The precise role of MLH1 in mammalian MMR is not clear. Using a genetically-matched HCT116 cell system the applicants recently demonstrated that cells lacking the MLH1 DNA mismatch repair (MMR) gene exhibited a """"""""damage-tolerant"""""""" phenotype and were unable to arrest at the G2/M cell cycle checkpoint following exposure to 5-fluoro-2'- deoxyuridine (FdUrd). They hypothesize that MLH-1 mediated MMR participates in the detection of FdUrd-induced DNA damage and that MLH1 expression is specifically required for both damage detection and G2/M checkpoint arrest responses. Arrest at G2/M may be the result of generation of DNA double strand breaks as a by-product to MMR activity, or through an as yet unidentified role of MLH1. The following Specific Aims will test this hypothesis:
Specific Aim 1 : To determine if MLH1 plans an unique role in relating G2/M cell cycle arrest, or if a defective G2/M arrest is common to cells with deficiencies in other MMR genes after FdUrd exposure. (Years 0-3).
Specific Aim 2 : To demonstrate that MLH1 plans a role in MMR activity and the regulation of G2/M cell cycle arrest after FdUrd treatment using cells from MLH1 knockout mice. (Years 0-3).
Specific Aim 3 : To determine if differences in G2/M arrest after FdUrd exposure between cells expressing or lacking MLH1, PMS2, or MSH2 are associated with MMR-mediated creating of DNA double-strand breaks (DSBs). (Years 2-5).
Specific Aim 4 : To determine if the DNA-directed cytotoxicity observed in cells expressing MLH1 following FdUrd treatment is due to the inhibition of thymidylate synthetase (TS) or to direct incorporation of FdUrd into DNA. (Years 1-5). 5-Fluorouracil (FUra), remains a standard drug used for colon cancer and many combined treatment modalities have been designed to increase its """"""""DNA-level"""""""" cytotoxicity. Their preliminary data strongly suggest that such DNA-targeted cytotoxicity may be contra-indicated when treating the tumors of patients with HNPCC. A better understanding of the mechanisms of FdUrd-mediated DNA lesion detection by MMR may allow them to (a) elucidate a major DNA repair mechanism within the cell for maintaining genomic stability following pool imbalances of FdUrd incorporation in DNA; (b) determine whether MMR in general, or MLH1 specifically, controls G2/M arrest responses after FdUrd treatment; and (c) eventually determine the signaling pathways which connect MMR detection of DNA damage to apoptotic cell death, since a significant apoptotic response was associated with MMR-specific killing after FdUrd treatment.
