Several lines of evidence have suggested that basic fibroblast growth factor (FGF2) plays a role in stimulating CNV. In this study, we tested this hypothesis using mice with targeted disruption of the FGF2 gene in a newly developed murine model of laser-induced CNV. One week after krypton laser photocoagulation in C57BL/6J mice, 34 of 60 burns (57%) showed fluorescein leakage and 13 of 16 (81%) showed histopathological evidence of CNV. At 2 weeks, CNV was detected in 9 of 10 burns (90%) in which a bubble had been observed at the time of the laser treatment. Electron microscopy showed fenestrated vessels with large lumens within choroidal neovascular lesions. Two weeks after laser-induced rupture of Bruch's membrane, 27 of 36 burns (75%) contained CNV in FGF2-deficient mice compared with 26 of 30 (87%) in wild-type control mice, a difference that is not statistically significant. This study demonstrates that FGF2 is not required for the development of CNV after laser-induced rupture of Bruch's membrane and provides a new model to investigate molecular mechanisms and anti-angiogenic therapy in CNV
— id: 7826, year: 1998, vol: 153, page: 1641, stat: Journal Article,

Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites
Ambrosetti DC; Basilico C; Dailey L
1997 Nov;17(11):6321-6329, Molecular & cellular biology
Octamer binding and Sox factors are thought to play important roles in development by potentiating the transcriptional activation of specific gene subsets. The proteins within these factor families are related by the presence of highly conserved DNA binding domains, the octamer binding protein POU domain or the Sox factors HMG domain. We have previously shown that fibroblast growth factor 4 (FGF-4) gene expression in embryonal carcinoma cells requires a synergistic interaction between Oct-3 and Sox2 on the FGF-4 enhancer. Sox2 and Oct-3 bind to adjacent sites within this enhancer to form a ternary protein-DNA complex (Oct-3*) whose assembly correlates with enhancer activity. We now demonstrate that increasing the distance between the octamer and Sox binding sites by base pair insertion results in a loss of enhancer function. Significantly, those enhancer 'spacing mutants' which failed to activate transcription were also compromised in their ability to form the Oct* complexes even though they could still bind both Sox2 and the octamer binding proteins, suggesting that a direct interaction between Sox2 and Oct-3 is necessary for enhancer function. Consistent with this hypothesis, Oct-3 and Sox2 can participate in a direct protein-protein interaction in vitro in the absence of DNA, and both this interaction and assembly of the ternary Oct* complexes require only the octamer protein POU and Sox2 HMG domains. Assembly of the ternary complex by these two protein domains occurs in a cooperative manner on FGF-4 enhancer DNA, and the loss of this cooperative interaction contributes to the defect in Oct-3* formation observed for the enhancer spacing mutants. These observations indicate that Oct-3* assembly results from protein-protein interactions between the domains of Sox2 and Oct-3 that mediate their binding to DNA, but it also requires a specific arrangement of the binding sites within the FGF-4 enhancer DNA. Thus, these results define one parameter that is fundamental to synergistic activation by Sox2 and Oct-3 and further emphasize the critical role of enhancer DNA sequences in the proper assembly of functional activation complexes
— id: 12266, year: 1997, vol: 17, page: 6321, stat: Journal Article,

Regulatory mechanisms governing FGF-4 gene expression during mouse development
Basilico C; Ambrosetti D; Fraidenraich D; Dailey L
1997 Nov;173(2):227-232, Journal of cellular physiology
— id: 12227, year: 1997, vol: 173, page: 227, stat: Journal Article,

Signaling through the ARK tyrosine kinase receptor protects from apoptosis in the absence of growth stimulation
Bellosta P; Zhang Q; Goff SP; Basilico C
1997 Nov 13;15(20):2387-2397, Oncogene
ARK (AXL) is the prototype of a distinctive family of receptor tyrosine kinases which contain in their extracellular domains features reminiscent of cell adhesion molecules. ARK is capable of homophilic binding, which results in a degree of receptor activation, but can also be activated by a heterophilic ligand, Gas6, a member of the family of vitamin K dependent proteins that is preferentially expressed in quiescent cells. Since a number of tissues and cell lines express both ARK and Gas6, we studied the effect of endogenous and exogenous Gas6 on the phenotype of ARK expressing cells. Here we show that constitutive expression of Gas6 in an NIH3T3 cell line that does not spontaneously express this protein does not result in cell transformation or uncontrolled growth, but protects from apoptosis induced by serum deprivation. Recombinant exogenous Gas6 was also capable of protecting cells from apoptosis at concentrations that did not result in significant induction of DNA synthesis. Activation of ARK phosphorylation and a weak but significant induction of MAP kinase activity accompanied the increased survival of cells treated with Gas6. The antiapoptotic effect of ARK signaling was confirmed by studies using fibroblasts from ARK knock-out mice, that showed that the absence of ARK resulted in higher levels of serum deprivation-induced apoptosis, that could not be rescued by the addition of Gas6. Interestingly ARK signaling protects from apoptosis induced by serum deprivation, myc overexpression, or by TNF alpha but not from u.v. irradiation or Staurosporine. These results suggest that a major function of Gas6-ARK signaling is that of increasing cell survival under conditions which do not allow cell proliferation
— id: 12211, year: 1997, vol: 15, page: 2387, stat: Journal Article,

FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression
Guthridge MA; Bellosta P; Tavoloni N; Basilico C
1997 Sep;17(9):5485-5498, Molecular & cellular biology
We have identified a novel type 2C serine-threonine phosphatase, FIN13, whose expression is induced by fibroblast growth factor 4 and serum in late G1 phase. The protein encoded by FIN13 cDNA includes N- and C-terminal domains with significant homologies to type 2C phosphatases, a domain homologous to collagen, and an acidic domain. FIN13 expression predominates in proliferating tissues. Bacterially expressed FIN13 and FIN13 expressed in mammalian cells exhibit serine-threonine phosphatase activity, which requires Mn2+ and is insensitive to inhibition by okadaic acid. FIN13 is localized in the nuclei of transiently transfected cells. Cotransfection of FIN13-expressing plasmids with a plasmid that expresses the neomycin resistance gene inhibits the growth of drug-resistant colonies in NIH 3T3, HeLa and Rat-1 cells. In transiently transfected cells, FIN13 inhibits DNA synthesis and results in the accumulation of cells in G1 and early S phases. Similarly, the induction of expression of FIN13 under the control of a tetracycline-regulated promoter in NIH 3T3 cells leads to growth inhibition, with accumulation of cells in G1 and early S phases. Thus, overexpression and/or unregulated expression of FIN13 inhibits cell cycle progression, indicating that the physiological role of this phosphatase may be that of regulating the orderly progression of cells through the mitotic cycle by dephosphorylating specific substrates which are important for cell proliferation
— id: 7161, year: 1997, vol: 17, page: 5485, stat: Journal Article,

Activation of FGF receptors by mutations in the transmembrane domain
Li Y; Mangasarian K; Mansukhani A; Basilico C
1997 Mar 27;14(12):1397-1406, Oncogene
Signaling through FGF receptors, which constitute a family of membrane-spanning tyrosine kinases, can stimulate cell proliferation, induce or inhibit cell differentiation and plays an important role in development. Recently, mutations in FGF receptors have been shown to be associated with a number of genetically dominant human skeletal disorders. A remarkably conserved mutation (Gly 380-->Arg) in the transmembrane region of FGFR-3 has been shown to be responsible for achondroplasia (ACH) but it was not clear whether such mutations result in loss of receptor function or constitutive activation. We have therefore made mutations in the transmembrane regions of murine FGFR-2 and FGFR-3 and studied their effect on receptor activity. We show here that the ACH mutation in FGFR-3 as well as two similar mutations in FGFR-2 result in constitutive activation of these receptors. This is manifested in their ability to become autophosphorylated in the absence of ligand in L6 cells, transforming activity on NIH3T3 fibroblasts, and the ability to inhibit myogenic differentiation in the absence of growth factor. Thus the transmembrane region of FGFR-2 and FGFR-3 plays a regulatory role in receptor function and the ACH mutation produces a dominant oversignaling receptor which is no longer regulated by FGF binding. These findings also support the newly identified role of FGF signaling as a negative regulator of bone growth
— id: 8208, year: 1997, vol: 14, page: 1397, stat: Journal Article,

Mutation associated with Crouzon syndrome causes ligand-independent dimerization and activation of FGF receptor-2
Mangasarian K; Li Y; Mansukhani A; Basilico C
1997 Jul;172(1):117-125, Journal of cellular physiology
FGF signaling is clearly important for proper bone development, and several autosomally dominant forms of genetic bone disorders have been mapped to FGF receptors 1, 2, and 3. We have studied the biological effects of the most commonly mutated cysteine residue in FGFR-2 which is detected in individuals with Crouzon syndrome, an autosomally dominant trait which causes premature fusion of the skull bones (craniosynostosis). This Crouzon mutation replaces the cysteine at position 342 with tyrosine, thus disrupting the formation of the third immunoglobulin (Ig)-like loop in the extracellular portion of the receptor. By transfecting mutated and wild-type receptors into a variety of cell lines, we have shown that the C342Y mutation in FGFR-2 produces a receptor which is constitutively activated and capable of transforming NIH3T3 cells and preventing the differentiation of C2 myoblasts in the absence of ligand. Constitutive activation appears to result from the ability of this receptor to form stable interreceptor dimers which involve disulfide bonds between the remaining free cysteine in the mutant receptor. The altered conformation of the third Ig-like domain in the mutated receptor also results in a drastically reduced ability to bind FGF-1 or FGF-2 and in a reduced level of receptor glycosylation. Thus it appears that Crouzon syndrome results from constitutive activation of FGFR-2 and that uncontrolled FGF signaling produces alterations of intramembranous bone development and premature closing of cranial sutures
— id: 8385, year: 1997, vol: 172, page: 117, stat: Journal Article,

Basic fibroblast growth factor is not necessary for the development of retinal neovascularization
Ozaki, H; Ortega, S; Basilico, C; Campochiaro, PA
1997 MAR 15 ;38(4):2848-2848, Investigative ophthalmology & visual science. IOVS
— id: 53236, year: 1997, vol: 38, page: 2848, stat: Journal Article,

Sodium salicylate induces apoptosis via p38 mitogen-activated protein kinase but inhibits tumor necrosis factor-induced c-Jun N-terminal kinase/stress-activated protein kinase activation
Schwenger P; Bellosta P; Vietor I; Basilico C; Skolnik EY; Vilcek J
1997 Apr 1;94(7):2869-2873, Proceedings of the National Academy of Sciences of the United States of America
In a previous study, we demonstrated that sodium salicylate (NaSal) selectively inhibits tumor necrosis factor (TNF)-induced activation of the p42 and p44 mitogen-activated protein kinases (MAPKs) (known as extracellular signal-regulated kinases). Here we show that in normal human FS-4 fibroblasts NaSal inhibits TNF-induced activation of another member of the MAPK family, the c-Jun N-terminal kinase/stress-activated protein kinase. c-Jun N-terminal kinase activation induced by interleukin 1 or epidermal growth factor was less strongly inhibited by NaSal. Unexpectedly, treatment of FS-4 cells with NaSal alone produced a strong activation of p38 MAPK and cell death by apoptosis. NaSal-induced apoptosis was blocked by the selective p38 MAPK inhibitor SB-203580, indicating that p38 MAPK serves as a mediator of NaSal-induced apoptosis in human fibroblasts. Activation of p38 MAPK and the resulting induction of apoptosis may be important in the demonstrated antineoplastic actions of nonsteroidal anti-inflammatory drugs
— id: 57523, year: 1997, vol: 94, page: 2869, stat: Journal Article,

Retinal degeneration in transgenic mice with photoreceptor-specific expression of a dominant-negative fibroblast growth factor receptor
Campochiaro PA; Chang M; Ohsato M; Vinores SA; Nie Z; Hjelmeland L; Mansukhani A; Basilico C; Zack DJ
1996 Mar 1;16(5):1679-1688, Journal of neuroscience
Mutant cDNAs coding for dominant-negative forms of the fibroblast growth factor receptors 1 (FGFR-1) and 2 (FGFR-2) that lack tyrosine kinase activity were ligated to a 2.2 kb DNA fragment containing the bovine rhodopsin promoter and used to generate transgenic mice. Six independent lines were generated with the FGFR-1 construct, and five were generated with the FGFR-2 construct. Five of the six FGFR-1 mutant lines and all five FGFR-2 mutant lines showed transgene expression in the retina by reverse transcription-PCR. By both in situ hybridization and immunohistochemistry, mutant FGFRs were found to be expressed specifically in photoreceptors of transgene-positive FGFR-1 and FGFR-2 mice. Lines expressing the FGFR-2 mutant showed progressive photoreceptor degeneration; the retinas showed minimal or no abnormalities at 1 month, but by 2 months they showed focal areas of thinning of the outer nuclear layer and disruption of photoreceptors. By 2-4 months, areas of complete loss of photoreceptors were seen. These abnormalities were not seen in control littermates not expressing the transgene. Mice from two FGFR-1 mutant lines showed focal areas of thinning of the outer nuclear layer and numerous photoreceptors with fragmented chromatin, whereas the other FGFR-1 lines showed minimal or no abnormalities. These data indicate that perturbation of FGF signaling in photoreceptors is associated with progressive photoreceptor degeneration, suggesting that one or more of the FGFs may act as a survival factor for photoreceptor cells
— id: 14410, year: 1996, vol: 16, page: 1679, stat: Journal Article,

Cleavage and release of a soluble form of the receptor tyrosine kinase ARK in vitro and in vivo
Costa M; Bellosta P; Basilico C
1996 Sep;168(3):737-744, Journal of cellular physiology
The receptor tyrosine kinase ARK (also called AXL or UFO) is the murine prototype of a small family of receptors with an extracellular domain resembling cell adhesion molecules and a conserved tyrosine kinase domain. ARK is capable of homophilic binding, as well as of binding of GAS6, a secreted member of the class of vitamin K dependent proteins whose expression is up-regulated in growth-arrested cells. To gain understanding of the physiological role of ARK signaling, we have investigated the ARK forms which are expressed by cells in culture as well as by mouse organs. We found that ARK is not only expressed as a transmembrane protein, but is also cleaved in the extracellular domain to generate a soluble ARK form of about 65 kDa, which is easily detected in conditioned media of ARK expressing cells, in serum and plasma and in mouse organs. Soluble ARK is also produced by tumor cells in vivo. The function of these molecules could be that of binding GAS6, thereby inhibiting the interaction of this ligand with its cell-associated receptor, or they could be involved in binding to ARK itself
— id: 12555, year: 1996, vol: 168, page: 737, stat: Journal Article,

