Alka Mansukhani, Ph.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,

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,

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,

Endothelium-targeted overexpression of constitutively active FGF receptor induces cardioprotection in mice myocardial infarction
Matsunaga, Shinsaku; Okigaki, Mitsuhiko; Takeda, Mitsuo; Matsui, Akihiro; Honsho, Shoken; Katsume, Asako; Kishita, Eigo; Che, Jishan; Kurihara, Tatsuya; Adachi, Yasushi; Mansukhani, Alka; Kobara, Miyuki; Matoba, Satoaki; Tatsumi, Tetsuya; Matsubara, Hiroaki
2009 May;46(5):663-673, Journal of molecular & cellular cardiology
Fibroblast growth factor receptor (FGFR) is expressed in a variety of cells and is involved in their proliferation/migration/survival. To elucidate FGFR-mediated specific action of vascular endothelial cells (ECs) on myocardial ischemia, we generated endothelium-targeted transgenic mice overexpressing constitutively active FGFR2 using Tie2 promoter (FGFR2-Tg). Infarct size, vessel formation and blood perfusion were significantly improved 28 days after myocardial infarction (MI) in FGFR2-Tg, compared with wild-type mice. Aortic ECs isolated from FGFR-Tg showed a marked increase in migratory capacity and tube formation. These in vitro angiogenic activities were blocked by PI3-kinase inhibitor. Whereas, parameters obtained from echocardiography were already improved at three days after MI. Cardiomyocyte apoptosis at the ischemic border zone was decreased in FGFR2-Tg (32.1%, p < 0.05) and cardiac mRNA expression of FGF2 (basic FGF) was also up-regulated (142%, p < 0.05) at 3 days after MI. 1% oxygen-mediated apoptosis was significantly inhibited in FGFR2-Tg-ECs and this inhibition was abolished by PI3-kinase inhibitor. FGFR2-Tg-ECs exposed to 1% oxygen exhibited enhanced phosphorylation of 416-Tyr-Src, 473-Ser-Akt, and HIF1alpha accumulation. The production of FGF2 was enhanced 2.1-fold in FGFR-Tg-ECs under 1% oxygen via the Src/Akt/HIF1alpha pathway, which induced the peri-vessel migration of vascular smooth muscle cells (VSMCs) and anti-apoptotic effects on VSMCs and cardiomyocytes. FGF receptor signaling in ECs promoted migration, survival and autocrine production of FGF2, leading to reduced infarct size, which is associated with anti-apoptotic action in the early stage and with enhanced angiogenesis in the late stage after MI
— id: 106003, year: 2009, vol: 46, page: 663, 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,

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,

Enhanced paracrine FGF10 expression promotes formation of multifocal prostate adenocarcinoma and an increase in epithelial androgen receptor
Memarzadeh, Sanaz; Xin, Li; Mulholland, David J; Mansukhani, Alka; Wu, Hong; Teitell, Michael A; Witte, Owen N
2007 Dec;12(6):572-585, Cancer cell
Enhanced mesenchymal expression of FGF10 led to the formation of multifocal PIN or prostate cancer. Inhibition of epithelial FGFR1 signaling using DN FGFR1 led to reversal of the cancer phenotype. A subset of the FGF10-induced carcinoma was serially transplantable. Paracrine FGF10 led to an increase in epithelial androgen receptor and synergized with cell-autonomous activated AKT. Our observations indicate that stromal FGF10 expression may facilitate the multifocal histology observed in prostate adenocarcinoma and suggest the FGF10/FGFR1 axis as a potential therapeutic target in treating hormone-sensitive or refractory prostate cancer. We also show that transient exposure to a paracrine growth factor may be sufficient for the initiation of oncogenic transformation
— id: 95356, year: 2007, vol: 12, page: 572, 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,

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,

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,

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 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,

Role of fibroblast growth factor receptor signaling in prostate cancer cell survival
Ozen, M; Giri, D; Ropiquet, F; Mansukhani, A; Ittmann, M
2001 Dec 5;93(23):1783-1790, Journal of the National Cancer Institute
BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies
— id: 106004, year: 2001, vol: 93, page: 1783, 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,

