Microarray analysis of promoter methylation in lung cancers

Abstract

Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp Icleaved (unmethylated + methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated + methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer.

Interferon Regulatory Factors IRF5 and IRF7 Inhibit Growth and Induce Senescence in Immortal Li-Fraumeni Fibroblasts

“…Together with IRF3 and IRF5, IRF7 regulates the late wave (or phase) of transcription of IFN-regulated genes after viral infection. The lack of IRF7 expression in 2fTGH fibrosarcoma cells and several lung cancer cell lines correlated with CpG island hypermethylation in its promoter region and indicated that epigenetic promoter silencing of the IRF7 gene does occur in cancer cells (35,36). Similarly, we also found that IRF7 was downregulated in preneoplastic immortal cells, and its expression could be restored by DNA demethylation (26).…”

Epigenetic Inactivation of RASSF1a in Uveal Melanoma

“…14 -16 Hypermethylation of the CpG island in the promoter region of a major alternative transcript of this gene, RASSF1a, occurs frequently in various carcinomas, including those of the breast, prostate, and lung, and in cutaneous malignant melanoma. [17][18][19][20] In this study, we investigated RASSF1a gene hypermethylation in uveal melanoma. We demonstrated that RASSF1a methylation of its promoter CpG island in uveal melanoma cell lines and primary tumors is a common event.…”

Alteration of Runt-related Transcription Factor 3 Gene Expression and Biologic Behavior of Esophageal Carcinoma TE-1 Cells after 5-Azacytidine Intervention

“…The CpG islands are located at various positions, such as exons and introns, not confined in promoter region of the TSGs. Although 79% of CpG islands are located in the promoter and the first exon, others are outside of these regions (Fukasawa et al, 2006). So data of methylation status of RUNX3 gene in promoter region is limited.…”