Unique acceptors for poly(ADP-ribose) in resting, proliferating and DNA-damaged human lymphocytes

Biochimica et Biophysica Acta. 740 (1983) 8- 18

8

Elsevier BBA 91202

U N I Q U E ACCEPTORS FOR P O L Y ( A D P - R I B O S E ) IN R E S T I N G , P R O L I F E R A T I N G AND DNA-DAMAGED HUMAN LYMPHOCYTES CAROL S. SUROWY and NATHAN A. BERGER * The Hematology/ Oncology Division of the Department of Medicine, Washington University School of Medicine and The Jewish Hospital of St. Louis, 216 South Kingshighway, St. Louis, MO 63110 (U.S.A.)

(Received December 28th, 1982)

Key words: Poly(ADP-ribose)," DNA damage," Acceptor protein; (Human lymphocyte)

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N.methyI-N'-nitro-N-nitrosoguanidine-inducedDNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyI-N'-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that M N N G treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histunes. In MNNG-treated cells, an increase in ADP-ribosylation of histone HI was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96 000. M N N G treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to he due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.

Introduction Poly(ADP-ribose) is synthesized from N A D + in the nuclei of eukaryotic cells by the enzyme poly(ADP-ribose) polymerase. In vitro studies show that poly(ADP-ribose) can be covalently attached to a number of proteins including histones and the polymerase itself [1,2]. During recent years it has been shown that induction of D N A strand breaks by DNA-damaging agents results in an * To whom correspondence should be addressed. Abbreviations: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HMG, high-mobility group; MeESO, dimethylsulfoxide; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; poly(ADP-ribose), poly(adenosinediphosphoribose). 0167-4781/83/$03.00 © 1983 Elsevier Science Publishers B.V.

increase in poly(ADP-ribose) synthesis [3-7]. In addition, when normal lymphocytes are stimulated to proliferate by the mitogen phytohemagglutinin the increase in D N A synthesis is preceded by an increase in activity of poly(ADP-ribose) polymerase [8-10]. As one approach to study the function of the ADP-ribosylation reaction we sought to identify those proteins which act as acceptors for ADPribosylation under the different conditions of cell growth a n d / o r D N A damage outlined above. Human lymphocytes are particularly useful for this type of study since they can be obtained in a quiescent, G O state with very low levels of poly(ADP-ribose) synthesis. They can be treated with DNA-damaging agents which cause them to

undergo a marked increase in poly(ADP-ribose) synthesis in association with DNA repair synthesis [2,4], or they can be stimulated by mitogens such as phytohemagglutinin which cause them to undergo a marked increase in replicative DNA synthesis in association with an increase in poly(ADP-ribose) synthesis [9,10]. Thus, we used resting human lymphocytes and those treated with DNA-damaging agents or the mitogen phytohemagglutinin to identify acceptors for poly(ADP-ribose), and to determine whether any proteins were specifically ADP-ribosylated in response to DNA damage or mitogen stimulation. Methods

3-Aminobenzamide was obtained from Pfaltz & Bauer, Inc. L-phytohemagglutinin (Type IV from Phaseolus vulgaris) was from Sigma Chemical Co., and [adenylate-32p]nicotinamide adenine dinucleotide (spec. act. 1 • 105 cpm/pmol) was from New England Nuclear. Poly(ADP-ribose) polymerase purified from human lymphoid tissue was the same as previously described [11]. Normal human lymphocytes (95% pure) were isolated by Ficoll-Hypaque gradient centrifugation [12] and resuspended to 2.106 cells/ml in alpha modified Eagle's medium supplemented with 10% heat-treated fetal calf serum, 50 U / m l penicillin, 50 #g/ml streptomycin and 25 mM Hepes buffered to a final pH of 7.2, as previously described [9]. For mitogen stimulation, culture medium contained 2 mM L-glutamine in addition to the components mentioned above. Phytohemagglutininwas added at a final concentration of 2 # g / m l to suspensions containing 5.105 cells/ml. 20 ml aliquots of phytohemagglutinin-treated cells were incubated at 37°C for 3 days in 75 cm2 tissue culture flasks. MNNG treatment was performed by dissolving the drug in Me2SO and then immediately adding an appropriate aliquot to a cell suspension. A final concentration of 20 /~g/ml MNNG and 0.2% Me2SO was used in all experiments, and control cells were adjusted to contain the same final concentration of Me2SO. At this concentration, Me2SO was shown to have no effect on the level of poly(ADP-ribose) synthesis [13]. After addition of MNNG a n d / o r Me2SO all cells were incubated at

