Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

Abstract

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP(-/-) fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP(-/-) fibroblasts were exposed for 3 weeks to 20 microM GPI 6150 (1,11b-dihydro-[2H:]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (K(i) = 60 nM) and one of the most potent PARP inhibitors to date (IC(50) = 0.15 microM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by approximately 91%, to about the residual levels in PARP(-/-) cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.

Figure 1

PARP expression in immortalized wild-type and PARP^–/– fibroblasts. (A) Cell extracts of wild-type and PARP^–/– fibroblasts (30 µg protein) were subjected to immunoblot analysis with antibodies to PARP (upper) and PAR (middle). The blot was stained with Ponceau S to verify equal loading and transfer of proteins in both lanes (lower). (B) RT–PCR was performed with specific primers for the mPARP mRNA. The positions of PARP and PAR are indicated.

Figure 2

Determination of the IC₅₀ for GPI 6150 (A), effects of GPI 6150 on cell growth (B) and endogenous PARP activity (C) of wild-type and PARP^–/– fibroblasts. (A) PARP^+/+ cells were washed with ice-cold PBS, cell extracts were derived and equal amounts of protein (30 µg) were subjected to PARP activity assays in the presence of various concentrations of GPI 6150. PARP activity assays were performed by measurement of [³²P]NAD incorporation in vitro into acid-insoluble acceptors at 25°C for 1 min, with triplicate determinations per treatment. (B) Cells were grown in the presence of 20 µM GPI 6150 or with vehicle alone (DMSO) for 3 weeks, during which GPI 6150 was replenished when the culture medium was changed every other day and when the cells were passaged once a week. During the first week, effects of GPI 6150 on cell viability and cell growth were determined by cell counts with Trypan blue staining every 24 h for the 3 days of exposure to GPI 6150 and prior to passaging the cells on day 4. (C) Inhibition of endogenous PARP activity in the wild-type cells exposed to 20 µM GPI 6150 at the indicated times was also verified by PARP activity assays. Equal amounts of total cellular protein (30 µg) were subjected to PARP activity assays in triplicate determinations. All the data are presented as means ± SD of three replicates of a representative experiment; essentially the same results were obtained in three independent experiments.

Figure 3

Complete inhibition of transient poly(ADP-ribosyl)ation of nuclear proteins during Fas-mediated apoptosis in wild-type fibroblasts by GPI 6150. Immortalized wild-type fibroblasts were exposed to anti-Fas (50 ng/ml) and cycloheximide (10 µg/ml) for the indicated times in the presence of 20 µM GPI 6150 or with vehicle alone. Equal amounts of total cellular protein (30 µg) were then subjected to immunoblot analysis with monoclonal antibodies to PAR. The positions of molecular size standards (in kDa) are indicated.

Figure 4

(Above and opposite) Effects of inhibition of PARP activity by GPI 6150 on the development of tetraploidy in immortalized wild-type and PARP^–/– fibroblasts. FACS analysis was performed using unsynchronized immortalized wild-type (left) and PARP^–/– (right) fibroblasts (A). Cells were harvested and nuclei were prepared and stained with propidium iodide for FACS analysis. In addition to the two major peaks of nuclei at G₀/G₁ and G₂/M apparent in the DNA histograms of unsynchronized wild-type cells, the DNA histograms of PARP^–/– cells exhibit a third peak corresponding to the G₂/M peak of an unstable tetraploid cell population (arrows). FACS analysis was performed after continuous exposure of wild-type (B) or PARP^–/– (C) fibroblasts to GPI 6150 (20 µM) or to vehicle (DMSO) alone for 1, 2 or 3 weeks to detect the presence of an unstable tetraploid population in the GPI 6150-treated wild-type cells. The G₂/M peak of the unstable tetraploid cell population in the DNA histograms of PARP^–/– cells is indicated by arrows. Essentially identical results were obtained in three independent experiments.

Figure 4

(Above and opposite) Effects of inhibition of PARP activity by GPI 6150 on the development of tetraploidy in immortalized wild-type and PARP^–/– fibroblasts. FACS analysis was performed using unsynchronized immortalized wild-type (left) and PARP^–/– (right) fibroblasts (A). Cells were harvested and nuclei were prepared and stained with propidium iodide for FACS analysis. In addition to the two major peaks of nuclei at G₀/G₁ and G₂/M apparent in the DNA histograms of unsynchronized wild-type cells, the DNA histograms of PARP^–/– cells exhibit a third peak corresponding to the G₂/M peak of an unstable tetraploid cell population (arrows). FACS analysis was performed after continuous exposure of wild-type (B) or PARP^–/– (C) fibroblasts to GPI 6150 (20 µM) or to vehicle (DMSO) alone for 1, 2 or 3 weeks to detect the presence of an unstable tetraploid population in the GPI 6150-treated wild-type cells. The G₂/M peak of the unstable tetraploid cell population in the DNA histograms of PARP^–/– cells is indicated by arrows. Essentially identical results were obtained in three independent experiments.