Angel G Pellicer, M.D., Ph.D.

Wild type N-ras displays anti-malignant properties, in part by downregulating decorin
Benet M; Dulman RY; Suzme R; de Miera EV; Vega ME; Nguyen T; Zavadil J; Pellicer A
2011 Aug 1;:?-?, Journal of cellular physiology
Previously, we have shown that wild type N-ras (wt N-ras) harbors an anti-malignant effect against mutated Ras and in tumors without Ras mutations. To investigate the molecular bases of this anti-malignant activity, we have studied the potency of this anti-malignant effect in a model system against SV40 large T antigen (SV40T). We show that wild-type N-ras (wt N-ras) counteracts the effects of SV40T in NIH3T3 cells as seen by a decrease in proliferation, anchorage independence and changes in migration. We also show that wt N-ras elicits the same anti-malignant effects in some human tumor cell lines (HT1080 and MDA-MB-231). Through mRNA and microRNA (miRNAs) expression profiling we have identified genes (decorin) and miRNAs (mir-29A, let-7b) modulated by wt N-ras potentially responsible for the anti-malignant effect. Wt N-ras appears to mediate its anti-malignant effect by downregulating some of the targets of the TGFbeta pathway and decorin, which are able to reverse the inhibition of migration induced by wt N-ras. Our experiments show that the molecules that mediate the anti-malignant effect by wt N-ras appear to be different from those modulated by transforming N-ras. The components of the pathways modulated by wt N-ras mediating its anti-malignant effects are potential targets for therapeutic intervention in cancer. J. Cell. Physiol. (c) 2011 Wiley-Liss, Inc
— id: 137054, year: 2011, vol: , page: ?, stat: Journal Article,

The human Rgr oncogene is overexpressed in T-cell malignancies and induces transformation by acting as a GEF for Ras and Ral
Osei-Sarfo, K; Martello, L; Ibrahim, S; Pellicer, A
2011 Aug 25;30(34):3661-3671, Oncogene
The Ras superfamily of GTPases is involved in the modification of many cellular processes including cellular motility, proliferation and differentiation. Our laboratory has previously identified the RalGDS-related (Rgr) oncogene in a DMBA (7,12-dimethylbenz[alpha]anthracene)-induced rabbit squamous cell carcinoma and its human orthologue, hRgr. In this study, we analyzed the expression levels of the human hRgr transcript in a panel of human hematopoietic malignancies and found that a truncated form (diseased-truncated (Dtr-hrgr)) was significantly overexpressed in many T-cell-derived neoplasms. Although the Rgr proto-oncogene belongs to the RalGDS family of guanine nucleotide exchange factors (GEFs), we show that upon the introduction of hRgr into fibroblast cell lines, it is able to elicit the activation of both Ral and Ras GTPases. Moreover, in vitro guanine nucleotide exchange assays confirm that hRgr promotes Ral and Ras activation through GDP dissociation, which is a critical characteristic of GEF proteins. hRgr has guanine nucleotide exchange activity for both small GTPases and this activity was reduced when a point mutation within the catalytic domain (CDC25) of the protein, (cd) Dtr-hRgr, was utilized. These observations prompted the analysis of the biological effects of hRgr and (cd) hRgr expression in cultured cells. Here, we show that hRgr increases proliferation in low serum, increases invasion, reduces anchorage dependence and promotes the progression into the S phase of the cell cycle; properties that are abolished or severely reduced in the presence of the catalytic dead mutant. We conclude that the ability of hRgr to activate both Ral and Ras is responsible for its transformation-inducing phenotype and it could be an important contributor in the development of some T-cell malignancies
— id: 136936, year: 2011, vol: 30, page: 3661, stat: Journal Article,

Regulation of HMGA1 expression by microRNA296 affects prostate cancer growth and invasion
Wei JJ; Wu X; Peng Y; Shi G; Olca B; Yang X; Daniels G; Osman I; Ouyang J; Hernando E; Pellicer A; Rhim J; Melamed J; Lee P
2011 Mar 15;17(6):1297-1305, Clinical cancer research
PURPOSE: High mobility group AT-hook 1 (HMGA1) is a non-histone nuclear binding protein that is developmentally regulated. HMGA1 is significantly overexpressed in and associated with high grade and advance stage of prostate cancer (PC). The oncogenic role of HMGA1 is at least mediated through chromosomal instability and structural aberrations. However, regulation of HMGA1 expression is not well understood. Identification of microRNA mediated HMGA1 regulation will provide a promising therapeutic target in treating PC. Experimental Designs: In this study, we examined the functional relation between miR-296 and HMGA1 expression in several prostate cancer cell lines and a large prostate cancer cohort. We further examined the oncogenic property of HMGA1 regulated by miR-296. RESULTS: Here we report that miR-296, a microRNA predicted to target HMGA1, specifically represses HMGA1 expression by promoting degradation and inhibiting HMGA1translation . Repression of HMGA1 by miR-296 is direct and sequence-specific. Importantly, ectopic miR-296 expression significantly reduced prostate cancer cell proliferation and invasion, in part through the down-regulation of HMGA1. Examining prostate cancer patient samples, we found an inverse correlation between HMGA1 and miR-296 expression: high levels of HMGA1 were associated with low miR-296 expression and strongly linked to more advanced tumor grade and stage. CONCLUSIONS: Our results indicate miR-296 regulates HMGA1 expression and is associated with PC growth and invasion
— id: 115464, year: 2011, vol: 17, page: 1297, stat: Journal Article,

LEF1 in androgen-independent prostate cancer: regulation of androgen receptor expression, prostate cancer growth, and invasion
Li, Yirong; Wang, Longgui; Zhang, Miao; Melamed, Jonathan; Liu, Xiaomei; Reiter, Robert; Wei, Jianjun; Peng, Yi; Zou, Xuanyi; Pellicer, Angel; Garabedian, Michael J; Ferrari, Anna; Lee, Peng
2009 Apr 15;69(8):3332-3338, Cancer research
A major obstacle in treating prostate cancer is the development of androgen-independent disease. In this study, we examined LEF1 expression in androgen-independent cancer as well as its regulation of androgen receptor (AR) expression, prostate cancer growth, and invasion in androgen-independent prostate cancer cells. Affymetrix microarray analysis of LNCaP and LNCaP-AI (androgen-independent variant LNCaP) cells revealed 100-fold increases in LEF1 expression in LNCaP-AI cells. We showed that LEF1 overexpression in LNCaP cells resulted in increased AR expression and consequently enhanced growth and invasion ability, whereas LEF1 knockdown in LNCaP-AI cells decreased AR expression and, subsequently, growth and invasion capacity. Chromatin immunoprecipitation, gel shift, and luciferase assays confirmed LEF1 occupancy and regulation of the AR promoter. Thus, we identified LEF1 as a potential marker for androgen-independent disease and as a key regulator of AR expression and prostate cancer growth and invasion. LEF1 is highly expressed in androgen-independent prostate cancer, potentially serving as a marker for androgen-independent disease
— id: 99128, year: 2009, vol: 69, page: 3332, stat: Journal Article,

Uroplakins in urothelial biology, function, and disease
Wu, Xue-Ru; Kong, Xiang-Peng; Pellicer, Angel; Kreibich, Gert; Sun, Tung-Tien
2009 Jun;75(11):1153-1165, Kidney international
Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder, and prostatic urethra. Although morphologically similar, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition, and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies urine composition. Urothelial cells express a number of ion channels, receptors, and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade the bladder mucosa, but also provide a route by which the bacteria ascend through the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis, and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins' biochemistry, structure, assembly, intracellular trafficking, and their emerging roles in urothelial biology, function, and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.Kidney International (2009) 75, 1153-1165; doi:10.1038/ki.2009.73; published online 1 April 2009
— id: 98907, year: 2009, vol: 75, page: 1153, stat: Journal Article,

Lef1 Expression in Androgen-Independent Prostate Cancer
Zhang, M; Li, YR; Wang, LG; Melamed, J; Liu, XM; Wei, JJ; Peng, Y; Pellicer, A; Garabedian, MJ; Ferrari, A; Lee, P
2009 ;89:921-921, Laboratory investigation
— id: 104576, year: 2009, vol: 89, page: 921, stat: Journal Article,

Lef1 Expression in Androgen-Independent Prostate Cancer
Zhang, M; Li, YR; Wang, LG; Melamed, J; Liu, XM; Wei, JJ; Peng, Y; Pellicer, A; Garabedian, MJ; Ferrari, A; Lee, P
2009 ;22(Suppl 1):203A-203A 921, Modern pathology
— id: 104577, year: 2009, vol: 22, page: 203A, stat: Journal Article,

MicroRNA expression in the tumor-associated stroma of prostate cancer
Gellert, LL; Basturk, O; Zon, XY; Wei, JJ; Pellicer, A; Kong, XT; Melamed, J; Lee, P
2008 OCT ;130(4):5-519, American journal of clinical pathology
— id: 98126, year: 2008, vol: 130, page: 5, stat: Journal Article,

Origin of the tetraspanin uroplakins and their co-evolution with associated proteins: implications for uroplakin structure and function
Garcia-Espana, Antonio; Chung, Pei-Jung; Zhao, Xiaoqian; Lee, Andy; Pellicer, Angel; Yu, Jun; Sun, Tung-Tien; Desalle, Rob
2006 Nov;41(2):355-367, Molecular phylogenetics & evolution
Genome level information coupled with phylogenetic analysis of specific genes and gene families allow for a better understanding of the structure and function of their protein products. In this study, we examine the mammalian uroplakins (UPs) Ia and Ib, members of the tetraspanin superfamily, that interact with uroplakins UPII and UPIIIa/IIIb, respectively, using a phylogenetic approach of these genes from whole genome sequences. These proteins interact to form urothelial plaques that play a central role in the permeability barrier function of the apical urothelial surface of the urinary bladder. Since these plaques are found exclusively in mammalian urothelium, it is enigmatic that UP-like genomic sequences were recently found in lower vertebrates without a typical urothelium. We have cloned full-length UP-related cDNAs from frog (Xenopus laevis), chicken (Gallus gallus), and zebrafish (Danio rerio), and combined these data with sequence information from their orthologs in all the available fully sequenced and annotated animal genomes. Phylogenetic analyses of all the available uroplakin sequences, and an understanding of their distribution in several animal taxa, suggest that: (i) the UPIa/UPIb and UPII/UPIII genes evolved by gene duplication in the common ancestor of vertebrates; (ii) uroplakins can be lost in different combinations in vertebrate lineages; and (iii) there is a strong co-evolutionary relationship between UPIa and UPIb and their partners UPII and UPIIIa/IIIb, respectively. The co-evolution of the tetraspanin UPs and their associated proteins may fine-tune the structure and function of uroplakin complexes enabling them to perform diverse species- and tissue-specific functions. The structure and function of uroplakins, which are also expressed in Xenopus kidney, oocytes and fat body, are much more versatile than hitherto appreciated
— id: 115882, year: 2006, vol: 41, page: 355, stat: Journal Article,

Biochemical and biological analyses of Rgr RalGEF oncogene
Martello, LA; Pellicer, A
2006 ;407:115-+, Methods in enzymology
The Ras superfamily of GTP-binding proteins is involved in many cellular processes, including cell proliferation, movement, and morphology. One such member, Ral GTPase, activates downstream signaling molecules after a conversion to the active state on GTP binding. The RalGDS-related (Rgr) oncogene belongs to the RalGDS family of guanine nucleotide exchange factors (GEFs). RalGEFs activate Ral by stimulating the dissociation of GDP, allowing the binding of GTP and the initiation of downstream signaling events by Ral effectors. Rgr was first identified as a fusion between the rabbit homolog of the Rad 23 gene and the Rgr gene in a rabbit squamous cell carcinoma. The Rgr portion of the fusion was demonstrated to contain the oncogenic activity. The human form of the Rgr oncogene was identified recently, and expression was detected in human T-cell malignancies. This chapter describes the analysis of rabbit and human Rgr function using various methods. These assays may be used for the study of oncogene function in other systems
— id: 64271, year: 2006, vol: 407, page: 115, stat: Journal Article,

Inhibition of Ras oncogenic activity by Ras protooncogenes
Diaz, Roberto; Lue, Jeffrey; Mathews, Jeremy; Yoon, Andrew; Ahn, Daniel; Garcia-Espana, Antonio; Leonardi, Peter; Vargas, Marcelo P; Pellicer, Angel
2005 Jan 10;113(2):241-248, International journal of cancer
Point mutations in ras genes have been found in a large number and wide variety of human tumors. These oncogenic Ras mutants are locked in an active GTP-bound state that leads to a constitutive and deregulated activation of Ras function. The dogma that ras oncogenes are dominant, whereby the mutation of a single allele in a cell will predispose the host cell to transformation regardless of the presence of the normal allele, is being challenged. We have seen that increasing amounts of Ras protooncogenes are able to inhibit the activity of the N-Ras oncogene in the activation of Elk in NIH 3T3 cells and in the formation of foci. We have been able to determine that the inhibitory effect is by competition between Ras protooncogenes and the N-Ras oncogene that occurs first at the effector level at the membranes, then at the processing level and lastly at the effector level in the cytosol. In addition, coexpression of the N-Ras protooncogene in thymic lymphomas induced by the N-Ras oncogene is associated with increased levels of p107, p130 and cyclin A and decreased levels of Rb. In the present report, we have shown that the N-Ras oncogene is not truly dominant over Ras protooncogenes and their competing activities might be depending on cellular context
— id: 47760, year: 2005, vol: 113, page: 241, stat: Journal Article,

Differential expression of cell cycle regulators in phenotypic variants of transgenically induced bladder tumors: implications for tumor behavior
Garcia-Espana, Antonio; Salazar, Edgard; Sun, Tung-Tien; Wu, Xue-Ru; Pellicer, Angel
2005 Feb 15;65(4):1150-1157, Cancer research
Proteins controlling cell growth, differentiation, apoptosis, and oncogenic stress are often deregulated in tumor cells. However, whether such deregulations affect tumor behavior remains poorly understood in many tumor types. We recently showed that the urothelium-specific expression of activated H-ras and SV40 T antigen in transgenic mice produced two distinctive types of tumors strongly resembling the human superficial papillary tumors and carcinoma in situ of the bladder, respectively. Here we assessed the expression of a key set of cell cycle regulators in these mouse tumors and in a new transgenic line expressing a cyclin D1 oncogene in the urothelium. We found that urothelia of the wild-type and cyclin D1 transgenic mice exhibited a profile of cell cycle regulators found in quiescent (G(0)) cells, indicating that urothelium overexpressing the cyclin D1 (an 8-fold increase) is reminiscent of normal urothelium and remains slow-cycling. Low-grade superficial papillary tumors induced by activated H-ras had no detectable Rb family proteins (Rb, p107, and p130) and late cell cycle cyclins and kinases (cyclin A, E, and CDK1), but had increased level of p16, p53, and MDM2. These data suggest that the inactivation of the Rb pathway plays an important role in H-ras-induced superficial papillary tumors and that oncogenic H-ras can induce a compensatory activation of alternative tumor suppressor pathways. In contrast, carcinoma in situ of the bladder induced by SV40 T antigen had increased expression of cell cycle regulators mainly active in post-G(1) phases. The fact that phenotypically different bladder tumors exhibit different patterns of cell cycle regulators may explain why these tumors have different propensity to progress to invasive tumors. Our results indicate that the transgenic mouse models can be used not only for studying tumorigenesis but also for evaluating therapeutic strategies that target specific cell cycle regulators
— id: 48701, year: 2005, vol: 65, page: 1150, stat: Journal Article,

The effect of sindbis viral vectors on the N-nitroso-bis(2-oxopropyl)amine(BOP) induced hamster pancreatic ductal adenocarcinoma and cholangiocarcinoma
Lin, Q; West, AB; Merali, S; Levin, B; Meruelo, D; Pellicer, A
2005 ;85(Suppl 1):283A-284A, Laboratory investigation
— id: 50475, year: 2005, vol: 85, page: 283A, stat: Journal Article,

The effect of Sindbis viral vectors on the N-nitroso-bis(2-oxopropyl)amine(BOP) induced hamster pancreatic ductal adenocarcinoma and cholangiocarcinoma
Liu, Q; West, AB; Merali, S; Levin, B; Meruelo, D; Pellicer, A
2005 ;18(Suppl 1):283A-284A, Modern pathology
— id: 50445, year: 2005, vol: 18, page: 283A, stat: Journal Article,

Biochemical and Biological Analyses of Rgr RalGEF Oncogene
Martello, Laura A; Pellicer, Angel
2005 ;407:115-128, Methods in enzymology
The Ras superfamily of GTP-binding proteins is involved in many cellular processes, including cell proliferation, movement, and morphology. One such member, Ral GTPase, activates downstream signaling molecules after a conversion to the active state on GTP binding. The RalGDS-related (Rgr) oncogene belongs to the RalGDS family of guanine nucleotide exchange factors (GEFs). RalGEFs activate Ral by stimulating the dissociation of GDP, allowing the binding of GTP and the initiation of downstream signaling events by Ral effectors. Rgr was first identified as a fusion between the rabbit homolog of the Rad 23 gene and the Rgr gene in a rabbit squamous cell carcinoma. The Rgr portion of the fusion was demonstrated to contain the oncogenic activity. The human form of the Rgr oncogene was identified recently, and expression was detected in human T-cell malignancies. This chapter describes the analysis of rabbit and human Rgr function using various methods. These assays may be used for the study of oncogene function in other systems
— id: 69337, year: 2005, vol: 407, page: 115, stat: Journal Article,