37°C for specified time periods. At intervals after treatment with MNNG or phytohemagglutinin, cells were collected from control and treated cultures; they were made permeable to exogenously supplied nucleotides [9,14,15] and used to measure the synthesis of poly(ADPribose). Briefly, cells were collected from suspension culture by centrifugation at 3000 × g for 10 min at 4°C and then resuspended to 2.106 cells/ml in a hypotonic buffer consisting of 10 mM Tris-HC1 (pH 7.8)/1 mM EDTA/4 mM MgCI2/30 mM 2-mercaptoethanol at 0°C and incubated in an ice-water bath for 15 min. The cells were collected again by centrifugation and resuspended in the same buffer at (4-10). 107 cells/ml and 0°C (cell concentration dependent upon the experiment). To measure poly(ADP-ribose) synthesis, 50 #1 portions of the permeable cell suspension ((2-5). 10 6 cells) were added to tubes containing 25 #1 of the reaction mix so that the final concentration of components was 33 mM Tris-HC1 (pH 7.8)/20 mM 2-mercaptoethanol/0.67 mM EDTA/2.67 mM MgC12/0.8 #M [32p]NAD+ (spec. activity 1.105 cpm/pmol). In some experiments (indicated in relevant legends to figures) a concentration of 334 #M [32p]NAD+ (spec. act. 240 cpm/pmol) was used. 3-aminobenzamide, prepared in 100 mM Tris-HC1 and adjusted to a final pH of 7.8, was added to the reaction system to give a final concentration of 0.3-10 mM. Control samples received an equal amount of buffer. To measure poly(ADP-ribose) synthesis using purified human poly(ADP-ribose) polymerase from lymphoid tissue [11], 30 #1 of enzyme solution (25 /~g protein/ml) were added to a reaction mixture containing 10 #g calf thymus DNA, 10 #g histone H1, 1 mM dithiothreitol, 100 mM Tris-HCl (pH 8.0) and 0.8 #M (1 • 105 cpm/pmol) or 334 #M (240 cpm/pmol) [32p]NAD+, in a final volume of 100/~1. Reaction components were combined in tubes in an ice-water bath and reactions were started by shifting tubes to a 37°C water bath. Reactions were terminated by precipitation with an excess of cold 20% trichloroacetic acid, 2% Na4P20 7. Samples were sonicated, collected on Whatman G F / C filter discs and processed for scintillation counting as described previously [4]. Each experiment has

10 been performed at least three times and each point within each experiment was performed in duplicate. Results are presented as the means of these determinations. In cases where samples were to be analyzed by SDS-polyacrylamide gel electrophoresis, the reactions were stopped by addition of an equal volume of a sample solution composed of 4% ( w / v ) SDS/0.2 M dithiothreitol/20 mM potassium phosphate/40% ( v / v ) glycerol/0.006% ( w / v ) bromophenol blue (pH 7.0) [16] and the mixture immediately boiled for 2 min. SDS gel electrophoresis was carried out in 7.5% polyacrylamide gels, using a stacking gel of 5% polyacrylamide [17]. The buffer systems of the separating and stacking gels were 0.238 M Tris-HCl/2 mM EDTA (pH 8.8) and 62.5 mM Tris-HCl/2 mM EDTA (pH 6.8), respectively. The electrode buffer consisted of 50 mM Tris-HCl/0.39 M glycine/1.89 m M EDTA (pH 8.3). Both gel and electrode buffer systems contained SDS at a final concentration of

0.1% (w/v). Acid extraction of total histones was performed as described in [18]. After incubation of permeabilized cells with [32p]NAD + the reaction was stopped by addition of 2 N H2SO 4 at 4°C to give a final concentration of 0.4 N H zSO4 and the samples were briefly vortexed and left on ice for 1.5 h. Samples were then centrifuged at 10000 x g for 5 min at 4°C and the pellets re-extracted. Supernatants from both extractions were combined and precipitated with 3 vols. of 95% ethanol at - 20°C overnight. Precipitates were collected by centrifugation at 10000 x g for 10 min at 4°C and washed twice with 20% trichloroacetic acid and then twice with 95% ethanol. Acid-urea gel electrophoresis of histones was carried out as described in Ref. 19, using 12 cm long slab gels of 15% polyacrylamide/6.25 M urea/0.9 M acetic acid (pH 3.2). The electrode buffer consisted of 0.9 M acetic acid. Samples were dissolved at room temperature in 0.9 M acetic acid/15% ( w / v ) sucrose/0.05% (w/v) pyronin Y by briefly vortexing, and electrophoresis was carried out at 25 mA/gel. Results Poly(ADP-ribose) synthesis in permeabilized lymphocytes from normal human donors was mea-

sured at different times after M N N G treatment. In the experiments shown in Fig. 1, lymphocytes in suspension culture were treated with M N N G ; at subsequent time intervals they were removed from culture, permeabilized and the level of poly(ADP-ribose) synthesis measured in a 5 min reaction. Fig. 1 shows that synthesis increased within 10 min of M N N G treatment, reached a maximum within 1 h and continued at this level 2 and 3 h after treatment. We also conducted similar experiments with phytohemagglutinin-stimulated lymphocytes. In this experiment lymphocytes were removed from culture at 24 h intervals after stimulation with phytohemagglutinin. At each time point cells were permeabilized and the level of poly(ADP-ribose) synthesis measured in a 5 min reaction. These studies showed that there was a gradual increase in the level of poly(ADP-ribose) synthesis which reached a maximum after cells were incubated with phytohemagglutinin for 3 days. This finding is in agreement with previous studies [8,9] and is in contrast to the rapid stimulation in poly(ADP-ribose) synthesis observed in MNNG-treated cells. Fig. 2 shows the kinetics of the poly(ADPribose) synthesis that occurs in lymphocytes that were either treated with M N N G for 1 h, stimulated with phytohemagglutinin for 3 days or treated with both reagents. In these experiments, cells were removed from suspension culture 1 h after M N N G treatment or 3 days after phytohemagglutinin stimulation, then permeabilized and the permeabilized cells incubated for varying time periods in the poly(ADP-ribose) synthesis reaction system. Control cells showed a low level of synthesis with continuous accumulation of radioactivity in poly(ADP-ribose) during the l h incubation. Phytohemagglutinin-stimulated cells showed an increase in the amount of synthesis of poly(ADPribose) compared to control cells. The greatest, but most transient, increase in poly(ADP-ribose) synthesis occurred in MNNG-treated cells. After a 5 min incubation the amount of poly(ADP-ribose) in MNNG-treated cells was 10-fold greater than in control cells. However, the level of newly synthesized polymer in these cells decreased rapidly to a value approaching control by 60 min. Thus, MNNG-treated cells showed an increase in poly(ADP-ribose) followed by rapid polymer