Induction of expression of growth-related genes by FGF-4 in mouse fibroblasts
Guthridge MA; Seldin M; Basilico C
1996 Mar 21;12(6):1267-1278, Oncogene
Cells monitor and respond to extracellular signals from polypeptide growth factors by the induction of a genetic program. Although poorly understood at the molecular level, the biological activity of growth factors is believed to be mediated by the regulation of specific sets of genes. We have isolated a number of cDNAs, the expression of whose corresponding RNAs is induced by FGF-4 (K-FGF) in murine NIH3T3 fibroblasts. The cDNAs (FIN, for FGF-inducible) were isolated using a strategy of subtractive hybridization designed to yield 'late' genes which compared transformed 3T3 cells that constitutively produce FGF-4 with their normal counterpart. The 21 independent cDNAs isolated were found to correspond to known genes (FIN1-12), or novel genes (FIN13-21). Expression of the FIN genes is induced in response to FGF-4 as well as to serum in NIH3T3 cells with delayed kinetics, with maximum stimulation occurring 12-18h after growth factor treatment. Induction requires protein synthesis and is mostly transcriptional. FIN1-12 encode a broad range of previously described genes, some of which are proposed to have an important role in cell proliferation. The novel clones include a putative serine-threonine phosphatase (FIN13) and a gene with homology to NTP-binding proteins (FIN16). The distribution of expression of the novel FIN clones in adult mouse tissues was highly restricted, although most were expressed in embryos. While expression of novel FIN cDNAs was strongly regulated in NIH3T3 cells, induction of differentiation in PC-12 cells by FGF-4 (as well as by NGF) did not result in significant induction of expression, suggesting that most of the FIN genes are proliferation-specific. Chromosomal localization of novel FIN clones indicated that each segregated independently to separate mouse chromosomes
— id: 7001, year: 1996, vol: 12, page: 1267, stat: Journal Article,

Effects of fibroblast growth factor-4 (k-FGF) on long-term cultures of human bone marrow cells
Quito FL; Beh J; Bashayan O; Basilico C; Basch RS
1996 Feb 15;87(4):1282-1291, Blood
Fibroblast growth factor-4 (FGF-4), a highly mitogenic protein encoded by the k-fgf/hst oncogene, stimulates the growth of a variety of cells of mesenchymal and neuroectodermal origin. Addition of FGF-4 to human long-term bone marrow cultures increased both the cell density of the stromal layer and the number of hematopoietic colony forming cells in the cultures in a dose-dependent manner. Hematopoiesis in the stromal layer persisted for up to 8 months. Erythropoiesis was maintained for up to 4 weeks, but granulocytes were the predominant nonadherent cell type. Cultures treated with FGF had increased numbers of monocytes compared with control cultures and some CD14+, CD45+ monocytes could still be detected after 8 months of continuous culture. The addition of the growth factor increased the rate of growth of the stromal layer and appeared to delay its senescence. Subcultures made in the presence of FGF-4 had up to 10-fold increases in plating efficiency and grew as relatively uniform monolayers. These subcultures retained the capacity to support hematopoiesis for several months, while untreated subcultures, made without FGF-4, grew erratically and generally lost the capacity to support hematopoiesis within 4 to 6 weeks. The improved growth after subculture greatly enhanced the reliability of limit-dilution assays of multipotential hematopoietic stem cells that use stromal cell monolayers. The primary effect of FGF-4 appeared to be on the stromal cells of the long-term bone marrow cultures, but a direct effect on hematopoietic progenitors could not be ruled out
— id: 7034, year: 1996, vol: 87, page: 1282, stat: Journal Article,

Fibroblast growth factor receptors 1 and 2 are differentially regulated in murine embryonal carcinoma cells and in response to fibroblast growth factor-4
Ali J; Mansukhani A; Basilico C
1995 Nov;165(2):438-448, Journal of cellular physiology
We have studied the expression of two of the receptors for fibroblast growth factors, FGFR-1 and FGFR-2, in response to ligand binding and in embryonal carcinoma (EC cells). Exposure of mouse fibroblasts to FGF-4 or FGF-2 results in a drastic downregulation of the mRNA levels for FGFR-2, while expression of FGFR-1 mRNA appears unaffected. Furthermore, FGF-4 transformed cells display low levels of FGFR-2 mRNA and these levels are significantly increased by treatment with anti FGF-4 neutralizing antibodies. In undifferentiated F9 EC cells, the levels of FGFR-2 mRNA are very low and increase substantially upon induction of differentiation. The levels of mRNA for FGFR-1 are again unaffected. To gain information on the regulation of expression of the gene encoding FGFR-2 (bek) we have cloned the FGFR-2 promoter region and used it to drive the expression of plasmids encoding the bacterial CAT enzyme. Transfection of these plasmids into FGF treated and untreated cells did not produce significant variation in CAT activity, suggesting that FGFR-2 downregulation in response to ligand binding occurs mainly by a post-transcriptional mechanism. In contrast, plasmids containing as little as 140 nt of the FGFR-2 promoter region were regulated in F9 cells, showing substantially higher expression in differentiated than in undifferentiated cells. It appears therefore that FGFR-2 expression in fibroblasts and EC cells is regulated by somewhat different mechanisms. In contrast, FGFR-1 expression does not vary substantially under the conditions shown to affect FGFR-2 expression. The implications of these findings are discussed
— id: 12716, year: 1995, vol: 165, page: 438, stat: Journal Article,

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding
Bellosta P; Costa M; Lin DA; Basilico C
1995 Feb;15(2):614-625, Molecular & cellular biology
The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition
— id: 8207, year: 1995, vol: 15, page: 614, stat: Journal Article,

THE ANTICOAGULATION FACTOR PROTEIN-S AND ITS RELATIVE, GAS6, ARE LIGANDS FOR THE TYRO 3/AXL FAMILY OF RECEPTOR TYROSINE KINASES
STITT, TN; CONN, G; GORE, M; LAI, C; BRUNO, J; RADZIEJEWSKI, C; MATTSSON, K; FISHER, J; GIES, DR; JONES, PF; MASIAKOWSKI, P; RYAN, TE; TOBKES, NJ; CHEN, DH; DISTEFANO, PS; LONG, GL; BASILICO, C; GOLDFARB, MP; LEMKE, G; GLASS, DJ; YANCOPOULOS, GD
1995 FEB 24 ;80(4):661-670, Cell
We report the identification of ligands for Tyro 3 (alternatively called Sky, rse, brt, or tif) and Axl (alternatively, Ark or UFO), members of a previously orphan family of receptor-like tyrosine kinases. These ligands correspond to protein S, a protease regulator that is a potent anticoagulant, and Gas6, a protein related to protein S but lacking any known function. Our results are reminiscent of recent findings that the procoagulant thrombin, a protease that drives clot formation by cleaving fibrinogen to form fibrin, also binds and activates intracellular signaling via a G protein-coupled cell surface receptor. Proteases and protease regulators that also activate specific cell surface receptors may serve to integrate coagulation with associated cellular responses required for tissue repair and growth, as well as to coordinate protease cascades and associated cellular responses in other systems, such as those involved in growth and remodeling of the nervous system
— id: 87416, year: 1995, vol: 80, page: 661, stat: Journal Article,

Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3
Yuan H; Corbi N; Basilico C; Dailey L
1995 Nov 1;9(21):2635-2645, Genes & development
Fibroblast growth factor 4 (FGF-4) has been shown to be a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass and later in distinct embryonic tissues but is transcriptionally silent in the adult. In tissue culture FGF-4 expression is restricted to undifferentiated embryonic stem (ES) cells and embryonal carcinoma (EC) cell lines. Previously, we determined that EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins and an EC-specific factor, Fx, that bind adjacent sites on the FGF-4 enhancer. Through the cloning and characterization of an F9 cell cDNA we now show that the latter activity is Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 and for any of the Sox factors, and offer insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis
— id: 56811, year: 1995, vol: 9, page: 2635, stat: Journal Article,

Interaction between a novel F9-specific factor and octamer-binding proteins is required for cell-type-restricted activity of the fibroblast growth factor 4 enhancer
Dailey L; Yuan H; Basilico C
1994 Dec;14(12):7758-7769, Molecular & cellular biology
Understanding how diverse transcription patterns are achieved through common factor binding elements is a fundamental question that underlies much of developmental and cellular biology. One example is provided by the fibroblast growth factor 4 (FGF-4) gene, whose expression is restricted to specific embryonic tissues during development and to undifferentiated embryonal carcinoma cells in tissue culture. Analysis of the cis- and trans-acting elements required for the activity of the previously identified FGF-4 enhancer in F9 embryonal carcinoma cells showed that enhancer function depends on sequences that bind Sp1 and ubiquitous as well as F9-specific octamer-binding proteins. However, sequences immediately upstream of the octamer motif, which conform to a binding site for the high-mobility group (HMG) domain factor family, were also critical to enhancer function. We have identified a novel F9-specific factor, Fx, which specifically recognizes this motif. Fx formed complexes with either Oct-1 or Oct-3 in a template-dependent manner. The ability of different enhancer variants to form the Oct-Fx complexes correlated with enhancer activity, indicating that these complexes play an essential role in transcriptional activation of the FGF-4 gene. Thus, while FGF-4 enhancer function is octamer site dependent, its developmentally restricted activity is determined by the interaction of octamer-binding proteins with the tissue-specific factor Fx
— id: 14411, year: 1994, vol: 14, page: 7758, stat: Journal Article,

EXPRESSION OF INT-2 ONCOGENE IN KAPOSIS-SARCOMA LESIONS
HUANG, YQ; LI, JJ; MOSCATELLI, D; BASILICO, C; NICOLAIDES, A; ZHANG, WG; POIESZ, BJ; FRIEDMANKIEN, AE
1994 AUG ;10(3-4):S54-S54, AIDS research & human retroviruses
— id: 52370, year: 1994, vol: 10, page: S54, stat: Journal Article,

Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors
Li Y; Basilico C; Mansukhani A
1994 Nov;14(11):7660-7669, Molecular & cellular biology
Ligand-induced dimerization and transphosphorylation are thought to be important events by which receptor tyrosine kinases generate cellular signals. We have investigated the ability of signalling-defective, truncated fibroblast growth factor (FGF) receptors (FGFR-1 and FGFR-2) to block the FGF response in cells that express both types of endogenous FGF receptors. When these dominant negative receptors are expressed in NIH 3T3 cells transformed by the secreted FGF-4, the transformed properties of the cells can be reverted to various degrees, with better reversion phenotype correlating with higher levels of truncated receptor expression. Furthermore, truncated FGFR-2 is significantly more efficient at producing reversion than FGFR-1, indicating that FGF-4 preferentially utilizes the FGFR-2 signalling pathway. NIH 3T3 clones expressing these truncated receptors are more resistant to FGF-induced mitogenesis and also exhibit reduced tyrosine phosphorylation upon treatment with FGF. The block in FGF-signalling, however, can be overcome by the addition of excess growth factor. The truncated receptors have binding affinities that are four- to eightfold lower than those of wild-type receptors, as measured by Scatchard analysis. We also observed a partial specificity in the responses of truncated-receptor-expressing clones to FGF-2 or FGF-4. Our results suggest that the block to signal transduction produced by kinase-negative FGF receptors is achieved through a combination of dominant negative effects and competition for growth factor binding with functional receptors
— id: 6681, year: 1994, vol: 14, page: 7660, stat: Journal Article,

EXPRESSION OF FIBROBLAST GROWTH-FACTORS AND THEIR RECEPTORS IN AIDS-ASSOCIATED KAPOSIS-SARCOMA TISSUE AND DERIVED CELLS
LI, JJ; HUANG, YQ; MOSCATELLI, D; BASILICO, C; NICOLAIDES, A; ZHANG, WG; POIESZ, BJ; FRIEDMANKIEN, AE
1994 AUG ;10(3-4):S52-S52, AIDS research & human retroviruses
— id: 52369, year: 1994, vol: 10, page: S52, stat: Journal Article,

Expression and function of FGF-4 in peri-implantation development in mouse embryos
Rappolee DA; Basilico C; Patel Y; Werb Z
1994 Aug;120(8):2259-2269, Development
One of the earliest events in mammalian embryogenesis is the formation of the inner cell mass (ICM) and the subsequent delamination of primitive endoderm. We have found that mRNA for fibroblast growth factor (FGF)-4, but not FGF-3, is expressed in preimplantation mouse blastocysts and that the FGF-4 polypeptide is present in ICM cells. ICM-like embryonal carcinoma cells and embryonic stem cells also express FGF-4. Conversely, differentiated embryonal carcinoma cells in the endoderm lineage express FGF-3, but not FGF-4 mRNA. Although mouse embryos expressed FGF-4 mRNA from the 1-cell stage, embryos cultured from the 2-cell through the blastocyst stage in the presence of recombinant FGF-4 did not respond mitogenically. However, when ICMs that were isolated by immunosurgery were cultured with FGF-4, the number of morphologically distinct, differentiated parietal endoderm cells growing out onto the coverslip increased, without an increase in the number of undifferentiated ICM cells. ICM outgrowths cultured with FGF-4 increased their secretion of 92 x 10(3) M(r) gelatinase and tissue plasminogen activator, a hallmark of migrating cells. Receptors for FGF-4 (FGFR-3 and FGFR-4) are expressed in all cells of the mouse blastocyst. These findings indicate that FGF-4 produced by undifferentiated ICM cells acts in the peri-implantation period of embryogenesis to influence the production and behavior of endoderm cells derived from them
— id: 14412, year: 1994, vol: 120, page: 2259, stat: Journal Article,

Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding
Roghani M; Mansukhani A; Dell'Era P; Bellosta P; Basilico C; Rifkin DB; Moscatelli D
1994 Feb 11;269(6):3976-3984, Journal of biological chemistry
The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin
— id: 6499, year: 1994, vol: 269, page: 3976, stat: Journal Article,

Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity
Bellosta P; Talarico D; Rogers D; Basilico C
1993 May;121(3):705-713, Journal of cell biology
The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity
— id: 8206, year: 1993, vol: 121, page: 705, stat: Journal Article,

Involvement of the conserved acidic amino acid domain of FGF receptor 1 in ligand-receptor interaction
Chaudhuri MM; Moscatelli D; Basilico C
1993 Nov;157(2):209-216, Journal of cellular physiology
The fibroblast growth factor receptor 1 (flg) contains eight acidic amino acids between the first and second immunoglobulin domain. This report examines the role of the acidic domain in the interaction of the flg receptor with its ligands. We observed a marked inhibition of binding of bFGF to the receptor when the acidic domain was completely deleted, but mutants with two and four amino acids deleted (flg delta A2 and flg delta A4, respectively) still bound the ligand. After addition of a bifunctional cross-linking reagent, cross-linked complexes (between bFGF and receptor) with the expected size were observed in cells expressing mutants lacking two or four acidic residues, but not in cells expressing mutants lacking six or eight acidic residues. Immunoprecipitation with anti-flg antibody followed by electrophoresis produced a band of 90 Kd in tunicamycin-treated cells expressing the mutant as well as the wild-type receptors, indicating that the inhibition of binding was not due to defective expression of the protein. The ability of flg delta A8 to mediate a mitogenic response to FGFs was also greatly reduced when this mutated receptor was expressed in receptor-negative cells. The effect of replacing the acidic amino acids with lysine residues was also studied. Binding of bFGF to cells transfected with a plasmid encoding a mutated protein with four amino acid substitutions was totally inhibited, but an eight amino acid substitution did not alter ligand binding to the receptor. In this case the mutation with four amino acids substitution caused a drastic impairment of protein expression. Thus the acidic domain of the FGFR-1 plays an essential role in receptor function, either because it is important for a stable protein configuration or for ligand-receptor interaction
— id: 6332, year: 1993, vol: 157, page: 209, stat: Journal Article,

Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression
Guerrini L; Gong SS; Mangasarian K; Basilico C
1993 Jun;13(6):3202-3212, Molecular & cellular biology
We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically up-regulated by asparagine deprivation in ts11 cells, mutants of BHK hamster cells which encode a temperature-sensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Sp1, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNA-binding properties
— id: 13153, year: 1993, vol: 13, page: 3202, stat: Journal Article,

