Oncogene alterations in in vitro transformed rat tracheal epithelial cells.

10 derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 nontumorigenic cell line transformed in vitro by treatment with 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for oncogene alterations. No abnormalities of Ha-ras or Ki-ras were seen that were suggestive of amplification, rearrangement or the presence of RFLPs. Analysis of specific-point mutations in Ha-ras using Pst I digestion (codon 12, GGA to GCA) or Ha-ras and Ki-ras using Xba I (codon 61, CAA to CTA) were negative. In one cell line derived by DMBA treatment, changes in the c-myc restriction digest pattern were seen after incubation with Bam HI and Hind III. Northern analysis revealed consistent differences between normal and transformed cells when probed with Ha-ras; c-myc expression was of low intensity, and the expression of Ki-ras could not be detected. Transfection of RTE cell DNAs into NIH/3T3 cells did not result in the appearance of morphologic transformants. The studies suggest that Ha-ras or Ki-ras codon 61 A to T transversions (CAA to CTA) are not associated with the immortal/tumorigenic phenotype in RTE cells transformed by DMBA or TPA, and are in contrast to results reported in some other biological systems.     

Presence of specific mutation of Ha-ras oncogene in skin tumors of mice induced under different experimental conditions

The mutation was revealed with substitution of A for T in the second position of the 61 Ha-ras oncogene codon in the DNA of 31 skin tumours (26 papillomas and 5 carcinomas) and in 23 mice treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA). Part of these mice were F progeny (n-6) and F progeny (n-4) following DMBA administration during pregnancy, another part F progeny (n-5) following ENU action on males prior to mating, the rest mice (n-3) did not undergo additional actions. The mutation under study was revealed only in 3 out of 5 papillomas and in all 5 carcinomas of mice subjected to DMBA administration during pregnancy.     

Absence of mutations in codon 61 of the Ha-ras oncogene in epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene

Epithelial cells of the respiratory tract of rats were transformed in vitro by 7,12-dimethylbenz(a)anthracene (DMBA) which has been reported to cause A----T transversion mutations of the second position of Ha-ras in codon 61 in several biological models. In this study Ha-ras exon 2 was amplified by the polymerase chain reaction (PCR) and then sequenced directly. In 10 transformed cell lines, of which 5 are known to be tumorigenic, no mutations in codon 61 were found. The results suggest that Ha-ras codon 61 mutations are not associated with cell transformation initiated with DMBA in this particular cell transformation system. These data imply that other genes (oncogenes) are responsible for transformation of these cells. The results are discussed in relation to observations in various transformation systems in vivo and in vitro.     

Oncogene alterations in in vitro transformed rat tracheal epithelial cells

10 derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 nontumorigenic cell line transformed in vitro by treatment with 7,12-dimethyl-benz[a]anthracene (DMBA), benzo[a]pyrene (BP) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for oncogene alterations. No abnormalities of Ha-ras were seen that were suggestive of amplification, rearrangement or the presence of RFLPs. Analysis of specific-point mutations in Ha-ras using Pst I digestion (codon 12, GGA to GCA) or Ha-ras and Ki-ras using Xba I (codon 61, CAA to CTA) were negative. In one cell line derived by DMBA treatment, changes in the c-myc restriction digest pattern were seen after incubation with Bam HI and Hind III. Northern analysis revealed consistent differences between normal and transformed cells when probed with Ha-ras; c-myc expression was of low intensity, and the expression of Ki-ras could not be detected. Transfection of RTE cell DNAs into NIH/3T3 cells did not result in the appearance of morphologic transformants. The studies suggest that Ha-ras or Ki-ras codon 61 A to T transversions (CAA to CTA) are not associated with the immoral/tumorigenic phenotype in RTE cells transformed by DMBA or TPA, and are in contrast to results reported in some other biological systems.     

The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.

We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is also detected in one Ki-ras allele of the MDA-MB231 cell line.     

High susceptibility of transgenic rats carrying the human c-Ha-ras proto-oncogene to chemically-induced mammary carcinogenesis

A rat line carrying three copies of the human c-Ha-ras proto-oncogenes, including its own promoter region, was established and designated as Hras128. Expression of the transgene was detected in all organs by Northern blot analysis. To examine its influence on susceptibility to mammary carcinogenesis, female rats were treated with N-methyl-N-nitrosourea (MNU) or 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age. With MNU, all the transgenic rats rapidly developed multiple mammary carcinomas within as short as 8 weeks (14.1 tumors/rat), in contrast to 0.46 tumors/rat in non-transgenic rats. PCR-RFLP analysis and direct sequencing for the transgene indicated that the large majority of carcinomas (38/44, 86.4%) contained cells with mutations at codon 12 in exon 1. However, comparison of the signal densities of the mutated band to dilution scale bands revealed that the cells with the mutated transgene were not in the majority. By PCR-SSCP analysis for codons 12 and 61 of the rat endogenous c-Ha-ras gene, no mutations were detected. Similarly, with DMBA, almost all (13/14, 92.9%) the transgenic rats developed multiple mammary carcinomas (9.39 tumors/rat) within 16 weeks, and 4 out of 12 (33.3%) non-transgenic rats had only small tumors (0.83 tumors/rat). A lower incidence of mutation of the transgene was found in codon 12 (5/25, 25%) than in MNU-induced tumors, but mutations were detected in codon 61 (7/20, 35%). No mutations were detected in the rat endogenous gene. No mutation was found in the rat endogenous c-Ha-ras gene in non-transgenic rats. As observed in both the MNU- and DMBA-induced tumor cases, the population of cells with the mutated transgene were in the minority. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene. Furthermore, irrespective of the mechanism of enhanced susceptibility, the Hras128 transgenic rats can be utilized for the screening of mammary carcinogens.     

