EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 29, No 1, 35-43, March 1997 Protein methylation in cellular proliferation and differentiation: non-histone nuclear methyl acceptor protein(s) during 3'-methyl-4-dimethylaminoazobenzene- induced hepatocarcinogenesis Moon-Kee Paik,1,4 Yoo-Jeong Han,1 94.7% sequence homology with human chorionic Jung-Hee Hong,1 Jung-Sook Kim,1 somatomammotropin precursor A and B. Kwang-Sun Suh2 and Se-Jin Yoon1,3 Ke y w o r d s : protein methyltransferases, cell division, cell 1 Department of Biochemistry, School of Medicine, differentiation, nuclear proteins, rat, dimethylaminoazo- Wonkwang University, Ik-San, Korea benzene, hepatoma, carcinogens 2 Department of Pathology, School of Medicine, Chungnam University, Daejeon, Korea 3 Department of Internal Medicine, School of Medicine, Introduction Chungbook University, Chong-Ju, Korea Protein methylation is one of the posttranslational modi- 4 Corresponding author fication reactions which modulate the function of proteins. Among many protein-specific methylation reactions, Accepted 25 January 1997 enzymatic methylation of protein-arginine residues has been suggested to be involved closely in cellular growth and differentiation. These contentions are based on the following evidences. First, the enzyme S-adenosylme- thionine: protein-arginine N-methyltransferase (protein Abstract methylase I; PM-I; EC 2.1.1.23) which is responsible for the methylation increased significantly in fast growing An accelerating effect of methyl-deficient diet (MDD) tissues (Tidwell et al., 1968; Lee and Paik, 1972; Paik on hepatocarcinogenesis and methylation pattern and Kim, 1980; Wainfan et al., 1988). Secondly, many of nuclear protein(s) by S-adenosylmethionine: highly specialized chromosomal proteins are found to be protein arginine N-methyltransferase (protein arginine-methylated in vivo (Reporter and Corbin, 1971; methylase I, PM-I) have been studied with 3'-methyl- Karn et al., 1977). Thirdly, an increase of PM-I activity 4-dimethyl- aminoazobenzene(MeDAB)-treated rats. precedes the synthesis of DNA in synchronized cell The MDD+MeDAB-fed group produced typical cancer cultures (Paik and Kim, 1980; Choi, 1989). An evidence cells in the liver almost two weeks earlier than the also indicated that protein-arginine N-methylation might control synthetic diet (CSD)+MeDAB-fed group. play a role in carcinogenesis (Yoon, 1994; Duerre et al., Protein methylase I (PM-I) activity in the livers of 1994). MDD alone fed rats began to increase at around 2 Hepatocarcinogenesis has been found to be accele- weeks after MDD-feeding, reaching a peak at 4 rated in methyl-deficient experimental animals and this weeks and declining thereafter. When nuclei isolated effect of methyl-deficiency is attributed to insufficiency either from normal livers or from cholangiocarcinoma of methylation of newly formed DNA (Doerfler, 1983; cells were incubated with PM-I preparation from Hoffman, 1985) and/or chromosomal proteins (Duerre, normal liver, 16 and 23-kDa nuclear proteins were 1988). Obviously, chromosomal protein methylation the major methylated proteins, regardless of the includes arginine-methylation of non-histone chromosomal source of the nuclei. However, when the above proteins. In order to elucidate biochemical function and mentioned nuclei were incubated with PM-I prepa- significance of protein-arginine N-methylation, it appears rations either from MDD alone fed livers or MDD+ to be imperative to identify protein species methylated MeDAB-induced cholangiocarcinoma cells, the under specific biological situations. Najbauer and Aswad methylation of 23-kDa protein was not detected. The (1990) found over 90% of the endogenous proteins result suggests that there is a hitherto-unknown were arginine N-methylated in pheochromocytoma cells PM-I specific to 23 kDa nuclear protein which was (PC 12 cell line) which were initially hypomethylated lost during methyl deficient diet feeding and hepa- with a methylation inhibitor, followed by incubation with tocarcinogenesis. The N-terminal 20 amino a c i d s A d o [m e t h y l-3H]Met. However, they did not attempt to sequence of the 23-kDa protein was found to be identify the proteins involved. We recently identified a 1G l y - V a l - P r o - L e u -5X - A r g - L e u - P h e - A s p -1 0H i s - A l a - number of methyl acceptor proteins (68, 45, 38, 23 and Met- Leu-Gln-1 5A l a - H i s - A r g - A l a - H i s -2 0Glu, having 16 kDa) in human placental nuclear fraction methylated 36 Exp. Mol. Med. by PM-I (Paik et al., 1991; Choi et al., 1993). Furthermore, Experimental animals the extents of methylation of 16-kDa and 23 kDa proteins