Expression of int-2 oncogene in Kaposi's sarcoma lesions
Huang YQ; Li JJ; Moscatelli D; Basilico C; Nicolaides A; Zhang WG; Poiesz BJ; Friedman-Kien AE
1993 Mar;91(3):1191-1197, Journal of clinical investigation
Fibroblast growth factors (FGFs), such as basic FGF, have been implicated in the growth of Kaposi's sarcoma (KS) cells in vitro. In the evaluation of the expression of the various genes of the different members of the FGF family and their receptors in fresh KS tissue specimens, int-2 was found to be expressed in more than half of the KS tumors examined. Using reverse transcription PCR, the expression of int-2 was detected in 21 of 38 (55.2%) fresh KS biopsy specimens. In contrast, int-2 mRNA transcripts were not found in normal appearing skin from the same patients except in one sample which was obtained from an AIDS patient with disseminated KS lesions. Sequence data confirmed that the amplified sequences were derived from int-2 mRNA with proper splicing. In addition, 12 nucleic acid alterations were identified in eight out of nine KS tumor samples sequenced. Using immunohistochemical methods, int-2 protein was detected in some of the spindle-shaped tumor cells surrounding the abnormal endothelial-lined vascular slits histologically characteristic of KS. Int-2 specific immunostaining was shown to be present in both the nuclei and cytoplasm of these spindle cells but was more pronounced in the nuclei. Neither amplification nor gross rearrangement of the int-2 gene was detected in KS lesions by Southern blot analysis. These results suggest that the expression of int-2 may play a role in the pathogenesis KS by stimulating local angiogenesis and cell proliferation
— id: 13228, year: 1993, vol: 91, page: 1191, stat: Journal Article,

The gene complementing a temperature-sensitive cell cycle mutant of BHK cells is the human homologue of the yeast RPC53 gene, which encodes a subunit of RNA polymerase C (III)
Ittmann M; Ali J; Greco A; Basilico C
1993 Jun;4(6):503-511, Cell growth & differentiation
The temperature-sensitive BN51 cell cycle mutant of BHK cells arrests in G1 at the nonpermissive temperature (39.5 degrees C). We have previously reported cloning the gene which complements this mutation. The complementing gene encodes a highly charged protein with a basic amino-terminal domain and an acidic carboxyl-terminal domain. We have recently found that the predicted BN51 protein shows significant homology to the 53 kilodalton subunit of RNA polymerase C (III) from Saccharomyces cerevisiae. Consistent with this, antibodies raised to fusion proteins containing BN51 coding sequences and antipeptide antibodies reveal that the BN51 gene encodes a 48 kilodalton protein which appears to be located primarily in the nucleus following subcellular fractionation and by immunohistochemistry. Analysis of RNA polymerase III activity in temperature-sensitive BN51 cells by nuclear runoff transcription assay reveals a marked drop in RNA polymerase III transcription after 48 h at the nonpermissive temperature (39.5 degrees C). This is correlated with a significant decrease in low molecular weight RNAs after 48 h at 39.5 degrees C. In addition, RNA polymerase III activity in S100 extracts of BN51 cells is more sensitive to heat inactivation at 39 degrees C than control extracts from BHK cells. When the yeast gene is introduced into the BN51 cells in a mammalian expression vector, it weakly complements the BN51 mutation in that it prevents cell death at 39.5 degrees C. The mechanism by which inhibition of RNA polymerase III activity leads to arrest in G1 is unclear but is not due to a marked decrease in total protein synthesis
— id: 13147, year: 1993, vol: 4, page: 503, stat: Journal Article,

A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas
Talarico D; Ittmann MM; Bronson R; Basilico C
1993 Apr;13(4):1998-2010, Molecular & cellular biology
The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF
— id: 13207, year: 1993, vol: 13, page: 1998, stat: Journal Article,

FIBROBLAST GROWTH-FACTOR RECEPTOR-4 SHOWS NOVEL FEATURES IN GENOMIC STRUCTURE, LIGAND-BINDING AND SIGNAL TRANSDUCTION
VAINIKKA, S; PARTANEN, J; BELLOSTA, P; COULIER, F; BASILICO, C; JAYE, M; ALITALO, K
1993 JAN 9 ;53(5):243-243, Journal of cellular biochemistry
— id: 54365, year: 1993, vol: 53, page: 243, stat: Journal Article,

The FGF family of growth factors and oncogenes
Basilico C; Moscatelli D
1992 ;59:115-165, Advances in cancer research
— id: 13785, year: 1992, vol: 59, page: 115, stat: Journal Article,

Angiogenic activity of the K-fgf/hst oncogene in neural transplants
Brustle O; Aguzzi A; Talarico D; Basilico C; Kleihues P; Wiestler OD
1992 Jun;7(6):1177-1183, Oncogene
Using retrovirus-mediated gene transfer into neural transplants, we have expressed the human K-fgf/hst oncogene in the central nervous system. Single-cell suspensions of fetal rat brains were removed at embryonic days 13 and 14, exposed to a retroviral vector encoding the K-fgf oncogene and stereotaxically implanted into the caudate putamen of syngenic adult Fisher rats. Recipient animals were sacrificed at intervals of 6-16 months without evidence of neurological impairment. Mock-infected grafts showed the characteristic histopathological appearance of organotypically differentiated neural transplants. In contrast, grafts exposed to the K-fgf gene exhibited abundant capillary proliferation and capillary angiomas. By in situ hybridization analysis and immunohistochemistry, expression of K-fgf was detected in neural cells adjacent to vascular proliferations. Neurons and glia with abundant K-fgf transcripts were morphologically unaffected. In order to examine the transforming potential of the K-fgf gene in the nervous system, we combined retrovirus-mediated transfer of the K-fgf oncogene with a single transplacental exposure of the donor animals to the neurotropic carcinogen N-ethyl-N-nitrosourea (NEU). However, this combination of transforming agents did not result in tumor formation in the grafts. These results provide evidence for a powerful angiogenic effect of K-fgf on the developing brain in vivo
— id: 14414, year: 1992, vol: 7, page: 1177, stat: Journal Article,

Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin
Mansukhani A; Dell'Era P; Moscatelli D; Kornbluth S; Hanafusa H; Basilico C
1992 Apr 15;89(8):3305-3309, Proceedings of the National Academy of Sciences of the United States of America
The bek gene encodes a member of the high-affinity fibroblast growth factor receptor family. The BEK/FGFR-2 receptor is a membrane-spanning tyrosine kinase with the typical features of FGF receptors. We have cloned a murine bek cDNA and expressed it in receptor-negative Chinese hamster ovary cells and in 32D myeloid cells. The BEK receptor expressed in Chinese hamster ovary cells binds acidic FGF, basic FGF, and Kaposi FGF equally well but does not bind keratinocyte growth factor or FGF-5 appreciably. Upon treatment with basic FGF or Kaposi FGF, the BEK receptor is phosphorylated and a mitogenic response is achieved. Heparan sulfate proteoglycans have been shown to play an obligate role in basic FGF binding to the high-affinity FLG receptor. Unlike the BEK-expressing Chinese hamster ovary cells, 32D cells expressing the BEK receptor require the addition of exogenous heparin in order to grow in the presence of basic FGF or Kaposi FGF. We show that the addition of heparin greatly enhances the binding of radio-labeled basic FGF to the receptor. Thus the BEK receptor, like FLG, also requires an interaction with heparan sulfate proteoglycans to facilitate binding to its ligands
— id: 13631, year: 1992, vol: 89, page: 3305, stat: Journal Article,

Collagen-induced release of interleukin 1 from human blood mononuclear cells. Potentiation by fibronectin binding to the alpha 5 beta 1 integrin
Pacifici R; Basilico C; Roman J; Zutter MM; Santoro SA; McCracken R
1992 Jan;89(1):61-67, Journal of clinical investigation
PBMC express cell surface receptors for extracellular matrix components known as integrins. We have recently shown that ligand binding to one PBMC integrin, the collagen receptor alpha 2 beta 1, stimulates the secretion of interleukin 1 (IL-1). We have now investigated the role of fibronectin (Fn), an adherence protein that has binding sites for both PBMC and collagen, in the generation of the IL-1 response to collagen. In contrast to collagen, Fn did not stimulate IL-1 release but Fn-depleted serum decreased the release of IL-1 induced by collagen. A polyclonal antiserum directed against Fn also decreased the collagen-induced IL-1 secretion. The IL-1 response to collagen from cells incubated in Fn-depleted serum was restored by the addition of either purified Fn or the 120-kD cell-binding fragment of Fn, which contains the cell-binding site but not the collagen-binding domain. Smaller Arg-Gly-Asp (RGD) peptides failed to enhance the PBMC response to collagen but inhibited in a concentration-dependent fashion the potentiating effect Fn. As expected, a MAb against the alpha 2 beta 1 collagen receptor decreased collagen-induced IL-1 release. However collagen-induced IL-1 release was also inhibited by a MAb against the alpha 5 beta 1 Fn receptor. The effect of the two MAbs was not additive, suggesting that the occupancy of both receptors by ligands is required in order for collagen to induce an maximal response from PBMC. The mechanism by which Fn exerts its effect remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 14415, year: 1992, vol: 89, page: 61, stat: Journal Article,

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction [published erratum appears in EMBO J 1993 Feb;12(2):810]
Vainikka S; Partanen J; Bellosta P; Coulier F; Birnbaum D; Basilico C; Jaye M; Alitalo K
1992 Dec;11(12):4273-4280, EMBO journal
Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions
— id: 14413, year: 1992, vol: 11, page: 4273, stat: Journal Article,

Regulation of asparagine synthetase gene expression by amino acid starvation
Gong SS; Guerrini L; Basilico C
1991 Dec;11(12):6059-6066, Molecular & cellular biology
We have studied the regulation of expression of the asparagine synthetase (AS) gene in ts11 cells, a mutant of BHK hamster cells which encodes a temperature-sensitive AS and therefore does not produce endogenous asparagine at 39.5 degrees C. Incubation of ts11 cells at the nonpermissive temperature drastically increases the level of AS mRNA, and the stimulation of AS mRNA expression is effectively suppressed by the addition of asparagine to the medium. We show here that regulation of AS gene expression involves cis-acting elements which are contained in the mRNA as well as in the 5' genomic region. When a plasmid containing the human AS cDNA under the control of the human AS promoter region was stably transfected into ts11 cells, the expression of human AS RNAs was regulated as that of the endogenous hamster transcripts, indicating that this construct contained all cis elements necessary for regulation. Expression of the AS cDNA in ts11 cells under the control of a constitutive foreign promoter was also regulated by the concentration of asparagine, and this regulation required translation. When we introduced by mutagenesis a number of stop codons in the AS cDNA, the mutant mRNAs with short open reading frames were expressed at low levels that were not increased by asparagine deprivation. Inhibition of protein and RNA synthesis also prevented down-regulation of AS mRNA levels by high concentrations of asparagine. In a parallel series of experiments, we showed that an AS DNA fragment including the promoter and first exon can also regulate RNA expression in response to asparagine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 13834, year: 1991, vol: 11, page: 6059, stat: Journal Article,

Response
Kaner RJ; Baird A; Florklewicz RZ; Mansukhani A; Basilico C; Hajjar DP
1991 Jul 12;253(5016):209-210, Science
— id: 95117, year: 1991, vol: 253, page: 209, stat: Journal Article,

A putative receptor tyrosine kinase with unique structural topology
Rescigno J; Mansukhani A; Basilico C
1991 Oct;6(10):1909-1913, Oncogene
We have cloned a murine cDNA on the basis of homology to the tyrosine kinase domain of the bek fibroblast growth factor receptor. This cDNA encodes a putative tyrosine kinase receptor with a unique structural pattern in its extracellular domain. It is a new member of the immunoglobulin superfamily with two immunoglobulin-like domains. It also contains two fibronectin type III domains which are found on diverse proteins such as receptor tyrosine phosphatases and neural cell adhesion molecules. This protein tyrosine kinase called ark (adhesion-related kinase) is likely to represent a new class of receptor tyrosine kinase. Ark mRNA appears to be expressed in most cell lines and adult tissues examined except those of hematopoietic lineage. It is undetectable in undifferentiated teratocarcinoma cells, F9 and N Tera 2
— id: 13882, year: 1991, vol: 6, page: 1909, stat: Journal Article,

The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway
Talarico D; Basilico C
1991 Feb;11(2):1138-1145, Molecular & cellular biology
The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface
— id: 14144, year: 1991, vol: 11, page: 1138, stat: Journal Article,

Molecular cloning of the human gene, CCG2, that complements the BHK-derived temperature-sensitive cell cycle mutant tsBN63: identity of CCG2 with the human X chromosomal SCAR/RPS4X gene
Watanabe M; Furuno N; Goebl M; Go M; Miyauchi K; Sekiguchi T; Basilico C; Nishimito T
1991 Sep;100(Pt 1):35-43, Journal of cell science
A temperature-sensitive mutant tsBN63 cell line was isolated by the fluorodeoxyuridine method from the BHK21/13 cell line after mutagenesis with nitrosoguanidine. When cultures of tsBN63 cells growing asynchronously at 33.5 degrees C were shifted to 39.5 degrees C, a nonpermissive temperature, the ability for protein synthesis was rapidly reduced and cell proliferation stopped mainly at G1 phase, and partly at G2 phase. Synchronized cultures of tsBN63 cells did not commence DNA synthesis when shifted up in G1 phase. The human gene complementing the tsBN63 mutation was cloned by DNA-mediated gene transfer and its cDNA of 1.1 kb conferring ts+ phenotype on tsBN63 cells was isolated from the cDNA library of Raj (mer+) cells with a frequency of 10(-3). On the basis of the determined nucleotide sequence, the isolated human gene turned out to be the X chromosomal RPS4X encoding the ribosomal protein S4. The size of the CCG2 gene was estimated to be about 12 kb by complementation analysis of the tsBN63 mutation with cloned genomic DNA
— id: 14416, year: 1991, vol: 100, page: 35, stat: Journal Article,

Expression of the K-fgf proto-oncogene is controlled by 3' regulatory elements which are specific for embryonal carcinoma cells
Curatola AM; Basilico C
1990 Jun;10(6):2475-2484, Molecular & cellular biology
Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 14419, year: 1990, vol: 10, page: 2475, stat: Journal Article,

Antihistaminic/antiallergic activity of 2-dialkylaminoalkylthio(oxy)-1-substituted benzimidazoles: evaluation "in vitro" and "in vivo"
Dini S; Caselli GF; Basilico C; Lavezzo A; Giani R
1990 Apr;30(1-2):174-177, Agents & actions
A new series of 2-dialkylamino-alkylthio(oxy)-1-substituted benzimidazoles synthesized in our laboratories was found to have promising antihistaminic activity. The results of pharmacological screening ('in vitro': radioreceptor binding and isolated organs; 'in vivo': protection against mortality induced by histamine or by compound 48/80, passive cutaneous anaphylaxis, and prolongation of barbiturate-induced sleeping-time) gave clear-cut structure-activity relationships. This series of products has a general selectivity towards H1 receptors, weak antiallergic properties and negligible central effects. DF 10967 (1-ethoxyethyl-2-dimethyl-aminoethylthiobenzimidazole) was the most interesting compound, being very potent both 'in vitro' (Ki = 3.2 +/- 0.8 nM) and 'in vivo' (ID50 11 micrograms/kg, i.p. and 8 micrograms/kg, i.p. against histamine- and 48/80-induced mortality), with no central effects. The last finding is probably due to poor penetration into the brain (as confirmed by 'in vivo' binding test with [3H]-mepyramine) and to lack of interaction with other central receptors
— id: 14422, year: 1990, vol: 30, page: 174, stat: Journal Article,

