Ras p21 proteins with high or low GTPase activity can efficiently transform NIH3T3 cells (original) (raw)
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Activation of a human c-K-ras oncogene
Nucleic Acids Research
The human lung carcinomas PR310 and PR371 contain activated c-K-ras oncogenes. The oncogene of PR371 was found to present a mutation at codon 12 of the first coding exon which substitutes cysteine for glycine in the encoded p21 protein. We report here that the transforming gene of PR310 tumor contains a mutation in the second coding exon. An A-+T transversion at codon 61 results in the incorporation of histidine instead of glutamine in the c-K-ras gene product. By constructing c-K-ras/c-H-ras chimeric genes we show that this point mutation is sufficient to confer transforming potential to ras genes, and that a hybrid ras gene coding for a protein mutant at both codons 12 and 61 is also capable of transforming NIH3T3 cells. The relative transforming potency of p21 proteins encoded by ras genes mutant at codons 12, 61 or both has been analyzed. Our studies also show that the coding exons of ras genes, including the fourth, can be interchanged and the chimeric p21 ras proteins retain their oncogenic ability in normal rodent established cell lines.
Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation
Molecular and Cellular Biology
Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer c...
Detection of ras oncogenes by analysis of p21 proteins in human tumor cell lines
Urological Research, 1988
To detect mutationally activated ms oncogenes, we analyzed electrophoretic mobilities of ras p21 proteins utilizing the fact that many ms oncogenes produce abnormal p21 proteins that migrate at SDS/polyacrylamide gel electrophoresis as a fast-moving or slow-moving species in comparison to a normal p21 depending on the kind of mutation. Of 18 human tumor cell lines analyzed, four (SW480, SW620 and SW403 colon cancers, and SW626 ovary cancer) produced p21 belonging to the slow-moving species, suggesting a point mutation within codon 12 of a member of the three ms genes, H-, Ki-and N-ms. Subsequent DNA transfection analysis using NIH/3T3 cells as recipients identified activated Ki-ras oncogenes in the same four but not in other 14 cell lines. Thus, the analysis of p21 might serve as a rapid primary method to screen a large number of tumor materials for the presence of certain types of mutationally activated ras oncogenes.
Antisense inhibition of ras p21 expression that is sensitive to a point mutation
Biochemistry, 1991
Many genetic disorders result from a single point mutation, and many tumor oncogenes have been found to be altered by a point mutation. The ability to inhibit selectively the expression of the mutated form of a protein without affecting its normal counterpart is central to many therapeutic strategies, since the normal protein may serve indispensable functions. Antisense oligonucleoside methylphosphonates and their psoralen derivatives directed at either normal human H a m s p21 or ras p21 that is mutated at a single base in codon 61 have been examined for their efficacy and specificity as inhibitors of p21 expression. Mixed cultures of cells expressing both forms of p21 were treated with the antisense oligomer complementary to the normal p21 or with the antisense oligomer complementary to the point-mutated p21. Each of the antisense oligomers specifically inhibited expression of only the form of ras p21 to which it was completely complementary and left the other form of p21 virtually unaffected. ras p21 proteins resemble G proteins in sequence homology and function and have been considered as potential intermediaries in the signal transduction pathway. Point mutations appear to be mainly responsible for the association of ras with the initiation and development of many human neoplasias. A point mutation in the 12th or 61st amino acid codon of c-Ha-rus has been found in numerous chemical-and radiation-induced tumors as well as in naturally occurring malignant mammalian tumors. These mutations are found to occur in regions of the protein that are GTP/GDP binding domains (Bishop, 1987; Weinberg, 1988; Barbacid, 1987, 1990). Amplification of the ras genes and overexpression of the ras gene product, p2 1, have been detected in many human tumors derived from different tissue types (Heighway & Hasleton, 1986).
Mechanism of activation of an N-ras oncogene of SW-1271 human lung carcinoma cells
Proceedings of the National Academy of Sciences, 1984
An N-ras-related transforming gene was detected in the human lung carcinoma cell line SW-1271 and molecularly cloned. The lesion responsible for its acquisition of transforming activity was localized to a single nucleotide transition from A to G in codon 61 of the predicted protein. This lesion in the second exon results in the substitution of arginine for glutamine at this position. These findings, together with previous studies, indicate that the activation of ras oncogenes in human tumors is most commonly due to point mutations at one of two major "hot spots" in the ras coding sequence.
Blood, 1990
DNA from 161 patients with various forms of hematologic malignancies were investigated for mutations in exons 1 and 2 of the N-RAS. K-RAS and Ha-RAS gene by direct sequencing of DNA amplified in vitro by the polymerase chain reaction. Mutations involving either codons 11,12, or 13 of the N-RAS gene were identified in 18 of the 161 patients. The relative frequencies of N-RAS gene mutations in these hematologic disorders was as follows: acute myelogenous leukemia (AML), 25%; acute lymphoblastic leukemia (ALL), 14%; myelodysplastic syndromes, 24%; and myeloid and lymphoid blast crisis of chronic myelogenous leukemia (CML), 3%. No correlation was observed between the presence of mutations and cytologic features or immunophenotype of these malignancies. Mutations
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.