Complex effects of Ras proto-oncogenes in tumorigenesis
Roberto Diaz1,
Lluis Lopez-Barcons2,*,
Daniel Ahn1,*,
Antonio Garcia-Espana3,
Andrew Yoon1,
Jeremy Matthews1,
Ramon Mangues4,
Roman Perez-Soler2 and
Angel Pellicer1,5
1 Department of Pathology and NYU Cancer Institute, New York University School of Medicine, New York, NY 10016, USA, 2 Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA, 3 Endocrinology Research Unit, Joan XXIII University Hospital, Rovira i Virgili University School of Medicine, Dr Mallafre i Guasch 4, Tarragona, Spain and 4 Laboratori d'Investigació Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, E-08025 Barcelona, Spain
 |
Abstract
|
---|
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.
Abbreviations: GTP, guanosine triphosphate; MCA, 3-methylcholanthrene; MMTV-LTR, mammary murine tumor virus-long terminal repeat; MNU, N-methylnitrosourea
 |
Introduction
|
---|
There are three ras genes (H-ras, N-ras and K-ras) involved in human and animal tumors, and these genes encode four highly related proteins of 21 kDa in size that are ubiquitously expressed: H-Ras, N-Ras, K-RasA and K-RasB (13). Ras is localized in membranes regulating a variety of differentiation processes and signal transduction by commanding the activation of effectors that mediate cell proliferation, vesicle movement, cell survival, senescence, apoptosis and T-cell activation (1,48). Ras proto-oncogenes can quickly operate as molecular switches between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. The amount of GTP-bound Ras is controlled by the actions of two classes of regulators: the guanine nucleotide exchange factors, which activate Ras by facilitating the exchange of GDP for GTP; and the GTPase-activating proteins (GAPs), which deactivate Ras by stimulating the intrinsic Ras GTPase activity (1,3,9).
By interacting with different effectors, Ras is involved in the regulation of opposing activities: promotion of apoptosis through the Raf/MAPK pathway (10) and through the NORE1/MST1 pathway (11), and inhibition of apoptosis through the PI3K/AKT pathway (12). Likewise, the analyses of the reports in the literature have demonstrated that Ras proto-oncogenes exhibit complex roles in tumorigenesis. There are some reports that indicate that the lack of wild-type Ras can promote tumorigenesis (1315); on the other hand, other reports demonstrate that the absence of wild-type Ras can inhibit tumorigenesis (16,17). Furthermore, there are studies that show that over-expression of Ras proto-oncogenes are able to suppress tumorigenesis (14,18); paradoxically, there are other reports that indicate that over-expression of Ras proto-oncogenes are also able to promote tumorigenesis (1923).
Interestingly, these reports have focused on the effects the different Ras isoforms have on a variety of tumors. It has been noted that the level of oncogenic ras and the stromal microenvironment are able to substantially influence tumorigenicity (24). The possibility exists that ras proto-oncogenes are able to mediate opposing effects in a manner dependent of its regulation of carcinogen metabolism or the stromal cell microenvironment. In order to clarify these seemingly contradictory observations, we have focused on one Ras isoformN-Rasand its effects in two systems: fibrosarcomas and B-cell lymphomas.
 |
Materials and methods
|
---|
Mouse lines and genotype determinations
The mouse lines used in this work include: the N-ras knockout (25); the N-ras proto-oncogene under the whole mammary murine tumor virus-long terminal repeat (MMTV-LTR) [line 5; (19,26,27)]; and the N-ras oncogene under the control of a truncated MMTV-LTR [line A; (26)]. We crossed line A with line 5 and obtained an F1 that included a line we termed A5, in which the N-ras oncogene is co-expressed with high levels of the N-ras proto-oncogene. The animals were monitored and analyzed for tumor appearance as described previously (26). Mice that did not show any visible signs of tumor development were killed after 54 weeks of age.
Genomic DNA was extracted from mouse toes as described previously (28). The genotypes of all mouse lines were determined as described previously (14). Care for the animals in this study was performed in accord with our institution and NIH guidelines.
Carcinogen treatments
Wild-type and line 5 male mice, 57 weeks of age, were given i.p. injections once a week for 5 weeks with 30 mg/kg of N-methylnitrosourea (MNU) dissolved in PBS, as described previously (19). Wild-type and N-ras knockout male mice, 68 weeks of age, were injected s.c. in the back with 1 mg of 3-methylcholanthrene (MCA) (Sigma, St Louis, MO) dissolved in 0.1 ml of olive oil (Sigma) as described previously (14,29). The mice were killed when the tumor volume reached 1 cm3.
