Differential cellular responses to prolonged LDR-IR in MLH1-proficient and MLH1-deficient colorectal cancer HCT116 cells.
OBJECTIVE
MLH1 is a key DNA mismatch repair (MMR) protein involved in maintaining genomic stability by participating in the repair of endogenous and exogenous mispairs in the daughter strands during S phase. Exogenous mispairs can result following treatment with several classes of chemotherapeutic drugs, as well as with ionizing radiation. In this study, we investigated the role of the MLH1 protein in determining the cellular and molecular responses to prolonged low-dose rate ionizing radiation (LDR-IR), which is similar to the clinical use of cancer brachytherapy.


METHODS
An isogenic pair of MMR(+) (MLH1(+)) and MMR(-) (MLH1(-)) human colorectal cancer HCT116 cells was exposed to prolonged LDR-IR (1.3-17 cGy/h x 24-96 h). The clonogenic survival and gene mutation rates were examined. Cell cycle distribution was analyzed with flow cytometry. Changes in selected DNA damage repair proteins, DNA damage response proteins, and cell death marker proteins were examined with Western blotting.


RESULTS
MLH1(+) HCT116 cells showed greater radiosensitivity with enhanced expression of apoptotic and autophagic markers, a reduced HPRT gene mutation rate, and more pronounced cell cycle alterations (increased late-S population and a G(2)/M arrest) following LDR-IR compared with MLH1(-) HCT116 cells. Importantly, a progressive increase in MLH1 protein levels was found in MLH1(+) cells during prolonged LDR-IR, which was temporally correlated with a progressive decrease in Rad51 protein (involved in homologous recombination) levels.


CONCLUSIONS
MLH1 status significantly affects cellular responses to prolonged LDR-IR. MLH1 may enhance cell radiosensitivity to prolonged LDR-IR through inhibition of homologous recombination (through inhibition of Rad51).
Yan
Tao
T
Department of Radiation Oncology and the Case Integrative Cancer Biology Program, Case Comprehensive Cancer Center, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Seo
Yuji
Y
Kinsella
Timothy J
TJ
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D001918:Brachytherapy / Q000379:methods; D045744:Cell Line, Tumor; D004249:DNA Damage; D045325:HCT116 Cells; D006801:Humans; D000070957:MutL Protein Homolog 1; D009154:Mutation; D009369:Neoplasms / Q000188:drug therapy* / Q000532:radiotherapy*; D009687:Nuclear Proteins / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000378:metabolism; D011839:Radiation, Ionizing; D016196:S Phase
D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000070957:MutL Protein Homolog 1