The mouse is a good model for evaluating the efficacy of chemopreventive agents for lung cancer. Gene silencing by promoter hypermethylation is a critical component for the development and progression of lung cancer and an emerging target for preventive intervention by demethylating agents. Genes methylated in mouse lung tumors could serve as biomarkers to evaluate the effectiveness of demethylating agents for preventing lung cancer and causing gene reexpression in vivo. The purpose of the current study was to evaluate a panel of genes inactivated by promoter hypermethylation in human lung cancer for silencing by this epigenetic mechanism in murine lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), cigarette smoke, or arising spontaneously. Cadherin-13, estrogen receptor-α, progesterone receptor, and runt-related transcription factor-3 were frequently methylated in mouse lung tumor-derived cell lines, whereas cadherin-1 and suppressor of cytokine signaling-1 were not. Methylation within these four genes was associated with lack of expression that could be restored after treatment with 5-aza-2′-deoxycytidine and with methylation within the CpG island of each gene. Methylation-specific PCR revealed that methylation of these four genes occurred at prevalences of 24% to 69% in primary lung tumors arising spontaneously or induced by exposure to cigarette smoke or NNK. Estrogen receptor-α methylation was more frequent in spontaneously occurring lung cancer than cigarette smoke–induced or NNK-induced lung cancer, whereas runt-related transcription factor-3 showed the opposite relationship. Thus, genes can be targeted for inactivation by methylation, depending on exposure history. This study indicates that methylation events frequently observed in human lung cancer are recapitulated in the mouse model and identifies four potential biomarkers for assessing intervention approaches for reversing epigenetically mediated gene silencing. (Mol Cancer Res 2006;4(4):267–73)

A sensitive screening approach for lung cancer could markedly reduce the high mortality rate for this disease. Previous studies have shown that methylation of gene promoters is present in exfoliated cells within sputum prior to lung cancer diagnosis. The purpose of the current study is to conduct a nested case-control study of incident lung cancer cases from an extremely high-risk cohort for evaluating promoter methylation of 14 genes in sputum. Controls (n = 92) were cohort members matched to cases (n = 98) by gender, age, and month of enrollment. The comparison of proximal sputum collected within 18 months to > 18 months prior to diagnosis showed that the prevalence for methylation of gene promoters increased as the time to lung cancer diagnosis decreased. Six of 14 genes were associated with a > 50% increased lung cancer risk. The concomitant methylation of three or more of these six genes was associated with a 6.5-fold increased risk and a sensitivity and specificity of 64%. This is the first study to prospectively examine a large panel of genes for their ability to predict lung cancer and shows the promise of gene promoter hypermethylation in sputum as a molecular marker for identifying people at high risk for cancer incidence.

Purpose: Lung cancer is the leading cause of cancer mortality in the United States, due in part to the lack of a validated and effective screening approach for early detection. The prevalence for methylation of seven and three genes was examined in DNA from sputum and plasma, respectively, from women at different risk for lung cancer.Experimental Design: Lung cancer survivors (n = 56), clinically cancer-free smokers (n = 121), and never smokers (n = 74) comprised the study population. Plasma was collected from all three groups, whereas sputum was collected from lung cancer survivors and smokers.Results: Methylation was detected in plasma DNA from 10 of 74 women who never smoked. Prevalence for methylation of the p16 gene in plasma was highest in lung cancer survivors. Lung cancer survivors showed a significant increase in the odds of having at least one or more genes methylated in plasma (odds ratio, 3.6; 95% confidence interval, 1.9-9.1) than never smokers. The prevalence for methylation of the O-6-methylguanine-DNA methyltransferase, ras effector homologue 1, death associated protein kinase, and PAX5 alpha genes in sputum was significantly higher in lung cancer survivors compared with smokers. Lung cancer survivors had 6.2-fold greater odds (95% confidence interval, 2.1-18.5) for methylation of three or more genes in sputum compared with smokers. Methylation was more commonly detected in sputum than plasma for O-6-methylguanine-DNA methyltransferase and ras effector homologue 1, but not p16, in lung cancer survivors.Conclusion: Concomitant methylation of multiple. gene promoters in sputum is strongly associated with lung cancer risk.

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.

