Induction of mutations in Ki-ras and INK4a in liver tumors of mice exposed in utero to 3-methylcholanthrene

An understanding of the basic mechanisms responsible for the pathogenesis of liver neoplasms is needed in order to develop better therapeutic strategies. The present study utilized a pharmacogenetic mouse model to assess the role of cytochrome P4501A1 (Cyp1a1) in modulating genetic damage to oncogenic and tumor suppressor loci following in utero exposure to the polycyclic aromatic hydrocarbon, 3- methylcholanthrene (MC). Analysis of the Ha-ras, Ki-ras, INK4a and p53 genes was carried out with lysates from paraffin-embedded liver tissue from transplacentally-treated mice. The lysates were subjected to DNA amplification by the PCR technique followed by allele-specific oligonucleotide hybridization screening and SSCP analysis. All of the 26 neoplasms screened (23 hepatocellular carcinomas, two hepatocellular adenomas and one sarcoma) exhibited a GGC-->CGC (GLY13-->ARG13) transversion at the Ki-ras gene locus. None of the tumors had Ki-ras mutations at codon 12 of exon 1. Approximately 12% (3/26) of the liver tumors exhibited point mutations in exon 1 of the INK4a gene, with each of the three tumors exhibiting two point mutations. Analysis of exon 2 of the INK4a gene showed the presence of a CCG-->CTG (PRO73-->LEU73) transition in two of the 26 neoplasms. No mutations were found in exons 1 or 2 of the Ha-ras gene, or in exons 5-8 of the p53 gene. Analysis of tumor RNAs showed overexpression of Ha-ras, cip1 and c-jun in approximately 38% of the liver tumor samples. The results of this study suggest that mutagenic damage to oncogenes and tumor suppressor genes may be critical factors in mediating transplacentally-induced liver tumorigenesis. The fact that Ki-ras mutations were found in all of the tumors suggests that mutation at this gene locus may be an early event in liver tumor pathogenesis, while mutation in tumor suppressor genes may occur later during tumor progression. These combined results are consistent with the pathogenesis of cancer in humans.      

H-ras 61st codon activation in archival proliferative hepatic lesions isolated from female B6C3F1 mice exposed to the leukotriene D4-antagonist, LY171883

LY171883, a peroxisome proliferator and leukotriene D_4–antagonist,induced a statistically significant increase in the number ofhepatic lesions in B6C3F1 female mice in a 2 year oncogenicitystudy at dietary doses of 0.0225% and 0.075%. The mutation frequencyand spectrum of the 61st codon of H-ras was determined for 64independent, archived lesions from the LY171883 2 year oncogenicitystudy using the polymerase chain reaction (PCR), allele specificoligo hybridization (ASO) and DNA sequencing. Results showed41 (64%) of these lesions had mutations at the 61st codon (16/21hepatocellular carcinomas, 4/10 hepatocellular adenomas, 19/26focal hepatocellular hyperplasias and 2/7 focal hepatocellularatypia). These mutations consisted of 18 C-A transversions,16 A-G transitions and seven A-T transversions. Compared tothe mutation frequency for spontaneously occurring archivalB6C3F1 hepatic lesions (41%), the frequency of LY171883 lesions(64%) was significantly higher (P < 0.01). Hie frequenciesof H-ras 61st codon mutations among the LY171883 lesion types(hepatocellular carcinomas 76%, hepatocellular adenomas 40%,focal hepatocellular hyperplasias 73% and hepatocellular atypia29%) were also significantly different (P = 0.035). In contrast,spontaneous lesions showed no statistical difference in thefrequencies of mutation among lesion types (P > 0.5). Themutation spectrum of the LY171883 lesions was not significantlydifferent from the spontaneous spectra. It may be concludedthat based on the similarity in mutation spectrum and the increasein mutation frequency, LY171883 may selectively promote spontaneoushepatic lesions containing H-ras 61st codon mutations. In addition,the difference in mutation frequency among lesion types doesnot support a linear progression of all LY171883 lesions throughfocal atypia, focal hepatocellular hyperplasias, hepatocellularadenomas and hepatocellular carcinomas.     

