Rescue of cells from ras oncogene-induced growth arrest by a second, complementing, oncogene (original) (raw)
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Cell growth & differentiation: the molecular biology journal of the American Association for Cancer Research
Microinjection of the anti-ras antibody Mab Yl 3-259 modifies ras function and can induce temporary reversion of the transformed phenotype in mutant rastransformed cells. Intracellular production of neutralizing antibodies represents an approach to investigate the regulation of gene fundion. The genes coding for the heavy and light chains of Mab Yl 3-259 were isolated from a cDNA library. NIH3T3 cells transfeded with heavy and light chain expression vedors produced fundional anti-ras antibody. The produdion of functional antibody did not require glycosylation. To ensure that the antibody entered the cytoplasm and not the secretory pathway, the hydrophobic leader sequences of both chains were removed and replaced with synthetic initiator sequences. The modified heavy chain gene was cloned under the control of the murine sarcoma virus long terminal repeat, and the light chain gene under the control of the mouse mammary tumor virus long terminal repeat, which allows the indudion of light chain expression in the presence of dexamethasone. When both heavy and light chain genes were expressed in cells with activated ras (morphologically transformed) in the presence of dexamethasone, we observed phenotypic reversion to charaderistics of nontransformed cells. These experiments show that intracellular expression of antibodies can also be used as an alternative to analyze biological fundions of a given protein.
Molecular and cellular biology, 1986
Normal rat fibroblasts of the established cell line Rat 4 were cotransformed with activated human ras oncogenes and with a cloned chicken thymidine kinase (tk) gene. Linkage between tk and ras genes allowed the isolation of oncogene deletion revertants and of cell clones showing varying degrees of malignant phenotype. Southern and Northern experiments in concert with tumorigenicity assays show that the malignant transformation of these cells by mutant ras oncogenes is a gradual but reversible process that depends on the relative abundance of oncogene sequences and their corresponding transcripts. We also show that moderate amplification of a c-K-ras oncogene in these cells results in a clear increase in their tumorigenicity and that the mutant gene present in low copy numbers in cultured cells undergoes amplification in the corresponding in vivo induced tumors.
Inhibition of Ras oncogenic activity by Ras protooncogenes
International Journal of Cancer, 2005
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.
Complex effects of Ras proto-oncogenes in tumorigenesis
Carcinogenesis, 2003
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 protooncogenes 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.
Cellular ras gene activity is required for full neoplastic transformation by polyomavirus
Journal of Virology
To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.
Cancer Research
Four different human tissue-derived cell lines, each previously shown to express either a Ha-, Ki-, or N-ras-activated oncogene, were fused in four different paired combinations. The three combinations that involved the tumor line HT1080 (activated N-ras oncogene) were found to be tumorigenic in nude mice, but to different degrees. However, the fusion of the tumor lines EJ and SW480 (activated Ha-ras and Ki-ras, respectively) resulted in hybrid cells suppressed for tumorigenicity. The EJ x SW480 hybrids were found to harbor and express both of the activated ras oncogenes. The results suggest that tumorigenic suppression can occur in the presence of two transforming oncogenes of the ras family and that tumorigenicity associated with ras oncogene activation involves additional mechanisms that may differ among tumor cells.
Cooperative transformation of 32D cells by the combined expression of IRS-1 and V-Ha-Ras
Oncogene, 2000
32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of ®broblasts, we asked whether expression of IRS-1 or Shc (two of the major substrates of the IGF-1R) could co-operate with oncogenic Ras in transforming 32D cells. We ®nd that IRS-1, but not Shc, in combination with v-Ha-Ras generates a fully transformed phenotype in 32D cells. 32D cells expressing both IRS-1 and v-Ha-Ras (32D/IRS1/Ras) survive and proliferate in the absence of IL-3, do not undergo granulocytic dierentiation in the presence of G-CSF and form tumors in nu/nu and syngeneic mice. In contrast, 32D cells expressing singly IRS-1 or v-Ha-Ras exhibit only a block in dierentiation capacity. Over-expression of Shc proteins, by itself, promotes dierentiation of 32D cells. Concomitant expression of IRS-1 and v-Ha-Ras synergistically phosphorylates ERK-1 and ERK-2 whereas a MEK inhibitor rapidly induces death of 32D/IRS1/Ras transformed cells. Furthermore, transformed 32D/IRS1/Ras cells display high levels of PI3-K activation and undergo rapid apoptosis when exposed to PI3-K inhibitors. The data indicate that: (1) a fully transformed phenotype in 32D cells is generated when a block in dierentiation (v-Ha-Ras) is coupled with another dierentiation block (IRS-1); (2) PI3-K and MAPK activity are required for the survival of transformed cells; (3) the signals generated by IRS-1 and oncogenic Ras converge on ERK and PI3-K resulting in high levels of activation. Oncogene 19, 3245 ± 3255. IRS-1 permits transformation of 32D cells by oncogenic Ras B Cristofanelli et al 3246 Oncogene IRS-1 permits transformation of 32D cells by oncogenic Ras B Cristofanelli et al IRS-1 permits transformation of 32D cells by oncogenic Ras B Cristofanelli et al IRS-1 permits transformation of 32D cells by oncogenic Ras B Cristofanelli et al IRS-1 permits transformation of 32D cells by oncogenic Ras B Cristofanelli et al 3254 Oncogene
A molecular approach to leukemogenesis: mouse lymphomas contain an activated c-ras oncogene
Proceedings of the National Academy of Sciences, 1984
By inducing mouse thymomas with carcinogens and y-radiation, we have studied the potential of tumor DNA to induce foci in rodent fibroblasts. A high percentage of the tumors used transformed the cultured cells, and the oncogenic phenotype segregated with extra copies of the c-ras gene family. There appears to be selectivity in the activated gene because so far all analyzed tumors induced by carcinogen have activated the N-ras gene, and those induced by radiation have activated the K-ras gene. The K-ras gene is the cellular counterpart of the viral ras oncogene in Kirsten murine sarcoma virus, but the N-ras has not yet been found in a retrovirus. The transformed cells have a marked increase in expression of the oncogene at the RNA and protein level. This model system might be a powerful tool in the study of leukemogenesis.