The prevalence of methylation of the p16, DAPK and RASSFIA genes was investigated in lung adenocarcinoma from smokers, former uranium miners and never smokers. The association between a common genetic alteration in adenocarcinoma, mutation of the K-ras gene and methylation of these genes, as well as survival was examined. Adenocarcinomas from 157 smokers, 46 never smokers and 34 former uranium miners were evaluated for methylation of the p16, DAPK and RASSFIA genes using the methylation-specific PCR assay. Comparisons were also made to prevalences of methylation of the MGMT gene and mutation of the K-ras gene previously examined in these tumors. The prevalence of methylation for all genes was similar between adenocarcinomas from smokers and never smokers, although the prevalence for methylation of the p16 gene tended to be higher in smokers compared to never smokers. A significantly higher prevalence for p16 methylation was seen in central vs. peripheral lung tumors. At least 1 gene was methylated in 35% of stage I tumors, whereas 2 and :3 genes were methylated in 40% and 16% of tumors, respectively. Methylation of all genes was independent of K-ras mutation, whereas methylation of the DAPK and RASSFIA genes was positively associated. Environmental tobacco smoke, the strongest lung cancer risk factor among never smokers, induces adenocarcinoma in part through inactivation of the p16, DAPK and RASSFIA genes. Adenocarcinomas may develop through 2 distinct processes: multiple gene inactivations through promoter hypermethylation and activation of the K-ras gene. (C) 2004 Wiley-Liss, Inc.

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.

The transcription factor activator protein-2α (AP-2α) has recently been implicated as a tumor suppressor protein that can be lost during tumor progression and that exhibits growth-inhibitory properties when overexpressed in cancer cell lines. We now demonstrate that hypermethylation of a discrete 5′ region within a promoter CpG island of the gene is associated in breast cancer with the loss of AP-2α expression. Multiple CpG sites within the island become hypermethylated during breast cancer evolution. However, only hypermethylation of the most CpG-rich region, a small, ∼300-bp area at the 3′ end of exon 1, fully distinguishes neoplastic from normal breast tissue and correlates with transcriptional silencing. In cell culture, silenced AP-2α, associated with exon 1 hypermethylation, is re-expressed by 5-aza-2′deoxycytidine resulting in the restoration of a functional DNA sequence-specific binding protein. In vivo, as detected by a very sensitive nested PCR approach, methylation of the discrete AP-2α exon 1 region does not occur in normal breast epithelium and occurs in only 3 (16%) of 19 ductal carcinoma in situ (DCIS) lesions, but is present in 12 (75%) of 16 invasive breast tumors (P < 0.001; DCIS versus invasive cancers). Tumors unmethylated for this region expressed AP-2α protein throughout, whereas tumors with hypermethylation showed large areas of loss. Our studies then determine that hypermethylation of a small region of a CpG island correlates with silencing of AP-2α in breast cancer and suggest that inactivation of this gene could be a factor in, and a useful marker for, the progression of DCIS lesions.

The retinoic acid receptor beta (RAR-beta) gene encodes one of the primary receptors for retinoic acid, an important signaling molecule in lung growth, differentiation and carcinogenesis. RAR-beta has been shown to be down-regulated by methylation in human lung cancer. We have used previously lung tumors induced in mice to evaluate the timing and effect of specific carcinogen exposures on targeting genes altered in human lung cancer. These studies were extended to characterize the role of methylation of the RAR-beta gene in murine lung cancers. After treatment with the demethylating agent 5-aza-2'-deoxycytidine (DAC), RAR-beta was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism. Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the 5' untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by carcinogen exposures with the detection of methylated alleles in virtually all primary tumors associated with cigarette smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of tumors induced by methylene chloride or vinyl carbamate. RAR-beta methylation was also detected in 54% of preneoplastic hyperplasias induced by treatment with NNK. Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of adenocarcinoma development. These studies demonstrate that aberrant methylation of RAR-beta is an early and common alteration in murine lung tumors induced by several environmentally relevant exposures.

