Role of PARP-1 and PARP-2 in the expression of apoptosis-regulating genes in HeLa cells

Poly (ADP-ribose) polymerase-1 (PARP-1) is a DNA-binding enzyme involved in DNA damage processing, apoptosis, and genetic stability. Many lines of evidence suggest that PARP-1 is implicated in transcriptional regulation of various genes through the modulation of chromatin structure or through direct interaction with transcription factors and/or transcription factor-binding sites. In the present study, we applied TaqMan Low-Density Array analyses to investigate the expression of genes involved in apoptotic cell death induced by an alkylating agent. Using RNA interference, we determined the roles of PARP-1 and PARP-2 in transcriptional regulation during apoptosis in HeLa cells. Of the 93 genes monitored, 33 differentially expressed genes were identified after induction of apoptosis. Whereas the down-regulation of PARP-1 and PARP-2 had no impact on gene expression per se, we observed that Bcl10, c-Rel, and tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 are differentially expressed after induction of apoptosis in a PARP-1-dependent manner. These findings suggest that PARP-1—but not PARP-2—is required for proper expression of major genes involved in regulation of apoptosi

Rapid regulation of telomere length is mediated by poly(ADP-ribose) polymerase-1

Shelterin/telosome is a multi-protein complex at mammalian telomeres, anchored to the double-stranded region by the telomeric-repeat binding factors-1 and -2. In vitro modification of these proteins by poly(ADP-ribosyl)ation through poly(ADP-ribose) polymerases-5 (tankyrases) and -1/-2, respectively, impairs binding. Thereafter, at least telomeric-repeat binding factor-1 is degraded by the proteasome. We show that pharmacological inhibition of poly(ADP-ribose) polymerase activity in cells from two different species leads to rapid decrease in median telomere length and stabilization at a lower setting. Specific knockdown of poly(ADP-ribose) polymerase-1 by RNA interference had the same effect. The length of the single-stranded telomeric overhang as well as telomerase activity were not affected. Release of inhibition led to a fast re-gain in telomere length to control levels in cells expressing active telomerase. We conclude that poly(ADP-ribose) polymerase-1 activity and probably its interplay with telomeric-repeat binding factor-2 is an important determinant in telomere regulation. Our findings reinforce the link between poly(ADP-ribosyl)ation and aging/longevity and also impact on the use of poly(ADP-ribose) polymerase inhibitors in tumor therapy

Downregulation of PARP-1 expression by RNA interference

Poly(ADP)polymerasen (PARPs) sind Proteine, die die Synthese von ADP-Ribose Polymeren katalysieren und unterschiedliche Rollen bei verschiedenen zellulären Funktionen spielen. Ziel der vorliegenden Arbeit war es, ein System zu entwickeln, dass die Downregulation von PARP- Proteinen mittels RNA Interferenz (RNAi) in Zellkulturen ermöglicht. Die Bedingungen für ein effizientes Gen Silencing wurden zunächst für PARP-1 ermittelt. Die Verminderung der PARP-1 Proteinmenge in Fibroblasten von der Maus wurde mittels zweier unterschiedlicher Techniken getestet. Die erste Methode basiert auf der Verwendung von DNA Plasmiden, die die siRNA Moleküle in den Zellen exprimieren, die zweite Methode auf der direkten Transfektion von in vitro hergestellter siRNA. Die zweite Methode ergab die besseren Ergebnisse. Es wurden sechs siRNAs Sequenzen von PARP- 1 auf ihr Silencing Vermögen hin untersucht. Immunoblot Analysen zeigten, dass die unterschiedlichen siRNA Moleküle in der Lage waren, die PARP-1 Expression von 35 % bis mehr als 98 % der ursprünglichen Level zu reduzieren. Der Effekt wurde nach 24 Stunden sichtbar und hielt bis mindestens 72 Stunden nach der Transfection an. Das Silencing von PARP-1zeigte unter normalen Wachstumsbedingungen keinen offensichtlichen Phenotyp; jedoch waren die mit siRNA behandelten Zellen resistenter gegenüber oxidativem Stress als die Wildtyp Zellen. Die vorliegende Studie zeigt die notwendigen Bedingungen für ein effizientes Silencing von PARP-1 in Zellkulturen; diese Methode wird die Basis für weitere Untersuchungen sein, die letztendlich zur Identifikation der Funktion von PARP-1, und mehr allgemein, zur Funktion der Poly(ADP-Ribose) Synthese, als Antwort auf DNA Schäden, führen. Poly(ADP-ribose) polymerases (PARPs) are enzymes that catalyze the synthesis of ADP-ribose polymers, covalently bound to acceptor proteins and play important roles in various cellular functions. This thesis work was aimed at setting up conditions for specific silencing of individual PARPs in living cells using RNA interference (RNAi). To knock-down PARP-1, the most abundant member of the PARP family, in mouse embryonic fibroblasts (MEFs), two approaches out of the various techniques for RNAi were tested. One approach involved the use of DNA plasmids which express siRNA within the cells and the second approach was based on the direct transfection of in vitro transcribed siRNAs. IThe latter approach gave the best results. Six different PARP-1 targenting siRNAs were tested for their ability to downregulate PARP-1 expression. Immunoblotting analyses revealed that siRNA molecules, targeting different positions on PARP-1 mRNA, were able to reduce PARP-1 expression from 35 % to more than 98 % of basal levels within 24 hours and that the silencing effect persists at least up to 72 hours after transfection. Post-transcriptional silencing of PARP-1 by RNAi did not cause any obvious phenotype under normal growth conditions; however, silenced cells appeared to be more resistant against oxidative stress than wild-type cells. In conclusion, these studies establish conditions for effective PARP-1 silencing in cultured cells; this will provide an essential background for further investigations on the role of PARP-1 and, more generally, of poly (ADP-ribose) synthesis, in the response to DNA damage

Rapid regulation of telomere length is mediated by

poly(ADP-ribose) polymerase-

Role of PARP-1 and PARP-2 in the expression of apoptosis-regulating genes in HeLa cells

Poly (ADP-ribose) polymerase-1 (PARP-1) is a DNA-binding enzyme involved in DNA damage processing, apoptosis, and genetic stability. Many lines of evidence suggest that PARP-1 is implicated in transcriptional regulation of various genes through the modulation of chromatin structure or through direct interaction with transcription factors and/or transcription factor-binding sites. In the present study, we applied TaqMan Low-Density Array analyses to investigate the expression of genes involved in apoptotic cell death induced by an alkylating agent. Using RNA interference, we determined the roles of PARP-1 and PARP-2 in transcriptional regulation during apoptosis in HeLa cells. Of the 93 genes monitored, 33 differentially expressed genes were identified after induction of apoptosis. Whereas the down-regulation of PARP-1 and PARP-2 had no impact on gene expression per se, we observed that Bcl10, c-Rel, and tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 are differentially expressed after induction of apoptosis in a PARP-1-dependent manner. These findings suggest that PARP-1—but not PARP-2—is required for proper expression of major genes involved in regulation of apoptosis