Increased expression of fibroblast growth factor 6 in human prostatic intraepithelial neoplasia and prostate cancer
Ropiquet, F; Giri, D; Kwabi-Addo, B; Mansukhani, A; Ittmann, M
2000 Aug 1;60(15):4245-4250, Cancer research
Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells. In addition to their effects on proliferation, FGFs can promote cell motility, increase tumor angiogenesis, and inhibit apoptosis, all of which play an important role in tumor progression. To determine whether FGFs are overexpressed in human prostate cancers, we analyzed 26 prostate cancer RNAs by reverse transcription-PCR for expression of FGF3, FGF4, and FGF6, which cannot be detected in normal prostate tissue by this technique. Fourteen of 26 prostate cancers expressed FGF6 mRNA. No expression of FGF3 or FGF4 was detected. An ELISA of tissue extracts of normal prostate, high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer for FGF6 showed that this growth factor was undetectable in normal prostate but was present at elevated levels in 4 of 9 PIN lesions and in 15 of 24 prostate cancers. Immunohistochemical analysis with anti-FGF6 antibody revealed weak staining of prostatic basal cells in normal prostate that was markedly elevated in PIN. In the prostate cancers, the majority of cases revealed expression of FGF6 by the prostate cancer cells themselves. In two cases, expression was present in prostatic stromal cells. Exogenous FGF6 was able to stimulate proliferation of primary prostatic epithelial and stromal cells, immortalized prostatic epithelial cells, and prostate cancer cell lines in tissue culture. FGF receptor 4, which is the most potent FGF receptor for FGF6, is expressed in the human prostate in vivo and in all of the cultured cell lines. Thus, FGF6 is increased in PIN and prostate cancer and can promote the proliferation of the transformed prostatic epithelial cells via paracrine and autocrine mechanisms
— id: 106005, year: 2000, vol: 60, page: 4245, 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,

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,

Expression of fibroblast growth factors (FGFs) and FGF receptors in human prostate
Ittman M; Mansukhani A
1997 Jan;157(1):351-356, Journal of urology
PURPOSE: To evaluate the expression of fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in human prostate. MATERIALS AND METHODS: RNA was extracted from surgically excised human prostate glands and from primary cultures of human prostatic epithelial and stromal cells. Expression of FGFs and FGF receptors was evaluated by Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques. RESULTS: FGF7 (KGF) is the major FGF mRNA expressed in the human prostate, with smaller amounts of FGF2 and extremely small amounts of FGF1. For all three FGFs, the prostatic stromal cells were the primary site of expression. Prostatic epithelial cells express primarily the FGFR-3 IIIc isoform, which preferentially binds FGF1 over FGF2, with smaller amounts of the FGFR-2 IIIb (FGF7 binding) isoform. Prostatic stromal cells express primarily the FGFR-3 IIIc isoform and smaller amounts of FGFR-1 IIIc and FGFR-2 IIIc isoforms, which bind both FGF1 and FGF2. CONCLUSION: The pattern of expression of FGFs and FGF receptors in the prostate is consistent with a paracrine stimulation of epithelial growth by stromal-derived FGFs and potential autocrine stimulation of stromal cell proliferation by stromal FGFs
— id: 8461, year: 1997, vol: 157, page: 351, 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,

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,

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,

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,

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,

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,

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,

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,

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,

DNA-binding properties of the Drosophila melanogaster zeste gene product
Mansukhani, A; Crickmore, A; Sherwood, P W; Goldberg, M L
1988 Feb;8(2):615-623, Molecular & cellular biology
The ability of the zeste moiety of beta-galactosidase-zeste fusion proteins synthesized in Escherichia coli to bind specific DNA sequences was examined. Such fusion proteins recognize a region of the white locus upstream of the start of transcription; this region has previously been shown to be required for genetic interaction between the zeste and white loci. Another strong binding site was localized to a region between 50 and 205 nucleotides before the start of the Ubx transcriptional unit; expression of the bithorax complex is also known to be influenced by the zeste locus. Weaker binding sites were also seen in the vicinity of the bxd and Sgs-4 genes, but it is currently unclear whether these binding sites play a role in transvection effects. The DNA-binding activity of the zeste protein is restricted to a domain of approximately 90 amino acids near the N terminus. This domain does not appear to contain homeobox or zinc finger motifs found in other DNA-binding proteins. The DNA-binding domain is not disrupted by any currently characterized zeste mutations
— id: 106006, year: 1988, vol: 8, page: 615, stat: Journal Article,

Nucleotide sequence and structural analysis of the zeste locus of Drosophila melanogaster
Mansukhani, A; Gunaratne, P H; Sherwood, P W; Sneath, B J; Goldberg, M L
1988 Jan;211(1):121-128, Molecular & general genetics
The zeste locus plays a central role in transvection phenomena, where the synaptic pairing of chromosomes carrying genes with which zeste interacts influences the expression of these genes. To explore the possible functions of the zeste gene product in this process, we have determined the DNA sequences both of a fragment of Drosophila genomic DNA capable of rescuing mutant zeste phenotypes, and of a near full-length cDNA clone derived from the 2.4-kb zeste mRNA. These data show that the zeste gene is interrupted by two small introns, and suggest that the majority of zeste sequences are contained within an intron of another transcriptional unit of opposite polarity. A large region of the predicted zeste product is comprised almost exclusively of glutamine and alanine residues. A domain near the N terminus of this protein, which is sufficient for site-specific DNA binding, is highly charged, as is the C-terminal region of the protein. A breakpoint of the rearrangement In (1)e(bx), which is associated with a za-like phenotype, is found within sequences encoding the zeste product, and would produce a truncated protein. The neomorphic mutation zv77h is correlated with a 300-bp deletion of sequences determining the untranslated 5' leader of the zeste messenger, but may also remove the initiating ATG codon, resulting in a zeste protein with an altered N terminus
— id: 106007, year: 1988, vol: 211, page: 121, stat: Journal Article,

Molecular cloning, germ-line transformation, and transcriptional analysis of the zeste locus of Drosophila melanogaster
Gunaratne, P H; Mansukhani, A; Lipari, S E; Liou, H C; Martindale, D W; Goldberg, M L
1986 Feb;83(3):701-705, Proceedings of the National Academy of Sciences of the United States of America
Approximately 170 kilobase pairs (kb) of contiguous DNA sequences derived from bands 3A3,4 of the Drosophila melanogaster X chromosome have been isolated by molecular cloning. Sequences required for the wild-type expression of the zeste locus are located within a 6-kb fragment of this chromosomal region, as shown by phenotypic rescue of zeste mutants in P element-mediated germ-line transformation. Expression of zeste is correlated with a 2.2-kb poly(A)+ RNA species transcribed at all postzygotic stages of Drosophila development. Many zeste alleles, including several producing neomorphic phenotypes, are not associated with detectable rearrangements of DNA
— id: 106008, year: 1986, vol: 83, page: 701, stat: Journal Article,

Cell culture mutants as aminoacyl-tRNA synthetase complex probes
Hampel, A; Mansukhani, A; Condon, T
1984 Dec;43(15):2991-2993, Federation Proceedings (Federation of American Societies for Experimental Biology)
A number of Chinese hamster ovary cell culture mutants have been characterized by us for their high-molecular-weight aminoacyl-tRNA synthetase (aaRS) complexes. The results all support a model of aaRS complex function wherein the high-molecular-weight complexes form aminoacyl-tRNA by utilizing extracellular amino acids immediately on their transport and before they have equilibrated with the internal pool. The low-molecular-weight aaRS forms exclusively utilize amino acids from only the intracellular pool
— id: 106009, year: 1984, vol: 43, page: 2991, stat: Journal Article,

A Chinese hamster ovary leucyl-tRNA synthetase mutant with a uniquely altered high molecular weight leucyl-tRNA synthetase complex
Mansukhani, A; Condon, T; Hampel, A; Oxender, D L
1984 Apr;22(3-4):349-355, Biochemical genetics
The Chinese hamster ovary (CHO) cell culture temperature-sensitive mutant ts025Cl with a defect in leucyl-tRNA synthetase ( LeuRS ) does not have an inherently more thermolabile LeuRS , but instead the mutation causes the complete loss of the LeuRS high molecular weight complexes which are present in normal wild-type cells. The mutant cell LeuRS has a single 8 S enzyme form which corresponds hydrodynamically to the 8 S free form of wild-type enzyme. Both 8 S forms have the same thermostability and the same Km for leucine, indicating that there is no inherent defect in the catalytic activity of the enzyme. The temperature-sensitive phenotype can be explained by the lack of thermostable high molecular weight forms of LeuRS
— id: 106010, year: 1984, vol: 22, page: 349, stat: Journal Article,

Isolation of Chinese hamster ovary cell mutants defective in the regulation of leucine transport
Shotwell, M A; Collarini, E J; Mansukhani, A; Hampel, A E; Oxender, D L
1983 Jul 10;258(13):8183-8187, Journal of biological chemistry
We have isolated mutants defective in the regulation of leucine transport by selecting temperature-resistant revertants from the CHO-tsH1 strain, a temperature-sensitive leucyl-tRNA synthetase mutant of the Chinese hamster ovary cell line. In each revertant, there is a stable 2- to 3-fold enhancement in the activity of transport System L, which serves for the uptake of branched-chain amino acids. This increased transport is reflected by an increase in the Vmax for System L transport without a significant change in the Km value and results in increased intracellular pools of leucine. The thermal stability of the leucyl-tRNA synthetase activity in each of these revertants is not changed significantly from that of the starting strain, CHO-tsH1. We conclude from these studies that the temperature resistance in the revertants arises from alterations in the regulation of transport System L, leading to constitutively high System L transport and increased intracellular pools of leucine, complementing the leucyl-tRNA synthetase defect
— id: 106011, year: 1983, vol: 258, page: 8183, stat: Journal Article,