Mouse p10, an alternative spliced form of p15INK4b, inhibits cell cycle progression and malignant transformation
Perez de Castro, Ignacio; Benet, Marta; Jimenez, Maria; Alzabin, Saba; Malumbres, Marcos; Pellicer, Angel
2005 Apr 15;65(8):3249-3256, Cancer research
The INK4 family of proteins negatively regulates cell cycle progression at the G(1)-S transition by inhibiting cyclin-dependent kinases. Two of these cell cycle inhibitors, p16(INK4A) and p15(INK4B), have tumor suppressor activities and are inactivated in human cancer. Interestingly, both INK4 genes express alternative splicing variants. In addition to p16(INK4A), the INK4A locus encodes a splice variant, termed p12--specifically expressed in human pancreas--and ARF, a protein encoded by an alternative reading frame that acts as a tumor suppressor through the p53 pathway. Similarly, the human INK4B locus encodes the p15(INK4B) tumor suppressor and one alternatively spliced form, termed as p10. We show here that p10, which arises from the use of an alternative splice donor site within intron 1, is conserved in the mouse genome and is widely expressed in mouse tissues. Similarly to mouse p15(INK4B), p10 expression is also induced by oncogenic insults and transforming growth factor-beta treatment and acts as a cell cycle inhibitor. Importantly, we show that mouse p10 is able to induce cell cycle arrest in a p53-dependent manner. We also show that mouse p10 is able to inhibit foci formation and anchorage-independent growth in wild-type mouse embryonic fibroblasts, and that these antitransforming properties of mouse p10 are also p53-dependent. These results indicate that the INK4B locus, similarly to INK4A-ARF, harbors two different splicing variants that can be involved in the regulation of both the p53 and retinoblastoma pathways, the two major molecular pathways in tumor suppression
— id: 55798, year: 2005, vol: 65, page: 3249, stat: Journal Article,

Complex effects of Ras proto-oncogenes in tumorigenesis
Diaz, Roberto; Lopez-Barcons, Lluis; Ahn, Daniel; Garcia-Espana, Antonio; Yoon, Andrew; Matthews, Jeremy; Mangues, Ramon; Perez-Soler, Roman; Pellicer, Angel
2004 May;25(4):535-539, Carcinogenesis
Ras proteins have been found mutated in about one-third of human tumors. In vitro, Ras has been shown to regulate distinct and contradictory effects, such as cellular proliferation and apoptosis. Nonetheless, the effects that the wild-type protein elicits in tumorigenesis are poorly understood. Depending on the type of tissue, Ras proto-oncogenes appear to either promote or inhibit the tumor phenotype. In this report, we treated wild-type and N-ras knockout mice with 3-methylcholanthrene (MCA) to induce fibrosarcomas and found that MCA is more carcinogenic in wild-type mice than in knockout mice. After injecting different doses of a tumorigenic cell line, the wild-type mice exhibited a shorter latency of tumor development than the knockouts, indicating that there are N-ras-dependent differences in the stromal cells. Likewise, we have analyzed B-cell lymphomas induced by either N-methylnitrosourea or by the N-ras oncogene in mice that over-express the N-ras proto-oncogene and found that the over-expression of wild-type N-ras is able to increase the incidence of these lymphomas. Considered together, our results indicate that Ras proto-oncogenes can enhance or inhibit the malignant phenotype in vivo in different systems
— id: 46050, year: 2004, vol: 25, page: 535, stat: Journal Article,

p53 deficiency provokes urothelial proliferation and synergizes with activated Ha-ras in promoting urothelial tumorigenesis
Gao, Jing; Huang, Hong-Ying; Pak, Joanne; Cheng, Jin; Zhang, Zhong-Ting; Shapiro, Ellen; Pellicer, Angel; Sun, Tung-Tien; Wu, Xue-Ru
2004 Jan 22;23(3):687-696, Oncogene
Mutation and deletion of the p53 tumor suppressor gene are arguably the most prevalent among the multiple genetic alterations found in human bladder cancer, but these p53 defects are primarily associated with the advanced diseases, and their roles in bladder tumor initiation and in synergizing with oncogenes in tumor progression have yet to be defined. Using the mouse uroplakin II gene promoter, we have targeted into urothelium of transgenic mice a dominant-negative mutant of p53 that lacks the DNA-binding domain but retains the tetramerization domain. Urothelium-expressed p53 mutant binds to and stabilizes the endogenous wild-type p53, induces nuclear abnormality, hyperplasia and occasionally dysplasia, without eliciting frank carcinomas. Concurrent expression of the p53 mutant with an activated Ha-ras, the latter of which alone induces urothelial hyperplasia, fails to accelerate tumor formation. In contrast, the expression of the activated Ha-ras in the absence of p53, as accomplished by crossing the activated Ha-ras transgenic mice with the p53 knockout mice, results in early-onset bladder tumors that are either low-grade superficial papillary or high grade in nature. These results provide the first in vivo experimental evidence that p53 deficiency predisposes the urothelium to hyperproliferation, but is insufficient for bladder tumorigenesis; that the mere reduction of p53 dosage, as produced in transgenic mice expressing the dominant-negative p53 or in heterozygous p53 knockouts, is incapable of synergizing with Ha-ras to induce bladder tumors; and that the complete loss of p53 is a prerequisite for collaborating with activated Ha-ras to promote bladder tumorigenesis
— id: 42019, year: 2004, vol: 23, page: 687, stat: Journal Article,

The Rgr oncogene induces tumorigenesis in transgenic mice
Jimenez, Maria; Perez de Castro, Ignacio; Benet, Marta; Garcia, Juan F; Inghirami, Giorgio; Pellicer, Angel
2004 Sep 1;64(17):6041-6049, Cancer research
To study the oncogenic potential of Rgr in vivo, we have generated several transgenic Rgr mouse lines, which express the oncogene under the control of different promoters. These studies revealed that Rgr expression leads to the generation of various pathological alterations, including fibrosarcomas, when its transgenic expression is restricted to nonlymphoid tissues. Moreover, the overall incidence and latency of fibrosarcomas were substantially increased and shortened, respectively, in a p15INK4b-defective background. More importantly, we also have demonstrated that Rgr expression in thymocytes of transgenic mice induces severe alterations in the development of the thymocytes, which eventually lead to a high incidence of thymic lymphomas. This study demonstrates that oncogenic Rgr can induce expression of p15INK4b and, more importantly, that both Rgr and p15INK4b cooperate in the malignant phenotype in vivo. These findings provide new insights into the tumorigenic role of Rgr as a potent oncogene and show that p15INK4b can act as a tumor suppressor gene
— id: 44745, year: 2004, vol: 64, page: 6041, stat: Journal Article,

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus
Perez de Castro, Ignacio; Bivona, Trever G; Philips, Mark R; Pellicer, Angel
2004 May;24(8):3485-3496, Molecular & cellular biology
Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells
— id: 46001, year: 2004, vol: 24, page: 3485, stat: Journal Article,

Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models
Tseng, Jen-Chieh; Hurtado, Alicia; Yee, Herman; Levin, Brandi; Boivin, Christopher; Benet, Marta; Blank, Stephanie V; Pellicer, Angel; Meruelo, Daniel
2004 Sep 15;64(18):6684-6692, Cancer research
We studied the therapeutic value of Sindbis vectors for advanced metastatic ovarian cancer by using two highly reproducible and clinically accurate mouse models: a SCID xenograft model, established by i.p. inoculation of human ES-2 ovarian cancer cells, and a syngenic C57BL/6 model, established by i.p. inoculation of mouse MOSEC ovarian cancer cells. We demonstrate through imaging, histologic, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in the peritoneal cavity, leading to significant suppression of the carcinomatosis in both animal models. Use of two different bioluminescent genetic markers for the IVIS Imaging System permitted demonstration, for the first time, of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis vector infection and growth suppression of murine MOSEC tumor cells indicate that Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the M(r) 67,000 laminin receptor. Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. We show that incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer
— id: 44950, year: 2004, vol: 64, page: 6684, stat: Journal Article,

Systemic tumor targeting and killing by Sindbis viral vectors
Tseng, Jen-Chieh; Levin, Brandi; Hurtado, Alicia; Yee, Herman; Perez de Castro, Ignacio; Jimenez, Maria; Shamamian, Peter; Jin, Ruzhong; Novick, Richard P; Pellicer, Angel; Meruelo, Daniel
2004 Jan;22(1):70-77, Nature biotechnology
Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects
— id: 44812, year: 2004, vol: 22, page: 70, stat: Journal Article,

Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1
Bivona, Trever G; Perez De Castro, Ignacio; Ahearn, Ian M; Grana, Theresa M; Chiu, Vi K; Lockyer, Peter J; Cullen, Peter J; Pellicer, Angel; Cox, Adrienne D; Philips, Mark R
2003 Aug 7;424(6949):694-698, Nature
Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane
— id: 39161, year: 2003, vol: 424, page: 694, stat: Journal Article,

NF1 modulates the effects of Ras oncogenes: evidence of other NF1 function besides its GAP activity
Corral, Teresa; Jimenez, Maria; Hernandez-Munoz, Inmaculada; Perez de Castro, Ignacio; Pellicer, Angel
2003 Nov;197(2):214-224, Journal of cellular physiology
Neurofibromin (NF1) (the product of Nf1 gene) is a large cytosolic protein known as a negative regulator of Ras. A fragment of some 400 residues located at the center of the NF1 GAP-Related Domain (NF1-GRD) has strong identity with other molecules of the GAP family, which comprises, among others, the mammalian proteins NF1 and p120GAP, and the yeast proteins IRA1 and IRA2. GAP family members are known by their ability to promote the GTPase activity of Ras proteins, facilitating the transit of those proteins to their inactive state. Recent findings (Tong et al., 2002, Nat Neurosci 5:95-96) indicate that NF1 may be involved in the regulation of adenyl cyclase activity. Our results show that NF1-GRD cooperates with Ras in the anchorage-independent growth capacity of Ras-expressing fibroblasts, without affecting: (i) their ability to grow in low serum, (ii) their cellular adhesion capability, or (iii) the expression of key proteins involved in cell-cell and cell-matrix interactions. On the other hand, NF1 overexpression induces an increase in the expression levels of the focal adhesion kinase (FAK), and specific changes in the activation status of the mitogen-activated protein kinases (MAPKs). These results suggest the existence of a Ras-independent NF1-dependent pathway able to modify the levels of expression of FAK and the levels of activation of MAPKs. Because FAK and many proteins recently found to bind NF1 have a role in the cytoskeleton, this pathway may involve rearrangement of cytoskeletal components that facilitate anchorage independence
— id: 39060, year: 2003, vol: 197, page: 214, stat: Journal Article,

rgr oncogene: activation by elimination of translational controls and mislocalization
Hernandez-Munoz, Inmaculada; Benet, Marta; Calero, Miguel; Jimenez, Maria; Diaz, Roberto; Pellicer, Angel
2003 Jul 15;63(14):4188-4195, Cancer research
Previous studies have identified a novel oncogene, rgr, which has homology to the guanine nucleotide exchange factor (GEF) Ral guanine dissociation stimulator (RALGDS). To determine the mechanism of activation of rgr, the wild-type form was isolated. rgr is expressed physiologically at very low levels, due, at least in part, to a long 5'-untranslated region that contains eight AUGs, which inhibit translation of the main open reading frame. When these regulatory sequences are removed, the wild-type gene is expressed at high levels. An investigation of how this GEF could transform cells showed that RGR interacts with RAS, supporting its involvement as a RAS-GEF. Because RAL is localized mainly to the Golgi, the expression of the RGR protein was identified in RK13 cells, a cell line that expresses endogenous rgr. RGR localizes to endomembranes. To determine its location upon transformation, a green fluorescent protein-RGR fusion protein was used to track the movement of RGR. Increasing amounts of expression result in enhanced localization of RGR to the plasma membrane. These results indicate that rgr is activated when its tight translational controls are eliminated and increased expression allows its relocation to the plasma membrane, where efficient activation of RAS occurs
— id: 39133, year: 2003, vol: 63, page: 4188, stat: Journal Article,

Differences on the inhibitory specificities of H-Ras, K-Ras, and N-Ras (N17) dominant negative mutants are related to their membrane microlocalization
Matallanas, David; Arozarena, Imanol; Berciano, Maria T; Aaronson, David S; Pellicer, Angel; Lafarga, Miguel; Crespo, Piero
2003 Feb 14;278(7):4572-4581, Journal of biological chemistry
Ras GTPases include the isoforms H-Ras, K-Ras, and N-Ras. Despite their great biochemical and biological similarities, evidence is mounting suggesting that Ras proteins may not be functionally redundant. A widespread strategy for studying small GTPases is the utilization of dominant inhibitory mutants that specifically block the activation of their respective wild-type proteins. As such, H-Ras N17 has proved to be extremely valuable as a tool to probe Ras functions. However, a comparative study on the inhibitory specificities of H-, K-, and N-Ras N17 mutants has not been approached thus far. Herein, we demonstrate that H-, K-, and N-Ras N17 mutants exhibit markedly distinct inhibitory effects toward H-, K-, and N-Ras. H-Ras N17 can effectively inhibit the activation of all three isoforms. K-Ras N17 completely blocks the activation of K-Ras and is only slightly inhibitory on H-Ras. N-Ras N17 can mainly inhibit N-Ras activation. In light of the recent data on the compartmentalization of H-Ras and K-Ras in the plasma membrane, here we present for the first time a description of N-Ras cellular microlocalization. Overall, our results on Ras N17 mutants specificities exhibit a marked correlation with the localization of the Ras isoforms to distinct membrane microdomains
— id: 115895, year: 2003, vol: 278, page: 4572, stat: Journal Article,

Mice deficient for N-ras: impaired antiviral immune response and T-cell function
Perez de Castro, Ignacio; Diaz, Roberto; Malumbres, Marcos; Hernandez, Maria-Inmaculada; Jagirdar, Jaishree; Jimenez, Maria; Ahn, Daniel; Pellicer, Angel
2003 Apr 1;63(7):1615-1622, Cancer research
Ras proteins have a key role in the regulation of several cellular functions, and are involved in a significant percentage of human tumors. However, the specific functions of the different Ras isoforms are poorly understood. In this work, we show for the first time a specific role for N-ras in T-cell function and development. Mice defective for N-ras have low numbers of CD8 single positive thymocytes and decreased thymocyte proliferation in vitro. In Ras signaling and activation assays, KO-N-ras thymocytes showed a defective response to T-cell activation. In turn, these deficiencies resulted in a significant reduction in the production of interleukin 2 on thymocyte activation. We have also detected in vivo the functional consequences of N-ras deficiency. KO-N-ras mice showed an increased sensitivity to influenza infection, especially when low doses of virus were used. Finally, we have detected an abnormal activation pattern of downstream Ras molecules in T-cell receptor-activated KO-N-ras thymocytes that is consistent with the defective T-cell function found in these animals. All of the results derived from this work constitute a significant contribution to the knowledge of N-ras-specific functions
— id: 39256, year: 2003, vol: 63, page: 1615, stat: Journal Article,

Overexpression of epidermal growth factor receptor in urothelium elicits urothelial hyperplasia and promotes bladder tumor growth
Cheng, Jin; Huang, Hongying; Zhang, Zhong-Ting; Shapiro, Ellen; Pellicer, Angel; Sun, Tung-Tien; Wu, Xue-Ru
2002 Jul 15;62(14):4157-4163, Cancer research
Although urothelium is constantly bathed in high concentrations of epidermal growth factor (EGF) and most urothelial carcinomas overexpress EGF receptor (EGFr), relatively little is known about the role of EGFr signaling pathway in urothelial growth and transformation. In the present study, we used the uroplakin II gene promoter to drive the urothelial overexpression of EGFr in transgenic mice. Three transgenic lines were established, all expressing a higher level of the EGFr mRNA and protein in the urothelium than the nontransgenic controls. The overexpressed EGFr was functionally active because it was autophosphorylated, and its downstream mitogen-activated protein kinases were highly activated. Phenotypically, the urinary bladders of all transgenic lines developed simple urothelial hyperplasia that was strongly positive for proliferative cell nuclear antigen and weakly positive for bromodeoxyuridine incorporation. When coexpressed with the activated Ha-ras oncogene in double transgenic mice, EGFr had no apparent tumor-enhancing effects over the urothelial hyperplastic phenotype induced by Ha-ras oncogene. However, when coexpressed with the SV40 large T antigen, EGFr accelerated tumor growth and converted the carcinoma in situ of the SV40T mice into high-grade bladder carcinomas, without triggering tumor invasion. Our studies indicate that urothelial overexpression of EGFr can induce urothelial proliferation but not frank carcinoma formation. Our results also suggest that, whereas EGFr and Ha-ras, both of which act in the same signal transduction cascade, stimulated urothelial hyperplasia, they were not synergistic in urothelial tumorigenesis, and EGFr overexpression can cooperate with p53 and pRB dysfunction (as occurring in SV40T transgenic mice) to promote bladder tumor growth
— id: 32471, year: 2002, vol: 62, page: 4157, stat: Journal Article,

The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene
Diaz, Roberto; Ahn, Daniel; Lopez-Barcons, Lluis; Malumbres, Marcos; Perez de Castro, Ignacio; Lue, Jeffrey; Ferrer-Miralles, Neus; Mangues, Ramon; Tsong, Jerry; Garcia, Roberto; Perez-Soler, Roman; Pellicer, Angel
2002 Aug 1;62(15):4514-4518, Cancer research
ras proto-oncogenes have traditionally been associated with the regulation and promotion of cell growth. We have induced thymic lymphomas in N-ras(-/-) mice and in transgenic mice that overexpress wild-type N-ras and found that the lack of wild-type N-ras alleles favors the development of thymic lymphomas,whereas overexpression of wild-type N-ras protects against thymic lymphomagenesis in the presence or absence of its oncogene. To investigate the inhibitory role of wild-type N-ras in in vitro transformation, we introduced wild-type N-ras in N-ras-deficient tumor cells that lack ras activating mutations and found decreased growth in both low serum and soft agar. Taken together, our results indicate that wild-type N-ras has 'tumor suppressor' activity, even in the absence of its oncogenic allele
— id: 115896, year: 2002, vol: 62, page: 4514, stat: Journal Article,

