Activating mutations at codon 61 of the c-Ha-ras gene in thin-tissue sections of tumors induced by aristolochic acid in rats and mice

The plant extract aristolochic acid, which consists mainly of aristolochic acid I (AAI) and aristolochic acid II (AAII), induces tumors in rats and mice. Thin-tissue sections of rat tumors induced by AAI and of mouse tumors induced by aristolochic acid, were analyzed for c-Ha-ras mutations in codon 61. Areas of neoplastic and histologically normal tissue were manually scraped out and separated. Using the polymerase chain reaction (PCR) and mutation detection by selective oligonucleotide hybridization, we observed AT----TA transversion mutations in DNA of neoplastic portions, but not in DNA of adjacent normal tissue in both rat and mouse tumors.     

Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products

A simple, sensitive method of DNA analysis of nucleotide substitutions, namely, single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis), was used for detection of mutated ras genes in surgical specimens of human lung cancer. Of a total of 129 tumors analysed, 22 contained a mutated ras gene. Of the 66 adenocarcinomas analysed, 14 contained an activated c-Ki-ras2 gene (the mutations in codon 12 in 6, in codon 13 in 4, in codon 18 in one, and in codon 61 in 3), one contained a c-Ha-ras1 gene with a mutation in codon 61 and 3 contained N-ras genes with mutations (in codon 12 in one and in codon 61 in 2). Mutated rats genes were also found in 2 of 36 squamous cell carcinomas (c-Ha-ras1 genes with mutations in codon 61) and 2 of 14 large cell carcinomas (c-Ki-ras2 genes with mutations in codon 12). No mutation of the ras gene was detected in 8 small cell carcinomas and 5 adenosquamous cell carcinomas. These results indicate that activation of the ras gene was not frequent (17%) in human lung cancers, that among these lung cancers mutation of the ras gene was most frequent in adenocarcinomas (27%) and 73% of the point mutations were in the c-Ki-ras2 gene in codon 12, 13, 18 or 61.     

Activation of the cellular Harvey ras gene in mouse skin tumors initiated with urethane

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c-Ha-ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha-ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha-ras gene was shown to be activated by a specific A----T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha-ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.     

Dichloroacetic acid reduces Ha-ras codon 61 mutations in liver tumors from female B6C3F1 mice

Dichloroacetic acid (DCA), a disinfection by-product of chlorination found in drinking water, is a hepatocarcinogenic in the B6C3F1 mouse. Previous studies have shown that DCA does not significantly alter the incidence of Ha-ras codon 61 mutations in male mouse liver carcinomas from that observed in spontaneous tumors (approximately 50% have Ha-ras mutations) but it alters the proportions of mutations that occur in Ha- ras codon 61. Twenty-two tumors were produced in female B6C3F1 mice after treatment with 3.5 g DCA per liter of drinking water over a period of 104 weeks. To detect potential Ha-ras mutations in the liver tumor tissue of female B6C3F1 mice, genomic DNA was isolated from tumors that had been frozen. The polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) was used to screen tumor DNA for mutations in Ha-ras exon 2. In DNA from liver tumors in female B6C3F1 mice induced by DCA-treatment we found only one mutation in exon 2 among the 22 tumors analyzed (4.5%). Direct-sequencing of exon 2 revealed a CAA to CTA transversion in Ha-ras codon 61. The result of this study indicates that tumor formation in DCA-treated female B6C3F1 mice is, therefore, not associated with a mutationally activated Ha-ras codon 61. This result differs from previous results obtained in male B6C3F1 mice.      

Spontaneous Ha-ras gene activation in cultured primary murine keratinocytes: consequences of Ha-ras gene activation in malignant conversion and malignant progression

