Satadal Chatterjee

Poly(ADP-ribose) polymerase: A guardian of the genome that facilitates DNA repair by protecting against DNA recombination

We have studied the clonogenic survival response to X-rays and MNNG of V79 Chinese hamster cells and two derivative cell lines, ADPRT54 and ADPRT351, deficient in poly(ADP-ribose) polymerase (PARP) activity. Under conditions of exponential growth, both PARP-deficient cell lines are hypersensitive to X-rays and MNNG compared to their parental V79 cells. In contrast, under growth-arrested, confluent conditions, V79 and PARP-deficient cells become similarly sensitive to X-rays and MNNG suggesting that PARP may be involved in the repair of X-ray or MNNG-induced DNA damage in logarithmically growing cells but not in growth-arrested confluent cells. This suggestion, however, creates a dilemma as to how PARP can be involved in DNA repair in only selected growth phases while it is functionally active in all growth phases. To explain these paradoxical results and resolve this dilemma we propose a hypothesis based on the consistent observation that inhibition of PARP results in a significant increase in sister chromatid exchange (SCEs). Thus, we propose that PARP is a guardian of the genome that protects against DNA recombination. We have extended this theme to provide an explanation for our results and the studies done by many others.

Mutant cells defective in poly(ADP-ribose) synthesis due to stable alterations in enzyme activity or substrate availability

We used two different approaches to develop cell lines deficient in poly(ADP-ribose) synthesis to help determine the role of this reaction in cellular functions. One approach to this problem was to develop cell lines deficient in enzyme activity; the other approach was to develop cell lines capable of growing with such low nicotinamide adenine dinucleotide (NAD) levels so as to effectively limit substrate availability for poly(ADP-ribose) synthesis. The selection strategy for obtaining cells deficient in activity of poly(ADP-ribose) polymerase was based on the ability of this enzyme to deplete cellular NAD in response to high levels of DNA damage. Using this approach, we first obtained cell lines having 37-82% enzyme activity compared to their parental cells. We now report the development and characterization of two cell lines which were obtained from cells having 37% enzyme activity by two additional rounds of further mutagenization and selection procedures. These new cell lines contain 5-11% enzyme activity compared to the parental V79 cells. In pursuit of the second strategy, to obtain cells which limit poly(ADP-ribose) synthesis by substrate restriction, we have now isolated spontaneous mutants from V79 cells which can grow stably in the absence of free nicotinamide or any of its analogs. These cell lines maintain NAD levels in the range of 1.5-3% of that found in their parental V79 cells grown in complete medium. The pathway of NAD biosynthesis in these NAD-deficient cells is not yet known. Further characterization of these lines showed that under conditions that restricted poly(ADP-ribose) synthesis, they all had prolonged doubling times and increased frequencies of sister chromatid exchanges.

Camptothecin hypersensitivity in poly(adenosine diphosphate-ribose) polymerase-deficient cell lines.

Mutant cell lines, derived from the Chinese hamster V79 cell line, deficient in poly(adenosine diphosphate-ribose) polymerase activity, and previously shown to be resistant to topoisomerase II inhibitors, were found to be hypersensitive to camptothecin, a topoisomerase I inhibitor. In all the cell lines, camptothecin induced dose-dependent protein-associated DNA single-strand breaks and sister chromatid exchanges. The increased sensitivity to camptothecin-induced cytotoxicity was not associated with an increase in DNA single strand breaks or sister chromatid exchanges. These results suggest the absence of any direct causal relation between (1) camptothecin induced sister chromatid exchanges and cytotoxicity or (2) camptothecin induced DNA strand breaks and cytotoxicity. The hypersensitivity of these mutant cell lines to camptothecin suggests that poly(adenosine diphosphate-ribose) polymerase is involved with topoisomerase I in modulating camptothecin induced cytotoxicity.

Hypersensitivity to clinically useful alkylating agents and radiation in poly(ADP-ribose) polymerase-deficient cell lines

Mutant V79 Chinese hamster cell lines, deficient in poly(ADP-ribose) polymerase activity, were previously shown to be significantly resistant to etoposide, a topoisomerase II inhibitor, and hypersensitive to camptothecin, a topoisomerase I inhibitor (Chatterjee, S.; Trivedi, D.; Petzold, S.J.; Berler, N.A. Mechanism of epipophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose) synthesis-deficient V79 Chinese hamster cell lines. Cancer Res. 50:2713-2718, 1990 and Chatterjee, S.; Cheng, M.F.; Trivedi, D.; Petzold, S.J.; Berger, N.A. Camptothecin hypersensitivity in poly(adenosine diphosphate-ribose) polymerase-deficient cell lines. Cancer Commun. 1:389-394; 1990). We have now demonstrated hypersensitivity of these mutant cell lines, designated ADPRT 54 and ADPRT 351, to a variety of antitumor agents including melphalan, BCNU, mitomycin, and bleomycin. They are also hypersensitive to UV- and x-irradiation. These mutants, however, are significantly resistant to the topoisomerase II-targeted DNA intercalators, Adriamycin and m-AMSA. Our results strongly suggest that inhibition of poly(ADP-ribose) polymerase could be useful to potentiate the cytotoxicity of a variety of currently available antitumor drugs.