Ki-ras mutation and cell proliferation of lung lesions induced by 1-nitropyrene in A/J mice

In this study, lung lesions were found in male A/J mice 24 wk after intraperitoneal injection of 1-nitropyrene (1-NP). The lesions were classified into three categories: alveolar/bronchiolar hyperplasia, adenoma, and adenocarcinoma. The proliferation kinetics of cells in the lesions were evaluated by assessing proliferating cell nuclear antigen (PCNA) expression and silver-staining nucleolar organizer regions (AgNORs). Furthermore, the role of the Ki-ras gene in tumorigenesis was studied by detecting point mutations in Ki-ras codons 12, 13, and 61 by polymerase chain reaction and sequence analysis. The PCNA-positive rates (± standard deviations) in various samples were as follows: 0% for specimens from six untreated animals and six uninvolved areas, 4.26 ± 3.94% for 19 hyperplasias (hyperplasias vs normal lung tissue, P < 0.01), 13.24 ± 6.35% for 25 adenomas (adenomas vs hyperplasias, P < 0.01), and 38.0 ± 9.63% for four adenocarcinomas (adenocarcinomas vs adenomas, P < 0.01). The corresponding mean AgNOR scores were as follows: 1.10 ± 0.05 for the untreated animals, 1.32 ± 0.09 for the uninvolved areas, 1.72 ± 0.59 for the hyperplasias (hyperplasias vs normal lung tissue, P < 0.05), 2.74 ± 0.70 for the adenomas (adenomas vs hyperplasias, P < 0.01), and 5.22 ± 0.62 for the adenocarcinomas (adenocarcinomas vs adenomas, P < 0.01). Ki-ras gene mutations were identified in three of four (75%) adenocarcinomas, six of 23 (26%) adenomas, and two of 17 (12%) hyperplasias. No mutations were found in normal lung tissue. The most frequent Ki-ras mutation was an arginine (CGA) AT → GC transition at codon 61 in exon 2. The PCNA-positive rates and AgNOR scores of cases with Ki-ras mutations were higher than those without an identified mutation (P < 0.05). Ki-ras mutations at codon 61 (Arg) may therefore influence the growth or development of 1-NP–induced lung lesions in A/J mice. Mol. Carcinog. 22:258–264, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Distribution of p53 and K-ras mutations in human lung cancer tissues

Studies were performed to examine the mutational pattern of K-ras exons 1 and 2 and p53 exons 5-8 in lung cancer tissues from 27 Chinese patients (10 smokers, 17 non-smokers) using single-stranded conformational polymorphism and DNA sequencing. K-ras mutations were found in 13/27 tumors (48%); all mutations were clustered in exon 1 and distributed between codons 9 and 32. The frequency and number of patients with K-ras mutations between smokers and non-smokers were not different, except that a high frequency of G --> A transitions (11/11) was found in non-smokers. Among cell types, K-ras mutations were found in 7/13 (54%) squamous cell carcinoma (SC) and 5/12 (42%) adenocarcinoma (AC) patients. A --> T transversions (all six transversions) were present only in SC. In p53, 18/27 (67%) tumors contained mutations in exons 7 and 8, frequently at codons 226, 270, 275 and 281. The number of tumors with p53 mutations in smokers (70%) and in non-smokers (65%) was similar, and the mutation frequency did not differ except for a higher number of G --> A (6/7) and T --> C (5/6) transitions in non-smokers. Among cell types, the number of tumors with p53 mutations was 9/13 (69%) in SC and 8/12 (67%) in AC. The A --> G (11/16) transitions and A --> C (4/4) transversions in p53 were more frequent in SC than in AC (P < 0.04 for A --> G; P < 0.02 for A --> C). The varying mutation patterns in both the K-ras and p53 genes between smokers and non-smokers and among cell types suggest that other than cigarette smoke, environmental and dietary factors may also be involved in the genesis of lung cancer among these patients.      

Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene.

