Detection and identification of activated oncogenes in human skin cancers occurring on sun-exposed body sites

High-molecular-weight DNA isolated from eight fresh human skin cancers occurring on sun-exposed body sites were assayed for their ability to transform NIH 3T3 cells. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select cells that had taken up the transfected DNA. About 2 weeks after transfection, G418-resistant colonies were pooled and injected s.c. into athymic nude mice. The NIH 3T3 cells transfected with DNA from six of the human skin cancers induced tumors in nude mice. DNAs from all six tumor cell lines contained human alu sequences. Southern blot hybridization with ras-specific probes revealed that DNAs from the four alu-rich tumors contained the human Ha-ras oncogene, in addition to that of the NIH 3T3 controls. In contrast, DNAs from the other two tumors did not contain any of the known oncogenes tested, except those endogenous to NIH 3T3 cells. DNAs from three of four first cycle tumorigenic transformants gave rise to morphologically transformed foci when assayed in a second cycle of transfection. DNAs from all three secondary transformants contained discrete human alu sequences, and in addition, contained Ha-ras sequences similar to those present in their respective primary transformants. Interestingly, DNA from both primary and secondary transformants of one particular human squamous cell carcinoma contained highly amplified copies of the Ha-ras oncogene. These results suggest that activation of the Ha-ras oncogene may be common in human skin cancers originating on sun-exposed body sites. Further characterization of the Ha-ras oncogenes present in these human skin cancers may provide information on the molecular mechanisms by which UV radiation of the sun induces human neoplasms on exposed body sites.     

Transformation of NIH3T3 cells by transfection with UV-irradiated human c-Ha-ras-1 proto-oncogene DNA

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain G----T mutations at the second position of Ha-ras codon 12. In this study, we investigated whether the c-Ha-ras-1 proto-oncogene could be activated by in vitro UV-irradiation of pEC plasmid DNA, which contains the 6.6 kb BamHI fragment of the human c-Ha-ras-1 proto-oncogene. Focus formation and nude mouse tumorigenicity assays showed that UV-irradiated pEC DNA induced morphologic and tumorigenic transformation of NIH3T3 cells in multiple cycles of transfection, whereas unirradiated pEC DNA did not. DNAs from secondary cycle foci and tertiary cycle tumors were analyzed for mutations in Ha-ras codons 12 and 61 using the polymerase chain reaction and synthetic oligonucleotide probes. Eleven of 11 secondary cycle foci analyzed possessed a G----T mutation at the second position of Ha-ras codon 12. However, the nude mouse tumors exhibited a G----A mutation at position 1 of the Ha-ras codon 12. These results suggest that in vitro UV irradiation of the c-Ha-ras-1 proto-oncogene DNA can induce mutations that are similar to those found in human skin cancers that originated on sun-exposed body sites.     

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.     

Ultraviolet-B-light-induced mutagenesis of C-H-ras codons 11 and 12 in human skin fibroblasts

Mutations in the ras oncogene are detected with a high frequency in non-melanoma skin cancer. Approximately half of the squamous-cell carcinomas (SCC) and one third of the basal-cell carcinomas (BCC) carry mutations at the second position of Ha-ras codon 12 (GGC to GTC), whereas mutations in Ki-ras codon 12 occur less frequently. Since the mutations in the Ha-ras and Ki-ras oncogenes are located opposite potential pyrimidine dimer sites (C-C), it is likely that the mutations are induced by ultraviolet radiation present in sunlight. We studied the capacity of ultraviolet B (UVB) light to induce base-pair changes in Ha-ras codons 11 and 12 in human skin fibroblasts. UVB induced mostly C to T and G to A transitions and C to A and G to T transversions. The base-pair change with the highest relative abundance was C to T in the middle position of codon 11 followed by (in diminishing relative abundance) C to A in the middle position of codon 11, G to A and G to T in the middle position of codon 12. The C to T and G to A transitions are compatible with pyrimidine photodimers as pre-mutagenic lesions, whereas the C to A and G to T transversions could be generated due to the formation of 8-hydroxyguanine, which is the major oxidation product of guanine. The relative abundance of mutations induced by UVB in Ha-ras codons 11 and 12 does not correlate with mutations observed in the DNA from non-melanoma skin cancer, where the G to T transversion in the middle position of codon 12 is selected.     