An abasic site analogue activates a c-Ha-ras gene by a point mutation at modified and adjacent positions.

Synthetic c-Ha-ras genes with an analogue of an abasic site in the first or the second position of codon 12, or in the second position of codon 61 were constructed and transfected into NIH3T3 cells. The genes with the lesions in codon 12 exhibited more focus formation than a normal c-Ha-ras gene, while the gene with the lesion in codon 61 did not. Transformed cells were isolated from the foci, and the c-Ha-ras genes present in the transformants were analysed. A point mutation to A in the modified position was found most frequently in the cases of ras genes modified in codon 12. Surprisingly, point mutations in the adjacent position were also detected. These results indicate that dTMP, and not dAMP, was mainly incorporated into the sites opposite to the abasic site analogue, and that incorrect deoxynucleotides were incorporated in the position adjacent to the abasic site analogue.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N(2)dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early preneoplastic mutations. In conclusion, this study suggests that depurinating adducts play a major role in PAH mutagenesis.     

Three different mutations in codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization

The activation of ras genes in naturally occurring tumors has, thus far, been found to be due to mutations in codon 12 or 61 resulting in single amino acid substitutions. We have used highly labeled synthetic oligonucleotides to detect mutations in these codons and to determine the exact position of the mutation. Using this approach we have found three different mutations in codon 61 of the N-ras gene of various human tumor cell lines. In the fibrosarcoma line HT1080 the first nucleotide of the codon is mutated; in the promyelocytic line HL60 the second and in the rhabdomyosarcoma line RD301 the third nucleotide. For RD301 this implies that the normal glutamine residue at position 61 is replaced by histidine. In addition to the mutated N-ras gene the three cell lines have a normal N-ras gene which is indicative of the dominant character of the mutations.     

A human gastric carcinoma contains a single mutated and an amplified normal allele of the Ki-ras oncogene.

The DNA from various human tumors and tumor cell lines was screened for the presence of mutated ras oncogenes with synthetic oligonucleotide probes, as well as with the NIH/3T3 cell transfection assay. Among the various mutations found we discovered two novel Ki-ras mutations in codon 12: gly to ala and gly to ser. A gastric carcinoma was found to possess a single mutated Ki-ras allele (gly-12 to ser), as well as a 30-50 fold amplified normal allele. This implies that two activating steps must have occurred in this malignancy.     

Mutagenesis in monkey cells of a vector containing a single d(GPG) cis-diamminedichloroplatinum(II) adduct placed on codon 13 of the human H-ras proto-oncogene.

Cisplatin (cis-[Pt(NH3)2Cl2]) is a widely used antitumor agent whose mutagenic activity raises the possibility of the induction of secondary cancer as a result of treatment. Mutation of the proto-oncogene H-ras is found in more than 30% of all human tumors, where it has been postulated to contribute to the initiation and progression of human cancers. Activating mutations in the H-ras gene are predominantly single-base substitutions, most frequently at codons 12, 13 and 61. In the present work we have studied the mutational spectra induced by a single cis-[Pt(NH3)2d(GpG)] adduct, the most frequent DNA crosslink formed by cisplatin. We have constructed a 25-mer-Pt oligonucleotide singly modified at codon 13 (GGT) within the human H-ras DNA sequence and we have inserted it into a single-stranded SV40-based shuttle vector able to replicate in simian COS7 cells. After replication in the mammalian host, vectors were extracted, amplified in bacteria and DNA from 124 randomly chosen colonies was sequenced. The observed mutation frequency was 21%. Base substitutions were the most frequent modification. 92% of the mutagenic events occurred at one or both of the platinated guanines of codon 13. The single G-->T transversion accounted for 65% of the total mutations scored. All single base substitutions were located at the G in the 3' position showing, for the first time, that the guanine at the 3' side of a cis-[Pt(NH3)2d(GpG)] adduct may be a preferential site for cisplatin induced mutations. The substitution G-->T at this position of the codon 13 of the H-ras proto-oncogene is known to induce the oncogenic properties of the p21ras protein.     