The carcinogenic effects of in utero exposure to 3-methylcholanthrene (MC) have been demonstrated in the tumor-resistant C57BL/6 (B6) and DBA (D2) strains of mice. In this study, we determined the effects of in utero exposure to MC in BALB/c mice, a strain which demonstrates greater susceptibility to lung tumor induction, and compared our findings with those previously found in [D2xB6D2F(1)]F(2) mice. In addition, we assessed the molecular pathogenesis of the chemically induced tumors and examined the effects of the putative lung tumor promoter butylated hydroxytoluene (BHT) in BALB/c mice. BALB/c mice were treated on day 17 of gestation with 5, 15 or 45 mg/kg MC and 6 weeks after birth with BHT for 6 consecutive weeks. Mice were killed at 6 months of age. Ki-ras, p16Ink4a and p19ARF gene loci were amplified from paraffin-embedded lung tumor tissue and screened for the presence of point mutations via allele-specific oligonucleotide hybridization and single strand conformation polymorphism (SSCP) analyses. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung tumors, with 33% (2/6) of the hyperplasias, 58% (10/19) of the adenomas and 73% (8/11) of the carcinomas exhibiting point mutations at this gene locus. Similar incidences of Ki-ras mutations were previously found following transplacental exposure of [D2xB6D2F(1)]F(2) mice to MC and treatment of adult A/J mice with urethane. Interestingly, a strain-dependent difference was observed in the mutational spectrum. Sixty-two and 38% of the lung lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast to the 13 and 84% incidences previously observed in [D2xB6D2F(1)]F(2) mice. SSCP analysis of the tumor suppressor gene p16Ink4a showed a 6% incidence of point mutations, consistent with that found in [D2xB6D2F(1)]F(2) mice. No mutations were found in exon 1beta of the p19ARF gene of either strain. BHT, a lung tumor promoter in adult mice, had no statistically significant effects on either tumor incidence, tumor multiplicity or the mutational spectrum produced in the Ki-ras gene by in utero MC treatment. However, though not significant, there was an observable trend in increased tumor multiplicity in mice co-treated with BHT. These data demonstrate the transplacental carcinogenic effect of MC in BALB/c mice and show that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both the tumor-sensitive and tumor-resistant strains. Unlike what occurs when adult BALB/c mice are treated with MC, BHT does not appear to significantly promote the formation of lung tumors following transplacental exposure to MC, possibly due to the rapid growth and cell proliferation in the developing organism. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.     

Transitions and transversions in Ki-ras gene in colorectal cancers in Mexican patients

AIMS & BACKGROUND: An important increase in the incidence of colorectal cancers has been detected in the last 15 years in Mexico. This fact has been attributed to several causes, including the change in diet acquired from industrialized countries. Various groups have studied the mutational pattern of oncogenes, including Ki-ras gene, in colorectal cancers from different human populations. The aim of this work was to study the prevalence of mutations at codons 12, 13 and 61 of the Ki-ras gene in 37 colorectal tumors from Mexican patients and to correlate them with clinical data. METHODS: Point mutations were studied in 37 colorectal cancers at codons 12 and 13 of the Ki-ras gene, using PCR followed by RFLP. We also performed PCR-SSCP to identify mutations at codon 61. We confirmed mutations by sequence analysis in all the altered codons. RESULTS: Our results indicated that 24.3% of the tumors presented mutations at codon 12, 5.4% at codon 13, and 2.7% at codon 61 of the Ki-ras gene. We found that 75% of these mutations were transitions and 25% transversions. The overall results indicated that the frequency of Ki-ras mutations in colorectal cancers in a sample of a Mexican population (Mexico City) was 32.4%, which is similar to that reported in other populations. We did not find a correlation between the Ki-ras mutations and gender, location of the tumor, or Dukes' stage, but survival of the patient without recurrence was statistically significant. CONCLUSIONS: The study of colorectal cancer indicated that in a Mexican population Ki-ras mutations were present in tumors of patients who survived without tumor recurrence. Most of them were transitions in the first and second base of codon 12.     

Risk factors for Ki-ras protooncogene mutation in sporadic colorectal adenomas.