Human rgr: transforming activity and alteration in T-cell malignancies
Leonardi, Peter; Kassin, Ezra; Hernandez-Munoz, Inmaculada; Diaz, Roberto; Inghirami, Giorgio; Pellicer, Angel
2002 Aug 1;21(33):5108-5116, Oncogene
We have previously identified the oncogene rgr (ralGDS related) in DNA derived from a rabbit squamous cell carcinoma. Here we describe the identification of the human orthologue of the rabbit rgr gene termed hrgr (human ralGDS related). Four alternatively spliced full-length hrgr transcripts were isolated from normal human testes and liver libraries. Truncation of hrgr confers transforming ability to its cDNA. Using a RT-PCR assay we have been able to detect the expression of an abnormally truncated transcript in several human T-cell lymphoma lines, and in fresh tissue samples of patients with T-cell malignancies. In the DHL cell line, an Anaplastic Large Cell Lymphoma (ALCL) line, a DNA rearrangement was detected within the hrgr gene region. We propose that these T-cell lymphomas, at least in part, owe their malignant phenotypes to genetic alterations of the hrgr gene. These findings also raise the possibility that mutations in the hrgr gene are involved in other malignancies
— id: 32450, year: 2002, vol: 21, page: 5108, stat: Journal Article,

Lung-specific expression of dominant-negative mutant p53 in transgenic mice increases spontaneous and benzo(a)pyrene-induced lung cancer
Tchou-Wong, Kam-Meng; Jiang, Yixing; Yee, Herman; LaRosa, Jennifer; Lee, Theodore C; Pellicer, Angel; Jagirdar, Jaishree; Gordon, Terry; Goldberg, Judith D; Rom, William N
2002 Aug;27(2):186-193, American journal of respiratory cell & molecular biology
Mutations in the p53 gene have been implicated to play an important role in the development of various human cancers. To evaluate the importance of p53 in lung cancer, a transgenic mouse model was established by utilizing the Clara cell secretory protein (CCSP) promoter to target the expression of a dominant-negative mutant form of p53 (dnp53) in the lung. In two transgenic CCSP-dnp53 founder lines, the dnp53 protein was expressed exclusively in the lungs. The incidence of spontaneous lung cancer in 18-month-old transgenic mice was 45%, whereas that in age-matched control mice was 20%. The relative risk of lung tumors in CCSP-dnp53 mice was 2.3 times that of wild-type mice (exact confidence limits of 0.69, 17.5). In addition to the increased incidence of spontaneous lung tumor, these mice were more susceptible to the development of lung adenocarcinoma after exposure to benzo(a)pyrene (BaP). Six months after intratracheal instillation of benzo(a)pyrene, the tumor incidence in wild-type and CCSP-dnp53 mice was 39% and 73%, respectively. The risk of lung tumors was 25.3 times greater in BaP-treated mice adjusted for transgene expression (95% confidence limits of 3.29, 678, mid-p corrected). These results suggest that p53 function is important for protecting mice from both spontaneous and BaP-induced lung cancers
— id: 32452, year: 2002, vol: 27, page: 186, stat: Journal Article,

Neurofibromatosis 1 (NF1) heterozygosity results in a cell-autonomous growth advantage for astrocytes
Bajenaru, ML; Donahoe, J; Corral, T; Reilly, KM; Brophy, S; Pellicer, A; Gutmann, DH
2001 MAR 15 ;33(4):314-323, Glia
Individuals with neurofibromatosis 1 (NF1) develop low-grade astrocytomas at an increased frequency. To gain insight into the function of the Nf1 gene product as a growth regulator for astrocytes, we examined mice heterozygous for a targeted Nf1 mutation. In our previous studies, we demonstrated increased numbers of proliferating astrocytes in Nf1 heterozygote (Nf1(+/-)) mice in vivo. We now show that cultured Nf1(+/-) astrocytes exhibit a cell-autonomous growth advantage in vitro associated with increased p21-ras pathway activation. Furthermore, we demonstrate that Nf1(+/-);wild-type N-ras mice have a similar astrocyte growth advantage in vitro and in vivo as either oncogenic N-ras or Nf1(+/-);oncogenic N-ras mice. Lastly, mice heterozygous for targeted defects in both Nf1 and p53 as well as Nf1 and Rb exhibit 3- and 2.5-fold increases in astrocyte proliferation in vivo, respectively, suggesting that abnormalities in Nf1- and p53/Rb-regulated pathways cooperate in the heterozygous state to confer a growth advantage for brain astrocytes. Collectively, these results provide evidence for a cell-autonomous growth advantage in Nf1(+/-) astrocytes and suggest that some of the brain pathology in individuals with NF1 might result from reduced, but not absent, NF1 gene function. GLIA 33:314-323, 2001. (C) 2001 Wiley-Liss, Inc
— id: 55120, year: 2001, vol: 33, page: 314, stat: Journal Article,

Role of the F-box protein Skp2 in lymphomagenesis
Latres E; Chiarle R; Schulman BA; Pavletich NP; Pellicer A; Inghirami G; Pagano M
2001 Feb 27;98(5):2515-2520, Proceedings of the National Academy of Sciences of the United States of America
The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G(1)-S transition by controlling the stability of several G(1) regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas
— id: 21093, year: 2001, vol: 98, page: 2515, stat: Journal Article,

Role of Ha-ras activation in superficial papillary pathway of urothelial tumor formation
Zhang ZT; Pak J; Huang HY; Shapiro E; Sun TT; Pellicer A; Wu XR
2001 Apr 12;20(16):1973-1980, Oncogene
Urothelial tumors develop along two distinctive phenotypic pathways (superficial papillary non-invasive tumors versus flat carcinoma in situ lesions), with markedly different biological behavior and prognosis. Although multiple genetic alterations have been identified in human bladder cancer, their cause-effect relationship with the two pathways has not been firmly established. Using a urothelium-specific promoter of the uroplakin II gene, we showed earlier in transgenic mice that the urothelial expression of SV40T antigen, which inactivates p53 and pRb, induced carcinoma in situ and invasive and metastatic bladder cancer. In striking contrast, we demonstrate here that the urothelial expression of an activated Ha-ras in transgenic mice caused urothelial hyperplasia and superficial papillary non-invasive bladder tumors. These results provide strong, direct experimental evidence that the two phenotypical pathways of bladder tumorigenesis are caused by distinctive genetic defects. Our results indicate that Ha-ras activation can induce urothelial proliferation in vivo; and that urothelial hyperplasia is a precursor of low-grade, superficial papillary bladder tumors. Our transgenic models provide unique opportunities to study the detailed molecular events underlying different types of bladder neoplasms, and can serve as useful preclinical models for evaluating the in vivo efficacy of preventive and therapeutic agents that act on various signaling pathways in bladder cancer
— id: 20658, year: 2001, vol: 20, page: 1973, stat: Journal Article,

The Rgr oncogene (homologous to RalGDS) induces transformation and gene expression by activating Ras, Ral and Rho mediated pathways
Hernandez-Munoz I; Malumbres M; Leonardi P; Pellicer A
2000 May 25;19(23):2745-2757, Oncogene
The effects of the 5'-truncated Rgr oncogene, a previously shown specific guanine exchange factor for Ral in vitro, in stimulating proliferation, cell transformation and gene expression were investigated. We have established TetRgr cell lines in which expression of Rgr can be inhibited by the presence of tetracycline in the medium. Using this system, we show that Rgr overexpressing cells are morphologically transformed and grow in a disorganized manner. At the transcriptional level, Rgr enhances the activity of the serum response element and c-Jun. Rgr induces phosphorylation of ERKs, p38 and JNK kinases, and increases the levels of the GTP-bound forms of Ral and Ras. Ras activation could account for the broad spectra of effects displayed by Rgr. The important role of these pathways is confirmed by experiments in which the transcriptional activation events can be blocked by dominant negative versions of Ras, Ral and Rho. Among all the Rgr-induced pathways, the Ras-Raf-MEK-ERK cascade is essential for the transforming properties of Rgr. Additional analysis has shown that the activation of this pathway by Rgr is not due to a feed back mechanism mediated by the Grb2 adaptor protein. Oncogene (2000)
— id: 11656, year: 2000, vol: 19, page: 2745, stat: Journal Article,

Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b)
Malumbres M; Perez De Castro I; Hernandez MI; Jimenez M; Corral T; Pellicer A
2000 Apr;20(8):2915-2925, Molecular & cellular biology
The cell cycle inhibitor p15(INK4b) is frequently inactivated by homozygous deletion together with p16(INK4a) and p19(ARF) in some types of tumors. Although the tumor suppressor capability of p15(INK4b) is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16(INK4a) alterations. Here we show that, in vitro, p15(INK4b) is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15(INK4b) is induced in cultured cells by oncogenic Ras to an extent similar to that of p16(INK4a), and their expression is associated with premature G(1) arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15(INK4b) and p16(INK4a) promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15(INK4b), by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15(INK4b) knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15(INK4b)-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15(INK4b) is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell
— id: 11788, year: 2000, vol: 20, page: 2915, stat: Journal Article,

Characterization of the murine p19(ARF) promoter CpG island and its methylation pattern in primary lymphomas
Melendez, B; Malumbres, M; Perez de Castro, I; Santos, J; Pellicer, A; Fernandez-Piqueras, J
2000 APR ;21(4):817-821, Carcinogenesis
The INK4a/ARF locus encodes two different proteins involved in cell cycle control. Both molecules, p16(INK4a) and p19(ARF), inhibit cell cycle progression and have been shown to act as tumor suppressors in a variety of models, Their expression is controlled by separate promoters responding to different stimuli and they therefore show independent transcriptional regulation. We have cloned and characterized a 2.5 kb region upstream of the murine p19(ARF) gene to determine the role of DNA methylation in suppressing p19(ARF) transcription in a wide panel of murine primary T cell lymphomas, This region contains a DNA fragment with the characteristics of a CpG island similar to those described for the murine p16(INK4a) and p15(INK4b) genes, Expression of p19(ARF) is decreased in a significant number (20 %) of the murine lymphomas analyzed. Overexpression of the p19(ARF) transcript is also frequent, suggesting alterations in molecules of the retinoblastoma or p53 pathways that are involved in p19(ARF) regulation. Although hypermethylation of the INK4a and INK4b promoters is frequently involved in murine lymphomas, the p19(ARF) CpG island is infrequently methylated in the murine primary lymphomas studied in this work. Since loss of p19(ARF) expression cannot be explained as the result of homozygous deletions or hypermethylation of the ARF gene, other regulatory mechanisms seem to be altered in these malignancies
— id: 98307, year: 2000, vol: 21, page: 817, stat: Journal Article,

Primitive neuroectodermal tumors of the brainstem: investigation of seven cases
Zagzag D; Miller DC; Knopp E; Farmer JP; Lee M; Biria S; Pellicer A; Epstein FJ; Allen JC
2000 Nov;106(5):1045-1053, Pediatrics
OBJECTIVE: We discuss the clinical aspects, pathology, and molecular genetics of 7 patients with primitive neuroectodermal tumors (PNETs) arising in the brainstem that were treated at our institution from 1986 through 1995. Most neuro-oncologists avoid performing biopsies in children with pontine tumors. This article raises the question as to whether biopsies should be performed, because treatment recommendations might differ if a PNET was diagnosed rather than a pontine glioma. PATIENTS AND METHODS: We reviewed the clinical neuro-oncology database and the files of the Division of Neuropathology at New York University Medical Center from 1986 through 1995 and identified 7 histologically confirmed PNETs arising in the brainstem among 146 pediatric brainstem tumors. The clinical, neuroradiological, and neuropathological data were reviewed. Postmortem examinations were performed in 2 cases. Formalin-fixed, paraffin-embedded tumor tissues were also available in 6 of 7 patients that were tested for p53 gene mutations using single-strand conformation polymorphism analysis. We also tested 9 cerebellar PNETs, 9 brainstem gliomas, and 3 normal brains for p53 gene mutations as controls. RESULTS: All 7 patients presented with focal cranial nerve deficits, and 2 were also hemiparetic. The median age at diagnosis was 2.7 (1-8 years). Magnetic resonance imaging (MRI) characteristics included a focal intrinsic exophytic nonenhancing brainstem lesion that had low T1-weighted and high T2-weighted signals. Hydrocephalus was present in 5 patients at diagnosis, 3 of whom had leptomeningeal dissemination. Meningeal dissemination occurred later in the course of the disease in 3 other patients. Five children required shunts at diagnosis and another 2 at recurrence. Despite therapy, all 7 PNET patients died within 17 months of diagnosis with a mean survival of 8 (4-17) months. No mutation in the p53 gene was detected. CONCLUSIONS: Brainstem PNETs tend to arise at a younger age than brainstem gliomas and medulloblastomas. The MRI pattern suggests a localized rather than a diffuse intrinsic nonenhancing brainstem tumor. Like other PNETs, brainstem PNETs have a high predilection to disseminate within the central nervous system. The absence of p53 mutations is similar to other PNETs. Despite their origin close to the cerebellum, brainstem PNETs exhibit a more aggressive behavior and result in worse clinical outcomes than do cerebellar PNETs
— id: 26837, year: 2000, vol: 106, page: 1045, stat: Journal Article,

The unr gene: evolutionary considerations and nucleic acid-binding properties of its long isoform product
Ferrer N; Garcia-Espana A; Jeffers M; Pellicer A
1999 Mar;18(3):209-218, DNA & cell biology
The unr transcription unit is located just upstream of the N-ras gene in the genome of mammals, in which unr, like N-ras, is ubiquitously expressed. To determine at what point in evolution the unr/N-ras linkage was created, analysis of nucleic acids by Southern and Northern blotting was performed, allowing us to track the presence of the unr gene to the start of vertebrate evolution and the unr/N-ras linkage to the time at which the reptilian and bird lines diverged. We have investigated, with specific anti-unr antibodies, a potential relation between unr protein levels and cellular processes in which N-ras is implicated. A positive correlation in the proliferation of 3T3 cells, but not differentiation of PC12 cells induced by nerve growth factor (NGF), was found. To study the nucleic acid-binding properties of unr, a protein with multiple repeats of a nucleic acid-binding motif, we expressed the long splicing isoform in a eukaryotic cell line and purified it in native form. The results obtained-a high affinity of unr for single-stranded DNA and RNA and lower affinity for double-stranded DNA without regard to nucleic acid sequence, and its intracellular localization in both the nuclear and non-nuclear compartments, together with its ubiquious expression in mammalian tissues-provide molecular information about the function of one of the closest gene tandems in mammalian cells (unr-N-ras)
— id: 6072, year: 1999, vol: 18, page: 209, stat: Journal Article,

Targeted gene transfer system using a streptavidin-transforming growth factor-alpha chimeric protein
Garcia-Espana A; Biria S; Malumbres M; Levin B; Meruelo D; Pellicer A
1999 Oct;18(10):743-749, DNA & cell biology
The previously reported streptavidin-TGFalpha chimeric protein-based delivery system (Ohno and Meruelo, DNA Cell Biol. 15:401-406, 1996) could efficiently transfer protein molecules into A431 cells via the epidermal growth factor (EGF) receptor. We have modified this delivery system for the transfer of DNA. For this purpose, we have linked the chimeric protein ST-TGFalpha to DNA through biotinylated polylysine molecules. We show with this system, in the presence of the endosome-destabilizing reagent chloroquine, an average of 50-fold increase in reporter gene expression in comparison with polylysine DNA complexes alone. This gene expression is specific for EGF receptor-expressing cells and is blocked by EGF-binding molecules. These results suggest that the ST-TGFalpha biotinylated polylysine system could be used to deliver DNA to targeted cells
— id: 11938, year: 1999, vol: 18, page: 743, stat: Journal Article,

The use of transgenic mice in the development of farnesyltransferase inhibitors
Kohl, NE; Anthony, NJ; Conner, MW; Chen, HY; deSolms, SJ; Gibbs, JB; Graham, SL; Hartman, GD; Koblan, KS; Omer, CA; Pellicer, A; Windle, JJ; Oliff, A
1999 NOV ;5(4):3869S-3870S, Clinical cancer research
— id: 53804, year: 1999, vol: 5, page: 3869S, stat: Journal Article,