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Fig. 1. Stimulation of ADP-ribosylation in permeabilized, MNNG-treated lymphocytes. Freshly isolated normal human lymphocytes were treated for the indicated times with 20 # g / m l MNNG/0.2% Me2SO (©) or with 0.2% Me2SO alone in the case of controls (O). After the indicated treatment times, cells were permeabilized and poly(ADP-ribose) synthesis was determined by incubating 5.106 cells in a reaction mixture containing 0.8 /~M [32p]NAD+ (spec. act. 1.105 cpm/pmol) for 5 min at 37°C. The time scale refers to the duration of incubation with MNNG before the cells were permeabilized. Fig. 2. Time course of ADP-ribosylation in resting lymphocytes and in lymphocytes treated with phytohemagglutinin and/or MNNG. Control cells (o) were freshly isolated human lymphocytes. MNNG-treated cells (O) were freshly isolated cells that were treated for 1 h with 20/~g/ml MNNG. Phytohemagglutinin-treated cells (A) were incubated at 37°C for 3 days with phytohemagglutinin as described in Methods. Phytohemagglutinin+MNNG-treated cells (zx) were incubated at 37°C for 3 days with phytohemagglutinin then treated with 20 /.Lg/ml MNNG for 1 h before permeabilization. Human lymphocytes pretreated under the different conditions were permeabilized and 2-106 cells incubated at 37°C for the indicated time periods in a system containing 334 #M [32p]NAD+ (spec. act. 240 cpm/pmol) to measure the course of poly(ADP-ribose) synthesis in the permeable cells. The time scale refers to the length of time that permeabilized cells were incubated with [ 32P]NAD +.

degradation. In contrast, the increased amount of poly(ADP-ribose) synthesized in phytohemagglutinin-stimulated cells was, in most cases, sustained for the entire 60 min incubation period. When lymphocytes were first stimulated with phytohemagglutinin for 3 days and then treated with MNNG the maximum amount of poly(ADPribose) synthesized was 2-fold greater than that in MNNG-treated, resting lymphocytes and 7-fold greater than that in non-damaged, phytohemagg-

lutinin-stimulated cells. Thus the effects of MNNG and phytohemagglutinin on the level of poly(ADP-ribose) synthesis appear to be synergistic. This suggests that the mechanisms and/or consequences of poly(ADP-ribose) polymerase stimulation by DNA damage and phytohemagglutinin treatment may differ. To enumerate the proteins ADP-ribosylated under these conditions, we incubated permeabilized cells with [32p]NAD÷ of high specific activity (1 • 105 cpm/pmol) and then used SDS gel electrophoresis and autoradiography to identify these proteins. Preliminary experiments revealed that boiling the samples for 2 min in 2% SDS sample solution provided the optimal conditions for rapidly stopping the reaction, inactivating the degradative enzymes and solubilizing the sample for SDS gel electrophoresis. Although there have been reports of the lability of the poly(ADP-ribose) protein linkage at high temperatures and alkaline pH [20,21], our studies showed that this brief exposure to boiling was optimal since when samples were solubilized at 25 or 37°C many of the ADP-ribosylated bands were degraded. Furthermore, our findings are in agreement with previous studies which showed that the release of poly(ADP-ribose) from protein is very much reduced by the presence of SDS [22]. Fig. 3 shows the autoradiograph of proteins ADP-ribosylated in control cells and in cells treated with MNNG for 1 h before being permeabilized and incubated with [32p]NAD+ for 5 rain. Lanes 1 and 3 (exposed for 40 h) show the extremes in the pattern of ADP-ribosylated bands which occurred when lymphocytes from different donors were analyzed in the resting state and lanes 2 and 4 (exposed for 15 h) show the extremes in the pattern of ADP-ribosylation after MNNG treatment. In control cells, ADP-ribosylated bands were observed at molecular weights of 116000, 72000, 42000, 32000 and in the region 21000-15000 (21000, 19000 and 15000). On treatment with MNNG there was an increase in ADP-ribosylation of the bands at molecular weights of 116000, 42 000, 32000 and 21000-15 000. In addition, in MNNG-treated cells there was always a heavily ADP-ribosylated protein of molecular weight 62 000. This 62 000 molecular weight ADP-ribosylated band can also be seen in lanes 1-4 of Fig. 4

