title: Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines.
abstract: Loss of DNA mismatch repair (MMR) occurs in 10-15% of sporadic colorectal cancer, is usually caused by hMLH1 hypermethylation, and has been shown to confer resistance to various chemotherapeutic reagents, including 5-fluorouracil (5-FU). We tested the hypothesis that demethylation of the hMLH1 promoter in hypermethylated colorectal cancer cells would restore MMR proficiency and drug sensitivity to 5-FU. We used the MMR-deficient cell lines SW48, HCT116, HCT116+chr2 and the -proficient cell line HCT116+chr3. After treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5 aza-dC), hMLH1 mRNA and protein expression were determined by RT-PCR and immunoblots. The methylation status for hMLH1 was investigated by methylation-specific PCR. Cells were subsequently treated with 5-FU and the growth characteristics ascertained by clonogenic assays. hMLH1 hypermethylation was reverted in SW48 cells 24 hr after treatment with 5 aza-dC and was accompanied by hMLH1 mRNA and protein reexpression. While 5 aza-dC alone did not affect the growth of SW48 cells, all other cell lines responded with a pronounced growth inhibition. 5-FU treatment strongly reduced the colony formation of HCT116+chr3 cells. These effects were significantly less in the MMR-deficient cells. Combined treatment of SW48 cells resulted in a similar growth pattern as seen in 5-FU only treated HCT116+chr3 cells. We demonstrate that in vitro resistance to 5-FU can be overcome by reexpression of hMLH1 protein through 5 aza-dC-induced demethylation in hypermethylated cell lines. Induction of the expression of methylated tumor suppressor or MMR genes could have a significant impact on the development of future chemotherapy strategies.
authors.affiliation: Department of Medicine, University of California at San Diego, San Diego, CA, USA.
authors.forename: Christian N
authors.initials: CN
authors.lastname: Arnold
authors.forename: Ajay
authors.initials: A
authors.lastname: Goel
authors.forename: C Richard
authors.initials: CR
authors.lastname: Boland
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020137:Base Pair Mismatch; D015153:Blotting, Western; D002352:Carrier Proteins; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D004260:DNA Repair; D000077209:Decitabine; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D051741:Kruppel-Like Transcription Factors; D018895:Microsatellite Repeats; D000070957:MutL Protein Homolog 1; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; C096870:Hic1 protein, mouse; D051741:Kruppel-Like Transcription Factors; C501486:MLH1 protein, human; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D000077209:Decitabine; D000070957:MutL Protein Homolog 1; D001374:Azacitidine; D005472:Fluorouracil