DEMETHYLATION BY 5-AZA-2'-DEOXYCYTIDINE OF SPECIFIC 5-METHYLCYTOSINE SITES IN THE PROMOTER REGION OF THE RETINOIC ACID RECEPTOR-BETA GENE IN HUMAN COLON-CARCINOMA CELLS

The role of retinoic acid receptor beta (RAR beta), a putative tumor s uppressor gene, in the development of colon malignancy still remains t o be clarified, We reported previously that the expression of RAR beta in DLD-1 human colon adenocarcinoma cells was silenced by DNA methyla tion at the level of the promoter region (Anti-Cancer Drugs 1997; 8: 5 6), In addition, we observed that RARE expression could be activated b y the hypomethylating action of 5-aza-2'-deoxycytidine (5-Aza-CdR). In this report we have identified, by sequencing of bisulfite-modified D NA of DLD-1 colon tumor cells, the specific 5-methylcytosine positions in the region of -46 to +251 bp from the transcription start site of RAR beta(2). We observed that 5-Aza-CdR treatment demethylated these s pecific sites, Based on this sequence data, specific primers for the m ethylation-specific PCR (MSP) assay were designed to discriminate meth ylated from unmethylated CpG sites in the promoter region of RAR beta. This assay confirmed the changes in the methylation status of the RAR beta gene in DLD-1 colon tumor cells before and after treatment with 5-Aza-CdR. The methylation status of the promoter region of the RAR be ta gene was also examined in primary human colon adenocarcinomas using the MSP assay. Six of the 14 colon tumor samples showed signs of hype rmethylation of this gene. The MSP assay for RAR beta may be a useful tool to clarify the role of DNA methylation for this gene in colon tum origenesis. [(C) 1998 Lippincott Williams & Wilkins.].