Genotypes in matrix metalloproteinase 9 are risk factors for chronic obstructive pulmonary disease.
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Case-Control Studies↗
Biomedical subjects
S A Belinsky
Publications and source records attributed to S A Belinsky.
The XRCC1 399 glutamine allele is a risk factor for adenocarcinoma of the lung.
Defects in the repair and maintenance of DNA increase risk for cancer. X-ray cross-complementing group 1 protein (XRCC1) is involved with the repair of DNA single-strand breaks. A nucleotide substitution of guanine to adenine leading to a non-conservative amino acid change was identified in the XRCC1 gene at codon 399 (Arg/Gln). This change is associated with higher levels of aflatoxin B1-adducts and glycophorin A somatic mutations. A case-control study was conducted to test the hypothesis that the 399Gln allele is positively associated with risk for adenocarcinoma of the lung. XRCC1 genotypes were assessed at codon 399 in 172 cases of lung adenocarcinoma and 143 cancer-free controls. Two ethnic populations were represented, non-Hispanic White and Hispanic. The distribution of XRCC1 genotypes differed between cases and controls. Among cases, 47.7% were Arg/Arg, 35.5% were Arg/Gln, and 16.9% were Gln/Gln. Among controls, XRCC1 allele frequencies were 45.5% for Arg/Arg, 44.8% for Arg/Gln, and 9.8% for Gln/Gln. Logistic regression analysis was used to assess the association between lung adenocarcinoma and the G/G genotype relative to the A/A or A/G genotypes. In non-Hispanic White participants, the lung cancer risk associated with the G/G genotype increased significantly after adjustment for age (OR=2.81; 95% CI, 1.2-7.9; P=0.03) and increased further after adjustment for smoking (OR=3.25; 95% CI, 1.2-10.7; P=0.03). Among all groups, a significant association was found between the G/G homozygote and lung cancer (OR=2.45; 95% CI, 1.1-5.8; P=0.03) after adjustment for age, ethnicity, and smoking. This study links a functional polymorphism in the critical repair gene XRCC1 to risk for adenocarcinoma of the lung.
Adenocarcinoma↗
p16INK4a and beta-catenin alterations in rat liver tumors induced by NNK.
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.
Animals↗
Reduced DNA-dependent protein kinase activity is associated with lung cancer.
Reduced DNA repair capacity of carcinogen-induced DNA damage is now thought to significantly influence inherent susceptibility to lung cancer. DNA-dependent protein kinase (DNA-PK) is a serine-threonine kinase activated by the presence of double-strand breaks in DNA that appears to play a major role in non-homologous recombination and transcriptional control. The purpose of this study was to determine whether DNA-PK activity varies among individuals and how this affects lung cancer risk. DNA-PK activity in peripheral mononuclear cells from individuals with lung cancer (n = 41) was compared with lung cancer-free controls (n = 41). Interindividual variability was seen within each group, however, significant differences (P = 0.03) in DNA-PK activity between cases and controls were seen when comparing the distribution of enzyme activity among these two groups. The percentages of cases and controls with DNA-PK activity in the ranges 2.5-5.0 and 7.6-10.0 units were 39 versus 20% and 7 versus 29%, respectively. The enzyme activity in peripheral mononuclear cells reflected that seen in bronchial epithelial cells, one progenitor cell for lung cancer, supporting the use of peripheral mononuclear cells for larger population-based studies of DNA-PK activity. Its role as a potential modifier for lung cancer risk was supported by the fact that cell growth in bronchial epithelial cells exposed to bleomycin was directly associated with enzyme activity. The results of this study demonstrate that reduced DNA-PK repair activity is associated with risk for lung cancer.
Adult↗
Refinement of an orthotopic lung cancer model in the nude rat.
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.
Animals↗
Perspective: cell differentiation theory may advance early detection of and therapy for lung cancer.
Many of these deaths could be prevented if there were better screening methods to uncover the disease when it is limited and most responsive to intervention. Novel biomarkers of early-stage disease are therefore needed. By applying the principle of "oncology recapitulates ontogeny", we have discovered three homeobox (HOX) genes that are inappropriately expressed in the majority of lung tumors. Understanding the role of these inappropriately expressed genes in lung epithelial cell carcinogenesis may not only augment early detection, but may also offer new avenues of treatment of this disease.
Cell Differentiation↗
Predicting lung cancer by detecting aberrant promoter methylation in sputum.
Despite the promise of using DNA markers for the early detection of cancer, none has proven universally applicable to the most common and lethal forms of human malignancy. Lung carcinoma, the leading cause of tumor-related death, is a key example of a cancer for which mortality could be greatly reduced through the development of sensitive molecular markers detectable at the earliest stages of disease. By increasing the sensitivity of a PCR approach to detect methylated DNA sequences, we now demonstrate that aberrant methylation of the p16 and/or O6-methyl-guanine-DNA methyltransferase promoters can be detected in DNA from sputum in 100% of patients with squamous cell lung carcinoma up to 3 years before clinical diagnosis. Moreover, the prevalence of these markers in sputum from cancer-free, high-risk subjects approximates lifetime risk for lung cancer. The use of aberrant gene methylation as a molecular marker system seems to offer a potentially powerful approach to population-based screening for the detection of lung cancer, and possibly the other common forms of human cancer.
Carcinoma, Squamous Cell↗
Aberrant methylation of gene promoters in cancer---concepts, misconcepts, and promise.
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Animals↗
Cyclooxygenase-2 expression is abundant in alveolar type II cells in lung cancer-sensitive mouse strains and in premalignant lesions.
Overexpression of cyclooxygenase-2 (COX-2) is seen in a high percentage of human colon tumors, lung adenocarcinomas and other cancers. Inhibition of this enzyme represses human colon tumorigenesis and decreases lung tumor multiplicity in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-exposed A/J mice. The purpose of this investigation was to characterize the expression of cyclooxygenase-2 (COX-2) during tumor progression in the A/J mouse lung and to compare the results with expression in other cancer-susceptible and several cancer-resistant mouse strains. Analysis of normal A/J mouse lung showed that type II alveolar epithelial cells express high levels of COX-2 protein and mRNA, indicating that COX-2 is present constitutively in this tumor progenitor cell prior to any carcinogen exposure. Examination of lung-cancer-resistant (C3H/HeJ, C57BL/6J, DBA/2J) and other lung-cancer-susceptible (A/WySnJ, SWR/J) strains showed similar levels of COX-2 mRNA expression in the three susceptible strains and lower levels of expression in two of the resistant strains, indicating a possible correlation between COX-2 expression in type II cells and lung cancer susceptibility. COX-2 protein expression was observed in A/J lung tumors at all stages of development. Variation and occasional absence of protein expression were also observed in A/J lung tumors, particularly in adenomas and adenocarcinomas, suggesting that COX-2 is not obligatory for maintenance of the malignant phenotype. In support of this conclusion, treatment of xenografted cell lines derived from malignant murine pulmonary tumors with COX-2 inhibitors produced only a slight repression of growth. However, the frequent expression of COX-2 in early lesions in the A/J mouse lung combined with the known reduction in tumor number in animals treated with COX-2 inhibitors prior to carcinogen exposure indicate that COX-2 could be a promising target for lung cancer chemoprevention. In addition, high levels of COX-2 expression in the normal tumor-progenitor cells of lung-cancer-sensitive mice indicate that COX-2 may play a role in lung cancer susceptibility.
Animals↗
1,3-butadiene: cancer, mutations, and adducts. Part I: Carcinogenicity of 1,2,3,4-diepoxybutane.
Reports in the literature suggest that one reason for the greater sensitivity of mice to the carcinogenicity of 1,3-butadiene (BD) is that exposed mice metabolize much more of the BD to 1,2,3,4-diepoxybutane (BDO2) than do exposed rats. The purpose of this study was to determine the tumorigenicity of BDO2 in rats and in mice exposed to the same concentration of the agent. Female B6C3F1 mice and Sprague-Dawley rats, 10 to 11 weeks old, 56 per group, were exposed by inhalation to 0, 2.5, or 5.0 ppm BDO2, 6 hours/day, 5 days/week for 6 weeks. Preliminary dosimetry studies in rodents exposed for 6 hours to 12 ppm BDO2 indicated that blood levels would be expected to be approximately 100 and 200 pmol/g at the two exposure concentrations in the rat and twice those levels in the mouse. During the 6-week exposure, the mice at the high exposure level showed signs of labored breathing during the last week, and four mice died. In the others, however, the respiratory symptoms disappeared after exposure ended. Rats showed no clinical signs of toxicity during exposure but developed labored breathing after the end of the exposure leading to the death of 13 rats within 3 months. At the end of the exposure, some animals (8 per group) were evaluated for the acute toxicity resulting from the BDO2 exposure. The remaining exposed rats and mice were held for 18 months for observation of tumor development. At the end of the exposure, rats had no biologically significant alteration in standard hematological parameters, but mice had a dose-dependent increase in neutrophils and decrease in lymphocytes. In both species the significant histopathologic lesions were in the nose, concentrated around the main airflow pathway. Necrosis, inflammation, and squamous metaplasia of the nasal mucosa, as well as atrophy of the turbinates, were all present at the end of exposure to 5.0 ppm. Within 6 months, necrosis and inflammation subsided, but squamous metaplasia remained in the mice. In rats that died after exposure, squamous metaplasia was seen in areas of earlier inflammation and, in other rats, extended beyond those areas with time. The metaplasia was severe enough to restrict and occlude the nasopharyngeal duct. Later, keratinizing squamous cell carcinomas developed from the metaplastic foci in rats but not mice. At the end of 18 months, the only significant increase in neoplasia in the exposed rats was a dose-dependent increase in neoplasms of the nasal mucosa (0/47, 12/48, and 21/48 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). Neoplasia of the nasal mucosa did not increase significantly in the mice; neoplastic lesions in the mice were observed in reproductive organs, lymph nodes, bone, liver, Harderian gland, pancreas, and lung. The only significant increase in neoplasms in a single organ in the mice was in the Harderian gland (0/40, 2/42, and 5/36 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). This tumor accounts for the apparent trend toward an increase in total neoplastic lesions in mice as a function of dose (10/40, 7/42, and 16/36 for control, 2.5 ppm, and 5.0 ppm exposures, respectively). These findings indicate that the metabolite of BD, BDO2, is carcinogenic in the respiratory tract of rats. An increase in respiratory tract tumors was not observed in similarly exposed mice despite the fact that preliminary studies indicated mice should have received twice the dose to tissue compared with the rats. High cytosolic activity of detoxication enzymes in the mouse may account, in part, for the differences in response.
Administration, Inhalation↗
In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis.
We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D-Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D-Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.
Carcinoma, Squamous Cell↗
Carcinogenicity of inhaled butadiene diepoxide in female B6C3F1 mice and Sprague-Dawley rats.
Previous studies suggest that the greater sensitivity of mice, compared to rats, to the carcinogenicity of 1,3-butadiene (BD) is linked to higher rates of BD metabolism to butadiene diepoxide (BDO2) by mice than rats. The purpose of this study was to determine the tumorigenicity of BDO2 in mice and rats exposed by inhalation to the same concentrations of the agent. Female B6C3F1 mice and Sprague-Dawley rats, 10-11 weeks old, 56/group, were exposed to 0, 2.5, or 5.0 ppm BDO2, 6 h/day, 5 days/week for 6 weeks. At the end of the BDO2 exposure, 8 animals/group were evaluated for toxicity. The remainder of the exposed rats and mice were held for up to 18 months for observation of tumor development. At the end of the exposure, rats had no biologically significant alteration in standard hematological parameters, but mice had a dose-dependent increase in neutrophils and decrease in lymphocytes. Most of the significant lesions in both species were in the nose, concentrated around the main airflow pathway. Necrosis, inflammation, and squamous metaplasia of the nasal mucosa, as well as atrophy of the turbinates, were all present in animals exposed to 5.0 ppm. In mice, necrosis and inflammation subsided within 6 months, but squamous metaplasia remained. In rats that died after exposure, squamous metaplasia was seen in areas of earlier inflammation and extended beyond those areas with time. The metaplasia was severe enough to restrict and occlude the nasopharyngeal duct. Later, keratinizing squamous-cell carcinomas developed from metaplastic foci in rats, but these were not seen in mice. At the end of 18 months, the only significant increase in neoplasia in the exposed rats was a dose-dependent increase in neoplasms of the nasal mucosa (0/47, 12/48, and 21/48 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). Neoplasia of the nasal mucosa did not increase significantly in the mice. Neoplastic lesions in the mice were observed in reproductive organs, lymph nodes, bone, liver, Harderian gland, pancreas, and lung, but the only significant increase in neoplasms in a single organ in the mice was in the Harderian gland (0/40, 2/42, and 5/36 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). This tumor accounts for the apparent trend toward an increase in total neoplastic lesions in mice as a function of dose (10/40, 7/42, and 16/36 for control, 2.5 ppm, and 5.0 ppm, respectively). These findings indicate that the metabolite of BD, BDO2, is carcinogenic in the upper respiratory tract of rats. An increase in upper respiratory tract tumors was not observed in similarly exposed mice, despite the fact that preliminary studies indicated mice should have received twice the dose to tissue than did the rats. Higher cytosolic activity of detoxication enzymes has been reported in the liver and lung cells of the mouse compared to the rat, and this may account, in part, for the differences in response. The transport of externally administered BDO2, into the cell and through the cytoplasm, might allow detoxication of the molecule before it reaches critical sites on the DNA. The results indicate that the site of formation of the BDO2 is important for tumor induction.
Administration, Inhalation↗
Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis.
The p16(INK4a) (p16) tumor suppressor gene can be inactivated by promoter region hypermethylation in many tumor types including lung cancer, the leading cause of cancer-related deaths in the U.S. We have determined the timing of this event in an animal model of lung carcinogenesis and in human squamous cell carcinomas (SCCs). In the rat, 94% of adenocarcinomas induced by the tobacco specific carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone were hypermethylated at the p16 gene promoter; most important, this methylation change was frequently detected in precursor lesions to the tumors: adenomas, and hyperplastic lesions. The timing for p16 methylation was recapitulated in human SCCs where the p16 gene was coordinately methylated in 75% of carcinoma in situ lesions adjacent to SCCs harboring this change. Moreover, the frequency of this event increased during disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%) to carcinoma in situ (50%) lesions. Methylation of p16 was associated with loss of expression in both tumors and precursor lesions indicating that both alleles were functionally inactivated. The potential of using assays for aberrant p16 methylation to identify disease and/or risk was validated by detection of this change in sputum from three of seven patients with cancer and 5 of 26 cancer-free individuals at high risk. These studies show for the first time that an epigenetic alteration, aberrant methylation of the p16 gene, can be an early event in lung cancer and may constitute a new biomarker for early detection and monitoring of prevention trials.
Adenoma↗
Overexpression of hMTH1 mRNA: a molecular marker of oxidative stress in lung cancer cells.