The activation of the c-Ha-ras gene and its contribution to the tumorigenic phenotype were examined in cultured mouse keratinocytes and squamous tumors using transfection into NIH 3T3 cells and nucleic acid hybridization. When normal keratinocytes were cultured in medium with 0.05 mM Ca2+ (low Ca2+ medium), many cells died within 2-3 wk, while others formed rapidly growing foci that could be subcultured. These rapidly growing cells produced benign tumors when grafted to nude mice and possessed a heterozygous mutation in the c-Ha-ras gene with an A----T transversion in codon 61. Fibroblast-conditioned low Ca2+ medium prevented cell death, focus formation, c-Ha-ras gene mutation, and tumorigenicity. Thus, suboptimal culture conditions favored a spontaneous mutation in codon 61 of the c-Ha-ras gene of keratinocytes. When a v-Ha-ras gene was introduced into normal keratinocytes by a replication-defective retrovirus, the recipient cells produced papillomas in vivo, and after 2 mo, 60% of the tumors converted to squamos cell carcinomas. None of the 22 converted tumors had an endogenous c-Ha-ras gene mutation at codon 61. However, the A----T transversion mutation developed when these carcinoma cells were cultured in low Ca2+ medium but not in fibroblast-conditioned medium. Cells with both an exogenous v-Ha-ras and an activated c-Ha61-ras gene produced undifferentiated, rapidly lethal carcinomas, while cells with only v-Ha-ras maintained the squamous carcinoma phenotype. Undifferentiated carcinomas also developed when the v-Ha-ras gene was introduced into papilloma cells with a chemically induced endogenous c-Ha61-ras gene mutation. These results suggest that mutation in the c-Ha-ras gene can contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis, and gene dosage may determine the phenotype expressed.     

12th codon mutation resulting in c-N-ras activation in acute myelogenous leukemia

Activation of the cellular oncogene c-N-ras has been frequently observed in DNA from leukemic cells in acute myeloid leukemia (AML). Ras gene activation sufficient to mediate in vitro transformation and rodent tumorigenesis usually results from point mutations and amino acid substitutions in the 12th or 61st codons. In AML and the related myelodysplastic syndromes, amino acid substitution at the 13th codon has been observed. An activated c-N-ras gene from a 45-year-old patient with AML was isolated by transfection analysis and subjected to molecular cloning and sequence analysis. A point mutation of the 12th codon (GGT to GAT) resulting in aspartic acid substitution for glycine was observed. In other neoplasms such as colon cancer, specific ras mutations occur predominantly (e.g., K-ras, codon 12). This predominance has been of demonstrable value in analyzing large cohorts for ras activation with techniques that are rapid and economical, such as oligonucleotide hybridization. It had previously been thought that such a predominance for activation of c-N-ras at codon 13 existed in AML; however, this study in concert with others underscores the importance of 12th codon c-N-ras mutations, along with 13th and 61st codon mutations in the molecular pathogenesis of AML. Guanylate to adenylate transition mutations are commonly observed in AML and may provide insight into potential environmental leukemogens. Addressing all commonly prevalent ras activating mutations bears impact in the future design of molecular surveys of the role of ras activation in leukemogenesis.     

ras gene mutations in human prostate cancer

Point mutations at codons 12, 13, or 61 of the Ha-, Ki-, and N-ras genes are able to convert these normal cellular genes into activated oncogenes. Previous studies have shown that ras gene mutations occur in a variety of human solid tumors and may be important in the pathogenesis of some of these tumors. In order to test the hypothesis that ras gene mutations may be associated with prostate cancer, we have used an oligodeoxynucleotide hybridization assay to detect wild-type and mutant alleles in genomic DNA from prostate tumors and prostate tumor cell lines amplified using the polymerase chain reaction. Twenty-four primary prostate tumors (23 acinar tumors and one ductal tumor) and five prostate tumor cell lines were examined for mutations at codons 12, 13, and 61 of the Ki-ras, Ha-ras, and N-ras genes. Two mutations were detected: an A----G transition causing a glutamine to arginine amino acid substitution at codon 61 of the Ha-ras gene in a primary prostatic duct adenocarcinoma and a G----T transversion causing a glycine to valine amino acid substitution at codon 12 of the Ha-ras gene in a prostate tumor cell line (TSU-PR1) derived from a lymph node metastasis. While the overall frequency of ras gene mutations in prostate tumors is low, when these mutations do occur they may have a role in the progression of disease or the development of the unusual ductal variant of prostatic adenocarcinoma.     