12

Fig. 3. Autoradiographs of [32p]ADP-ribosylated proteins in control, MNNG a n d / o r phytohemagglutinin-treated normal human lymphocytes. Normal human lymphocytes were subjected to the treatments indicated under each lane then permeabilized, and 5.10 6 cells incubated with 0.8 ~ M [32 P]NAD + (spec. act. 1.105 cpm/pmol) at 37°C for either 5 min (lanes 1-5) or 60 min (lane 6). After addition of an equal volume of sample solution for SDS-polyacrylamide gel electrophoresis, as described in Methods, samples were solubilized, and then SDS gel electrophoresis and autoradiography carried out as described in Methods. MNNG-treated cells were incubated with 20 # g / m l for 1 h before permeabilization. Control cells were permeabilized at the same time as those which had been treated with MNNG. Phytohemagglutinin-treated cells were treated with the mitogen for 3 days before permeabilization. Each pair of lanes shows the ADP-ribosylated products of a different donor. Lanes 1, 3, 5 and 6 were exposed for autoradiography for 40 h and lanes 2 and 4 were exposed for 15 h. Molecular weights indicated on the left of each set of autoradiographs were determined by Coomassie brilliant blue staining of standards included in each slab gel. Molecular weight standards were as follows: myosin 200000; beta-galactosidase 116500; phosphorylase B 94000; bovine serum albumin 68000, ovalbumin 43000; carbonic anhydrase 30000; soybean trypsin inhibitor 21000; lysozyme 14300,

a n d l a n e 1 o f Fig. 6 w h e r e it is c l e a r l y d i s t i n c t from the ADP-ribosylated band of molecular w e i g h t 72 000. T h e s e e x a m p l e s s h o w t h a t t h e 6 2 0 0 0 molecular weight ADP-ribosylated band reproducibly occurs on M N N G treatment of lymphocytes from different donors. Analysis of ADPribosylation at successive intervals after MNNG treatment showed that ADP-ribosylation of the

Fig. 4. Comparison of ADP-ribosylated proteins synthesized by permeabilized cells and by purified human poly(ADP-ribose) polymerase. Human lymphocytes were treated for 1 h with 20 ~ g / m l MNNG, permeabilized and then 5.106 cells were incubated for 5 min in a reaction system containing the concentrations of [32p]NAD + listed below each of the first four lanes. The specific activity for 0.8 ,ttM [32p]NAD* was 1. l0 s cpm/pmol, for 10 ~M [32p]NAD + it was 8.103 cpm/pmol, for 100 #M [32P]NAD + it was 800 cpm/pmol and for 334/tM [-~2P]NAD + it was 240 cpm/pmol. In each case the total reaction system was subjected to SDS-polyacrylamide gel electrophoresis and autoradiographed as described in Methods. The reaction systems with purified poly(ADP-ribose) polymerase contained 0.8 t~M [32P]NAD+ (spec. act. 1.10 s cpm/pmol) or 334 #M [32p]NAD* (spec. act. 240 cpm/pmol) and were incubated for the time indicated below each of the lanes. The products of these reactions were run simultaneously and then analyzed by autoradiography, The length of exposure for autoradiography was 10 h for all lanes. Molecular weight standards included in the slab gel were identified as described in the legend to Fig. 3.

62 000 molecular weight band was coincident with the time of maximal poly(ADP-ribose) synthesis, t h a t is, it w a s p r i m a r i l y s e e n at 1 h a f t e r M N N G t r e a t m e n t . A D P - r i b o s y l a t i o n o f t h e 62 0 0 0 m o l e c u l a r w e i g h t b a n d w a s b a r e l y d e t e c t a b l e a t 10 m i n o r at 2 h after treatment, even though a substantial i n c r e a s e i n A D P - r i b o s y l a t i o n o f t h e 116 0 0 0 m o l e c ular weight protein and histones was apparent at these time points. Fig. 3, l a n e 5 ( e x p o s e d f o r 40 h ) s h o w s t h e r e s u l t s w h e n t h e s a m e t e c h n i q u e s w e r e u s e d to i d e n t i f y t h e p r o t e i n s t h a t a r e A D P - r i b o s y l a t e d in phytohemagglutinin-stimulated cells. T h e s e cells also showed an increase in ADP-ribosylation of

13 the 116000 molecular weight and 21000-15000 molecular weight proteins. In addition, phytohemagglutinin-stimulated cells showed ADP-ribosylation of a protein of molecular weight 96000 and, to a lesser extent, ADP-ribosylation of aband of molecular weight 37000. In contrast to the stimulation in d~,maged cells, the ADP-ribosylation of the bands at molecular weights 42 000 and 32 000 did not appear to increase in phytohemagglutinin-stimulated cells. When phytohemagglutinin-stimulated cells were incubated with [32p]NAD+ for 60 rain (Fig. 3, lane 6; exposed for 40 h), ADP-ribosylation of the 96000 molecular weight band could be seen even more clearly. ADP-ribosylation of this band and the 37000 molecular weight band was always observed on phytohemagglutinin stimulation. When phytohemagglutinin-stimulated cells were treated with MNNG both the 62000 and 96000 molecular weight ADP-ribosylated proteins were apparent in addition to other proteins associated with the response to MNNG treatment or phytohemagglutinin stimulation. All bands that were ADPribosylated in control cells were also found to be ADP-ribosylated after MNNG treatment or phytohemagglutinin stimulation. It is interesting to note that while MNNG or phytohemagglutinin treatment caused an increase in ADP-ribosylation of many bands such as the polymerase and histones, there was no apparent change in the degree of ADP-ribosylation of the 72 000 molecular weight band. In the course of these studies, we have analyzed the patterns of ADP-ribosylated proteins at serial time points during the 60 min period following cell permeabilization and have found that the same patterns of ADP-ribosylation occur after short or long incubations. Most of the figures are, therefore, presented to show the patterns after 5 rain, at which time the bands usually showed optimal labelling and resolution. While the intensities of some of these bands varied with longer incubations, their patterns remained constant. For example, Fig. 3, lanes 5 and 6 shows that the same pattern of ADP-ribosylation existed in the phytohemagglutinin-stimulated cells after 5 or 60 min incubation with [32p]NAD +. However, in this case the 60 rain incubation served to enhance the autoradiographic demonstration of ADP-ribosylation

