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National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage-inducible gene 45 beta (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80-90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response.

A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg of 3'-azido-3'-deoxythymidine (AZT)/kg bw/day, through a 18 to 19-day gestation [National Toxicology Program, NIH Pub. No. 04-4458, 2004]. The incidences of alveolar/bronchiolar adenomas and carcinomas, in the 200 and 300 mg/kg male treatment groups, were significantly greater than that of the controls. In the present study, we evaluated the benign and malignant lung neoplasms from this bioassay for point mutations, in the K-ras and p53 cancer genes that are often mutated in human lung tumors. K-ras and p53 mutations were detected by cycle sequencing of polymerase chain reaction-amplified DNA, isolated from formalin-fixed, paraffin-embedded neoplasms. K-ras mutations were detected in 25 of 38 (66%) of the AZT-induced lung tumors, and the predominant mutations were codon 12 G-->T transversions. p53 mutations were detected in 32 of 38 (84%) of the AZT-induced lung tumors, with the predominant mutations being exon 8, codon 285 A-->T transversions, and exon 6, codon 198 T-->A transversions. No K-ras or p53 mutations were detected in five tumors, examined from control mice. The patterns of mutations identified in the lung tumors suggest that incorporation of AZT or its metabolites into DNA, oxidative stress, and genomic instability may be the contributing factors to the mutation profile and development of lung cancer in these mice., ((c) 2006 Wiley-Liss, Inc.)

1,3-Butadiene and chloroprene are multisite carcinogens in B6C3F1 mice with the strongest tumor response being the induction of lung neoplasms in females. Incidence of brain tumors in mice exposed to 1,3-butadiene was equivocal. This article reviews the efforts of our laboratory and others to uncover the mechanisms of butadiene and chloroprene induced lung and brain tumor responses in the B6C3F1 mouse. The formation of lung tumors by these chemicals involved mutations in the K-ras cancer gene and loss of heterozygosity in the region of K-ras on distal chromosome 6, while alterations in p53 and p16 were implicated in brain tumorigenesis.

Lung cancer is the leading cause of cancer death worldwide, and the need to develop better diagnostic techniques and therapies is urgent. Mouse models have been utilized for studying carcinogenesis of human lung cancers, and many of the major genetic alterations detected in human lung cancers have also been identified in mouse lung tumors. The importance of mouse models for understanding human lung carcinogenic processes and in developing early diagnostic techniques, preventive measures and therapies cannot be overstated. In this report, the major known molecular alterations in lung tumorigenesis of mice are reviewed and compared to those in humans.

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCR on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/beta-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.

Inbred strains of mouse show various susceptibilities to spontaneous and chemical-induced lung tumorigenesis. Genetic analyses have revealed that lung tumor susceptibilities of inbred mouse strains are governed by quantitative trait loci (QLTs) located on multiple chromosomes. A major lung tumor resistance QLT, designated pulmonary adenoma resistance 2 (Par2), was mapped to the mouse chromosome 18 independently by several groups and accounted for up to 60% phenotype variance between susceptible A/J and more resistant BALB/c strains. The authors recently conducted studies to positionally clone the Par2 gene. This review summarizes the effort and progress towards the identification of Par2 candidates.

Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.

The nervous system of the B6C3F1 mouse has rarely been a target for chemical carcinogenesis in the National Toxicology Program (NTP) bioassays. However, 6 malignant gliomas and 2 neuroblastomas were observed in B6C3F1 mice exposed to 625 ppm 1,3-butadiene (NTP technical reports 288 and 434). These mouse brain tumors were evaluated with regard to the profile of genetic alterations that are observed in human brain tumors. Alterations in the p53 tumor suppressor gene were common. Missense mutations were observed in 3/6 malignant gliomas and 2/2 neuroblastomas and were associated with loss of heterozygosity. Most of the mutations occurred in exons 5-8 of the p53 gene and were G-->A transitions, and did not involve CpG sites. Loss of heterozygosity at the Ink4a/Arf gene locus was observed in 5/5 malignant gliomas and 1/1 neuroblastoma, while the PTEN(phosphatase and tensin homologue) gene locus was unaffected by deletions. One of 2 neuroblastomas had a mutation in codon 61 of H-ras, while H-ras mutations were not observed in the malignant gliomas examined. Only 1 brain tumor has been reported from control mice of over 500 NTP studies. This malignant glioma showed no evidence of alterations in the p53 gene or K- and H-ras mutations. It is likely that the specific genetic alterations observed were induced or selected for by 1,3-butadiene treatment that contributed to the development of mouse brain tumors. The observed findings are similar in part to the genetic alterations reported in human brain tumors.

Treatment with 8-methoxypsoralen (8-MOP) and ultraviolet radiation (primarily UVA), called PUVA therapy, has been used to treat different chronic skin diseases but led to a significant increased risk for skin cancer. The National Toxicology Program (NTP) performed a study in mice treated with PUVA that showed a significant increase in squamous cell carcinomas of the skin. In the present study, we evaluated the protein expression of p53 and PCNA and DNA mutations of p53 and H-ras genes in both hyperplastic and neoplastic squamous cell lesions from the NTP study. By immunohistochemical staining, protein expression of both p53 and PCNA was detected in 3/16 (19%) of hyperplastic lesions and 14/17 (82%) of SCCs in groups treated with both 8-MOP and UVA. The mutation frequency of p53 in SCCs from mice administered 8-MOP plus UVA was 15/17 (88%) with a predominant distribution of mutations in exon 6 (14/15 - 93%). No H-ras mutations were detected in the hyperplastic lesions/tumors. The mutagenic effect of PUVA on the p53 tumor suppressor gene may lead to a conformational modification and inactivation of the p53 protein, which are considered critical steps in PUVA-induced skin carcinogenesis. The p53 mutational frequency and patterns from our study were different from those reported in human PUVA-type tumors.

Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2'-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.

Genotoxic carcinogens exert their tumorigenic effects in part by inducing genomic instability. We recently showed that loss of heterozygosity (LOH) on chromosome 12 associates significantly with the induction of chromosome instability (CIN) by the likely human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate (VC) during mouse lung carcinogenesis. Here, we demonstrate the carcinogen specificity of this event and its effect on lung tumor evolution. LOH on chromosome 12 was observed in 45% of NNK-induced, 59% of VC-induced, 58% of aflatoxin B1 (AFB1)-induced, 14% of N-ethyl-N-nitrosourea (ENU)-induced and 12% of spontaneous lung adenocarcinomas. The frequency of LOH in each of the carcinogen-induced groups, except ENU, was significantly higher than in the spontaneous group (P<0.001). Deletion mapping revealed four potential candidate regions of 1-4 centiMorgans suspected to contain targeted tumor suppressor genes, with at least one expected to have a role in CIN. The relationship between LOH on chromosome 12 and additional chromosomal alterations occurring during lung tumor progression was also examined. LOH on chromosomes 1 and 14 were moderately frequent during malignant progression in tumors from all treatment groups, occurring in 21-35 and 18-33% of tumors. However, these alterations showed significant concurrence with LOH on chromosome 12 in VC-, NNK- and AFB1-induced tumors (P<0.05). The results suggest that a carcinogen-selective mechanism of lung cancer induction involves the frequent inactivation of genes on chromosome 12, including a stability gene that evidently promotes the evolutionary selection of additional chromosomal alterations during malignant progression.

In the previous 500 2-year chemical bioassays within the National Toxicology Program, large intestinal tumors (cecal carcinomas) related to chemical exposure have not been observed in B6C3F1 mice. The recently completed o-nitrotoluene study provided the first cecal tumor response and an opportunity to evaluate the morphology and molecular profile of oncogenes and tumor suppressor genes that are relevant to humans. Morphologically, the carcinomas were gland-forming tumors lined by tall columnar epithelial cells that were positive for cytokeratin 20 and negative for cytokeratin 7. Using immunohistochemistry beta-catenin (encoded by Catnb) protein accumulation was detected in 80% (8/10) of the cecal carcinomas, while increased cyclin D1 and p53 protein expression was detected in 73% (8/11), respectively. There was no difference in adenomatous polyposis protein expression between normal colon and cecal carcinomas. All tumors examined exhibited mutations in exon 2 (corresponds to exon 3 in humans) in the Catnb gene. Mutations in p53 were identified in nine of 11 carcinomas, and all were in exon 7. Analysis of the K-ras gene revealed mutations in 82% (9/11) of carcinomas; all had specific G --> T transversions (Gly --> Val) at codons 10 or 12. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations that activate signal transduction pathways (K-ras and Catnb) and changes that disrupt the cell-cycle and bypass G(1) arrest (p53, cyclin D1). These alterations, which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene exposure.

In this study, we performed systematic candidate gene analyses of the Pulmonary adenoma resistance 2 locus. Differential gene expression in lung tissues and nucleotide polymorphisms in coding regions between A/J and BALB/cJ mice were examined using reverse transcription-PCR and direct sequencing. Although not all genes in the interval were analyzed at this moment due to the recent database updating, we have found that the Pol iota gene, encoding the DNA polymerase iota, contains 25 nucleotide polymorphisms in its coding region between A/J and BALB/cJ mice, resulting in a total of ten amino acid changes. Primer extension assays with purified BALB/cJ and A/J proteins in vitro demonstrate that both forms of Pol iota are active but that they may differ in substrate discrimination, which may affect the formation of Kras2 mutations in mouse lung tumors. Altered expression of POL iota protein and an amino acid-changing nucleotide polymorphism were observed in human lung cancer cells, suggesting a possible role in the development of lung cancer. Thus, our data support the Pol iota gene as a modifier of lung tumorigenesis by altering DNA polymerase activity.

We have performed oligonucleotide array analysis on various murine lung tissues [normal lungs, lung adenomas, and lung adenocarcinomas (ACs)] using Affymetrix U74Av2 GeneChips to examine the complex genetic changes occurring during lung carcinogenesis. Analysis yielded 20 novel genes differentially expressed in both lung adenomas and ACs versus normal lungs, including the tumor suppressor APC2 and the oncogene Ros 1. In addition, 50 genes were found to be differentially expressed in lung adenomas versus lung ACs, including the differentiation factor Hox C6, the oncogene Ets 2, and the Ras nuclear transport factor, nuclear transport factor 2. To understand the potential relationship between genes expressed in murine lung tumors and its relationship to altered gene expression observed during embryogenesis and postnatal development, tissues from embryonic lungs and from lungs of mice up to 4 weeks following birth were examined using Affymetrix U74Av2 GeneChips. From this analysis, approximately 1300 genes were determined to exhibit differential expression in fetal lung versus postnatal lung. When we compared lung adenomas, lung ACs, and normal lung parenchyma, 24 developmentally regulated genes were found aberrantly expressed in lung tumors; these included the cell cycle control factor CDC5, the cellular differentiation factor TEA domain 4, and the proapoptotic factor BNIP 2. Finally, we compared the murine lung tumor gene expression data to the expression of genes in human lung cancer, in order to assess the relevance of murine lung cancer models in the study of human AC formation. When the 17 human lung ACs and six human lung large cell carcinomas were examined, it was found that 13 of the 17 human lung ACs clustered tightly together in a pattern that was different from the remaining four human lung ACs and six large cell carcinomas, which exhibited a different pattern. Interestingly, the mouse lung adenomas appeared similar to 13 clustered ACs, while mouse lung ACs appeared more similar in pattern to the group consisting of four ACs and six large-cell carcinomas (LCCs). Nevertheless, when compared with the combined human ACs, 39 genes with similar expression changes in murine lung tumors and human ACs/LCCs were identified, such as the oncogene-related BCL7B, the cell cycle regulator CDK4, and the proapoptotic Endophilin B1. Overall, we have determined, for the first time, the expression profiles during murine lung tumor progression and have established, at the molecular level, an association between murine lung tumorigenesis and lung development. We have also attempted to compare the expression profiles found in mouse lung cancers and those in human lung ACs.

Based on long-term toxicity and carcinogenicity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP- and TNM-induced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G --> A transitions at the second base of the K-ras codon 12 (GGT --> GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP- and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G --> A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.

The present study characterized the immunohistochemical localization of beta-catenin protein in hepatocellular neoplasms and hepatoblastomas in B6C3F(1) mice exposed to diethanolamine (DEA) for 2 years and evaluated genetic alterations in the Catnb and H-ras genes which are known to play important roles in the pathogenesis of liver malignancies. Genomic DNA was isolated from paraffin sections of each liver tumor. Catnb exon 2 (corresponds to exon 3 in human) genetic alterations were identified in 18/18 (100%) hepatoblastomas from DEA exposed mice. Deletion mutations (15/18, 83%) were identified more frequently than point mutations (6/18, 33%) in hepatoblastomas. Eleven of 34 (32%) hepatocellular adenomas and carcinomas from DEA treated mice had mutations in exon 2 of the beta-catenin gene, while only 1 of 10 spontaneous neoplasms had a deletion mutation of codon 5-6. Common to all liver neoplasms (hepatocellular adenomas, carcinomas and hepatoblastomas) was membrane staining for the beta-catenin protein, while cytoplasmic and nuclear staining was observed only in hepatoblastomas. The lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas suggests that the ras signal transduction pathway is not involved in the development of liver tumors following DEA exposure which is different from that of spontaneous liver tumors that often contain H-ras mutations.

The most prominent neoplastic lesions in mice in the 2-year studies of o-nitrotoluene and riddelliine were hemangiosarcomas. Fifteen o-nitrotoluene-induced hemangiosarcomas of the skeletal muscle, subcutaneous tissue, and mesentery; 12 riddelliine-induced hemangiosarcomas of the liver; and 15 spontaneous subcutaneous hemangiosarcomas were examined for genetic alterations in ras, p53, and beta-catenin genes. Mutations in at least one of these genes were identified in 13 of 15 (87%) of the o-nitrotoluene-induced hemangiosarcomas with missense mutations in p53 exons 5-8 detected in 11 of 15 (73%) of these neoplasms. Seven of 15 (47%) hemangiosarcomas from mice exposed to o-nitrotoluene had deletions at exon 2 splice sites or smaller deletions in the beta-catenin gene. K-ras mutation was detected in only 1 of the 15 (7%) o-nitrotoluene-induced hemangiosarcomas. In contrast to the o-nitrotoluene study, 7/12 (58%) riddelliine-induced hemangiosarcomas had K-ras codon 12 GTT mutations and, when screened by immunohistochemistry, 9/12 (75%) had strong staining for the p53 protein in malignant endothelial cells, the cells of origin of hemangiosarcomas. Riddelliine-induced hemangiosarcomas were negative for the beta-catenin protein. Spontaneous hemangiosarcomas from control mice lacked both p53 and beta-catenin protein expression and ras mutations. Our data indicated that p53 and beta-catenin mutations in the o-nitrotoluene-induced hemangiosarcomas and K-ras mutations and p53 protein expression in riddelliine-induced hemangiosarcomas most likely occurred as a result of the genotoxic effects of these chemicals. It also suggests that these mutations play a role in the pathogenesis of the respective hemangiosarcomas in B6C3F1(1) mice.

