[CANCER RESEARCH 46, 3756-3761, August 1986] Regulation of Hypoxanthine DNA Glycosylase in Normal Human and Bloom's Syndrome Fibroblasts1 Philip Dehazya2 and Michael A. Sirover3 Fels Research institute and the Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 ABSTRACT cells appears to be dependent on the proliferative state of the cell. Previous studies described the relationship between the The regulation of the base excision repair enzyme hypoxanthine DNA increase in the activity of base excision repair enzyme activities glycosylase was examined in normal human skin fibroblasts (NHS) and to the induction of DNA replication and to cell division (9-11). fibroblasts from a patient with Bloom's syndrome. Using randomly In base excision repair, the initial enzymatic step is performed proliferating cells and those synchronized at specific intervals in the cell cycle, enzyme levels were shown to become elevated severalfold in a by a DNA glycosylase, which catalyzes the hydrolysis of the proliferation-associated manner. In NHS synchronized in ( .„byserum glycosyl bond releasing the modified base (2-4). Subsequent deprivation or in (., by isoleucine deprivation, maximal enzyme levels nuclease and DNA polymerase activities cleave and extend the were reached prior to maximal rates of DNA synthesis. In Bloom's resultant apurinic or apyrimidinic site in DNA and resynthesize syndrome cells synchronized in this manner, these two activities were the appropriate nucleotide sequences. Lastly, DNA ligase re coincident. Cells synchronized at the (.,-S border by hydroxyurea exhibit establishes the covalent linkage of the repaired DNA. an initial wave of DNA synthesis upon removal of the drug. The cells In normal human cells, uracil DNA glycosylase and the base then undergo another DNA synthetic cycle climaxing 18-21 h after excision repair pathway were enhanced prior to the induction release. Maximal hypoxanthine glycosylase activity of hydroxyurea- of DNA replication and DNA polymerase in serum-stimulated synchronized Bloom's cells was observed during the second round of cells (12, 13). We suggested that this enhancement of repair DNA synthesis. However, in NHS the peak of enzyme activity was enzyme activity might serve as a proofreading or prescreening observed as early as 9 h prior to the second round of DNA synthesis. To determine if hypoxanthine glycosylase could be induced in the absence mechanism to help prevent the fixation of mutagenic alterations of DNA synthesis, serum-synchronized NHS were released in the pres in the genome (14). Recently, we reported that hypermutable cells from Bloom's syndrome patients (15, 16) were character ence of hydroxyurea. The results showed that inhibition of DNA synthesis did not diminish glycosylase induction which demonstrated that DNA ized by an inability to correctly regulate the proliferation de replication was not required for glycosylase induction. pendent enhancement of base excision repair (17). In particular, uracil DNA glycosylase and the base excision repair pathway were enhanced coordinately with DNA replication. This would INTRODUCTION suggest that these individuals might be unable to efficiently repair carcinogen-modified DNA prior to replication. To ex Numerous studies have established the relationship between the initiation of neoplasia and the presence of chemical- or amine the specificity of this alteration in human gene expression radiation-induced DNA damage. DNA-carcinogen adducts we examined whether the regulation of another base excision such as 06-alkylguanine or <74-alkylthymidine are promutagenic repair enzyme, hypoxanthine DNA glycosylase, showed similar alterations in Bloom's syndrome cells. This enzyme excises and result in GC to AT transitions (1). Further, spontaneous deamination of cytidine to uracil and adenine to hypoxanthine hypoxanthine residues from DNA produced by the mutagenic represents mutational alterations in DNA. Enzymes which deamination of adenine (18). We now report that normal hu man cells enhanced this base excision repair enzyme prior to remove these lesions have been identified in mammalian cells DNA replication. However, in Bloom's syndrome cells, there (2-4). For the most part, their specific activities in diverse tissues correlate inversely with the susceptibility of such tissues was a temporal alteration such that the enzyme was induced to the action of carcinogenic agents. concomitantly with DNA replication. This altered temporal Eukaryotic cells specifically regulate the activities of diverse sequence was identical to that previously observed with the uracil DNA glycosylase. These results suggest that there is an DNA replicative enzymes during cell proliferation. In some intrinsic genetic defect in Bloom's syndrome cells which results instances, enzyme activities are induced to supply the increased quantities of metabolic intermediates for DNA synthesis. Thus, in the altered regulation of at least two individual base excision dihydrofolate reducíase, thymidine synthase, and thymidine repair enzymes during