DNA Mismatch Repair–Dependent and –Independent G2 Cell Cycle Arrest and Apoptotic Signaling Pathways After Alkylating Damage

 
 
 
 

DNA Mismatch Repair–Dependent and –Independent G2 Cell Cycle Arrest and Apoptotic Signaling Pathways After Alkylating Damage

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Title: DNA Mismatch Repair–Dependent and –Independent G2 Cell Cycle Arrest and Apoptotic Signaling Pathways After Alkylating Damage
Author: Wagner, Mark W
Description: Cells deficient in MMR display a loss of G2 cell cycle checkpoint arrest and resistance to the alkylating agent, N–methyl–N′–nitro–N–nitrosoguanidine (MNNG), or the fluorinated thymidine analog, 5–fluoro–2′–deoxyuridine (FdUrd). Here, we investigated signaling pathways involved in differential G2 arrest and apoptotic responses using isogenic MMR–proficient and —deficient cells exposed to either MNNG or FdUrd. Contrary to published data, we demonstrated that these cell lines exhibited equal Chk1 phosphorylation in response to MNNG in spite of MMR status. Furthermore, both caffeine and Gö6976, that abrogate G2 arrest by inhibiting ATM/ATR and Chk1, respectively, only partially abrogated G2 arrest, but did so in a MMR–independent manner. Similarly, disruption of Chk1 by shRNA partially abrogated G2 arrest, but did so in an MMR–independent manner, completely abrogating G2 arrest in MMR–deficient cells and only partially affecting MMR–competent cells. Collectively, our data strongly suggest that while the ATR/Chk1 pathway is activated in response to MNNG exposure, it is not facilitated by MMR signaling. These data strongly suggested a separate MMR–dependent G2 arrest pathway. We then examined the role of c–Abl kinase in MMR–dependent cell cycle signaling and apoptosis. Disruption of c–Abl activity, either by STI571 (Gleevec™, a c–Abl inhibitor) addition or infection with c–Abl–specific shRNA, abolished MMR–dependent G2 arrest and apoptosis. Decreased MMR–dependent p73α stabilization and Gadd45α protein expression, in response to MNNG exposure noted in c–Abl knockdown cells. Blocking both ATR/Chk1 and c–Abl–dependent G2 arrest signaling pathways completely abrogated G2 arrest in MMR–proficient cells in response to MNNG exposure. Our data strongly suggest that two overlapping G2 arrest pathways are triggered after MNNG exposures: (i) a MMR–dependent hMLH1/c–Abl/p73α/Gadd45α signaling pathway leading from G2 arrest responses to eventual apoptotic cell death; and (ii) a separate but simultaneously functioning, MMR-independent ATR/Chk1 signaling pathway leading to G2 cell cycle checkpoint arrest responses. Understanding these separate signaling pathways may allow their manipulation to avoid error–prone (i.e., MMR–independent mediated) from error–free (MMR–mediated) G2 arrest responses and apoptosis that are important in carcinogenesis.
Permanent Link: http://rave.ohiolink.edu/etdc/view?acc_num=case1181137349
http://hdl.handle.net/2374.OX/17655
Date: 2007

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