In this study we used liver neoplasms induced by several chemical carcinogens to investigate potential nuclear targets associated with beta-catenin/Wnt signaling and potential membrane-associated beta-catenin binding partners. Strong expression of cyclin D1, in a pattern similar to that observed previously for beta-catenin, was observed by Western analysis for all five hepatoblastomas examined regardless of treatment. Increased expression of cyclin D1 was also detected in 12 of 35 (34%) hepatocellular neoplasms. Ten of 15 tumors (67%) that had mutations in the Catnb gene had upregulation of cyclin D1, while only 2 of 20 tumors (10%) without Catnb mutations had increased cyclin D1 expression. Immunohistochemical analysis confirmed strong expression of cyclin D1 in most nuclei of hepatoblastomas and scattered nuclear staining in hepatocellular tumors that had Catnb mutations. Increased c-Jun expression was observed in 19 of 30 (63%) hepatocellular tumors and all hepatoblastomas, although upregulation was not completely correlated with Catnb mutation. C-Myc expression was not increased in the tumors. Reduced expression of E-cadherin, which interacts with beta-catenin at the membrane, was observed in some tumors, but this did not correlate with Catnb mutation. Expression of the epidermal growth factor receptor, which may have a role in beta-catenin tyrosine phosphorylation, was lower in some tumors than in normal tissue depending on chemical treatment. The results provide evidence that increased expression of cyclin D1 and c-Jun may provide an advantage during tumor progression and in the transition from hepatocellular neoplasms to hepatoblastomas. Moreover, it is likely increased cyclin D1 expression results at least in part from Catnb mutation, beta-catenin accumulation, and increased Wnt signaling.

We hypothesized that the mouse liver tumor response to non-genotoxic carcinogens would involve some common early gene and protein expression changes that could ultimately be used to predict chemical hepatocarcinogenesis. In order to identify a panel of genes to test, we analyzed global differences in gene and protein expression in livers from B6C3F1 mice following dietary treatment with two rodent carcinogens, the benzodiazepine anti-anxiety drug oxazepam (2500 p.p.m.) and the hypolipidemic agent Wyeth (Wy)-14,643 (500 p.p.m.) compared with livers from untreated mice. Male mice were exposed for 2 weeks and 1, 3 or 6 months to oxazepam or Wy-14,643 in an age-matched study design. By histopathological evaluation, no liver preneoplastic foci or tumors were detected at 6 months in treated or control groups. By cDNA microarray analysis [NIEHS Mouse Chip (8700 genes); n = 3 individual livers/group, four hybridizations/sample], expression of 36 genes or 220 genes were changed relative to control livers following 6 months of oxazepam or Wy-14,643 treatment, respectively. To obtain a more comprehensive picture of gene/protein expression changes, we also conducted a proteomics study by 2D-gel electrophoresis followed by matrix assisted laser desorption/ionization-mass spectrometry on cytoplasmic, nuclear, and microsomal subcellular fractions of the same liver samples utilized for the cDNA microarray analysis. Real-time PCR, western blot analysis and immunohistochemistry were utilized for validation and to expand the results to other time points. Cyp2b20, growth arrest- and damage-inducible gene beta (Gadd45beta), tumor necrosis factor alpha-induced protein 2 and insulin-like growth factor binding protein 1 (Igfbp5) genes and proteins were upregulated by oxazepam, and Cyp2b20, Cyclin D1, proliferating cell nuclear antigen, Igfbp5, Gadd45beta and cell death-inducing DNA fragmentation factor alpha subunit-like effector A exhibited higher expression after Wy-14,643 treatment. Most of these genes/proteins were also deregulated at 2 weeks. There appeared to be more distinct than common changes in the expression of carcinogenesis-related genes/proteins between the two compounds, suggesting that the major carcinogenic pathways are different for these compounds and may be distinct for different chemical classes.

Lung tumors from AC3F1 mice treated with aflatoxin B(1) (AFB(1)), were examined for loss of alleles, point mutations and hypermethylation of CpG sites within the promoters of the two genes in the Ink4a/Arf gene locus. Loss of microsatellite alleles in the Ink4a/Arf region occurred in 22 of 74 (30%) AFB(1)-induced lung tumors. Fifty-one of 61 (83%) tumors had at least partial methylation of CpG sites within the p16Ink4a promoter-exon 1alpha region. At least partial methylation of CpG sites was observed in 43 of 49 (88%) tumors analyzed for p19Arf promoter hypermethylation, with methylation of identified transcription factor binding sites or consensus sequences occurring in 21 tumors (DMP1/Ets in two tumors, CTCF in four tumors, E2F in three tumors, Sp1 in 16 tumors). Two tumors contained point mutations in the p19Arf promoter. Nuclear staining for p19(Arf) was decreased by 80-100% in 41 of 71 (58%) tumors. The concordance between p19Arf molecular perturbations and altered protein expression was 63%. However, upon comparing p19Arf promoter perturbations (i.e. methylation of functional transcription factor binding sites and point mutations) and altered p19(Arf) expression, the concordance was 86%, suggesting a mechanism for changes in protein expression in some tumors. There was an absence of a mutually exclusive relationship between disruption of p53 and p19(Arf), since the concordance was 62%. Similarly, no evidence was found of inverse relationships between perturbation of p16Ink4a and p19(Arf) (43% concordance) or p16Ink4(a) and p53 (37% concordance), suggesting that inactivation of these genes occurs independently and provides evidence that, although these genes may participate in cooperative cellular pathways, they also have functions in independent pathways that are important in mouse lung tumorigenesis.

Recent evidence suggests that genome-wide allelic imbalances are inducible by carcinogens and may occur as cells adapt to carcinogenic exposure during tumorigenesis. We investigated the role of carcinogenic exposure on global and selected loss of heterozygosity (LOH) during mouse lung adenocarcinogenesis. Tumor induction by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or vinyl carbamate (VC) resulted in a significant overall increase in the number of chromosomes affected by LOH per tumor when compared with spontaneous lung tumors. Allelic loss on chromosome 12 occurred at a frequency of 35% and 40% in NNK- and VC-induced tumors, respectively, compared with 8.3% in spontaneous tumors (P < 0.01). In contrast, spontaneous lung adenocarcinomas displayed LOH on chromosome 4 at a frequency of 77%, whereas a frequency of only 36% (P < 0.001) was observed in tumors induced by NNK. Sixty-four percent of the VC-induced tumors displayed LOH on chromosome 4. In addition, allelic losses on chromosomes 12 and 14 were significantly associated with an increase in chromosomal instability, suggesting that genes inactivated on these chromosomes may contribute to this effect. The results from this study demonstrate that genotoxic carcinogens increase chromosome instability, as evidenced by a significant increase in global LOH frequency, and significantly alter the selection of chromosomal alterations during lung tumor development.

