[CANCER RESEARCH 46, 1110-1113, March 1986] Changes in de Novo DNA (Cytosine-5-)-methyltransferase Activity in Oncogenically Susceptible Rat Target Tissues Induced by A/-Methyl-A/-nitrosourea1 Annie Pfohl-Leszkowicz and Guy Dirheimer Institut de Biologie Moléculaireet Cellulaire du CNRS et Facultéde Pharmacie, UniversitéLouis Pasteur, 15 rue Descartes, 67084 Strasbourg, France ABSTRACT The activity of de novo DNA (cytosine-5-)-methyltransferase (DNA methylase) in various rat tissues after administration of a single dose of A/-methyl-A/-nitrosourea (MNU) has been analyzed. The total and specific activities of the DNA methylase of the brain, where tumor induction is important, are increased. In kidney, the DNA methylase activity first increases up to 16 h and decreases afterwards. Liver DNA methylase activity does not change. This organ is not susceptible to MNU induced cancers. Because organs in which the DNA methylase activity is high or increased after MNU are more prone to carcinogenesis by this compound, we argue that there is a relationship between the effects of MNU and DNA methylase activity. INTRODUCTION Nitroso compounds induce tumors in many animals (1). The principal site of tumor induction has been shown to vary with strains (2), with dose (3), with route of administration, and with age or developmental stage (4, 5). These compounds react with various nucleophilic sites in cellular macromolecules and partic ularly with DNA to give alkylated bases, principally 7-methyl- guanine, followed by 6-methylguanine, 3-methyladenine, 4-meth- ylthymine, and 2-methylcytosine and also phosphotriesters (6). Increasing evidence indicates that O6-alkylation in DNA is a promutagenic lesion (1, 2). Other modifications, such as the O4- alkylation of thymine, have also been implicated in mutagenesis and carcinogenesis (7) but phosphotriesters and depurinated sites arising from 7-methylguanine may also be highly significant. The alkyl in O6 of guanine can be removed from DNA by a O6- alkyltransferase, and in several rat tissues tumor induction has been shown to correlate with a relative repair deficiency in the target organ (8, 9). Changes in DNA methylation are an important event leading to carcinogenesis (for a recent review, see Ref. 10). In fact, a number of carcinogens can directly affect DNA methylation in vitro. DNAs modified by A/-acetoxy-W-2-acetylaminofluorene or by MNU2 are methylated in vitro by DNA methylase to a lesser extent than native DNA (11-15). On the other hand, previous modification of DNA by 4-acetoxyaminoquinoline 1-oxide or by A/-hydroxyaminofluorene increases the rate and overall extent of methylation of these modified DNAs in vitro, compared with native DNA (16, 17). Some of these agents have been shown to ' Received 3/4/85; revised 7/24/85, 10/18/85; accepted 11/14/85. ' This work was partly supported by Institut National de la Santéet de la Recherche Médicale(INSERM. Contrat 85/2009) and théLigue Nationale Française contre le Cancer, ComitéDépartementaldu Haut-Rhin. 2 The abbreviations used are: MNU, N-methyl-N-nitrosourea; DNA methylase, DNA-(cytosine-5-)-methyltransferase. decrease DNA methylation in vivo (18-21). In fact methylation patterns are tissue specific and are clonally inherited. In the somatic tissue, the pattern is conserved by the "maintenance" DNA methylase. The formation of a novel pattern involves changes in the methylation pattern by passive demethylation to generate unmethylated sites and/or by de novo methylation of unmethylated sites. It should be noted that no evidence exists for the presence of two different methylases in eukaryotic cells. It presently cannot be ruled out that a de novo methylase and a "maintenance" methylase both operate in the cell side by side; it is more likely that, under suitable conditions, the maintenance methylase shows de novo activity as well. Recently Adams et al. (22) had shown that de novo methylation can be stimulated following limited proteolysis of DNA methylase, but the maintenance DNA methylase is not stimulated. We have shown previously the existence of a correlation between the carcinogenic effect of N- hydroxy-A/-2-acetylaminofluorene and the activity of de novo DNA methylase in vivo (23). This paper reports the effect of administration of MNU to rats on the de novo activity of the DNA methylase in target and nontarget tissues. MATERIALS AND METHODS Male Sprague-Dawley rats weighing 120 g were obtained from Iffa Credo, Lyon (France) and were adapted to a 12-h light-dark cycle. MNU was a gift from Professor Kleihues (Freiburg, Federal Republic of Germany) and was dissolved in ethanol/trioctanoin (60/40, v/v). Trioc- tanoin was from Sigma. S-Adenosyl-L-[mef/)y/-3H]methionine (specific activity, 15-20 Ci/mmol) was from Amersham (Amersham, United King dom). Nonradiolabeled S-adenosyl-L-[mef/iy/-3H]methionine was from Boehringer (Mannheim, Federal Republic of Germany). Pancreatic RNase was from Worthington. Chicken erythrocyte DNA was a gift from Profes sor Daune and prepared as in Ref. 24. All other reagents were of analytical grade. A single pulse of MNU (50 mg/kg of body weight) was administered i.p. At 6, 16, 40, and 48 h, respectively, after MNU pulse, 6 animals/time point were killed. Livers, brains, kidneys, and spleens were excised and DNA methylase activity was extracted as follows from the nuclei of the organs which were not pooled. For preparation of the nuclei, the organs were excised and homogenized with 10 volumes of 0.32 M sucrose-4 HIM MgCI2 in a Potter homogenizer with 5 strokes at 1,400 rpm. After centrifugation at 800 x g for 15 min, the crude nuclear sediment was taken up in 2.8 M sucrose-1 HIM MgCfe to a final concentration of 2.1 M sucrose and centrifuged for 90 min at 19,000 rpm in a Beckman No. 19 rotor. The nuclei were taken up in 0.25 M sucrose-3 mw MgCI2 and sedimented again at 1,000 x g for 20 min. The DNA methylase from the nuclei was extracted with 0.8 M KCI in 50 mw Tris-HCI buffer, pH 7.6- 0.5% Triton X-100 for 30 min in a total volume of 9 ml. The KCI concentration was lowered to 0.3 M by addition of 10 HIM Tris-HCI, pH 7.6. After occasional stirring for 30 min, the insoluble chromatin was sedimented at 20,000 x g for 15 min. The supernatant was first dialyzed CANCER RESEARCH VOL. 46 MARCH 1986 1110 Research. on December 8, 2021. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from