Lifestyle factors and Ki-ras mutations in colon cancer tumors

Heterogeneity in colon tumors implies that environmental, lifestyle, or genetic factors influence the type of mutations seen in tumors. In this study we evaluate the association between previously identified risk factors for colon cancer and Kirsten-ras (Ki-ras) mutations in tumors. The presence of Ki-ras mutations in codons 12 and 13 were determined in a population-based case-control study of colon cancer. Participants were between 30 and 79 years of age at time of diagnosis and include both men and women. Questionnaire data were used to obtain information on lifestyle factors. Valid study data and Ki-ras mutational status were available from 1428 cases of colon cancer, data from 2410 controls were available for comparative purposes. Participants with Ki-ras mutations were more likely to have proximal rather than distal tumors. Cigarette smoking, use of aspirin and/or NSAIDs, use of vitamin/mineral supplements, and consumption of caffeine were associated with both Ki-ras+ and Ki-ras- tumors; the associations were not confounded by dietary intake or other lifestyle factors. Among men, but not among women, those with low levels of physical activity were more likely to have a tumor with a Ki-ras mutation than one without a Ki-ras mutation. However, among women, those with a larger BMI were more likely to have a Ki-ras mutation in their tumor. Given the limited information available on what causes Ki-ras mutations, the information generated from this study indicates that these factors previously associated with colon cancer work through other disease pathways.     

Differential selection for B-raf and Ha-ras mutated liver tumors in mice with high and low susceptibility to hepatocarcinogenesis

Activation of the Ras/Raf/MEK/ERK pathway is frequently observed in animal and human tumors. In our study, we analyzed B-raf codon 637 (formerly 624) and Ha-ras codon 61 mutations in liver tumors from C3H, B6C3F1 and C56BL mice which differ considerably with regard to their susceptibility to hepatocarcinogenesis. In total, 73% (102/140) of tumors induced by a single application of N-nitrosodiethylamine or 7,12-dimethylbenz[a]anthracene contained either B-raf or Ha-ras mutations and only <3% (4/140) were mutated in both genes. In addition, B-raf mutations were present in 76% (19/25) of early precancerous liver lesions. The prevalence of Ha-ras mutated tumors was significantly higher in the susceptible C3H and B6C3F1 mouse strains (39-50%) than in the comparatively resistant C57BL mouse (7%). B-raf mutated tumors, by contrast, were more frequent in C57BL mice (68%) than in the other two strains (17-45%). Taken together, our findings indicate that alterations affecting the Ras/Raf/MEK/ERK signalling pathway are a hallmark of carcinogen-induced liver tumors in mice. Moreover, our results show that mutational activation of B-raf in liver tumors of different mouse strains is, by contrast to Ha-ras, inversely related to their susceptibility to hepatocarcinogenesis. Although activated Ras and Raf proteins are assumed to have similar biological effects because they feed into the same signalling pathway, there seem to be subtle strain-specific differences in selection processes favouring the preferential outgrowth of either B-raf or Ha-ras mutated tumor populations in mouse liver.     

Influence of Ki-ras-driven oncogenic transformation on the protein network of murine fibroblasts

Ki-ras gene mutations that specifically occur in codons 12, 13 and 61 are involved in the carcinogenesis of acute myeloid leukemia, melanoma and different carcinomas. In order to define potential mutation-specific therapeutic targets, stable transfectants of NIH3T3 cells carrying different Ki-ras4B gene mutations were generated. Wild type Ki-ras transformants, mock transfectants and parental cells served as controls. These in vitro model systems were systematically analyzed for their protein expression pattern using two-dimensional gel electrophoresis followed by mass spectrometry and/or protein sequencing. Using this approach, a number of target molecules that are differentially but coordinately expressed in the ras transfectants were identified next to other proteins that exhibit a distinct regulation pattern in the different cell lines analyzed. The differentially expressed proteins predominantly belong to the families of cytoskeletal proteins, heat shock proteins, annexins, metabolic enzymes and oxidoreductases. Their validation was assessed by real-time quantitative RT-PCR and/or Western blot analysis. Our results suggest that the Ki-ras-transformed cells represent a powerful tool to study Ki-ras gene mutation-driven protein expression profiles. In addition, this approach allows the discovery of ras-associated cellular mechanisms, which might lead to the identification of physiological targets for pharmacological interventions of the treatment of Ki-ras-associated human tumors.     

Concurrent mutations in two different ras genes in acute myelocytic leukemias.

DNA transfection analyses (tumorigenicity assay) and hybridization to mutation specific oligonucleotide probes established point mutations in codon 61 of both, N-ras and Ki-ras genes in fresh leukemic cells of an AML patient. Concurrent activation of N-ras and Ki-ras sequences by point mutations in codons 12 were demonstrated for AML cell line Rc2a. Moreover, using a rapid and sensitive dot-blot screening procedure based on the combination of in vitro amplification of ras specific sequences and oligonucleotide hybridization we could show that ras gene activation was not present in primary leukemic cells of the patient this cell line had been derived from, but rather occurred during later passages of Rc2a.     

Activation of a human c-K-ras oncogene

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.