N-methyl-N'-nitro-N-nitrosoguanidine activates multiple cell death mechanisms in human fibroblasts.
Response to genotoxic stress may trigger the activation of distinct mechanisms that serve to promote cell death, including apoptosis and necrosis. In this study we examined the response of human fibroblasts, either proficient or deficient for the damage-activated protein kinase ataxia telangiectasia-mutated (ATM), to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Analysis of both long- and short-term viability shows that both ATM-proficient YZ-5 and ATM-deficient EBS-7 fibroblasts display a cytotoxic response to MNNG. Consistent with activation of apoptosis in response to MNNG, we observed increased caspase-3 cleavage and activity, appearance of fragmented nuclei, and increased staining with annexin V in both ATM-proficient and -deficient fibroblasts. Flow cytometry demonstrated that these cell lines also display a nonapoptotic cell death in response to MNNG. This form of cell death is associated with activation of poly-ADP ribose polymerase (PARP), and analysis of PARP activity indicated increased protein poly(ADP-ribosylation) in YZ-5 when compared to EBS-7. This PARP activity was accompanied by apoptosis-inducing factor release and translocation from the mitochondria to the nucleus. Finally, the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) or the caspase-3 inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically diminished the cytotoxic response to MNNG, reinforcing the roles for apoptotic and nonapoptotic cell death in human fibroblasts treated with MNNG. From these findings, we conclude that MNNG induces a heterogeneous death response in human fibroblasts.
Lee
Michael W
MW
Department of Biochemistry and Molecular Biology and the UF-Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Kim
Wan-Ju
WJ
Beardsley
Dillon I
DI
Brown
Kevin D
KD
D017304:Annexin A5 / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D003114:Colony-Forming Units Assay; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000502:physiology*
D017304:Annexin A5; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D025521:Tumor Suppressor Proteins; D008769:Methylnitronitrosoguanidine; D011065:Poly(ADP-ribose) Polymerases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D020169:Caspases