Direct stimulation of poly(ADP ribose) polymerase in permeabilized cells by double-stranded DNA oligomers

ANALYTICAL

193,236-239

BIOCHEMISTRY

(19%)

Direct Stimulation of Poly(ADP Ribose) Polymerase in Permeabilized Ceils by Double-Stranded DNA Oligomers Karlheinz Institut

Received

Grube,

Jan-Heiner

fiir VirusforschunglATV,

September

Kiipper,

and Alexander

Deutsches Krebsforschungszentrum,

Poly(ADP ribosyl)ation, a post-translational modification of nuclear proteins catalyzed by poly(ADP ri-

236

requests

Im Neuenheimer

Feld 506, D-6900 Heidelberg

1, Germany

‘7, 1990

Poly(ADP ribosyl)ation, a post-translational modification of nuclear proteins catalyzed by poly(ADP ribose) polymerase, is an immediate response of most eukaryotic cells to DNA strand breaks and has been implicated in DNA repair and other cellular phenomena associated with DNA strand breakage. Poly(ADP ribose) polymerase activity levels have been frequently assayed by incubating permeabilized cells with radioactively labeled NAD+ as substrate. In such assays enzyme activation has routinely been achieved indirectly by prior exposure of living cells to carcinogens or by adding DNase I to permeabilized cells, thereby introducing strand breaks in chromosomal DNA. Here we show that, as an alternative method, the direct activation of purified poly(ADP ribose) polymerase by double-stranded oligonucleotides (N. A. Berger and S. I. Petzold, 1985, Biochemistry 24, 4352-4355) can be adopted for permeabilized cell systems. The inclusion of a palindromic decameric deoxynucleotide in the reaction buffer stimulated the enzyme activity in permeabilized Molt-3 human lymphoma cells up to 30-fold (at 5 fig/ml oligonucleotide concentration) in a concentration-dependent manner. The activating effect of oligonucleotides was also evident when ethanol-fixed HeLa cells were postincubated with NAD+ to allow poly(ADP ribose) synthesis to occur in situ, which was detected as specific anti-poly(ADP ribose) immunofluorescence. We conclude that double-stranded oligonucleotides can be conveniently used as chemically and stoichiometritally well-defined poly(ADP ribose) polymerase activators in permeabilized or ethanol-fixed mammalian 0 1991 Academic Press, Inc. cells.

’ To whom

Biirkle’

for reprints

should

be addressed.

bose) polymerase (EC 2.4.2.30)*, is an immediate response of most eukaryotic ceils to DNA strand breakage (see Refs. (1,2) for review). Upon binding to a DNA strand break, mediated by the second of two zinc fingers within the poly(ADP ribose) polymerase DNA-binding domain (3), the enzyme catalyzes the transfer of ADP ribose residues from NAD+ to acceptor proteins, elongation to form poly(ADP ribose) chains, and branching of such polymers. Poly(ADP ribosyl)ation has been implicated in DNA repair [e.g., (4-6)] and other cellular responses to DNA damage, such as cell cycle perturbations (7), mutagenesis, DNA amplification (8,9), malignant transformation [e.g., (lO,ll)], but also in DNA replication (12), integration of transfected foreign DNA into the cellular genome (13,14), signal transduction (15), differentiation [e.g., (16,17)], and aging [e.g., (18-20)]. For many experimental systems it is of interest to determine the level of cellular poly(ADP ribose) polymerase activity. This has frequently been accomplished in cells which were rendered nucleotide-permeable by either hypotonic cold shock (4,16,17,19) or digitonin (5), and incubated with radioactively labeled NAD+ whose incorporation into acid-insoluble material was taken as a measure for enzyme activity. To activate the enzyme above the endogenous level, strand breaks have been introduced in the chromosomal DNA either by pretreatment of cells with chemical or physical carcinogens or by incubating permeabilized cells with DNase I. There are, however, drawbacks when enzyme activity is assayed after introducing DNA breaks in chromosomal DNA, especially when different cell types or cells from different animal species are compared: There might exist a differential chromatin accessibility for DNA-damaging agents, and/or varying DNA-repair endonuclease ’ Enzyme Nomenclature. Recommendations clature Committee of the International Union demic Press, Orlando, FL.