Cell culture and tumorigenicity assays
The 4G21 N-ras-/- tumor cell line (14) was maintained in 10% fetal bovine serum (Invitrogen, Carlsbad, CA) in RPMI 1640 (Cell Gro, Herndon, VA) without antibiotics and incubated in 5% CO2 and 95% air at 37°C in a humidified incubator. This cell line was assayed with a Mycoplasma Plus PCR primer set (Stratagene, La Jolla, CA) and found to be free of mycoplasma. The tumorigenicity assays were performed as described previously (30). Subconfluent cultures (5070%) were harvested by a short treatment with Trypsin (Invitrogen). The cells were washed in supplemented medium and then resuspended in plain RPMI medium. Only single-cell suspensions of >90% viability, as determined by Trypan blue dye exclusion (Invitrogen), were used. Tumor cell suspensions were prepared at concentrations of 5 x 105 or 5 x 106 viable cells/ml. Single inoculations in a final volume of 0.1 ml were performed s.c. in the back using a hypodermic needle. Tumor growth was monitored twice a week, and tumor volumes were calculated from caliper measurements of two orthogonal diameters and using the formula: volume = (1/2)xy2, where x is the large diameter and y is the small diameter. The mice were killed after the tumor volume reached <1 cm3.
Statistical analyses
All statistical tests used in these studies are two-sided log-rank statistical test, except for the tumorigenesis assays where the statistical test used is the Student's t-test.
 |
Results
|
---|
Presence of wild-type N-Ras can enhance tumorigenesis in fibrosarcomas
To determine if N-Ras has a cooperative, neutral or inhibitory effect in the development of fibrosarcomas, we injected wild-type and N-ras-/- mice with the MCA carcinogen. We found that the wild-type mice show a higher incidence of tumor development than the knockout mice (80 versus 40%, P < 0.005), suggesting that the presence of the wild-type N-ras gene product appears to promote tumorigenesis compared with animals lacking the gene (Figure 1). The greater potency of the MCA carcinogen in wild-type mice could be attributable to N-Ras-dependent differences in the metabolism of MCA, in the tumor cells and/or in the stromal cells.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1. The presence of N-ras proto-oncogene produces an increase in MCA-induced fibrosarcomas. Ten wild-type and 10 N-ras-/- male mice, 7 weeks of age, were treated with MCA as described previously (14,29). Fibrosarcomas induced by MCA were more frequent in wild-type than in N-ras-/- mice (80 and 40%, respectively). These differences in incidence are significant (P < 0.005). There were no differences in tumor latency: 20.7 (±4.2) weeks for wild-type and 20.7 (±3.7) weeks for N-ras-/- mice.
|
|
In order to provide mechanistic insights into these results, we injected both N-ras+/+ and N-ras-/- mice with different doses of a highly tumorigenic cell line that had been derived from these tumors (14) and monitored for the incidence and latency of tumors (Table I). A shorter latency was associated with the presence of N-ras alleles (P < 0.01), although there were no significant differences in the final incidence of tumorigenesis, indicating that there are N-Ras-dependent differences in the stromal cells. These two independent results suggest that the presence of N-ras in mice could enhance the susceptibility to the in vivo formation of certain tumors via a mechanism dependent upon the microenvironment of the stromal cells.