Lung cancer from radon or (239)plutonium exposure has been linked to alpha-particles that damage DNA through large deletions and point mutations. We investigated the involvement of an epigenetic mechanism, gene inactivation by promoter hypermethylation in adenocarcinomas from plutonium-exposed workers at MAYAK, the first Russian nuclear enterprise established to manufacture weapons plutonium. Adenocarcinomas were collected retrospectively from 71 workers and 69 non-worker controls. Lung adenocarcinomas were examined from workers and non-worker controls for methylation of the CDKN2A (p16), O(6)-methylguanine-DNA methyltransferase (MGMT), death associated protein kinase (DAP-K), and Ras effector homolog 1 genes (RASSF1A). The prevalence for methylation of the MGMT or DAP-K genes did not differ between workers and controls, while a higher prevalence for methylation of the RASSF1A gene was seen in tumors from controls. In marked contrast, the prevalence for methylation of p16, a key regulator of the cell cycle, was increased significantly (P = 0.03) in tumors from workers compared with non-worker controls. Stratification of plutonium exposure into tertiles also revealed a striking dose response for methylation of the p16 gene (P = 0.008). Workers in the plutonium plant where exposure to internal radiation was highest had a 3.5 times (C.I. 1.5, 8.5; P = 0.001) greater risk for p16 methylation in their tumors than controls. This increased probability for methylation approximated the 4-fold increase in relative risk for adenocarcinoma in this group of workers exposed to plutonium. In addition, a trend (P = 0.08) was seen for an increase in the number of genes methylated (> or =2 genes) with plutonium dose. Here we demonstrate that exposure to plutonium may elevate the risk for adenocarcinoma through specifically targeting the p16 gene for inactivation by promoter methylation.

An animal model of lung carcinogenicity induced by chronic inhalation of mainstream cigarette smoke would be useful for research on carcinogenic mechanisms, smoke composition-response relationships, co-carcinogenicity, and chemoprevention. A study was conducted to determine if chronic whole-body exposures of rats would significantly increase lung tumor incidence. Male and female F344 rats (n = 81 to 178/gender) were exposed whole-body 6 h/day, 5 days/week for up to 30 months to smoke from 1R3 research cigarettes diluted to 100 (LS) or 250 (HS) mg total particulate matter/m(3), or sham-exposed to clean air (C). Gross respiratory tract lesions and standard lung and nasal sections were evaluated by light microscopy. A slight reduction of survival suggested that the HS level was at the maximum tolerated dose as commonly defined. Cigarette smoke exposure significantly increased the incidences of non-neoplastic and neoplastic proliferative lung lesions in females, while nonsignificant increases were observed in males. The combined incidence of bronchioloalveolar adenomas and carcinomas in females were: HS = 14%; LS = 6%; and C = 0%. These incidences represented minima because only standard lung sections and gross lesions were evaluated. Mutations in codon 12 of the K-ras gene occurred in 4 of 23 (17%) tumors. Three mutations were G to A transitions and one was a G to T transversion. The incidence of neoplasia of the nasal cavity was significantly increased at the HS, but not the LS level in both males and females (HS = 6%, LS = 0.3%, C = 0.4% for combined genders). These results demonstrate that chronic whole-body exposure of rats to cigarette smoke can induce lung cancer.

Adenocarcinoma (AC) is the most common type of lung cancer diagnosed in the United States, comprising up to 40% of tumors in smokers and 50-80% of tumors in never-smokers. Exposures to cigarette smoke, direct or second-hand, and radiation in the form of radon progeny are the major risk factors for lung AC in both smokers and never-smokers. The goal of the current study was to determine the prevalence for O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in a large sample of central or peripheral ACs from smokers (n = 157), former uranium miners (n = 34), and never-smokers (n = 46). The mutation rate at codon 12 of the K-ras gene was determined to assess whether activation of this oncogene was associated with MGMT methylation. The overall prevalence for MGMT methylation was 51%. Significantly more tumors from never-smokers than smokers exhibited MGMT methylation (66 versus 47%, respectively). In contrast, exposure to radon through uranium mining did not affect the prevalence for methylation. The frequency of MGMT methylation was increased significantly in association with tumor stage. K-ras mutations were detected in 24% of all ACs and 22, 24, and 28% of tumors from never-smokers, smokers, and miners, respectively. Alterations in both the K-ras and MGMT genes were seen in only 11% of ACs. Kaplan-Meier survival estimates did not reveal any difference between patient survival with or without MGMT methylation. In contrast, survival was significantly reduced over the initial 60 months after diagnosis for patients with a transition mutation in the K-ras gene compared with those with a transversion mutation. This investigation demonstrates that MGMT promoter hypermethylation is a common event in the progression of early stage AC of the lung. We have shown that the incidence of MGMT methylation was significantly higher in never-smokers than smokers and have detected a higher frequency of mutations within the K-ras gene than previously reported in never-smokers. This study also suggests that K-ras activation is independent of MGMT methylation.