(1984) of the Nomenof Biochemistry, Aca-

0003-2697/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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activities in different cells (21-23). Thus, physical or chemical carcinogens might not induce the same number of DNA breaks if applied to different types of living cells. Likewise, differences in chromatin accessibility might also be a problem when DNase I is employed to saturate poly(ADP ribose) polymerase with DNA strand breaks. To provide a direct stimulus for poly(ADP ribose) polymerase in permeabilized cells, we adopted the finding of Berger and Petzold (24) who showed with activity assays of purified poly(ADP ribose) polymerase that double-stranded deoxyoligonucleotides are even better enzyme activators than nicked calf thymus DNA, obviously by providing a large number of DNA ends. MATERIALS

AND

METHODS

Materials. Benzamide, bovifie serum albumin (grade V), dithiothreitol, P-NADf (grade V-C), and sodium pyrophosphate were purchased from Sigma (Munich, FRG). [adenine-2,8-3H]NAD+ (1.33 T Bq/ mmol [=36 Ci/mmol]) was from Amersham (Braunschweig, FRG). Trichloroacetic acid was from Roth (Karlsruhe, FRG). All other chemicals were from Merck (Darmstadt, FRG). Deoxyoligonucleotides were synthesized according to the phosphoramidite method on an Applied Biosystems 380A DNA synthesizer, HPLCpurified, dried by evaporation, and resuspended in 15 mM NaCl. The 3’- and 5’-termini of the oligonucleotides were unphosphorylated. Mouse monoclonal antibody HlO raised against poly(ADP ribose) was a kind gift of Dr. H. Kawamitsu (Tokyo, Japan) (25). Fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins were from Bio-Yeda (Rehovot, Israel). Cell culture. Molt-S, a human T-cell-lymphoma-derived cell line, was maintained as suspension culture in RPM1 1640 medium (Biochrom, Berlin, FRG) supplemented with 100 U penicillin, 100 pg/ml streptomycin, 2 mM glutamine, and 10% heat-inactivated fetal calf serum (Biochrom). HeLa cells were grown as monolayers in Dulbecco’s minimum essential medium (Biochrom), supplemented with 5% fetal calf serum and further additives as above. Cultures were incubated at 37°C in an atmosphere of 5% CO, in air. Cell permeabilization and poly(ALlP ribose) polymerase activity assay. Molt-3 cells were washed in 0.9% NaCl, pelleted, resuspended in ice-cold hypotonic permeabilization buffer (10 mM Tris-HCl, pH 7.8,l mM EDTA, 4 mM MgCl,, 30 mM P-mercaptoethanol) at 2 X lo6 cells/ ml, and left on ice for 15 min. Then cells were pelleted at 2OOg,O”C, for 10 min and resuspended in ice-cold permeabilization buffer at lo6 cells/53 ~1. Microscopic examination revealed that more than 90% of the cells were rendered permeable to trypan blue. To samples of lo6 cells on ice were added 13 ,crlof oligonucleotide dilutions

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in 15 mM NaCl as indicated, 1 ~1 [3H]NAD’ (18.5 kBq [=0.5 &i]), and 33 ~1 3~ reaction mix (100 mM TrisHCl, pH 7.8, 1 mM NAD+, 120 mM MgCl,), respectively, giving a total volume of 100 ~1per reaction. The reaction was carried out in triplicate for the times indicated at 30°C and stopped by adding 1 ml ice-cold 10% trichloroacetic acid, 2% sodium pyrophosphate. After overnight incubation at 4’C, precipitates were collected on Whatman GF/C filters, washed with 3 X 5 ml 10% trichloroacetic acid, 2% sodium pyrophosphate, and 2~ 5 ml 96% ethanol. Filters were air-dried and liquid scintillation counting was carried out with 10 ml Optifluor 0 (Packard, Frankfurt/Main, FRG), using a Packard TriCarb 1500 liquid scintillation counter. Indirect immunofluorescence. HeLa cells were grown on coverslips, fixed in ethanol for 10 min at -20°C and postincubated with 100 mM Tris-HCl, pH 8.0, 0.4 mM NAD+, 1 mM dithiothreitol, 10 mM MgCl, for 1 h at 30°C to allow in situ poly(ADP ribose) synthesis, as described by Ikai et al. (26). Oligonucleotides or benzamide were included as indicated. Coverslips were then incubated with mouse ascites containing antibody HlO (25) (diluted 1:lOO in PBS3/1% bovine serum albumin) for 45 min at 37°C in a humid chamber, followed by washing for 15 min in PBS. Fluorescein isothiocyanate-conjugated anti-mouse immunoglobulins (diluted 1:50 in PBS/l% bovine serum albumin) were applied for 30 min at 37°C. Coverslips were again washed for 15 min in PBS, and immunofluorescence was evaluated using a Leitz Dialux 22EB microscope. RESULTS