View this table:
[in this window]
[in a new window]
|
Table I. Wild-type male mice are more susceptible to tumorigenesis than N-ras-/- mice after injecting different doses of MCA-induced fibrosarcomas in an N-ras null backgrounda
|
|
Over-expression of the N-Ras proto-oncogene enhances B-cell lymphomagenesis
In thymic lymphomagenesis induced by either MNU or the N-ras oncogene, we reported that the lack of N-ras in mice increases the susceptibility to tumor formation and that the over-expression of N-ras protects against the development of these types of tumors (14). In the formation of fibrosarcomas, we found that the presence of N-ras is associated with the promotion of tumorigenesis (Figure 1). Therefore, we investigated if over-expressing the N-ras proto-oncogene can cooperate in the formation of other types of tumors. Our laboratory has characterized previously a transgenic mouse line, line 5, that expresses the N-ras proto-oncogene >10-fold under the control of the MMTV-LTR (19,26,27). The wild-type and line 5 mice were treated with MNU and were monitored for the formation of lymphomas. It was originally determined that over-expression of the N-ras proto-oncogene did not affect the incidence of lymphomas induced by MNU (19), but after focusing on thymic lymphoma induction, we reported a significant decrease in thymic lymphomagenesis when the N-ras proto-oncogene was over-expressed (14). Interestingly, it was observed that high expression of wild-type N-ras cooperated with MNU in the formation of B-cell lymphomas (Figure 2). We found that after MNU treatment, the line 5 mice developed B-cell lymphomas with an incidence of 21.2% wheras the wild-type mice had an incidence of just 4.8% (P < 0.05).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2. Over-expression of wild-type N-ras cooperates with MNU in the induction of B-cell lymphomas. Wild-type and line 5 mice were treated with MNU as described previously (19). The transgenic mice from line 5 express the N-ras proto-oncogene at high amounts (19,26,27). Line 5 mice treated with MNU developed B-cell lymphomas with an incidence of 21.2% and a latency of 40.9 (±14.1) weeks. Treating wild-type mice with MNU resulted in the induction of B-cell lymphomas with an incidence of 4.8% and a latency of 43.5 (±0.7) weeks. These differences in incidence are significant (P < 0.05).
|
|
In order to confirm and expand the promoting role of wild-type N-ras over-expression in the formation of B-cell lymphomas, these types of tumors were induced by another approach, the N-ras oncogene. Our laboratory has also characterized a transgenic mouse line, line A, that expresses the N-ras oncogene at low levels under the control of the truncated MMTV-LTR (26). Line A and line 5 were crossed, producing line A5, a transgenic line that expresses low levels of the N-ras oncogene with high levels of the N-ras proto-oncogene. Analysis of the types of tumors that these animals developed revealed no significant differences in the overall incidence of lymphomas between lines A and A5 (82.1 and 88.2%, respectively). However, after separating lymphomas of T- and B-cell origins, a significant decrease in the formation of thymic lymphomas was noted in line A5 (14). In contrast, a >2-fold increase in the incidence of the B-cell lymphomas was found in line A5 when compared with lines A (P < 0.001, Figure 3). By inducing B-cell lymphomas with two distinct approaches, we have shown that over-expression of the N-ras proto-oncogene is able to enhance the malignant phenotype in these types of tumors.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3. B-cell lymphomagenesis induced by the N-ras oncogene is enhanced by high over-expression of the N-ras proto-oncogene. We crossed line A, a transgenic mouse line that expresses the N-ras oncogene at low levels (26), with line 5, producing line A5. Line A produced B-cell lymphomas with an incidence of 35.9% and a latency of 39.5 (±11.5) weeks. Line A5 produced B-cell lymphomas with an incidence of 67.6% and a latency of 33.7 (±9.6) weeks. The differences in incidence between lines A and A5 are significant (P < 0.001).
|
|
 |
Discussion
|
---|
The reports in the literature and our results presented here show that Ras proto-oncogenes exhibit complex roles in tumorigenesis (Tables II and III). There are studies that show that the lack of endogenous Ras could either enhance or inhibit the malignant phenotype (Table II). For instance, there are reports that indicate that the presence of endogenous Ras could inhibit lung tumors and thymic lymphomas (13,14). Paradoxically, other studies suggest that the absence of Ras proto-oncogenes inhibits malignant properties (16,17).
In experiments using transgenic animals that over-express the different Ras proto-oncogenes, the results are just as complex (Table III). For example, some studies indicate that over-expression of Ras proto-oncogenes leads to an enhancement in the malignant phenotype (1923). Nonetheless, Thompson et al. (18) have reported that the over-expression of H-Ras and K-Ras in rats treated with MNU resulted in a decrease in the incidence of mammary tumors. These results are in direct contradiction to those of another group (20,22). Thompson et al. (18) attribute these inter-study differences to the different promoters involved in the generation of the transgenic rats, to probable differences in the protein's expression levels, and/or to differences in the timing of ras gene expression in the rat mammary cells.
