H-ras mutations at codon 61 or 13 in tumors initiated with a NO donor in mouse skin

The tumor-initiating activity of nitric oxide (NO) in carcinogenesis was assessed using (+/-)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1), a synthetic NO donor. Topical application of NOR1 followed by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment twice a week for 20 weeks resulted in the development of papillomas in mice. All of the papillomas examined contained H-ras mutations at codons 61 or 13. At codon 61, CAA-CTA and CAA-TTA mutations were seen in 42/46 and 1/46 of the papillomas, respectively. Three papillomas without a mutation at codon 61 were mutated at codon 13. A GGC-CGC mutation was found in two of these samples while the third possessed a GGC-GTC mutation. These results suggest that NO possesses tumor-initiating activity through a process that induces mutation in H-ras.     

Transplacental mutagenicity of cisplatin: H-ras codon 12 and 13 mutations in skin tumors of SENCAR mice

Cisplatin is an anticancer agent sometimes used in pregnantwomen. It is also a potent initiator of skin tumors in micewhen administered transplacentally. For characterization ofthe transplacental mutagenicity of cisplatin, tumors initiatedin fetal skin by cisplatin or 7,12-dimethyI-benz[     

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.     

Metastasis from squamous cell carcinomas of SENCAR mouse skin produced by complete carcinogenesis

The incidence of metastasis was evaluated in female SENCAR mice after induction of squamous cell carcinomas by repetitive applications of either benzo [a] pyrene (B [a] P) or N-methyl-N'-nitro-N-nitrosogaunidine (MNNG). Between 41 and 50 weeks 50% of the animals with carcinomas in the B [a] P group had metastases, whereas 20% had metastases in the MNNG group. Very few metastases were observed before 40 weeks of treatment. The major site of metastasis was the lungs; however, metastatic tumors were also found in lymph nodes, adrenal glands and kidneys.     

Inhibition of chrysarobin skin tumor promotion in SENCAR mice by antioxidants

The present study was designed to further investigate the roleof reactive oxygen species in the mechanism of action of anthronetumor promoters. To accomplish this, the effects of severalantioxidants on the induction of epidermal ornithine decarboxylase(ODC) activity, epidermal hyperplasia, skin edema, and skintumor promotion by chrysarobin [1, 8-di-hydroxy-3-methyl-9-anthrone)were tested. Ascorbyl palm-itate (AP), given 5 min prior tothe promoter at 1 and 4 µmol doses, effectively inhibitedthe induction of ODC activity (28% and 59%, respectively) by220 nmol of chrysarobin. Using a similar protocol,     

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.     

Dietary fat effects on the initiation and promotion of two-stage skin tumorigenesis in the SENCAR mouse

We investigated the influence of dietary corn oil on initiation of skin tumors in SENCAR mice with 7,12-dimethylbenz(a)anthracene (DMBA) (10 nmol at 8 to 9 wk of age) and the promotion of these tumors with 12-O-tetradecanoylphorbol-13-acetate (TPA) (3.2 nmol twice weekly for 20 wk). Diet high in corn oil (24.6%) was fed, in comparison with control diet (5%), to mice during two time schedules: (a) high-fat diet was fed preceding and for 1 wk following DMBA to assess the effects of high corn oil diet on initiation; and (b) high-fat diet was fed starting at the time of the first TPA treatment (1 wk following DMBA initiation) until the end of the experiment to assess effects of high corn oil diet on promotion. Mice were trained to consume equivalent caloric allotments of the low- and high-fat diets to ensure that the observed effects on tumor development were for dietary fat at constant calorie intake. Feeding high corn oil diet during DMBA treatment did not influence the incidence of skin papilloma or carcinoma, but the number of papillomas per effective mouse was reduced in mice fed the high-fat diet during initiation. Consumption of the high corn oil diet during and following TPA treatment resulted in an increase in the incidence of papillomas up until Wk 14 of the experiment, an increase in the number of papillomas per effective mouse throughout the experiment, and an increase in the number of carcinomas per effective mouse during Wk 25 to 34. However, cumulative carcinoma yield (Wk 25-44) did not differ between the diet groups. Dietary treatment did not influence food consumption, body weight, or survival in the mice treated with DMBA and TPA. Northern blot hybridization studies were carried out on RNA purified from tumors of high- and low-fat mice to determine if diet influenced the pattern of Ha-ras oncogene expression. The results of this experiment indicated that elevated levels of Ha-ras-specific RNA, in comparison with normal epidermal RNA, were present in papillomas and carcinomas from DMBA-initiated, TPA-promoted mice irrespective of the diet the mice were fed.     

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.     

