title: Parp1 activation in mouse embryonic fibroblasts promotes Pol beta-dependent cellular hypersensitivity to alkylation damage.
abstract: Alkylating agents induce cell death in wild-type (WT) mouse embryonic fibroblasts (MEFs) by multiple mechanisms, including apoptosis, autophagy and necrosis. DNA polymerase beta (Pol beta) knockout (KO) MEFs are hypersensitive to the cytotoxic effect of alkylating agents, as compared to WT MEFs. To test the hypothesis that Parp1 is preferentially activated by methyl methanesulfonate (MMS) exposure of Pol beta KO MEFs, we have examined the relationship between Pol beta expression, Parp1 activation and cell survival following MMS exposure in a series of WT and Pol beta deficient MEF cell lines. Consistent with our hypothesis, we observed elevated Parp1 activation in Pol beta KO MEFs as compared to matched WT MEFs. Both the MMS-induced activation of Parp1 and the MMS-induced cytotoxicity of Pol beta KO MEFs are attenuated by pre-treatment with the Parp1/Parp2 inhibitor PJ34. Further, elevated Parp1 activation is observed following knockdown (KD) of endogenous Pol beta, as compared to WT cells. Pol beta KD MEFs are hypersensitive to MMS and both the MMS-induced hypersensitivity and Parp1 activation is prevented by pre-treatment with PJ34. In addition, the MMS-induced cellular sensitivity of Pol beta KO MEFs is reversed when Parp1 is also deleted (Pol beta/Parp1 double KO MEFs) and we observe no MMS sensitivity differential between Pol beta/Parp1 double KO MEFs and those that express recombinant mouse Pol beta. These studies suggest that Parp1 may function as a sensor of BER to initiate cell death when BER is aborted or fails. Parp1 may therefore function in BER as a tumor suppressor by initiating cell death and preventing the accumulation of cells with chromosomal damage due to a BER defect.
authors.affiliation: Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine & University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Jelezcova
authors.forename: Ram N
authors.initials: RN
authors.lastname: Trivedi
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Wang
authors.forename: Jiang-Bo
authors.initials: JB
authors.lastname: Tang
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Brown
authors.forename: Eva M
authors.initials: EM
authors.lastname: Goellner
authors.forename: Sandy
authors.initials: S
authors.lastname: Schamus
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Fornsaglio
authors.forename: Robert W
authors.initials: RW
authors.lastname: Sobol
mesh_terms: D000478:Alkylation; D000818:Animals; D016923:Cell Death; D002460:Cell Line; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta