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Induction of the DNA Repair Enzyme Uracil-DNA Glycosylase in Stimulated Human Lymphocytes―

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Induction of the DNA Repair Enzyme Uracil-DNA Glycosylase in Stimulated Human Lymphocytes― [CANCER RESEARCH 39. 2090-2095, June 1979] 0008-5472/79/0039—0000$02.0O Induction of the DNA Repair Enzyme Uracil-DNA Glycosylase in Stimulated Human Lymphocytes― Michael A. Sirover Fels Research Institute and the Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 ABSTRACT induced by PHA, but nucleotide kinase activities remain con stant despite cell proliferation. The capacity of human cells to modulate the synthesis of The uracil-ONA glycosylase was induced 10-fold during lym DNA repair enzymes has been investigated by measuring the phocyte stimulation by PHA. Glycosylase stimulation was co induction of the umacil-ONAglycosylase during lymphocyte ordinate with the induction of DNA synthesis and DNA polym stimulation. Treatment of peripheral lymphocytes with phyto erase activity. Furthermore, treatment with either actinomycin hemagglutinin increased glycosylase activity 10-fold. Glyco 0 or cycloheximide at maximal stimulation diminished enzyme sylase stimulation was coordinate with the activation of DNA activity after an appreciable interval. These results suggest that synthesis and DNA polymerase activity. Two chnomatographi stable base modifications which persist in quiescent cells may cally distinct species of the glycosylase have been resolved; be miscopied during cell activation. only one species is induced during phytohemagglutinin stimu lation. The effect of actinomycin 0 and cycloheximide on gly MATERIALS AND METHODS cosylase induction was determined. Treatment with either in hibiton at 96 hr after phytohemagglutinin addition (maximal Lymphocyte Culture. Lymphocytes were isolated by sedi induction) decreased glycosylase activity after an appreciable mentation through Ficoll-Hypaque gradients (6). Final pmepa lag period. This suggested that induction of the umacil-ONA nations contained greater than 90% lymphocytes (all concen glycosylase requires transcription and translation although the trations referred to are final concentrations in the reaction enzyme may be quite stable once induced. mixture or in the buffer utilized); 99% were viable as defined by trypan blue dye exclusion. Cells were diluted to a final INTRODUCTION density of 0.75 to 1.0 x 106 cells/mI in Eagle's minimum essential medium (spinner modification) which contained 20% The excision-repair of DNA modified by chemical carcino fetal calf serum, 20 mr@i4-(2-hydroxyethyl)-1-piperazineeth gens may occur through 2 pathways: nucleotide excision-re anesulfonic acid (pH 7.4), 2 mr@iL-glutamine,100 @tgofstrep pair in which the initial event is an endonucleolytic cleavage of tomycin, and 100 units of penicillin per ml. Cells were cultured DNA (12); or base excision-repair in which the first step is the in 1-ml aliquots with 50 @lofPHA penml of cells. DNA synthesis cleavage of the base-sugar glycosyl bond producing an apu was determined by measuring the incorporation of [3H]thymi rinic or apyrimidinic site (1 7). DNA glycosylases which excise dine (6.7 Ci/mmol; 2.5 MCi/culture) for 2 hr before collection. N3-methyladenine, hypoxanthine, and uracil from DNA have These cell pellets were used to measure concurrently total been identified (9, 13, 18, 24, 27). The uracil-DNA glycosylase DNA polymenase activity as described previously (20). excises uracil residues in DNA which may be formed by the Formation of GlycosylaseSubstrates. Substrateswere pre mutagenic deamination of cytosine (3) or by the incorporation pared by copying either activated calf thymus DNA or poly of umacilduring DNA replication (5, 34). deoxyadenylate .oligodeoxythymidylate with homogeneous Although some of the enzymes comprising the excision Escherichia coli DNA polymerase I, using [3H]dUTP (15,000 repair pathways have been characterized, the mechanisms by dpm/pmol) and other appropriate deoxyribonucleoside tn which human cells regulate their synthesis and the relationship phosphates as precursors. After 30 mm incubation at 37°,the of such regulation to carcinogenesis remain unknown (8, 22, reaction mixture was extracted with phenol, dialyzed 4 times 23, 31). Synthesis of repair enzymes may be constitutive (10, with 3.5 liters of 1 M NaCI plus 50 m@iTnis-HCI(pH 8.0), and 33); alternatively, cells may modulate the activity of one or then dialyzed 4 times with 3.5 liters of 50 mMTmis-HCI(pH8.0). several repair enzymes (4, 25). Furthermore, the temporal Assay for Uracil-DNA Glycosylase in Cell Extracts. Uracil sequence between the induction of enzymes involved in DNA DNA glycosylase was measured by determining the release of repair and those involved in DNA replication during the cell ethanol-soluble or acid-soluble radioactivity from [3H}uracil-Ia cycle has not been established. To consider these questions, beled DNA templates. Cell pellets were freeze-thawed 3 times we have asked initially whether the uracil-DNA glycosylase is in a solution (total volume, 100 @l)whichcontained 20 mM induced in human cells which are quiescent but which can be K2PO4(pH 7.5) pIus 1 mM dipotassium EDTA, 2.5 mM OTT, stimulated to proliferate. Lymphocytes activated by PHA2were and 20% glycerol. chosen as a model system, inasmuch as PHA stimulation Uracil-DNA glycosylase activity was measured in a reaction results in a sequential activation of selective enzymatic activi mixture (total volume, 200 @sl)whichalso contained 100 mM @ ties (19, 20). For example, DNA polymerases and /@are Tnis-HCI(pH 8.0), 7.5 mM OTT, 15 mM dipotassium EDTA (pH @ 7.0), 1 of bovine serum albumin, and 3 @gof[3Hjumacil labeled DNA (19,000 dpm/pmol). Incubations were performed I This study was supported by NIH Grant ES-Ol 735 and CA-i 2227. 