The Ki-ras protooncogene frequently is mutated in colorectal adenocarcinomas, but the etiology of this molecular event is uncertain. We investigated the association between variables known or suspected to be related to risk for colorectal cancer and the occurrence of Ki-ras mutations in colorectal adenomas. This study was conducted among 678 male and female participants, 40-80 years of age, enrolled in a phase III trial testing the effects of a wheat bran fiber supplement on adenoma recurrence. Exposure information on the risk factors of interest was assessed through self-administered questionnaires. Mutations in codons 12 and 13 of the Ki-ras protooncogene were analyzed in baseline adenomas 0.5 cm or larger by PCR amplification followed by direct sequencing. Eighteen percent (120 of 678) of the participants had one or more adenoma(s) with Ki-ras mutations. A higher risk of Ki-ras mutations was associated with increasing age and a lower intake of total folate. The odds ratio (OR) for Ki-ras mutations for individuals >72 years of age was 1.98 [95% confidence interval (CI) = 1.19-3.27; P for trend = 0.008] compared with those less than 65 years of age. Compared with individuals in the lower tertile of total folate, those in the upper tertile had an approximately 50% lower risk of having Ki-ras mutation-positive adenomas (OR = 0.52; 95% CI = 0.30-0.88; P for trend = 0.02). There was a suggestion of a stronger inverse association of total folate with G-->T transversions (OR = 0.41; 95% CI = 0.20-0.87) than G-->A transitions (OR = 0.61; 95% CI = 0.31-1.21), although the CIs for the associations overlap. The results of these analyses suggest that the protective effect of folate in colon cancer observed in published studies may be mediated through folate's effect on Ki-ras mutations.     

Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986.

In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.     

K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster.

K-ras oncogene mutation has been shown to be a frequent event in pancreatic ductal adenocarcinomas induced by the carcinogen N-nitroso-bis(2-oxopropyl)amine in the hamster. The present study examines the mutational status of the K-ras oncogene in lesions that precede the appearance of invasive ductal adenocarcinomas. Syrian golden hamsters (80-100 g) received 12 weekly doses of 15 mg/kg N-nitroso-bis(2-oxopropyl)amine and were serially sacrificed at 8, 12, 14, 16, or 24 weeks following the initiation of treatment. Ten microns-thick sections of formalin-fixed paraffin-embedded pancreas were examined for hyperplasia, papillary hyperplasia, carcinoma in situ, and invasive and metastatic ductal carcinoma. Marked lesions of interest were scraped from the slide, subjected to polymerase chain reaction-mediated amplification of the first exon of the K-ras gene, and probed by oligonucleotide-specific hybridization for mutations at codon either 12 or 13. Of 186 samples assayed, K-ras codon 12 mutations were detected in 26% of hyperplasias, 46% of papillary hyperplasias, 76% of carcinoma in situ, 80% of adenocarcinomas, and 43% of lymph node metastases. Codon 12 mutations were exclusively G to A changes at the second position. Codon 13 mutations were only detected in 9 of 168 samples. These results suggest that K-ras activation is an early event in N-nitroso-bis(2-oxopropyl)amine-induced pancreatic carcinogenesis in the hamster.     

Differences in the p53 gene mutational spectra of prostate cancers between Japan and Western countries

Mutations of the p53 gene are related to development of human cancers and their frequencies and spectra, the latter representing fingerprints left by carcinogens, provide information about the molecular epidemiology of the disease. Prostate cancer is the most common neoplasm in American males and although its incidence is still relatively low in Japanese people, it has recently been increasing with the westernization of life style. To assess the frequency and spectrum of p53 gene mutations in Japanese prostate cancers, we examined a series of 90 lesions using polymerase chain reaction (PCR)-single- strand conformation polymorphism (SSCP) analysis. The patients' mean age was 69.3 years (range 57-87). Of the total, six were well-, 34 moderately- and 50 poorly-differentiated adenocarcinomas, and the median Gleason score was 7.9. Eleven of the 90 cases (12%) had mutations in exons 2-11 of the p53 gene: none of the five clinical- stage A, one of 25 stage B (4%), three of 35 stage C (9%) and seven of 25 stage D (28%) cancers. The correlation with an advanced stage was statistically significant. One insertion and 10 base pair substitutions were encountered, comprising six transversions (55%) and four transitions (36%). Two of the latter involved methylated cytosine- guanine (CpG). These 11 mutations were combined with 18 other mutations in previous reports concerning Japanese prostate cancers to facilitate comparison of the p53 gene mutational spectrum with those reported for American and European prostate cancers. In the latter, 61% were transitions and 33% were transversions. The greater proportion of transversions in the Japanese population suggests that there are different factors responsible for carcinogenesis of the prostate glands in the various countries.      