Hypermethylation of the cell cycle inhibitor p15INK4b 3'-untranslated region interferes with its transcriptional regulation in primary lymphomas
Malumbres M; Perez de Castro I; Santos J; Fernandez Piqueras J; Pellicer A
1999 Jan 14;18(2):385-396, Oncogene
The cyclin-dependent kinase inhibitor p15INK4b has been shown to be involved in human and rodent tumors and seems to act as a tumor suppressor gene in hematological malignancies. Alterations of this gene in tumors include mainly homozygous deletions and hypermethylation of the CpG island in the promoter region. In this work, we describe a new area sensitive to methylation in the 3' untranslated region (UTR) of the murine p15INK4b gene. This region shows different levels of methylation depending on the tissues, being relatively highly methylated in brain and gut, and weakly methylated in liver, spleen or thymus. DNA methylation and expression is similar in both maternal and paternal alleles indicating no imprinting effect. Although methylation of the p15INK4b 3'-UTR is low in normal thymus, increased levels (up to 100%) of specific methylation in this region are found in up to 30% of radiation- or carcinogen-induced thymic lymphomas, correlating with decreased gene expression. Hypermethylation of the p15INK4b 3'-UTR frequently occurs in tumors with loss of heterozygosity (LOH) but without methylation of the promoter CpG island or intragenic mutations. Furthermore, in vitro CpG methylation of the 3'-UTR produces reduced levels of a luciferase reporter in cultured cells. Methylation of two CpG sites in a 120 bp region is sufficient to interfere with transcription of the reporter gene. These data suggest that although the levels of p15INK4b in normal tissues can be mainly determined by promoter regulatory elements, strong hypermethylation of the 3'-UTR can interfere with transcription. Thus, hypermethylation of the 3'-UTR may explain the lack of p15INK4b gene expression in a subset of tumors with no promoter methylation and could be a new alternative mechanism for tumor suppressor gene inactivation in tumorigenesis
— id: 7398, year: 1999, vol: 18, page: 385, stat: Journal Article,

Antineoplastic effect of a farnesyl-transferase inhibitor in N-Ras overexpressing tumors
Mangues, R; Corral, T; Khol, NE; Gibbs, JB; Oliff, A; Guerrero, S; Casanova, I; Farre, L; Capella, G; Pellicer, A
1999 APR ;35(6):S13-S13, European journal of cancer
— id: 54021, year: 1999, vol: 35, page: S13, stat: Journal Article,

Cooperative alterations of Rb pathway regulators in mouse primary T cell lymphomas
Perez de Castro IP; Malumbres M; Santos J; Pellicer A; Fernandez-Piqueras J
1999 Sep;20(9):1675-1682, Carcinogenesis
Alterations in the Rb pathway have been described in many different tumors. In order to study this cell cycle regulatory mechanism in murine T cell lymphomas, we have analyzed the RNA and protein expression of the cyclin D1, cdk4 and retinoblastoma genes in primary tumor samples. We have detected overexpression of the cyclin D1 gene and deficient expression of the retinoblastoma gene in 42 and 28% of these tumors, respectively. The immunohistochemical analysis showed that these RT-PCR results are correlated with a significant increase in the number of positive cells for cyclin D1 and a moderate decrease in the expression of Rb protein, respectively. The analysis of cyclin D1, Rb, p15(INK4b) and p16(INK4a) showed that 75% of lymphomas had alterations in these genes and indicates that the Rb pathway is frequently altered in mouse primary T cell lymphomas. Moreover, 31% of lymphomas presented simultaneous alterations in at least two of these genes, suggesting the importance of concurrent alteration of different Rb pathway regulators. In addition, we have characterized these samples for mutational status of the N-ras and K-ras genes. We have only detected mutations in codon 12 of K-ras in six of 49 lymphomas (12%). Interestingly, five of these lymphomas also showed alterations in at least one of the Rb pathway regulators analyzed here. Taken together, these data suggest that deregulation of the Rb pathway regulators and/or oncogenic activation of K-ras may represent a common important clue in progression of murine T cell lymphomas
— id: 6192, year: 1999, vol: 20, page: 1675, stat: Journal Article,

RAS pathways to cell cycle control and cell transformation
Malumbres M; Pellicer A
1998 Aug 6;3:d887-d912, Frontiers in biosciences
Ras genes are among the most frequently activated oncogenes in cancer. The corresponding protooncogenes are proteins expressed in the majority of tissues in mammals and have a signal transduction activity. Ras proteins interact with a wide spectrum of regulators and downstream effectors producing different cellular responses, including proliferation, differentiation or apoptosis. This review deals with the most recent advances on the role of Ras in the signal transduction pathway from external signals to the cell cycle and gene expression control. We specially address the new developments on the effect of Ras activation in the regulation of different molecules driving the cell cycle progression. Both positive and negative regulators of the cyclin-dependent kinases (CDK), cyclins and CDK inhibitors, are targets of Ras, giving rise to different effects in the cell cycle progression. These Ras-mediated interactions are an extraordinary example of the complexity of the signal transduction networks and the diversity of pathways used by Ras to propagate molecular signals
— id: 47854, year: 1998, vol: 3, page: d887, stat: Journal Article,

An AC-repeat adjacent to mouse Cdkn2B allows the detection of specific allelic losses in the p15INK4b and p16INK4a tumor suppressor genes
Malumbres M; Perez de Castro I; Santos J; Perez-Olle R; Fernandez-Piqueras J; Pellicer A
1998 Mar;9(3):183-185, Mammalian genome
The cyclin-dependent kinase inhibitors p15INK4b and p16INK4a are involved in the development of a wide range of human and murine tumors. These tumor suppressor genes are inactivated by deletions frequently associated to point mutations in the coding regions or hypermethylation of their promoters. In this work, we describe a simple-sequence length polymorphism located in mouse Chromosome (Chr) 4, between the Cdkn2B (p15INK4b) and Cdkn2A (p16INK4a) genes, only 700 bp downstream of the Cdkn2B locus. This DNA region was analyzed in different inbred strains showing a variable AC-repetitive DNA sequence. We used this microsatellite to detect loss of heterozygosity of the Cdkn2A and Cdkn2B loci in gamma-irradiation-induced thymic lymphomas of C57BL/6J x RF/J F1 hybrids. Using this specific marker, we were able to locate additional allelic losses not detected by other microsatellites. Since the allelic losses can be detected by a simple PCR amplification, this AC-repetitive sequence is specially useful as a genetic marker for these Cdkn2 genes and specifically for the p15INK4b cell cycle inhibitor
— id: 57202, year: 1998, vol: 9, page: 183, stat: Journal Article,

Antitumor effect of a farnesyl protein transferase inhibitor in mammary and lymphoid tumors overexpressing N-ras in transgenic mice
Mangues R; Corral T; Kohl NE; Symmans WF; Lu S; Malumbres M; Gibbs JB; Oliff A; Pellicer A
1998 Mar 15;58(6):1253-1259, Cancer research
We tested the antineoplastic effect of the farnesyltransferase inhibitor L-744,832 in mammary and lymphoid tumors overexpressing the N-ras proto-oncogene in transgenic mice. Mice bearing mammary tumors were randomly assigned to receive daily 40 mg/kg s.c. injections of this compound (experimental group, n = 6) or vehicle (control group, n = 6) per day for 5.5 weeks. Treatment with the compound significantly reduced the mammary tumor mean growth rate in the experimental group (-0.7 mm3/day), as compared with the control group (+28.2 mm3/day; P < 0.001). There was a significant difference in lymphoma incidence at the end of the treatment between the experimental (0 of 6) and the control (3 of 6) groups (P < 0.05). Therefore, this compound is effective in treating in vivo mammary carcinomas and lymphomas in which an activated N-Ras pathway drives tumorigenesis. The number of apoptotic figures in mammary tumors was significantly higher (P = 0.04) in the experimental (14.7 +/- 8.1) than it was in the control (5.7 +/- 3.5) group, indicating that apoptotic induction could contribute to the mechanism of antitumor activity of this compound. We analyzed the level of processing of N-Ras and H-Ras after immunoprecipitation and Western blotting of protein extracts obtained from mammary tumors treated with L-744,832 or vehicle, either in vivo or in vitro (after primary culture of the same tumors), and from several in vitro treated control cell lines. In all compound-treated mammary tumors and cell lines, H-Ras was mostly unprocessed (more so after in vitro than after in vivo treatment), whereas N-Ras remained mostly processed. Both H-Ras and N-Ras remained fully processed in all vehicle-treated samples. These findings are consistent with a less intense antineoplastic effect of the treatment with the compound in our N-ras model than the effect previously reported for the same compound in H-ras transgenics. In addition, the finding that, in compound-treated mammary tumors, the N-Ras protein remains mainly processed suggests that, in our model, other proteins in addition to Ras may be a target for the compound. Our results and the previous findings of frequent N-ras activation in human hematopoietic malignancies support a role for L-744,832 in the treatment of lymphomas and of mammary carcinomas with an activated N-Ras pathway, as well as the testing of a farnesyl protein transferase inhibitor in humans to establish its clinical relevance
— id: 57203, year: 1998, vol: 58, page: 1253, stat: Journal Article,

NF1 inactivation cooperates with N-ras in in vivo lymphogenesis activating Erk by a mechanism independent of its Ras-GTPase accelerating activity
Mangues R; Corral T; Lu S; Symmans WF; Liu L; Pellicer A
1998 Oct 1;17(13):1705-1716, Oncogene
We crossed transgenic mice overexpressing the N-ras proto-oncogene (RasTg) with mice carrying one inactivated copy of the NF1 tumor suppressor gene (NF1+/-) to assess their possible cooperation in tumorigenesis. We have found a significant increase in the incidence of lymphomas in animals with both lesions (RasTg NF1+/-), as compared with animals with single lesions. The mechanism of this cooperation appears to be independent of the NF1 GTPase activating activity since the level of Ras-GTP in primary cultures of tumor tissue do not differ among animals with double and with single lesions. Nevertheless, the finding of significantly higher levels of Erk-1 and Erk-2 activation in lymphomas in the RasTg NF1+/- than in the RasTg group suggests that this cooperative effect may be in part explained by increased signaling through the Erk pathways. Consistent with a role for Erk activation in transformation is the additional observation that Erk-1 and Erk-2 activation is significantly increased in lymphomas as compared with normal spleen. This activation is likely to occur by phosphorylation of previously synthesized and inactive Erk proteins since, despite differences in activation, Erk-1 and Erk-2 expression is similar in normal and lymphoid tissue in all groups. The observed cooperation in in vivo lymphomagenesis between N-ras overexpression and NF1 inactivation emphasizes the importance of searching for additional functions for the NF1 protein and of intensifying the screening for NF1 mutations in human lymphomas
— id: 7672, year: 1998, vol: 17, page: 1705, stat: Journal Article,

A new candidate site for a tumor suppressor gene involved in mouse thymic lymphomagenesis is located on the distal part of chromosome 4
Santos J; Herranz M; Perez de Castro I; Pellicer A; Fernandez-Piqueras J
1998 Aug 20;17(7):925-929, Oncogene
In a previous work we provided preliminary evidence of the existence of a putative tumor suppressor gene region on the distal part of chromosome 4 in gamma-radiation-induced T-cell lymphomas of (C57BL/6J x RF/J) F1 hybrid mice. This region, named as TLSR2 (Thymic Lymphoma Suppressor Region 2), was located centered at D4Mit54. A more detailed allelotype analysis in the mentioned tumors with new informative microsatellites on distal chromosome 4 region, as well as in thymic lymphomas induced with gamma-rays in F1 hybrids generated from reciprocal crosses between the strains C57BL/6J and BALB/cJ, and having in mind the new map position of D4Mit205b, allowed us to confirm the existence of TLSR2 and to define it more precisely as centered at D4Mit205b. In addition, we identified a new candidate region, named as TLSR3 (Thymic Lymphoma Suppressor Region 3), located between the Mom-1 locus and D4Mit68, as the site of another putative tumor suppressor region involved in thymic lymphomagenesis
— id: 7973, year: 1998, vol: 17, page: 925, stat: Journal Article,

rsc: a novel oncogene with structural and functional homology with the gene family of exchange factors for Ral
D'Adamo DR; Novick S; Kahn JM; Leonardi P; Pellicer A
1997 Mar 20;14(11):1295-1305, Oncogene
A novel oncogene, rsc (rabbit squamous cell carcinoma), has been identified from a DMBA-induced rabbit squamous cell carcinoma using gene transfer and the nude mouse tumorigenesis assay. A full-length cDNA has been isolated and sequenced. rsc has potent tumorigenic activity in nude mice (latency <4 weeks), but does not induce focus formation or anchorage independent growth. The oncogene resulted from the fusion of rHR 23A (a rabbit homologue of yeast Rad 23) with a member of the ral-GDS family which we named rgr (ral-GDS related). Deletion analysis demonstrated that the oncogenic potential resides in the Rgr portion of the gene. Rgr is 40% identical overall to Ral-GDS, with identity increasing to 72% over a 100 amino acid region of the catalytic domain. Biochemical experiments indicate that Rgr has GTP/GDP exchange activity for Ral, providing evidence that this pathway is associated with tumorigenesis. The linkage between the Ral pathway and tumorigenesis by a molecule in the Ral-GDS gene family (Ral-GDS being a known effector for Ras) will open the way for the characterization of this pathway and provide an important tool to understand its biological function
— id: 7136, year: 1997, vol: 14, page: 1295, stat: Journal Article,

Anti-tumor activities of farnesyltransferase inhibitors
Kohl, NE; Anthony, NJ; Conner, MW; deSolms, SJ; Gibbs, JB; Graham, L; Hartman, GD; Koblan, KS; Omer, CA; Pellicer, A; Williams, TM; Windle, JJ; Oliff, A
1997 JUL 31 ;11(9):A993-A993, FASEB journal
— id: 53455, year: 1997, vol: 11, page: A993, stat: Journal Article,

Isolation of high molecular weight DNA for reliable genotyping of transgenic mice
Malumbres M; Mangues R; Ferrer N; Lu S; Pellicer A
1997 Jun;22(6):1114-1119, Biotechniques
A fast and reliable method for the PCR characterization of DNA from mouse toes is described. The toes biopsied to tag the mice are incubated for 2 h in proteinase K and heated for 15 min at 95 degrees C. This DNA solution is directly used as a template for PCR amplification. The same procedure can be used for PCR analysis of DNA from other tissues in adult mice, mouse embryos and cultured cells. Because of minimal tissue manipulation, high-quality and high-molecular-weight DNA (fragments larger than 100-200 kb) is isolated. This procedure is performed in a single tube and requires no organic solvent extraction or centrifugation, allowing the isolation of high-molecular-weight DNA suitable for PCR amplification in a fast and reproducible way. Only the tissue excised during mice tagging is used and a large number of animals can be quickly and simultaneously analyzed as required to maintain a transgenic mice colony. In addition, this rapid and efficient procedure represents an alternative to other methods in which, in our experience, inhibition of the PCR amplification occurs when DNA from tail tissues is used
— id: 7209, year: 1997, vol: 22, page: 1114, stat: Journal Article,

Inactivation of the cyclin-dependent kinase inhibitor p15INK4b by deletion and de novo methylation with independence of p16INK4a alterations in murine primary T-cell lymphomas
Malumbres M; Perez de Castro I; Santos J; Melendez B; Mangues R; Serrano M; Pellicer A; Fernandez-Piqueras J
1997 Mar 20;14(11):1361-1370, Oncogene
A wide panel of murine induced T-cell lymphomas have been analysed for p16INK4a or p15INK4b alterations. Only one gamma-radiation-induced lymphoma showed p16INK4a homozygous deletion and no other intragenic mutations were found in these INK4 genes. However, de novo methylation of the 5' CpG islands of the murine p15INK4b and p16INK4a genes was found to be highly frequent. While p16INK4a hypermethylation was found in 36% of the neutron-radiation-induced lymphomas and 15% of the gamma-radiation-induced lymphomas, de novo methylation of p15INK4b occurs in 88% and 42% of these tumors respectively, correlating with deficient expression of the corresponding mRNA and allelic losses in the p15INK4b and p16INK4a chromosome location. These data represent, to our knowledge, the first report on the significant involvement of hypermethylation of these INK4 genes in murine primary tumors. Moreover, they show the importance of allelic losses and CpG island methylation of p15INK4b gene inactivation and support a tumor suppressor role for p15INK4b in T-cell lymphomas independent of p16INK4a
— id: 7210, year: 1997, vol: 14, page: 1361, stat: Journal Article,

P53 oncoprotein expression and gene mutations in some keratoacanthomas
Perez MI; Robins P; Biria S; Roco J; Siegel E; Pellicer A
1997 Feb;133(2):189-193, Archives of dermatology
OBJECTIVE: To analyze the relationship of p53 oncoprotein overexpression in most keratoacanthomas (KAs) with gene mutations. DESIGN: Expression of p53 oncoprotein in immunohistochemical staining and its correlation to gene mutations in DNA extracted from KAs and tested in single-strand conformational polymorphism (SSCP) analysis and direct sequencing. SETTING: A micrographic surgery unit and a dermatopathology unit at a university medical center. PATIENTS: Sixteen formalin-fixed, paraffin-embedded skin biopsy specimens were retrieved from dermatopathology archives. Biopsies were performed to establish the diagnosis of KA before surgical treatment. MAIN OUTCOME MEASURES: Intensity of staining in immunohistochemical testing for p53 oncoprotein expression and sequencing of gene mutations. RESULTS: Immunohistochemical staining of 16 KA specimens detected p53 oncoprotein in 15 (94%), distributed as strong in 4 (25%), moderate in 2 (12%) mild in 9 (56%), and negative in 1 (6%), compared with control specimens. Specimens were screened by SSCP for mutations in the p53 gene, and 1 specimen showed a potential mutation in exon 7. Direct sequencing of the samples revealed 2 point mutations. One specimen showed a change of G:A for A:G in codon 146 of exon 5, predicting an amino acid substitution of tryptophan for a stop codon. Another specimen revealed a change of T:A for A:T in codon 234 of exon 7, predicting an amino acid substitution of tyrosine for asparagine. CONCLUSIONS: Ninety-four percent of KA specimens evaluated had detectable p53 oncoprotein. This protein was associated with a point mutation in the p53 gene in 2 of 16 KAs evaluated. In a small fraction of KAs, overexpression of p53 oncoprotein may be associated with point mutations in the p53 gene
— id: 16874, year: 1997, vol: 133, page: 189, stat: Journal Article,