A mammalian temperature-sensitive mutation affecting G1 progression results from a single amino acid substitution in asparagine synthetase
Gong SS; Basilico C
1990 Jun 25;18(12):3509-3513, Nucleic acids research
ts11 is a temperature-sensitive (ts) mutant isolated from the BHK-21 Syrian hamster cell line that is blocked in the G1 phase of the cell cycle at the non-permissive temperature (39.5 degrees C). We previously showed that the human gene encoding asparagine synthetase (AS) transformed ts11 cells to a ts+ phenotype and that ts11 cells were auxotrophic for asparagine at 39.5 degrees C. We show here that ts11 cells exhibit a ts phenotype for AS activity, and that the ts11 AS was much heat-labile than the wt enzyme. We have isolated AS cDNAs from wt BHK and ts11 cells and found that wt, but not ts11 AS cDNAs were capable of transformation. The deduced amino acid sequence of Syrian hamster AS showed 95% identity to the human protein as well as the same number of residues. The inability of the ts11 AS cDNAs to transform was due to a single base change, a C to T transition, that would result in the substitution of leucine with phenylalanine at a residue located in the C-terminal fourth of the enzyme. Thus the ts11 mutation identifies a mutated, thermolabile AS
— id: 14417, year: 1990, vol: 18, page: 3509, stat: Journal Article,

Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis
Hebert JM; Basilico C; Goldfarb M; Haub O; Martin GR
1990 Apr;138(2):454-463, Developmental biology (Orlando)
To initiate a study of the role of the fibroblast growth factor (FGF) family in mammalian development, we have isolated cDNAs encoding four mouse FGF family members, aFGF, bFGF, kFGF, and FGF-5. This was achieved by a process that circumvents the use of cDNA libraries: for each family member, a cDNA fragment containing the conserved portion of the coding region was amplified from a pool of embryonic and teratocarcinoma cell cDNAs using the polymerase chain reaction (PCR) and cloned; the remaining coding sequences 5' and 3' to the conserved region were cloned using the RACE method. The cDNA clones obtained were used as probes to analyze the expression of these genes at the RNA level in teratocarcinoma cells and embryos at 10.5 to 17.5 days of gestation. Fgfk appears to be specific to undifferentiated teratocarcinoma stem cells. Fgf5 transcripts were detected at every stage and in every tissue tested, but showed a dramatic 15-fold increase in abundance as teratocarcinoma stem cells differentiated to simple embryoid bodies. Fgfb expression showed the greatest tissue-specific variability in abundance, with the highest levels detected in the developing limbs and tail. Fgfa showed the least variable pattern of expression, with transcripts detected at roughly equivalent levels in almost all samples analyzed. On the basis of these data we speculate on some possible roles that the different FGF family members may play in the developing embryo
— id: 14421, year: 1990, vol: 138, page: 454, stat: Journal Article,

Fibroblast growth factor receptor is a portal of cellular entry for herpes simplex virus type 1 [see comments]
Kaner RJ; Baird A; Mansukhani A; Basilico C; Summers BD; Florkiewicz RZ; Hajjar DP
1990 Jun 15;248(4961):1410-1413, Science
Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen responsible for considerable morbidity in the general population. The results presented herein establish the basic fibroblast growth factor (FGF) receptor as a means of entry of HSV-1 into vertebrate cells. Inhibitors of basic FGF binding to its receptor and competitive polypeptide antagonists of basic FGF prevented HSV-1 uptake. Chinese hamster ovary (CHO) cells that do not express FGF receptors are resistant to HSV-1 entry; however, HSV-1 uptake is dramatically increased in CHO cells transfected with a complementary DNA encoding a basic FGF receptor. The distribution of this integral membrane protein in vivo may explain the tissue and cell tropism of HSV-1
— id: 14418, year: 1990, vol: 248, page: 1410, stat: Journal Article,

A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF
Mansukhani A; Moscatelli D; Talarico D; Levytska V; Basilico C
1990 Jun;87(11):4378-4382, Proceedings of the National Academy of Sciences of the United States of America
We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family
— id: 14420, year: 1990, vol: 87, page: 4378, stat: Journal Article,

Protection of mice against tumor growth by immunization with an oncogene-encoded growth factor
Talarico D; Ittmann M; Balsari A; Delli-Bovi P; Basch RS; Basilico C
1990 Jun;87(11):4222-4225, Proceedings of the National Academy of Sciences of the United States of America
The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family that is secreted and transforms cells through a mechanism of autocrine cell proliferation. K-fgf-transformed cells are highly tumorigenic in immunocompetent allogeneic and syngeneic animals. BALB/c mice were immunized with a bacterial fusion protein consisting of a portion of the MS2 polymerase and of the human K-FGF precursor lacking only the first 4 amino acids or with a recombinant protein corresponding to the mature, secreted form of K-FGF (176 amino acids). They were then challenged with syngeneic K-fgf- or H-ras-transformed cells. Vaccinated animals exhibited a significant degree of protection against tumor induction, which was specific for K-fgf-transformed cells and correlated with the ability of the immunized mice to produce high titers of anti-K-FGF antibodies. Thus immunization with a single oncogene product can protect animals against tumor cells expressing this oncogene
— id: 14365, year: 1990, vol: 87, page: 4222, stat: Journal Article,

Autocrine growth stimulation by secreted Kaposi fibroblast growth factor but not by endogenous basic fibroblast growth factor
Wellstein A; Lupu R; Zugmaier G; Flamm SL; Cheville AL; Delli Bovi P; Basilico C; Lippman ME; Kern FG
1990 Feb;1(2):63-71, Cell growth & differentiation
We studied the different potentials of a secreted and a nonsecreted member of the fibroblast growth factor (FGF) family to induce autocrine growth stimulation in human adrenal cortex carcinoma cells (SW-13). These epithelial cells express basic FGF (bFGF) cell surface receptors, and picomolar concentrations of bFGF suffice to induce anchorage-independent growth. The requirement for exogenously added bFGF contrasts with the intracellular storage of biologically active bFGF in SW-13 cells greater than 10,000-fold in excess of the concentration needed to stimulate anchorage independent growth. To study whether the expression of a secreted FGF would alter the growth phenotype of these cells, we transfected them with an expression vector coding for the Kaposi-fgf (K-fgf) oncogene. In contrast to controls, K-fgf-transfected cells secrete significant amounts of biologically active K-fgf protein into the growth media, show up to 50-fold increased colony formation in soft agar, and grow into rapidly progressing, highly vascularized tumors in athymic nude mice. A reversible inhibition of the autocrine growth stimulation in vitro is brought about by the polyanionic compound suramin. We conclude that FGF has to be released from SW-13 cells to function fully as a growth stimulator in vitro and in vivo
— id: 14423, year: 1990, vol: 1, page: 63, stat: Journal Article,

EXPRESSION OF FIBROBLAST GROWTH-FACTOR RECEPTOR GENE IN HUMAN-MELANOMA CELLS
Zouzias, DC; Mansukhani, A; Basilico, C
1990 Apr;38(2):A663-A663, Clinical research
— id: 32082, year: 1990, vol: 38, page: A663, stat: Journal Article,

EXPRESSION OF FIBROBLAST GROWTH-FACTOR RECEPTOR GENE IN HUMAN-MELANOMA CELLS
Zouzias, DC; Mansukhani, A; Basilico, C
1990 Apr;94(4):594-594, Journal of investigative dermatology
— id: 32101, year: 1990, vol: 94, page: 594, stat: Journal Article,

Expression and activation of the K-fgf oncogene
Basilico C; Newman KM; Curatola AM; Talarico D; Mansukhani A; Velcich A; Delli-Bovi P
1989 ;567:95-103, Annals of the New York Academy of Sciences
— id: 10786, year: 1989, vol: 567, page: 95, stat: Journal Article,

Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase
Greco A; Gong SS; Ittmann M; Basilico C
1989 Jun;9(6):2350-2359, Molecular & cellular biology
The human ts11 gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle ts11 mutant, which is blocked in G1 at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the ts11 block. The ts11 (AS) mRNA has a size of about 2 kilobases and is induced in mid-G1 phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the ts11 gene. The human ts11 gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts11 P1) contained in a 280-base-pair region upstream of the first exon and the other (ts11 P2) contained in the first intron. ts11 P1 produced about sixfold more chloramphenicol acetyltransferase activity than did ts11 P2 and had features of the promoters of housekeeping genes: high G + C content, multiple transcription start sites, absence of a TATA box, and presence of putative Sp1 binding sites. ts11 P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The ts11 gene was overexpressed in ts11 cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented G1 induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition
— id: 10604, year: 1989, vol: 9, page: 2350, stat: Journal Article,

Chromosomal localization of human genes required for G1 progression in mammalian cells
Greco A; Ittmann M; Barletta C; Basilico C; Croce CM; Cannizzaro LA; Huebner K
1989 Apr;4(3):240-245, Genomics
Specific probes derived from the human genes that complement the mutations of two independent temperature-sensitive (ts) mutants of the BHK-21 hamster cell line were used to determine the chromosomal locations of the loci in the human genome. The ts11 gene, which complements a mutation that blocks progression through the G1 phase of the cell cycle and which has now been identified as the structural gene for asparagine synthetase, is a member of a small gene/pseudogene family with four members. In a rodent-human somatic cell hybrid panel, the ts11 genomic locus from which the genomic probe derives segregates with human chromosome region 7cen----7q35, proximal to the TCR beta locus. In situ hybridization maps this locus more precisely to the q21-31 region of chromosome 7. Two other members of the gene family detected by the ts11 probe segregate concordantly with chromosome region 8pter----8q24 and chromosome region 21pter----21q22. Similar experiments using the same rodent-human hybrid panel conducted with a probe identifying the tsBN51 gene, which also encodes a function necessary for G1 progression, mapped this locus to human chromosome 8, proximal to the large amplification unit encompassing the c-myc gene of Colo320 cells. Chromosomal in situ hybridization of the tsBN51 probe confirmed the localization of this gene to chromosome 8, with the most likely location of the gene being 8q21
— id: 10677, year: 1989, vol: 4, page: 240, stat: Journal Article,

Transformation by basic fibroblast growth factor requires high levels of expression: comparison with transformation by hst/K-fgf
Quarto N; Talarico D; Sommer A; Florkiewicz R; Basilico C; Rifkin DB
1989 ;5(2):101-110, Oncogene research
Basic fibroblast growth factor is a potent mitogen for a variety of cell types and has been suggested to have transforming activity. To test this hypothesis, we have introduced a human bFGF cDNA into NIH 3T3 cells either by DNA transfection or by retrovirus infection. We have compared the properties of cell lines obtained with cells prepared similarly but expressing the hst/K-fgf growth factor. While bFGF does not contain an amino terminal signal sequence and is not secreted from cells in which it is synthesized, hst/K-fgf does contain a signal sequence and is secreted from cells. Our results show that the transformed phenotype correlates directly with the level of bFGF expression, since all transformed clones expressed high levels of bFGF, while nontransformed clones expressed comparatively low levels of bFGF. In contrast, even low levels of hst/K-fgf expression resulted in a transformed phenotype. These results suggest that bFGF is an inefficient transforming protein and that this may relate to its lack of secretion
— id: 10813, year: 1989, vol: 5, page: 101, stat: Journal Article,

Expression of the K-fgf protooncogene is repressed during differentiation of F9 cells
Velcich A; Delli-Bovi P; Mansukhani A; Ziff EB; Basilico C
1989 ;5(1):31-37, Oncogene research
Utilizing F9 embryonal carcinoma cells as a model system for early mammalian development, we have studied the pattern of expression of the endogenous murine homolog of the human K-fgf/hst oncogene, which encodes a new member of the fibroblast growth factors (FGFs) family. The K-fgf mRNA is expressed in undifferentiated F9 cells and its level becomes undetectable upon the induction of differentiation. Furthermore, a growth-promoting activity with properties identical to those of K-FGF is present in the conditioned medium of F9 cells, but absent in that of differentiated cells. Shut-off of K-fgf expression is mediated at the transcriptional level. The acidic FGF gene is also expressed in undifferentiated F9 cells and down modulated once differentiation is induced. In contrast, int-2, another member of the FGF gene family, is transcriptionally induced in differentiated F9 cells. Our data suggest that single members of the FGF gene family may perform distinct functions in vivo, and that the physiological role of K-FGF may be related to early development
— id: 10762, year: 1989, vol: 5, page: 31, stat: Journal Article,

Cell cycle control in eukaryotes
Beach, David; Basilico, Claudio; Newport, John
Cold Spring Harbor NY : Cold Spring Harbor Laboratory, 1988,
— id: 2090, year: 1988, vol: , page: , stat: ,

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential
Delli-Bovi P; Curatola AM; Newman KM; Sato Y; Moscatelli D; Hewick RM; Rifkin DB; Basilico C
1988 Jul;8(7):2933-2941, Molecular & cellular biology
We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences
— id: 11053, year: 1988, vol: 8, page: 2933, stat: Journal Article,

The FGF-related oncogene, K-FGF, maps to human chromosome region 11q13, possibly near int-2
Huebner K; Ferrari AC; Delli Bovi P; Croce CM; Basilico C
1988 ;3(3):263-270, Oncogene research
The protein encoded in a novel human oncogene isolated by transfection of Kaposi's sarcoma DNA is a growth factor with significant homology to basic and acidic FGFs. The genomic structure of this oncogene (designated K-FGF), as originally isolated, carried DNA rearrangements upstream and downstream of the coding region. The normally discontinuous sequence upstream of the K-FGF coding region derived from the 3' end of the c-fms gene and thus originated from human chromosome 5. In order to determine the normal chromosomal location of the K-FGF gene and of the DNA sequences adjacent to its 3' end, we have correlated the presence of these sequences with retention of specific human chromosome regions in rodent-human somatic cell hybrids. These experiments mapped the K-FGF gene to human chromosome region 11q13----11q23, and in situ hybridization localized it more precisely to region 11q13 near int-2, which also belongs to the FGF family. The sequence downstream of the gene in transfectants and discontinuous with K-FGF in normal human DNA derives from chromosome region 12p12----12q13, possibly near the int-1 locus
— id: 14424, year: 1988, vol: 3, page: 263, stat: Journal Article,

Isolation of a rearranged human transforming gene following transfection of Kaposi sarcoma DNA
Delli Bovi P; Basilico C
1987 Aug;84(16):5660-5664, Proceedings of the National Academy of Sciences of the United States of America
By transfecting high molecular weight DNA from a Kaposi sarcoma lesion into murine NIH 3T3 cells, we have identified and molecularly cloned a set of human DNA sequences capable of inducing focus formation, growth in agar, and tumorigenicity in these cells. The human DNA sequences present in primary, secondary, and tertiary NIH 3T3 transformants encompass about 32 kilobases (kb) and contain four rearrangements with respect to normal human DNA and a portion of the c-fms protooncogene (FMS in human gene nomenclature). However, the minimal transforming region (6.6 kb) identified in our cloned DNA borders on the c-fms DNA region but does not contain c-fms coding sequences. The fms sequences are also not represented in the two transcripts (approximately equal to 1.2 and 3.5 kb) detected in NIH 3T3 transformants; however, they might provide elements regulating expression. Hybridization to several known oncogene probes and preliminary sequencing data indicate that we have identified a previously unrecognized 'activated' oncogene. Since the rearrangements present in our cloned DNA sequences are not detectable in the original Kaposi tumor DNA used for transfection, it is possible that this oncogene was generated during gene transfer
— id: 14427, year: 1987, vol: 84, page: 5660, stat: Journal Article,