of the 96000 molecular weight band. Thus, the differences in ADP-ribosylation of proteins we observed do not merely reflect differences in the time of incubation; rather they appear to represent true differences in the response of the poly(ADPribose) system to different conditions of cell growth and DNA damage. As shown in Fig. 4, the 116 000 molecular weight band in lymphocytes corresponds to poly(ADPribose) polymerase which was purified from normal human lymphoid tissue Ill]. Radioactive labelling of a band that corresponds to poly(ADPribose) polymerase has previously been shown in preparations of chromatin from HeLa nuclei [1] and in permeabilized human lymphocytes [2]. Using purified poly(ADP-ribose) polymerase from rat, calf, sheep and human tissues, it has been clearly shown that this band is due to auto-ADPribosylation of the enzyme [11,23-26]. The increase in ADP-ribosylation of poly(ADP-ribose) polymerase after MNNG treatment coincided with a reduced mobility and broadening of the radioactively labelled band on SDS gels. This is probably the result of an increase in chain length of the covalently bound poly(ADP-ribose) molecule and a consequent increase in the molecular weight of the enzyme [1]. The broadening of the band may represent heterogeneity in the length of poly(ADP-ribose) chains on the enzyme molecules. Alternatively, it is possible that the increase in molecular weight of the polymerase is the result of an increase in the number of poly(ADP-ribose) chains on the enzyme and/or an aggregation of enzyme molecules. As shown in Fig. 4, similar effects were produced when cells or purified poly(ADP-ribose) polymerase were incubated with either increasing concentrations of NAD + or for increasing lengths of time. Both of these conditions resulted in a progressive increase in molecular weight of the ADPribosylated polymerase. As shown in Fig. 4, lanes 8, 9 and 10, when the purified polymerase was incubated with 334 /tM NAD ÷, the increase in molecular weight of modified enzyme was so great that the labelled enzyme was no longer observed at its normal position on SDS gels. Rather, after incubation in the presence of 334/~M NAD +, the ADP-ribosylated enzyme was located on top of the separating gel (exclusion limit approx. 250 000 Da)

14 after a 30 s reaction (lane 8), and, after a 5 min incubation, it was located on top of the stacking gel (lane 10). Similar effects on the mobility of poly(ADP-ribose) polymerase on SDS gels have been shown in a purified enzyme system, in chromatin [1] and in permeabilized cells [2]. When the labelled bands in Fig. 3 were compared to standards on the Coomassie brilliant blue stained gels, it was apparent that the ADP-ribosylated band at 32 000 corresponded to the position of histone H1, and the bands at 21 000-15 000 to the region where core histones and H M G proteins migrated. In order to obtain more precise information on the identity of the 32 000 and 21 000-15 000 molecular weight ADP-ribosylated proteins we carried out acid extraction of control, M N N G treated or phytohemagglutinin-stimulated cells which had been permeabilized and then incubated with 0.8 ~ M [32p]NAD+ for 5 min. Fig. 5 shows the distribution of radioactivity in the acid-extracted histones as revealed by acid-urea gel electrophoresis and autoradiography. Under all treatment conditions there was ADP-ribosylation of histone H1 and to a lesser extent of H 2 A / H 2 B . N o difference could be observed in ADP-ribosylation of histone H1 in phytohemagglutinin-stimulated lymphocytes (Fig. 5, lane 2) compared with control, resting lymphocytes (Fig. 5, lane 1) which is in agreement with our results obtained by SDS gel electrophoretic analysis (Fig. 3, lane 5 compared to Fig. 3, lanes 1 and 3). Fig. 5, lane 3 shows that M N N G treatment caused a marked increase in A D P - r i b o s y l a t i o n of histones H1 and H 2 A / H 2 B . The M N N G - t r e a t e d cells also showed a low level of ADP-ribosylation of H3 and H4. The increase in ADP-ribosylation of core histones on M N N G treatment was much greater than that observed on phytohemagglutinin stimulation which is in agreement with the results obtained on SDS gel electrophoresis. In addition to the histones, which could be identified, some minor ADPribosylated proteins were also observed in the M N N G - t r e a t e d cells. These may represent H M G proteins or highly ADP-ribosylated histone species, including H 1 dimer [27]. In subsequent studies we evaluated the effect of 3 - a m i n o b e n z a m i d e , a p o t e n t i n h i b i t o r of poly(ADP-ribose) polymerase [28,29], on the patterns of ADP-ribosylated proteins that occurred in

Fig. 5. ADP-ribosylationof histones in resting lymphocytesand in lymphocytes treated with PHA or MNNG. Human lymphocytes were subjected to the treatments indicated under each lane, then permeabilized and 5.10 6 cells incubated with 0.8 p.M [32P]NAD÷ (spec. act. 1.105 cpm/pmol) for 5 min at 37°C. Acid extraction and acid-urea gel electrophoresis of histones was carried out as described in Methods, followed by autoradiography of the dried gels. The length of exposure for autoradiography was 24 h for all lanes. The positions of histones were determined by Coomassie brilliant blue staining of internal standards included in each slab gel.

