cytidylyl-3--5--guanosine and Lung-Neoplasms

The gene encoding the tumour suppressor protein p53 is one of the most commonly mutated genes in human cancers. Analysis of the mutational events that target the p53 gene has revealed evidence for both exogenous and endogenous mutational mechanisms. For example, the p53 mutational spectrum reveals evidence for a direct causal effect of ultraviolet radiation in skin cancer, of aflatoxin B1 in liver cancer and of tobacco smoke in lung cancer. This novel field, molecular epidemiology of human cancer risk, has added a new dimension to classical associative epidemiology by providing a direct link between human cancer and carcinogen exposure.

Topics: Aflatoxin B1; Animals; Carcinogens; Carcinoma, Hepatocellular; Dinucleoside Phosphates; Genes, p53; Genes, Tumor Suppressor; Genetic Markers; Genome; Humans; Lung Neoplasms; Molecular Epidemiology; Mutation; Skin Neoplasms; Smoking; Ultraviolet Rays

We present a straightforward and comprehensive approach for DNA methylation analysis in mammalian genomes. The methylated-CpG island recovery assay (MIRA), which is based on the high affinity of the MBD2/MBD3L1 complex for methylated DNA, has been used to detect cell type-dependent differences in DNA methylation on a microarray platform. The procedure has been verified and applied to identify a series of novel candidate lung tumor suppressor genes and potential DNA methylation markers that contain methylated CpG islands. One gene of particular interest was DLEC1, located at a commonly deleted area on chromosome 3p22-p21.3, which was frequently methylated in primary lung cancers and melanomas. Among the identified methylated genes, homeodomain-containing genes were unusually frequent (11 of the top 50 hits) and were targeted on different chromosomes. These genes included LHX2, LHX4, PAX7, HOXB13, LBX1, SIX2, HOXD3, DLX1, HOXD1, ONECUT2, and PAX9. The data show that MIRA-assisted microarray analysis has a low false-positive rate and has the capacity to catalogue methylated CpG islands on a genome-wide basis. The results support the hypothesis that cancer-associated DNA methylation events do not occur randomly throughout the genome but at least some are targeted by specific mechanisms.

Topics: Chromosome Mapping; Chromosomes, Human, Pair 3; Dinucleoside Phosphates; DNA; DNA Methylation; DNA, Neoplasm; False Positive Reactions; Humans; Lung Neoplasms; Oligonucleotide Array Sequence Analysis; Plasmids; Tumor Suppressor Proteins

Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.

Topics: Binding Sites; Blotting, Western; Cell Line, Tumor; Dinucleoside Phosphates; DNA Methylation; DNA-Binding Proteins; Genes, Tumor Suppressor; HeLa Cells; Humans; Lung Neoplasms; Lymphoma; Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in several types of cancers. In non-small cell lung cancer (NSCLC), several genes are known to be frequently methylated and the correlation of their methylation with clinical features has been studied. We determined the methylation of p16, CDH13 and RAR-beta which were reported to be methylated frequently in NSCLCs and HPP-1 which was known to be methylated in other types of cancers. The correlation between methylation and clinicopathological features were examined. The frequencies of methylation in NSCLCs were 20% for p16, 37% for CDH13, 34% for RAR-beta, and 13% for HPP1. The methylation of p16 is correlated with smoking history and methylation of HPP1 was significantly more frequent in adenocarcinomas than in squamous cell carcinomas. This is the first description of aberrant methylation of the HPP1 gene in lung cancers and our data support the previous reports on methylation in NSCLCs and association with smoking.

