Elevated expression and altered pattern of activity of DNA methyltransferase in liver tumors of rats fed methyl-deficient diets.

N.G.Lopatina 0 2 B.F.Vanyushin 0 1 2 G.M.Cronin 0 2 L.A.Poirier 0 2 0 Abbreviations: E-64 , trans-epoxy succinyl- 1 Present address: A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University , Moscow 119899 , Russia 2 Division of Nutritional Toxicology, National Center for Toxicological Research , NCTR Drive, Jefferson, AR 72079 , USA 3 C , 5-methyldeoxycytidine; MDD, methyl-deficient diet; MSD, methyl- sufficient diet; MTase, DNA(cytosine-5)-methyltransferase; PMSF, phenyl- methylsulfonyl fluoride; SAM, S-adenosylmethionine; TLCK, N-tosyl- 2To whom correspondence should be addressed Email: DNA methyltransferase (MTase) activity in nuclear extracts from neoplastic and preneoplastic livers of rats fed a methyl-deficient diet (MDD) is elevated compared with that seen in the livers of control rats. Nuclear proteins were prepared in the presence of protease inhibitors including trans-epoxy succinyl-L-leucylamido-(4-guanido)butane and were fractionated by isoelectric focusing. In normal, control liver, two distinct MTase fractions were observed. In MDDinduced malignant liver, a third fraction, in addition to the previous two, was also seen. Both the DNA substrate and the cytosine site specificities of the third MTase fraction differ from those of the other two fractions. The distinct MTase activity in liver tumor has significantly more de novo MTase activity than do the MTase fractions of normal, control liver. Thus, normal and neoplastic rat livers differ in DNA MTase fractionation patterns and site specificities. The altered DNA MTase activity observed in rat liver tumors caused by MDDs may be one of the critical factors contributing to cancer formation through abnormal DNA methylation. - Methyl-deficient diets (MDDs) cause cancer (13) and DNA under-methylation in rodent liver (410). Since DNA methylation plays a significant role in the regulation of transcription, DNA replication and DNA repair (11), it is postulated that abnormal DNA methylation may play an essential role in carcinogenesis (1215). Currently, little is known about the nature and specificity of DNA (cytosine-5)-methyltransferase (EC 2.1.1.37) (MTase) in malignant tissues (1417) and about the extent to which the enzyme may be responsible for the abnormal DNA methylation found in cancer cells. Furthermore, the effects of stringent dietary methyl deficiency on hepatic DNA MTase require further investigation. Previous studies have shown that the chronic administration of methyl-deficient diets to rats leads to increased MTase activity both in preneoplastic liver and in the resultant tumors (7,9,10). The present study provides evidence showing that tumors produced by MDD display elevated expression and activity of DNA Eight microgram DNA aliquots were hydrolysed with 10 U of HpaII restriction endonuclease (Gibco BRL, Gaithersburg, MD). DNA hydrolysates were fractionated by electrophoresis in 0.9% agarose (16) and transferred to Hybond N1 (Amersham, Amersham, UK) by alkaline blotting as recommended by the manufacturer. The membranes were prehybridized for 1 h as described (16), and then incubated with a random primed [a -32P]dCTP-labeled probe at 60C for 18 h. The 4423 bp C-terminal murine cytosine DNA MTase gene fragment cut from the pBlueScript construct containing a full copy of this gene (a gift from Dr T.H.Bestor) by BamHI restriction endonuclease (Gibco BRL) and isolated by electrophoresis in a 0.9% polyacrylamide gel was used as a probe. The membranes were washed (16) and exposed to Kodak X-Omat AR film for 14 days at 70C. The progressive loss of high molecular weight material with the accumulation of smaller molecular weight bands in DNA samples hydrolyzed by HpaII enzyme is indicative of DNA demethylation at Cm5CGG sites. RNA isolation and northern blots mRNA was isolated from 0.5 g frozen liver samples using a FastTrack mRNA isolation kit (Invitrogen, San Diego, CA). RNA was measured by its absorbance at 260 nm. Aliquots of 1 m g liver mRNA from control and experimental rats were applied to Hybond as described by the manufacturer. Hybridization with a random primed [a -32P]dCTP-labeled 4423 bp C-terminal murine DNA MTase gene fragment was performed in 0.25 M Na2HPO4 (pH 7.2), 7% SDS at 64C for 18 h, and the membranes were washed twice for 15 min each in 0.02 M Na2HPO4, 5% SDS and twice for 15 min each in 0.02 M Na2HPO4 1% SDS at 64C. The blots were exposed at 70C to Kodak X-Omat film for 12 days. Isolation and determination of DNA MTase activity Rat liver nuclei and nuclear extracts were isolated as described (17,18). The protein concentrations both in nuclear extracts and in samples obtained by isoelectric focusing were determined as previously described (19). The DNA MTase reaction conditions were such that both the methyl donor and the DNA template were in excess, so that the extent of methyl group incorporation was dependent on the level of DNA MTase activity. Extracts were incubated for 4 h at 37C in a 130 m l reaction mixture containing 10 mM Tris-HCl buffer (pH 7.4), 5 mM EDTA, 10 m g of calf thymus DNA (Sigma, St Louis, MO), and 1 m Ci of S-adenosyl-L-[methyl-3H]methionine (SAM, sp. act. 7.9 Ci/mmol; NEN, Boston, MA). The samples were applied to Whatman DE filters, and DNA radioactivity was quantified by liquid scintillation counting; enzyme activity was expressed as cpm/mg protein. DNA MTase fractionation and the site specificity assay To fractionate DNA MTases, the nuclear proteins were precipitated by the addition of solid ammonium sulfate to the nuclear extracts obtained above (600 mg/ml, or 80% saturation) (17). The precipitates were collected by centrifugation at 105 000 g for 1 h and dialyzed as previously described (20). The resulting protein preparations were processed on a Rotofor Cell column (Bio-Rad, Hercules, CA) as described by the manufacturer. Twenty 4 ml fractions of each protein preparation applied were obtained as the result of the isoelectric focusing for 6 h in the presence of ampholytes at pH 310. All procedures on DNA MTase isolation (tissue homogenation, nuclei isolation, protein precipitation and dialysis, and electrophoresis) were carried out at 4C and in the presence of our standard set of protease inhibitors, i.e. 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 1 m g/ml leupeptin (17,20). In a second set of studies, MTase was prepared from tumors derived from rats fed the semi-synthetic MDD (9); in this case the DNA MTase isolation procedure was modified to include, in addition to the PMSF and leupeptin used as above, the protease inhibitors trans-epoxy succinyl-L-leucylamido(4-guanido)butane (E-64), N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), each at 10 m g/ml as previously described (18). The studies with the additional protease inhibitors were prompted by the findings of Xu et al. (18), showing that they effectively inhibited proteolysis during the isolation and partial purifica (...truncated)