Site-specific Mutation of the Human c-Ha-ras Transgene Induced by Dimethylbenzanthracene Causes Tissue-specific Tumors in Mice

Forestoraach squamous cell carcinomas, lung adenocarcinomas and spleen angiosarcomas were induced by dimethylbenzanthracene (DMBA) in the rasH2 transgenic mouse line carrying human c-Ha-ras genes with their own promoter, encoding the prototype p21 gene product. Fifteen out of 21 mice (71%) developed forestomach squamous cell carcinomas, while 15 out of 21 (71%) had lung adenocarcinomas and 3 out of 21 (14%) showed spleen angiosarcomas within 8 weeks after a single administration of 50 mg/kg DMBA intraperitoneally. Somatic mutation at the 61st codon of the transgenes, from CAG(Gln) to CTG(Len), was detected in all these newly developed tumors. However, non-transgenic littermates demonstrated no tumors at all. These findings provide strong evidence that the somatic mutational activation of human c-Ha- ras genes is a critical event in tumorigenesis and a close relationship is therefore strongly suggested between the tissue-specific development of tumors and the somatic mutation of human c-Ha-ras genes in these rasH2 transgenic mice.     

Demonstration of ras and p53 Gene Mutations in Carcinomas in the Forestomach and Intestine and Soft Tissue Sarcomas Induced by N-Methyl-N-nitrosourea in the Rat

The presence of ras family and p53 gene mutations in rat forestomach, intestine and liver tumors and soft tissue sarcomas induced by N methyl- N -nitrosourea (MNU) was examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by direct sequencing analysis. In the forestomach squamous cell carcinomas (SCC), Ha- ros and p53 mutations were detected in 2 (40%) and 4 (80%) of 5 cases, respectively. The figures for Ki- ras and p53 gene mutations in adenocarcinomas of the large and small intestines were 3 (18.8%) and 5 (31.3%) of 16 cases. Soft tissue sarcomas in different sites were found to have mutations of Ki- ras in 7 (23.3%)and of p53 in 9 (30%) of 30 cases. One forestomach SCC and 2 soft tissue sarcomas had double p53 mutations in different exons. Single cases of forestomach SCC and intestinal adenocarcinoma had mutations in both Ki- ras and p53 genes. No mutations were found in counterpart benign tumors or hepatocellular adenomas. The p53 mutation spectrum revealed preferential clustering within exon 8 for the forestomach SCCs, and exons 5 and 8 for the intestinal adenocarcinomas, whereas the distribution was evenly spread through exons 5 to 8 in soft tissue sarcomas. All the detected ras or p53 mutations were G:C to A:T transitions. These results indicate firstly that specific Ki- ras , Ha- ras and p53 gene mutations in MNU-induced lesions are related to particular alkylation sites (G:C to A:T transitions) and secondly, although not essential, Ki- ras , Ha- ras or p53 gene mutations may be involved in the progression stage of forestomach, intestine and soft tissue neoplasms induced by MNU.     

Glycine to aspartic acid mutations at codon 13 of the c-Ki-ras gene in human gastrointestinal cancers

Point mutations of c-ras genes were analyzed in human gastrointestinal cancers. DNA obtained from the tissues was amplified by polymerase chain reaction and then analyzed by dot blot hybridization assay with oligonucleotide probes to detect mutations at codons 12, 13, and 61 of c-Ki-ras, c-Ha-ras, and c-N-ras. In two of 25 cases of stomach cancer point mutations at codon 13 of c-Ki-ras were found. In colorectal cancer, eight of 30 cases showed mutations: four cases of codon 12 and one case at codon 13 of c-Ki-ras and two cases at codon 61 and one case at codon 13 of c-N-ras. These results may indicate involvement of a wide variety of c-ras gene point mutations, in addition to those at codon 12 of c-Ki-ras, in oncogenesis of human gastrointestinal cancers. In all three mutations of c-Ki-ras at codon 13 which had been seldom found in human cancers, glycine to aspartic acid mutations due to identical G to A transition at the second nucleotide were observed.     

Analysis of point mutations in murine c-Ha-ras of skin tumors initiated with dibenz[a,j]anthracene and derivatives.