Alkylating agent hypersensitivity in poly(adenosine diphosphate-ribose) polymerase deficient cell lines

Starting with the V79 cell line, two poly(ADP-ribose) polymerase deficient mutants, designated ADPRT 54 and ADPRT 351, had been shown to be hypersensitive to x- and UV-irradiation and to topoisomerase I inhibitors but to be resistant to topoisomerase II inhibitors (Chatterjee, S.; Cheng, M. F.; Berger, N. A. Hypersensitivity to clinically useful alkylating agents and radiation in poly(ADP-ribose) polymerase-deficient cell lines. Cancer Commun. 2:401-407;1990). We now report that these mutants were hypersensitive to a series of different alkylating agents, including alkylsufonates, alkylnitrosoureas, and nitrosoguanidine. In addition, they were hypersensitive to the UV-mimetic agent 4-nitroquinoline-1-oxide. Our findings provide strong evidence that poly(ADP-ribose) polymerase was involved in the repair of alkylating agent induced DNA damage as well as in the damage induced by UV- and x-irradiation and radiomimetic agents. The poly(ADP-ribose) polymerase deficient cell lines showed a marked decrease in the shoulder region of their survival curves, suggesting that poly(ADP-ribose) polymerase was involved in the repair of alkylating agent induced sublethal damage.

Catalytic activity of poly(ADP-ribose) polymerase is necessary for repair of N-methylpurines in nontranscribed, but not in transcribed, nuclear DNA sequences

The role of poly(ADP-ribose) polymerase (PADPRP) in nuclear DNA repair and other nuclear processes has been intensely studied and debated for decades. Recent studies have begun to shed new light on these arguments with firm experimental data for its role, primarily, as a remodeler of chromatin structure. Those studies imply that PADPRP plays an indirect role in DNA repair, serving to expose DNA to repair enzymes through chromatin remodeling. Only DNA that is tightly packaged would require PADPRP activity for its repair; while DNA in an open conformation would be accessible to DNA repair enzymes and not require PADPRP activity. The purpose of the current studies was to address the above hypothesis directly. Using quantitative Southern blot analysis, we studied repair in transcribed and nontranscribed nuclear DNA sequences in ADPRT 351 cells 95% deficient in PADPRP activity. Cells were exposed to methylnitrosourea (MNU) for 1 h and allowed to repair for 8 or 24 h. Densitometric scans of autoradiographs revealed that, when compared to their parental V79 cell line, ADPRT 351 cells 95% deficient in PADPRP activity were equally as efficient in repair of N-methylpurines in the transcribed sequence containing the dihydrofolate reductase gene. However, the ADPRT 351 cells were deficient in the ability to repair these lesions in the nontranscribed sequence containing the IgE gene compared to repair of the same sequence in the parental V79 cells. Nucleoid sedimentation assays demonstrated that the ADPRT 351 cells are deficient in repair across the entire genome when compared to the parental V79 cells. These studies indicate that PADPRP activity is not required for repair of N-methylpurines in transcribed nuclear DNA sequences but is necessary for the repair of these lesions in nontranscribed nuclear DNA sequences as well as across the entire genome since the DNA in a given cell is predominantly nontranscribed.

Poly(ADP-Ribose) Polymerase in Response to DNA Damage

The first observation that paved the way for the discovery of poly(ADP-ribose) and poly(ADP-ribose) polymerase (PARP) was reported by Chambon, et al. in 1963 (30). They found that a particulate fraction from nuclei of chicken liver catalyzed the nicotinamide mononucleotide (NMN)-dependent incorporation of 14C-adenine-labeled ATP into trichloroaceticacid-insoluble material and that the reaction was markedly inhibited by deoxyribonuclease, but not by ribonuclease. It was later found that the DNasedependent inhibition of the catalytic activity could be restored by DNA, establishing a dependency on DNA. Furthermore, incorporation of 14C-adenine-ATP in the presence of NMN was drastically decreased by addition of unlabeled NAD, suggesting that NAD was an obligatory intermediate.

V-79 Cell Variants Deficient in Poly(ADP-Ribose) Polymerase

We devised a strategy to select cells with reduced levels of poly(ADP-ribose) polymerase based on our demonstration of the involvement of this enzyme in the suicide response after high levels of DNA damage (1“ 2). We reasoned that high levels of DNA damage would activate the enzyme to deplete NAD and ATP pools in normal cells resulting in the cessation of all energy dependent processes and rapid cell death, whereas, cells deficient in the enzyme activity would avoid these metabolic perturbations and therefore be more likely to survive. Employing the strategy we isolated variants from V-79 Chinese hamster cells having 5-11% of the normal enzyme activity.