Increased DNA methylation in the HoxA5 promoter region correlates with decreased expression of the gene during tumor promotion

✍ Scribed by Rebecca E. Watson; Geoffrey M. Curtin; Gary M. Hellmann; David J. Doolittle; Jay I. Goodman

• Publisher: John Wiley and Sons
• Year: 2004
• Tongue: English
• Weight: 478 KB
• Volume: 41
• Category: Article
• ISSN: 0899-1987
• DOI: 10.1002/mc.20043

✦ Synopsis

Abstract

Promoter‐region DNA methylation inhibits transcription. A two‐stage SENCAR (__sen__sitive to mouse __car__cinogenesis) mouse skin carcinogenicity model was used to examine gene‐specific changes in methylation during skin tumor promotion. Analysis was performed on 7,12‐dimethylbenz[a]anthracene (DMBA)‐initiated skin promoted with 9, 18, 27, or 36 mg cigarette smoke condensate (CSC) for 9 wk, or 27 mg CSC for 9 wk and sacrificed 6 wk afterwards (recovery group). Additionally, tumors arising following promotion with 27 mg CSC for 29 wk were assessed. Gene array analysis identified differentially expressed genes. Expression of HoxA5, a tumor suppressor gene, was decreased following 9 wk of treatment with 27 mg CSC, and returned to control levels during recovery. HoxA5 promoter methylation was measured with the enzymatic regional methylation assay (ERMA). DNA was bisulfite‐modified, PCR‐amplified with primers containing dam sites, incubated with [^14^C‐methyl] S‐adenosyl‐l‐methionine (SAM) and dam methyltransferase for DNA quantification, then incubated with [^3^H‐methyl] SAM and SssI methylase to quantify methylation status. Higher ^3^H/^14^C ratios indicate increased methylation. The ^3^H/^14^C ratios of animals promoted with 27 or 36 mg CSC (48.2 ± 6.9 and 24.2 ± 6.1, respectively) were higher than the control or recovery group ratios (12.3 ± 0.1 and 12.6 ± 0.3, respectively); sequence analysis supported these findings. Increased methylation of p16 or O^6^ methylguanine methyltranferase (MGMT) was detected in 4/8 (50%) of the tumor samples from mice promoted with 27 mg CSC. These data suggest that increased DNA methylation contributes to the downregulation of HoxA5, and combined with hypermethylation of p16 or MGMT, this might facilitate the clonal expansion of increasingly aberrant cells during promotion. © 2004 Wiley‐Liss, Inc.