The role of poly(ADP-ribose) polymerase-1 in the MDM2-p53 DNA damage response pathway.

Abstract

p53 is a tumour suppressor protein that is stabilised and activated by DNA damage. DNA damage-induced p53 is able to bring about either cell cycle arrest or apoptosis by the induction of p53-responsive genes such as mdm2 and p21 waf-I. Mdm2 regulates p53 function by blocking the transcriptional transactivation domain of p53 and also by targeting p53 for degradation via an ubiquitin-mediated pathway. Increases in the levels and activity of p53 are brought about by post-translational modifications. The most widely studied modification of p53 is phosphorylation, mediated by several DNA damageactivated kinases. Poly(ADP-Ribose) Polymerase-l (PARP-l) is also a DNA damageactivated enzyme which covalently modifies several target proteins by poly(ADPribosylation). It is well established that PARP-1 plays a key role in DNA base excision repair. More recently, several studies have implicated PARP-1 in the regulation of p53 function in response to DNA damage, although the nature of this relationship has been controversial. This study aimed to clarify and investigate further the role of PARP-1 in p53 regulation using PARP-1 proficient and PARP-1 deficient mouse embryonic fibroblasts (MEFs) as well as a novel potent PARP-1 inhibitor (AGI4361; Ki < 6nM). In this study, both primary and immortalised PARP-l MEFs were used. Initial experiments revealed a tendency for PARP-l +/+ MEFs to develop p53 mutations during immortalisation. Interestingly. PARP-1 -/- MEFs retained wild-type p53, suggesting that the absence of PARP-l bypasses the requirement for p53 to be mutated during the immortalisation of MEFs. As these cells could not be used to analyse p53 responses, experiments were perfonned on primary PARP-l MEFs. However. the primary PARP-l- - MEFs were found to grow very slowly compared to their PARP-1 proficient counterparts. Interestingly. treatment of primary PARP-1+1+ MEFs with AG14361 had a similar effect on cellular growth. This growth inhibition in the absence of PARP-1 was only evident in primary and not immortalised cells. It was therefore decided to stably transfect immortalised PARP-l-- MEFs, expressing wild-type p53, with a plasmid construct containing PARP-l to produce an isogenic cell line pair. These cells have been used, together with a human colorectal carcinoma cell line (HCT-116) and the potent PARP-1 inhibitor AG14361 to analyse the p53 response to different DNA damaging agents. In response to ionising radiation and ultra violet radiation, the absence of PARP-1 did not alter the induction or activity of p53. In response to the alkylating agent temozolomide, treatment of PARP-l proficient MEFs with AG14361 potentiated the increase in p53 protein levels without affecting the transcriptional transactivation activity of p53, possibly due to an impaired repair of the DNA damage and hence increased signalling to p53 due to the persistence of DNA strand breaks. However, similar results were not obtained in the absence of PARP-1 protein (P ARP-1-/- MEFs) or in HCT -116 cells treated with AG 14361 The data presented do not support the hypothesis that PARP-1 is directly involved in the DNA damage induced regulation of p53. There may, however, be an altered p53 response in the absence of PARP-l when cells are treated with particular DNA damaging agents, due to an impaired DNA repair pathway.