An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family
Delli Bovi P; Curatola AM; Kern FG; Greco A; Ittmann M; Basilico C
1987 Aug 28;50(5):729-737, Cell
We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi's sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family
— id: 14426, year: 1987, vol: 50, page: 729, stat: Journal Article,

Molecular cloning of a gene that is necessary for G1 progression in mammalian cells
Greco A; Ittmann M; Basilico C
1987 Mar;84(6):1565-1569, Proceedings of the National Academy of Sciences of the United States of America
We have cloned a human cDNA that complements the mutation of ts11, a temperature-sensitive (ts) mutant of the BHK hamster cell line that at the nonpermissive temperature is blocked in progression through the G1 phase of the cell growth cycle. After transfecting human chromosomal DNA into ts11 cells and selecting for cells that had acquired a non-ts phenotype, we screened a genomic library constructed in the EMBL3 lambda vector from a secondary non-ts transformant and isolated a recombinant phage containing human DNA sequences that were uniformly present in primary and secondary non-ts transformants. Genomic probes that recognized an mRNA of about 2 kilobases in human cells were used to isolate from a cDNA expression library two cDNA plasmids that could efficiently transform ts11 cells to a non-ts phenotype. Sequencing of one of these cDNAs revealed a single open reading frame, which could encode a 540 amino acid protein. The ts11 gene has at least two other homologs in human DNA and thus it appears to be part of a small gene/pseudogene family. Experiments with serum-synchronized cells indicate that the expression of the ts11 gene, which is necessary for G1 progression, is itself cell-cycle regulated, being induced in approximately mid-G1
— id: 14429, year: 1987, vol: 84, page: 1565, stat: Journal Article,

Isolation of the human gene that complements a temperature-sensitive cell cycle mutation in BHK cells
Ittmann M; Greco A; Basilico C
1987 Oct;7(10):3386-3393, Molecular & cellular biology
We have cloned the human genomic DNA and the corresponding cDNA for the gene which complements the mutation of tsBN51, a temperature-sensitive (Ts) cell cycle mutant of BHK cells which is blocked in G1 at the nonpermissive temperature. After transfecting human DNA into TsBN51 cells and selecting for growth at 39.5 degrees C, Ts+ transformants were identified by their content of human AluI repetitive DNA sequences. Following two additional rounds of transfection, a genomic library was constructed from a tertiary Ts+ transformant and a recombinant phage containing the complementing gene isolated by screening for human AluI sequences. A genomic probe from this clone recognized a 2-kilobase mRNA in human and tertiary transformant cell lines, and this probe was used to isolate a biologically active cDNA from the Okayama-Berg cDNA expression library. Sequencing of this cDNA revealed a single open reading frame encoding a polypeptide of 395 amino acids. The deduced BN51 gene product has a high proportion of acidic and basic amino acids which are clustered in four hydrophilic domains spaced at 60- to 80-amino-acid intervals. These domains have strong sequence homology to each other. Thus, the tsBN51 protein consists of periodic repetitive clusters of acidic and basic amino acids
— id: 11347, year: 1987, vol: 7, page: 3386, stat: Journal Article,

A reiterated leader sequence is present in polyomavirus late transcripts produced by a transformed rat cell line
Kern FG; Bovi PD; Basilico C
1987 Dec;61(12):4055-4059, Journal of virology
In cells transformed by polyomavirus, the viral genome is integrated into the host DNA, and in the absence of excision, viral gene expression is limited to the early region. We report here that the ability of a unique transformed rat cell line, designated SS1A, to produce readily detectable levels of late mRNAs is due to rearrangements of the integrated viral sequences. The structure of the SS1A insertion, resulting from amplification and deletion events, allows for the formation of a primary late transcript that can subsequently be spliced to generate a reiterated leader attached to the body of the late mRNA coding sequences. The presence of transcripts containing such a leader was confirmed by sequencing the 5' end of cDNA copies of late mRNAs isolated from a library constructed with SS1A mRNA. These results suggest that a reiterated leader sequence is necessary to stabilize late mRNA
— id: 11315, year: 1987, vol: 61, page: 4055, stat: Journal Article,

The transcription of B2 repeated sequences is regulated during the transition from quiescent to proliferative state in cultured rodent cells
Lania L; Pannuti A; La Mantia G; Basilico C
1987 Jul 27;219(2):400-404, FEBS letters
The RNA polymerase III-dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus-induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus-transformed cells. In both systems, nuclear run-off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2-specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation
— id: 14428, year: 1987, vol: 219, page: 400, stat: Journal Article,

Amplification and expression of foreign genes in cells producing polyoma virus large T-antigen
Pellegrini S; Basilico C
1987 Jun;1(1):23-41, Oncogene research
Polyoma virus (Py) large T-Antigen (LT) can promote the amplification of viral genomes integrated in the chromosomal DNA of rat fibroblasts, and this phenomenon requires the interaction of the LT protein with the viral origin of DNA replication. To compare the rate and the modality of selectable amplification events promoted by the Py LT with cellular-driven events, we constructed a double expression vector containing a murine dihydrofolate reductase (dhfr) cDNA and the bacterial chloramphenicol acetyl transferase (cat) gene controlled by the viral regulatory region. The plasmid was introduced into a rat cell line constitutively producing a temperature sensitive LT (cl 2), and clones were selected in low concentration of methotrexate (MTX). Three cl 2 transformants and one control cell line lacking LT were propagated at temperatures permissive (33 degrees C) and non permissive (39 degrees C) for LT function and, subsequently, challenged in one step with high MTX dosage at 39 degrees C. While the control line produced the same number of colonies irrespective of the temperature of propagation, the three LT positive lines yielded between 2 and greater than 100 times more colonies following propagation at permissive temperature, indicating that the presence of Py LT considerably increased the rate of amplification of integrated sequences linked to the viral origin of replication. In all cases the amplification event involved the exogenous and not the endogenous dhfr gene, and overexpression of the cat gene occurred as a result of co-amplification with the selectable dhfr sequences. Analysis of the structure of the amplified domain in the various resistant derivatives revealed that, in the presence of the viral protein, amplification occurred within the boundaries of the primary plasmid insert. In the absence of a functional LT protein, amplification both internal or involving adjacent host DNA were observed
— id: 11395, year: 1987, vol: 1, page: 23, stat: Journal Article,

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro
Prives C; Murakami Y; Kern FG; Folk W; Basilico C; Hurwitz J
1987 Oct;7(10):3694-3704, Molecular & cellular biology
Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro
— id: 14425, year: 1987, vol: 7, page: 3694, stat: Journal Article,

A subclass of polyomavirus middle tumor antigen binds to DNA cellulose
Bolen JB; Cary K; Scheller A; Basilico C; Israel MA; Prives C
1986 Apr;58(1):157-164, Journal of virology
We examined the binding of polyomavirus large (L-T)-, middle (M-T)-, and small-tumor antigens to DNA cellulose. At pH 6.0, the majority of L-T bound to calf thymus DNA cellulose, while little or no small tumor antigen was retained under these conditions. Unexpectedly, a small but reproducible proportion of M-T bound to both native and denatured DNA cellulose. M-T encoded by polyomavirus mutant dl 8, which expressed shortened L-T and M-T, bound to DNA, indicating that the deleted sequences are not required for DNA binding. Also, M-T from transformed BMT-1 rat cells, which synthesize exclusively this polyomavirus tumor antigen, bound to DNA, indicating that its binding is not due to association with other polyomavirus-encoded proteins. Using the DNA fragment immunoassay, we found that, under conditions in which L-T bound specifically to DNA fragments containing viral regulatory sequences, no viral DNA fragments were bound by M-T. The existence of distinct subpopulations of M-T that differ in their DNA-binding properties was indicated by rebinding experiments in which M-T that had bound to DNA cellulose rebound very efficiently, while that which had not been originally retained by DNA cellulose rebound poorly. Furthermore, the M-T-pp60 c-src complex did not bind to DNA cellulose. These data suggest that polyomavirus M-T is heterogeneous, consisting of populations of molecules that differ in their interactions with DNA cellulose
— id: 14434, year: 1986, vol: 58, page: 157, stat: Journal Article,

Presence of chromosomal abnormalities and lack of AIDS retrovirus DNA sequences in AIDS-associated Kaposi's sarcoma
Delli Bovi P; Donti E; Knowles DM 2d; Friedman-Kien A; Luciw PA; Dina D; Dalla-Favera R; Basilico C
1986 Dec;46(12 Pt 1):6333-6338, Cancer research
The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mice but contained a number of chromosomal rearrangements which were often monoclonal within the same culture. However, these abnormalities were different from culture to culture and even in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity for endothelial markers. Taken together these results indicate that neither the AIDS retrovirus, CMV, or hepatitis B virus is directly responsible for the altered growth of KS cells, that KS may be polyclonal even within the same lesion, and that KS cells have a tendency to karyotypic rearrangements
— id: 14431, year: 1986, vol: 46, page: 6333, stat: Journal Article,

ISOLATION AND STUDY OF THE HUMAN-GENE WHICH COMPLEMENTS THE MUTATION IN A TEMPERATURE-SENSITIVE MUTANT OF THE BHK CELL-LINE
GRECO, A; ITTMANN, M; BASILICO, C
1986 NOV ;103(5):A150-A150, Journal of cell biology
— id: 41319, year: 1986, vol: 103, page: A150, stat: Journal Article,

An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen
Kern FG; Basilico C
1986 ;43(3):237-245, Gene
We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA
— id: 14437, year: 1986, vol: 43, page: 237, stat: Journal Article,

Regulation of polyomavirus late promoter activity by viral early proteins
Kern FG; Pellegrini S; Cowie A; Basilico C
1986 Oct;60(1):275-285, Journal of virology
To assess the effect of the polyomavirus (Py) early proteins, the large T (LT), middle T (MT), and small T (ST) antigens, on gene expression from the Py late promoter, replication-defective plasmid constructs with the bacterial chloramphenicol acetyltransferase (cat) gene linked to this promoter were cotransfected into mouse or rat cells with plasmids capable of producing either LT, MT, or all three early proteins. When target CAT plasmids contained a truncated early region and thus had the coding potential for MT and ST, base-line CAT activities were low, whereas cotransfection with an LT plasmid resulted in up to 70-fold stimulation of CAT activity that was also reflected in similar increases in the level of steady-state mRNA. Studies with target plasmids with deletions within the Py regulatory region indicated that at least the major LT-binding site C and a functional enhancer region were both required for maximal stimulation of CAT activity. However, although enhancer deletions totally suppressed the ability of target plasmids to be trans activated, a consistent two- to fourfold stimulation of CAT activity by LT was still observed with a plasmid in which all three major LT-binding sites were deleted. Of four mutant LTs incapable of binding Py DNA but retaining immortalization potential, only one showed a low but significant trans-activating ability. When the early coding region was completely eliminated from the target plasmid, base-line CAT activity was increased 10-fold. LT failed to stimulate CAT activity to the same levels observed with target plasmid containing the truncated early region, but this limited response could be enhanced by supplying, in addition, MT and ST. Our results suggest that LT trans activation may involve the formation of a complex of transcriptional factors which interacts with the enhancer, an interaction that is facilitated both by the binding of LT to the Py regulatory region and by the presence of MT or ST or both, and that a significant portion of LT stimulation of late gene expression is a result of the removal of the competing early transcriptional unit via autoregulation. In addition, our results suggest that LT trans activation involves a second indirect component acting independently of LT binding and that the immortalization and trans activation functions of LT can be dissociated
— id: 14433, year: 1986, vol: 60, page: 275, stat: Journal Article,

[Biological action of contaminating fungi of food for chickens]
Lura MC; Basilico C
1986 ;37(4):437-440, Acta cientifica venezolana
— id: 14436, year: 1986, vol: 37, page: 437, stat: Journal Article,

Multiple monoclonal B cell expansions and c-myc oncogene rearrangements in acquired immune deficiency syndrome-related lymphoproliferative disorders. Implications for lymphomagenesis
Pelicci PG; Knowles DM 2d; Arlin ZA; Wieczorek R; Luciw P; Dina D; Basilico C; Dalla-Favera R
1986 Dec 1;164(6):2049-2060, Journal of experimental medicine
AIDS (acquired immune deficiency syndrome) and ARC (AIDS-related complex) are associated with a spectrum of lymphoproliferative disorders ranging from lymphadenopathy syndrome (LAS), an apparently benign polyclonal lymphoid hyperplasia, to B cell non-Hodgkin's lymphoma (B-NHL), i.e., malignant, presumably monoclonal B cell proliferations. To gain insight into the process of lymphomagenesis in AIDS and to investigate a possible pathogenetic relationship between LAS and NHL, we investigated the clonality of the B or T lymphoid populations by Ig or T beta gene rearrangement analysis, the presence of rearrangements involving the c-myc oncogene locus, and the presence of human immunodeficiency virus (HIV) sequences in both LAS and B-NHL biopsies. Our data indicate that multiple clonal B cell expansions are present in a significant percentage of LAS (approximately 20%) and B-NHL (60%) biopsies. c-myc rearrangements/translocations are detectable in 9 of our 10 NHLs, but not in any of the LAS cases. However, only one of the B cell clones, identified by Ig gene rearrangements carries a c-myc gene rearrangement, suggesting that only one clone carries the genetic abnormality associated with malignant B cell lymphoma. Furthermore, the frequency of detection of c-myc rearrangements in AIDS-associated NHLs of both Burkitt and non-Burkitt type suggest that the biological alterations present in AIDS favor the development of lymphomas carrying activated c-myc oncogenes. Finally, our data show that HIV DNA sequences are not detectable in LAS nor in NHL B cell clones, suggesting that HIV does not play a direct role in NHL development. Taken together, these observations suggest a model of multistep lymphomagenesis in AIDS in which LAS would represent a predisposing condition to NHL. Immunosuppression and EBV infection present in LAS can favor the expansion of B cell clones, which in turn may increase the probability of occurrence of c-myc rearrangements leading to malignant transformation
— id: 14430, year: 1986, vol: 164, page: 2049, stat: Journal Article,

Rat fibroblasts expressing high levels of human c-myc transcripts are anchorage-independent and tumorigenic
Pellegrini S; Basilico C
1986 Jan;126(1):107-114, Journal of cellular physiology
The c-myc oncogene has been implicated in the genesis of tumors as it has been found to undergo rearrangements and structural alterations in several types of neoplasms. However, the molecular mechanism of its involvement in transformation is still unclear as is the function of its protein product. We have studied the biological activity of the normal human c-myc oncogene in a well-characterized rat fibroblast line, F2408. The human gene, deleted of its 5' non-coding exon, was placed under the control of the Polyoma virus DNA regulatory region, linked to a dominant selectable marker (neomycin-resistance gene) and transfected into F2408 cells. Several neoR colonies were assayed for ability to grow in agar suspension, and the steady-state levels of mRNA transcripts of the exogenous c-myc gene were analyzed by Northern blot. While the greatest majority of these cell populations are phenotypically normal and express low levels of human myc transcripts, the rare neoR clones which display anchorage-independence express the human gene at high levels. This correlation was confirmed by the analysis of two neoR lines which contain low levels of c-myc RNA but gave rise to agar colonies at a frequency of about 10(-5). These agar derivatives were found to have greatly increased levels of c-myc mRNA when compared to the respective parental lines. The clones expressing high levels of myc RNA were found to be tumorigenic upon inoculation into young syngeneic rats. These results suggest that constitutive high expression of the normal c-myc gene is sufficient to confer anchorage-independence and tumorigenic potential to established rat fibroblasts
— id: 14435, year: 1986, vol: 126, page: 107, stat: Journal Article,