response to the different treatments outlined above. Fig. 6 shows the distribution of ADP-ribosylated proteins derived from M N N G - t r e a t e d lymphocytes that were incubated in the presence of increasing concentrations of 3-aminobenzamide. With increasing concentrations of 3-aminobenzamide, a progressive inhibition of ADP-ribosylation of proteins was detectable on autoradiography. Similar results were obtained with resting lymphocytes and with lymphocytes treated with phytohemagglutinin. An interesting observation was that ADP-ribosylation of a protein of 72 000 molecular weight appeared to be resistant to the

15 a strong inhibitor of ADP-ribosylation and that, with the exception of the 72 000 molecular weight protein, the inhibition is a general one.

Discussion

Fig. 6. Effect of 3-aminobenzamide(3-AB)on poly(ADP-ribose) acceptors. Lymphocytes which had been treated with MNNG for 1 h and then permeabilizedwere incubated (5.106 cells/reaction) for 60 min with 0.8 tiM [32p]NAD+ (spec. act. 1.105 cpm/pmol) in the absence or presence of increasing concentrations of 3-AB. The concentration of 3-AB is indicated below the corresponding lane. The products of each reaction were analyzed by SDS gel electrophoresisand subsequent autoradiography. The length of exposure for autoradiography was 20 h for all lanes. The positions of molecular weight markers were determined as described in the legend to Fig. 3.

inhibitory action of 3-aminobenzamide. This protein was radioactively labelled in permeabilized cells under all the different treatments described above and always to the same extent. The data shown in Fig. 6 confirm that 3-aminobenzamide is

Previous studies to identify proteins that are ADP-ribosylated under different conditions have dealt with analyzing the different extents of ADPribosylation of poly(ADP-ribose) polymerase a n d / o r histones, for example [2,30,31]. A few studies using chromatin or isolated nuclei have identified other ADP-ribosylated proteins such as a 70000 molecular weight ADP-ribosylated protein [32], ADP-ribosylated protein A24 [33] and ADP-ribosylated H M G proteins [34-36]. In contrast to the studies outlined above which concentrated on determining whether specific proteins were ADP-ribosylated, in the present study we were concerned with determining the range of proteins that were ADP-ribosylated in resting, proliferating and D N A - d a m a g e d human lymphocytes. Table I provides a summary of the proteins that were ADP-ribosylated in lymphocytes from 30 different normal donors. For each ADP-ribosylated band the average molecular weight is given together with the range observed in different donors. Control, resting lymphocytes were characterized by ADP-ribosylation of poly(ADPribose) polymerase (116000), histone H1 (32000) ~nd core histones (21000-15000). In addition, there was also ADP-ribosylation of bands at molecular weights of 72000 and 42000. On M N N G treatment, a substantial increase in ADPribosylation of the polymerase, histone H1 and core histones occurred. This is in agreement with earlier studies in isolated nuclei and permeabilized cells [7,25]. In addition, there was a small increase in ADP-ribosylation of the 42 000 molecular weight protein and a marked increase in ADP-ribosylation of a protein of molecular weight 62 000. This 62 000 molecular weight protein was occasionally ADP-ribosylated in control or phytohemagglutinin-stimulated cells but only to a very small extent. It is possible that ADP-ribosylation of this protein in control cells reflects a small degree of D N A damage incurred during the preparation of these cells. ADP-ribosylation of the 72 000 molecu-

16 TABLE I ACCEPTORS OF POLY(ADP-RIBOSE) IN PERMEABILIZED HUMAN LYMPHOCYTES Human lymphocytes pre-treated under different conditions were permeabilized and 5.10 6 cells incubated at 37°C for 5 min in a system containing 0.8 p,M [32p]NAD + followed by SDS gel electrophoresis and autoradiography. Control cells were freshly isolated human lymphocytes. M N N G treated lymphocytes were treated for 1 h with 20 #g/ml M N N G before permeabilization. Phytohemagglutinin-treated cells were incubated at 37°C for 3 days with phytohemagglutinin (PHA) as described in Methods. PHA+ MNNG treated cells were incubated at 37°C for 3 days with PHA then treated with 20 ktg/ml MNNG for 1 h before permeabilization. The range in molecular weights is given in parentheses. +, ADP-ribosylated; + +, increase in extent of ADP-ribosylation; + / - , ADP-ribosylated in a small percentage of donors, but only weakly; - , not ADP-ribosylated. M r of ADPribosylated protein ( × 10- 3) 116(110-126) 96 (92-100) 72 (66- 75) 62 (57- 66) 52 (51- 52) 45 (44- 48) 42 (40- 43) 37 (36- 39) 32 (30- 34) 27 (25- 27) 21 ( 1 9 -

24)

19 (17- 22) 15 (14- 16)

