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.

Fig. 1. WT and Pol β KO MEFs used in this study

Expression of the DNA repair proteins Pol β, Ape1, Xrcc1, Parp1 and PCNA in MEFs as determined by immunoblot analysis of nuclear proteins isolated from the following MEF cells: 92TAg (lane 1), 88TAg (lane 2), 53TAg (lane 3), 50TAg (lane 4), 36.3 (lane 5), 38Δ4 (lane 6), 16tsa (lane 7) and 19tsA (lane 8).

Fig. 2. Preferential Parp1 activation in Pol β KO cells following MMS exposure

Parp1 activation was determined in MEF cell extract by immunoblot analysis, measuring the synthesis of PAR in the absence (left panel) or in the presence of PJ34 (2 μM, right panel), after exposure with MMS (1.25 mM) for 0, 15 or 30 minutes in [92TAg (WT) and 88TAg (Pol β KO); Panel-A], [53TAg (WT) and 50TAg (Pol β KO); Panel-B], [36.3 (WT) and 38Δ4 (Pol β KO); Panel-C] and [16tsa (WT) and 19tsA (Pol β/Pol ι double KO); Panel-D] MEFs. Parp1 and PCNA expression was determined by immunoblot. PCNA expression is shown as a loading control.

Fig. 3. Chemical inhibition of Parp1 sensitizes WT MEFs yet provides partial resistance to MMS in Pol β KO MEFs

Wild type (92TAg, 53TAg, 36.3 and 16tsa; open squares), Pol β KO (88TAg, 50TAg and 38Δ4; open circles) and Pol β/Pol ι double KO (19tsA; open circles) MEFs were seeded in 96-well plates in two sets. After 24 hours, cells were pre-treated with media (open squares or open circles) or PJ34 (filled squares or filled circles) at a final concentration of 2 μM. After 30 minutes of media or PJ34 pre-treatment, cells were treated with a range of concentrations of MMS for one hour (A, B, C and D). A second set was treated with media (open squares or open circles) or media supplemented with PJ34 (2 μM) and MMS (0.25-2.0 mM) simultaneously (filled squares or filled circles) for one hour (E, F, G and H). Drug-containing medium was replaced with fresh media or media containing PJ34 and the plates were incubated at 37°C for 48 hours and viable cells were determined using a modified MTT assay. Plots show the % viable cells as compared to untreated (control) cells. Means are calculated from quadruplicate values in each experiment. Results indicate the mean ± S.E. of four independent experiments.

Fig. 4. Mouse Pol β knockdown causes robust Parp1 activation and Parp1 dependent cell death following MMS exposure in MEFs

(A & B) BER protein expression as determined by immunoblot analysis of nuclear proteins isolated from the 92TAg cells or 92TAg cells transduced with a mouse Pol β shRNA lentiviral vector (A) or the HCC384 cells or HCC384 cells transduced with a mouse Pol β shRNA lentiviral vector (B). (A) Proteins isolated from the shRNA expressing clone (92TAg/Polβ-KD(3); lane 2) as compared to proteins isolated from control cells (92TAg; lane 1) are shown in (A) and from the shRNA expressing clone (HCC384/Polβ-KD(1); lane 2) as compared to proteins isolated from control cells (HCC384; lane 1) are shown in (B). Pol β, Ape1, Xrcc1 and Parp1 expression was determined by immunoblot. PCNA expression is shown as a loading control. (C & D) Parp1 activation was determined in cell extracts by immunoblot analysis, measuring the synthesis of PAR after exposure with MMS (1.25 mM) for 0, 15 or 30 minutes in (C) 92TAg cells [WT] or 92TAg cells transduced with a mouse Pol β shRNA lentiviral vector, clone 3 [Pol β KD] or (D) HCC384 cells [WT] or HCC384 cells transduced with a mouse Pol β shRNA lentiviral vector, clone 1 [Pol β KD]. PCNA expression is shown as a loading control. (E & F) MMS-induced cytotoxicity following PJ34 pre-treatment was evaluated by culturing (E) 92TAg cells (open squares) or 92TAg cells transduced with a mouse Pol β shRNA lentiviral vector (92TAg/Polβ-KD(3); open circles) or (F) HCC384 cells (open squares) or HCC384 cells transduced with a mouse Pol β shRNA lentiviral vector (HCC384/Polβ-KD(1); open circles), in 96-well plates for 24 hours prior to exposure with media (open squares or open circles) or PJ34 (filled squares or filled circles; 2 μM) for 30 minutes. After PJ34 pre-treatment, cells were then treated with MMS and viable cells were determined using a modified MTT assay as described in Fig. 3.

