Characterization of Human Poly(ADP-ribose) Polymerase with Autoantibodies*

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poIy(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immu- noblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in hu- man lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10’ enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10’ poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. PoIy(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed

and the regulation of enzyme synthesis and degradation have been difficult to measure precisely. The catalytic activity of poly(ADP-ribose) polymerase absolutely requires DNA and is stimulated strongly by DNA with strand breaks (10). Furthermore, NAD poorly penetrates cell membranes and is also metabolized by routes other than poly(ADP-ribosy1)ation. For all these reasons, the relation between cellular levels of poly(ADP-ribose) polymerase and the catalytic activity of the enzyme has not been clearly established.
Poly(ADP-ribose) polymerase has been purified from several sources and characterized extensively. Polyclonal or monoclonal antibodies have been raised against the purified calf enzyme (11,12) and the human enzyme (13). Although the catalytic properties of poly(ADP-ribose) polymerase are well conserved among mammals, the antigenic specificities differ from species to species (13). Thus, previous reports showed that polyclonal rabbit antibodies against calf thymus poly(ADP-ribose) polymerase reacted only weakly with the human enzyme (13).
Recently, we have identified specific human autoantibodies to poly(ADP-ribose) polymerase in the sera of patients with rheumatic disorders (14). The human autoantibodies recognized multiple determinants on the protein and reacted indistinguishably with the calf and human enzymes. These high titer and high affinity human autoantibodies allowed us to isolate poly(ADP-ribose) polymerase-specific cDNA from a human placental expression library.' In this study, we have used the anti-poly(ADP-ribose) polymerase autoantibodies to quantitate the immunoreactive levels of poly(ADP-ribose) polymerase in various human cells under changing culture conditions.