Aberrant methylation of 5'CpG islands is a key epigenetic event in many human cancers. A PCR-based technique of methylated CpG island amplification followed by representational difference analysis was used to identify genes methylated in cancer. Two of the CpG islands identified mapped to the 5' untranslated region of the PAX5 alpha and beta genes. These genes, located on chromosome 9p13, are transcribed from two distinct promoters and form two alternative first exons that are subsequently spliced to the common exons 2-10. The resulting splice variants encode two distinct transcription factors important in cell differentiation and embryonic development. Examination of the methylation status of each gene using methylation-specific PCR revealed that both genes are methylated in approximately 65% of breast and lung tumors. Bisulfite sequencing revealed dense methylation patterns within each 5'CpG island, strongly correlating with transcriptional silencing. Expression in cell lines with dense methylation of either the PAX5 alpha or beta promoter region was restored after treatment with the demethylating agent 5-Aza-2'-deoxycytidine. The PAX5 beta gene encodes for the transcription factor B cell-specific activating protein that, in turn, directly regulates CD19, a gene shown to negatively control cell growth. A strong association was observed between PAX5 beta methylation and loss of expression of the CD19 gene demonstrating that inactivation of the PAX5 beta gene likely contributes to neoplastic development by inhibiting growth regulation through effects on CD19 gene expression. Recent studies have demonstrated the importance of PAX5 gene alterations in human cancer. Our results are the first to identify aberrant promoter methylation as a common mechanism for dysregulation of these genes in solid tumors.

Disruption of one allele for the cytosine-DNA methyltransferase 1 (DNMT1) gene in mice with a germ-line mutation in a tumor suppressor gene was shown previously to reduce tumor formation in juvenile animals. This effect is now reproduced in our studies of mature mice where this genetic DNMT1 reduction leads to a 50% decrease in tobacco carcinogen-induced lung cancer and a similar reduction in DNMT activity in type II pneumocytes that give rise to the tumors. Short-term treatment of DNMT wild-type female mice with low doses of the demethylating agent 5-aza-2'-deoxycytidine decreased the incidence of neoplasms by 30%. Importantly, when 5-aza-2'-deoxycytidine was combined with the histone deacetylase inhibitor sodium phenylbutyrate, lung tumor development was significantly reduced by >50%; no effect was seen with phenylbutyrate alone. This identical combination of inhibitors also acts synergistically to cause re-expression of densely hypermethylated and transcriptionally silenced tumor suppressor genes in human cancer cells. Thus, reduction in DNMT and histone deacetylase activities that likely block epigenetically mediated gene silencing might provide a novel clinical strategy to help prevent the leading cause of cancer death in the United States.

Recent studies by our laboratory indicate that the p16(INK4a) gene is frequently methylated in lung tumors induced by genotoxic carcinogens and that the frequency for methylation of the estrogen receptor alpha (ER) gene varies as a function of carcinogenic exposure. The purpose of the current investigation was to define the role of these two genes in lung tumors induced by the particulate carcinogens carbon black (CB), diesel exhaust (DE) or beryllium metal. Methylation of p16 was observed in 59 and 46% of DE and CB tumors, respectively. In contrast, the ER gene was inactivated in only 15% of DE or CB tumors. Methylation of the p16 and ER genes was very common (80 and 50%, respectively) in beryllium-induced lung tumors; both genes were methylated in 40% of the tumors. Bisulfite sequencing revealed dense methylation throughout exon 1 of the ER gene. The inhibitory effect of methylation on gene transcription was confirmed through RT-PCR expression studies in which p16 gene expression was 30-60-fold lower in methylated than unmethylated tumors. Residual expression in methylated tumors was consistent with contamination by stromal and inflammatory cells. Results indicate that tumors induced by these particulate carcinogens arise, in part, through inactivation of the p16 and ER genes. Furthermore, the inactivation of the p16 gene by these carcinogenic exposures supports a possible role for oxidative stress and inflammation in the etiology of human lung cancer.