We have two reasons to believe that our conclusions could be generalized to other systems in which the N-ras gene is involved. First, the experiments in this manuscript, used previously, generated mouse lines [described in reference (26)] in which we studied the effect of clonal variability. For each transgene, we generated several mouse lines. Clonal variability induced only quantitative differences in tumor incidence or latency without qualitatively changing the nature of the tumor phenotypes. Moreover, in some transgenic lines we used a truncated construct, missing a 5' fragment of the promoter that contains an enhancer, leading to diminished transgene expression and tumor incidence, but, again, no qualitative changes were observed (26). Secondly, in the same mouse model (line 5), the expression of a unique transgene was able to generate three different tumor types (T-cell lymphomas, B-cell lymphomas and mammary gland carcinomas) in which N-ras wild-type had opposite effects depending on the tissue. Thus, we observed that, after crossing this line with mice expressing the N-ras oncogene or after carcinogen treatment, the wild-type N-ras gene had a positive effect on B-cell lymphomagenesis (Figures 2 and 3) or mammary tumorigenesis (19) whereas it had a negative effect on T-cell lymphomagenesis (14).
Interestingly, N-ras wild-type over-expression, in combination with genetic inactivation of a negative regulator of N-Ras (the GAP protein NF1) leads to similar effects to its combination with N-ras oncogene expression or with carcinogens (31). Both combinations lead to an increased incidence of B-cell lymphomas (this manuscript) and mammary tumors (19,31), associated with enhanced activation of the N-Ras downstream ERK pathway (31). In contrast, while N-ras wild-type over-expressing mice had a low incidence of T-cell lymphomas, we did not detect any T-cell lymphoma in NF1+/-/N-ras+/+ mice (31).
These findings indicate that depending on the type of tissue, Ras proto-oncogenes appear to either promote or inhibit the tumor phenotype. Interestingly, treating N-ras-/- mice with MNU (14) and MCA (Figure 1) also led to different outcomes in thymic lymphomas (which involve T cells) and in fibrosarcomas (which involve fibroblasts), respectively. These differences could be attributed to the possibility that N-ras could mediate a specific signal that would favor tumor development in fibroblasts and B cells after treatment with MCA and MNU, respectively. The greater potency of MCA in wild-type mice could be attributable to N-ras-dependent differences in the tumor cells, in the metabolism of MCA, and/or in the stromal cells. Elenbaas et al. (24) have noted that the efficiency or latency of tumor formation in a human breast cancer cell line is modulated by the tumor microenvironment. The results shown in Table I, where the lack of N-ras in the host mice affects tumor progression after injecting lower doses of a tumorigenic cell line, indicate that there are N-ras-dependent differences in the stromal cells. The possibility then exists that N-ras in host cells could mediate a specific signal that would facilitate tumor growth in these tissues.
Alternatively, N-ras could be playing a unique and specific tumor suppressor role in T cells so that its absence enhances and its high over-expression inhibits thymic lympomagenesis induced by MNU or the N-ras oncogene (14). Interestingly, the opposite is observed with B-cell lymphomas (Figures 2 and 3) and mammary carcinomas (19). This is not surprising since it has been reported that oncogenic N-ras induces apoptosis in thymocytes in vivo, yet transforms other hematopoietic cell types (32). The possibility exists that the N-ras proto-oncogene could act through the same pathways as the N-ras oncogene for tumorigenesis (26) in B-cell lymphomas and mammary carcinomas. The mechanisms via which endogenous N-ras is able to suppress the malignant phenotype in T cells and not in B cells, fibroblasts or mammary epithelial cells still remain to be fully elucidated, but it is evidence for tissue-specific functions for the Ras isoforms.
All these paradoxical observations reflect the many different functions of the Ras molecule: Ras has been shown to regulate and promote cellular proliferation and to induce cells to undergo differentiation, senescence or apoptosis [reviewed in (1,2,58)]. The knowledge generated by the present studies should help to elucidate the complex functions of ras genes in tumorigenesis and should open the possibility to manipulate Ras expression levels as a therapeutic strategy in specific tumor types.
 |
Notes
|
---|
5 To whom correspondence should be addressed Email: pellia01{at}med.nyu.edu 
* These authors contributed equally to this paper. 
 |
Acknowledgments
|
---|
We thank T.Tunney for excellent technical assistance. R.D. was supported in part by training grant T32 CA09161. R.M. was also supported by the Spanish Ministry of Science and Technology through grants SAF03-07437 and by the Spanish Ministry of Health through grants FIS C03/10 and FIS 01/0853. This work was supported by grant CA 36327 from the National Institutes of Health.