This study was designed to evaluate the point mutations in the murine c-Ha-ras gene of skin papillomas induced by initiation with dibenz[a,j]anthracene (DB[a,j]A), its bay-region anti-diol epoxide ((+/-)anti-DB[a,j]A-DE), and a 7,14-dimethyl analogue (7,14-diMeDB[a,j]A). Recent studies (Nair RV, et al., Chem Res Toxicol 4:115-122, 1991) in our laboratory have revealed both deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from the anti- and syn-diol epoxides of DB[a,j]A in cultured mouse epidermal cells after exposure to this hydrocarbon. Using PCR amplification and direct sequencing, we found specific A182----T transversion mutations (eight of 10 tumors) in codon 61 of c-Ha-ras in papillomas induced by initiation with DB[a,j]A. Analysis of papillomas generated by initiation with the more biologically potent analogue 7,14-diMeDB[a,j]A revealed that five of five tumors exhibited A182----T transversions in codon 61. The nature of the changes in the two DB[a,j]A tumors not showing codon 61 mutations in Ha-ras is currently not known since these tumor DNAs also did not possess c-Ha-ras mutations at codons 12, 13, or 59. Interestingly, papillomas produced by initiation with (+/-)anti-DB[a,j]A-DE also possessed A182----T transversion mutations in codon 61 of c-Ha-ras (five of five tumors). These data suggest that dAdo adducts derived from both parent hydrocarbons may play an important role in their tumor-initiating activity and possibly implicate a specific diol epoxide-dAdo adduct in this process.     

H-ras mutations in rat urinary bladder carcinomas induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide and sodium saccharin, sodium ascorbate, or related salts

Male F344 rats were fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazoly]formamide for 6 weeks and then fed 3% or 5% sodium saccharin, 5% sodium ascorbate, 3.12% calcium saccharin, 1.34% sodium chloride, 5.2% calcium saccharin plus 1.34% sodium chloride, or basal diet alone for 72 weeks. Protein and DNA were extracted from 89 bladder tumors [87 transitional cell carcinomas (TCC), 1 papilloma, and 1 sarcoma] from 86 rats p21 expression was examined by Western blotting using a monoclonal antibody against p21 (NCC-RAS-004). H-ras mutations in exons 1 and 2 were examined by direct sequencing of DNA amplified by polymerase chain reaction. Sequencing results demonstrated mutations at codon 61 (CAA to CGA in 15 TCCs; CAA to CTA in 2 TCCs), at codon 12 (GGA to TGG in 1 TCC), and at codon 13 (GGC to GTC in 3 TCCs). Mutations at codon 61 were confirmed by faster mobility of the p21 band in Western blots. The level of p21 expression varied among samples, but many TCCs appeared to express more p21 than controls. The overall incidence of H-ras mutations was 24.4% (21 of 86 rats). The type of chemical used for the promoting phase had essentially no effect on H-ras mutation, suggesting that the effects observed were related to FANFT administration. The frequency of H-ras mutation in each group was negatively related to the incidence of carcinoma (r = -0.85; P less than 0.01). Two groups of tumors (with or without the mutated ras gene) were compared for tumor size (reflected by the bladder weight), histological grading, and the presence of invasion. The size of tumors with mutated ras was significantly smaller than those without mutated ras. There was no difference in the histological grading between the two groups. Although not statistically significant, histological invasion was more frequently observed in tumors with mutated ras (14.3%) than in tumors without mutation (3.1%).     

Mutations in ras genes in experimental tumours of rodents.

Studies of carcinogenesis in rodents are valuable for examining mutagenesis in vivo. An advantage of evaluating the frequency and spectra of ras mutations in chemically induced neoplasms is that the additional data at the molecular level indicate whether the carcinogenic effect is due to the chemical and is not a spontaneous event, as illustrated by the numerous examples in Appendices 1 and 2. In addition, data on the frequency and spectra of ras mutations in spontaneous and chemically induced neoplasms clearly expand the toxicological database by providing information helpful for understanding the pathogenesis of carcinogenesis. For example: (1) ozone-induced lung neoplasms had two unique mutations, one (codon 61 K-ras CTA mutation) consistent with a direct genotoxic event and a second (codon 12 K-ras G --> T transversion) consistent with an indirect genotoxic effect; (2) isoprene-induced Harderian gland neoplasms had a unique K-ras A --> T transversion at codon 61 which provided evidence that formation of an epoxide intermediate was involved; (3) 1,3-butadiene-induced neoplasms had a characteristic K-ras G --> C transversion mutation at codon 13 which was also consistent with a chemical-specific effect; (4) methylene chloride-induced liver neoplasms had an H-ras mutation profile at codon 61 similar to that of spontaneous tumours, suggesting that methylene chloride promotes cells with 'spontaneously initiated' ras mutations and (5) oxazepam-induced liver neoplasms had a low frequency of ras mutations, suggesting a nonmutagenic pathway of carcinogenesis. By extending the evaluation of rodent tumours to include molecular studies on ras mutation spectra and abnormalities in other cancer genes with human homologues, a number of hypotheses can be tested, allowing the most complete understanding of carcinogenesis in rodents and in potential extrapolation to the human risk situation.     

