title: The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine triggers apoptosis through p53-dependent and -independent pathways.
abstract: One of the cellular responses to DNA damaging events is the activation of programmed cell death, also known as apoptosis. Apoptosis is an important process in limiting tumorigenesis by eliminating cells with damaged DNA. This view is reinforced by the finding that many genes with pro-apoptotic function are absent or altered in cancer cells. The tumor suppressor p53 performs a significant role in apoptotic signaling by controlling expression of a host of genes that have pro-apoptotic or pro-survival function. The S(N)1 DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers apoptosis and the upregulation/phosphorylation of p53; however, the mechanism(s) governing MNNG-induced cell death remain unresolved. We observed that the human lymphoblastoid cell line WTK-1, which expresses mutant p53, shows far less sensitivity to the cytotoxic effects of MNNG than the closely related, p53-normal line TK-6. Exposure to 15 muM MNNG (LD50 at 24 h in TK-6) leads to a kinetically slower rate of apoptotic onset in WTK-1 cells compared to TK-6 as judged by viability assays and approaches that directly examine apoptotic onset. Similar results were obtained using an unrelated human lymphoblastoid line B310 expressing reduced levels of p53 due to E6 oncoprotein expression, indicating that MNNG activates both p53-dependent and -independent apoptotic mechanisms and that these two mechanisms are discernable by the rates which they trigger apoptotic onset. We document, during time points corresponding to peak apoptotic response in TK6, WTK-1, B310, and B310-E6, that these cell lines show marked decreases in mitochondrial transmembrane potential and increases in cytochrome c within the cytosolic fraction of MNNG-treated cells. Consistent with these events, we observed that both caspase-9 and -3 are activated in our panel of lymphoblastoid cells after MNNG exposure. We also found, using both broad spectrum and specific inhibitors, that blocking caspase activity in TK-6 and B310 cells had a significant effect on apoptotic advance, but that this treatment had no effect on entry of WTK-1 or B310-E6 cells into apoptosis. Finally, the PARP inhibitors benzamide and 6(5H)-phenanthridinone exerted notable inhibition of PARP activity and the nuclear translocation of the mitochondrial protein AIF (apoptosis-inducing factor) in MNNG-treated cells; however, these compounds exhibited no detectable inhibitory effects on MNNG-induced death in human lymphoblastoid cells. These observations suggest that PARP activity is not required during MNNG-triggered apoptosis in this cell type. Taken together, our observations support the conclusion that MNNG activates multiple apoptogenic pathways that contain both common and unique mechanisms.
authors.affiliation: Department of Biochemistry and Molecular Biology, and the Stanley S. Scott Cancer Center, LSU Health Sciences Center, New Orleans, LA 70112, USA.
authors.forename: Wan-Ju
authors.initials: WJ
authors.lastname: Kim
authors.forename: Dillon I
authors.initials: DI
authors.lastname: Beardsley
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Adamson
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Brown
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001549:Benzamides / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D064447:Checkpoint Kinase 2; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects; D010616:Phenanthrenes / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
chemical_list: D001549:Benzamides; D010616:Phenanthrenes; D016159:Tumor Suppressor Protein p53; D008769:Methylnitronitrosoguanidine; C037689:benzamide; C040601:phenanthridone; D011065:Poly(ADP-ribose) Polymerases; D064447:Checkpoint Kinase 2; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases