sodium-bisulfite and Carcinoma--Squamous-Cell

Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element 1 L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. LINE-1 methylation is a useful marker for predicting cancer prognosis and monitoring efficacy of adjuvant therapy. Nonetheless, no study has examined LINE-1 methylation in esophageal squamous cell carcinoma (ESCC). The aim of this study is to assess the precision of sodium bisulfite conversion and polymerase chain reaction (PCR) pyrosequencing assay for evaluating LINE-1 methylation in ESCC.. To measure assay precision, we performed bisulfite conversion on 5 different DNA specimen aliquots (bisulfite-to-bisulfite) and repeated PCR pyrosequencing five times (run to run). Second, to assess heterogeneity of LINE-1 methylation levels within tumor, we made 5 different tissue sections from one tumor and examined LINE-1 methylation level of each section (section to section). Third, to evaluate LINE-1 methylation status in ESCC, we applied this assay to 30 ESCCs and 30 matched normal esophageal mucosa.. Bisulfite-to-bisulfite standard deviation (SD) ranged from 1.44 to 2.90 (median 2.32) in ESCCs; and 0.57 to 4.02 (median 1.23) in normal esophagus. Run-to-run SD ranged from 0.63 to 3.25 (median 1.54) in ESCCs. Section-to-section SD ranged from 1.37 to 3.31 (median 1.94). ESCC tissues showed significantly lower levels of LINE-1 methylation than matched normal mucosa (P < .0001; n = 30). There was no significant relationship between LINE-1 methylation level and tumor stage (P = 0.14).. Bisulfite conversion and PCR pyrosequencing assay can measure LINE-1 methylation in ESCC, and may be useful in clinical and research settings.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; DNA; DNA Methylation; Esophageal Neoplasms; Esophagus; Humans; Long Interspersed Nucleotide Elements; Neoplasm Staging; Paraffin Embedding; Polymerase Chain Reaction; Prognosis; Sulfites

Tissue inhibitor of metalloproteinase-3 (TIMP-3) has an inhibitory effect on tumor development, growth and metastasis due to its interaction with matrix metalloproteinases (MMPs). We investigated the prognostic significance of TIMP-3 gene promoter methylation in esophageal squamous cell carcinoma (ESCC). TIMP-3 methylation was analyzed by MethyLight, a quantitative and methylation-specific PCR method. Hypermethylation was found in 9/51 (18%) surgically resected ESCC samples and was associated with reduced disease-free (p=0.0039) and overall survival (p=0.0047). Upon multivariate analysis, it was found to be an independent prognostic parameter for poor survival and was associated with earlier recurrence after surgery (p=0.0238), especially via pleural dissemination (p=0.001). Expression levels for TIMP-3 protein and for several MMPs were analyzed by Western blot analysis in 20 pairs of ESCC and adjacent normal tissue. The expression of MMP-2, -7 and -9 proteins in tumor tissue was significantly higher than in corresponding normal mucosa (p=0.0051, 0.0064 and 0.0004, respectively). In contrast, the expression of TIMP-3 protein in tumor tissue was significantly lower than in matched normal mucosa (p=0.0152). Tumors with TIMP-3 hypermethylation expressed lower levels of TIMP-3 protein compared to those without hypermethylation (p=0.0357). These results demonstrate that TIMP-3 hypermethylation is associated with lower TIMP-3 protein expression in ESCC and with poor patient survival due to a high frequency of recurrence by pleural dissemination.

Topics: Carcinoma, Squamous Cell; Disease-Free Survival; DNA Methylation; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Mucous Membrane; Neoplasm Invasiveness; Neoplasm Metastasis; Polymerase Chain Reaction; Prognosis; Proportional Hazards Models; Sulfites; Tissue Inhibitor of Metalloproteinase-3; Treatment Outcome

Head and neck squamous cell carcinomas (HNSCC) often metastasise to the cervical lymph nodes. It is known for HNSCC as well as other cancers that progression from normal tissue to primary tumour and finally to metastatic tumour is characterised by an accumulation of genetic mutations. DNA methylation, an epigenetic modification, can result in loss of gene function in cancer, similar to genetic mutations such as deletions and point mutations. We have investigated the DNA methylation phenotypes of both primary HNSCC and metastatic tumours from 13 patients using restriction landmark genomic scanning (RLGS). With this technique, we were able to assess the methylation status of an average of nearly 1300 CpG islands for each tumour. We observed that the number of CpG islands hypermethylated in metastatic tumours is significantly greater than what is found in the primary tumours overall, but not in every patient. Interestingly, the data also clearly show that many loci methylated in a patient's primary tumour are no longer methylated in the metastatic tumour of the same patient. Thus, even though metastatic HNSCC methylate a greater proportion of CpG islands than do the primary tumours, they do so at different subsets of loci. These data show an unanticipated variability in the methylation state of loci in primary and metastatic HNSCCs within the same patient. We discuss two possible explanations for how different epigenetic events might arise between the primary tumour and the metastatic tumour of a person.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cloning, Molecular; CpG Islands; DNA Fingerprinting; DNA Methylation; Female; Genetic Markers; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Mouth Neoplasms; Pharyngeal Neoplasms; Phenotype; Restriction Mapping; Sequence Analysis, DNA; Sulfites

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT-PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Topics: Aged; Blotting, Southern; Bronchi; Carcinoma, Squamous Cell; Cell Line; Cells, Cultured; CpG Islands; DNA; DNA Methylation; Endothelin-1; Epithelial Cells; Female; Gene Silencing; Humans; Immunohistochemistry; Introns; Lung Neoplasms; Male; Middle Aged; Models, Genetic; Physical Chromosome Mapping; Promoter Regions, Genetic; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sulfites; Transcription, Genetic; Tumor Cells, Cultured