FUNCTIONAL OVEREXPRESSION OF HUMAN POLY(ADP-RIBOSE) POLYMERASE IN TRANSFECTED RAT-TUMOR CELLS

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a nuclear enzyme po ssibly involved in DNA base excision repair, The presence of single- o r double-strand breaks in DNA stimulates this enzyme to covalently mod ify acceptor proteins with poly(ADP-ribose) in a reaction that uses NA D(+) as substrate, To test the hypothesis that increased PARP activity could promote resistance towards DNA-damaging agents and gamma-radiat ion, 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 diff erent clonal origin. PARP activity was determined in permeabilized cel ls after maximal stimulation with a short, double-stranded oligonucleo tide, Activity in different human PARP-expressing subclones was increa sed 1.6- to 3.1-fold compared with non-expressing subclones, In vivo l abeling of poly(ADP-ribose) was performed in one of these subclones, r evealing that the level of poly(ADP-ribose) accumulation after the sam e 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 su rvival assays revealed a sensitization upon treatment with gamma-radia tion (up to 1.4-fold) or MNNG (up to 2.7-fold) of several subclones ex pressing human PARP; in some others survival was not changed. Survival after cisplatin (DDP) treatment remained essentially unchanged, A pro tective 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 gamma-radi ation or treatment with the DNA-damaging agents MNNG or DDP.