Previous work showed a correlation between K-ras mutation and loss of heterozygosity (LOH) on chromosome 6 in the region of K-ras in lung carcinomas from B6C3F1 mice. We hypothesized that mitogen-activated protein kinase (MAPK) would be activated only in those lung neoplasms with both K-ras mutation and LOH. As MAPK activity can be correlated directly with signal detection using antibodies to phosphorylated MAPK, we were able to analyze lung carcinomas from B6C3F1 mice for the presence or absence of MAPK activity by western analysis. Vanadium pentoxide-induced mouse lung carcinomas, which had been shown to have a high frequency of K-ras mutations and LOH on chromosome 6 and for which frozen tumor tissue was available, were used for this study. Total MAPK expression levels were similar between normal lung and lung carcinomas. Phospho-MAPK was elevated in five of six lung carcinoma samples examined in which K-ras mutations and chromosome 6 LOH were identified and in four of five carcinomas with K-ras mutations that lacked LOH. Phospho-MAPK was undetectable or weakly expressed in seven carcinomas examined without K-ras mutations and in normal lung. By immunohistochemistry three K-ras positive/LOH negative samples exhibited multifocal areas of nuclear and cytoplasmic staining for phospho-MAPK. Large amounts of non-staining fibroblasts, lymphocytes and macrophages were also observed in these tumors. Two of these lung carcinomas were microdissected and chromosome 6 LOH was detected in regions of phospho-MAPK positive cells. These results suggest that MAPK is activated during vanadium pentoxide-induced B6C3F1 mouse lung tumorigenesis following K-ras mutation and loss of the wild-type K-ras allele.

Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal-regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

Gastric cancers are commonly subdivided into intestinal and diffuse subtypes on a morphologic basis, supported by corollary evidence of differences at the pathogenetic and molecular levels. Chronic atrophic gastritis with intestinal metaplasia is a common precursor lesion for the intestinal type of carcinoma. To identify early molecular changes, in this study we have examined 13 surgical specimens both for the expression of E-cadherin, p53 and beta-catenin by immunohistochemistry and for methylation of the CDH1 promoter (E-cadherin) by bisulfite genomic sequencing of laser capture microdissected samples. Each specimen examined contained areas of normal (nonmetaplastic) gastric mucosa, as well as areas of intestinal metaplasia and/or carcinoma. Reduced or absent E-cadherin and partial to complete methylation of one to multiple CpG sites examined in the CDH1 promoter were observed in all of the metaplasia samples. Thus, the methylation status of the CDH1 promoter and expression of E-cadherin together provide strong evidence that loss of E-cadherin is an early event in intestinal type gastric carcinogenesis. In contrast, expression of p53, assumed to be mutant p53, was generally not detected (except for isolated cells) until the carcinoma stage in tissues from these patients. These results suggest that mutation of p53 is a late event in intestinal type gastric cancer. The level of beta-catenin expression did not appear to change between normal, metaplastic and carcinoma cells of intestinal type, and no nuclear staining was visible in any of the tissues. These results suggest that the Wnt signaling pathway is not upregulated in this type of cancer.

1-Amino-2,4-dibromoanthraquinone (ADBAQ) is an anthraquinone-derived vat dye, and a potent carcinogen in laboratory animals. In a 2-year study with dietary exposure to 10,000 or 20,000 ppm ADBAQ, increased incidence of forestomach and lung tumors were observed in B6C3F1 mice. The present study indentified genetic alterations in H-ras and K-ras proto-oncogenes in ADBAQ-induced tumors. Point mutations in ras proto-oncogenes were identified by restriction fragment length polymorphism, single-stranded conformational polymorphism analysis and cycle sequencing of polymerase chain reaction-amplified DNA isolated from paraffin-embedded squamous cell papillomas and carcinomas in the forestomach, and alveolar/bronchiolar adenomas and carcinomas in the lung. A higher frequency of ras mutations was identified in ADBAQ-induced forestomach (23/32, 72%) and lung tumors (16/23, 70%) than in spontaneous forestomach (4/11, 36%) and lung tumors (26/86, 30%). H-ras codon 61 CTA mutations were detected in (4/8, 50%) ADBAQ-induced forestomach squamous cell papillomas and (10/24, 42%) squamous cell carcinomas, but not in the spontaneous forestomach tumors examined. H-ras codon 61 CGA mutation (6/24, 25%) was also detected in ADBAQ-induced forestomach squamous cell carcinomas. K-ras codon 61 A to T transversions and A to G transitions were prominent in ADBAQ-induced lung alveolar/bronchiolar adenomas and alveolar/bronchiolar carcinomas. The major finding of A to T transversions or A to G transitions in forestomach and lung tumors suggests that ADBAQ or its metabolites target adenine bases in the ras proto-oncogenes and that these mutations play a dominant role in multi-organ

1,3 Butadiene (BD), isoprene (IP) and chloroprene (CP) are structural analogs. There were significantly increased incidences of forestomach neoplasms in B6C3F1 mice exposed to BD, IP or CP by inhalation for up to 2-years. The present study was designed to characterize genetic alterations in K- and H-ras proto-oncogenes in a total of 52 spontaneous and chemically induced forestomach neoplasms. ras mutations were identified by restriction fragment length polymorphism, single strand conformational polymorphism analysis, and cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded forestomach neoplasms. A higher frequency of K- and H-ras mutations was identified in BD-, IP- and CP-induced forestomach neoplasms (83, 70 and 57%, respectively, or combined 31/41, 76%) when compared to spontaneous forestomach neoplasms (4/11, 36%). Also a high frequency of H-ras codon 61 CAA-->CTA transversions (10/41, 24%) was detected in chemically induced forestomach neoplasms, but none were present in the spontaneous forestomach neoplasms examined. Furthermore, an increased frequency (treated 13/41, 32% versus untreated 1/11, 9%) of GGC-->CGC transversion at K-ras codon 13 was seen in BD-, and IP-induced forestomach neoplasms, similar to the predominant K-ras mutation pattern observed in BD-induced mouse lung neoplasms. These data suggest that the epoxide intermediates of the structurally related chemicals (BD, IP, and CP) may cause DNA damage in K-ras and H-ras proto-oncogenes of B6C3F1 mice following inhalation exposure and that mutational activation of these genes may be critical events in the pathogenesis of forestomach neoplasms induced in the B6C3F1 mouse.

beta-Catenin plays a key role in the Wnt signaling pathway, and mutations of CTNNB1, the gene that encodes beta-catenin, have been identified in about one-fourth of human hepatocellular carcinomas from regions of low aflatoxin B1 exposure. In this study 62 hepatocellular carcinomas (HCCs) from people highly exposed to aflatoxin B1 in Guangxi, People's Republic of China, were laser-capture microdissected and examined for CTNNB1 mutations. In addition, 41 of the HCCs were evaluated for the presence of the beta-catenin protein by immunohistochemical methods. Twenty of the HCCs showed positive results for beta-catenin, with strong membrane staining, while adjacent non-neoplastic liver tissue lacked or showed only weak membrane staining. One HCC, in which a CTNNB1 mutation was not detected, showed nuclear staining for the beta-catenin protein. Mutations of CTNNB1 were identified in five HCCs. These consisted of four point mutations in the glycogen serine kinase-3beta phosphorylation region of codons 32-45 and one deletion of codons 32-38. These mutations were similar to those previously reported for human HCC, although at a lower frequency. A signature mutation profile associated with aflatoxin B1 exposure could not be identified. The immunohistochemical findings indicate a role for accumulation of beta-catenin and possibly increased Wnt signaling in aflatoxin B1-associated HCC. The low frequency of CTNNB1 mutations, however, suggests that mutation of another Wnt signaling component, such as the Wnt scaffolding protein axin or the adenomatous polyposis coli protein, both of which modulate beta-catenin stability, also may be involved in aflatoxin-associated HCC. Published 2001 Wiley-Liss, Inc.

