Frequent activation of the Ki-ras oncogene at codon 12 in N-methyl-N-nitrosourea-induced rat prostate adenocarcinomas and neurogenic sarcomas.

Rat neoplasms induced by methylating carcinogens frequently contain ras genes activated by a single point mutation. Rat prostatic tumors induced by a combination of a single injection of N-methyl-N-nitrosourea (MNU) and long-term treatment with testosterone were examined for the presence of such activating point mutations in ras genes. These tumors, which arose exclusively in the dorsolateral prostate, included both adenocarcinomas and sarcomas. Activating mutations in codon 12 of the Ki-ras gene were found in 7 of 10 carcinomas and 4 of 5 sarcomas, using selective oligonucleotide hybridization analysis of DNA amplified by the polymerase chain reaction (PCR). However, no mutated Ha-ras oncogenes were detected. The presence of PCR-engineered Hphl restriction sites created by the existence of a G35----A mutation in the rat Ki-ras oncogene identified the mutation as a GC----AT transition at the second position of codon 12. Production of O6-methylguanine adducts in the Ki-ras codon 12 followed by base mispairing during replicative DNA synthesis is thus the likely molecular mechanism of initiation of prostatic carcinogenesis by MNU in the rat. Three of the four sarcomas positive for the Ki-ras G35----A mutation were immunohistochemically defined as of Schwann cell origin, indicating that involvement of the ras gene family is possible in tumorigenesis of this cell lineage. Loss of the wild-type Ki-ras allele was also observed in all four of these sarcomas.     

Activated Ki-ras genes in bladder epithelial cell lines transformed by treatment of primary mouse bladder explant cultures with 7,12-dimethylbenz[a]anthracene

DNA from five lines of transformed bladder epithelial cells derived from cultures of primary cells that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) can transform NIH 3T3 mouse fibroblasts in DNA transfection experiments. Southern analysis of DNA from NIH 3T3 primary and secondary transformants established that four of the DMBA-transformed cell lines contained activated cellular Ki-ras, while the remaining cell line contained a transforming gene that is unrelated to Ki-ras, N-ras, and Ha-ras. The point mutations responsible for Ki-ras activation were detected using oligonucleotide probes following selective amplification of Ki-ras specific sequences using the polymerase chain reaction. The results showed that activation of Ki-ras invariably involved a GC----AT transition mutation of the first position of codon 12. Surprisingly, a Ki-ras gene that was activated by a GC----AT transition mutation at the same position was also detected in a single transformed bladder urothelial cell line derived from control cultures of mouse bladder cells. Together, our results indicate that Ki-ras activation in the DMBA-transformed bladder cell lines may not be a direct consequence of interaction of activated DMBA metabolites with the Ki-ras gene.     

Incidence of ras oncogene activation in lung carcinomas in Hong Kong.

BACKGROUND. In Hong Kong, lung carcinomas contribute to the majority of cancer deaths among Chinese. Point mutational activation of ras oncogenes has been observed in several populations. The incidence of these mutations in Hong Kong lung carcinomas was investigated. METHODS. Lung resections obtained from 52 Chinese patients whose conditions were newly diagnosed as non-small cell lung cancer, paraffin sections from 29 Chinese patients with previously diagnosed adenocarcinoma of the lung, and paraffin sections from 49 squamous cell carcinomas were examined for the presence of point mutations in Ki-ras codon 12, N-ras codon 61, and Ha-ras codon 12 oncogenes by allele-specific hybridization after specific amplification of appropriate regions of the DNA using the polymerase chain reaction. RESULTS. Among the 130 lung carcinomas investigated, Ki-ras point mutations were detected in seven cases, of which six were adenocarcinomas and one a squamous cell carcinoma. No mutations were detected in the N-ras and Ha-ras codons. CONCLUSIONS. The incidence of Ki-ras codon 12 point mutational activation in Chinese patients with adenocarcinomas was 6 of 63 (9.5%). The incidence of Ki-ras 12 point mutational activation among men with lung adenocarcinomas in Hong Kong (6 of 32 patients, 18.8%) is significantly different from that in women in Hong Kong (0 of 31 patients, 0%). Although ras oncogenes are implicated as having a role in the development of lung adenocarcinomas, especially among smokers, it is clear from these data that they are not associated with the unusually high incidence of lung adenocarcinomas among women in Hong Kong.     

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.     

