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.     

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.     

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.     

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.     

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.     

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.     

Murine p53 intron sequences 5-8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors.

Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor--derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5-8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5-8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5-8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5-8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.     

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.     

Dose-related changes in the profile of ras mutations in chemically induced CD-1 mouse liver tumors

We investigated the role od dosing regimen on ras mutationsin chemically induced CD-1 mouse liver tumors. The spectra ofras gene mutations in liver tumors that were induced by 15 dailyi.p. injections of 7,12-dimethyl-benz[a]anthracene (DMBA), 4-aminoazobenzene(AAB), N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or N-nitrosodiethylamine(DEN) were compared to those previously obtained for tumorsinduced by a single but higher dose of each carcinogen. Theprincipal assay used was a direct tumor analysis involving sequencingof polumerase chain reaction (PCR)-amplified tumor DNA; additionalmutations that were present in only a small fraction of tumorcells were detected using a transfection assay or a PCR-engineeredrestriction fragment length polymorphism method. Spontaneousliver tumors had a relatively low frequency of ras mutations,all found in Ha-ras codon 61, and most of these mutations werepresent in only a small fraction of tumor cells. With the exceptionof multiple-dose DEN, each group of single- and multiple-dosecarcinogen-induced tumors exhibited a higher frequency of rasmutations compared with spontaneous tumors. For AAB, N-OH-AAFand DEN, the dosing regimen was found to affect significantlythe profile of rasmutations. For each of these carcinogens,the multiple-dose tumor group (versus single-dose group) hadfewer Ki-ras codon 61 (c     

Mutations at codon 61 of the Ha-ras proto-oncogene in precancerous liver lesions of the B6C3F1 mouse

Liver tumors of the B6C3F1 mouse frequently contain mutations at specific sites of codon 61 of the Ha-ras proto-oncogene. To address whether these mutations occur early or late during carcinogenesis, we analyzed mutations in the Ha-ras gene in small precancerous liver lesions of the B6C3F1 mouse. For this purpose, 10-microns frozen liver sections were prepared and stained for glucose-6-phosphatase activity. Using punching cannuli, we then took small tissue samples of approximately 5-30 micrograms from enzyme-deficient liver lesions and from normal parts of the liver. These tissue samples were analyzed for mutations in the Ha-ras gene by in vitro amplification of DNA via the polymerase chain reaction combined with selective oligonucleotide hybridization. By this approach we were able to analyze mutations in the Ha-ras gene within lesions with diameters of less than 0.5 mm. Our results demonstrate that approximately 15% of the glucose-6-phosphatase-negative lesions that occurred 24-28 wk after a single injection of diethylnitrosamine contain either C----A transversions at the first base or A----G transitions and A----T transversions at the second base of codon 61 of the Ha-ras gene. The same types of mutations, although with a somewhat higher frequency (33%), were found in liver tumors taken 68 wk after diethylnitrosamine treatment. These findings demonstrate that Ha-ras mutations can be detected even in very small precancerous liver lesions, suggesting that these mutations may be an early, perhaps even the first, critical event during murine hepatocarcinogenesis.     

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.     

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.     

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.     

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.     

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.     

Ras gene mutation and amplification in human nonmelanoma skin cancers

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain activated Ha-ras oncogenes capable of inducing morphologic and tumorigenic transformation of NIH 3T3 cells. In this study, we analyzed human primary squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) occurring on sun-exposed body sites for mutations in codons 12, 13, and 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 the primary human skin cancers, we also analyzed Ha-ras-positive NIH 3T3 transformants for mutations in the Ha-ras oncogene. The results indicated that all three NIH 3T3 transformants, 11 of 24 (46%) SCCs, and 5 of 16 (31%) BCCs contained mutations at the second position of Ha-ras codon 12 (GGC----GTC), predicting a glycine-to-valine amino acid substitution, whereas only 1 of 40 skin cancers (an SCC) displayed a mutation in the first position of Ki-ras codon 12 (GGT----AGT), predicting a glycine-to-serine amino acid change. In addition, three of the SCCs contained highly amplified copies of the N-ras oncogene in their genomic DNA. Interestingly, two of the SCCs containing amplified N-ras sequences also had G----T mutations in codon 12 of the Ha-ras oncogene. These studies demonstrate that mutations in codon 12 of the Ha-ras oncogene occurred at a high frequency in human skin cancers originating on sun-exposed body sites, whereas mutation in codon 12 of Ki-ras or amplification of N-ras occurred at a low frequency. Since the mutations in the Ha-ras and Ki-ras oncogenes were located opposite potential pyrimidine dimer sites (C-C), it is likely that these mutations were induced by ultraviolet radiation present in sunlight.     

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.      

Suppression of apoptosis in C3H mouse liver tumors by activated Ha-ras oncogene

Liver tumors were induced in male C3H mice by a single injection of N-nitrosodiethylamine and characterized with respect to the presence of base substitutions in the hot-spot position at codon 61 of the Ha-ras proto-oncogene. An increase in Ha-ras mutation prevalence was found with time after induction of tumors, suggesting that the activated ras gene provides a selective growth advantage. However, no significant differences in 5-bromodeoxyuridine labeling indices were evident between ras mutated and ras wild-type tumors, demonstrating that cell division rates in the two tumor populations were very similar. Apoptotic indices were determined by counting eosinophilic apoptotic bodies. The frequency of occurrence of apoptotic bodies was found to be approximately five times lower in tumors with Ha-ras mutations when compared with tumors not showing the mutation. This demonstrates that the activated p21(Ras) protein has anti-apoptotic activity in transformed mouse hepatocytes in vivo and suggests that the preferential outgrowth of Ha-ras-mutated hepatoma cells is mediated by suppression of apoptosis rather than by stimulation of cell division.     

Identification and quantification of a carcinogen-induced molecular initiation event in cell transformation.

All transformed foci of Balb/c 3T3 clone A31-1-1 cells induced by 7,12-dimethylbenz[a]anthracene (DMBA) (42 out of 42 examined) contained an A to T transversion at codon 61 (A182 to T) of the Ki-ras gene. The transformants induced by other carcinogens tested did not contain such a mutation, except one out of nine 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced transformed foci. Thus, we hypothesized that this mutation is a specific DMBA-induced initiating event in Balb/c 3T3 cell transformation and we have measured its frequency of induction before transformation occurs, employing our recently developed method. Such mutations can be detected in the cell population as early as 3 days after exposure to DMBA. The same mutation was also detected in the Ha-ras gene. No detectable level (< 10(-6) of these mutations was induced by other carcinogens tested. The mutation frequency of the Ha-ras gene reached a plateau after 1 week's exposure, but that of the Ki-ras gene continued to increase. These results suggest that the A182 to T mutation of the Ki-ras gene, but not that of the Ha-ras gene, contributes to morphological transformation of Balb/c 3T3 cells. We have demonstrated that the level of expression of ras genes determines the rate of recruitment of cells into transformation. Quantitative analysis of the frequencies of ras gene mutations (initiation) and of transformation suggests that about 25% of those cells with the Ki-ras mutation were recruited into the full transformation process and that, in the presence of the tumor promoter TPA, about 56% of them completed morphological cell transformation.     

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.