AND

DISCUSSION

As shown in Fig. 1, the addition of a palindromic (and hence double-stranded) decameric deoxyoligonucleotide (CGGAATTCCG) into the reaction buffer stimulated poly(ADP ribose) polymerase activity in permeabilized Molt-3 human lymphoma cells. Enzyme activation was concentration-dependent, reaching saturation levels at 5 pg oligonucleotide per lOO-~1 reaction volume. After background subtraction (zero time controls without oligonucleotide yielding 1275 + 238 cpm) maximal enzyme stimulation was about 30-fold. Very similar results were obtained with a human rhabdomyosarcoma cell line (RD176), with maximal enzyme activities being in the same range as obtained when saturating doses of y irradiation (1000 Gy [ = 100,000 rad]) were applied to living cells on ice immediately before the assay (data not shown). As is shown in Fig. 2 for Molt-3 cells, the oligonucleotide-stimulated reaction is almost linear during the first 10 min, in good agreement with the results on the purified enzyme (24). 3 Phosphate-buffered disodium hydrogen phate.

saline: phosphate,

128.3 mM sodium 10.4 mM potassium

chloride, 18.4 mM dihydrogen phos-

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4

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a

KijPPER,

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BiiRKLE

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FIG. 1. Poly(ADP ribose) polymerase activity in permeabilized Molt-3 cells as a function of double-stranded oligonucleotide concentration. Molt-3 cells were permeabilized and 100~~1 enzyme assays were run for 10 min as detailed under Materials and Methods. Oligonucleotides were included as indicated. Results are given as crude cpm (means of triplicate f one standard deviation).

FIG. bose) pg/ml

FIG. 3. Stimulation of poly(ADP ribose) polymerase activity in ethanol-fixed procc ?ssed for immunofluorescence as detailed under Materials and Methods. NAD I+/benzamide (10 mM); (D) with NAD+/oligonucleotide (100 pg/ml).

2.5

5.0

7.5

,

10.0 min

2. Time course of oligonucleotide-stimulated poIy(ADP synthesis in permeabilized Molt-3 cells. Assays containing oligonucleotide were run for the times indicated.

cells by double-stranded (A) postincubation without

oligonucleotides. NAD+; (B) with

ri6

HeLa cells were NAD+; (C) with

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The activating effect of the same double-stranded oligonucleotide was also evident when HeLa cells were fixed with ethanol and postincubated with NAD+ to allow poly(ADP ribose) synthesis to occur in situ, a method described by Ikai et al. (26). Figure 3 shows immunofluorescence data obtained with monoclonal antibody HZ0 (25) directed against poly(ADP ribose). In Fig. 3A (control) cells were postincubated in buffer without NAD+. Only a nonspecific cytoplasmic background and a very faint nucleolar staining is visible. In contrast, cells subjected to NAD+ postincubation (Fig. 3B) show an intensification of nuclear fluorescence as compared with controls (in this case mostly in the peripheral region of nuclei), indicative of enzyme activity. The presence of benzamide (10 mM), a competitive inhibitor of poly(ADP ribose) polymerase, during NAD+ postincubation prevented poly(ADP ribose) formation completely (Fig. 3C). However, when 100 pg/ml doublestranded oligonucleotides were included in the postincubation buffer (Fig. 3D), a marked further increase in nuclear fluorescence intensity (and hence enzyme activity) was observed as compared to Fig. 3B. Identical results were obtained with CV-1 monkey cells (data not shown). Our results show that including a double-stranded oligonucleotide in poly(ADP ribose) polymerase assays provides a chemically and stoichiometrically well-defined enzyme activator in permeabilized or ethanolfixed mammalian cells. This procedure obviates the need for cell manipulations before the assay (e.g., irradiation or chemical carcinogen treatment) and, more importantly, circumvents problems arising from differential DNA repair activities or differential chromatin accessibility for strand break-inducing carcinogens or DNase I, when different cell types or species are to be compared. Finally, the immunofluorescence assay described here could be helpful for a qualitative or semiquantitative in situ monitoring of large differences in enzyme activity, e.g., in cell cultures manipulated by molecular genetic means toward higher or lower than normal enzyme activity levels.

ACKNOWLEDGMENTS We thank Prof. H. zur Hausen for his continuous support and critical reading of the manuscript, Dr. H. Kawamitsu (Tokyo, Japan) for antibody HlO, Th. Rupp, W. Weinig, and M. Thome for oligonucleotide synthesis and purification, and Dr. Gilbert de Murcia (Strasbourg, France) for critical reading of the manuscript.

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