Frequent codon 12 Ki-ras mutations in mouse skin tumors initiated by N-methyl-N'-nitro-N-nitrosoguanidine and promoted by mezerein

The skin tumor initiators N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz[a]anthracene (DMBA) differ in effectiveness when tumor formation is promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Even at high doses, MNNG is less effective, producing fewer benign and malignant tumors with a longer latent period. In DMBA-initiated skin, 10 wk of TPA promotion produced a maximal tumor response. With MNNG, 20 wk of TPA promotion was required, producing nearly four times as many papillomas as 10 wk of promotion. Promotion of MNNG-initiated skin with mezerein induced the appearance of very rapidly-growing papillomas within 5 wk, 3 wk earlier than the first TPA-promoted papillomas. Thus, MNNG may induce a novel mutation resulting in a population of initiated cells that respond especially well to mezerein. Since ras mutations are common in experimental tumors in many tissues, we determined the frequency of activating mutations in the Ha-ras, Ki-ras, and N-ras oncogenes. Activating Ha-ras mutations were present in essentially all DMBA-initiated tumors and about 70% of MNNG-initiated tumors. No N-ras mutations were found in tumors lacking other ras mutations. Surprisingly, 41% of the papillomas arising in the first 11 wk in MNNG-initiated, mezerein-promoted mice bore mutations in codon 12 of the Ki-ras oncogene. Activating Ki-ras mutations were also found in more than 60% of squamous cell carcinomas and 40% of keratoacanthomas. Although mutations in Ha-ras are frequently detected in mouse skin tumors, mutations in Ki-ras are rare. This is the first report of mutated Ki-ras in skin tumors from mice initiated by MNNG.     

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.     

Analysis of ras DNA sequences in rat renal cell carcinoma

The DNA sequences for Ha-, Ki-, and N-ras were determined in six cell lines derived from independent rat hereditary renal cell carcinomas (RCC). Genomic regions encompassing codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras, and codon 117 of Ha-ras were PCR amplified and directly sequenced. The DNA sequences of Ha-ras and Ki-ras were normal in all lines tested, as were the codon 12 and 61 sequences of N-ras. However, DNA sequence variations that could code for amino acid substitutions were observed in codons 13, 14, and 18 of N-ras in all the lines. The codon 13 Gly----Val alteration observed was consistent with activating N-ras mutations previously reported. When normal kidney DNA from rats with the hereditary tumor syndrome was sequenced, the same N-ras sequence variations observed in the tumor lines were found. DNA from outbred Long-Evans and inbred Fischer rats also had the altered N-ras sequences. The variant N-ras sequence was not observed in PCR-amplified N-ras cDNA from the RCC lines. Thus, tumor-associated activation of ras oncogene appears to be an infrequent event in spontaneous rat RCC. In addition, these data indicate that rats contain an N-ras DNA polymorphism that appears to be a species-specific anomaly.     

Detection of activated ras oncogenes in human thyroid carcinomas

Focus formation following DNA transfection of mouse 3T3-Vill cells was used to search for the presence of activated oncogenes in human thyroid tumors. Oncogenes belonging to the ras family were detected in four out of six thyroid carcinomas (Ki-ras in one anaplastic tumor and one follicular moderately differentiated tumor and Ha-ras and N-ras in two papillary tumors). Normal thyroid tissue samples obtained from two patients, one with an anaplastic tumor and one with a benign adenoma, and samples from 4 benign adenomas and from one toxic goiter of a patient with Graves' disease gave negative results. In one case, restriction enzyme analysis demonstrated the presence of a mutation in codon 12 of the activated Ha-ras oncogene. Our data show that all three ras proto-oncogenes can become activated in malignant thyroid tumors.     

Activating point mutation in Ki-ras codon 63 in a chemically induced rat renal tumor.

