Role of c-Abl Kinase in DNA Mismatch Repair-dependent G2 Cell Cycle Checkpoint Arrest Responses

Mark W Wagner,Long Shan Li,Julio C Morales,Cristi L Galindo,Harold R Garner,William G Bornmann,David A Boothman

doi:10.1074/jbc.m709953200

Abstract

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.

Highlights

Primarily due to acquisition of mutations in both copies of the human (h) MLH1 or MSH2 genes [1,2,3], are directly linked to hereditary nonpolyposis colon cancer
Using MMR-deficient human RKO colon cancer cells corrected by single replacement and expression of hMLH1 protein at endogenous wildtype (WT) levels, we found that the ATR/Chk1 pathway was activated after MNNG treatment; its downstream phosphorylation of Chk1 was not MMR-dependent because equivalent consensus site Chk1 phosphorylation was noted regardless of MMR status
Restoration of hMLH1 Expression and MMR Function in MMR-deficient RKO Cells—RKO cells lack hMLH1 expression due to promoter hypermethylation [4]. hMLH1 expression was restored by stably transfecting RKO cells with cytomegalovirus-driven hMLH1 cDNA

Summary

Introduction

Primarily due to acquisition of mutations in both copies of the human (h) MLH1 (mutL homolog-1) or MSH2 (mutS homolog-2) genes [1,2,3], are directly linked to hereditary nonpolyposis colon cancer. MNNG causes a spectrum of specific DNA lesions, including methylation of dG at the O6-position in dG:dC base pairs These DNA lesions are mutagenic because replication over O6-methylguanine base pairs causes preferential pairing with Thy. mutagenic O6-methylguanine:Thy mispairs are excellent MMR substrates [9]; the exact signal transduction processes that regulate G2 arrest responses remain ill defined. G2 arrest can be mediated by DNA damage activation of ATM (Ataxia telangiectasia mutated) or ATR (Ataxia telangiectasia-Rad3-related kinase) phosphatidylinositol 3-like kinases These kinases phosphorylate and activate p53 and downstream Chk and Chk checkpoint kinases. The phosphorylationmediated functional activation of the p53 tumor suppressor by the ATM/Chk and/or ATR/Chk pathway leads to its transcriptional regulation of several downstream genes involved in cell cycle checkpoint arrest, including p21, GADD45␣ (growth arrest- and DNA damage-inducible-45␣), and 14-3-3␴ [10]. MMR-dependent GADD45␣ protein increases, in particular, were shown after FdUrd exposure [5], and GADD45␣ can mediate G2 arrest by direct binding to and inhibiting Cdc2 [10]

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