Genetic analysis of lung tumours of non-smoking subjects: p53 gene mutations are constantly associated with loss of heterozygosity at the FHIT locus.

Lung cancer is strictly associated with tobacco smoking. Tumours developed in non-smoking subjects account for less than 10% of all lung cancers and show peculiar histopathological features, being prevalently adenocarcinomas. A number of genetic data suggest that their biological behaviour may be different from that of lung tumours caused by smoking, however the number of cases investigated to date is too low to draw definitive conclusions. We have examined the status of p53 and K-ras genes and the presence of loss of heterozygosity (LOH) at the FHIT locus in a series of 35 lung adenocarcinomas that developed in subjects who had never smoked. Results were compared with those obtained in a series of 35 lung adenocarcinomas from heavy-smoking subjects. In the group of non-smoking subjects p53 mutations and LOH at the FHIT locus were present in seven (20%) cases, and the two alterations were constantly associated (P < 0.0001), whereas they were not related in the series of carcinomas caused by smoking. In tumours developed in heavy-smoking subjects, the frequency of LOH at the FHIT locus was significantly higher (P = 0.006) than in tumours from non-smoking subjects. The frequency of p53 mutations in adenocarcinomas caused by smoking was not different from that seen in non-smoking subjects. However, in the group of smoking subjects we observed mostly G:C --> T:A transversions, whereas frameshift mutations and G:C --> A:T transitions were more frequently found in tumours from non-smoking subjects. No point mutations of the K-ras gene at codon 12 were seen in subjects who had never smoked, whereas they were present (mostly G:C --> T:A transversions) in 34% of tumours caused by smoking (P = 0.002). Our data suggest that lung adenocarcinomas developed in subjects who had never smoked represent a distinct biological entity involving a co-alteration of the p53 gene and the FHIT locus in 20% of cases.     

Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA

Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single- stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G- ->T transversions predominated, followed by G-->C transversions and G-- >A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N- (deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.      

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.     

Carcinogen dose-dependent variation in the transgene mutation spectrum in urethane-induced lung tumors in transgenic mice carrying the human prototype c-Ha-ras gene

Urethane-induced lung tumors and their genetic changes were investigated in transgenic (Tg) mice carrying a human prototype c-Ha-ras gene (rasH2 mice). Male and female rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1000 mg/kg of urethane once or three times at 2-day intervals. Hyperplasias and adenomas of the lung were observed in all animals of each group from week 10, and carcinomas were observed in male and female rasH2 mice of the triple injection group from week 10 and female non-Tg mice of the single injection group at 15/20 weeks. The multiplicities of lung proliferative lesions including hyperplasias, adenomas and carcinomas, in treated rasH2 mice were significantly higher than those in treated non-Tg mice. CAG to CTG transversions were observed in the c-Ha-ras gene in these lung proliferative lesions of rasH2 mice of the single injection group at high incidence (male: 58.3%, female: 62.5%), but no mutations of the mouse c-Ki-ras gene were evident in either rasH2 or non-Tg mice. In the triple injection group, transgene mutations were detected at a relatively low incidence, and mouse c-Ki-ras gene mutations(CAA to CGA) were observed in both rasH2 and non-Tg mice. These results suggest that the variation of the lesions induced by different doses of urethane was not the cause of the variation of the mutation spectrum and mutations of both transgene and mouse c-K-ras gene are not principal genetic events in urethane-induced lung proliferative lesions in rasH2 mice.     