Renal mesenchymal tumors induced in F344 rats with methyl(methoxymethyl)nitrosamine (DMN-OMe) have previously been shown by our laboratory to contain transforming Ki-ras sequences, activated most commonly by a variety of codon 12 mutations. Further sequence analysis of the one DMN-OMe-induced tumor with transforming Ki-ras sequences detected by NIH 3T3 transfection assay but with no mutation in codon 12 detected by selective oligonucleotide hybridization has now revealed an activating point mutation in codon 63. The observed GAG----AAG transition in codon 63, which replaces glutamic acid with lysine, was the only detectable mutation in exon 1 and 2 hotspot regions of Ki-ras in this tumor. The same mutation was also detected in Ki-ras sequences derived from first- and second-cycle transformants in NIH 3T3 transfection assays. Although random mutagenesis studies of cloned Ha-ras sequences by Fasano et al. (Proc Natl Acad Sci USA 81:4008-4012, 1984) had already indicated that GAG----AAG mutations in codon 63 of ras are transforming, this is the first demonstration of the natural occurrence of this particular activating mutation in a tumor.     

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.     

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.     

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.     

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.     

ras gene activation in a minor proportion of the blast population in acute myeloid leukemia

DNAs from 22 acute myeloid leukaemia (AML) patients were screened for activated transforming genes using NIH3T3 transfection followed by assay for tumor formation in Nude mice. In four samples an activated N-ras oncogene, and in two samples an activated Ha-ras oncogene were detected in transfectants. Synthetic oligonucleotide probes were used to characterise the mutations in the ras genes. Three samples were found to be mutated to N-ras codon 12 ASP, one to N-ras codon 13 ASP and two to Ha-ras codon 12 VAL. When the corresponding AML DNAs were screened using direct gel hybridisation, the mutant ras genes were detectable in only one case. In two AML samples (82 and 84) with very low percentage blasts (3% and 19%), the absence of mutant ras signal from direct gel hybridisation may be due to the lack of sensitivity of this technique in detecting activated ras in such small fractions of the total DNA. These results illustrate the sensitivity of the in vivo tumour assay in detecting activated ras. When DNA from one of the remaining three AML DNAs was amplified using the polymerase chain reaction method, the mutation present in the transfectant was detected. These findings suggest that even in AMLs with high percentage blasts (40%, 70% and 90%), cells containing mutant ras may comprise only a minor proportion of the major leukaemic clone.     

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.     

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.     

Ras mutations in United Kingdom examples of oral malignancies are infrequent

Point mutations in codons 12, 13 or 61 of the oncogenes Ha-ras, Ki-ras or N-ras have been identified in human malignancies of many types. Using the PCR (polymerase chain reaction) technique for DNA amplification in vitro and stringent probing of the amplified DNA on dot blots with a library of specific oligonucleotides, we have screened for the presence of ras mutations in oral and para-oral malignancies and some associated lesions. The material, from UK patients, consisted of 22 oral squamous-cell carcinomas including 5 neck metastases, 1 oral mucosal dysplasia, 1 proliferative verrucous leukoplakia, 1 antral and 1 tonsillar carcinoma, 1 basal-cell carcinoma, 1 salivary adenocarcinoma, 1 salivary adenoid cystic carcinoma and 1 lung adenocarcinoma metastatic to the gingiva. Genomic DNA was extracted from tissues which were fresh or preserved in liquid nitrogen. Two DNA samples contained point mutations in codon 61 of Ki-ras. One of these mutations was in the lymphocytes infiltrating a retromolar SCC. The other mutation (CAA to CAU; substitution of glutamine by histidine) was in the lung adenocarcinoma metastasis. The absence of ras mutations in the epithelium of primary oral squamous-cell carcinomas is of considerable interest as other work in our Department on Indian cases of oral carcinomas associated with chewing tobacco (quid) revealed that 35% of these had a codon 12, 13 or 61 mutation in Ha-ras. While ras activations arising from point mutations may occur in a high proportion of oral malignancies associated with chewing tobacco (quid), this was not the case in UK oral malignancies, even where tobacco was smoked.