Functional overexpression of human poly(ADP-ribose) polymerase in transfected rat tumor cells

Abstract

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a nuclear enzyme possibly involved in DNA base excision repair. The presence of single- or double-strand breaks in DNA stimulates this enzyme to covalently modify acceptor proteins with poly(ADP-ribose) in a reaction that uses NAD+ as substrate. To test the hypothesis that increased PARP activity could promote resistance towards DNA-damaging agents and γ-radiation, we established stable rat cell transfectants that constitutively express human PARP. A number of subclones that showed different levels of PARP activity were isolated from two primary transfectants of different clonal origin, PARP activity was determined in permeabilized cells after maximal stimulation with a short, double-stranded oligonucleotide. Activity in different human PART-expressing subclones was increased 1.6- to 3.1-fold compared with non-expressing subclones. In vivo labeling of poly(ADP-ribose) was performed in one of these subclones, revealing that the level of poly(ADP-ribose) accumulation after the same treatment with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) was four times higher in the human PARP-expressing subclone compared with both non-expressing transfected control cells and parental cells. Clonal survival assays revealed a sensitization upon treatment with γ-radiation (up to 1.4-fold) or MNNG (up to 2.7-fold) of several subclones expressing human PARP; in some others survival was not changed. Survival after cisplatin (DDP) treatment remained essentially unchanged. A protective effect against DNA-damage was never observed. We conclude that human PARP overexpression in rodent cells leads to increased poly(ADP-ribosyl)ation capacity and does not promote survival after γ-radiation or treatment with the DNA-damaging agents MNNG or DDP.