Fig. 5. Impact of Parp1 expression on MMS sensitivity in the absence of Pol β

(A & B) BER protein expression as determined by immunoblot analysis of nuclear proteins isolated from the (A) 88TAg cells or 88TAg cells complemented with mouse Pol β or (B) Pol β/Parp1 double KO cells or Pol β/Parp1 double KO cells complemented with mouse Pol β. Proteins isolated from two separate mouse Pol β (pIRES-Puro-mBeta) expressing clones are shown: (A) [88TAg/mPol β (27) and 88TAg/mPol β (28); lanes 4 and 5] as compared to proteins isolated from control cell (88TAg; lane 1) or pIRES-Puro-EGFP transfected in 88TAg cell [88TAg/EGFP (1) and 88TAg/EGFP (2); lanes 2 and 3]. (B): [Pol β/Parp1 double KO/mPol β (6) and Pol β/Parp1 double KO/mPol β (7); lanes 4 and 5] as compared to proteins isolated from control cells (Pol β/Parp1 double KO cell; lane 1) or pIRES-Puro-EGFP transfected in Pol β/Parp1 double KO cells [Pol β/Parp1 double KO/EGFP (1) and Pol β/Parp1 double KO/EGFP (2); lanes 2 and 3]. Pol β, Ape1, Xrcc1 and Parp1 expression was determined by immunoblot. PCNA expression is shown as a loading control. (C & D) Parp1 activation was determined in cell extract by immunoblot analysis, measuring the synthesis of PAR after exposure with MMS (1.25 mM) for 0 or 15 minutes in (C) 88TAg cells (group 1) or 88TAg cell transfected with pIRES-Puro-EGFP (group 2) or 88TAg cells transfected with pIRES-Puro-mBeta (group 3) or (D) Pol β/Parp1 double KO cells (group 1) or Pol β/Parp1 double KO cells transfected with pIRES-Puro-EGFP (group 2) or Pol β/Parp1 double KO cells transfected with pIRES-Puro-mBeta (group 3). PCNA expression is shown as a loading control. (E) MMS-induced cytotoxicity following PJ34 pre-treatment was evaluated by culturing 88TAg cells [88Tag], 88TAg cells transfected with pIRES-Puro-EGFP [88Tag/GFP] or 88TAg cells complemented with mouse Pol β [88Tag/mPolβ], in 96-well plates for 24 hours prior to exposure with media [open bars] or PJ34 (2 μM) [filled bars] for 30 minutes. After PJ34 pre-treatment, cells were then treated with MMS (1.5 mM) and viable cells were determined using a modified MTT assay as described in Fig. 3. (F) MMS-induced cytotoxicity following PJ34 pre-treatment was evaluated by culturing Pol β/Parp1 double KO cells transfected with pIRES-Puro-EGFP or Pol β/Parp1 double KO cell complemented with mouse Pol β, in 96-well plates for 24 hours prior to exposure with media [open bars] or PJ34 (2 μM) [filled bars] for 30 minutes. After PJ34 pre-treatment, cells were then treated with MMS (1.5 mM) and viable cells were determined using a modified MTT assay as described in Fig. 3.