MLH1 deficiency enhances radiosensitization with 5-fluorodeoxyuridine by increasing DNA mismatches.
The antitumor drug 5-fluoro-2'-deoxyuridine (FdUrd) also sensitizes tumor cells to ionizing radiation in vitro and in vivo. Although radiosensitization with FdUrd requires dTTP depletion and S-phase arrest, the exact mechanism by which these events produce radiosensitization remains unknown. We hypothesized that the depletion of dTTP produces DNA mismatches that, if not repaired before irradiation, would result in radiosensitization. We evaluated this hypothesis in mismatch repair (MMR)-deficient HCT116 0-1 cells that lack the expression of the required MMR protein MLH1 (inactive MLH1), and in MMR-proficient (wild-type MLH1) HCT116 1-2 cells. Although HCT116 0-1 cells were less sensitive to FdUrd (IC(50) = 3.5 microM) versus HCT116 1-2 cells (IC(50) = 0.75 microM), when irradiation followed FdUrd (IC(50)) the MLH1-inactivated cells exhibited greater radiosensitization compared with MMR-wild-type cells [radiation enhancement ratio (RER) = 1.8 +/- 0.28 versus 1.1 +/- 0.1, respectively] and an increase (> or =8-fold) in nucleotide misincorporations. In SW620 cells and HCT116 1-2 MLH1-wild-type cells, FdUrd (IC(50)) did not produce radiosensitization nor did it increase the mutation frequency, but after short hairpin RNA-directed suppression of MLH1 this concentration produced excellent radiosensitization (RER = 1.6 +/- 0.10 and 1.5 +/- 0.06, respectively) and an increase in nucleotide misincorporations (8-fold and 6-fold, respectively). Incubation with higher concentrations of FdUrd (IC(90)) after suppression of MLH1 produced a further increase in ionizing radiation sensitivity in both SW620 and HCT116 1-2 cells (RER = 1.8 +/- 0.03 and 1.7 +/- 0.13, respectively) and nucleotide misincorporations (>10-fold in both cell lines). These results demonstrate an important role for MLH1 and implicate mismatches in radiosensitization by FdUrd.
Flanagan
Sheryl A
SA
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI 48109-0504, USA.
Krokosky
Christina M
CM
Mannava
Sudha
S
Nikiforov
Mikhail A
MA
Shewach
Donna S
DS
D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency*; D020137:Base Pair Mismatch / Q000187:drug effects* / Q000528:radiation effects; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D016923:Cell Death / Q000187:drug effects / Q000528:radiation effects; D005467:Floxuridine / Q000494:pharmacology*; D045325:HCT116 Cells; D006801:Humans; D000070957:MutL Protein Homolog 1; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000172:deficiency*; D009711:Nucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects* / Q000528:radiation effects; D011839:Radiation, Ionizing
D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D009711:Nucleotides; D034741:RNA, Small Interfering; D005467:Floxuridine; D000070957:MutL Protein Homolog 1