Resistance to mammary tumorigenesis in Copenhagen rats is associated with the loss of preneoplastic lesions

The resistance of Copenhagen (Cop) rats to mammary tumor development has recently been linked to three loci, but the genes have yet to be cloned and the mechanism of resistance is still largely unknown. In order to determine the cellular events associated with resistance, we prepared mammary whole mounts from Cop and susceptible Wistar Furth (WF) rats 0, 15, 30, 45 and 60 days after treatment with 50 mg/kg N-methyl-N-nitrosourea (MNU). At 15 days, treated rats of both strains had significantly more undifferentiated structures [terminal end buds (TEBs)] and significantly fewer differentiated structures [alveolar buds (ABs)] than untreated rats. Treated Cop rats, however, had significantly more TEBs and fewer ABs than age-matched, treated WF rats. Histological analysis of preneoplastic lesions tentatively identified from the whole mounts showed that like WF rats, Cop rats developed early preneoplastic lesions [intraductal proliferations (IDPs)] by 15 days post-MNU treatment. Unlike IDPs from WF rats, however, the IDPs in Cop rats then decreased in number until they were absent 60 days post-MNU treatment. Furthermore, they failed to progress into more advanced lesions such as ductal carcinomas in situ (DCIS). Finally, we found G-->A activating mutations in codon 12 of the Ha-ras gene in 60% of IDPs from Cop rats and 75% of IDPs from WF rats. Our results show that resistance in Cop rats is not due to a target cell population for the carcinogen that is smaller than in susceptible rats or to the failure of the carcinogen to inhibit mammary gland differentiation. Furthermore, we have shown that Cop rats develop preneoplastic IDPs that harbor Ha-ras mutations but, unlike IDPs in susceptible strains, they fail to progress and ultimately disappear.     

Activation of the Ki-ras gene in spontaneous and chemically induced lung tumors in CD-1 mice.

As part of an evaluation of the effectiveness of using ras mutation analysis for distinguishing carcinogen-induced from spontaneous tumors, we examined the profile of ras gene point mutations in spontaneous, 7,12-dimethylbenz[a]anthracene (DMBA)-induced, and N-nitrosodiethylamine (DEN)-induced lung tumors from Crl:CD-1(ICR)BR (CD-1) mice. Although all of the lung tumors were assayed for mutations in the Ha-ras, Ki-ras, and N-ras genes (codons 12, 13, and 61), only Ki-ras mutations were found, which is consistent with other studies that have noted a strong preference for Ki-ras gene activation in mouse, rat, and human lung tumors. We found that spontaneous CD-1 mouse lung tumors had a very high frequency of Ki-ras gene activation (17 of 20 tumors; 85%), distributed among codons 12 (5 of 20), 13 (1 of 20), and 61 (11 of 20). DMBA-induced lung tumors had a slightly higher frequency of Ki-ras gene mutations (16 of 16; 100%), again distributed among codons 12 (5 of 16), 13 (2 of 16), and 61 (9 of 16). However, seven of the DMBA tumors had mutations qualitatively different from those found in spontaneous tumors. In contrast to DMBA-induced tumors, DEN-induced tumors had a lower frequency of Ki-ras mutations (36%) when compared with spontaneous lung tumors, suggesting that DEN primarily induces lung carcinogenesis by a mechanism other than ras gene activation. Thus, although spontaneous and induced CD-1 mouse lung tumors have a strong tissue-specific preference for carrying an activated Ki-ras gene, the nature of the initiating carcinogen can influence the frequency or profile of Ki-ras mutations.     

Codon 12 Ki-ras mutation in non-small-cell lung cancer: comparative evaluation in tumoural and non-tumoural lung.

Ki-ras activation by point mutation on codon 12 has been reported in non-small-cell lung carcinomas and in various models of experimental lung tumours induced by chemical carcinogens. The cellular targets for carcinogenic compounds of tobacco smoke are usually considered to be the cells of the bronchial mucosa or alveolar epithelium. However, little is known about preneoplastic events in bronchopulmonary carcinogenesis. The hypothesis of the presence of widespread target cells containing Ki-ras mutation was investigated by evaluating concurrent neoplastic and non-neoplastic bronchial and alveolar samples from 51 patients with non-small-cell lung carcinomas. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method used can detect one cell with a mutation on codon 12 among 10(2) normal cells. In tumour samples, a mutation was detected in 20% of adenocarcinomas, but in none of the adenosquamous or squamous cell carcinomas. No mutation was detected in the non-neoplastic bronchial or parenchymal samples. When using an enriched PCR-RFLP method detecting one mutated allele among 10(3) normal alleles a mutation was detected in 23% of adenocarcinomas. In conclusion, Ki-ras activation by mutation on codon 12 was not observed in non-neoplastic bronchial or parenchymal tissues in patients with bronchopulmonary cancers and does not appear to be a genetic event present in non-malignant epithelial target cells exposed to tobacco smoke.     