In a number of recent studies, a lung tumor resistance locus designated either Par2 or Pas7 was mapped to distal chromosome 18 in crosses between susceptible A/J and more resistant BALB/c mice. This locus is important in that it accounts for as much as 60% of the difference in lung tumor susceptibility between the A/J and BALB/c mice, both of which contain the susceptible allele of Kras2, a marker and strong candidate for the major lung tumor susceptibility gene on mouse chromosome 6. We have now fine-mapped the Par2 locus by using congenic mice that were constructed by placing part of chromosome 18 from the susceptible A/J onto the genetic background of lung tumor-resistant BALB/c mice. After 7 generations of backcrossing, N7 mice that carried 28 cM of the A/J quantitative trait locus (QTL) region were crossed to the BALB/c to generate the N8 generation. Congenic strains (N8) that contain various QTL regions were generated. N9 mice, generated from N8 males x 3 BALB/c females, were genotyped in the region of the Par2 locus and treated with an initiating dose of urethane and allowed to form lung tumors over 6 months. The mice were killed and the lung tumors counted. With this cross the Par2 locus was narrowed to a 6-cM region. Potential candidate genes in this region include Smad4, Smad2, and Dcc. Previously, we excluded Smad4 and Smad2 as candidates for Par2 based on the lack of functional polymorphism(s) and differential expression in lungs from A/J and BALB/c mice. In this study, no polymorphism of the coding sequence of Dcc was observed between A/J and BALB/c mice. Further fine mapping and positional cloning are required for the identification of the Par2 gene.

Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examined to investigate the role of transcriptional silencing of the p16 (Ink4a) tumor suppressor gene by DNA hypermethylation during mouse lung carcinogenesis. Hypermethylation (>/=50% methylation at two or more of the CpG sites examined) of the p16 (Ink4a) promoter region was detected in DNA from 12 of 17 (70%) of the B6C3F1 primary mouse lung adenocarcinomas examined, whereas hypermethylation was not detected in normal B6C3F1, C57BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 protein within and among the tumors. Laser capture microdissection was employed to collect cells from immunostained sections of four tumors displaying areas of relatively high and low p16 expression. The methylation status of the microdissected samples was assessed by sodium bisulfite genomic sequencing. The pattern of p16 expression correlated inversely with the DNA methylation pattern at promoter CpG sites in nine of 11 (82%) of the microdissected areas displaying variable p16 expression. To provide further evidence that hypermethylation is involved in the loss of p16 (Ink4a) gene expression, three mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete methylation at seven promoter CpG sites and no p16 (Ink4a) expression were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Re-expression of p16 (Ink4a) and partial demethylation of the p16 (Ink4a) promoter were observed in two cell lines (C10 and sp6c) following treatment. These are the first reported studies to provide strong evidence that DNA methylation is a mechanism for p16 inactivation in mouse lung tumors.

1,3-Butadiene is a multisite carcinogen in rodents. Incidences of cardiac hemangiosarcomas were significantly increased in male and female B6C3F1 mice that inhaled 1,3-butadiene (BD) for 2 years. Eleven BD-induced cardiac hemangiosarcomas were examined for genetic alterations in ras protooncogenes and in the p53 tumor suppressor gene. Nine of 11 (82%) BD-induced hemangiosarcomas had K-ras mutations and 5 of 11 (46%) had H-ras mutations. All of the K-ras mutations were G-->C transversions (GGC-->CGC) at codon 13; this pattern is consistent with reported results in BD-induced lung neoplasms and lymphomas. Both K-ras codon 13 CGC mutations and H-ras codon 61 CGA mutations were detected in 5 of 9 (56%) hemangiosarcomas. The 11 hemangiosarcomas stained positive for p53 protein by immunohistochemistry and were analyzed for p53 mutations using cycle sequencing of polymerase chain reaction (PCR) amplified DNA isolated from paraffin-embedded sections. Mutations in exons 5 to 8 of the p53 gene were identified in 5 of 11 (46%) hemangiosarcomas, and all of these were from the 200- or 625-ppm exposure groups that also had K-ras codon 13 CGC mutations. Our data indicate that K-ras, H-ras, and p53 mutations in these hemangiosarcomas most likely occurred as a result of the genotoxic effects of BD and that these mutations may play a role in the pathogenesis of BD-induced cardiac hemangiosarcomas in the B6C3F1 mouse.

The molecular pathogenesis of hepatoblastomas in the B6C3F1 mouse is unclear but may involve alterations in the beta-catenin/Wnt signaling pathway as was recently described for chemically induced hepatocellular neoplasms and human liver cancers. The objective of this study was to characterize the mutation frequency and spectrum of beta-catenin mutations and the intracellular localization of beta-catenin protein accumulation in chemically induced hepatoblastomas. In this study, beta-catenin mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblastomas examined. Although several hepatoblastomas had multiple deletion and/or point mutations, the pattern of mutations in the hepatoblastomas did not differ from that identified in hepatocellular neoplasms. In a majority of the hepatoblastomas (six of seven) examined by immunohistochemical methods, both nuclear and cytoplasmic localization of beta-catenin protein were detected, whereas in hepatocellular adenomas, carcinomas, and normal liver only membrane staining was observed. Our data suggest that beta-catenin mutations and the subsequent translocation of beta-catenin protein from the cell membrane to the cytoplasm and nucleus may be critical steps in providing hepatocellular proliferative lesions with the growth advantage to progress to hepatoblastoma.

The promoter of the hTERT gene encoding the catalytic subunit of telomerase was recently cloned and has a dense CG-rich CpG island, suggesting a role for methylation in regulation of hTERT expression. In this study, we have initiated the analysis of the regulation of hTERT expression by examining the methylation status of up to 72 CpG sites extending from 500 bases upstream of the transcriptional start site of the hTERT gene into the first exon in 37 cell lines. These cell lines represent a variety of cell and tissue types, including normal, immortalized, and cancer cell lines from lung, breast, and other tissues. Using bisulfite genomic sequencing, we did not find a generalized pattern of site-specific or region-specific methylation that correlated with expression of the hTERT gene: most of the hTERT-negative normal cells and about one-third of the hTERT-expressing cell lines had the unmethylated/hypomethylated promoter, whereas the other hTERT-expressing cell lines showed partial or total methylation of the promoter. The promoter of one hTERT-negative fibroblast cell line, SUSM-1, was methylated at all sites examined. Treatment of SUSM-1 cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A induced the cells to express hTERT, suggesting a potential role for DNA methylation and/or histone deacetylation in negative regulation of hTERT. This study indicates that there are multiple levels of regulation of hTERT expression in CpG island methylation-dependent and -independent manners.

beta-catenin activation, and subsequent upregulation of Wnt-signaling, is an important event in the development of certain human and rodent cancers. Recently, mutations in the beta-catenin gene in the region of the serine-threonine glycogen kinase (GSK)-3beta phosphorylation target sites have been identified in hepatocellular neoplasms from humans and transgenic mice. In this study we examined 152 hepatocellular neoplasms from B6C3F1 mice included in five chemical treatment groups and controls for mutations in the beta-catenin gene. Twenty of 29 hepatocellular neoplasms from mice treated with methyleugenol had point mutations at codons 32, 33, 34 or 41, sites which are mutated in colon and other cancers. Likewise, nine of 24 methylene chloride-induced hepatocellular neoplasms and 18 of 42 oxazepam-induced neoplasms exhibited similar mutations. In contrast, only three of 18 vinyl carbamate-induced liver tumors, one of 18 TCDD-induced liver tumors, and two of 22 spontaneous liver neoplasms had mutations in beta-catenin. Thus, there appears to be a chemical specific involvement of beta-catenin activation in mouse hepatocellular carcinogenesis. Expression analyses using Western blot and immunohistochemistry indicate that beta-catenin protein accumulates along cell membranes following mutation. The finding of mutations in both adenomas and carcinomas from diverse chemical treatment groups and the immunostaining of beta-catenin protein in an altered hepatocellular focus suggest that these alterations are early events in mouse hepatocellular carcinogenesis.

