A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Abstract

leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version ofthe DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNAbinding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: Evidence for increased tumor cell apoptosis in vivo

“…Our results, showing that the absence of polymer production by expression of dominant negative PARP increases DNA damageinduced apoptosis, favor the hypothesis that poly(ADP-ribosyl)ation is a negative regulator of this form of apoptosis. Our results are in line with published work showing that there is increased MNNG-induced apoptosis in stably transfected HeLa cell cultures expressing the PARP DBD (Schreiber et al, 1995). It is not yet clear by which mechanism the DBD facilitates apoptosis in vivo, but it is obvious to speculate that this e ect is due to increased sensitivity to oxidative stress caused by the attack of immunocompetent cells other than T-cells.…”

“…In these so-called COM3 cells, we could show that expression of dominant negative PARP sensitizes cells against g-irradiation and the alkylating agent MNNG, while there was neither an in¯uence detectable on DNA replication nor on cellular proliferation in the absence of genotoxic stress (KuÈ pper et al, 1995). This sensitization is associated with a block in DNA base excision repair (Molinete et al, 1993) and increased gene ampli®cation and sister chromatid exchanges (KuÈ pper et al, 1995Schreiber et al, 1995). We were then interested to see whether PARP DBD overexpression a ects tumor formation in nude mice.…”

Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor

“…We attributed this e ect to the co-regulated inhibition of the expression of PARP, an enzyme implicated in DNA-repair (Satoh and Lindahl, 1992). This suggestion is consistent with ®ndings by others that PARP levels are not rate limiting for the proliferation of normal (undamaged) cells and that downregulation of PARP expression results in increased cell susceptibility to DNA-damaging treatments (Ding et al, 1992;Wang et al, 1995;KuÈ pper et al, 1995;Schreiber et al, 1995). Another explanation may involve the potential role of the ETS protein itself in the radiation response of EWS cells.…”

Novel role for the transient receptor potential channel TRPM2 in prostate cancer cell proliferation

“…17 Over-activation of PARP can cause cell necrosis because of the depletion of cytoplasmic ATP. 18,19 To examine the (Figure 4c). PC-3 cells were seeded in a 96-well plate at B1000 cells per well in triplicate.…”