Rapid induction of skin tumors in human but not mouse c-Ha-ras proto-oncogene transgenic mice by chemical carcinogenesis

The rasH2 transgenic mice carry human c-Ha-ras proto-oncogene, and are highly susceptible to chemical carcinogenesis. Previous studies showed that the mutation of c-Ha-ras induced by DMBA in the tumors of rasH2 were detected only in transgenes. To examine if the difference between the codons of the c-Ha-ras gene in human and mouse contributed to the tissue-specific sensitivity to DMBA, we generated a line of transgenic mice, mras, carrying mouse c-Ha-ras genome with its own promoter. Western blot analysis showed that the protein expression of H-RAS in the skin was increased in both rasH2 and mras compared with wild-type. Chemical skin carcinogenesis was induced by DMBA and TPA. In rasH2 mice, the latency of tumor formation was shorter than wild-type littermates. Both the number and the volume of skin tumors were increased in rasH2 than those of wild-type. However, in mras mice, enhancement of tumor formation was not observed as compared with wild-type. The mean number of tumors and the latency of tumor development was almost the same between mras and wild-type littermates. Mutational analysis showed only A to T transversion in human c-Ha-ras transgenes at codon 61 but not in murine endogenous c-Ha-ras gene in the tumors of rasH2. In the tumors of wild-type littermates and mras, A to T transversion in murine c-Ha-ras at codon 61 were detected. These results indicate that the differences in the codon of the c-Ha-ras gene between mouse and human might contribute to the tissue-specific sensitivity of DMBA.     

Point mutation analysis of ras genes in spontaneous and chemically induced C57Bl/10J mouse liver tumours.

The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.     

Incidence of mutations at codon 61 of the Ha-ras gene in liver tumors of mice genetically susceptible and resistant to hepatocarcinogenesis

By selective oligonucleotide hybridization of polymerase chain reaction (PCR) amplified tumor DNAs we have analysed the incidence of mutations at codon 61 of the Ha-ras gene in 42 liver tumors spontaneously developed in Balb/c, C3Hf and B6C3 male mice, and in 79 liver tumors induced by the chemical carcinogens diethylnitrosamine (NDEA) and urethan in B6C3 and B6C male and female mice. The incidence of Ha-ras gene mutations in both spontaneously developed and urethan-induced liver tumors was 50%-63% in mice genetically susceptible to hepatocarcinogenesis (C3Hf, B6C3) and 7%-9% in mice genetically resistant (Balb/c, B6C). Urethan-induced tumors showed about the same incidence of ras mutations in male and in female B6C3 mice. NDEA-induced tumors showed a low incidence of Ha-ras mutations in both the hybrid mice (3/18 and 1/13 in B6C3 and B6C male mice, respectively). The most frequently found mutations were a C----A transversion at the 1st base of codon 61 in spontaneous tumors, and an A----T transversion at the 2nd base in urethan-induced tumors. Our results indicate that liver tumors induced by NDEA or urethan or spontaneously arisen have a different pattern of Ha-ras mutations at codon 61 and that these mutations constitute a rare molecular alteration in the pathogenesis of liver tumors in genetically resistant mice.     

N-ras mutation in ultraviolet radiation-induced murine skin cancers.