Mutagenic effects of tumorigenic neutron radiation
Garcia-Espana A; Kahn JM; Saez G; Pellicer A
1996 Mar 1;65(5):677-681, International journal of cancer
A fraction of thymic lymphomas induced by high LET neutron radiation contains activating mutations (single-base substitutions) in the ras genes. To determine whether such mutations are the result of the interaction of high LET radiation with cellular DNA, we have utilized an in vitro model system to screen and isolate neutron-radiation-induced mutants. With that aim, we irradiated the PL61 hamster cell line with 0.4 MeV neutrons. This cell line contains linked copies of the gpt and neo(r) genes, which permits selection for large or small alterations, depending on the selection imposed. Mutants selected for large alterations represented 98.2% of the total. When selection for small mutations was imposed, 9 clones grew. The molecular and biochemical analysis of these clones revealed that 5 of them had identifiable mutations in the gpt gene, consisting of small insertions and deletions, but no single-base substitutions were detected. This represents the first sequence characterization of neutron-induced mutants. The results obtained are consistent with the notion that the ras point mutations identified in the neutron-induced tumors are most likely detected due to the strong selective advantage that they confer to the host cell, but they probably arose during tumour evolution, since they represent a negligible proportion of the total number of alterations induced by neutron radiation
— id: 56813, year: 1996, vol: 65, page: 677, stat: Journal Article,

Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis
Mangues R; Symmans WF; Lu S; Schwartz S; Pellicer A
1996 Sep 5;13(5):1053-1063, Oncogene
We compared the tumorigenic effects of the N-ras oncogene and the N-ras proto-oncogene in lymphoid and mammary tissues in an in vivo model. For this purpose, we generated transgenic mice with high levels of N-ras oncogene or N-ras proto-oncogene expression, driven by the complete mouse mammary tumor virus LTR (MMTV-LTR) (MMTV/N-rasT and MMTV/N-rasN constructs) and transgenic mice with low levels of N-ras oncogene or N-ras proto-oncogene expression, driven by a truncated MMTV-LTR (TMTV/N-rasT and TMTV/N-rasN constructs). We show that both, the N-ras proto-oncogene and the N-ras oncogene with a C:G-->A:T mutation at codon 61, lead to identical tumor types: lymphoblastic T-cell lymphomas, cleaved B-cell lymphomas and poorly differentiated mammary carcinomas. Nevertheless, there were quantitative differences in tumor incidence and latency and in transgene expression among N-ras oncogene and N-ras proto-oncogene transgenics. Despite these differences in tumor kinetics, the predisposition to identical tumor types is in agreement with the idea that the N-ras oncogene and the N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis in B-cells, T-cells or mammary epithelial cells
— id: 7080, year: 1996, vol: 13, page: 1053, stat: Journal Article,

Allelic losses on chromosome 4 suggest the existence of a candidate tumor suppressor gene region of about 0.6 cM in gamma-radiation-induced mouse primary thymic lymphomas
Santos, J; deCastro, IP; Herranz, M; Pellicer, A; FernandezPiqueras, J
1996 FEB 1 ;12(3):669-676, Oncogene
Mouse chromosome 4 was investigated to assess the involvement of tumor suppressor genes in primary thymic lymphomas induced by gamma-irradiation. PCR analysis using microsatellite DNA polymorphic markers was performed in F1 animals generated from a cross between the strains C57BL/6J and RF/J. Microsatellite markers were selected with focus on chromosome 4 around the region containing the interferon alpha gene cluster (D4Mit17, D4Wsm1, D4Mit9, and D4Mit205 and a more distal region (D4Mit12, D4Mit54, and D4Mit13). Allelic losses were detected in 21/47 (44.7%) gamma-radiation-induced thymic lymphomas, Analysis of markers located on six other chromosomes as well as on the proximal region of chromosome 4 illustrates the specificity of the occurrences of LOH appearing on the former markers. This analysis clearly suggests the existence of a candidate tumor suppressor gene region on mouse chromosome 4 of about 0.6 cM between the markers D4Wsm1 and D4Mit9 (TLSR1, Thymic Lymphoma Suppressor Region 1). In addition, another more distal region centered at the marker D4Mit58 could be also exist (TLSR2), In most tumors, allelic losses on these chromosome regions involved the paternal RF/J allele (19 of 20)
— id: 53068, year: 1996, vol: 12, page: 669, stat: Journal Article,

Promoter demethylation in MMTV/N-rasN transgenic mice required for transgene expression and tumorigenesis
Mangues R; Schwartz S; Seidman I; Pellicer A
1995 Oct;14(2):94-102, Molecular carcinogenesis
We studied demethylation within the transgene promoter in transgenic mice carrying the N-ras proto-oncogene driven by the mouse mammary tumor long terminal repeat (MMTV/N-rasN) and the relationship of demethylation to transgene overexpression and tumorigenesis. Demethylation at Fspl or Clal sites correlated with age of the animal and transgene expression in nontumorous mammary gland. Demethylation preceded expression in this tissue. In lymphomas and mammary tumors, the promoter Fspl and Clal sites were significantly more demethylated than in nontumorous control tissues. The Aval, Cfol, and Hpall sites were also found to be undermethylated in older animals and showed differences between tumor and control tissues. Two additional sites (Eagl and Narl) remained fully methylated in all tissues. In contrast with normal tissue, demethylation at the Fspl and Clal sites and expression were not correlated in tumor tissue. An increase in expression in normal tissue initially occurred and was correlated with the level of promoter demethylation; this increase was followed by a further increment in transgene expression when tumors developed. Thus, promoter demethylation leading to transgene overexpression was associated with long-latency tumorigenesis in MMTV/N-rasN transgenic mice. Demethylation of proto-oncogene promoters may therefore be a mechanism of carcinogenesis that requires further investigation in human tumors
— id: 6932, year: 1995, vol: 14, page: 94, stat: Journal Article,

Differential expression of the H-ras mutated and normal alleles in rabbit DMBA-induced keratoacanthomas
Matesanz F; Oliva MR; Villamarin A; Kamino H; Pellicer A
1995 May 29;61(5):679-682, International journal of cancer
Keratoacanthomas (KAs) are benign and self-regressing tumors in which a high incidence of the mutated H-ras oncogene has been observed both in humans and in experimental models. To determine the level of expression of the mutated H-ras allele with respect to its normal counterpart in 7,12-dimethylbenz(a)anthracene (DMBA)-induced KAs in rabbit skin, we have utilized a quantitative technique based on reverse transcription polymerase chain reaction (RT-PCR) and selective cleavage of the mutated molecules of the H-ras gene. Analysis of 16 KAs showed that the mutated H-ras transcripts were up to 3-fold more abundant than the non-mutated H-ras transcript in the different tumors. This higher expression of the mutated allele appears to correlate with increased differentiation in the KAs and in turn may contribute to tumor regression
— id: 56683, year: 1995, vol: 61, page: 679, stat: Journal Article,

IN-VIVO AND IN-VITRO ANALYSIS OF RETROVIRAL VECTORS CARRYING THE N-RAS ONCOGENE
MATESANZ, F; PELLICER, A
1995 SEP ;7(3):443-451, International journal of oncology
We have analyzed, in vivo and in vitro, the behavior of two retroviral vectors carrying the genomic or cDNA N-ras oncogene to study the role of N-ras in the initiation and development of thymic lymphomas. The vector bearing the genomic gene produced an array of transcripts originating from the LTR and the oncogene promoter. The majority of the transcripts initiated at the LTR did not carry the packaging signal producing low titer clones. The cDNA vector produced two transcripts correctly spliced and the titers obtained were as high as 10(6) pfu/ml. Bone marrow cell infection and grafting of lethally irradiated mice was performed. The integrated vector in blood cells was followed at different times, observing that the provirus can disappear and reappear in peripheral blood cells during the course of the experiment. This observation fits with the hypothesis of clonal contribution of small number of stem cells in the renewal of blood cells. No tumors were detected in the infected animals, probably due to low expression of the integrated provirus. These experiments provide information on the advantages and disadvantages of genomic versus cDNA constructs in retroviral vectors
— id: 87243, year: 1995, vol: 7, page: 443, stat: Journal Article,

THE MURINE N-RAS GENE IS NOT ESSENTIAL FOR GROWTH AND DEVELOPMENT
UMANOFF, H; EDELMANN, W; PELLICER, A; KUCHERLAPATI, R
1995 FEB 28 ;92(5):1709-1713, Proceedings of the National Academy of Sciences of the United States of America
The mammalian ras gene family encodes key cell-signaling, cell growth-related proteins that have been highly conserved in species from yeast to man. Specific point mutations in the ras genes are associated with various mammalian tumors. To understand the developmental role of the N-ras protooncogene in the mouse, we have disrupted its gene function by homologous recombination in embryonic stem cells. Mice derived from these cells that are homozygous for the N-ras mutation do not produce any detectable N-Ras protein and are morphologically and histologically indistinguishable from their heterozygous and wild-type siblings. Since N-ras is expressed at high levels in hematopoietic cells, we examined different populations of cells in peripheral blood and found no differences between mutant and normal animals. Our results show that N-ras gene function is dispensable for normal mouse development, growth, and fertility
— id: 87408, year: 1995, vol: 92, page: 1709, stat: Journal Article,

BRAIN INTERLEUKIN-1-BETA IN ALZHEIMERS-DISEASE AND VASCULAR DEMENTIA
CACABELOS, R; ALVAREZ, XA; FERNANDEZNOVOA, L; FRANCO, A; MANGUES, R; PELLICER, A; NISHIMURA, T
1994 MAR ;16(2):141-151, Methods & findings in experimental & clinical pharmacology
Recent investigations indicate that a neuroimmune reaction, associated with inflammatory mechanisms, can contribute in Alzheimer's disease (AD) to cell damage and neurodegeneration. Activation of microglial cells. expression of immunohistochemical markers of brain immune function, the presence of complement proteins in brain tissue and changes in cytokine production have been reported in AD. We have studied the concentration of interleukin-1beta (IL-1beta) in different regions of the central nervous system (CNS) in post-mortem samples from patients with AD or vascular dementia (VD) and in age-matched control subjects (CS). IL-1beta levels were significantly higher in AD than in VD or CS in the frontal cortex, parietal cortex, temporal cortex, hypothalamus, thalamus and hippocampus. The highest increases in IL-1beta levels were observed in the frontal cortex (CS = 0.75 +/- 0.045,- AD = 2.47 +/- 0.12, p < 0. 001; VD = 1.52 +/- 0.078 pg/mg, p < 0.001) and hippocampus (CS = 0.71 +/- 0.042, +/- AD = 2.63 +/- 0.19, p < 0.001; VD = 1.21 +/- 0.23 pg/mg, p < 0.01). No significant changes were detected in the occipital cortex and cerebellum in either AD or VD. These results clearly demonstrate that demented patients show a generalized increment of IL-1beta production in the CNS, with maximum response in those brain regions where AD neuropathology is most prominent. This overall increase in cytokine production might represent an early event in the activation of a neuroimmune cascade leading to cell death and neurodegeneration in brain regions where a primary cause (e.g., genetic, toxic, vascular) facilitates the induction of resting microglia for firing brain immune function
— id: 52377, year: 1994, vol: 16, page: 141, stat: Journal Article,

P53 MUTATIONS IN HUMAN BLADDER-CANCER - GENOTYPIC VERSUS PHENOTYPIC PATTERNS
CORDONCARDO, C; DALBAGNI, G; SAEZ, GT; OLIVA, MR; ZHANG, ZF; ROSAI, J; REUTER, VE; PELLICER, A
1994 FEB 1 ;56(3):347-353, International journal of cancer
The objective of this study was to characterize the pattern of p53 mutations in bladder cancer. The sensitivity and specificity to detect these mutations using clinical material was assessed for the following assays: immunohistochemistry, restriction-fragment-length polymorphism, single-strand-conformation polymorphism, and sequencing. Discrepancies of reported results aimed at the identification of genetic alterations in the p53 gene may be due to differences in methodology, as well as to deficient morphological evaluation of the source of tissue utilized. In order to address these critical issues, we have implemented a novel experimental design that permits analysis by molecular genetics and immunopathology techniques in any given tissue specimen, allowing morphological correlation with genotypic and phenotypic characteristics of the tissue analyzed. Forty-two patients affected with bladder tumors in whom paired normal and tumor tissues were available were used for the present study. Nuclear immunoreactivities were observed in 26 out of 42 bladder tumors analyzed. Abnormal shifts in mobility were noted in 14 of the 42 cases in distinct exons, with one tumor revealing 3 mutations. There was a strong association between p53 nuclear over-expression and detection of mutations by SSCP and sequencing. According to receiver-operating-curve statistical analysis, the accuracy of detecting p53 mutations by IHC was estimated to be 90.3%. It is our conculsion that, when properly used, this is a highly sensitive and specific method with simple application using clinical material. (C) 1994 Wiley-Liss, Inc
— id: 52581, year: 1994, vol: 56, page: 347, stat: Journal Article,

Identification of multiple promoters within the N-ras proto-oncogene
Jeffers M; Pellicer A
1994 Nov 22;1219(3):623-635, Biochimica & biophysica acta
N-ras possesses a 'housekeeping' promoter, being G + C-rich and devoid of a TATA-box. Transcription initiates at a number of locations within this gene, a phenomena that is generally attributed to the absence of a TATA-box. In this report we investigate the possibility that multiple promoters, which could potentially contribute to the observed 5' end heterogeneity, exist within the murine N-ras gene. The 5' region of the gene was subdivided into several fragments, each corresponding to a region in which one or more transcription initiation site(s) had been mapped, and the ability of each fragment to express a reporter gene was assessed. Promoter activity was found associated with three independent, non-overlapping fragments, two of which were located entirely within transcribed regions of the gene. We found that these intragenic promoters were able to express the N-ras gene itself, as well as the reporter gene. In addition, we found that the activity of an intragenic promoter fragment was dependent upon the presence of regions encompassing initiation sites, and that a small fragment (approximately 40 bp) encompassing several initiation sites possessed promoter activity. These data support the existence of an 'initiator' element within the N-ras gene. Overall, our results demonstrate that multiple promoters reside within N-ras and suggest that they may play a role in generating the observed mRNA 5' end heterogeneity. The identification of multiple promoters within N-ras may have important implications regarding the regulation of expression of this gene in normal and malignant tissues. In addition, since a number of other genes with housekeeping promoters also initiate transcription at multiple locations, it is possible that the utilization of multiple promoters may represent a common feature of this class of genes
— id: 6652, year: 1994, vol: 1219, page: 623, stat: Journal Article,

An overexpressed N-ras proto-oncogene cooperates with N-methylnitrosourea in mouse mammary carcinogenesis
Mangues R; Kahn JM; Seidman I; Pellicer A
1994 Dec 15;54(24):6395-6401, Cancer research
The induction of tumors with chemicals and the production of transgenic animals are two experimental approaches to study oncogene involvement in carcinogenesis. The combination of both strategies offers an excellent model system to study tumor development. This study analyzes the potential cooperation of N-methylnitrosourea (MNU) treatment and N-ras proto-oncogene overexpression in tumorigenesis in transgenic mice. The overexpression of the N-ras proto-oncogene in these animals is associated with development of mammary tumors and lymphomas. After MNU treatment we analyzed tumor incidence and latency, levels of transgene expression, and pattern of ras mutations in codons 12, 13, and 61 of H-, K-, and N-ras genes in both tumor types. Transgenic mice treated with MNU had significantly (P < 0.001) shorter latency of appearance of mammary tumors [8.6 +/- 3.0 (SD) months] than phosphate-buffered saline-treated transgenics (12.8 +/- 2.3 months). All mammary tumors overexpressed the N-ras transgene and lacked ras mutations. Moreover, MNU-treated transgenics had an incidence and latency of lymphomas similar to that of MNU-treated nontransgenic mice. No significant differences in incidence of point mutations (K-ras codon 12 or 13 and N-ras codon 61) in lymphomas were seen between these two groups. All lymphomas overexpressed the N-ras transgene, except for those carrying a K-ras point mutation. Overexpression of the N-ras proto-oncogene cooperates with non-ras genes mutated by MNU in mouse mammary carcinogenesis. Conversely, N-ras proto-oncogene overexpression does not show cooperation with MNU in lymphomagenesis in our system. This study suggests that proto-oncogene overexpression may be a mechanism of activation of the ras pathway, alternative to point mutation. Similarly to actions for ras genes activated by point mutation, overexpression of the N-ras protooncogene predisposes to tumorigenesis and cooperates with a carcinogen in tumorigenesis. The possibility that ras overexpression plays a role in human breast tumorigenesis requires active investigation
— id: 56679, year: 1994, vol: 54, page: 6395, stat: Journal Article,

Absence of MDM-2 gene amplification in experimentally induced tumors regardless of p53 status
Saez GT; Oliva MR; Mangues R; Pellicer A
1994 Jan;9(1):40-45, Molecular carcinogenesis
To assess the generality of the hypothesis that murine double-minute-2 (MDM-2) gene amplification complements the absence of p53 mutation during tumor development, we analyzed 143 murine tumors induced by a variety of carcinogenic agents in two different mouse strains. Only three of 143 tumors showed p53 genetic alterations and none showed MDM-2 amplification, indicating the existence of alternative pathways that permit tumor cells to bypass p53-MDM-2 control
— id: 56580, year: 1994, vol: 9, page: 40, stat: Journal Article,

Existence of at least five interleukin-2 molecules in different mouse strains
Matesanz F; Alcina A; Pellicer A
1993 ;38(4):300-303, Immunogenetics
— id: 13326, year: 1993, vol: 38, page: 300, stat: Journal Article,