cell proliferation. kinase activities are increased as a function of growth (5-7). In contrast, in other instances, DNA replication and subsequent MATERIALS AND METHODS cell division would require the diminution of specific DNA Cell Culture. Normal human skin fibroblasts (CRL 1222; American metabolic enzymes; thus TTP dephosphorylase activity is di Type Culture Collection, Rockville, MD) and Bloom's syndrome fibro minished during cell proliferation (8). In each instance, the blasts (GM 2548; NIGMS Human Genetic Mutant Cell Repository, Camden, NJ) were obtained at passage 10 or lower. Cells were propa regulation of each gene would be an essential requirement for gated at 37°Cin 5% CO2 in air in 150-cm2 tissue culture flasks in DNA synthesis. Dulbecco's minimal essential medium (Gibco) containing glutamine, Similarly, the quantity of DNA repair enzymes in human antibiotics, and 10% fetal bovine serum unless specified. Cells were Received 10/25/85; revised 2/10/86, 4/1/86; accepted 4/16/86. subcultured after attaining confluence but were not maintained beyond The costs of publication of this article were defrayed in part by the payment passage IS. of page charges. This article must therefore be hereby marked advertisement in Methods of Cell Synchronization. Cells were synchronized in early accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This study was supported by grants to M. A. S. from the W. W. Smith GI by serum, middle GI by isoleucine deprivation, or at the Gi-S border Charitable Trust, the National Science Foundation (DCB-8416295), and the NIH by incubation with 0.5 IHMHU.4 Cells to be synchronized by serum (CA-29414) and by grants to the Fels Research Institute from the NIH (CA 12227) and from the American Cancer Society (SIG-6). 4 The abbreviations used are: HU, hydroxyurea; NHS, normal human skin 2 Present address: Merrell Dow Research Institute, Cincinnati, OH 45215. fibroblasts; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; 3To whom requests for reprints should be addressed. HPLC, high-pressure liquid chromatography. 3756 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1986 American Association for Cancer Research. HYPOXANTHINE DNA GLYCOSYLASE REGULATION deprivation were seeded into 100 itiM plastic dishes at a density of IO6 cells per dish in DMEM containing 0.3% serum. After 5 days in culture, cells were released into the cell cycle by the addition of 20% serum containing medium. Synchronization by isoleucine deprivation was accomplished by seeding cells as above in 10% dialyzed fetal bovine serum containing DMEM without isoleucine (Gibco). After 3 days in culture, cells were released by the readdition of complete DMEM. HU synchronization of cultures was accomplished by the addition of a stock solution of HU to a final concentration of 0.5 HIM.After 18 h cells were rinsed twice with prewarmed Hanks' balanced salt solution (Gibco) and incubated with DMEM. For some experiments cells were given 2 miviHU immediately after release. For asynchronous growth, confluent cells in 150-cm2 tissue culture flasks were trypsinized and seeded in 100 mM dishes at a density of 1- 40 50 60 70 80 2 x 10s cells/dish. Cells were grown for 6 days without medium change. FRACTION NUMBER Cell Extracts. Cells to be used for various assays were rinsed twice Fig. 1. HPLC of ethanol-soluble products from hypoxanthine glycosylase with cold Dulbecco's phosphate-buffered saline (Gibco) and harvested assay. A typical chromatogram is shown. [3H]Deoxyinosine-containing substrate with a rubber policeman. After sedimentation at 300 x g, cell pellets was incubated with 10 ng of cell extract protein for 120 min at 37'C as described were frozen at -20°C.Prior to assay, frozen cells were resuspended in in "Materials and Methods." After precipitation with ethanol and carrier DNA, 1 ml of 20 mM Tris-HCl (pH 8.0)-1 mM DTT-20% (v/v) glycerol and the supernatant was concentrated under nitrogen. Authentic hypoxanthine was sonicated at 70 W for 30 s at 0°C,with a Bransonic sonicator equipped added, and the sample was injected directly on a Waters Ci8-f Bondapak reversed- phase column eluted isocratically at I ml/min with 10 mM ammonium formate, with a needle probe. Extracts were centrifuged at 300 x g to remove pH 3.8. Fractions were collected at 30-s intervals. The arrow indicates the position unbroken cells. of the hypoxanthine marker. Insel, release of hypoxanthine-specific radioactivity Preparation of Hypoxanthine DNA Glycosylase Substrate. Hypoxan- as a function of time of incubation (A. lit,.] of substrate and 10 ng of cell protein) or quantity of cell protein in assay mixture (O, 10 n\ of substrate, 10 »igofprotein, thine DNA glycosylase was assayed using a substrate prepared by a incubation for 120 min at 37"C). modi fil-aïionof a method reported by Karran (19). [3H]dATP (Amer- sham or ICN; specific activity, 26 Ci/mmol, 5.0 mCi) was deaminated say. Therefore, 1-2 Mgof hypoxanthine was used as a marker for 5 h with freshly prepared nitrous acid (3.0 Msodium nitrate adjusted to pH 3.9 with nitric acid).