Topics: Adenocarcinoma; Aged; Base Sequence; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Male; Membrane Proteins; Middle Aged; Neoplasm Proteins; Receptors, Retinoic Acid

Fragile histidine triad (FHIT) gene plays an important role in the pathogenesis of lung cancer. However, the clinicopathological significance of CpG island hypermethylation of FHIT gene in non-small cell lung cancer (NSCLC) remains to be elucidated. We studied FHIT methylation in 254 NSCLCs in order to further understand the clinicopathological and prognostic significance of FHIT methylation in NSCLC. Methylation status of the FHIT gene was examined using Methylation-Specific PCR. All statistical analyses were two-sided, with a 5% type I error rate. Hypermethylation of the FHIT gene occurred more frequently in squamous cell carcinoma than adenocarcinoma. For 93 adenocarcinomas there was no statistically significant association between FHIT methylation and age, gender, smoking history, pathologic stage and p16 methylation. However, FHIT methylation in 125 squamous cell carcinomas was associated with exposure to tobacco smoke and p16 methylation, but not with age, gender and pathologic stage. Hypermethylation of FHIT in squamous cell carcinomas occurred more frequently in current smokers (45%) than in never-smokers (13%). FHIT methylation was significantly associated with p16 methylation in current- and ex-smokers (P = 0.02 and P = 0.01, respectively) with squamous cell carcinoma and in patients with pathologic stage I squamous cell carcinoma (P = 0.001). Patients with p16 methylation were 3.74 times [95% confidence interval (CI) = 1.62 - 7.95; P = 0.001] more likely to have FHIT methylation in squamous cell carcinoma. FHIT methylation in squamous cell carcinoma occurred at a 4.62 times (95% CI = 1.26 - 34.97; P = 0.02) higher prevalence in current smokers than in never-smokers. No prognostic effect of FHIT methylation was observed in stage I and stage II NSCLCs. In conclusion, hypermethylation of the FHIT gene did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinoma, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.

Topics: Acid Anhydride Hydrolases; Aged; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Dinucleoside Phosphates; DNA Methylation; Female; Genes, p16; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Polymerase Chain Reaction; Smoking; Survival Analysis

Methylation of 5' CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Dinucleoside Phosphates; DNA Methylation; DNA, Neoplasm; Female; Follow-Up Studies; Genes, APC; Humans; Lung; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Promoter Regions, Genetic; Survival Rate; Time Factors

Loss of heterozygosity at 3p21.3 occurs in more than 90% of small cell lung carcinomas (SCLCs). The Ras association domain family 1 (RASSF1) gene cloned from the lung tumor suppressor locus 3p21.3 consists of two major alternative transcripts, RASSF1A and RASSF1C. Epigenetic inactivation of isoform A (RASSF1A) was observed in 40% of primary non-small cell lung carcinomas and in several tumor cell lines. Transfection of RASSF1A suppressed the growth of lung cancer cells in vitro and in nude mice. Here we have analysed the methylation status of the CpG island promoters of RASSF1A and RASSF1C in primary SCLCs. In 22 of 28 SCLCs (=79%) the promoter of RASSF1A was highly methylated at all CpG sites analysed. None of the SCLCs showed evidence for methylation of the CpG island of RASSF1C. The results suggest that hypermethylation of the CpG island promoter of the RASSF1A gene is associated with SCLC pathogenesis.

Topics: Base Sequence; Carcinoma, Small Cell; Dinucleoside Phosphates; DNA Methylation; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Isoforms; Tumor Cells, Cultured; Tumor Suppressor Proteins

The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.

Topics: Adult; Breast Neoplasms; Carrier Proteins; Cell Cycle Proteins; Cell Line; Child; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 9; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Glioma; Homozygote; Humans; Leukemia; Leukemia, Myeloid, Acute; Lung Neoplasms; Lymphocytes; Male; Methylation; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prostatic Neoplasms; Reference Values; Restriction Mapping; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Bronchi; Carcinogens; Cells, Cultured; Codon; Dinucleoside Phosphates; DNA Adducts; Exons; Fibroblasts; Genes, p53; HeLa Cells; Humans; Lung Neoplasms; Mutagens; Mutation; Nicotiana; Plants, Toxic; Smoke

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Alleles; Bone Marrow; Breast Neoplasms; Cell Line; Child; Child, Preschool; Colon; Colonic Neoplasms; Dinucleoside Phosphates; DNA Methylation; DNA Primers; Female; Humans; Insulin-Like Growth Factor II; Leukemia; Lung Neoplasms; Lymphocytes; Male; Middle Aged; Neoplasms; Polymerase Chain Reaction; Promoter Regions, Genetic; Tumor Cells, Cultured