Inactivation of the p16(INK4a) tumor suppressor gene and O(6)-methylguanine-DNA methyltransferase (MGMT) DNA repair gene by aberrant promoter methylation appears to be an important step in respiratory carcinogenesis after exposure to tobacco smoke and radon progeny. The determinants of aberrant promoter methylation are not well characterized. Polymorphic variants of genes of which the products are involved in pathways that modulate and repair DNA damage after carcinogen exposure may affect the occurrence of de novo promoter methylation. On the basis of their associations with risk of lung cancer, we hypothesized that functional polymorphic variants of the NADPH quinone oxidoreductase, glutathione S-transferases P1 and M1, myeloperoxidase, and XRCC1 genes are associated with p16 and/or MGMT promoter methylation in sputum from cancer-free subjects at high risk for developing lung cancer. This hypothesis was tested by conducting a cross-sectional study of 70 former uranium miners from the Uranium Epidemiological Study cohort who were at high risk for lung cancer. The polymorphic variant genotypes were characterized through PCR-RFLP on DNA isolated from peripheral lymphocytes, and the methylation status of the p16 and MGMT promoters was determined by methylation-specific PCR on DNA isolated from sputum. Subjects who had at least one GSTP1 polymorphic allele (A-to-G at bp 104) had an increased risk for MGMT methylation [odds ratio (OR), 4.8; 95% confidence interval (CI), 1.2-18.6] or for either p16 or MGMT methylation (OR, 4.4; 95% CI, 1.3-14.2). Lack of a wild-type NADPH quinone oxidoreductase allele (C at bp 609) was also associated with methylation of either p16 or MGMT (OR, 3.1; 95% CI, 1.0-9.2). These results provide the first link between germ-line functional deficits in pathways that protect the cell from tobacco- and radon-induced DNA damage, and the development of aberrant promoter methylation of the p16 and MGMT genes in the respiratory epithelium of individuals at high risk for lung cancer.

Recent studies from our laboratory suggest that gene-specific methylation changes in sputum could be good intermediate markers for the early detection of lung cancer and defining the efficacy of chemopreventive interventions. The purpose of our study was to determine the prevalence for aberrant promoter methylation of the p16, O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein (DAP) kinase, and Ras effector homologue (RASSFIA) genes in nonmalignant bronchial epithelial cells from current and former smokers in a hospital-based, case control study of lung cancer. The relationship between loss of heterozygosity, at 9p and p16 methylation in bronchial epithelium and the prevalence for methylation of these four genes in sputum from cancer-free, current and former smokers were also determined. Aberrant promoter methylation of p16 was seen in at least one bronchial epithelial site from 44% of cases and controls. Methylation of the DAP kinase gene was seen in only 1 site from 5 cases and 4 controls, whereas methylation of the RASSFIA was not detected in the bronchial epithelium. Promoter methylation for p16 and DAP kinase was seen as frequently in bronchial epithelium from current smokers as from former smokers. No promoter methylation of these genes was detected in bronchial epithelium from never-smokers. Methylation of the p16 gene was detected in sputum from 23 of 66 controls. DAP kinase gene promoter methylation was also seen in sputum from 16 controls, and 8 of these subjects were positive for p16 methylation. Methylation of the MGMT gene was seen in sputum from 9 controls, whereas RASSFIA promoter methylation was only seen in 2 controls. The correlation between p16 status in the bronchial epithelium obtained from lung lobes that did not contain the primary tumor and the tumor itself was examined. Seventeen of 18 tumors (94%) showed an absolute concordance, being either methylated in the tumor and at least 1 bronchial epithelial site, or unmethylated in both tumor and bronchial epithelium. These results indicate that aberrant promoter hypermethylation of the p16 gene, and to a lesser extent, DAP kinase, occurs frequently in the bronchial epithelium of lung cancer cases and cancer-free controls and persists after smoking cessation. The strong association seen between p16 methylation in the bronchial epithelium and corresponding primary tumor substantiates that inactivation of this gene, although not transforming by itself, is likely permissive for the acquisition of additional genetic and epigenetic changes leading to lung cancer.

