cytidylyl-3--5--guanosine and Carcinoma--Squamous-Cell

Our group has initiated experiments to epigenetically profile CpG island hypermethylation in genomic DNA from tissue specimens of head and neck squamous cell carcinoma (HNSCC) using a microarray of 12,288 CpG island clones. Our technique, known as a methylation-specific restriction enzyme (MSRE) analysis, is a variation of the differential methylation hybridization (DMH) technique, in that it is not an array comparison of two DNA samples using methylation-specific restriction enzymes. Instead, it is a comparison of a single DNA sample's response to a methylation-sensitive restriction enzyme (HpaII) and its corresponding methylation-insensitive isoschizomer (MspI). Estimation of the reproducibility of this microarray assay by intraclass correlation (ICC) demonstrated that in four replicate experiments for three tumor specimens, the ICC observed for a given tumor specimen ranged from 0.68 to 0.85 without filtering of data. Repeated assays achieved 87% concordance or greater for all tumors after filtering of array data by fluorescence intensity. We utilized hierarchical clustering on a population of 37 HNSCC samples to cluster tumor samples with similar DNA methylation profiles. Supervised learning techniques are now being utilized to allow us to identify associations between specific epigenetic signatures and clinical parameters. Such techniques will allow us to identify select groups of CpG island loci that could be used as epigenetic markers for both diagnosis and prognosis in HNSCC.

Topics: Blotting, Southern; Carcinoma, Squamous Cell; Dinucleoside Phosphates; DNA Methylation; DNA, Neoplasm; Genome, Human; Genomics; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Oligonucleotide Array Sequence Analysis

The unmethylated or methylated status of individual CpG sites is faithfully copied into daughter cells. Here, we analyzed the fidelity in replicating their methylation statuses in cancer cells. A single cell was clonally expanded, and methylation statuses of individual CpG sites were determined for an average of 12.5 DNA molecules obtained from the expanded population. By counting the deviation from the original methylation patterns inferred, the number of errors was measured. The analysis was done in four gastric cancer cell lines for five CpG islands (CGI), and repeated six times (total 1,495 clones sequenced). HSC39 and HSC57 showed error rates <1.0 x 10(-3) errors per site per generation (99.90-100% fidelity) for all the five CGIs. In contrast, AGS showed significantly elevated error rates, mainly due to increased de novo methylation, in three CGIs (1.6- to 3.2-fold), and KATOIII showed a significantly elevated error rate in one CGI (2.2-fold). By selective amplification of fully methylated DNA molecules by methylation-specific PCR, those were stochastically detected in KATOIII and AGS but never in HSC39 and HSC57. When methylation of entire CGIs was examined for eight additional CGIs, KATOIII and AGS had frequent methylation, whereas HSC39 and HSC57 had few. KATOIII and AGS had four and eight times, respectively, as high expression levels of DNMT3B as HSC39. These data showed that some cancer cells have decreased fidelity in replicating methylation patterns in some CGIs, and that the decrease could lead to methylation of the entire CGIs.

Topics: Breast; Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Dinucleoside Phosphates; DNA Methylation; DNA Replication; Epithelial Cells; Humans

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

Cell cycle progression is monitored by checkpoint mechanisms to ensure the integrity of the genome and the fidelity of sister chromatid separation. Failure of such checkpoint functions results in genomic instability, a condition that predisposes cells to neoplastic transformation and tumor progression. Recently, Scolnick and Halazonetis defined a new mitotic checkpoint that acts at prophase and delays chromosome condensation in response to mitotic stress, and identified a gene, named checkpoint with FHA and ring finger (Chfr), that seems to be required for delaying prophase in human cells. In the present study, we examined human Chfr mRNA expression in 15 human esophageal cancer cell lines and 43 primary esophageal cancers to investigate the potential involvement of Chfr in the pathogenesis of esophageal cancers. We report here that a significant proportion of human esophageal cancer has loss of expression of Chfr gene. Furthermore, we found aberrant hypermethylation of the promoter region of this checkpoint gene in four of 15 (26.7%) esophageal cancer cell lines and in seven of 43 (16.3%) primary cancers.

Topics: Adenocarcinoma; Aged; Base Sequence; Carcinoma, Squamous Cell; Cell Cycle Proteins; Dinucleoside Phosphates; DNA Methylation; DNA Primers; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Proteins; Poly-ADP-Ribose Binding Proteins; Polymerase Chain Reaction; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

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