Transplacental and transgeneration carcinogenic effect of 7,12-dimethylbenz[a]anthracene: relationship with ras oncogene activation.

Transgeneration transmission of the carcinogenic action of 7,12-dimethylbenz[a]anthracene (DMBA) was studied in two generations of mice using transplacental DMBA initiation followed by postnatal skin tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the first generation (F0) and only promotion in the second generation (F1). Local application of TPA resulted in increased skin tumor yield in both the in utero DMBA-exposed mice and their progeny (P = 0.0002 and P = 0.0941 respectively compared to control). Similarly, lung tumor incidence was increased in the two generations of mice (P less than 0.0001 and P = 0.0080 respectively). The results suggest transgeneration transfer of the effect of DMBA. A to T mutation at the second base of codon 61 of the Ha-ras oncogene was found in skin tumors of DMBA-exposed mice, but not in tumors induced by TPA without initiation. Analysis of Ki-ras codon 61 in seven lung tumors from DMBA-treated mice revealed three types of mutation: two cases with CA[C or G or T], one case with CCA and one case with CTA (the remaining cases having only the wild type). Six of these mice also had skin tumors, which contained A to T mutation at the second base of codon 61 of the Ha-ras gene in five cases. Thus mutations of different ras genes were found in skin and lung tumors from the same animals. In the progeny (F1) of DMBA-exposed F0 mice, only skin tumor samples were available for oncogene analysis and none contained the Ha-ras mutation. The results confirm our previous finding that initiation of skin and lung tumorigenesis can be transmitted transgenerationally. On the other hand, our data from a limited number of skin tumors suggests that ras gene mutation may not be critically involved in this transmission.     

Identification of genes preferentially expressed in mammary epithelial cells of Copenhagen rat using subtractive hybridization and microarrays

Rats, like humans, vary considerably in susceptibility for mammary cancer development among different strains. The Copenhagen (Cop) rat is extremely resistant to mammary cancer development induced by a variety of carcinogens. Multiple genetic loci have been linked to the resistant phenotype, but the genes have yet to be cloned and the mechanisms underlying the resistance still remain unknown. Transplantation experiments, however, have demonstrated that these genes act only in the epithelial cells of mammary parenchyma; they do not act systemically. In the present study, we analyzed genes differentially expressed in mammary epithelial cells obtained from pubescent female Cop and susceptible Buffalo (Buf) rats, using PCR-based suppressive subtractive hybridization and cDNA microarray approaches. Our results showed a high degree of similarity in the expression profiles of about 4000 genes between Cop and Buf rats, with a few exceptions. We found that the interleukin-2 receptor alpha (IL-2Ralpha) chain gene and claudin-6 gene were preferentially expressed in mammary epithelial cells purified from Cop rats. We further demonstrated that IL-2Ralpha message was undetectable in two rat mammary cancer cell lines and in two human breast cancer cell lines. The level of claudin-6 mRNA was undetectable in two rat mammary cancer cell lines and was lower in two human breast cancer cell lines and one breast cancer sample than that in normal breast tissues. These results suggest that IL-2Ralpha and claudin-6 may function as tumor suppressors, particularly for breast cancer. However, this possibility needs further investigation.     

Tissue-specific activating mutations of Ha- and Ki-ras oncogenes in skin, lung, and liver tumors induced in mice following transplacental exposure to DMBA

Transplacental carcinogenesis represents a good model in which to study the involvement of tissue-specific oncogene activation in carcinogenesis because a single exposure to a carcinogen induces tumors at various sites. We tested tumors of the skin, liver, and lung produced in mice after transplacental 7,12-dimethylbenz[a]-anthracene (DMBA) exposure for possible activation of ras genes. XbaI restriction fragment-length polymorphism analysis has shown that exposure to DMBA in utero may result in appearance of A----T transversion at the second position of codon 61 of Ha-ras oncogene in skin and liver tumors but not in lung tumors. Moreover, DNA samples isolated from spontaneous and DMBA-induced lung and liver tumors were analyzed for mutations at the same position of Ki-ras oncogene using differential hybridization with specific oligonucleotides. Among five spontaneous lung tumors, three cases of A----G transition, and one case of A----T transversion were found, whereas four of ten lung tumors of DMBA-treated animals were positive for A----T mutation. No Ki-ras mutation was detected in one spontaneous and four DMBA-induced hepatomas. In two cases, we revealed Ki-ras A----T mutation in the lung tumor and Ha-ras mutation in the liver tumor taken from the same animal. These results indicate first that DMBA treatment may induce A----T mutation at the second position of codon 61 both in Ha-ras and in Ki-ras and, second, that the role of different activated oncogenes in carcinogenesis may differ, depending on the tissue in which the tumor develops.     

