Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines

Int J Cancer. 2003 Aug 10;106(1):66-73. doi: 10.1002/ijc.11176.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Antimetabolites, Antineoplastic / pharmacology
  • Azacitidine / analogs & derivatives*
  • Azacitidine / pharmacology
  • Base Pair Mismatch
  • Blotting, Western
  • Carrier Proteins
  • Cloning, Molecular
  • Colorectal Neoplasms / drug therapy*
  • Colorectal Neoplasms / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics
  • DNA Methylation*
  • DNA Repair
  • Decitabine
  • Drug Resistance, Neoplasm
  • Enzyme Inhibitors / pharmacology
  • Fluorouracil / pharmacology*
  • Humans
  • Immunoblotting
  • Kruppel-Like Transcription Factors
  • Microsatellite Repeats
  • MutL Protein Homolog 1
  • Neoplasm Proteins / genetics*
  • Nuclear Proteins
  • Promoter Regions, Genetic*
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Time Factors
  • Transcription Factors / genetics
  • Transcriptional Activation
  • Tumor Cells, Cultured


  • Adaptor Proteins, Signal Transducing
  • Antimetabolites, Antineoplastic
  • Carrier Proteins
  • Cyclin-Dependent Kinase Inhibitor p16
  • Enzyme Inhibitors
  • Hic1 protein, mouse
  • Kruppel-Like Transcription Factors
  • MLH1 protein, human
  • Mlh1 protein, mouse
  • Neoplasm Proteins
  • Nuclear Proteins
  • RNA, Messenger
  • Transcription Factors
  • Decitabine
  • MutL Protein Homolog 1
  • Azacitidine
  • Fluorouracil