DNA Mismatch Repair-dependent Response to Fluoropyrimidine-generated Damage

Abstract

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G 2 arrest in HCT116 human colon cancer cells, as well as Mlh1 ؊/؊ murine embryonic fibroblasts, after treatment with 5-fluoro-2-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2 In addition to its roles in correcting DNA replication errors and editing recombination intermediates, DNA mismatch repair (MMR) 1 can process numerous DNA lesions (1-4). In fact, an intact MMR system is required for the lethality of specific DNA-damaging agents such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), 6-thioguanine (6-TG), and cisplatin (5-7). MMR also mediates the lethality of fluoropyrimidines (FPs) such as 5-fluorouracil (FU) and 5-fluoro-2Ј-deoxyuridine (FdUrd) (8, 9). Inactivation of MMR allows resistance to the cytotoxic effects of these agents, a phenomenon referred to as "damage tolerance" (10 -13). Importantly, this enables cancer cells to uncouple persistent DNA damage from cell death, resulting in increased drug resistance (14 -16).The two major gene products that comprise MMR are MSH2 (which heterodimerizes with MSH3 or MSH6 to recognize mispairs and loops in DNA) and MLH1 (which heterodimerizes with PMS2 or MLH3 to act as a molecular matchmaker between the MSH2 complex and other DNA repair/replication and perhaps cell cycle factors) (17, 18). Defects in these two genes account for most cases of hereditary non-polyposis colorectal cancer, a familial condition with a predisposition to cancers of the colon, endometrium, stomach, ovary, and biliary tracts (19), as well as sporadic tumors of the colon (20), endometrium (21), stomach (22), head and neck (23), and prostate (24).Others and we (8, 9) have demonstrated that cells deficient in MLH1 are resistant to the cytotoxic effects of FU and FdUrd. Because FPs are the agents of choice in the treatment of colorectal cancer, understanding potential resistance mechanisms is important. FPs exert cytotoxic effects through incorporation into RNA and/or DNA, as well as inhibition of thymidylate synthase (TS). The inhibition of TS, which is the central enzyme of de novo pyrimidine synthesis, leads to decreases in intracellular dTTP pools; this depletion results in immediate cytostatic effects (via inhibition of DNA synthesis) and alters dNTP pool sizes (thus increasing the error rate of DNA polymerase) (25). A hallmark of MMR deficiency is instability in the length of repetitive sequences in DNA, referred to as microsatellite instability (MSI). This reflects the inability of MMRdeficient cells to correct insertions and deletions in their DNA that result from polymerase slippage at these sequences (26). It is also an easily measured clinical marker. Due to the resistance of MMR-deficient (i.e. MSI ϩ ) cancer cells to FU and FdUrd, one woul...

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…Arrest in G2 is observed within the first cell cycle in MMR-proficient cells, but not in MMR-deficient cells (Carethers et al, 1999;Meyers et al, 2003Meyers et al, , 2005. Inhibition of TS without FU treatment elicits a G2 arrest regardless of MMR status suggesting that the contribution of MMR to DNA damage signalling is via incorporation of FPs into DNA as opposed to the inhibition of TS and the resulting imbalance in nucleotide pools.…”

“…For example, Pani et al (2007) have shown that the MMR pathway contributes to the cytoxicity of cisplatin in human cells; however, other pathways are also critical in mediating the response to cisplatin particularly homologous recombination. In the case of FdU, widely used as an adjuvant therapy in advanced colorectal cancer, MMR-mediated apoptosis (Meyers et al, 2005) may be one of several contributors to the overall cytotoxicity of FdU as base excision repair (BER) enzymes also target FdU (Meyers et al, 2004). The BER enzyme SMUG1 removes FdU from DNA and confers protection from cell killing .…”

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil

“…Base excision repair of individual misincorporated nucleotides would probably not generate an effective Chk1-activating signal, since it is unlikely to create significant tracts of single-stranded DNA (Zou and Elledge, 2003). However, processing and repair of DSBs, which are thought to arise as a consequence of simultaneous BER of multiple, closely spaced lesions in 5FU-treated cells (Ingraham et al, 1986), likely would suffice, as might longer single-strand repair tracts arising from MMR-mediated excision of individual misincorporations of FdUTP (Meyers et al, 2005). We further speculate that the purpose of Chk1-dependent S-phase slowing may be to limit the accumulation of misincorporated FdUTP/dUTP under conditions of nucleotide pool perturbation by allowing time for multiple cycles of excision, re-synthesis, and repair at individual replication forks.…”

“…Fluorodeoxyuridine monophosphate is also converted into fluorodeoxyuridine triphosphate (FdUTP) which itself is a substrate for DNA polymerases and readily misincorporated into DNA (Sampath et al, 2003). FdUTP or dUTP misincorporation is potentially mutagenic and miscoding, however, both can be excised through the action of base excision repair (BER) (Ingraham et al, 1980;Mauro et al, 1993) and, in the case of FdUTP, potentially mismatch repair (MMR) (Meyers et al, 2001(Meyers et al, , 2005Tajima et al, 2004). Finally, conversion of 5FU to fluorouridine triphosphate allows incorporation into both messenger and structural RNAs with potentially deleterious effects on stability, processing, modification, and coding potential (Longley et al, 2003).…”

Aberrant DNA Methylation and Epigenetic Inactivation of hMSH2 Decrease Overall Survival of Acute Lymphoblastic Leukemia Patients via Modulating Cell Cycle and Apoptosis

“…Defects in MMR proteins have been found be associated with reduced or absent benefit from 5-FU adjuvant chemotherapy in clinical trials (Ribic et al, 2003). MMR impairment appears to cause reduced incorporation of 5-FU metabolites into DNA, leading to reduced G2/M arrest and apoptosis after 5-FU treatment (Meyers et al, 2005;Hewish et al, 2010). The deficiencies of MMR genes are also associated with MNNG, 6-thioguanine and cisplatin (Fiumicino et al, 2000;Meyers et al, 2001;Loo et al, 2005).…”