Lymphocyte Treatment Control

MNNG

PHA

PHA + MNNG

+ +/+ +/+/+ +/+ +/-

++ +/+ ++ +/++ +/++ +/-

++ ++ + +/+/+/+ + + -

++ ++ + ++ +/+/++ + ++ -

+

+ +

+ +

+ +

+ +

++ ++

++ ++

++ ++

lar weight b a n d d i d not increase on M N N G treatment. O n p h y t o h e m a g g l u t i n i n s t i m u l a t i o n there was an increase in A D P - r i b o s y l a t i o n of the p o l y m e r a s e a n d core histones b u t not of histone H I . In a d d i tion, there was A D P - r i b o s y l a t i o n of a b a n d at 96 000 m o l e c u l a r weight a n d a s o m e w h a t smaller degree of A D P - r i b o s y l a t i o n of a b a n d at 37000 m o l e c u l a r weight. A l t h o u g h these latter two b a n d s were o c c a s i o n a l l y o b s e r v e d in resting cells they were always p r e s e n t in p h y t o h e m a g g l u t i n i n - s t i m u lated cells a n d always to a m u c h greater extent. A D P - r i b o s y l a t i o n of these b a n d s in a p o p u l a t i o n o f u n t r e a t e d l y m p h o c y t e s m a y b e a t t r i b u t a b l e to the presence of a small p e r c e n t a g e of cells which exist in the proliferative state [9]. M N N G treat-

m e n t of p h y t o h e m a g g l u t i n i n - s t i m u l a t e d cells showed A D P - r i b o s y l a t i o n of b o t h the 6 2 0 0 0 m o l e c u l a r weight b a n d characteristic of M N N G t r e a t e d cells a n d the 96 000 m o l e c u l a r weight b a n d c h a r a c t e r i s t i c of p h y t o h e m a g g l u t i n i n - s t i m u l a t e d cells. Since the r a d i o a c t i v e b a n d s d e t e c t e d in resting l y m p h o c y t e s were quite n a r r o w a n d restricted these studies i n d i c a t e that A D P - r i b o s y l a t i o n in resting [ y m p h o c y t e s results in only one or a few A D P ribose residues being a t t a c h e d to each protein. As n o t e d above, t r e a t m e n t of cells with M N N G or p h y t o h e m a g g l u t i n i n resulted in an increase in enz y m e activity a n d an increase in the A D P - r i b o s y l a tion of p o l y ( A D P - r i b o s e ) p o l y m e r a s e , which coinc i d e d with a s u b s t a n t i a l increase in its m o l e c u l a r weight as shown b y the r e d u c e d m o b i l i t y a n d b r o a d e n i n g of the labelled b a n d on a u t o r a d i o graphs. T h e increase in m o l e c u l a r weight suggests that long a n d / o r m u l t i p l e chains of p o l y ( A D P ribose) were a t t a c h e d to the protein. These findings are in a g r e e m e n t with those of Benjamin and G i l l [7] a n d L e h m a n n et al. [8], who r e p o r t e d that the increase in p o l y ( A D P - r i b o s e ) synthesis that o c c u r r e d in response to D N A d a m a g e was associa t e d with p o l y m e r s of greater chain length [7], a n d that that caused b y p h y t o h e m a g g l u t i n i n stimulation was a s s o c i a t e d with an increase in chain numb e r rather than chain length [8]. Both of these events w o u l d increase the m o l e c u l a r weight of the p o l y ( A D P - r i b o s e ) p o l y m e r a s e a n d account for the a p p e a r a n c e of the e n z y m e b a n d seen on a u t o r a d i o g r a p h s of M N N G and phytohemagglutinins t i m u l a t e d cells. The finding that an increase in A D P - r i b o s y l a tion of p o l y ( A D P - r i b o s e ) p o l y m e r a s e a n d core histones o c c u r r e d in cells treated with M N N G , as well as those treated with p h y t o h e m a g g l u t i n i n , suggests that some similarities exist in the activation of p o l y ( A D P - r i b o s e ) p o l y m e r a s e a n d its consequences in b o t h conditions. F o r example, the A D P - r i b o s y l a t i o n of histones m a y be involved in a l t e r a t i o n s in c h r o m a t i n structure necessary for access of enzymes of b o t h D N A replication a n d repair. In a d d i t i o n , the finding that some unique p r o t e i n s are A D P - r i b o s y l a t e d u n d e r these c o n d i tions suggests that there m a y also be some specific differences in the m e c h a n i s m a n d / o r results of e n z y m e activation.

17

In conclusion, this study has revealed that there are several common proteins which are ADPribosylated under different conditions of cell growth and DNA damage. In addition, unique acceptors for ADP-ribosylation have been identified in proliferating and in DNA-damaged human lymphocytes. In particular ADP-ribosylation of a protein of 62 000 molecular weight appears to be damage-dependent and ADP-ribosylation of a protein of 96 000 molecular weight appears to be proliferation-stimulated. Thus, unique as well as common proteins appear to become ADP-ribosylated in response to DNA damage or to mitogen stimulation. It is possible that some proteins which are ADP-ribosylated in vivo are not determined by this technique since permeable cells have been shown to leak some proteins [37]. However, the permeable cell system has been shown to reflect in vivo conditions more accurately than nuclear preparations, and the permeable cell technique allows the use of radioactive NAD + precursors for rapid and direct labelling studies. The current findings demonstrate the usefulness of the permeable cell system to show that different proteins are ADPribosylated under different conditions of cell growth. Techniques using affinity columns to extract ADP-ribosylated proteins from intact cells have recently been described [38,39] and the combination of these techniques should be useful in identifying the nature of the different acceptors in resting, proliferating and DNA-damaged cells. Such investigations should provide important insights into the mechanism by which the poly(ADP-ribose) system affects the processes of DNA replication and repair.