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human lung cancers. However, p53 mutations are more rarely detected in chemically induced mouse lung tumors. In this study, 62 female AC3F1 (A/J x C3H/HeJ) mice were treated with aflatoxin B1 (AFB1; 150 mg/kg i.p. divided into 24 doses over 8 weeks). At 6-14 months after dosing, mice were killed, and tumors were collected. A total of 71 AFB1-induced lung tumors were examined for overexpression of p53 protein by immunohistochemical staining. Positive nuclear p53 staining was observed in 79% of the AFB1-induced tumors, but the pattern was highly heterogeneous. In approximately 73% of the positively stained tumors, fewer than 5% of cells demonstrated positive staining; in the other 27%, between 10% and 60% of the cells stained positively, with staining localized to the periphery of the tumors in many cases. Single-strand conformational polymorphism analysis of the evolutionarily conserved regions of the p53 gene (exons 5-8) from AFB1-induced whole lung tumor DNA revealed banding patterns consistent with point mutations in 20 of 76 (26%) tumors, with 85% of the mutations in exon 7 and 15% of the mutations in exon 6. Identification of point mutations could not be confirmed by direct sequence analysis because bands representing putative mutations appeared only weakly on autoradiograms. This was presumably due to the heterogeneous nature of the DNA analyzed. Single-strand conformational polymorphism analysis of DNA from laser capture microdissected cells of paraffin-embedded AFB1-induced tumor tissue sections stained for p53 produced banding patterns consistent with point mutations in 18 of 30 (60%) DNA samples. Direct sequencing of the microdissected samples revealed mutations at numerous different codons in exons 5, 6, and 7. Of 26 mutations found in microdissected regions from adenomas and carcinomas, 9 were G:C-->A:T transitions, 11 were A:T-->G:C transitions, and 5 were transversions (2 G:C-->T:A, 2 T:A-->A:T, and 1 A:T-->C:G), whereas 1 deletion mutation was identified. The concordance between immunostaining and molecular detection of p53 alterations was 72% when laser capture microdissection was used versus 17% based on whole tumor analysis. The high mutation frequency and heterogeneous staining pattern suggest that p53 mutations occur relatively late in AFB1-induced mouse lung tumorigenesis and emphasize the value of analyzing different staining regions from paraffin-embedded mouse lung tumors.

Chloroprene is the 2-chloro analog of 1,3-butadiene, a potent carcinogen in laboratory animals. Following 2 years of inhalation exposure to 12.8, 32 or 80 p.p.m. chloroprene, increased incidences of lung and Harderian gland (HG) neoplasms were observed in B6C3F1 mice at all exposure concentrations. The present study was designed to characterize genetic alterations in the K- and H-ras proto-oncogenes in chloroprene-induced lung and HG neoplasms. K-ras mutations were detected in 80% of chloroprene-induced lung neoplasms (37/46) compared with only 30% in spontaneous lung neoplasms (25/82). Both K- and H-ras codon 61 A-->T transversions were identified in 100% of HG neoplasms (27/27) compared with a frequency of 56% (15/27) in spontaneous HG neoplasms. The predominant mutation in chloroprene-induced lung and HG neoplasms was an A-->T transversion at K-ras codon 61. This mutation has not been detected in spontaneous lung tumors of B6C3F1 mice and was identified in only 7% of spontaneous HG neoplasms. In lung neoplasms, greater percentages (80 and 71%) of A-->T transversions were observed at the lower exposures (12.8 and 32 p.p.m.), respectively, compared with 18% at the high exposure. In HG neoplasms, the percentage of A-->T transversions was the same at all exposure concentrations. The chloroprene-induced ras mutation spectra was similar to that seen with isoprene, where the predominant base change was an A-->T transversion at K-ras codon 61. This differed from 1,3-butadiene, where K-ras codon 13 G-->C transitions and H-ras codon 61 A-->G transitions were the predominant mutations. The major finding of K-ras A-->T transversions in lung and Harderian gland neoplasms suggests that this mutation may be important for tumor induction by this class of carcinogens.

To understand the causes of cancer, it is necessary to elucidate the molecular basis and environmental factors that influence the carcinogenesis process. Cancers are progressive diseases characterized by the accumulation of defects in many different genes. The patterns of mutation of some genes identified in tumours suggest a direct action of chemicals binding to and altering DNA. Other cancer-associated genes may be altered as a consequence of endogenous mutagens, germ-line mutations, spontaneous mutations that occur during cell replication or increased genetic instability in precancerous cells. Recent advances in molecular biology and genetics have provided new tools and concepts for studying the causes of cancer. We know that cancers are caused by a combination of environmental and genetic factors, and the discovery of the molecular alterations that occur at various stages in different tumours is increasing our understanding of these causes. Thus, we are now beginning to discover which genes are involved, how they function normally and in tumour tissues and why cancers develop after a series of genetic and epigenetic changes in certain cells. As data from studies on cancer-associated genes have accrued, the categories of genes and molecular pathways that have been found to play a role in carcinogenesis have also increased. Genes involved in development and other normal cellular processes have been implicated in cancer. These include genes involved in signal transduction, cell cycle control, DNA repair, cell growth and differentiation (growth factors and growth factor receptors), transcriptional regulation, senescence and apoptosis. Genes involved in angiogenesis, immune regulation, cellular responses to stress, motility, adhesion and invasion are also involved, but less is known about their relationship to carcinogenesis, and these processes are not discussed in this review. The diverse nature of these categories of cancer-related genes indicates the variety of processes that must be disrupted in order for tumours to develop. Many of the genes have several functional domains, and the functions of some have only recently been proposed. In this review, we describe some of the major classes of genes implicated in human cancers and some of the major findings on genetic alterations and dysfunction in human tumours. Comparisons are made with certain rodent models.

Studies of carcinogenesis in rodents are valuable for examining mutagenesis in vivo. An advantage of evaluating the frequency and spectra of ras mutations in chemically induced neoplasms is that the additional data at the molecular level indicate whether the carcinogenic effect is due to the chemical and is not a spontaneous event, as illustrated by the numerous examples in Appendices 1 and 2. In addition, data on the frequency and spectra of ras mutations in spontaneous and chemically induced neoplasms clearly expand the toxicological database by providing information helpful for understanding the pathogenesis of carcinogenesis. For example: (1) ozone-induced lung neoplasms had two unique mutations, one (codon 61 K-ras CTA mutation) consistent with a direct genotoxic event and a second (codon 12 K-ras G --> T transversion) consistent with an indirect genotoxic effect; (2) isoprene-induced Harderian gland neoplasms had a unique K-ras A --> T transversion at codon 61 which provided evidence that formation of an epoxide intermediate was involved; (3) 1,3-butadiene-induced neoplasms had a characteristic K-ras G --> C transversion mutation at codon 13 which was also consistent with a chemical-specific effect; (4) methylene chloride-induced liver neoplasms had an H-ras mutation profile at codon 61 similar to that of spontaneous tumours, suggesting that methylene chloride promotes cells with 'spontaneously initiated' ras mutations and (5) oxazepam-induced liver neoplasms had a low frequency of ras mutations, suggesting a nonmutagenic pathway of carcinogenesis. By extending the evaluation of rodent tumours to include molecular studies on ras mutation spectra and abnormalities in other cancer genes with human homologues, a number of hypotheses can be tested, allowing the most complete understanding of carcinogenesis in rodents and in potential extrapolation to the human risk situation.