Life-span inhalation exposure to mainstream cigarette smoke induces lung cancer in B6C3F1 mice through genetic and epigenetic pathways

Although cigarette smoke has been epidemiologically associated with lung cancer in humans for many years, animal models of cigarette smoke-induced lung cancer have been lacking. This study demonstrated that life time whole body exposures of female B6C3F1 mice to mainstream cigarette smoke at 250 mg total particulate matter/m(3) for 6 h per day, 5 days a week induces marked increases in the incidence of focal alveolar hyperplasias, pulmonary adenomas, papillomas and adenocarcinomas. Cigarette smoke-exposed mice (n = 330) had a 10-fold increase in the incidence of hyperplastic lesions, and a 4.6-fold (adenomas and papillomas), 7.25-fold (adenocarcinomas) and 5-fold (metastatic pulmonary adenocarcinomas) increase in primary lung neoplasms compared with sham-exposed mice (n = 326). Activating point mutations in codon 12 of the K-ras gene were identified at a similar rate in tumors from sham-exposed mice (47%) and cigarette smoke-exposed mice (60%). The percentages of transversion and transition mutations were similar in both the groups. Hypermethylation of the death associated protein (DAP)-kinase and retinoic acid receptor (RAR)-beta gene promoters was detected in tumors from both sham- and cigarette smoke-exposed mice, with a tendency towards increased frequency of RAR-beta methylation in the tumors from the cigarette smoke-exposed mice. These results emphasize the importance of the activation of K-ras and silencing of DAP-kinase and RAR-beta in lung cancer development, and confirm the relevance of this mouse model for studying lung tumorigenesis.     

Mutations of adenomatous polyposis coli and beta-catenin genes during progression of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats

In the present study, we investigated mutations of the adenomatous polyposis coli (APC) and beta-catenin genes to clarify possible molecular mechanisms underlying development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in drinking water for 12 weeks and then maintained without further treatment until sacrifice at week 25 DNA was extracted from paraffin-embedded tissues, and PCR-single-strand conformation polymorphism analysis, followed by nucleotide sequencing, was performed. No APC mutations were detected in 17 hyperplasias, but 2 of 15 adenomas (13.3%) and 8 of 20 adenocarcinomas (40.0%) showed changes within exon 1 to the mutation cluster region in exon 15. For beta-catenin, no mutations were detected in 17 hyperplasias, but 3 of 15 adenomas (20.0%) and 5 of 20 adenocarcinomas (25.0%) had alterations within or flanking codons corresponding to important phosphorylation sites. Immunohistochemical staining showed beta-catenin protein localized in the cell membranes in the surrounding normal-appearing lung and 216 hyperplasias and localized mainly in the cytoplasm and/or nucleus in 10 of 37 adenomas (27.0%) and 21 of 40 adenocarcinomas (52.5%). These results suggest that the APC-beta-catenin-T-cell factor signaling pathway is involved in the acquisition of growth advantage from adenomas to adenocarcinomas in BHP-induced rat lung carcinogenesis.     

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Associations of Ki-ras proto-oncogene mutation and p53 gene overexpression in sporadic colorectal adenomas with demographic and clinicopathologic characteristics.

In colorectal tumorigenesis, Ki-ras proto-oncogene mutation often occurs early in the adenoma-adenocarcinoma sequence, whereas mutation of the p53 gene is associated with late progression to carcinoma. We evaluated the relationship of demographic and clinicopathologic characteristics to Ki-ras mutation and p53 gene product overexpression in 1,093 baseline sporadic colorectal adenomas from 926 individuals enrolled in a phase III recurrence prevention trial. Ki-ras mutation was found in 14.7% of individuals and p53 overexpression was found in 7.0% of those tested. Multivariate analysis found older age, rectal location, and villous histology to be independently associated with Ki-ras mutation. Individuals with an advanced adenoma (>or=1 cm or high-grade dysplasia or villous histology) had a 4-fold higher likelihood of Ki-ras mutation [odds ratios (OR), 3.96; 95% confidence intervals (CI), 2.54-6.18]. Ki-ras mutations in codon 12 and of the G-to-A transition type were more frequent in older individuals, whereas G-to-T transversion was more frequent in rectal adenomas than in the colon. Multivariate analysis showed that previous history of a polyp (P = 0.03) was inversely associated with p53 overexpression. Large adenoma size (>or=1 cm), high-grade dysplasia, and villous histology were independently associated with p53 overexpression, with the strongest association for advanced adenomas (OR, 7.20; 95% CI, 3.01-17.22). Individuals with a Ki-ras mutated adenoma were more likely to overexpress p53 (OR, 2.46; 95% CI, 1.36-4.46), and 94.8% of adenomas with both alterations were classified as advanced (P 相似文献