Acknowledgements These studies were supported by NIH Grants CA24986, GM26563 and American Cancer Society Grant CH-134. Some cell culture media used in these experiments were prepared in a Cancer Center Facility supported by NIH Grant No. 2 P30 CA16217. A travel grant to C.S.S. from The Wellcome Trust, England is acknowledged. N.A.B. is a Leukemia Society of America Scholar.

References 1 Butt, T.R. and Smulson, M. (1980) Biochemistry 19, 5235-5242 20gata, N., Kawaichi, M., Ueda, K. and Hayaishi, O. (1980) Biochem. Int. 1,229-236 3 Miller, E.G. (1975) Biochim. Biophys. Acta 395, 191-200 4 Berger, N.A., Sikorski, G.W., Petzoid, S.J. and Kurohara, K.K. (1979) J. Clin. Invest. 63, 1164-1171 5 Juarez-Salinas, H., Sims, J.L. and Jacobson, M.K. (1979) Nature 282, 740-741 6 Durkacz, B.W., Omidiji, O., Gray, D.A. and Shall, S. (1980) Nature 283, 593-596 7 Benjamin, R.C. and Gill, D.M. (1980) J. Biol. Chem. 255, 10493-10508 8 Lehmann, A.R., Kirk-Bell, S., Shall, S. and Whish, W.J.D. (1974) Exp. Cell Res. 83, 63-72 9 Berger, N.A., Adams, J.W., Sikorski, G.W., Petzold, S.J. and Shearer, W.T. (1978)J. Clin. Invest. 62, 111-118 10 Rochette-Egly, C., Ittel, M.E., Bilen, J. and Mandel, P. (t980) FEBS Lett. 120, 7-11 11 Carter, S.G. and Berger, N.A. (1982) Biochemistry 21, 5475-5481 12 Mendelsohn, J., Skinner, S.A. and Kornfeld, S. (1971) J. Clin. Invest. 50, 818-826 13 Berger, N.A., Petzold, S.J. and Berger, S.J. (1979) Biochim. Biophys. Acta 564, 90-104 14 Berger, N.A. (1978) Methods Cell Biol. 20, 325-339 15 Berger, N.A., Weber, G. and Kaichi, A.S. (1978) Biochim. Biophys. Acta 519, 87-104 16 Ito, S., Shizuta, Y. and Hayaishi, O. (1979) J. Biol. Chem. 254, 3647-3651 17 Studier, F.W. (1973) J. Mol. Biol. 79, 237-248 18 Whitby, A.J., Stone, P.R. and Whish, W.J.D. (1979) Biochem. Biophys. Res. Commun. 90, 1295-1304 19 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346 20 Riquelme, P.T., Burzio, L.O. and Koide, S.S. (1979) J. Biol. Chem. 254, 3018-3028 21 Dam, V.T., Faribault, G. and Poirier, G.G. (1981) Anal. Biochem. 114, 330-335 22 Burzio, L.O., Riquelme, P.T. and Koide, S.S. (1979) J. Biol. Chem. 254, 3029-3037 23 Kawaichi, M., Ueda, K. and Hayaishi, O. (1981) J. Biol. Chem. 256, 9483-9489 24 Yoshihara, K., Hashida, T., Yoshihara, H., Tanaka, Y. and Ohgushi, H. (1977) Biochem. Biophys. Res. Commun. 78, 1281-1288 25 Ogata, N., Ueda, K., Kawaichi, M. and Hayaishi, O. (1981) J. Biol. Chem. 256, 4135-4137 26 Booth, B.A., Petzold, S.J., Sims, J.L. and Berger, N.A. (1982) Fed. Proc. 41, Abstr. 4736 27 Stone, P.R., Lorimer, W.S. and Kidwell, W.R. (1977) Eur. J. Biochem. 81, 9-18 28 Purnell, M.R. and Whish, W.J.D. (1980) Biochem. J. 185, 775-777 29 Sims, J.S., Sikorski, G.W., Catino, D.M., Berger, S.J. and Berger, N.A. (1982) Biochemistry 21, 1813-1821

18 30 Jump, D.B., Butt, T.R. and Smulson, M. (1979) Biochemistry 18, 983-990 31 Nolan, N.L., Butt, T.R., Wong, M., Lambrianidou, A. and Smulson, M.E. (1980) Eur. J. Biochem. 113, 15-25 32 Jump, D.B., Butt, T.R. and Smulson, M. (1980) Biochemistry 19, 1031-1037 33 Okayama, H. and Hayaishi, O. (1978) Biochem. Biophys. Res. Commun. 84, 755-762 34 Levy-Wilson, B. (1981) Arch. Biochem. Biophys. 208, 528-534

35 Dam, V.T., Miclette, J.G., Bronssean, Y. and Poirier, G.G. (1981) Proc. Can. Fed. Biol. Soc. 24, 211 36 Reeves, R., Chang, D. and Chung, S.-C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6704-6708 37 Berger, N.A., Erickson, W.P. and Weber, G. (1976) Biochim. Biophys. Acta 447, 65-75 38 Minaga, T., Romaschin, A.D., Kirsten, E. and Kun, E. (1979) J. Biol. Chem. 254, 9663-9668 39 Romaschin, A.D., Kirsten, E., Jackowski, G. and Kun, E. (1981) J. Biol. Chem. 256, 7800-7805