Four putative quantitative trait loci (QTLs) that influence susceptibility to the induction of lung adenomas by urethane in an F2 cross between A/J and BALB/cOlaHsd have been mapped. Following microsatellite typing of mice with resistant and susceptible phenotypes at 97 microsatellite marker loci, a major locus was identified on chromosome 18 with a lod score of 15. This was responsible for an 8- to 10-fold increase in tumor multiplicity in males and females, respectively, having the AA and CC genotypes at the D18Mit188 marker locus. It mapped close to Dcc (deleted in colorectal cancer). A locus on chromosome 4 (lod score 6.5) had the resistant allele in strain A/J and the susceptible allele in BALB/c, with a 14-fold difference in tumor multiplicity between mice of the AA and CC genotypes. This mapped close to the Cdkn2a (cyclin-dependent kinase inhibitor 2A) locus, which is commonly deleted in mouse lung tumors. Two loci with smaller effects (lod scores 3.03 and 3.25) were identified on chromosomes 1 and 11. There was also significant sexual dimorphism in tumor multiplicity both among 151 F2 hybrids and among 52 mice resulting from a backcross to strain A/J, with males having higher tumor counts than females., (Copyright 1998 Academic Press.)

Tetrafluoroethylene (TFE) was evaluated for carcinogenicity in inhalation studies because of its high use in the production of Teflon. There was clear evidence of hepatocarcinogenic activity in B6C3F1 mice after 2 yr of TFE exposure. The present study was designed to characterize the mutation profiles of H- and K-ras oncogenes in liver neoplasms in mice after exposure to 0, 312, 625, or 1,250 ppm TFE. ras mutations were identified by restriction fragment length polymorphism, single-stranded conformation polymorphism analysis, and direct sequencing of polymerase chain reaction amplified-DNA isolated from frozen or paraffin-embedded liver neoplasms. A low frequency (15%, 9/59) of H-ras codon 61 mutations was detected in hepatocellular neoplasms when compared with the higher frequency (59% of this study and 56% of historical data) in spontaneously occurring liver neoplasms. There was no difference in the mutation frequency or spectrum among exposure groups or between benign and malignant hepatocellular neoplasms. K-ras mutations at codons 12, 13, and 61 and H-ras mutations at codon 117 were not detected in hepatocellular neoplasms. These data suggest that TFE-induced hepatocellular neoplasms are developed by pathways that are mostly independent of ras mutations. The ras mutation frequency and spectrum were similar to those of the structurally related chemical tetrachloroethylene.

A locus for mouse pulmonary adenoma susceptibility, Pas-1, has been mapped on distal chromosome 6, where the K-ras gene is located. Allele-specific activation and expression of the K-ras allele from the susceptible parent has been observed in lung tumors from F1 hybrid mice. We report here genetic mapping of lung tumor susceptibility genes in urethane-treated A x B and B x A recombinant inbred (RI) mice using microsatellite markers to seek further evidence for the K-ras gene as candidate for Pas-1. The K-ras genotype differs between the A/J and C57BL/6J progenitors of the RI strains, and distal chromosome 6 contained a major lung tumor susceptibility determinant in the RI mice. Additional evidence that Pas-1 is K-ras involved linkage analysis of (A/JOLaHsd x BALB/ cOLaHsd) F2 intercross mice whose parents shared the same K-ras genotype. In contrast to the results with the A x B and B x A RI strains, no distal chromosome 6 site was significantly associated with tumor development in these F2 mice. In addition to this major locus, linkage analysis of the RI mice revealed additional quantitative trait loci for susceptibility on chromosomes 10, 17, and 19. These loci may serve as modifiers of Pas-1. The relationship between the K-ras genotype and the frequency of K-ras mutations in urethane-induced lung tumors from the RI mice was also explored. All 18 tumor DNAs from RI mice with high susceptibility contained an AT-->TA transversion at the second base of K-ras codon 61. This was also true for DNAs from 27 of 27 (100%) tumors in mice with high intermediate susceptibility. In RI strains with a low intermediate susceptibility, the DNA from 39 of 47 (83%) tumors contained an AT-->TA transversion at codon 61, and only 13 of 21 (62%) tumors had this mutation in the most resistant group. This reflects a positive correlation between the frequency of K-ras mutations in lung tumors of A x B or B x A RI strains and their susceptibility to lung carcinogenesis. Since K-ras appears to be Pas-1, these results suggest that some RI mice that have the resistant K-ras or Pas-1 allele undergo tumor development by a K-ras-independent route.

During the last decade new methods for mapping quantitative trait loci (QTLs) have helped geneticists uncover disease-associated genes. Genetic dissection of complex multigenic diseases such as cancer is being accomplished in part by mapping QTLs in experimental crosses of mice [1]. With the recent construction of dense genetic linkage maps for the mouse, mapping of quantitative trait loci has become practical [2]. Over 6000 polymorphic simple sequence length repeat markers (microsatellite markers) have been mapped in the mouse genome [3], and new analytical approaches to linkage analysis have made QTL mapping a powerful technique for identifying cancer genes [4-7]. In this overview we discuss the design of QTL mapping studies and some of the findings from studies on the mapping of murine lung tumor susceptibility loci.

Mouse uterine tumors were examined for genetic alterations in the ras proto-oncogene and p53 tumor suppressor gene and for other biologically relevant immunohistochemical markers that may increase our understanding of the events that occur in uterine cancer. Fourteen dimethylhydrazine (DMH)-induced uterine sarcomas, including 3 primary malignant fibrous histiocytomas (MFH), 7 transplanted MFH, 3 stromal sarcomas, and 1 undifferentiated sarcoma, were first screened by immunohistochemistry for p53 missense mutations, followed by single strand conformation polymorphism analysis and DNA sequencing for the identification of point mutations. There was 100% correlation between p53 protein immunopositivity and subsequent detection of p53 mutations in DMH-induced malignant fibrous histiocytomas. All MFH had a characteristic p53 G:C-->A:T transition mutation, consistent with O6-methylguanine mispairing with thymine, the most common DNA lesion caused by alkylating agents. DMH-induced uterine MFH with p53 mutations also had a higher proliferative rate (qualitatively evaluated by immunohistochemical detection of proliferating cell nuclear antigen) when compared with other DMH-induced sarcomas. Uterine sarcomas were further evaluated for biological end points, such as estrogen receptor and desmin. Neoplastic cells from stromal sarcomas (SS), undifferentiated sarcomas (US), and MFH did not stain for desmin. The estrogen receptor was detected in normal uteri and a small portion of MFH, SS, and US. Our data suggest that DMH-induced uterine sarcomas are not consistent with smooth muscle cell origin and that a subset of these tumors, specifically DMH-induced malignant fibrous histiocytomas, have unique p53 G:C-->A:T transitions and a high proliferative rate.

Establishing the diagnosis of Helicobacter hepaticus infection in mouse liver has recently become important for the interpretation of rodent carcinogenicity bioassays. A seminested primer polymerase chain reaction (PCR) amplification of the bacterial 16S ribosomal RNA gene in combination with a restriction fragment length polymorphism (RFLP) assay was designed to identify and distinguish H. hepaticus from H. muridarum and H. bilis in mouse liver. The PCR-RFLP assay was applied to formalin-fixed, paraffin-embedded and, when available, corresponding frozen liver tissues from male and female B6C3F1 mice with or without histologic evidence of infection from various National Toxicology Program 2-yr bioassay studies. PCR products consistent with H. hepaticus were detected in 10-80% of livers from mice in studies with other evidence of infection that were frozen or fixed for less than 24 hr but not in liver fixed for several weeks. The sensitivity of the PCR-RFLP assay for H. hepaticus on formalin-fixed, paraffin-embedded mouse liver varied between studies from markedly decreased when compared to the results from frozen liver or histologic evaluation to nearly equivalent or more sensitive than histologic evaluation. The PCR-RFLP results appeared dependent on the duration of fixation and bacterial load but not on the presence of hepatitis, sampling from neoplastic or nonneoplastic liver, or sex of the mouse.