SIGNAL TRANSDUCTION PATHWAYS INTERACTIONS - NEUROTROPHIC FACTORS, ONCOGENES, D2 RECEPTOR
SAEZ, G; THOMSON, T; GOLDSTEIN, M; PELLICER, A
1993 FEB 19 ;7(3):A376-A376, FASEB journal
— id: 98466, year: 1993, vol: 7, page: A376, stat: Journal Article,

Phylogenetic relationships among laboratory and wild-origin Mus musculus strains on the basis of genomic DNA RFLPs
Santos J; Cole Y; Pellicer A
1993 Sep;4(9):485-492, Mammalian genome
Genetic distance measures between the laboratory mouse strains C57BL/6J and RF/J and the wild-origin Mus musculus mouse strains CAST/Ei, MOLF/Ei, POSCH I, and CZECH II were estimated by allelic patterns revealed by RFLP analysis. These results suggest phylogenetic relationships indicating that the mouse strains related to the subspecies M.m. domesticus (RF/J, POSCH I and C57BL/6J) are more closely related to the CAST/Ei strain (derived from M.m. castaneus) than to the strains CZECH II (M.m. musculus) and MOLF/Ei (M.m. molossinus). Furthermore, the hybrid strain C57BL/6J is more closely related to POSCH I (M.m. poschiavinus) than to RF/J as calculated by the method distance measures of Cavalli-Sforza and Edwards (Evolution 21,550, 1967), Nei's minimum (Am. Natural. 106,283, 1972) and unbiased minimum (Genetics 89,583, 1978), Edwards (Biometrics 27,873, 1971; Genetic Distance, p. 41, 1974) and Rogers modified (1986)
— id: 13086, year: 1993, vol: 4, page: 485, stat: Journal Article,

Novel RFLPs at protooncogene and cancer-related gene loci on mouse chromosomes
Santos J; Pellicer A
1993 ;62(4):217-219, Cytogenetics & cell genetics
DNA probes for the NRAS, HRAS, KRAS2, LCK, RAF1, MET, MYCL1, MYCN, MYB, ERBB2, FOS, CSF1R, and SRC protooncogene loci; the retinoblastoma gene locus (RB1); the tumor virus integration sites INT2, PVT1, and MLV12; and the locus of the tumor-specific antigen T1A were used to screen mouse genomic DNAs from RF/J, CAST/Ei, MOLF/Ei, Mus musculus musculus, M. m. poschiavinus, and M. spretus. Polymorphic DNA fragments for the 18 DNA probes have been identified using Southern blot hybridization and restriction fragment length polymorphism (RFLP) analysis
— id: 13332, year: 1993, vol: 62, page: 217, stat: Journal Article,

A METHOD TO EXTRACT DNA FOR MOLECULAR STUDIES FORM CELLS FIXED IN CARNOY
BARRIOS, L; MIRO, R; COROMINAS, M; PELLICER, A; EGOZCUE, J
1992 APR ;59(2):217-218, Cancer genetics & cytogenetics
We describe a simple method for isolation of high-molecular-weight DNA from cells fixed in Carnoy's solution for cytogenetic studies. DNA samples were extracted from NIH3T3 cells, and no apparent degradation was noticed. This method should be useful for molecular analysis of cells fixed for cytogenetic studies
— id: 51948, year: 1992, vol: 59, page: 217, stat: Journal Article,

Multiple intragenic elements regulate the expression of the murine N-ras gene
Jeffers M; Pellicer A
1992 Nov;7(11):2115-2123, Oncogene
We have identified two separate regions within the murine N-ras transcription unit which may participate in the regulation of N-ras gene expression. One of these regions, localized to the first half of intron 1, contains a site of premature transcriptional arrest. We propose that premature transcription termination, which has been identified as an important control mechanism for several genes, may also play a role in the regulation of the N-ras gene. In addition, we have identified a positively acting region within the N-ras transcription unit. This region, localized to the end of intron 1/beginning of exon 1, was found to greatly increase expression from the N-ras promoter
— id: 13374, year: 1992, vol: 7, page: 2115, stat: Journal Article,

ras activation in experimental carcinogenesis
Mangues R; Pellicer A
1992 Aug;3(4):229-239, Seminars in cancer biology
We review experimental models of carcinogenesis in which the role of ras activation has been most thoroughly studied: skin, thymus, mammary gland and liver. Qualitative changes (point mutations), as well as quantitative changes (over-expression, increased gene dosage) contribute to the transforming phenotype induced by ras genes. The activation of the three different ras family members is associated with particular tumor types, carcinogenic agents, and carcinogenic stages, suggesting the ras proteins may be involved in different biological functions. Depending on the system, ras activation has been shown to be an early and/or a late event in the multi-step process of carcinogenesis. These data underscore the possible relationship between ras activation and cell type specificity, proliferation, differentiation or cell-cell interaction
— id: 13500, year: 1992, vol: 3, page: 229, stat: Journal Article,

Overexpression of the N-ras proto-oncogene, not somatic mutational activation, associated with malignant tumors in transgenic mice
Mangues R; Seidman I; Gordon JW; Pellicer A
1992 Oct;7(10):2073-2076, Oncogene
We have produced transgenic mice that carry a foreign gene construct consisting of the N-ras proto-oncogene driven by the mouse mammary tumor virus (MMTV) long terminal repeat. Overexpression of the normal N-ras gene is associated with development of hyperplasias and tumors in a variety of tissues. The tumors are clearly malignant, as evidenced by the presence of metastatic lesions. Extensive analysis of the foreign ras gene in these tumors by use of polymerase chain reaction and sequencing demonstrates in all cases the absence of somatically acquired mutations at those codons normally associated with activation of the ras genes. Thus, these tumors develop from overexpression of the proto-oncogene rather than the presence of the mutated oncogene. These data demonstrate that overexpression of a protooncogene of the ras family can predispose cells in vivo to fully malignant behavior
— id: 13431, year: 1992, vol: 7, page: 2073, stat: Journal Article,

A new cDNA sequence for the murine interleukin-2 gene
Matesanz F; Alcina A; Pellicer A
1992 Oct 20;1132(3):335-336, Biochimica & biophysica acta
We have amplified by PCR and sequenced the first exon of the interleukin 2 gene from the RF/J mouse strain DNA. When we compared the RF/J first exon sequence with the one reported previously, we found several differences. These differences are also reflected in the deduced amino acid sequence and they have been localized in the first 23 amino acids of the mature polypeptide. The finding of this new IL-2 sequence shows that there is more than one allele for the mouse IL-2 molecule and raises the possibility of functional differences between alleles
— id: 13393, year: 1992, vol: 1132, page: 335, stat: Journal Article,

THE EFFECTS OF NEUROTROPHIC FACTORS (NF) ON MORPHOLOGY AND CATECHOLAMINE LEVELS OF U-7 CELLS
SAEZ, GT; PELLICER, A; LEW, JY; TANG, D; GOLDSTEIN, M
1992 FEB 26 ;6(4):A1275-A1275, FASEB journal
— id: 52081, year: 1992, vol: 6, page: A1275, stat: Journal Article,

Activation of the ras oncogene in gamma radiation and neutron radiation induced thymic lymphomas
Sloan SR; Pellicer A
1992 ;374:1-18, Progress in clinical & biological research
— id: 13738, year: 1992, vol: 374, page: 1, stat: Journal Article,

Oncogene involvement in tumor regression: H-ras activation in the rabbit keratoacanthoma model
Corominas M; Leon J; Kamino H; Cruz-Alvarez M; Novick SC; Pellicer A
1991 Apr;6(4):645-651, Oncogene
Activated H-ras genes are present in a number of skin tumors induced in animals by carcinogen treatment. The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, benign and self-regressing tumors, as well as malignant squamous cell carcinomas. Both tumors were induced in rabbit ears by repeated applications of 7,12 dimethylbenz(a)anthracene (DMBA). The rabbit H-ras gene was cloned and sequenced. PCR analysis revealed that approximately 82% of the keratoacanthoma DNAs contained an A:T to T:A transversion in codon 61. The relative levels of H-ras transcript were increased in keratoacanthomas compared to normal skin and the activated allele was expressed in tumors, even during the regressing phase. Although a G:C to A:T mutation in codon 12 of the H-ras and an activated N-ras gene were found in two squamous cell carcinomas, the frequency of H-ras activation in codon 61 was much lower (40%) in the malignant tumours induced by the same carcinogen treatment. Therefore, DMBA induced at least two types of genetic lesions in this system: H-ras activation, present in most regressing keratoacanthomas, and activation of other unidentified oncogenes which may result in the development of malignant tumors. Our observations indicate that expression of an activated H-ras gene, in this system, is neither sufficient to induce a malignant phenotype nor even capable of maintaining the growth of a benign tumor and suggest that it could be involved in tumor regression
— id: 14088, year: 1991, vol: 6, page: 645, stat: Journal Article,

Differential expression of the normal and mutated K-ras alleles in chemically induced thymic lymphomas
Corominas M; Perucho M; Newcomb EW; Pellicer A
1991 Oct 1;51(19):5129-5133, Cancer research
The presence of point mutations in the K-ras gene was examined in murine thymic lymphomas induced by a single dose of N-methylnitrosourea by the RNase A mismatch cleavage method and by allelic-specific oligonucleotide hybridization of in vitro amplified DNA by polymerase chain reaction. The results show that the frequency of mutations is lower than that of tumors induced by multiple N-methylnitrosourea treatments. Four mutations identified were the aspartic acid at codon 12, a G:C to A:T transition in its second position. A G:C to T:A transversion in codon 146 was also found in one thymic lymphoma, changing the amino acid alanine to serine. The use of the RNase A assay allowed an estimation of the relative expression levels of both normal and mutant K-ras alleles. The results show that in approximately one half of the tumors the mutant allele is predominantly expressed, suggesting that the normal allele has been lost or that the mutant allele has been amplified relative to the normal. Altogether, these findings are consistent with ras mutations occurring in some instances during tumor development and with a ras effect being not strictly dominant but favoring selection for increasing levels of expression from the oncogenic allele
— id: 13892, year: 1991, vol: 51, page: 5129, stat: Journal Article,

ras activation in human tumors and in animal model systems
Corominas M; Sloan SR; Leon J; Kamino H; Newcomb EW; Pellicer A
1991 Jun;93:19-25, Environmental health perspectives
Environmental agents such as radiation and chemicals are known to cause genetic damage. Alterations in a limited set of cellular genes called proto-oncogenes lead to unregulated proliferation and differentiation. We have studied the role of the ras gene family in carcinogenesis using two different animal models. In one case, thymic lymphomas were induced in mice by either gamma or neutron radiation, and in the other, keratoacanthomas were induced in rabbit skin with dimethylbezanthracene. Human keratoacanthomas similar to the ones induced in rabbits were also analyzed. We found that different types of radiation such as gamma rays and neutrons, induced different point mutations in ras genes. A novel K-ras mutation in codon 146 has been found in thymic lymphomas induced by neutrons. Keratoacanthomas induced in rabbit skin by dimethylbenzanthracene show a high frequency of H-ras-activated genes carrying a mutation in codon 61. The same is observed in human keratoacanthomas, although mutations are in both the 12th and the 61st codons of the H-ras gene. H-ras activation is less frequent in human squamous cell carcinomas than in keratoacanthomas, suggesting that ras genes could play a role in vivo in differentiation as well as in proliferation
— id: 14011, year: 1991, vol: 93, page: 19, stat: Journal Article,

GENETIC ALTERATIONS OF THE P53 GENE ARE A FEATURE OF MALIGNANT MESOTHELIOMAS
Cote, RJ; Jhanwar, SC; Novick, S; Pellicer, A
1991 Oct 1;51(19):5410-5416, Cancer research
A putative tumor suppressor gene, p53, has been shown to be altered in a variety of human tumor types. The primary mechanism of p53 inactivation is believed to be mutation of one allele followed by loss of the second allele. Malignant mesothelioma is a tumor that has been highly associated with exposure to asbestos fibers, which are known to cause chromosomal abnormalities in mesothelial cells. We have examined four mesothelioma cell lines for genetic abnormalities in p53. Cytogenetic analysis revealed that two of the four tumors had abnormalities (numerical and/or structural) of chromosome 17 (the locus of the p53 gene). Restriction fragment length polymorphism analysis using a chromosome 17p- specific probe (pYNZ22) revealed that two tumors had loss of heterozygosity in the region of 17p13. The relative level of p53 mRNA expression was examined by Northern analysis, with one tumor showing negligible expression of p53 mRNA. The complementary DNA of p53 was generated from the three tumors showing detectable mRNA expression, and the region between codons 70 and 319 was amplified by the polymerase chain reaction and sequenced. DNA single-base substitutions were detected in two of the tumor cell lines, each resulting in amino acid substitutions. One tumor had an arginine to histidine substitution at position 175, and one tumor had a glycine to aspartic acid substitution at position 245. The observed mutations took place in regions of high cross-species sequence homology, indicating that these regions may be functionally important. The correlation of chromosomal loss in 17p on the cytogenetic and molecular level along with p53 mRNA expression and DNA sequence data indicate that genetic alterations in p53 could be a feature of malignant mesotheliomas and may reveal an important role of asbestos fibers in tumor suppressor gene inactivation
— id: 32150, year: 1991, vol: 51, page: 5410, stat: Journal Article,

Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element
Paciucci R; Pellicer A
1991 Mar;11(3):1334-1343, Molecular & cellular biology
The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene
— id: 14118, year: 1991, vol: 11, page: 1334, stat: Journal Article,

Staging of T-cell receptor beta chain gene rearrangements and ras oncogene mutations in the development of murine thymic lymphomas
Sloan SR; Pellicer A
1991 Mar 15;51(6):1627-1631, Cancer research
While it is known that the T-cell receptor beta chain gene is rearranged in fully developed murine thymic lymphomas induced by N-nitrosomethylurea and that the ras gene is activated in approximately 50% of these tumors (L. E. Diamond et al., Mol. Cell. Biol., 8: 2233-2236, 1988), it is unknown when these events occur or where the cells committed to a malignant phenotype are first located. We have studied these questions by treating mice with N-nitrosomethylurea, extracting thymocytes and bone marrow cells from the treated mice before they would have developed tumors, transferring the cells into recipient mice, monitoring those mice until they developed lymphoid tumors, and analyzing those tumors. This analysis showed that the initial cells committed to becoming malignant can be located in either the bone marrow or thymus and that both activation of the ras oncogene and rearrangements of the T-cell receptor gene can occur earlier than 30 days after N-nitrosomethylurea treatments. Furthermore, these results suggest that the T-cell receptor beta chain gene can undergo additional rearrangements during progression of a tumor
— id: 62166, year: 1991, vol: 51, page: 1627, stat: Journal Article,

Workshop report from the Division of Research Grants, National Institutes of Health. Molecular genetic approaches to the analysis of malignant transformation--a pathology B study section workshop
Furmanski P; Chisari F; Pellicer A; Fausto N; Padarathsingh M
1990 Jun 15;50(12):3805-3806, Cancer research
— id: 10198, year: 1990, vol: 50, page: 3805, stat: Journal Article,

CHARACTERIZATION OF UNR - A GENE CLOSELY LINKED TO N-RAS
Jeffers, M; Paciucci, R; Pellicer, A
1990 Aug 25;18(16):4891-4899, Nucleic acids research
— id: 31849, year: 1990, vol: 18, page: 4891, stat: Journal Article,

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells
Lacal, J C; Cuadrado, A; Jones, J E; Trotta, R; Burstein, D E; Thomson, T; Pellicer, A
1990 Jun;10(6):2983-2990, Molecular & cellular biology
Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional
— id: 134714, year: 1990, vol: 10, page: 2983, stat: Journal Article,

Tumorigenesis and male sterility in transgenic mice expressing a MMTV/N-ras oncogene
Mangues, R; Seidman, I; Pellicer, A; Gordon, J W
1990 Oct;5(10):1491-1497, Oncogene
Transgenic mice carrying the activated N-ras oncogene under the transcriptional control of the mouse mammary tumor virus (MMTV) long terminal repeat were produced. The transgene is expressed in a tissue distribution consistent with the fact that it is driven by the MMTV-LTR, and similarly to MMTV/H-ras constructs, its presence elicits tumors in Harderian, mammary and salivary glands. In addition it appears to compromise male reproductive function, which has not been described with the other ras transgenes. This finding is consistent with the existence of distinct physiological actions for each of the ras family members
— id: 116463, year: 1990, vol: 5, page: 1491, stat: Journal Article,

COMPARATIVE-ANALYSIS AND ANATOMIC DISTRIBUTION OF RAS P21, IL-2R, AND MEL-14 IN MALIGNANT AND HYPERPLASTIC MURINE THYMUS
NEWCOMB, EW; PELLICER, A; CORDON, C
1990 FEB ;136(2):307-317, American journal of pathology
— id: 98511, year: 1990, vol: 136, page: 307, stat: Journal Article,

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation
Sloan, S R; Newcomb, E W; Pellicer, A
1990 Jan;10(1):405-408, Molecular & cellular biology
Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA
— id: 134712, year: 1990, vol: 10, page: 405, stat: Journal Article,