 |
References
|
---|
- Malumbres,M. and Pellicer,A. (1998) RAS pathways to cell cycle control and cell transformation. Front. Biosci., 3, d887912.[Medline]
- Adjei,A.A. (2001) Blocking oncogenic Ras signaling for cancer therapy. J. Natl Cancer Inst., 93, 10621074.[Abstract/Free Full Text]
- Barbacid,M. (1987) ras genes. Annu. Rev. Biochem., 56, 779827.[CrossRef][ISI][Medline]
- Bos,J.L. (1997) Ras-like GTPases. Biochim. Biophys. Acta, 1333, M1931.[CrossRef][ISI][Medline]
- Spandidos,D.A., Sourvinos,G., Tsatsanis,C. and Zafiropoulos,A. (2002) Normal ras genes: their onco-suppressor and pro-apoptotic functions (Review). Int. J. Oncol., 21, 237241.[ISI][Medline]
- Campbell,S.L., Khosravi-Far,R., Rossman,K.L., Clark,G.J. and Der,C.J. (1998) Increasing complexity of Ras signaling. Oncogene, 17, 13951413.[CrossRef][ISI][Medline]
- Rebollo,A. and Martinez,A.C. (1999) Ras proteins: recent advances and new functions. Blood, 94, 29712980.[Free Full Text]
- Ayllon,V. and Rebollo,A. (2000) Ras-induced cellular events (review). Mol. Membr. Biol., 17, 6573.[CrossRef][ISI][Medline]
- Khosravi-Far,R., Campbell,S., Rossman,K.L. and Der,C.J. (1998) Increasing complexity of Ras signal transduction: involvement of Rho family proteins. Adv. Cancer Res., 72, 57107.[ISI][Medline]
- Kauffmann-Zeh,A., Rodriguez-Viciana,P., Ulrich,E., Gilbert,C., Coffer,P., Downward,J. and Evan,G. (1997) Suppression of c-Myc-induced apoptosis by Ras signalling through PI (3)K and PKB. Nature, 385, 544548.[CrossRef][ISI][Medline]
- Khokhlatchev,A., Rabizadeh,S., Xavier,R., Nedwidek,M., Chen,T., Zhang,X.F., Seed,B. and Avruch,J. (2002) Identification of a novel Ras-regulated proapoptotic pathway. Curr. Biol., 12, 253265.[CrossRef][ISI][Medline]
- Kennedy,S.G., Wagner,A.J., Conzen,S.D., Jordan,J., Bellacosa,A., Tsichlis,P.N. and Hay,N. (1997) The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev., 11, 701713.[Abstract]
- Zhang,Z., Wang,Y., Vikis,H.G. et al. (2001) Wildtype Kras2 can inhibit lung carcinogenesis in mice. Nature Genet., 29, 2533.[CrossRef][ISI][Medline]
- Diaz,R., Ahn,D., Lopez-Barcons,L. et al. (2002) The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene. Cancer Res., 62, 45144518.[Abstract/Free Full Text]
- James,R.M., Arends,M.J., Plowman,S.J., Brooks,D.G., Miles,C.G., West,J.D. and Patek,C.E. (2003) K-ras proto-oncogene exhibits tumor suppressor activity as its absence promotes tumorigenesis in murine teratomas. Mol. Cancer Res., 1, 820825.[Abstract/Free Full Text]
- Ise,K., Nakamura,K., Nakao,K., Shimizu,S., Harada,H., Ichise,T., Miyoshi,J., Gondo,Y., Ishikawa,T., Aiba,A. and Katsuki,M. (2000) Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis. Oncogene, 19, 29512956.[CrossRef][ISI][Medline]
- Liu,M.L., Shibata,M.A., Von Lintig,F.C., Wang,W., Cassenaer,S., Boss,G.R. and Green,J.E. (2001) Haploid loss of Ki-ras delays mammary tumor progression in C3 (1)/SV40 Tag transgenic mice. Oncogene, 20, 20442049.[CrossRef][ISI][Medline]
- Thompson,T.A., Haag,J.D., Lindstrom,M.J., Griep,A.E., Lohse,J.K. and Gould,M.N. (2002) Decreased susceptibility to NMU-induced mammary carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter. Oncogene, 21, 27972804.[CrossRef][ISI][Medline]