2 The abbreviations used are: PHA, phytohemagglutinin; OTT, dithiothreitol. at 37°for 60 mm. Reactions were terminated by adding se Received December 21, 1976, accepte'@March 13, 1979. quentially 300 @lofethanol, 60 @iIof2 M NaCI, and 100 @gof 2090 CANCERRESEARCHVOL. 39 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1979 American Association for Cancer Research. Induction of DNA Repair Enzymes denatured calf thymus DNA (1 mg/mI in 20 mt@iKCI,pH 6.8). + After a minimum of 60 mm at —20°,themixtures were centri U' In r') @ fuged at 3000 rpm for 10 mm. The ethanol supemnatant(200 IC I.#U ‘@ @.tl)wasremoved and counted. In some experiments, reactions E -4 were terminated by adding 10% tnichloroacetic acid (300 gil) U U C 3 and 100 i@gof denatured calf thymus DNA. These mixtures C) were centrifuged after 20 mm at 5°.[3H]Uracil released in the C @ supemnatantwas identified as a reaction product by thin-layer 50 E chromatography. I- Partial Purification of the Uracil-DNA Glycosylase. Lym phocytes were cultured with PHA as described. Cells were In cultured for 4 days before collection. Cell pellets were frozen and thawed 3 times and then suspended in 7.6 ml of 20 mt,i S Tris-HCI (pH 8.0)-i 0 mM MgCl2-1 m@OTT (Buffer 1). Triton X 3 l( 100 (0.4 ml) was added. The solution was kept at 0°for 30 U3 mm before homogenizing with 20 strokes in a Oounce homog 0 E enizem.The cell extract was adjusted to 20% glycerol (Buffer 2) and then centrifuged at 9000 rpm for 20 mm. The super a) 0 natant contained greater than 90% of the glycosylase activity. a) Nucleic acids were extracted by adding to the supemnatant 2.5 S ml of DEAE-cellulose previously equilibrated with 20 mr@iTris U0 HCI (pH 8.0)-i mM DTT-20% glycerol (Buffer 3). The suspen sion was stirred gently for 30 mm and then centrifuged at 3000 In rpm for 10 mm. The supemnatantwas collected and absorbed to a Sephadex G-100 column (2.5 x 42 cm) equilibrated in Buffer 3 plus 100 mM NaCI. Uracil-DNA glycosylase was as Time after PHA Stimulation (Days) sayed using 20 @slofcolumn effluent. Fractions containing Chart 1. Induction of the uracil-DNA glycosylase during lymphocyte stimula tion by PHA. Lymphocytes were isolated and cultured as previously described. glycosylase activity were pooled, dialyzed overnight against 4 DNA synthesis was determined by measuring the incorporation (Inc.) of liters of Buffer 3, and absorbed to a phosphocellulose column [3H]thymidine (6.7 Ci/mmol; 2.5 MCi/culture) for 2 hr before collection. Total DNA polymerase activity and urscil-DNA glycosylase activity were measured as (1.5 x 17 cm) equilibrated with Buffer 3. The column was previously described. Each point represents the average of quintuplicate cultures. washed with 20 ml of Buffer 3; a linear gradient of 0 to 0.5 M L@,PHA-stimulatedcells;A,unstimulatedcells. KCI plus Buffer 3 (total volume, 60 ml) was applied. Enzyme activity was monitored using 50 @lofcolumn effluent. taming [3HJdAMP,[3H]dCMP, or [3H]dGMP, increasing concen RESULTS trations of the glycosylase preparation failed to release detect able radioactivity (Chart 2). Using reaction conditions in which Lymphocyte stimulation by PHA was monitored by measuring the glycosylase released 61 .5% of umacilfrom DNA, less than [3Hjthymidine incorporation and by measuring DNA polymenase 1.3% of the other bases were rendered soluble. Using a tem activity in vitro (Chart 1A). DNA synthesis and DNA polym plate containing [3Hjuracil and [32Plphosphates, the glycosy erase activity were coordinately induced by PHA; maximal lass released 51% of uracil from DNA; less than 1% of 32Pwas activities were observed 3 to 4 days after PHA addition. Umacil solublized. Similar results were observed using cell-free ex DNA glycosylase activity was measured in parallel cultures tracts. Furthermore, the glycosylase preparation did not me (Chart 1B). In this experiment, the uracil-ONA glycosylase was lease N@-methyladeninefrom DNA (results not shown). induced 10-fold. Furthermore, glycosylase stimulation was The increase in glycosylase activity during PHA stimulation temporally coordinate to that of DNA synthesis and of DNA was also observed during enzyme purification. Low-speed su polymemaseactivity. No glycosylase induction was observed in pemnatantswere applied to a DEAE-cellulose column; umacil parallel cultures which were incubated without PHA.
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