Sprague-Dawley rats, weighing approximately 150 g, were very different in opposing manner during hepatic were fed for 2 weeks with normal diet before the start of regeneration of rat. It was suggested that methylation of experiments. After acclimatization, the animals were the 16 kDa protein might be involved in cellular growth divided into MDD alone, CSD+0.07% MeDAB, and whereas that of the 23 kDa in cellular differentiation MDD+0.07% MeDAB groups, and the animals were fed (Lee et al., 1994). with the diets for 12weeks. MeDAB was dissolved in corn In the present study, we fed rats with methyl deficient oil. The composition of the diets are listed in Table 1. diet (MDD), and confirmed that the induction of cholan- giocarcinoma by 3'-methyl-4-dimethylaminoazobenzene Enzyme assay (MeDAB) was accelerated in these animals. During the induction, however, the 23-kDa protein methylation was Protein methylase I activity was assayed according to lost. The amino acid sequence analysis of N-terminal 20 the method of Paik and Kim (1980). Incubation mixture μ amino acids showed 94.7% sequence homology with containing 100 l of 0.2 M phosphate buffer (pH 7.6), 50 μ μ human chorionic somatomammotropin. l of histone II-AS (40 mg/ml) and 50 l of enzyme preparation was preincubated at 37˚C for 3 min, followed by the addition of 20 μl of Ado[me t h y l -14 C]Met (2.5 nmol, Materials and Methods 0.125 μCi). Incubation was carried out for 30 min at 37˚C. The enzyme preparation boiled for 5 min served Materials as a blank for the enzyme. The reaction was terminated 1 4 by adding 4 ml of 15% trichloroacetic acid to the mixture. S - A d e n o s y l -L- [m e t h y l- C]methionine (specific activity, 14 3 Unreacted Ado[me t h y l - C]Met, nucleic acid, phospholipid 49 mCi/mmol) and S-adenosyl-L- [m e t h y l- H ] m e t h i o n i n e and the product formed by contaminating protein (specific activity, 73 Ci/mmol) were purchased from methylase II (S-adenosyl-methionine: protein carboxyl Amersham International. Acrylamide, glycine, hydroxy- O-methyltransferase; EC 2.1.1.24) were removed, and apatite, and PVDF protein sequence membrane were the final precipitate was dissolved in 10 ml of liquid obtained from Bio-Rad Laboratories, and DEAE-Sephacel scintillation fluid for radioactivity counting. from Pharmacia. Histone type II-AS (a mixture of Protein concentration was estimated by the method various subtypes of calf thymus histone), histone H2A, of Bradford (1976), using bovine serum albumin as the histone H3, dithiothreitol, phenylmethylsulfonyl fluoride standard. (PMSF), trizma base and 2,5-diphenyloxazole were from Sigma Chemical Co. MDD and control synthetic Partial purification of protein methylase I diet (CSD) were prepared by Harlan Teklad, Wisconsin, and 3'-4-dimethylaminoazobenzene from Tokyo Whasei All the procedures were carried out at 4˚C. Ten g of Co., Japan. The rest of the reagents were obtained from liver from MDD+MeDAB fed rats or normal rats was various commercial sources and of the highest purity homogenized in 40 ml of buffer A containing 0.5 mM available. EDTA, 0.5 mM PMSF, 1 mM dithiothreitol, 5 mM NH4Cl, 10% glycerol, 5 mM phosphate buffer (pH 7.4) by polytron Brinkman homogenizer for 10 s five times. The homogenate was centrifuged at 39,000 g for 30 min and finely powdered (NH4)2SO4 was added to the superna- Table 1. The composition of diets (per kg) tant at 58% saturation. The mixture was left in ice-bath Diet Constituents for 30 min and then centrifuged at 39,000 g for 30 min. Control MDD The precipitate was dissolved in 7 ml of buffer A (pH 8.0), and (NH4)2SO4 was added at 55% saturation. The Amino acid mixture 180 g 180 g enzyme preparation was dialyzed against buffer A (pH Corn oil 50 g 50 g 8.0), the volume was made up to 50 ml, and the sample Glucose monohydrate 770 g 770 g was loaded onto DEAE-Sephacel chromatography Salt mixture 40 g 40 g column (1.4ለ4.5 cm; packing volume, 7 ml) which had Riboflavin 0.001g 0.001 g been equilibrated with buffer A. The column was a Vitamin mixture 5 ml 5 ml washed with 50 mM buffer A (pH 8.0) until no 280 nm MeDAB 0.7 g 0.7 g absorbing material was detected. The column was eluted with a linear gradient formed by 35 ml each of a Contains cod liver oil 20 g, choline chloride 1.5 g and pteroylglutamic acid 0.6 g, biotin buffer A (pH 8.0) containing 0 and 500 mM NaCl. 1.5 mg, thiamine-HCl 20 mg, pyridoxine-HCl 20 mg, menadione 50 mg(in 20 g of cod Fraction volume was 50 drops (2.5 ml). The enzyme liver oil), nicotinamide 50 mg, potassium parabenzoate 50 mg, calcium pantothenic acid activity was detected in fractions 7 to 14, and the 60 mg, inositol 10 mg and cyanocobalamin 40 μg.