Adenovirus E1a proteins repress expression from polyomavirus early and late promoters
Velcich A; Kern FG; Basilico C; Ziff EB
1986 Nov;6(11):4019-4025, Molecular & cellular biology
We have examined the effects of the E1a products of adenovirus types 5 and 12 on the expression of polyomavirus early and late promoters. In cotransfection experiments in HeLa cells, plasmids expressing the E1a region of adenovirus type 5 or 12 repressed both the early and late promoters of polyomavirus, and deletion analysis indicates that the polyomavirus enhancers were the target of the E1a repression. With mutants lacking enhancer sequences, the polyomavirus early promoter but not the late promoter was trans-activated by E1a. Chimeric mutant plasmids with deletions in the regulatory region that contained either the A enhancer or the B enhancer were repressed to the same extent, indicating that E1a can repress both elements. Polyomavirus variant plasmids with rearrangements in the regulatory region conferring activity in embryonal carcinoma stem cells were repressed by E1a as was the wild type, suggesting that the repressor function is quite general. We discuss a model in which the influence of E1a on the transcriptional activity of a gene is the sum of positive and negative effects on promoter and enhancer elements and discuss possible mechanisms of negative regulation of enhancer function
— id: 14432, year: 1986, vol: 6, page: 4019, stat: Journal Article,

Sequences in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression
Dailey L; Basilico C
1985 Jun;54(3):739-749, Journal of virology
We constructed and analyzed a series of deletion mutants in the noncoding regulatory region of tsa polyomavirus DNA to identify some of the sequences critical to the DNA replication origin and to the expression of the viral early genes in vivo. By using both transient and long-term assays under conditions where the influence of large T antigen (T-Ag) in replication or autoregulation was minimized, we observed no more than a 30% reduction in early gene expression upon removal of the CAAT or TATA elements or both. These assays demonstrated a predominant effect of upstream promoter or enhancer elements and indicated that removal of the CAAT or TATA boxes did not significantly affect viral early gene expression. Studies on the replicative ability of these mutants in mouse cells constitutively expressing the polyoma early proteins revealed that the removal of DNA sequences contained within a previously identified T-Ag high-affinity binding site (nucleotides 39 to 64) abolished viral DNA replication, whereas removal of two other high-affinity sites, closer to the early mRNA cap sites, did not. Furthermore, a deletion including this same high-affinity site plus a low-affinity binding site within the 32-base-pair palindrome of the origin core sequences eliminated the ability of the viral large T-Ag to efficiently repress early gene transcription. It is thus possible that the origin-proximal high-affinity T-Ag binding site is involved in both of the functions of large T-Ag, i.e., the initiation of viral DNA replication and the autoregulation of early gene transcription
— id: 14439, year: 1985, vol: 54, page: 739, stat: Journal Article,

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids
Kern FG; Basilico C
1985 Apr;5(4):797-807, Molecular & cellular biology
We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events
— id: 14440, year: 1985, vol: 5, page: 797, stat: Journal Article,

Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids
Kern FG; Dailey L; Basilico C
1985 Aug;5(8):2070-2079, Molecular & cellular biology
In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression
— id: 14438, year: 1985, vol: 5, page: 2070, stat: Journal Article,

The mechanism of cell transformation by SV40 and polyoma virus
Basilico C
1984 ;26(2):235-272, Pharmacology & therapeutics
— id: 14445, year: 1984, vol: 26, page: 235, stat: Journal Article,

Deletion of the origin of replication impairs the ability of polyomavirus DNA to transform cells and to form tandem insertions
Dailey L; Pellegrini S; Basilico C
1984 Mar;49(3):984-987, Journal of virology
We examined the transforming properties of polyomavirus DNA molecules which can produce a functional large T-antigen but which are cis defective for viral DNA replication. The inability of these molecules to replicate results from the deletion of sequences comprising the viral replication origin. We found that even in the presence of a functional large T-antigen, transformation of rat cells by these viral DNAs was greatly reduced when compared with replication-competent parental DNA, and cells transformed by origin-minus mutants generally contained the integrated viral DNA in a nontandem arrangement. Therefore, polyomavirus large T-antigen promotes the establishment of transformation and tandem integration by interacting with the viral origin of DNA replication. This indicates that viral DNA synthesis is directly involved in these processes
— id: 14444, year: 1984, vol: 49, page: 984, stat: Journal Article,

Inhibition of polyoma gene expression in transformed mouse cells by hypermethylation
Liboi E; Basilico C
1984 Jun;135(2):440-451, Virology
The evolution of mouse cells transformed by a recombinant plasmid containing the genome of the tsA mutant of polyoma virus (Py) cloned at the BamHI site into the plasmid pML, whose sequences therefore interrupt the Py late region, has been studied. Clones of transformed cells were selected at 39 degrees (nonpermissive temperature for large T antigen). Under these conditions viral DNA integration is stable and the cells display a uniformed transformed phenotype. Also studied in detail was the evolution of one of these cell lines (A4) upon shift to a temperature permissive for large T-Ag function (33 degrees); immediately after shift, 90% of the population became intensely positive for T-Ag and a considerable amount of free-viral DNA was produced, accompanied by a clear cytopathic effect. Surviving cells proliferated actively after 4 weeks at 33 degrees and showed a decreased expression of large T-Ag (only 2-3% of the population was T-Ag positive by immunofluorescence), a drastic reduction in the amount of free-viral DNA produced, but no apparent change in the pattern of integration of Py DNA in the host chromosomes. Analysis of the high-molecular-weight DNA with the restriction enzymes HpaII and MspI revealed that the cytosines in the recognition sequences of these enzymes were methylated. Accordingly, treating the cells with 5-Azacytidine, a methylation inhibitor, results in the expression of viral T-Ags in more than 80% of the cell population. Analysis of DNA transcription revealed a dramatic reduction of virus-specific poly(A)+ mRNA in the methylated cells; in addition, the phenotype of the 33 degrees A4 populations was much less transformed than that of the original cultures. The block of Py expression by methylation is not complete; approximately 2% of the cells remain T-Ag positive and viral transcription is not completely suppressed. This could be explained by an incomplete methylation which randomly leaves unmethylated sequences essential for Py gene expression, or by the fact that methylation is not sufficient to block transcription completely. Possible mechanisms underlying this type of evolution are discussed
— id: 14442, year: 1984, vol: 135, page: 440, stat: Journal Article,

New rat cell line that is highly susceptible to transformation by several oncogenes
Liboi E; Caruso M; Basilico C
1984 Dec;4(12):2925-2928, Molecular & cellular biology
We describe here a new cell line, EL2, which spontaneously arose from primary rat embryo fibroblasts and has the distinctive property of being highly susceptible to a number of different transforming genes. The high susceptibility is expressed not only in high transformation frequencies but, most importantly, in an unusually high rate of growth of EL2 transformants under selective conditions, i.e., in soft agar or as foci. The biological characteristics of EL2 cells greatly accelerate the isolation of transformants from known oncogenes and could be useful to detect new transforming genes
— id: 14441, year: 1984, vol: 4, page: 2925, stat: Journal Article,

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication
Pellegrini S; Dailey L; Basilico C
1984 Apr;36(4):943-949, Cell
Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination
— id: 14443, year: 1984, vol: 36, page: 943, stat: Journal Article,

Changes in the levels of viral and cellular gene-transcripts in the cell cycle of SV40 transformed mouse cells
La Bella F; Brown EH; Basilico C
1983 Oct;117(1):62-68, Journal of cellular physiology
We have analyzed the regulation of transcription of integrated SV40 DNA and of five cellular genes during the cell cycle of two lines of SV40 transformed mouse 3T3 cells. These cells (ts SV3T3) are temperature sensitive for the expression of the transformed phenotype and at the nonpermissive temperature (39 degrees C) become arrested in G1 at low serum concentrations. SV40 specific RNAs are not detected either in the nuclear or in the cytoplasmic poly(A+)RNA of quiescent cells, suggesting control at the level of transcription. After serum stimulation, however, viral transcription increases and reaches its maximum during S-phase. The expression of a group of selected housekeeping genes has received parallel analysis to determine whether other cellular genes, beside the integrated SV40, are shut off in G1 arrested cells or are expressed in restricted periods of the cell cycle. We have found that, while the mRNAs for collagen, adenosinphosphoribosiltransferase (APRT) and the mouse major histocompatibility complex (H2) are present throughout the cell cycle, the genes coding for the multifunctional protein CAD and dehydrofolate reductase are cell-cycle regulated
— id: 14446, year: 1983, vol: 117, page: 62, stat: Journal Article,

Induction of sister chromatid exchange by polyoma large viral tumor antigen in transformed rat fibroblasts
Brown EH; Basilico C
1982 May;42(5):1909-1912, Cancer research
The frequency of sister chromatid exchange (SCE) was determined in rat fibroblasts transformed by wild-type polyoma virus or by a mutant temperature sensitive for viral large tumor antigen function (ts-a). Elevated SCE frequencies were observed in two wild-type transformed cell lines growing at 37 degrees and in four ts-a-transformed lines upon growth at the permissive temperature for large viral tumor antigen (33 degrees). The increase in SCE frequency in ts-a-transformed cells at 33 degrees was reversed by growth at 39 degrees (nonpermissive for T-antigen function). An increase in SCE at 33 degrees was not observed in untransformed cells or in a ts-a-transformed cell line which makes a defective large viral tumor antigen. These results suggest that large viral tumor antigen can induce SCEs. Since large viral tumor antigen is also responsible for amplification of integrated viral DNA sequences (4), we tried to correlate this phenomenon with the increased SCE frequency. However, increasing SCE artificially by growing cells in the presence of 12-O-tetradecanoylphorbol-13-acetate did not result in amplification of integrated viral DNA in the absence of large viral tumor antigen function. Thus, there is no simple causal relationship between increased SCE and amplification
— id: 14450, year: 1982, vol: 42, page: 1909, stat: Journal Article,

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells
Colantuoni V; Dailey L; Valle GD; Basilico C
1982 Aug;43(2):617-628, Journal of virology
— id: 14449, year: 1982, vol: 43, page: 617, stat: Journal Article,

The evolution of polyoma-transformed rat cell lines during propagation in vitro
Dailey L; Colantuoni V; Fenton RG; La Bella F; Zouzias D; Gattoni S; Basilico C
1982 Jan 15;116(1):207-220, Virology
— id: 14451, year: 1982, vol: 116, page: 207, stat: Journal Article,

Changes in the topography of early region transcription during polyoma virus lytic infection
Fenton RG; Basilico C
1982 Dec;79(23):7142-7146, Proceedings of the National Academy of Sciences of the United States of America
We have studied the pattern of transcription of the early region of polyoma virus DNA after the onset of the late phase of lytic infection of mouse cells. Following initiation of viral DNA synthesis, the early/late switch is accompanied not only by efficient production of late mRNAs but also by the appearance of previously unidentified early-strand RNAs which have certain structural features in common with the classical early mRNAs. Stable poly(A)+RNAs have been identified by blot analysis and S1 nuclease mapping that are not detected early during infection or in polyoma virus-transformed cells. One group consists of transcripts whose 5' ends map 150-200 nucleotides upstream from the major early 5' ends (at positions 148 and 153 on the polyoma virus genome) but whose splicing pattern and poly(A) addition sites are indistinguishable from those of mRNAs produced early in infection. The 5' exons of these early region transcripts contain an open translational reading frame that extends from nucleotide positions 5,255 to 124 and is capable of encoding a basic protein of 53 amino acids. Transcription of these RNAs does not appear to be negatively regulated by large tumor antigen. A transcript of 1,800 nucleotides appears to map predominantly between 93 and 26 map units and does not contain sequences present in the early mRNA 5' exons. These data suggest that, after the onset of polyoma DNA replication, the activation of new early-strand promoters leads to the expression of previously untranscribed viral DNA sequences
— id: 14447, year: 1982, vol: 79, page: 7142, stat: Journal Article,

Regulation of polyoma virus early transcription in transformed cells by large T-antigen
Fenton RG; Basilico C
1982 Sep;121(2):384-392, Virology
— id: 14448, year: 1982, vol: 121, page: 384, stat: Journal Article,

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells
Della Valle G; Fenton RG; Basilico C
1981 May;1(5):418-425, Molecular & cellular biology
To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to 'position' effects or to the high efficiency of recombination of large DNA fragments
— id: 11453, year: 1981, vol: 1, page: 418, stat: Journal Article,

Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells
Della Valle G; Fenton RG; Basilico C
1981 Feb;23(2):347-355, Cell
— id: 14453, year: 1981, vol: 23, page: 347, stat: Journal Article,

Viral gene expression in polyoma virus-transformed rat cells and their cured revertants
Fenton RG; Basilico C
1981 Oct;40(1):150-163, Journal of virology
We have studied transcription of integrated viral DNA sequences in a variety of ts-a polyoma virus-transformed rat cells and cured revertants (which had undergone excision of variables amounts of integrated viral DNA) to characterize the structure of viral mRNA's produced in these lines under conditions in which integrated DNA is stable. Our results indicate that cells containing intact early region sequences, either in single-copy or tandem insertions, produce mRNA's indistinguishable from those observed early in lytic infections; sequences complementary to the polyoma late region were not transcribed from integrated viral DNA. Cured revertants no longer encoded full-length early mRNA's , but produced viral transcripts whose 3' ends mapped at an alternative early region polyadenylic acid attachment site at 99 map units or extended in to flanking host sequences. The phenotype of these revertant cells correlated with the abundance of these transcripts, suggesting that the transforming function(s) of polyoma virus controls the cellular phenotype in a dose-dependent manner. Unexpected results were obtained from studies of cells containing tandem repeats of defective viral DNA in which the polyadenylic acid attachment signal at 25.8 map units and surrounding sequences were deleted. In these cases, polyadenylated mRNA's were observed that contained sequences complementary to the early strand of the polyoma late region. These mRNA's (some larger than 8 kilobases) originated at the viral early promoter, extended into the late region, and continued into the early region of the contiguous repeat in the tandem. The multimeric mRNA's produced contained defective early regions in tandem with late region sequences. S1 analysis indicated that whereas the 5' early region sequences of readthrough transcripts were spliced in the usual manner, internal early region repeats were either unspliced or used only one of the small early region splices. When deletions in the viral readthrough transcripts were observed. This suggests that sequences nearby the AAUAAA sequence at 26 map units may control transcription termination of the polyoma early region
— id: 14452, year: 1981, vol: 40, page: 150, stat: Journal Article,

Integration and excision of polyoma virus genomes
Basilico C; Zouzias D; Della-Valle G; Gattoni S; Colantuoni V; Fenton R; Dailey L
1980 ;44 Pt 1:611-620, Cold Spring Harbor symposia on quantitative biology
— id: 14459, year: 1980, vol: 44 Pt 1, page: 611, stat: Journal Article,