Cyclooxygenase-2 (COX-2) is frequently expressed in cancer cells, contributing to tumor development. Most studies of COX-2 expression have examined artificially induced expression in noncancer cells rather than basal expression in cancer cells. Therefore, basal COX-2 expression and its regulation were examined in cell lines derived from a murine model of lung adenocarcinoma. The presence of COX-2 protein in these cells was demonstrated by Western analysis. COX-2 promoter activity was repressed by U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], a mitogen-activated protein kinase kinase inhibitor, as well as SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole], an inhibitor of p38 mitogen-activated protein kinase, substantiating the involvement of these signal transduction pathways in the regulation of basal COX-2 expression. Retinoic acid also repressed promoter activity, yet increased activity significantly in one cell line after 18 and 30 h of treatment. Deletions of the murine COX-2 promoter revealed that the 5' transcription factor binding sites were not required for basal expression, including the only nuclear factor-kappaB sites of the promoter. Site-directed mutagenesis of the 3' C/EBP (CCAAT/enhancer-binding protein) sites inhibited promoter activity by 20 to 55%, while mutation of the 3' ATF/CREB/AP-1 (activating transcription factor/cAMP response element-binding protein/activator protein-1) site inhibited activity by 70%. Mutation of the 3' upstream stimulatory factor site did not affect promoter activity. Electrophoretic mobility shift assays indicated that the AP-1 transcription factor does not bind to the 3' ATF/CREB/AP-1 site, leaving C/EBP and ATF/CREB as the major transcriptional regulators of basal expression of COX-2 in these lung tumor-derived cell lines and identifying new targets for the prevention/treatment of lung cancer through the modulation of COX-2 expression.

Over 85% of people with lung cancer eventually succumb to this disease, largely because current chemotherapies are ineffective. The testing and validation of promising new approaches generally rely on achieving responses with cell lines in vitro or in tumor xenografts in nude mice. However, quite often the results seen with these models are not recapitulated in the clinic, thus necessitating the need for better animal models of lung cancer for preclinical testing of new therapies. One promising model is that of orthotopic lung cancer, where xenografts of human lung cancer are established in lungs of immunodeficient rodents. The problems associated with this model include poor rates of engraftment, limited tumor multiplicity, and a heightened risk for surgical trauma. The purpose of our study was to develop an efficient approach to engraftment of orthotopic tumors throughout the lungs of the Rowett nude rat. Initially, we augmented immunosuppression in the rats with whole-body X-irradiation and then used orotracheal cannulas to intratracheally instill human cancer cells from the Calu-6 cell line. This protocol produced a low rate of engraftment and low tumor multiplicity. The hypothesis that slight disruption of the pulmonary epithelium or the surfactant layer would allow better tumor engraftment was tested by coadministration of either pancreatic elastase or ethylenediaminetetraacetic acid (EDTA) along with the cell instillations. Lung tumor engraftment was evaluated 8 weeks after instillation. The inclusion of elastase or EDTA with the Calu-6 cells resulted in an 80-100% engraftment rate, respectively. Coadministration of EDTA resulted in significantly larger and greater numbers of tumors/lung than those in elastase-treated animals. Temporal studies demonstrated that small nodules were scattered throughout the lung parenchyma 5 weeks after instilling Calu-6 cells and EDTA. These nodules grew to coalesce and form large masses that effaced > 75% of the parenchyma at 9 weeks postinstillation. The refinements made through our studies have led to the development of an orthotopic lung cancer model that should facilitate the evaluation of novel therapies designed to treat or impede lung cancer development.

Inactivation of the p16INK4a (p16) tumor suppressor gene by promoter hypermethylation and mutation within exon 3 of beta-catenin represent two of the more common gene alterations in human hepatocellular carcinoma (HCC). One exposure implicated in the development of liver cancer is hepatitis B or C viral infection, which causes chronic destruction and regeneration of liver parenchyma. Treatment of rats with high doses of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes liver toxicity and a high incidence of tumors. The purpose of the current investigation was to define the prevalence of genetic alterations in p16 and beta-catenin in NNK-induced rat liver cancer to determine if the molecular mechanisms seen in human tumors are the same in this animal model. DNA isolated from 15 adenomas and 14 carcinomas was examined for methylation of p16 by methylation-specific PCR. p16 methylation was detected in five of 15 adenomas and eight of 14 carcinomas (45% of all tumors). Methylation of p16 was extensive within the 5'-untranslated region and exon 1alpha, areas shown to correlate with loss of gene transcription. Liver tumors were also screened for mutations within exon 3 of beta-catenin. Single strand conformation polymorphism and DNA sequencing revealed five mutations in four of 29 tumors (14%). Mutations were present in three adenomas and one carcinoma and were located within codons 33, 36 or 37. All mutations resulted in amino acid substitutions; three of these mutations occurred at potential serine phosphorylation sites. Our results link two important regulatory pathways altered in human HCC to cancer induced in the rat NNK model. The fact that common genetic alterations are observed between rodent and human HCC suggests that the rat NNK model could be useful for identifying additional genetic alterations critical to the initiation of HCC.