Quantitation of inhibition of DNA methylation of the retinoic acid receptor beta gene by 5-aza-2 '-deoxycytidine in tumor cells using a single-nucleotide primer extension assay

The expression of several cancer-related genes has been reported to be sile nced by DNA methylation of their promoter region, 5-Aza-2'-deoxycytidine (5 -AZA-CdR), a potent and specific inhibitor of DNA methylation, can reactiva te the in vitro expression of these genes. In future clinical trials in tum or therapy with 5-AZA-CdR a method to quantitate its inhibition of methylat ion of specific tumor suppressor genes would provide important data for the analysis of the therapeutic efficacy of this analogue, We have modified th e methylation-sensitive single-nucleotide primer extension assay reported b y Gonzalgo and Jones (Nucleic Acids Res. 25, 2529-2531, 1997), Genomic DNA was treated with bisulfite and a fragment of the promoter region of the hum an retinoic acid receptor beta (RAR beta) gene, a tumor suppressor gene, wa s amplified using seminested PCR, Using two different primers we quantitate d the inhibition of methylation produced by 5-AZA-CdR at two specific CpG s ites in the RAR beta promoter in a human colon and a breast carcinoma cell line, The results obtained with the modified assay show a precise and repro ducible quantitation of inhibition of DNA methylation produced by 5-AZA-CdR in tumor cells, (C) 2000 Academic Press.