Assessment of Unique and Combined Functions of Poly(ADP-Ribose) Polymerases by Using RNA Interference

Abstract

Poly(ADP-ribose) polymerases (PARP) catalyze the synthesis of poly (ADP-ribose) (PAR), a reversible modification of proteins, using NAD+ as a substrate. Poly(ADP-ribosyl)ation produced by PARP-1 and PARP-2 is involved in cytoplasmic and nuclear processes, such as chromatin remodeling, DNA damage signaling and repair, RNA processing, and regulation of cell death. Genetic knockout mouse models of PARP-1 and PARP-2 have revealed a degree of redundancy in cellular PARP functions. However, advancements in elucidating this redundancy have been hindered by the embryonic lethality of the combined PARP-1 and PARP-2 genetic knockout in mice. To date there are several in vitro studies on the cellular depletion of PARP-1 and PARP-2, but these reports did neither aim to investigate this degree of redundancy nor try to provide detailed understanding of the consequences of a combined knockdown of PARP-1 and PARP-2.In the present work, a first systematic study on the unique and combined functions of PARP-1 and PARP-2 was provided by RNA interference of both proteins in two different cellular approaches. The first approach using a doxycycline inducible microRNA-adapted shRNA (shRNAmir) system had revealed design difficulties in the expression of a polycistronic head to tail configuration to achieve concurrent expression of two shRNAmir sequences, a design formerly reported to be successful but also problematic in some instances. Here, expression of only the second shRNAmir sequence (i.e. PARP-1) was successful, whereas PARP-2 shRNAmir expression could not be demonstrated in stable PARP-1 and PARP-2 shRNAmir expressing HeLa S3 clonal cell populations. Thus, this first approach using concurrent expression of two shRNAmir sequences was not successful in generating a combined knockdown of PARP-1 and PARP-2 in a cellular in vitro system. Moreover, the study design also cautioned before use of older published target siRNA sequences (likely to express off-target effects), although being successful in generating a stable PARP-1 shRNAmir HeLa S3 clonal cell populations. Therefore, in an alternative approach, previously observed difficulties were addressed and concurrent knockdown of PARP-1 and PARP-2 was performed in transient siRNA transfections in two different human cancer cell lines.To deplete PARP-1 and PARP-2 protein expression in the alternative approach, new and effective PARP-1 and PARP-2 siRNA were generated for use in transient siRNA transfections. Here, single and combined transfections of PARP-1 and PARP-2 siRNA demonstrated a strong knockdown of PARP-1 and/or PARP-2 protein expression in western blot analysis and quantifications of relative mRNA levels in HeLa S3 and U2OS cells. Furthermore, both PARP-1 and PARP 2 siRNA were able to strikingly reduce poly(ADP ribose) formation after oxidative stress, demonstrating a functional loss of poly(ADP-ribosyl)ation capacities in cells.In following analyses of population doubling, cell proliferation after genotoxic stress, clonogenic survival, cell death, and finally cell cycle phase distributions in HeLa S3 and U2OS cells, no functional redundancies between PARP-1 and PARP-2 could be observed. In contrast, a novel function of PARP-2 during cellular proliferation in HeLa S3 and U2OS cell lines was demonstrated. Depletion of PARP-2, but not PARP-1, significantly reduced cellular proliferation dependent processes as examined by population doubling, cell proliferation after genotoxic stress and clonogenic survival. Moreover, this new PARP-2 function during cellular proliferation was also independent of oxidative or genotoxic stress and could not be attributed to alterations in cell death. Changes in cell cycle have been found instead to mediate this new PARP-2 function, demonstrating a cell-type and p53 independent G1 phase cell cycle arrest. Finally, this G1 phase cell cycle arrest was shown to be independent of PARP catalytic activity, which might be due the reported function of PARP-2 as a transcriptional repressor of cell cycle related promoters, such as c-MYC, which regulate the G1 phase cell cycle checkpoint.In summary, this first systematic study on unique and combined functions of PARP-1 and PARP-2 demonstrated no functional redundancies of PARP-1 and PARP-2 in the endpoints analyzed. In contrast, a novel catalytic- and PARP-1-independent function of PARP-2 during cellular proliferation was demonstrated within the present work, which might advance understanding of targeting PARP-2 in cancer therapy to suppress tumor growth.