Oncogene N-ras mediates selective inhibition of c-fos induction by nerve growth factor and basic fibroblast growth factor in a PC12 cell line
Thomson, T M; Green, S H; Trotta, R J; Burstein, D E; Pellicer, A
1990 Apr;10(4):1556-1563, Molecular & cellular biology
A cell line was generated from U7 cells (a subline of PC12 rat pheochromocytoma cells) that contains a stably integrated transforming mouse N-ras (Lys-61) gene under the control of the long terminal repeat from mouse mammary tumor virus. Such cells, designated UR61, undergo neuronal differentiation upon exposure to nanomolar concentrations of dexamethasone, as a consequence of expression of the activated N-ras gene (I. Guerrero, A. Pellicer, and D.E. Burstein, Biochem, Biophys. Res. Commun. 150:1185-1192, 1988). Exposure of UR61 cells to either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) results in a marked induction of c-fos RNA, with kinetics paralleling those of NGF- or bFGF-induced expression of c-fos RNA in PC12 cells. Dexamethasone-induced expression of activated N-ras p21 results in blocking of c-fos RNA induction by NGF or bFGF in a time-dependent manner. Activated N-ras p21-mediated inhibition of c-fos RNA induction in UR61 cells is selective for NGF and bFGF and is not due to selective degradation of c-fos RNA. Normal and transforming N-ras can trans activate the chloramphenicol acetyltransferase gene linked to mouse c-fos regulatory sequences when transient expression assays are performed. Our observations suggest that N-ras p21 selectively interacts with pathways involved in induction of c-fos expression which initiate at the receptors for NGF and bFGF
— id: 134713, year: 1990, vol: 10, page: 1556, stat: Journal Article,

POTENTIATION OF ONCOGENIC N-RAS-INDUCED NEURITE OUTGROWTH AND ORNITHINE DECARBOXYLASE ACTIVITY BY PHORBOL DIBUTYRATE AND PROTEIN-KINASE INHIBITOR H-8
Trotta, RJ; Thomson, TM; Lacal, JC; Pellicer, A; Burstein, DE
1990 Apr;143(1):68-78, Journal of cellular physiology
— id: 32088, year: 1990, vol: 143, page: 68, stat: Journal Article,

Oncogene activation in human benign tumors of the skin (keratoacanthomas): is HRAS involved in differentiation as well as proliferation?
Corominas M; Kamino H; Leon J; Pellicer A
1989 Aug;86(16):6372-6376, Proceedings of the National Academy of Sciences of the United States of America
In vitro DNA amplification followed by oligonucleotide mismatch hybridization was used to study the frequency of HRAS mutations in the benign self-regressing skin tumors keratoacanthomas and in squamous cell carcinomas. We used freshly obtained keratoacanthomas as well as Formalin-fixed paraffin-embedded tissues from both types of tumors. DNA from 50 samples of each tumor type was analyzed for activating mutations involving codons 12 and 61. A relatively high percentage (30%) of HRAS mutations was found in the keratoacanthomas compared with 13% in the squamous cell carcinomas. The most frequent mutation identified is the A-T-to-T.A transversion in the second position of codon 61. The present findings demonstrate the involvement of the HRAS oncogene in human benign tumors. Moreover, they indicate that an activated HRAS oncogene is not sufficient to maintain a neoplastic phenotype and argue against a role of HRAS in the progression of skin tumorigenesis
— id: 10536, year: 1989, vol: 86, page: 6372, stat: Journal Article,

Response: Angiotensin II: Does It Have a Direct Obligate Role in Ovulation?
Naftolin, F; Andrade-Gordon, P; Pellicer, A; Palumbo, A; Apa, R; Zreik, T; Ki Yoon, T; Decherney, A
1989 Aug 25;245(4920):871-871, Science
— id: 139700, year: 1989, vol: 245, page: 871, stat: Journal Article,

Angiotensin II: does it have a direct obligate role in ovulation?
Naftolin, F; Andrade-Gordon, P; Pellicer, A; Palumbo, A; Apa, R; Zreik, T; Yoon, T K; DeCherney, A
1989 Aug 25;245(4920):870-871, Science
— id: 102811, year: 1989, vol: 245, page: 870, stat: Journal Article,

Multistage carcinogenesis in murine thymocytes: involvement of oncogenes, chromosomal imbalances and T cell growth factor receptor
Newcomb EW; Corominas M; Bayona W; Pellicer A
1989 Sep-Oct;9(5):1407-1415, Anticancer research
An animal model of carcinogenesis has been exploited to analyze the various events involved in carcinogen-induced T cell lymphomagenesis. Two carcinogenic agents, the alkylating agent N-methylnitrosourea (NMU) and ionizing gamma-radiation, induce tumors in C57BL/6J mice that are phenotypically and histologically identical. Are the genetic events similar or different in the T cell tumors produced by these two carcinogenic agents? NMU treatment produced a different spectrum of activated oncogenes from gamma-irradiation. The K-ras oncogene was preferentially activated in all of the NMU-induced tumors, most frequently by a GGT to GAT transition in codon 12. Ionizing gamma-radiation produced two different transforming activities. Approximately half of the radiation-induced tumors contained activated N-ras genes and half contained a novel non-ras transforming activity. Analysis of NMU- and gamma-irradiated treated animals for chromosomal abnormalities showed anomalies early in the disease. Although both agents produce tumors containing trisomy of chromosome 15, the timing of this event appears to be different occurring early in NMU-induced tumors and later in gamma-radiation induced tumors. In addition, a unique marker chromosome consisting of a translocation between chromosomes one and five appears to be involved in the early stages of radiation-induced disease and may be associated with the novel transforming activity detected in these same tumors. Expression of receptors for the T cell growth factor (IL-2R) is similar in both NMU- and gamma-irradiation induced tumors. Changes in the expression of IL-2R on different T cell populations with disease progression may account for thymus dependent and thymus independent phases of malignant T cell growth
— id: 10506, year: 1989, vol: 9, page: 1407, stat: Journal Article,

Radiation and chemical activation of ras oncogenes in different mouse strains
Newcomb EW; Diamond LE; Sloan SR; Corominas M; Guerrerro I; Pellicer A
1989 May;81:33-37, Environmental health perspectives
A survey of a large series of radiation- or chemically induced thymic lymphomas in (AKR X RF)F1, RF/J, 129/J, and C57BL/6J mouse strains for activated ras oncogenes showed that of the tumors containing transforming activity, in more than 75% of the cases this activity segregated with either K-ras or the N-ras gene. H-ras activity was never detected. The genetic background of the host influenced susceptibility to tumor induction and oncogene activation. The K-ras gene was preferentially activated over the N-ras gene (approximately 2:1) whether the inducing agent was radiation or the chemical N-nitrosomethylurea. The activating mutation for the K-ras gene was consistently identified as a GGT to GAT transition in codon 12. In contrast, several different mutations of the N-ras gene were identified and localized to codons 12, 13, or 61. Assessment of the allelic composition of the ras locus shows that some proportion of the tumors lost the normal ras allele
— id: 10650, year: 1989, vol: 81, page: 33, stat: Journal Article,

Functional receptors for nerve growth factor on Ewing's sarcoma and Wilm's tumor cells
Thomson TM; Pellicer A; Greene LA
1989 Oct;141(1):60-64, Journal of cellular physiology
Quantification of changes in levels of c-fos RNA was used as an indicator of the presence of functional responses to nerve growth factor in several human non-neuronal cell lines which have previously been shown to express high levels of NGF receptors. Four Ewing's sarcomas, one Wilm's tumor, and one melanoma were examined. Of these cell lines, the Ewing's sarcoma IARC-EW1 showed greatly increased levels (10-20-fold) of c-fos RNA after 1 hour of exposure to NGF. Except for the melanoma line, the other tumor lines exhibited small, but reproducible, elevation of c-fos RNA expression. In IARC-EW1 cells, this induction was analyzed for kinetics, dose-response, and suppression by selective inhibitors of NGF action. The results indicate that these cells bear high-affinity receptors for NGF, which utilize signal pathways similar to NGF receptors on PC12 cells. Thus, we report new types of cells with functional responses to NGF and indicate that these may constitute a new model which will usefully complement those presently used for studying the mechanism of action of NGF
— id: 10484, year: 1989, vol: 141, page: 60, stat: Journal Article,

Concomitant K- and N-ras gene point mutations in clonal murine lymphoma
Diamond LE; Guerrero I; Pellicer A
1988 May;8(5):2233-2236, Molecular & cellular biology
We have surveyed a panel of induced murine lymphomas for c-ras gene mutations. The K-ras gene seems to be preferentially activated in our system, and there are at least two examples of concomitant K- and N-ras gene mutations in the same tumor. This indicates that in some cases additional ras mutations may contribute to tumorigenesis and is evidence for a role of ras activation in tumor progression
— id: 11109, year: 1988, vol: 8, page: 2233, stat: Journal Article,

T-CELL RECEPTOR GENE REARRANGEMENT IN PRIMARY TUMORS - EFFECT OF GENETIC BACKGROUND AND INDUCING AGENT
DIAMOND, LE; SLOAN, SR; PELLICER, A; HAYDAY, AC
1988 AUG ;28(2):71-80, Immunogenetics
— id: 98521, year: 1988, vol: 28, page: 71, stat: Journal Article,

DISSOCIATION OF C-FOS FROM ODC EXPRESSION AND NEURONAL DIFFERENTIATION IN A PC12 SUBLINE STABLY TRANSFECTED WITH AN INDUCIBLE N-RAS ONCOGENE
GUERRERO, I; PELLICER, A; BURSTEIN, DE
1988 FEB 15 ;150(3):1185-1192, Biochemical & biophysical research communications
— id: 41856, year: 1988, vol: 150, page: 1185, stat: Journal Article,

H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system
Leon J; Kamino H; Steinberg JJ; Pellicer A
1988 Feb;8(2):786-793, Molecular & cellular biology
The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis
— id: 11200, year: 1988, vol: 8, page: 786, stat: Journal Article,

Identification of a specific marker chromosome early in tumor development in gamma-irradiated C57BL/6J mice
McMorrow LE; Newcomb EW; Pellicer A
1988 Feb;2(2):115-119, Leukemia
Chromosomal changes such as aneuploidies, translocations, and gene amplification occur in many murine tumors. In this study, we have analyzed the changes in chromosomes at different stages of tumor development in C57BL/6J mice treated with gamma-irradiation or the chemical carcinogen, N-methylnitrosourea. Trisomy 15 occurred in both groups of mice regardless of inducing agent. The frequency of this event differed significantly in radiation-treated animals between stage I and stage II of the disease. A specific marker chromosome occurred only in the gamma-irradiated mice and not in the mice treated with N-methylnitrosourea. This marker consists of a translocation between chromosomes 1 and 5. It occurred in 43% of the gamma-irradiated animals at stage 1 of the disease and did not vary markedly during tumor development. In contrast, trisomy 15 increased in frequency between stages I and II of the disease in the same animals. These results suggest that the translocation event may be an early event in tumor development, whereas trisomy 15 may contribute to tumor progression
— id: 27817, year: 1988, vol: 2, page: 115, stat: Journal Article,

ras oncogenes and phenotypic staging in N-methylnitrosourea- and gamma-irradiation-induced thymic lymphomas in C57BL/6J mice
Newcomb EW; Steinberg JJ; Pellicer A
1988 Oct 1;48(19):5514-5521, Cancer research
We have investigated stages of thymic lymphoma development in radiation and N-methylnitrosourea (NMU)-treated C57BL/6J mice. The lymphoma cell was identified serologically as a cortical population bearing MEL-14hi, H-2Khi, and IL-2R+ surface markers. According to these parameters in C57BL/6J mice the lymphoma cell was the same regardless of inducing agent or activated oncogene (ras or non-ras). Transforming activity in the radiation and NMU-induced tumors was analyzed using both the nude mouse tumorigenicity assay and the focus-forming assay. 8/10 NMU-induced tumors and 12/15 radiation-induced tumors showed transforming activity in the tumorigenicity assay. Southern blot analysis of the nude mouse transformants demonstrated K-ras transforming sequences in eight of eight NMU-induced lymphoma DNAs, two of 12 radiation-induced lymphoma DNAs and N-ras transforming sequences in five of 12 radiation-induced lymphoma DNAs. The non-ras transforming activity in five DNAs from radiation-induced thymic lymphomas indicates the presence of an unidentified oncogene(s) in these tumors. Staging of thymic lymphoma development in this animal model system will allow the study of oncogene activation early in the course of carcinogen-induced disease. These results also emphasize the high sensitivity of the nude mouse assay to score for activated oncogenes and might also indicate a high frequency of K-ras activation in NMU-induced lymphomas in C57BL/6J mice
— id: 10954, year: 1988, vol: 48, page: 5514, stat: Journal Article,

Follicular development is impaired by inhibitors of serine proteases in the rat
Pellicer, A; Lightman, A; Ariza, A; DeCherney, A H; Naftolin, F; Littlefield, B A
1988 Mar;158(3 Pt 1):670-676, American journal of obstetrics & gynecology
Serine proteases such as plasminogen activators are produced by granulosa cells both in vivo and in vitro and have been implicated in the process of ovulation. For a study of potential roles of serine proteases in early follicular development, immature rats were injected with pregnant mare serum gonadotropin, followed 2 hours later by laparotomy and injection of the serine protease inhibitors benzamidine and epsilon-aminocaproic acid into the bursa of one ovary. As a control, saline solution was injected into the contralateral bursa. Animals were put to death 48 hours after injection of serine protease inhibitors, and three to five randomly selected longitudinal sections were evaluated by computerized morphometry. The area occupied by antral follicles relative to the total cross-sectional area of each section was computed. Resultant ratios from serine protease inhibitor-treated ovaries were compared with those from contralateral control ovaries. Ninety-three percent of serine protease inhibitor-treated ovaries showed a reduction in antral follicular size when compared with corresponding control ovaries, which is indicative of inhibitory effects of serine protease inhibitor treatment on folliculogenesis. To further investigate this effect, ovulation was induced by human chorionic gonadotropin administration 48 hours after pregnant mare serum gonadotropin and 46 hours after serine protease inhibitor or saline solution treatment. Animals were put to death 20 hours later and the number of oocytes ovulated into oviducts was determined. Oviducts from serine protease inhibitor-treated ovaries contained 51% fewer oocytes than their control counterparts. Artifacts of surgical stress or vascular diffusion of serine protease inhibitor from treated to control sides were ruled out by appropriate control experiments. We conclude that early serine protease inhibitor treatment of pregnant mare serum gonadotropin-stimulated rat ovaries impairs folliculogenesis. Thus, in addition to involvement in ovulation, serine proteases appear to play important roles throughout follicular development
— id: 102801, year: 1988, vol: 158, page: 670, stat: Journal Article,

Blockage of ovulation by an angiotensin antagonist
Pellicer, A; Palumbo, A; DeCherney, A H; Naftolin, F
1988 Jun 17;240(4859):1660-1661, Science
Angiotensin II (Ang II) is present in high concentrations in preovulatory follicular fluid, and ovarian follicular cells have specific Ang II receptors. To investigate the possible direct involvement of Ang II in ovulation the specific receptor antagonist of Ang II, saralasin, was administered by intraperitoneal injection to immature rats in which follide development and ovulation had been induced with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG), respectively. Saralasin halved the number of oocytes found in the fallopian tubes 17 to 20 hours after administration of hCG. The antiovulatory effect was observed when saralasin was given 1 hour before hCG or 1 or 3 hours after hCG but not when given 5 hours after hCG. Simultaneous administration of Ang II reversed the saralasin blockage of ovulation. These results indicate a direct, obligate role for Ang II in ovulation and raise the possibility of contraceptive and profertility applications for agonists or antagonists of the renin-angiotensin system that are aimed at the ovulatory process
— id: 139688, year: 1988, vol: 240, page: 1660, stat: Journal Article,

INDUCTION OF ORNITHINE DECARBOXYLASE ACTIVITY (ODC) BY ONCOGENE N-RAS IN A RAS-RESPONSIVE NEURODIFFERENTIATING CELL-LINE
TROTTA, R; GUERRERO, I; THOMSON, T; PELLICER, A; BURSTEIN, D
1988 MAR 15 ;2(4):A805-A805, FASEB journal
— id: 41814, year: 1988, vol: 2, page: A805, stat: Journal Article,

ISOLATION OF A CELL-LINE WHICH CEASES DIVISION AND DIFFERENTIATES IN RESPONSE TO TRANSFECTED ONCOGENE N-RAS
Burstein, DE; Guerrero, I; Trotta, RJ; Pellicer, A
1987 Mar;28(3):47-47, Proceedings (American Association for Cancer Research)
— id: 31213, year: 1987, vol: 28, page: 47, stat: Journal Article,

Mouse N-ras genes: organization of the functional locus and of a truncated cDNA-like pseudogene
Chang HY; Guerrero I; Lake R; Pellicer A; D'Eustachio P
1987 Jul;1(2):129-136, Oncogene research
The N-ras gene was first identified in both humans and mice as a mutationally activated oncogene found in tumors. The nucleotide sequence and intron/exon structure of the coding region of the mouse gene have been determined previously. We have now determined the sequence and intron/exon structure of the 5' and 3' untranslated regions of mouse N-ras. Like its human homolog, the 3' untranslated region of the gene is encoded by two exons, and the 5' region is encoded by one. In addition, we have isolated and sequenced a second mouse gene homologous to N-ras. This locus, which we have named N-ras-2ps, resides at a chromosomal site distinct from N-ras and appears to be a truncated cDNA-like pseudogene
— id: 11391, year: 1987, vol: 1, page: 129, stat: Journal Article,

Cloning of a full-length complementary DNA for an Artemia salina glycine-rich protein. Structural relationship with RNA binding proteins
Cruz-Alvarez M; Pellicer A
1987 Oct 5;262(28):13377-13380, Journal of biological chemistry
Overlapping cDNAs have been isolated containing all the coding sequences for Artemia salina protein GRP33, a glycine-rich protein (16.6 mol % glycine), with a molecular weight of 32,992. GRP33 is closely related to HD40, the major protein component of Artemia heterogeneous nuclear ribonucleoprotein particles, and shares certain characteristics with other RNA binding proteins. The C-terminal region (123 amino acids) contains 39 glycine residues. This region has multiple arginine residues flanked by glycines, resembling the glycine-dimethylarginine clusters present in other RNA binding proteins. Secondary structure predictions for the protein reveal two distinct domains: a hydrophilic C-terminal domain with an extended conformation and a larger N-terminal domain with a number of alpha-helices and beta-sheets
— id: 11345, year: 1987, vol: 262, page: 13377, stat: Journal Article,