Amplification of integrated viral DNA sequences in polyoma virus-transformed cells
Colantuoni V; Dailey L; Basilico C
1980 Jul;77(7):3850-3854, Proceedings of the National Academy of Sciences of the United States of America
Polyoma virus (Py) transformation of rat cells requires integration of viral genomes into the host DNA, which generally occurs in a partial or full head-to-tail tandem arrangement. The instability of this structure was previously demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show that integrated Py DNA sequences can also undergo amplification. We studied two rat cell lines transformed by the ts-a Py mutant, which codes for a thermolabile large T antigen. In a derivative of the ts-a H6A cell line, we have observed loss of full-length Py DNA molecules from the integrated tandem ('curing'), accompanied by the creation of new tandem repeats of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA replication. In the ts-a H3A cell line, which contains an integrated partial tandem of about 1.3 viral genomes with three distinct deletions, propagation at 33 degrees C resulted in the generation of full tandem repeats of a 94% Py DNA 'unit' (including two 3% deletions), an 85% 'unit' (including a 3% and the 12% deletion), or both. Amplification of integrated viral DNA was not observed in cells propagated at 39.5 degrees C, the nonpermissive temperature for large T antigen function. Amplification of integrated Py DNA sequences thus requires an active large T antigen and can generate a full tandem of integrated viral DNA molecules long after the initial integration event
— id: 14456, year: 1980, vol: 77, page: 3850, stat: Journal Article,

Decreased initiation of DNA synthesis in a temperature-sensitive mutant of hamster cells
Eilen E; Hand R; Basilico C
1980 Nov;105(2):259-266, Journal of cellular physiology
We have analyzed ongoing DNA replication in ts BN-2, a dna- mutant of BHK-21 cells (Nishimoto et al. '78). At the non-permissive temperature of 39.5 degrees C, inhibition of 3H-thymidine into acid-precipitable material begins 1 to 2 h after the cells are released from a block at the start of the S-phase. The fraction of nuclei incorporating 3H-thymidine is similar to that of wild-type cells through the synchronized S-phase of 8 h. Alkaline sucrose gradient analysis shows that pulse-labeled DNA from mutant cells is incorporated into high molecular weight material after 3 h at either the permissive or non-permissive temperature. DNA fiber autoradiograms reveal that, at 39.5 degrees C, the rate of replication fork movement is about 30% increased in the mutant as compared to wild-type cells. In the mutant cells, however, the interval between adjacent initiation sites is increased and the relative frequency of initiation events is decreased at the restrictive temperature. The results indicate that there is a block to ongoing replication in ts BN-2 at the level of initiation of synthesis on individual replication units; elongation of nascent chains is not inhibited
— id: 14454, year: 1980, vol: 105, page: 259, stat: Journal Article,

Relationship between integrated and nonintegrated viral DNA in rat cells transformed by polyoma virus
Gattoni S; Colantuoni V; Basilico C
1980 Jun;34(3):615-626, Journal of virology
Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose presence is under the control of the viral A gene. To understand the mechanism of production of the 'free' viral DNA, we have characterized the DNA species produced by several rat lines transformed by wild-type virus or by ts-a polyoma virus and compared them with the integrated viral sequences. Every cell line tested yielded a characteristic number of discrete species of viral DNA. The presence of defectives was a very common occurrence, and these molecules generally carried deletions mapping in the viral 'late' region. The production of multiple species of free viral DNA was not due to heterogeneity of the transformed rat cell population, and its pattern did not change upon fusion with permissive mouse cells. Analysis of the integrated viral DNA sequences in the same cell lines showed, in most cases, a full head-to-tail tandem arrangement of normal-size and defective molecules. The free DNA produced by these lines faithfully reflected the integrated species. This was true also in the case of a cell line which contained a viral insertion corresponding to approximately 1.3 polyoma genomes, with each of the repeated portions of the viral DNA molecule carrying a different-size deletion. These results support the hypothesis that the free DNA derives from the integrated form through a mechanism of homologous recombination leading to excision and limited replication
— id: 14458, year: 1980, vol: 34, page: 615, stat: Journal Article,

DECREASED INITIATION OF DNA-REPLICATION IN A TEMPERATURE- SENSITIVE MUTANT OF HAMSTER-CELLS
Hand, R; Eilen, E; Basilico, C
1980 ;28(2):A526-A526, Clinical research
— id: 28121, year: 1980, vol: 28, page: A526, stat: Journal Article,

A temperature-sensitive mutation affecting S-phase progression can lead to accumulation of cells with a G2 DNA content
Nishimoto T; Takahashi T; Basilico C
1980 Jul;6(4):465-476, Somatic cell genetics
Cultures of ts BN75, a temperature-sensitive mutant of BHK 21 cells, show a gradual biphasic drop in [3H]thymidine incorporation together with an accumulation of cells having a G2 DNA content when incubated at 39.5 degrees. However, when higher (41 degrees - 42 degrees) nonpermissive temperatures were used, the major block was in S-phase DNA synthesis. The cultures of ts BN75 shifted to 42 degrees at the start of the S phase, cell-cycle progress was arrested in the middle of S, while under these conditions wild-type BHK cells underwent at least one cycle of DNA synthesis. When ts BN75 cells growth-arrested at high temperature with a G2 DNA content were shifted to the permissive temperature (33.5 degrees C), the restart of DNA synthesis preceded the appearance of mitotic cells. These data suggest that the ts defect of ts BN75 cells might affect primarily the S phase of the cycle rather than the G2 phase
— id: 14457, year: 1980, vol: 6, page: 465, stat: Journal Article,

Human fibroblasts transformed by the early region of SV40 DNA: analysis of "free" viral DNA sequences
Zouzias D; Jha KK; Mulder C; Basilico C; Ozer HL
1980 Jul 30;104(2):439-453, Virology
— id: 14455, year: 1980, vol: 104, page: 439, stat: Journal Article,

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function
Basilico C; Gattoni S; Zouzias D; Valle GD
1979 Jul;17(3):645-659, Cell
Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the 'curing' of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for 'curing' by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo 'curing' with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable
— id: 14460, year: 1979, vol: 17, page: 645, stat: Journal Article,

T-antigen expression in proliferating and non-proliferating simian virus 40-transformed mouse cells
Zouzias D; Basilico C
1979 Jun;30(3):711-719, Journal of virology
Previous studies with simian virus 40-transformed mouse 3T3 cells which are temperature sensitive for the expression of the transformed phenotype (ts SV3T3 cells) have shown that T-antigen expression and viral DNA transcription are under cell cycle control. Using these ts SV3T3 cells, we studied the expression of the viral genome under proliferating and non-proliferating conditions, in the presence and absence of inhibitors of macromolecular synthesis and of the tumor promoter phorbol myristate acetate. ts SV3TE cells which are growth arrested at 39 degrees C by low serum concentration or saturation density accumulated in G1 and did not express T-antigen. When these cells were induced to proliferate, at either 32 or 39 degrees C, T-antigen synthesis preceded the entry of the cells into the S-phase and was not coupled to DNA replication. G1-arrested ts SV3T3 cells were induced to synthesize T-antigen by phorbol myristate acetate treatment, but T-antigen alone was not sufficient to induce cellular DNA synthesis. Isoleucine deprivation arrested growth of ts SV3T3 cells, but these cells, as well as normal 3T3, did not accumulate in G1 and continued to express T-antigen. The temperature-sensitive expression of the transformed phenotype in the ts SV3T3 cells does not appear to be due to a lack of transcription of specific regions of the integrated simian virus 40 genome at 39 degrees C
— id: 14461, year: 1979, vol: 30, page: 711, stat: Journal Article,

Selective production of cell cycle specific ts mutants
Basilico C
1978 Jun;95(3):367-372, Journal of cellular physiology
— id: 14465, year: 1978, vol: 95, page: 367, stat: Journal Article,

G1 ARREST AND PREMATURE CHROMOSOME CONDENSATION IN A TS DNA MUTANT OF BHK CELLS
Eilen, E; Nishimoto, T; Basilico, C
1978 ;79(2):A4-A4, Journal of cell biology
— id: 29869, year: 1978, vol: 79, page: A4, stat: Journal Article,

Analysis of a method for selecting temperature-sensitive mutants of BHK cells
Nishimoto T; Basilico C
1978 May;4(3):323-340, Somatic cell genetics
A procedure for the isolation of temperature sensitive mutants of BHK cells is described. Mutagenized cells were synchronized in G1 by serum starvation at 33 degrees, and, following release, shifted to 37.5 degrees in the presence of FUdR, to kill cells entering the DNA synthetic phase. Eight independently mutagenized series of cells were carried through four cycles of this selection, and surviving cells were tested for ability to grow at 33 degrees and 39.5 degrees after each cycle. The results suggest that such procedure is effective in enriching for non-leaky ts mutants and favors the isolation of mutants which at 39.5 degrees are inhibited in the processes necessary for DNA synthesis and lose viability rapidly. The effectiveness of repeated selection cycles and the general characteristics of the ts mutants isolated by this method were also evaluated
— id: 14466, year: 1978, vol: 4, page: 323, stat: Journal Article,

Premature of chromosome condensation in a ts DNA- mutant of BHK cells
Nishimoto T; Eilen E; Basilico C
1978 Oct;15(2):475-483, Cell
A temperature-sensitive mutant of BHK, designated ts BN-2, shows a rapid drop in 3H-thymidine incorporation along with accumulation of the cells in the G1 phase of the cycle when asynchronous cultures are shifted from 33.5 degrees C to the nonpermissive temperature of 39.5 degrees C. Synchronized cultures of ts BN-2 cells did not enter DNA synthesis when shifted up in G1. Shift-up of cultures at the beginning of the S phase resulted in an approximately normal rate of DNA synthesis for about 2 hr. The rate of DNA synthesis then quickly declined, and the cells became arrested in mid-S after completion of approximately 0.5 rounds of DNA replication. At the same time, the majority of the cells were observed to lose the nuclear membrane and displayed premature chromosome condensation. These events were followed by the appearance of cells containing several micronuclei and eventual cell disruption and death. The nonpermissive temperature appeared to have no effect on either the elongation of short fragments of DNA or the execution of mitosis after the completion of the S phase under permissive conditions. The ts defect in this mutant may directly limit the initiation of DNA synthesis or alter the regulation of chromatin condensation
— id: 14463, year: 1978, vol: 15, page: 475, stat: Journal Article,

Nonintegrated viral DNA in rat cells doubly transformed by SV40 and polyoma virus
Prasad I; Zouzias D; Basilico C
1978 Mar;85(1):328-331, Virology
— id: 14468, year: 1978, vol: 85, page: 328, stat: Journal Article,

Requirements of BHK cells for the exit from different quiescent states
Talavera A; Basilico C
1978 Dec;97(3 Pt 2 Suppl 1):429-439, Journal of cellular physiology
We have investigated the kinetics of exit from the resting state of BHK cells which had been arrested by isoleucine deprivation, serum starvation, or high temperature in the case of three ts G1 mutants. In addition, we have studied the effect of imposing a secondary deprivation on cells which had been released from one of the above mentioned blocks. The results obtained show that the quiescent states reached by BHK cells following serum or isoleucine deprivation cannot be differentiated on the basis of the exit kinetics from Smith and Martin's probabilistic A-state. Nevertheless, the response of cells to secondary deprivation is different, depending on the nature of the primary arresting condition used, reflecting physiological differences between the different resting states. A model is presented which postulates that cycle transition specific genes require the presence of different proliferative agents for their expression
— id: 14462, year: 1978, vol: 97, page: 429, stat: Journal Article,

Synthesis of H1 histones by BHK cells in G1
Tarnowka MA; Baglioni C; Basilico C
1978 Sep;15(1):163-171, Cell
The synthesis of histones and DNA was examined in BHK cells arrested in G1 by isoleucine starvation and in cells progressing into the S phase upon isoleucine refeeding. Approximately 2-3% of the cells were not arrested in G1 and synthesized DNA. The rate of synthesis of DNA and nucleosomal histones observed in cells starved for isoleucine could be accounted for by the presence of these asynchronous cells. Synthesis of H1 histones by cells in G1, however, was 3 times that of the nucleosomal histones and approximately 15% of the rate of H1 histone synthesis in mid-S. Upon entry into S, the histones were synthesized in the same molar ratio in which they are present in chromatin. The possible biological significance of H1 histone synthesis in G1 cells and its implications for the regulatory mechanisms controlling histome synthesis are discussed
— id: 14464, year: 1978, vol: 15, page: 163, stat: Journal Article,

Regulation of viral functions in simian virus 40-transformed cells
Zouzias D; Basilico C
1978 May;(48):239-244, National Cancer Institute monograph
To define the relationship between simian virus 40 (SV40)-specific T-antigen and cell growth and to look for regulatory mechanisms that might control T-antigen synthesis in transformed cells, we studied the expression of T-antigen and the viral transcription in SV40-transformed cells that were exponentially growing or arrested in the G1-phase of the cell cycle. We took advantage of the behavior of two lines of SV40-transformed mouse 3T3 cells (ts SV3T3), which, although transformed by wild-type SV40, are temperature sensitive for the expression of the transformed phenotype. At 32 degrees C, ts SV3T3 cells behave like standard transformants, whereas at 39 degrees C, they become arrested in G1 after reaching saturatio n density or under serum starvation. At 32 degrees C or growing at 39 degrees C, ts SV3T3 were 100% T-antigen positive and contained virus-specific mRNA. However, after G1 arrest at 39 degrees C, most of the cells became T-antigen negative. This seems to be caused by a lack of transcription of the integrated viral DNA, since these cells contain no appreciable amounts of SV40-specific RNA. Induction of proliferation in resting, T-antigen-negative ts SV3T3 cultures results in the reappearance of T-antigen a few hours before the cells enter DNA synthesis. These results suggest that transcription of the viral genome and T-antigen expression in SV40-transformed cells is subjected to a cell cycle control
— id: 14467, year: 1978, vol: , page: 239, stat: Journal Article,

REGULATION OF VIRAL FUNCTIONS IN SV40-TRANSFORMED MOUSE CELLS
Zouzias, DC; Basilico, C
1978 ;37(6):1746-1746, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29910, year: 1978, vol: 37, page: 1746, stat: Journal Article,

Temperature-sensitive mutations in animal cells
Basilico C
1977 ;24:223-266, Advances in cancer research
— id: 14473, year: 1977, vol: 24, page: 223, stat: Journal Article,

Suppression of production of mouse 28S ribosomal RNA in mouse-human hybrids segregating mouse chromosomes
Croce CM; Talavera A; Basilico C; Miller OJ
1977 Feb;74(2):694-697, Proceedings of the National Academy of Sciences of the United States of America
Mouse-human somatic cell hybrids that lose (segregate) human chromosomes produce only mouse 28S ribosomal RNA even when they retain copies of the human chromosomes that contain the genes for 28S ribosomal RNA. In contrast, mouse-human hybrid cells that segregate mouse chromosomes produce only human 28S ribosomal RNA even when they have retained copies of mouse chromosomes that contain the 28S ribosomal RNA genes
— id: 14472, year: 1977, vol: 74, page: 694, stat: Journal Article,

Expression of the SV40-transformed phenotype in hybrids between conditional and wild-type transformants
Rovigatti U; Basilico C
1977 May;106(2):277-284, Experimental cell research
— id: 14471, year: 1977, vol: 106, page: 277, stat: Journal Article,

Temperature sensitive mutants of BHK cells affected in cell cycle progression
Talavera A; Basilico C
1977 Sep;92(3):425-436, Journal of cellular physiology
— id: 14470, year: 1977, vol: 92, page: 425, stat: Journal Article,

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations
Zouzias D; Prasad I; Basilico C
1977 Oct;24(1):142-150, Journal of virology
F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication
— id: 14469, year: 1977, vol: 24, page: 142, stat: Journal Article,

