thioguanine-anhydrous and Endometrial-Neoplasms

We investigate the roles of DNA mismatch repair (MMR) and p53 in mediating the induction of autophagy in human tumor cells after exposure to 6-thioguanine (6-TG), a chemotherapy drug recognized by MMR. We also examine how activation of autophagy affects apoptosis (type I cell death) after MMR processing of 6-TG.. Using isogenic pairs of MLH1(-)/MLH1(+) human colorectal cancer cells (HCT116) and MSH2(-)/MSH2(+) human endometrial cancer cells (HEC59), we initially measure activation of autophagy for up to 3 days after 6-TG treatment using LC3, a specific marker of autophagy. We then assess the role of p53 in autophagic signaling of 6-TG MMR processing using both pifithrin-alpha cotreatment to chemically inhibit p53 transcription and small hairpin RNA inhibition of p53 expression. Finally, we use Atg5 small hairpin RNA inhibition of autophagy to assess the effect on apoptosis after MMR processing of 6-TG.. We find that MMR is required for mediating autophagy in response to 6-TG treatment in these human tumor cells. We also show that p53 plays an essential role in signaling from MMR to the autophagic pathway. Finally, our results indicate that 6-TG-induced autophagy inhibits apoptosis after MMR processing of 6-TG.. These data suggest a novel function of MMR in mediating autophagy after a chemical (6-TG) DNA mismatch damage through p53 activation. The resulting autophagy inhibits apoptosis after MMR processing of 6-TG.

Topics: Adaptor Proteins, Signal Transducing; Autophagy; Cell Line, Tumor; Colorectal Neoplasms; DNA Mismatch Repair; Endometrial Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, p53; HCT116 Cells; Humans; MutL Protein Homolog 1; Nuclear Proteins; Thioguanine; Tumor Suppressor Protein p53

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.

Topics: Adaptor Proteins, Signal Transducing; Base Pair Mismatch; Carrier Proteins; CDC2 Protein Kinase; Cell Survival; Colorectal Neoplasms; DNA Repair; Endometrial Neoplasms; Female; G2 Phase; Humans; MutL Protein Homolog 1; Neoplasm Proteins; Nuclear Proteins; Ovarian Neoplasms; Phosphorylation; S Phase; Signal Transduction; Thioguanine; Tumor Cells, Cultured

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Antineoplastic Agents; Carboplatin; Carrier Proteins; Cisplatin; Colorectal Neoplasms; Cyclophosphamide; DNA Adducts; DNA Damage; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; Doxorubicin; Drug Resistance, Neoplasm; Endometrial Neoplasms; Etoposide; Female; Fluorouracil; Humans; Melphalan; Methylnitronitrosoguanidine; Mutagenesis; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nuclear Proteins; Paclitaxel; Proto-Oncogene Proteins; Thioguanine; Tumor Cells, Cultured