Mismatch Repair Protein Deficiency Compromises Cisplatin-induced Apoptotic Signaling

Ryan P Topping,John C Wilkinson,Karin Drotschmann Scarpinato

doi:10.1074/jbc.m809303200

Abstract

Mismatch repair (MMR) proteins participate in cytotoxicity induced by certain DNA damage-inducing agents, including cisplatin (cis-diamminedichloroplatinum(II), CDDP), a cancer chemotherapeutic drug utilized clinically to treat a variety of malignancies. MMR proteins have been demonstrated to bind to CDDP-DNA adducts and initiate MMR protein-dependent cell death in cells treated with CDDP; however, the molecular events underlying this death remain unclear. As MMR proteins have been suggested to be important in clinical responses to CDDP, a clear understanding of MMR protein-dependent, CDDP-induced cell death is critical. In this report, we demonstrate MMR protein-dependent relocalization of cytochrome c to the cytoplasm and cleavage of caspase-9, caspase-3, and poly(ADP-ribose) polymerase upon treatment of cells with CDDP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent cytotoxicity, suggesting that a caspase-dependent signaling mechanism is required for the execution of this cell death. p53 protein levels were up-regulated independently of MMR protein status, suggesting that p53 is not a mediator of MMR-dependent, CDDP-induced death. This work is the first indication of a required signaling mechanism in CDDP-induced, MMR protein-dependent cytotoxicity, which can be uncoupled from other CDDP response pathways, and defines a critical contribution of MMR proteins to the control of cell death.

Highlights

Recruitment of repair machinery is performed by the MutS␣ complex or MutS␤ complex
Cisplatin Induces Loss of Cell Viability in an MSH2/MSH6dependent Manner—To assess MMR protein-dependent cytotoxicity and pro-death signaling events in cells treated with CDDP, we utilized the paired endometrial carcinoma cell lines HEC59/HEC59(2) and HHUA/HHUA[2]
To confirm previously described MMR protein-dependent sensitivity to CDDP, we examined MMR protein-dependent decrease in cell viability in cells treated with CDDP using two methods: the MTT assay, and the propidium iodide (PI) exclusion assay

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Chemicals—Human endometrial carcinoma cell lines HEC59, HEC59(2), HHUA, and HHUA[2] and human colon cancer cell lines HCT116 and HCT116(2) cells were a gift from T. For MTT assays, 3 ϫ 103 cells/well were seeded into wells of 96-well plates and incubated overnight before treatment with indicated drugs. Goat anti-mouse (170-6515) and -rabbit (1706516) secondary antibodies were purchased from Bio-Rad. Isolation of Whole Cell or Cytoplasmic Extracts and Immunoblot Analysis—Cells were plated at 4 ϫ 105 cells/dish in 60-mm dishes, incubated overnight, and treated with indicated drugs. Cells were harvested at various times post drug treatment by trypsinization, transferred with media into a conical tube, and centrifuged at 250 ϫ g. Cells were incubated on ice 10 min and centrifuged at 1000 ϫ g, and supernatant containing cytoplasmic proteins was removed to a clean microcentrifuge tube and stored at Ϫ80 °C until use. Protein concentrations for each sample were acquired using the Bio-Rad Protein Assay and relative fluorescence units/min values were standardized to milligrams of protein per sample

RESULTS

PI exclusion assay

DISCUSSION