Regulation of viral transciption and tumor antigen expression in cells transformed by simian virus 40
Basilico C; Zouzias D
1976 Jun;73(6):1931-1935, Proceedings of the National Academy of Sciences of the United States of America
We have studied the expression of simian virus 40 (SV40) specific tumor antigen (T-antigen) and viral RNA in SV40-transformed mouse 3T3 cells that are temperature-sensitive for the expression of the transformed phenotype (ts SV3T3). Although transformed by wild-type SV40, ts SV3T3 cells at 32 degrees behave like standard transformants, while at 39 degrees they became arrested in G1 after reaching saturation density or under conditions of serum starvation. ts SV3T3 cells at 32 degrees or exponentially growing at 39 degrees are uniformly T-antigen positive. However, after G1 arrest at 39 degrees the majority of the cells becomes T-antigen negative. Induction of proliferation in the resting cultures results in the reappearance of T-antigen in most of the cells, concomitant with the induction of DNA synthesis. The reason for the disappearance of T-antigen from ts SV3T3 cells arrested in G1 seems to reside in a transcriptional control operating on the integrated viral DNA, since these cells contain no appreciable amounts of SV40 specific RNA. Viral RNA can be easily detected in cells growint at 32 degrees or at 39 degrees. The results suggest that transcription of the viral genome in SV40-transformed cells is cell-cycle-dependent
— id: 14474, year: 1976, vol: 73, page: 1931, stat: Journal Article,

Transcriptional activity and chromatin structural changes in a temperature-sensitive mutant of BHK cells blocked in early G1
Kane G; Basilico C; Basterga R
1976 Apr;99(1):165-173, Experimental cell research
— id: 14477, year: 1976, vol: 99, page: 165, stat: Journal Article,

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules
Prasad I; Zouzias D; Basilico C
1976 May;18(2):436-444, Journal of virology
The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). 'Free' viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives
— id: 14475, year: 1976, vol: 18, page: 436, stat: Journal Article,

STATE OF FREE VIRAL-DNA IN RAT CELLS TRANSFORMED BY POLYOMA- VIRUS
Prasad, I; Zouzias, D; Basilico, C
1976 ;70(2):A67-A67, Journal of cell biology
— id: 29449, year: 1976, vol: 70, page: A67, stat: Journal Article,

Imperfect complementation in human-hamster somatic cell hybrids
Talavera A; Basilico C; Croce CM
1976 Feb 26;259(5545):667-670, Nature
— id: 14478, year: 1976, vol: 259, page: 667, stat: Journal Article,

ISOLATION AND CHARACTERIZATION OF CELL-CYCLE MUTANTS OF BHK
Talavera, A; Nishimoto, T; Basilico, C
1976 ;70(2):A341-A341, Journal of cell biology
— id: 28741, year: 1976, vol: 70, page: A341, stat: Journal Article,

Processing of ribosomal RNA in a temperature sensitive mutant of BHK cells
Toniolo D; Basilico C
1976 Apr 2;425(4):409-418, Biochimica & biophysica acta
The processing of ribosomal RNA has been studied in a temperature sensitive mutant of the Syrian hamster cell line BHK 21. At 39 degrees C, these cells are unable to synthesize 28S RNA, and 60S ribosomal subunits, while 18S RNA, and 40S subunits are produced at both temperatures. At 39 degrees C the 45S RNA precursor is transcribed and processed as in wild type cells. The processing of the RNA precursors becomes defective after the cleavage of the 41S RNA, and the separation of the 18S and 28S RNAs sequences in two different RNA molecules. The 36S RNA precursor, which is always present in very small quantity in the nucleoli of wild type cells and of the mutant at 33 degrees C, is found in very large amounts in the mutant at 39 degrees C. The 36S RNA can be, however, slowly processed to 32S RNA. The 32S RNA cannot be processed at 39 degrees C, and it is degraded soon after its formation. Only a small proportion accumulates in the nucleoli. The 32S RNA synthesized at 39 degrees C cannot be processed to 28S RNA upon shift to the permissive temperature, even when the processing of the newly synthesized rRNA has returned to normal. The data suggest that the 36S and 32S RNAs are contained in aberrant ribonucleoprotein particles, leading to a defective processing of the particles as a whole
— id: 14476, year: 1976, vol: 425, page: 409, stat: Journal Article,

REGULATION OF T-ANTIGEN AND VIRAL-RNA SYNTHESIS IN SV40- TRANSFORMED CELLS
Zouzias, DC; Basilico, C
1976 ;35(7):1688-1688, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29475, year: 1976, vol: 35, page: 1688, stat: Journal Article,

Transformation by polyoma virus alters expression of a cell mutation affecting cycle traverse
Burstin SJ; Basilico C
1975 Jul;72(7):2540-2544, Proceedings of the National Academy of Sciences of the United States of America
A temperature-sensitive mutant of hamster BHK 21/13 cells, tsAF8, which at 39 degrees becomes arrested in the G1 (G0) phase of the cell cycle, is phenotypically altered with respect to temperature sensitivity after transformation with polyoma virus. Polyoma transformation does not produce reversion to a non-temperature-sensitive phenotype but causes increased entry into S and increased rate of cell death at the nonpermissive temperature, compared to untransformed tsAF8 cells. The increased frequency of cells synthesizing DNA is not accompanied by an increased frequency of mitosis, since most of the polyoma-transformed tsAF8 cells that synthesize DNA at the nonpermissive temperature do not divide. At the permissive temperature, polyoma-transformed tsAF8 cells, unlike tsAF8, also lose viability when exposed to other methods of arresting cells in G1. The most likely explanation for this phenomenon is that polyoma virus transformation interferes with the cellular response to this mutation as well as to other conditions that cause cell cycle arrest in G1
— id: 14479, year: 1975, vol: 72, page: 2540, stat: Journal Article,

Temperature-sensitive cell mutations that inhibit adenovirus 2 replication
Nishimoto T; Raskas HJ; Basilico C
1975 Jan;72(1):328-332, Proceedings of the National Academy of Sciences of the United States of America
Five temperature-sensitive growth mutants of the hamster cell line BHK-21 were tested for the ability to support adenovirus 2 multiplication at 39 degrees and 33 degrees. Wild-type BHK-21 and mutants ts 422E and ts BCH yielded comparable amounts of virus at 33 degrees and 39 degrees, whereas in three other mutants, ts T22, ts T23, and ts AF8, virus production at 39 degrees was reduced to about 1% of that at 33 degrees. Virus yield in the three mutants was not reduced because of a delay in virus production; for all cells tested maximal virus yield at 39 degrees was obtained by 40-50 hr after infection. Normal yields of infectious virus were not obtained from ts AF8 even with a very high multiplicity of infection. In contrast, the virus yield from ts T22 and ts T23 was multiplicity-dependent. Shiftup experiments demonstrated that in ts AF8, a cell cycle mutant which at 39 degrees becomes arrested in G1, virus multiplication was thermosensitive for the first 40 hr of infection. In ts T22 and ts T23, the thermosensitivity was only for the first 3-4 hr of the infection. In all three mutants viral DNA synthesis was reduced by at least 95% at the higher temperature. The cell function specified by the ts AF8 mutation seems to be required for the early period of adenovirus 2 replication, after virus entry into the cell but before the onset of viral DNA replication
— id: 14481, year: 1975, vol: 72, page: 328, stat: Journal Article,

INTEGRATION OF SV40
Prasad, I; Basilico, C
1975 ;16(MAR):20-20, Proceedings (American Association for Cancer Research)
— id: 28665, year: 1975, vol: 16, page: 20, stat: Journal Article,

SV40-transformed cells with temperature-dependent serum requirements
Toniolo D; Basilico C
1975 Mar;4(3):255-262, Cell
We have isolated temperature-sensitive SV40-transformed 3T3 cells which are unable to grow in low or depleted serum at the nonpermissive temperature. At 39 degrees C, these cells do not grow in 1 percent serum, but they grow if the serum concentration is raised to 10 percent. At 32 degrees they grow in both serum concentrations. This phenotype seems to be due to a cellular mutation, as the virus rescued from these cells is wild-type. We tested whether other characteristics of transformed cells were expressed in a temperature sensitive way. While high saturation density is ts in these cells, other parameters of transformation are expressed at both temperatures. In addition, when these cells are incubated in low serum at 39 degrees C, they keep synthesizing DNA and lose viability very fast, while under the same conditions normal 3T3 cells remain viable for long times and are unable to initiate DNA synthesis. These cells therefore do not appear to revert to a normal phenotype at the high temperature, and they are more likely to represent transformed cell variants with a temperature-dependent serum requirement
— id: 14480, year: 1975, vol: 4, page: 255, stat: Journal Article,

Mutant of polyoma virus with impaired adsorption to BHK cells
Basilico C; DiMayorca G
1974 Apr;13(4):931-934, Journal of virology
— id: 14484, year: 1974, vol: 13, page: 931, stat: Journal Article,

Methods for selecting and studying temperature-sensitive mutants of BHK-21 cells
Basilico C; Meiss HK
1974 ;8(0):1-22, Methods in cell biology
— id: 14486, year: 1974, vol: 8, page: 1, stat: Journal Article,

A temperature-sensitive cell cycle mutant of the BHK cell line
Burstin SJ; Meiss HK; Basilico C
1974 Dec;84(3):397-408, Journal of cellular physiology
— id: 14482, year: 1974, vol: 84, page: 397, stat: Journal Article,

Cyclic AMP levels in temperature sensitive SV40 transformed cell lines
Burstin SJ; Renger HC; Basilico C
1974 Aug;84(1):69-73, Journal of cellular physiology
— id: 14483, year: 1974, vol: 84, page: 69, stat: Journal Article,

TEMPERATURE SENSITIVE MUTATION OF BHK 21 CELLS
Burstin, SJ; Meiss, HK; Basilico, C
1974 ;63(2):A44-A44, Journal of cell biology
— id: 28421, year: 1974, vol: 63, page: A44, stat: Journal Article,

Complementation of a defect in the production of ribosomal RNA in somatic cell hybrids
Toniolo D; Basilico C
1974 Mar 29;248(447):411-413, Nature
— id: 14485, year: 1974, vol: 248, page: 411, stat: Journal Article,

TEMPERATURE-SENSITIVE MUTANTS OF MAMMALIAN SOMATIC-CELLS
BASILICO, C
1973 ;35(2):181-182, Transactions of the New York Academy of Sciences
— id: 39808, year: 1973, vol: 35, page: 181, stat: Journal Article,

Surface changes in temperature-sensitive Simian virus 40-transformed cells
Noonan KD; Renger HC; Basilico C; Burger MM
1973 Feb;70(2):347-349, Proceedings of the National Academy of Sciences of the United States of America
— id: 14489, year: 1973, vol: 70, page: 347, stat: Journal Article,

Temperature-sensitive simian virus 40-transformed cells: phenomena accompanying transition from the transformed to the "normal" state
Renger HC; Basilico C
1973 May;11(5):702-708, Journal of virology
— id: 14487, year: 1973, vol: 11, page: 702, stat: Journal Article,

A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells
Toniolo D; Meiss HK; Basilico C
1973 Apr;70(4):1273-1277, Proceedings of the National Academy of Sciences of the United States of America
— id: 14488, year: 1973, vol: 70, page: 1273, stat: Journal Article,

Temperature sensitive mutants of BHK 21 cells
Meiss HK; Basilico C
1972 Sep 20;239(90):66-68, Nature: new biology
— id: 14490, year: 1972, vol: 239, page: 66, stat: Journal Article,

Mutation causing temperature-sensitive expression of cell transformation by a tumor virus (SV40-3T3 mouse cells-growth control)
Renger HC; Basilico C
1972 Jan;69(1):109-114, Proceedings of the National Academy of Sciences of the United States of America
— id: 14491, year: 1972, vol: 69, page: 109, stat: Journal Article,

Multiplication of polyoma virus in mouse-hamster somatic hybrids: a hybrid cell line which produces viral particles containing predominantly host deoxyribonucleic acid
Basilico, C; Burstin, S J
1971 Jun;7(6):802-812, Journal of virology
— id: 14492, year: 1971, vol: 7, page: 802, stat: Journal Article,

Susceptibility to superinfection of hybrids between polyoma "transformed" BHK and "normal" 3T3 cells
Basilico, C; Wang, R
1971 Mar 24;230(12):105-107, Nature: new biology
— id: 14494, year: 1971, vol: 230, page: 105, stat: Journal Article,

Mammalian somatic cell hybrids and their susceptibility to viral infection
Green, H; Wang, R; Basilico, C; Pollack, R; Kusano, T; Salas, J
1971 May-Jun;30(3):930-934, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 14493, year: 1971, vol: 30, page: 930, stat: Journal Article,

Induction of mitochondrial DNA synthesis by polyoma virus
Vesco, C; Basilico, C
1971 Jan 29;229(5283):336-338, Nature
— id: 14495, year: 1971, vol: 229, page: 336, stat: Journal Article,

The interaction of polyoma virus with mouse-hamster somatic hybrid cells
Basilico C; Matsuya Y; Green H
1970 Jun;41(2):295-305, Virology
— id: 14496, year: 1970, vol: 41, page: 295, stat: Journal Article,

Origin of the thymidine kinase induced by polyoma virus in productively infected cells
Basilico C; Matsuya Y; Green H
1969 Feb;3(2):140-145, Journal of virology
— id: 14498, year: 1969, vol: 3, page: 140, stat: Journal Article,

Electron microscopic studies of polyoma DNA released in protein monolayers
Vasquez C; Kleinschmidt AK; Basilico C
1969 Jul 28;43(2):317-325, Journal of molecular biology
— id: 14497, year: 1969, vol: 43, page: 317, stat: Journal Article,

Studies on a temperature-sensitive mutant of vaccinia virus strain WR
Basilico C; Joklik WK
1968 Dec;36(4):668-677, Virology
— id: 14500, year: 1968, vol: 36, page: 668, stat: Journal Article,

Properties and uses of human-mouse hybrid cell lines
Matsuya Y; Green H; Basilico C
1968 Dec 21;220(173):1199-1202, Nature
— id: 14499, year: 1968, vol: 220, page: 1199, stat: Journal Article,

Effect of the inhibition of protein synthesis on the establishment of transformation by polyoma virus
Marin G; Basilico C
1967 Feb;1(1):120-127, Journal of virology
— id: 14502, year: 1967, vol: 1, page: 120, stat: Journal Article,

Transformation of polyploid hamster cells by polyoma virus
Marin G; Basilico C
1967 Oct 7;216(110):62-63, Nature
— id: 14501, year: 1967, vol: 216, page: 62, stat: Journal Article,

Requirement for the integrity of the viral genome for the induction of host DNA synthesis by polyoma virus
Basilico C; Marin G; Mayorca G di
1966 Jul;56(1):208-215, Proceedings of the National Academy of Sciences of the United States of America
— id: 14504, year: 1966, vol: 56, page: 208, stat: Journal Article,

Infection of thymidine kinase-deficient BHK cells with polyoma virus
Littlefield JW; Basilico C
1966 Jul 16;211(46):250-252, Nature
— id: 14503, year: 1966, vol: 211, page: 250, stat: Journal Article,

Radiation target size of the lytic and the transforming ability of polyoma virus
Basilico C; Di Mayorca G
1965 Jul;54(1):125-127, Proceedings of the National Academy of Sciences of the United States of America
— id: 14505, year: 1965, vol: 54, page: 125, stat: Journal Article,