DISCUSSION
These experiments have analyzed for the first time the content of poly(ADP-ribose) polymerase in human cells using specific immunologic methods. The poly(ADP-ribose) polymerase levels were determined by a sensitive immunoblotting procedure with high affinity human autoantibodies. Prior to immunoblotting, cells were rapidly solubilized by boiling in Laemmli's sample buffer containing 2% SDS3 Under these conditions, poly(ADP-ribose) polymerase was released from Portions of this paper (including "Experimental Procedures," "Results," and Figs. 1-7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
The abbreviations used are: SDS, sodium dodecyl sulfate; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; kb, kilobase pairs. 3879 the nuclear matrix, and enzyme proteolysis was minimal. This result is consistent with earlier findings that demonstrated the complete release of proteins from the nuclear matrix following SDS extraction (15). Furthermore, the rapid solubilization procedure minimized DNA damage and thereby prevented the attachment of high molecular weight ADPribose polymers to the poly(ADP-ribose) polymerase protein.
For these reasons, the immunoblotting technique was preferable to immunoprecipitation or enzyme-linked immunosorbent assay for the quantitation of enzyme protein in crude extracts. However, immunoprecipitation was useful for the comparative measurements of poly(ADP-ribose) polymerase turnover under changing culture conditions. With these methods, we were able to show that 1) poly(ADP-ribose) polymerase is an abundant nuclear protein, 2) the total immunoreactive cellular content of poly(ADP-ribose) polymerase varies by not more than %fold during the growth cycle of proliferating cells, and 3) the turnover rate of poly(ADP-ribose) polymerase is approximately equivalent to the average turnover rate of total cellular protein.
Previous experiments have measured changes in the cellular content of poly(ADP-ribose) chains during the cell growth cycle. These experiments showed that levels of the polymer reached a maximum during G2/M phase (1,(15)(16)(17)(18)(19). However, the rate of poly(ADP-ribose) synthesis does not necessarily reflect poly(ADP-ribose) polymerase content, but also depends upon the state of enzyme activation by DNA. The activity of poly(ADP-ribose) polymerase in permeabilized cells following DNase I treatment has been used to estimate the total intracellular poly(ADP-ribose) polymerase, because nicked DNA highly stimulates enzyme activity (3). We also observed a significant correlation between the amounts of poly(ADP-ribose) polymerase determined by immunoblotting and the DNase I inducible poly(ADP-ribose) polymerase activity. However, the changes in DNase I activatable poly(ADP-ribose) polymerase during the cell growth cycle were approximately %fold more than the changes in the total immunoreactive enzyme. Recent reports have suggested that some poly(ADP-ribose) polymerase molecules may be tightly bound to the nuclear matrix (20,21). Changes in the structure and composition of chromatin during the cell cycle would be expected to influence poly(ADP-ribose) polymerase activity. Thus, it may be the interaction of poly(ADP-ribose) polymerase with DNA, rather than the levels of enzyme, that principally regulates poly(ADP-ribose) synthesis during the cell growth cycle.
The amounts of poly(ADP-ribose) polymerase per cell increased to a much greater degree following stimulation of peripheral blood lymphocytes with phytohemagglutinin than during the passage of cultured CEM or WI-L2 lymphoblastoid cells from G1 to G2/M phase.
Moreover, the poly(ADPribose) polymerase content in the mitogen-stimulated lymphocytes rose just prior to new DNA synthesis, and the rise continued during S phase (3). These results suggest that the production of poly(ADP-ribose) polymerase is necessary for quiescent lymphocytes to re-enter the cell growth cycle, but is not strictly dependent on DNA synthesis. The minimal amounts of poly(ADP-ribose) polymerase in neutrophils and monocyte-derived macrophages may therefore be related to the terminal differentiation of the two cell types.
Based upon immunoblotting'experiments, we estimate that CEM lymphoblasts contain approximately 2 X lo6 poly(ADPribose) polymerase molecules per cell. This value is in the same order of magnitude as DNA topoisomerase I1 (1 X lo6 copies/cell) (22), individual nuclear lamins (22), U1 or U2 small nuclear ribonucleoproteins (approximately 1 X lo6 cop-ies/cell) (23), and the Ku DNA-binding protein (at least 4 X lo5 copies/HeLa cell) (24), although representing only 1% of total histone content. If poly(ADP-ribose) polymerase is distributed evenly among chromatin, one enzyme molecule would be present for every 1000 base pairs of DNA and for every five nucleosomes. Although compartmentalization of poly-(ADP-ribose) polymerase within the nucleus may occur, these results do suggest that poly(ADP-ribose) polymerase is an abundant nuclear protein.
The maximal rate of NAD utilization by purified poly(ADPribose) polymerase has been estimated to be 1.4 nmol/min/ pg protein (13). Since lo6 CEM cells contain 0.4 pg of poly(ADP-ribose) polymerase, the lymphoblasts can potentially consume 560 pmol of NAD/min/106 cells. In comparison, cultured mammalian cells under standard growth conditions have been reported to contain less than 1 pmol of poly(ADP-ribose) per lo6 cells (1,25,26). Moreover, the total rate of NAD turnover in rapidly dividing HeLa cells has been reported to be 78,000 molecules/s/cell (27), which is equal to 7.7 pmol of NAD/min/106 cells. Thus, it appears that the pace of NAD turnover in human cells is only about 1% of the maximal rate of poly(ADP-ribose) synthesis. The relative abundance of cellular poly(ADP-ribose) polymerase is in accord with the slow turnover rate of the enzyme and with the apparent stability of poly(ADP-ribose) polymerase following activation of the enzyme by irradiation of DNA. During balanced growth, cultured lymphoblasts contain more than enough poly(ADP-ribose) polymerase than that which is required for poly(ADP-ribose) synthesis.
Poly(ADP-ribose) polymerase has been postulated to function as an emergency enzyme that is stimulated specifically by DNA damage (28). The activation of the enzyme has been shown to influence DNA-histone interactions. A sudden increase in poly(ADP-ribose) synthesis can also lead to the rapid consumption of NAD and ATP, and thereby can prevent or delay cell proliferation (28,29). Perhaps the large storage pool of poly(ADP-ribose) polymerase, which is maintained in lymphoblasts throughout the cell cycle, facilities the rapid activation of the enzyme at individual sites of DNA strand break formation. Considering the relatively large amounts of poly(ADP-ribose) polymerase in the nucleus, it is also possible that the enzyme plays a structural role in the formation of chromatin and that the poly(ADP-ribosy1)ation reaction modifies chromatin structure by changing the interaction of the protein with DNA.