- Mangues,R., Kahn,J.M., Seidman,I. and Pellicer,A. (1994) An overexpressed N-ras proto-oncogene cooperates with N-methylnitrosourea in mouse mammary carcinogenesis. Cancer Res., 54, 63956401.[Abstract]
- Asamoto,M., Ochiya,T., Toriyama-Baba,H., Ota,T., Sekiya,T., Terada,M. and Tsuda,H. (2000) Transgenic rats carrying human c-Ha-ras proto-oncogenes are highly susceptible to N-methyl-N-nitrosourea mammary carcinogenesis. Carcinogenesis, 21, 243249.[Abstract/Free Full Text]
- Ota,T., Asamoto,M., Toriyama-Baba,H., Yamamoto,F., Matsuoka,Y., Ochiya,T., Sekiya,T., Terada,M., Akaza,H. and Tsuda,H. (2000) Transgenic rats carrying copies of the human c-Ha-ras proto-oncogene exhibit enhanced susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine bladder carcinogenesis. Carcinogenesis, 21, 13911396.[Abstract/Free Full Text]
- Tsuda,H., Asamoto,M., Ochiya,T., Toriyama-Baba,H., Naito,A., Ota,T., Sekiya,T. and Terada,M. (2001) High susceptibility of transgenic rats carrying the human c-Ha-ras proto-oncogene to chemically-induced mammary carcinogenesis. Mutat. Res., 477, 173182.[ISI][Medline]
- Asamoto,M., Toriyama-Baba,H., Ohnishi,T., Naito,A., Ota,T., Ando,A., Ochiya,T. and Tsuda,H. (2002) Transgenic rats carrying human c-Ha-ras proto-oncogene are highly susceptible to N-nitrosomethylbenzylamine induction of esophageal tumorigenesis. Jpn. J. Cancer Res., 93, 744751.[ISI][Medline]
- Elenbaas,B., Spirio,L., Koerner,F., Fleming,M.D., Zimonjic,D.B., Donaher,J.L., Popescu,N.C., Hahn,W.C. and Weinberg,R.A. (2001) Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev., 15, 5065.[Abstract/Free Full Text]
- Umanoff,H., Edelmann,W., Pellicer,A. and Kucherlapati,R. (1995) The murine N-ras gene is not essential for growth and development. Proc. Natl Acad. Sci. USA, 92, 17091713.[Abstract]
- Mangues,R., Symmans,W.F., Lu,S., Schwartz,S. and Pellicer,A. (1996) Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis. Oncogene, 13, 10531063.[ISI][Medline]
- Mangues,R., Seidman,I., Pellicer,A. and Gordon,J.W. (1990) Tumorigenesis and male sterility in transgenic mice expressing a MMTV/N-ras oncogene. Oncogene, 5, 14911497.[ISI][Medline]
- Malumbres,M., Mangues,R., Ferrer,N., Lu,S. and Pellicer,A. (1997) Isolation of high molecular weight DNA for reliable genotyping of transgenic mice. Biotechniques, 22, 11141119.[ISI][Medline]
- Niwa,O., Enoki,Y. and Yokoro,K. (1989) Overexpression and amplification of the c-myc gene in mouse tumors induced by chemicals and radiations. Jpn. J. Cancer Res., 80, 212218.[ISI][Medline]
- Llorens,A., Rodrigo,I., Lopez-Barcons,L., Gonzalez-Garrigues,M., Lozano,E., Vinyals,A., Quintanilla,M., Cano,A. and Fabra,A. (1998) Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinase expression. Lab. Invest., 78, 11311142.[ISI][Medline]
- Mangues,R., Corral,T., Lu,S., Symmans,W.F., Liu,L. and Pellicer,A. (1998) NF1 inactivation cooperates with N-ras in in vivo lymphogenesis activating Erk by a mechanism independent of its Ras-GTPase accelerating activity. Oncogene, 17, 17051716.[CrossRef][ISI][Medline]
- MacKenzie,K.L., Dolnikov,A., Millington,M., Shounan,Y. and Symonds,G. (1999) Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice. Blood, 93, 20432056.[Abstract/Free Full Text]
Received August 21, 2003;
revised November 10, 2003;
accepted November 11, 2003.