Codon 61 mutations in the c-Harvey-ras gene in mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene plus okadaic acid class tumor promoters

Three okadaic acid class tumor promoters, okadaic acid, dinophysistoxin-1, and calyculin A, have potent tumor-promoting activity in two-stage carcinogenesis experiments on mouse skin. DNA isolated from tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and each of these tumor promoters revealed the same mutation at the second nucleotide of codon 61 (CAA----CTA) in the c-Ha-ras gene, determined by the polymerase chain reaction procedure and DNA sequencing. Three potent 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, TPA, teleocidin, and aplysiatoxin, showed the same effects. These results provide strong evidence that this mutation in the c-Ha-ras gene is due to a direct effect of DMBA rather than a selective effect of specific tumor promoters.     

Characterization of skin tumor promotion and progression by chrysarobin in SENCAR mice

The characteristics of the skin tumor promotion response with anthrone derivatives has been further examined in SENCAR mice. Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) was an effective skin tumor promoter when applied twice weekly with dose-dependent increases in both papillomas and squamous cell carcinomas between 25 and 100 nmol/mouse. A similar dose-response relationship for papilloma and carcinoma formation was observed when chrysarobin was applied once weekly. Interestingly, chrysarobin was approximately twice as active as a skin tumor promoter when applied once weekly versus twice weekly. Doses of 25,100, and 220 nmol/mouse gave maximal papilloma responses of 2.90, 8.15, and 9.38 versus 0.73, 4.70, and 5.42 papillomas/mouse, respectively, in mice initiated with 25 nmol 7,12-dimethylbenz(a)anthracene. Thus, unlike 12-O-tetradecanoylphorbol-13-acetate (TPA), where a twice weekly application frequency is optimal, application of anthrone promoters such as chrysarobin once weekly is a more optimal frequency for papilloma development. Chrysarobin was also a much more effective skin tumor promoter when the start of promotion was delayed by an additional 10 weeks. Thus, groups of mice initiated with 10 nmol 7,12-dimethylbenz(a)anthracene and having promotion started in either the 3rd or the 13th week after initiation had maximal responses of 5.6 or 11.0 papillomas/mouse, respectively. In addition, the rate of papilloma development was faster in the delayed promotion group. The progression of papillomas to carcinomas was examined in all chrysarobin-treated groups and compared with three groups of mice treated with 3.4 nmol TPA. After 60 weeks of promotion, the anthrone promoter-treated groups had carcinoma:papilloma ratios 2.5 to 5.0 times higher than the TPA-treated groups. This was due primarily to the fact that similar carcinoma responses were observed in both anthrone- and TPA-treated mice at optimal promoting doses whereas the papilloma responses were significantly lower in the former groups. The data suggest that anthrone derivatives are very efficient tumor promoters. The results are further discussed in terms of mechanisms of skin tumor promotion.     

Epidermal papillomas and carcinomas induced in uninitiated mouse skin by tumor promoters alone contain a point mutation in the 61st codon of the Ha-ras oncogene

Previous results in a number of laboratories have demonstratedthat epidermal papillomas and carcinomas induced by the two-stageprotocol of initiation and promotion contain a point mutationin the 61st codon of the c-Ha-ras oncogene when the initiatingagent used is 7,12-dimethylbenz-[a]anthracene (DMBA). In thepresent report, we have analyzed DNA purified from ‘spontaneouslyinitiated’ papillomas and carcinomas induced in SENCARmouse epidermis after repetitive treatments with a tumor-promotingagent. Southern blot hybridization studies of tumor DNA digestedwith diagnostic restriction endonudeases demonstrated that sevenof nine papillomas and carcinomas contained a point mutationin the 61st codon of one allele of the c-Ha-ras oncogene. Theimplications of our findings with respect to the role whicha point-mutated Ha-ras protooncogene plays in initiation ofskin tumorigenesis are discussed.     

Further characterization of skin tumor promotion and progression by mezerein in SENCAR mice