DIFFERENTIAL EXPRESSION OF SURFACE-MARKERS ON THYMIC LYMPHOMAS INDUCED BY 2 CARCINOGENIC AGENTS IN DIFFERENT MOUSE STRAINS
DIAMOND, LE; BERMAN, JW; PELLICER, A
1987 JUN ;107(1):115-120, Cellular immunology
— id: 41701, year: 1987, vol: 107, page: 115, stat: Journal Article,

GENETIC MECHANISMS IN TUMOR INITIATION AND PROGRESSION .4. MUTATIONAL ACTIVATION OF ONCOGENES IN ANIMAL-MODEL SYSTEMS OF CARCINOGENESIS
Guerrero, I; Pellicer, A
1987 May;185(3):293-308, Mutation research. DNA repair reports
— id: 31176, year: 1987, vol: 185, page: 293, stat: Journal Article,

DIFFERENTIAL EXPRESSION OF THE RAS GENE FAMILY IN MICE
Leon, J; Guerrero, I; Pellicer, A
1987 Apr;7(4):1535-1540, Molecular & cellular biology
— id: 31252, year: 1987, vol: 7, page: 1535, stat: Journal Article,

MOLECULAR AND GENETIC-HETEROGENEITY IN ACID ALPHA GLUCOSIDASE DEFICIENCY
Martiniuk, F; Meredith, G; Mehler, M; Pellicer, A; Hirschhorn, R
1987 Apr;35(3):A650-A650, Clinical research
— id: 31204, year: 1987, vol: 35, page: A650, stat: Journal Article,

CHROMOSOME CHANGES IN X-RAY AND NMU-TREATED C57BL/6J MICE AT DIFFERENT STAGES OF TUMOR-DEVELOPMENT
Mcmorrow, LE; Newcomb, EW; Pellicer, A
1987 Apr;82(4):100-100, Journal of cellular biochemistry
— id: 31228, year: 1987, vol: 82, page: 100, stat: Journal Article,

Intraovarian markers of follicular and oocyte maturation
Pellicer, A; Diamond, M P; DeCherney, A H; Naftolin, F
1987 Aug;4(4):205-217, Journal of in vitro fertilization & embryo transfer
The use of ovulation induction for multiple follicular growth in in vitro fertilization (IVF) has introduced the problem of follicular asynchrony. As a consequence of the asynchrony, the parameters most commonly used by IVF groups to assess follicular and oocyte quality within those follicles are not sufficiently sensitive or specific. Thus, each follicle must be considered separately, and specific markers of follicular and/or oocyte maturation must be sought from within the follicle. In this review we analyze previous reports of potential markers of follicular and oocyte maturation. In regards to the follicular fluid constituents, the level of estradiol in follicular fluid correlates with fertilization and pregnancy in stimulated cycles. Other steroids are only helpful when specific stimulation protocols are used. The level of some follicular proteins such as alpha-1-antitrypsin and fibrinogen also correlates with fertilization and pregnancy outcome. Cyclic AMP levels in follicular fluid are significantly reduced in follicles leading to conception. Regulators of oocyte maturation, such as the Oocyte Maturation Inhibitor (OMI) or the Meiosis Inducing Substance (MIS) have also been correlated with IVF outcome, but their exact structure remains still unknown. In addition, other sophisticated parameters, such as chemotactic activity of human leukocytes, or simple methods, such as the presence of intrafollicular echoes, have also been used as successful markers in predicting IVF outcome
— id: 102793, year: 1987, vol: 4, page: 205, stat: Journal Article,

ACTIVATED N-RAS GENE INDUCES NEURONAL DIFFERENTIATION OF PC12 RAT PHEOCHROMOCYTOMA CELLS
BURSTEIN, DE; GUERRERO, I; WONG, H; PELLICER, A
1986 MAR 5 ;45(4):938-938, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 41496, year: 1986, vol: 45, page: 938, stat: Journal Article,

ONCOGENES AND SURFACE-MARKERS IN MOUSE-TUMORS
GUERRERO, I; VILLASANTE, A; DIAMOND, L; BERMAN, J; NEWCOMB, E; MCMORROW, L; STEINBERG, JJ; PELLICER, A
1986 MAR ;49(3):540-541, International journal of radiation biology
— id: 41477, year: 1986, vol: 49, page: 540, stat: Journal Article,

ONCOGENE ACTIVATION AND SURFACE-MARKERS IN MOUSE LYMPHOMAS INDUCED BY RADIATION AND NITROSOMETHYLUREA
GUERRERO, I; VILLASANTE, A; DIAMOND, L; BERMAN, JW; NEWCOMB, EW; STEINBERG, JJ; LAKE, R; PELLICER, A
1986 AUG ;10(7):851-858, Leukemia research
— id: 41567, year: 1986, vol: 10, page: 851, stat: Journal Article,

ACTIVATED N-RAS GENE INDUCES NEURONAL DIFFERENTIATION OF PC12 RAT PHEOCHROMOCYTOMA CELLS
GUERRERO, I; WONG, H; PELLICER, A; BURSTEIN, DE
1986 OCT ;129(1):71-76, Journal of cellular physiology
— id: 41550, year: 1986, vol: 129, page: 71, stat: Journal Article,

Isolation of a cDNA for human acid alpha-glucosidase and detection of genetic heterogeneity for mRNA in three alpha-glucosidase-deficient patients
Martiniuk F; Mehler M; Pellicer A; Tzall S; La Badie G; Hobart C; Ellenbogen A; Hirschhorn R
1986 Dec;83(24):9641-9644, Proceedings of the National Academy of Sciences of the United States of America
Lysosomal acid alpha-glucosidase (EC 3.2.1.3) hydrolyzes 1,4-linked alpha-D-glucose polymers present in glycogen. Genetic deficiency of acid alpha-glucosidase results in glycogen-storage disease type II, encompassing a spectrum of disorders of varying severity. To study the molecular basis for this heterogeneity, we sought to clone the coding sequence for human acid alpha-glucosidase. We screened 10(6) recombinant phage from a human liver cDNA expression library with an affinity-purified polyclonal antibody to human acid alpha-glucosidase. When we retested positive phage for reactivity to monoclonal antibodies, we identified a single phage, containing a 2-kilobase (kb) cDNA insert, that reacted with both polyclonal and monoclonal antibodies. The 2-kb cDNA hybridized to a 20-kb EcoRI fragment of human genomic DNA. This 20-kb EcoRI fragment was present only in DNA from somatic cell hybrids that retained the human chromosome 17 segment q21-q23, which contains the gene for human acid alpha-glucosidase. The cDNA also hybridized to a 3.4-kb mRNA, consistent with the size (approximately 105 kDa) of the acid alpha-glucosidase protein. Finally, in one of two infantile-onset acid alpha-glucosidase-deficient cell lines tested, the 3.4-kb mRNA was not detectable, whereas in an adult-onset cell line, an mRNA of reduced size and amount was found. Examination of DNA digested with restriction enzymes did not reveal any major deletions in the genomic DNA of these patients
— id: 15208, year: 1986, vol: 83, page: 9641, stat: Journal Article,

Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes
Martiniuk F; Pellicer A; Mehler M; Hirschhorn R
1986 Jan;12(1):1-12, Somatic cell & molecular genetics
We cotransformed mouse 3T3 cells with total genomic human DNA and the dominant selectable bacterial gene Neo and analyzed 121 NeoR clones for expression of 15 human 'housekeeping' enzymes which can be distinguished from their murine homologs. The estimated frequency of expression of unlinked human genes was 1 in 360 NeoR clones and at least three different human enzymes (peptidase D, phosphoglucomutase 1, and acid alpha glucosidase) were detected. We further examined the frequency and stability of cotransformation for one of these enzymes, acid alpha glucosidase (GAA). We tested approximately 4000 NeoR clones and found 25 clones expressing human GAA, as determined by rocket immunoelectrophoresis (RIE) specific for human GAA. Transformants progressively became negative on continued growth and retesting by RIE, with only two clones still expressing GAA at the eighth testing. This apparent loss of expression was not due to nonclonality of the original isolates. In one subclone examined, loss of expression was accompanied by loss of both Neo-derived pBR322 and human Alu repetitive sequence DNA. Thus, under the conditions utilized, cotransformants expressing homomeric housekeeping enzymes were found at relatively high frequency but were progressively lost even under conditions selective for expression of the dominant vector
— id: 15209, year: 1986, vol: 12, page: 1, stat: Journal Article,

ISOLATION OF A CDNA FOR HUMAN ACID ALPHA-GLUCOSIDASE AND DETECTION OF GENETIC-HETEROGENEITY FOR MESSENGER-RNA IN 2 PATIENTS DEFICIENT FOR ALPHA-GLUCOSIDASE
MARTINIUK, F; MEHLER, M; PELLICER, A; TZALL, S; LABADIE, G; HIRSCHHORN, R
1986 DEC ;25(4):710-711, American journal of medical genetics
— id: 41328, year: 1986, vol: 25, page: 710, stat: Journal Article,

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution
Cruz-Alvarez M; Szer W; Pellicer A
1985 Jun 11;13(11):3917-3930, Nucleic acids research
A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved
— id: 17472, year: 1985, vol: 13, page: 3917, stat: Journal Article,

ACTIVATION OF C-RAS ONCOGENES IS ASSOCIATED WITH TUMOR- INDUCTION IN EXPERIMENTAL-ANIMALS
Guerrero, I; Villasante, A; Corces, V; Diamond, L; Altman, R; Pellicer, A
1985 ;32(1):72-85, Progress in medical virology
— id: 30879, year: 1985, vol: 32, page: 72, stat: Journal Article,

LOSS OF THE NORMAL N-RAS ALLELE IN A MOUSE THYMIC LYMPHOMA INDUCED BY A CHEMICAL CARCINOGEN
GUERRERO, I; VILLASANTE, A; CORCES, V; PELLICER, A
1985 ;82(23):7810-7814, Proceedings of the National Academy of Sciences of the United States of America
— id: 41178, year: 1985, vol: 82, page: 7810, stat: Journal Article,

Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells
Martiniuk F; Pellicer A; Hirschhorn R
1985 Nov 15;260(26):14351-14354, Journal of biological chemistry
To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a 'rocket' which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection
— id: 15210, year: 1985, vol: 260, page: 14351, stat: Journal Article,

SUCCESSFUL TRANSIENT EXPRESSION OF ENZYME-ACTIVITY BY CELLS TRANSFECTED WITH TOTAL GENOMIC AND RECOMBINANT PHAGE DNA
Martiniuk, F; Pellicer, A; Hirschhorn, R
1985 ;33(2):A604-A604, Clinical research
— id: 30934, year: 1985, vol: 33, page: A604, stat: Journal Article,

Gene transfer in lymphoid cells: expression of the Thy-1.2 antigen by Thy-1.1 BW5147 lymphoma cells transfected with unfractionated cellular DNA
Berman JW; Basch RS; Pellicer A
1984 Nov;81(22):7176-7179, Proceedings of the National Academy of Sciences of the United States of America
We have transferred the gene coding for the Thy-1.2 alloantigen into a Thy-1.1-bearing T-cell lymphoma. BALB/c thymocyte DNA, precipitated with calcium phosphate, was used to effect the transfer. We report the stable transformation of lymphoid cells by total cellular DNA. To our knowledge, this has not been previously reported. Transient expression of the transfected gene could be detected by flow cytofluorometry, and 5% (range, 1.5%-11%) of the recipient cells had Thy-1.2 antigen detectable on their surface 48 hr after transfer. 'Stable' transformants were isolated by repeatedly selecting for cells expressing the Thy-1.2 antigen, by use of fluorescence-activated cell sorting or 'panning.' No metabolic selection was required. The transferred gene, detected by Southern blotting, encoded a product that is indistinguishable from the normal antigen by immunoprecipitation and NaDod-SO4/PAGE
— id: 14370, year: 1984, vol: 81, page: 7176, stat: Journal Article,

EXPRESSION OF CLONED VACCINIA VIRUS-DNA SEQUENCES INTRODUCED INTO ANIMAL-CELLS
BONI, C; ESTEBAN, M; PELLICER, A
1984 ;65(JUL):1245-1251, Journal of general virology
— id: 40919, year: 1984, vol: 65, page: 1245, stat: Journal Article,

IDENTIFICATION OF SEQUENCES INVOLVED IN THE TRANSCRIPTIONAL CONTROL OF A DROSOPHILA HEAT-SHOCK GENE
CORCES, V; PELLICER, A
1984 ;259(23):4812-4817, Journal of biological chemistry
— id: 41037, year: 1984, vol: 259, page: 4812, stat: Journal Article,

Isolation, characterization, and chromosome assignment of mouse N-ras gene from carcinogen-induced thymic lymphoma
Guerrero I; Villasante A; D'Eustachio P; Pellicer A
1984 Sep 7;225(4666):1041-1043, Science
Treatment of mice with the carcinogen N-methylnitrosourea results in the development of thymic lymphomas with frequent involvement of the N-ras oncogene. The activated mouse N-ras gene was isolated from one of these lymphomas and, by transformation in concert with restriction digestion, a map of the gene was prepared and its approximate boundaries were determined. By means of somatic cell hybrids the normal N-ras gene was found to be unlinked to other members of the ras gene family
— id: 17266, year: 1984, vol: 225, page: 1041, stat: Journal Article,

A MOLECULAR APPROACH TO LEUKEMOGENESIS - MOUSE LYMPHOMAS CONTAIN AN ACTIVATED C-RAS ONCOGENE
GUERRERO, I; CALZADA, P; MAYER, A; PELLICER, A
1984 ;81(1):202-205, Proceedings of the National Academy of Sciences of the United States of America
— id: 41121, year: 1984, vol: 81, page: 202, stat: Journal Article,

ACTIVATION OF A C-K-RAS ONCOGENE BY SOMATIC MUTATION IN MOUSE LYMPHOMAS INDUCED BY GAMMA-RADIATION
GUERRERO, I; VILLASANTE, A; CORCES, V; PELLICER, A
1984 ;225(4667):1159-1162, Science
— id: 41060, year: 1984, vol: 225, page: 1159, stat: Journal Article,

DETECTION AND FREQUENCY OF DNA MEDIATED TRANSFORMANTS EXPRESSING NON-SELECTABLE HUMAN INTRACELLULAR ENZYMES
MARTINIUK, F; PELLICER, A; HIRSCHHORN, R
1984 ;32(2):A549-A549, Clinical research
— id: 40983, year: 1984, vol: 32, page: A549, stat: Journal Article,

Murine leukemia virus sequences are encoded in the murine major histocompatibility complex
Meruelo D; Kornreich R; Rossomando A; Pampeno C; Mellor AL; Weiss EH; Flavell RA; Pellicer A
1984 Mar;81(6):1804-1808, Proceedings of the National Academy of Sciences of the United States of America
The studies reported here localize murine leukemia viral sequences to the TL region of the major histocompatibility complex, H-2. We examined a battery of 38 cosmids, isolated from two large genomic libraries constructed from C57BL/10 spleen DNA, that define 25 class I gene sequences. The viral probes used hybridized with only four cosmids, containing overlapping mouse sequences, that define four class I gene-related sequences in a region of 90 kilobases of DNA. The data show that two distinct viral envelope sequences are contained in the cluster. One of these sequences is situated with its 3' end next to the 3' end of a class I sequence. The other sequence, which does not contain the entire viral envelope, is proximal to the 3' end of a different class I sequence. Hybridization of the viral probes with the H-2 cosmid clones does not appear to be due to homology between viral and H-2 sequences. Rather, the viral sequences detected appear to be linked to or inserted amid class I genes. These findings may be significant in understanding molecular mechanisms involved in the generation of H-2 class I gene diversity
— id: 15257, year: 1984, vol: 81, page: 1804, stat: Journal Article,

Association of endogenous viral loci with genes encoding murine histocompatibility and lymphocyte differentiation antigens
Meruelo D; Rossomando A; Offer M; Buxbaum J; Pellicer A
1983 Aug;80(16):5032-5036, Proceedings of the National Academy of Sciences of the United States of America
Several polymorphic DNA restriction endonuclease fragments hybridizing with xenotropic and ecotropic envelope virus probes map adjacent to minor histocompatibility and lymphocyte (H/Ly) antigen-encoding loci. Viral DNA restriction fragments are associated with Ly-17 on chromosome 1, H-30, H-3, and H-13 on chromosome 2, Ly-21 on chromosome 7, H-28 on chromosome 3, and H-38 (chromosomal location as yet undetermined). In each case no recombinant can be found between the H/Ly locus in question and the virus-related restriction fragment, suggesting that linkage is very tight. Although some viral loci map to locations where no H/Ly has yet been mapped, the frequency and tightness of linkage in the seven instances described, coupled with the large number of as yet unmapped H/Ly loci, suggests that the associations found are significant
— id: 15258, year: 1983, vol: 80, page: 5032, stat: Journal Article,

GENE-TRANSFER, STABILITY, AND BIOCHEMICAL-PROPERTIES OF ANIMAL-CELLS TRANSFORMED WITH VACCINIA DNA
PELLICER, A; ESTEBAN, M
1982 ;122(2):363-380, Virology
— id: 40370, year: 1982, vol: 122, page: 363, stat: Journal Article,