In this study we investigated the mouse mad-related genes, Smad4/Dpc4 and Smad2 (homolog of JV18-1), as candidates for involvement in lung tumor resistance and suppression. These genes are located in a region of mouse chromosome 18 that is syntenic with human 18q21.1, where several genes that are mutated in various cancers have been mapped. A newly identified murine lung tumor resistance locus, Par2 has also been mapped to this region of chromosome 18. We found no mutations in the coding regions of either gene in 11 lung tumors from B6CF1 (C57BL/6 x BALB/c) mice by RT-PCR and SSCP/RFLP, suggesting that these genes are not mutated in lung carcinogenesis in this strain. Moreover, loss of heterozygosity in this region of chromosome 18 was not detected in 28 lung adenocarcinomas from B6CF1 mice, 17 lung adenocarcinomas from B6C3F1 mice or 18 lung adenocarcinomas from AB6F1 mice. These data provide evidence that a 'classical' tumor suppressor gene for mouse lung carcinogenesis in these strains does not reside in this region. In order to investigate Smad4/Dpc4 and Smad2 as candidates for the Par2 resistance locus mapped to this region, we also sequenced the coding regions of both genes in cDNA from normal lungs of A/J, BALB/c and C57BL/6 inbred strains of mice. No polymorphisms were detected in the coding region of Smad4. In Smad2, two sequence polymorphisms were identified that are not in the conserved regions of the gene. Northern blot analysis revealed no differential expression in normal lung tissue among the three strains for either gene. Thus, in this study we found no evidence that either Smad4 or Smad2 represents the Par2 lung tumor resistance locus or is a lung tumor suppressor gene in the B6CF1 mice.

Uterine leiomyomas are the most prevalent tumor type in women of reproductive age and are the most common reason for hysterectomies. Although uterine leiomyomas are considered to be benign, they are a major public health concern for women. In contrast, leiomyosarcomas are rare but highly malignant uterine tumors. They may arise in uteri with preexisting leiomyomas and histologically sometimes resemble leiomyomas, thus causing controversy about whether leiomyosarcomas arise within leiomyomas. In this study, we used comparative genomic hybridization (CGH) to identify genetic alterations unique to each tumor type and alterations that are common between the two tumors. We analyzed 14 cases of uterine leiomyomas and eight cases of uterine leiomyosarcomas. Only two of the 14 leiomyomas exhibited genetic alterations, and those were restricted to gains on chromosomes 14 and 19 and losses on chromosomes 1 and 4. In addition, 68 leiomyomas were examined for loss of heterozygosity on chromosomes 1 and 4, and only three tumors exhibited any losses. In contrast, all eight leiomyosarcomas showed gains and losses of DNA by CGH, and in many cases multiple changes were observed. The most commonly observed genetic aberration, occurring in five tumors, was gains on both arms of chromosome 1, suggesting that this chromosome contains loci involved in the development of leiomyosarcoma. Our results do not provide evidence for the progression from benign leiomyoma to malignant leiomyosarcoma. Moreover, the large number of random chromosomal alterations in the leiomyosarcomas suggests that increased genetic instability plays a role in the formation of these tumors.

Isoprene is the 2-methyl analog of 1,3-butadiene, a genotoxic and carcinogenic compound in rats and mice. Male B6C3F1 mice were exposed to 0, 2200 or 7000 ppm isoprene by inhalation (6 h/day; 5 days/week) for 26 weeks. Following a 26-week recovery period, an increased incidence of Harderian gland (HG) neoplasms was observed at both concentrations. The present study was designed to characterize genetic alterations in the K-ras and H-ras protooncogenes in HG neoplasms. Mutations in K-ras and H-ras were identified by single-strand conformational analysis and direct sequencing of polymerase chain reaction (PCR) amplified DNA, isolated from paraffin-embedded sections of HG neoplasms. A higher frequency of ras mutations, in particular K-ras mutations, was detected in isoprene-induced neoplasms than in 1,3-butadiene-induced or control HG neoplasms. All of the isoprene-induced HG neoplasms exhibited activated K-ras (60%) or H-ras (40%) mutations. In contrast, ras mutations were detected in 69% of HG neoplasms from 1,3-butadiene exposed mice (14% K-ras and 55% H-ras) and in 56% of HG neoplasms obtained from control B6C3F1 mice (8% K-ras and 48% H-ras). The predominant mutations in isoprene-induced HG neoplasms, but not in previously or newly analysed 1,3-butadiene-induced HG neoplasms, consisted of A-->T transversions (CAA-->CTA) at K-ras codon 61 (15/30) and C-->A transversions (CAA-->AAA) at H-ras codon 61 (8/30). Two-thirds of the K-ras CTA mutations were detected in HG neoplasms from the 2200 ppm exposure group while one-third was present in the 7000 ppm group. Isoprene-induced HG neoplasms with K-ras or H-ras mutations had an elevated proliferating cell nuclear antigen (PCNA) index, compared to spontaneous HG neoplasms without ras mutations. The high frequency and specificity of the ras mutation profile suggest that ras protooncogene activation contributes to isoprene-induced HG tumorigenesis.

In addition to being a potent hepatocarcinogen, aflatoxin B1 (AFB1) is a pulmonary carcinogen in experimental animals and epidemiological studies have shown an association between AFB1 exposure and lung cancer in humans. Since point mutations at codons 12, 13 and 61 of the K-ras protooncogene are often implicated in chemically induced mouse lung tumors and in human lung adenocarcinomas, we undertook an investigation of the role of K-ras activation in AFB1-induced pulmonary carcinogenesis. Female AC3F1 (A/J x C3H/HeJ) mice were treated with AFB1 (150 mg/kg i.p., divided into 24 doses over 8 weeks), and 6-14 months after the completion of dosing mice were killed and pulmonary adenomas and carcinomas removed. Of the 76 AFB1-induced lung tumors analyzed by single strand conformation polymorphism (SSCP) and direct sequencing, 75 possessed K-ras codon 12 mutations (46 GTT, 14 GAT, 13 TGT and 2 TTT; normal, GGT) and one had a GGC-->CGC mutation in codon 13. The observation that K-ras mutations occurred only at G:C base pairs is in agreement with N7-guanine being the primary site of AFB1-DNA adduct formation and with guanine residues being targets for AFB1-induced oxidative DNA damage via formation of 8-hydroxydeoxyguanosine (8-OHdG). The AFB1-specific nature of the observed K-ras mutation spectrum and the fact that 100% of the tumor samples examined contained K-ras mutations is consistent with K-ras activation being an early, critical event in AFB1-induced pulmonary carcinogenesis in AC3F1 mice. The parental origin of the observed K-ras mutations was determined by allele-specific PCR amplification of AFB1-induced lung tumor DNA followed by SSCP analysis. In the vast majority of tumors (73/76), the mutated K-ras allele was derived from the lung tumor susceptible A/J parent. This finding supports the existence of a link between K-ras and differences in mouse lung tumor susceptibility.