MLH1 mediates PARP-dependent cell death in response to the methylating agent N-methyl-N-nitrosourea.
BACKGROUND
Methylating agents such as N-methyl-N-nitrosourea (MNU) can cause cell cycle arrest and death either via caspase-dependent apoptosis or via a poly(ADP-ribose) polymerase (PARP)-dependent form of apoptosis. We wished to investigate the possible role of MLH1 in signalling cell death through PARP.


METHODS
Fibroblasts are particularly dependent on a PARP-mediated cell death response to methylating agents. We used hTERT-immortalised normal human fibroblasts (WT) to generate isogenic MLH1-depleted cells, confirmed by quantitative PCR and western blotting. Drug resistance was measured by clonogenic and cell viability assays and effects on the cell cycle by cell sorting. Damage signalling was additionally investigated using immunostaining.


RESULTS
MLH1-depleted cells were more resistant to MNU, as expected. Despite having an intact G(2)/M checkpoint, the WT cells did not initially undergo cell cycle arrest but instead triggered cell death directly by PARP overactivation and nuclear translocation of apoptosis-inducing factor (AIF). The MLH1-depleted cells showed defects in this pathway, with decreased staining for phosphorylated H2AX, altered PARP activity and reduced AIF translocation. Inhibitors of PARP, but not of caspases, blocked AIF translocation and greatly decreased short-term cell death in both WT and MLH1-depleted cells. This MLH1-dependent response to MNU was not blocked by inhibitors of ATM/ATR or p53.


CONCLUSIONS
These novel data indicate an important role for MLH1 in signalling PARP-dependent cell death in response to the methylating agent MNU.
McDaid
J R
JR
Stem Cells and Epigenetics Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK.
Loughery
J
J
Dunne
P
P
Boyer
J C
JC
Downes
C S
CS
Farber
R A
RA
Walsh
C P
CP
D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D000477:Alkylating Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D020169:Caspases / Q000502:physiology; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000502:physiology; D006657:Histones / Q000378:metabolism; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology*; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D019098:Telomerase / Q000502:physiology; D013866:Thioguanine / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D000477:Alkylating Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D007546:Isoquinolines; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D010880:Piperidines; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C108729:3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone; D008770:Methylnitrosourea; D011065:Poly(ADP-ribose) Polymerases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; C509186:TERT protein, human; D019098:Telomerase; D020169:Caspases; D000070957:MutL Protein Homolog 1; D013866:Thioguanine