UV radiation is a potent DNA-damaging agent and a known inducer of skin cancer in experimental animals. To elucidate the role of oncogenes in UV carcinogenesis, we analyzed UV-induced murine skin tumors for mutations in codon 12, 13, or 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to UV-induced C3H mouse skin tumors, we also analyzed skin tumors induced in the same strain of mice by other carcinogenic agents such as 8-methoxypsoralen + UVA, angelicin + UVA, dimethylbenz-[a]anthracene + UV + croton oil, and 4-nitroquinoline-1-oxide. We found that 4 of 20 UV-induced skin tumors contained either C----A or A----G base substitutions at N-ras codon 61. In addition, 2 of 5 melanomas possessed a G----A transition in N-ras codon 13 and an A----T transversion in N-ras codon 61, respectively. Interestingly, none of the 8-methoxypsoralen + UVA- or angelicin + UVA-induced tumors we analyzed contained mutations in any of the ras genes. However, 1 of 4 4-nitroquinoline-1-oxide-induced tumors exhibited a G----T transversion at Ki-ras codon 12, a potential site for formation of a 4-nitroquinoline-1-oxide adduct with a guanine residue. We also found that 2 nonmelanoma tumors induced by dimethylbenz[a]anthracene + UV + croton oil contained an A----T transversion at Ha-ras codon 61 position 2, which is characteristic of most dimethylbenz[a]anthracene-induced tumors. These results suggest that UV-induced C3H mouse tumors display mutations preferentially in the N-ras oncogene. Since most N-ras mutations in UV-induced tumors occurred opposite dipyrimidine sequences (T-T or C-C), one can infer that these sites are the targets for UV-induced mutation and transformation.     

Infrequent ras activation in chronic myelogenous leukemia (CML): activating 61st codon mutation in the CML-derived cell line, IM-9

The proto-oncogene c-N-ras frequently bears point mutations in ANLL cell DNA which endow it with the capacity to transform NIH/3T3 cells in vitro. Chronic myelogenous leukemia (CML) is a neoplasm highly related to ANLL since it involves the same hematopoietic progenitor cells and ultimately transforms to a neoplasm virtually indistinguishable from acute nonlymphoblastic leukemia (ANLL). Thus, we and others have examined ras genes in CML. This report confirms that ras gene activation is a very infrequent event in CML. However, a lymphoblastic cell line derived from a patient with CML did exhibit a novel second exon 61st codon activating mutation of c-N-ras.     

p53 Mutations in human hepatocellular carcinomas from Germany.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined 13 cases of human hepatocellular carcinomas from Germany for the presence of p53 aberrations in exons 4 to 8 of the gene by single-strand conformation polymorphism and restriction fragment-length polymorphism analyses and by sequencing of polymerase chain reaction products. Single base substitutions occurred in two human hepatocellular carcinomas: a C:G----T:A transition at a CpG site in codon 257, and a T:A----A:T transversion at codon 273. One of these point-mutated tumors and two additional tumors without point mutations demonstrated a loss of one p53 allele. None of the tumors was mutated in codons 12 or 61 of the c-Ha-ras gene.     

Mutational activation of c-Ha-ras gene in squamous cell carcinomas of rat Zymbal gland induced by carcinogenic heterocyclic amines

The activation of the c-Ha-ras gene and its carcinogen specificity were examined in squamous cell carcinomas (SCCs) induced by the mutagenic heterocyclic amines 2-amino-3-methylimidazo [4,5-f]quinoline (IQ),2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in the Zymbal gland in rats. DNA fragments of the c-Ha-ras gene were amplified from formalin-fixed and paraffin-embedded tissues by polymerase chain reaction and analyzed for activating mutations involving codons 12, 13, and 61 by oligonucleotide differential hybridization and sequencing. c-Ha-ras mutations were found in four of seven and two of six Zymbal gland SCCs induced by IQ and MeIQx, respectively. These mutations were located in either codon 13 or 61. In the case of MeIQ, point mutations at the second nucleotide of codon 13 were found in nine of the total 14 Zymbal gland SCCs and in one papilloma. Of the nine SCCs that had mutations in codon 13, two possessed mutations at the second nucleotide of codon 12 as well. Most reported mutations in c-Ha-ras are located at codon 12 or 61, but the heterocyclic amines in this study induced mutations not only at codons 12 and 61 but also in codon 13. Transversions were the dominant mutation induced by these heterocyclic amines.