High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinomas

The frequency of Ki-ras gene mutations was studied in 100 paraffin-embedded sections obtained from 63 pancreatic adenocarcinomas by in vitro amplification of target sequences via polymerase chain reaction (PCR) and selective oligonucleotide hybridization. Forty-seven (75%) of the tumors contained a Ki-ras mutation at codon 12. No predominant amino acid substitution or nucleotide transition at this codon was observed. Two carcinomas exhibited 2 distinct Ki-ras mutations. No particular correlation could be established between the incidence of Ki-ras mutation and clinical parameters (sex, age, survival), tumor grade or tumor stage.     

Mammary tumor induction by N-methyl-N-nitrosourea in genetically resistant Copenhagen rats.

The Copenhagen rat is completely resistant to mammary cancer induction by N-methyl-N-nitrosourea (MNU) when the carcinogen is administered during sexual development, a period when other strains of rats are normally susceptible to mammary gland carcinogenesis. Here we administered 30 mg/kg MNU i.p. to two groups of neonatal (2-3-day-old) Copenhagen rats. One group (group B, 18 animals) received no further treatment, while the other group (group C, 17 animals) received a second dose of 30 mg/kg MNU via the tail vein at 50 days of age. About 30% of the rats in group B and about 70% of those in group C developed mammary carcinomas before they were 1 year of age. About one-half of the tumors in both groups were cribriform adenocarcinomas and one-half were adenosquamous carcinomas. The latter tumor type has not been observed previously in susceptible rat strains. The ability to induce these mammary tumors in the Copenhagen rat suggests that the putative mammary carcinoma suppressor gene is functionally inactive in neonatal animals or is inactivated when these animals are treated with MNU.     

Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets

Rats treated with the alkylating agent methylnitrosourea (MNU) develop multiple, hormonally dependent mammary tumors. Roughly 50% of the tumors have Ha-ras mutation, whereas 50% do not. The MNU-induced rat mammary tumor model was employed to examine the therapeutic efficacy of the farnesyltransferase inhibitor (FTI), R115777, and to examine the use of genomics in identifying susceptible tumors as well as identifying genes whose expression are modulated by FTI treatment. In animals bearing palpable mammary tumors (< 7 mm diameter), we performed a surgical biopsy, and 3 days following the biopsy, rats were treated with R115777 (50 mg/kg body wt/day) by gavage. Tumors with Ha-ras mutations underwent profound regression, with nearly 90% showing complete regressions within 4 weeks. In contrast, the non-Ha-ras mutation-bearing tumors yielded a more variable response, although roughly half of the non-Ha-ras mutation tumors underwent significant regression. These results show that although all tumors appear to respond to the FTI inhibitor the tumors with Ha-ras mutations were exquisitely sensitive. We employed a microarray approach to define potential targets and the mechanism of action of R115777 in Ha-ras mutant or wildtype tumors following treatment with FTI. In addition, we determined whether gene expression prior to FTI treatment can be used to differentiate highly sensitive tumors (Ha-ras mutant) and tumors with variable sensitivity (Ha-ras wildtype). Untreated or FTI-treated (4 days at 50 mg/kg body wt) tumors (Ha-ras mutant or wildtype) were examined using oligonucleotide arrays. A significant number of genes were differentially expressed in control rat mammary tumors with or without an activated Ha-ras mutation, suggesting that a microarray analysis might differentiate highly sensitive and variably sensitive tumors. Most of the genes whose expressions were modulated by FTI in tumors were independent of Ha-ras status and were presumably modulated by effects on farnesylation of proteins other than Ha-ras. However, treatment of Ha-ras-mutated mammary tumors with R155777 results in preferential modulation of genes involved in ras-MAP kinase signal transduction pathway and in decreased expression of many genes involved with cell proliferation. In contrast, several classes of genes are altered in rat mammary tumors without a mutated Ha-ras, suggesting that non-ras targets are involved. Ras pathway related genes, p53, WT1 and PCNA, were preferentially modulated in Ha-ras-mutated tumors, whereas modulation of genes in the G-protein pathway, various cytochrome p450s and RB1 are involved in Ha-ras wildtype tumors. Elucidation of gene expression changes in FTI-treated or control rat mammary adenocarcinomas will help in identifying potential pharmacodynamic markers of FTI treatment as well as potential molecular targets of R115777 and other FTIs.     