Human MutT homologue (hMTH1) mRNA was overexpressed in SV-40-transformed non-tumorigenic human bronchial epithelial cells (BEAS-2B cells) and in 11 out of 12 human lung cancer cell lines relative to normal human bronchial epithelial cells. Expression levels of hMTH1 mRNA were inversely proportional to cellular levels of 8-oxo-deoxyguanosine. Together, these results suggest that hMTH1 gene expression may represent a molecular marker of oxidative stress that could ultimately be used to elucidate the temporal relationships between oxidative stress, genomic instability and the development of lung cancer.
Adaptor Proteins, Signal Transducing↗
Effect of promoter and intron 2 polymorphisms on murine lung K-ras gene expression.
Differences in tumor formation among inbred mouse strains with high (A/J) and low (C3H) susceptibility for lung cancer have been linked to a repetitive element within the second intron of the K-ras gene. The purpose of this investigation was to determine whether differences within the K-ras gene promoter region or the intron 2 polymorphism affect K-ras gene expression in lung tumors and target alveolar type II cells isolated from A/J and C3H mice. Ribonuclease protection assays were performed using RNA isolated from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors from each mouse strain and alveolar type II cells isolated from A/J and C3H mice. An 838 bp fragment of the murine K-ras gene promoter region was amplified by PCR, cloned and sequenced from both mouse strains. Promoter regions from both mouse strains were inserted into a luciferase reporter gene vector, with and without the second intron polymorphism, and transfected into sensitive, intermediate and resistant lung tumor cell lines. No significant differences in K-ras gene promoter activity was found between the two strains using these specific reporter gene constructs. Consistent with these results, levels of K-ras expression did not differ between alveolar type II cells, whole lung or tumors induced by NNK in A/J or C3H mice. Moreover, in lung tumor cell lines derived from mice with differing susceptibility for lung cancer, K-ras expression did not correlate with the growth rate of tumors induced in nude mice from these cell lines. These results indicate that factors involved in modulating the rapid clonal expansion of the mutated K-ras allele from A/J mice are not directly linked to expression of this gene. Other genetic changes or losses in conjunction with hypothesized modifier loci, such as the Par1 locus, must play a significant role in establishing the phenotypic strain differences for lung tumor formation.
3T3 Cells↗
Frequency of trisomy 20 in nonmalignant bronchial epithelium from lung cancer patients and cancer-free former uranium miners and smokers.
Lung cancer is the leading cause of cancer-related deaths. The development of sensitive screening methods to identify at-risk individuals before emergence of clinical disease would permit early intervention that could decrease this mortality. Our previous studies have shown that cells with trisomy 7 can be detected in bronchial epithelium from cancer-free smokers and former uranium miners. However, the use of more than one molecular marker could increase the chance of identifying at-risk individuals. Trisomy 20, which is found in 43-57% of non-small cell lung cancers, is a candidate marker. The purpose of the current investigation was to determine the percentage of cells with trisomy 20 in persons with a high risk for lung cancer. Bronchial epithelial cells that had been assayed for trisomy 7 were assayed for trisomy 20 by fluorescence in situ hybridization. Trisomy 20 was detected in bronchial epithelial cells from lung cancer patients and from smokers and ex-uranium miners without lung cancer. In some cases, patients who were negative for trisomy 7 exhibited trisomy 20. Consequently, more people with field cancerization were identified using both markers. However, the two markers combined did not appear to stratify the risk for lung cancer.
Adult↗
Molecular identification of individuals at high risk for lung cancer.
The objective of the work reviewed herein was to evaluate whether a cancerization field-consisting of cells with genetic alterations can be detected within normal-appearing bronchial epithelium. By using fluorescence in situ hybridization (FISH) for trisomy 7, cancerization fields were detected in the majority of cancer patients and also in significant percentages of cancer-free tobacco smokers and former uranium miners. These results suggest that molecular analyses may enhance the power of detecting premalignant changes in bronchial epithelium and may ultimately lead to identifying persons at greatest risk for developing lung cancer.
Bronchi↗
Genetic changes in lung cancer: potential biomarkers for early detection and prevention.
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Biomarkers, Tumor↗