This study evaluated the skin tumor-promoting activity of mezerein in SENCAR mice. The effect of initiation dose of 7,12-dimethylbenz(a)anthracene (DMBA) on tumor promotion by mezerein was examined. Excellent dose-response relationships were observed for initiation with DMBA at 0.2-20 micrograms per mouse with mezerein as a complete promoter. None of the mezerein-only promotion groups had papilloma responses similar to those of the corresponding groups receiving two-stage promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) followed by mezerein, even when a 40-micrograms initiating dose of DMBA was used. The effect delaying promotion with mezerein for 10 weeks was also examined in mice initiated with either 0.2, 2, 20, or 40 micrograms of DMBA per mouse. The 10-week delay led to a slight increase in the number of papillomas per mouse in some but not all treatment groups. Again, none of the delayed-mezerein-treatment groups had papilloma responses similar to those of the corresponding two-stage promotion (TPA-mezerein) groups at any corresponding initiating dose of DMBA. Finally, the progression of papillomas to carcinomas during promotion with mezerein was examined in groups of mice initiated with either 2 or 20 micrograms of DMBA. Higher ratios of carcinomas to papillomas were observed in mice promoted with mezerein than in mice receiving TPA promotion or two-stage promotion (TPA-mezerein). However, the presence of two to four times more papillomas in some mezerein-treated groups did not lead to greater numbers of carcinomas than in the groups with fewer papillomas. The data do not support the idea that spontaneous stage I promotion can be induced by delaying mezerein treatment for 10 weeks. Furthermore, the data suggest that the higher ratio of carcinomas to papillomas observed with mezerein promotion may be a function of the lower tumor burdens obtained after promotion with this compound rather than a specific property of the chemical.     

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.     

Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR

Mutation of the Kirsten ras (K-ras) gene is one of most common alterations in solid tumors including lung and colorectal cancers. We developed new enriched PCR-RFLP assay to detect mutations of K-ras codon 61 at the 1st and 2nd letters and non-enriched PCR-RFLP assay to detect the 3rd letter mutation. One mutant allele among 10(3) wild-type alleles was detected by enriched PCR-RFLP assay, while one mutant in 10 wild-type alleles was detected by non-enriched PCR-RFLP assay for codon 61 3rd letter. We then examined K-ras codon 12, 13 and 61 mutations in lung and colorectal cancers using these assays. K-ras codon 12 mutation was detected in 10 of 109 (9%) lung cancer and 19 of 83 (23%) colorectal cancer cases. K-ras codon 13 mutation was detected in 2 of 83 (2%) colorectal and 0 of 109 NSCLC cases, respectively. There was no K-ras codon 61 mutation in either type of cancer. Our results demonstrate that enriched PCR-RFLP is a sensitive assay to detect K-ras codon 61 mutation, however, it was extremely rare in lung and colorectal cancers, suggesting organ-specific pathways in mutagenesis of the ras gene family.     

Influence of diet and calorie restriction on the initiation and promotion of skin carcinogenesis in the SENCAR mouse model

Diets were restricted to 60% of the intake of the control mice by feeding less diet (total diet restriction, TDR) or by feeding fewer calories from fat and carbohydrate (calorie restriction, CR) during the initiation or promotion phases of skin tumorigenesis in female SENCAR mice. Skin cancer was initiated by topical treatment with 10 nmol of 7,12-dimethylbenzanthracene in acetone and promoted by twice weekly treatments with 12-O-tetradecanoylphorbol-13-acetate in acetone for 20 wk. Dietary restriction preceding and during 7,12-dimethylbenzanthracene treatment did not influence skin papilloma or carcinoma yield. Papilloma incidence and the number of papillomas per effective mouse were reduced in mice restricted by both TDR and CR protocols during and following promotion with 12-O-tetradecanoylphorbol-13-acetate. Papilloma size was reduced at experimental wk 16 and 20 in both TDR and CR groups fed these diet regimens during promotion. However, by wk 28 and 32, papilloma sizes were similar in the control and TDR groups, and smaller papillomas were observed only in the CR group. The average carcinoma latency was extended by 26% in the groups restricted during promotion, and incidence was reduced in both groups. The reduction, however, was statistically significant only in the CR group. Body weight gain was reduced during the times when dietary restriction was enforced, and in a short-term study, both restricted diet treatments reduced the percentage of carcass protein.     

Bryostatin 1, an activator of protein kinase C, inhibits tumor promotion by phorbol esters in SENCAR mouse skin

Bryostatin 1, like the phorbol esters, activates protein kinaseC. However, bryostatin 1 induces only some of the effects incultured cells which result from phorbol ester treatment, whereasit blocks other responses to the phorbol esters. In mouse keratinocytesin particular, bryostatin 1 induces ornithine decarboxylase,a marker of proliferation, but blocks induction of markers ofdifferentiation. Because of the postulated role of inductionof differentiation in tumor promotion, we have now examinedbryostatin 1 as a tumor promoter and as an inhibitor of phorbolester tumor promotion in the initiation—promotion modelof skin carcinogenesis. After initiation with 7,12-dimethylbenz[a]anthracene,weekly topical treatments of the backs of mice with 1 µg(1.1nmol) bryostatin 1 induced epidermal hyperplasia and inflammation,although not to the extent seen after treatment with the promoter12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment with bryostatin15 min before each TPA exposure reduced the phorbol ester-inducedhyperplasia. Bryostatin 1 was ineffective as a complete tumorpromoter and displayed very weak activity as a second stagepromoter upon treatment of initiated mice for 30 weeks. Combinedexposure of mice to bryostatin 1 and TPA resulted in a substantialinhibition of promotion by TPA. Our in vivo results extend earlierin vitro findings that bryostatin 1 acts as a partial inhibitorof protein kinease C function.     

Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane

The beta-catenin gene is frequently mutated at codons 33, 41 and 45 of the glycogen synthase kinase-3beta phosphorylation motif in human colon cancers in patients without APC mutations. Frequent mutations at codons 32 and 34, as well as 33 and 41, have been detected in rat colon tumors induced by azoxymethane (AOM), with the second G of CTGGA sequences being considered as a mutational hot-spot. In the present study, exon 3 of the beta-catenin gene in mouse colon tumors induced by AOM was amplified by PCR and mutations were detected by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. All 10 colon tumors tested were found to have beta-catenin mutations, four in codon 34, three in codon 33, two in codon 41 and one in codon 37, nine being G:C-->A:T transitions. However, no mutations were found in codon 32 of the mouse beta-catenin gene. On immmunostaining, beta-catenin was observed in the cytoplasm and nucleus of the tumor cells. The cytoplasmic staining was homogeneous, while both homogeneous and heterogeneous patterns were noted for the nuclei. Highly frequent mutations of the beta-catenin gene in AOM-induced mouse colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.     

Analysis of c-Ha-ras gene mutations in skin tumors induced in carcinogenesis-susceptible and carcinogenesis-resistant mice by different two-stage protocols or tumor promoter alone

In the present study we describe the molecular analysis of c-Ha-ras gene mutations in 47 papillomas and 17 carcinomas developed in two lines of mice, carcinogenesis-susceptible (Car-S) and carcinogenesis-resistant (Car-R), selectively bred for extreme susceptibility or resistance to chemical skin carcinogenesis initiated and promoted with different doses of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This study also presents the analysis of c-Ha-ras gene mutations in 22 papillomas and 22 carcinomas in Car-S mice initiated with DMBA and promoted with benzoyl peroxide (BzPo) and in seven papillomas and one carcinoma from a group of uniniated Car-S mice that received only BzPo treatment. The data showed that a A(182)-->T transversion in the c-Ha-ras gene was present in 100% and 81% of the skin tumors developed in Car-S and Car-R mice, respectively, after DMBA initiation and TPA promotion, suggesting that differences in genetic susceptibility can influence the frequency of c-Ha-ras mutations in the skin tumors produced. The same A(182)-->T mutation with an incidence of 68% was found in papillomas from DMBA-initiated and BzPo-promoted Car-S mice. The difference in the mutation frequency between DMBA/BzPo and DMBA/TPA papillomas suggested that the promotion step contributes to the final mutation pattern. The tumor induction experiment with BzPo alone showed that this compound can induce tumor development in 26% of Car-S mice, and the molecular analysis of the tumors showed a broad mutation spectrum, including mutations in codons 12, 13, and 61 of the c-Ha-ras gene. Mol. Carcinog. 30:111-118, 2001.     

Deficiencies in mouse Myh and Ogg1 result in tumor predisposition and G to T mutations in codon 12 of the K-ras oncogene in lung tumors

Oxidative DNA damage is unavoidably and continuously generated by oxidant byproducts of normal cellular metabolism. The DNA damage repair genes, mutY and mutM, prevent G to T mutations caused by reactive oxygen species in Escherichia coli, but it has remained debatable whether deficiencies in their mammalian counterparts, Myh and Ogg1, are directly involved in tumorigenesis. Here, we demonstrate that deficiencies in Myh and Ogg1 predispose 65.7% of mice to tumors, predominantly lung and ovarian tumors, and lymphomas. Remarkably, subsequent analyses identified G to T mutations in 75% of the lung tumors at an activating hot spot, codon 12, of the K-ras oncogene, but none in their adjacent normal tissues. Moreover, malignant lung tumors were increased with combined heterozygosity of Msh2, a mismatch repair gene involved in oxidative DNA damage repair as well. Thus, oxidative DNA damage appears to play a causal role in tumorigenesis, and codon 12 of K-ras is likely to be an important downstream target in lung tumorigenesis. The multiple oxidative repair genes are required to prevent mutagenesis and tumor formation. The mice described here provide a valuable model for studying the mechanisms of oxidative DNA damage in tumorigenesis and investigating preventive or therapeutic approaches.