Analysis of oncogene alterations in human endometrial carcinoma: prevalence of ras mutations

The molecular genetics of human endometrial carcinoma have yet to be defined to any significant extent. Cell lines from 11 endometrial carcinomas were examined for alterations in proto-oncogenes that might predictably be present, based on existing data from the better-characterized human carcinomas of the uterine cervix, ovary, and breast. Codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes were examined for possible point mutations, and the c-erbB2/neu, c-myc, and epidermal growth factor receptor (EGFR) genes were examined for amplification or overexpression. Ras mutations were found in seven of 11 (64%) tumors, including three in codon 61 of Ha-ras (CAG----CAT) and four in codon 12 of Ki-ras (GGT----GAT in two and GGT----GTT in two). No evidence was found for amplification or overexpression of the c-erbB2 or EGFR genes in any tumor. One tumor contained amplified c-myc sequences and exhibited relative overexpression of c-myc. These data suggest that the amplification or overexpression of several proto-oncogenes frequently observed in other human gynecologic and breast tumors are not prevalent in endometrial carcinoma and that ras gene mutations are relatively common in this tumor type.     

Activation of the K-ras protooncogene in lung tumors from rats and mice chronically exposed to tetranitromethane

Dominant transforming genes were detected in lung tumors from Fischer 344 rats and C57BL/6 X C3H F1 mice chronically exposed by inhalation to tetranitromethane, a highly volatile compound used in several industrial processes. The rat lung neoplasms were classified as adenocarcinomas, squamous cell carcinomas (epidermoid carcinomas), or adenosquamous carcinomas. The mouse lung tumors were classified as papillary adenocarcinomas or adenomas. In both species, the tumors were morphologically similar to lung tumors in humans. The transfection assay using NIH/3T3 mouse fibroblasts detected transforming genes in 74% (14 of 19) of the rat lung tumors and in 100% (4 of 4) of the mouse lung tumors. Southern blot analysis indicated that transforming gene was an activated K-ras protooncogene in both species. The first exon of the K-ras gene in normal DNA and in DNA from two cell lines transformed by tumor DNA was compared by cloning and sequencing the gene. Experiments showed that there was a GC----AT transition in the second base of the 12th codon of the K-ras oncogene in the two transfectant DNAs. Oligonucleotide hybridization indicated that all of the rat and mouse transfectants had this activating lesion. Additional tumor DNA was then tested for the presence of a mutated allele with the GC----AT transition. All of the rat tumors tested and all of the mouse tumors tested had this mutation present. Hybridization using the normal oligonucleotide sequence around the 12th codon indicated that the normal allele was also present in the majority of the tumors, suggesting that the loss of normal allele is not necessary for the development of neoplasia. One rat lung tumor had no normal allele present, possibly suggesting that this tumor could have been in a more advanced stage than the other tumors. This is the first study to detect activated protooncogenes in rodent tumors induced under conditions which mimic human exposure to a chemical in the workplace. Tetranitromethane may exert its carcinogenic action by both activation of the K-ras oncogene and stimulation of cell proliferation by its irritant properties.     

Possible involvement of c-myc but not ras genes in hepatocellular carcinomas developing after spontaneous hepatitis in LEC rats.

LEC (Long-Evans with a cinnamon-like coat color) rats develop hepatocellular carcinomas (HCCs) spontaneously. We examined mutations of codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes in four HCCs by the polymerase chain reaction (PCR)-single-stranded DNA direct sequencing method. No ras gene mutations were observed, suggesting that ras activation is not involved in spontaneous hepatocarcinogenesis in LEC rats. The expression of mRNAs for c-myc, Ha-ras, c-raf, and the protein phosphatase 2A alpha gene (PP-2A alpha) was also examined in the four HCCs by northern blot analysis. Three of the four HCCs had c-myc expression levels approximately 30-fold higher than that in the liver of control Long-Evans rats with an agouti coat color (LEA), a sibling line of LEC rats, while the remaining HCC had an expression level sevenfold higher than that of control. In contrast, the expression levels of the Ha-ras, c-raf, and PP-2A alpha genes were the same as those in the livers of control rats. Studies of c-myc expression and mitotic index in five other HCCs, two hyperplastic nodules, and two nontumorous portions of livers of HCC-bearing LEC rats that had chronic-phase hepatitis suggested that the high level of c-myc gene expression was not due only to increased cell proliferation but might possibly be more integrally involved in hepatocarcinogenesis.     

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC-->AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA-->CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type.