Increased Activity of Polynucleotide Ligase from Rat Hepatoma Induced by 7V-2-Fiuorenylacetamide1

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[CANCER RESEARCH 32, 886-888, May 1972] Increased Activity of Polynucleotide Ligase from Rat Hepatoma Induced by 7V-2-FIuorenylacetamide1

Kinji Tsukada, Shigeru Hokari, Nobuko Hayasaki, and Nobuyuki Ito Drug Research Institute, Faculty of Pharmaceutical Science, Toyama University, Toyama Â¡K.T., S. H., and N. H.], and First Department of Pathology, NaraMedical University, Kashihara, Nara, [N. I./, Japan

SUMMARY Chemicals, Rochester, N. Y.) followed by 3 months of stock diet (Oriental East Co., Osaka, Japan). Four rats were Polynucleotide ligase in both the soluble fraction and the sacrificed 6 months after the start of the experiment. The liver nuclear extract, which catalyzes the covalent joining of 2 of each rat was examined macroscopically and divided into segments of an interrupted strand in a DNA duplex, was nonhepatoma and hepatoma areas. Tissue samples were taken studied with normal rat liver and hepatoma induced by from areas adjacent to tissues used for biochemical studies. yV-2-fluorenylacetamide. The activity of this enzyme in For histological studies, tissues were fixed in 10% neutral hepatoma was about 5 times greater in the soluble fraction and buffered formaldehyde solution and stained with hematoxylin about 3 times greater in the nuclear fraction than in normal rat and eosin. Histologically, hepatoma areas showed findings of liver. typical hepatocellular carcinoma. In this experiment, typical hepatocellular carcinoma was used as hepatoma. INTRODUCTION Rat liver nuclei and nuclear extract were prepared by the methods described previously (6). The nuclei extract and It has been postulated that in DNA replication relatively soluble fraction (105,000 X g supernatant) were dialyzed small polynucleotides are formed as intermediates (12). In the against 2 liters of 0.01 M Tris-HCl buffer (pH 7.7) for 2 hr at course of this process, polynucleotide ligase catalyzes the 2 . Subcellular fractionation was carried out in the usual covalent joining of interrupted deoxyribonucleotide strands of manner by centrifugation in Medium A (5). Protein was a bihelix (14). Polynucleotide ligase was isolated from bacteria determined by the method of Lowry et al. (10), and DNA was by several groups of investigators (2â€”4,13, 17, 18). Recently, determined by the procedure of Burton (1). the presence of this enzyme was demonstrated in mammalian The substrate of polynucleotide ligase, nicked tissues (9); however, its function remains to be elucidated. Our 5'-phosphoryl-DNA-32P, was prepared as previously described laboratory reported that the activity of the ligase from rat liver (6), with polynucleotide kinase from rat liver nuclear extract. after partial hepatectomy is several times greater than that The standard assay measures the conversion of from normal rat liver (15). In this communication, we wish to 5'-phosphomonoesters-32P in nicked DNA into a form which describe the increased activity of this enzyme in rat hepatoma remains acid insoluble after incubation with phosphatase. The induced by 2-FAA.2 incubation mixture (0.2 ml) contained 5 jug of nicked 5'-phosphoryl-DNA-32P (about 15,000 cpm), 10 Â¿/molesof Tris-HCl buffer (pH 8.0), 2 Â¿zmolesof MgCl2, 2 /amÃ³les of MATERIALS AND METHODS mercaptoethanol, 0.04 jumÃ³leof ATP or NAD* and nuclear extract or soluble fractions. After incubation at 37" for 20 ATP^y-32P (sodium salt in 50% aqueous ethanol, 1960 mCi/mmole) was obtained from the Radiochemical Center min, 0.1 ml of 1 M glycine buffer (pH 9.5) was added and (Amersham, England); thymidine-2-14C was obtained from heated at 100Â°for 10 min. The mixture was cooled in ice, and 5 /ug of alkaline phosphatase (1.5 units) was added. Each New England Nuclear (Boston, Mass.); calf-thymus DNA, reaction mixture was incubated for 20 min at 65Â°. The pancreatic DNase, alkaline phosphatase from Escherichia coli, and venom 5'-nucleotidase were obtained from Sigma mixture was cooled in ice, and 0.1 ml of 0.1 M sodium Chemical Company, (St. Louis, Mo.); and other enzymes were pyrophosphate was added, followed by 5% trichloroacetic purchased from Worthington Biochemical Corp. (Freehold, acid. The precipitate was collected on a glass filter. After N.J.). extensive washes with trichloroacetic acid, ethanol, and ether, the samples were counted in a Packard Tri-Carb liquid Wistar male rats (Fuji Animal Farm, Tokyo, Japan) scintillation spectrometer. weighing 165 g were used. The basal diet was the same as DNA-14C was prepared by extracting DNA from thymidine- described previously (11). Animals were fed for 3 months with 2-14C-labeled nuclei from regenerating rat liver (16). the basal diet containing 0.025% 2-FAA (Eastman Organic

1This work was supported in part by the Scientific Research Fund of the Ministry of Education of Japan. RESULTS 2The abbreviations used are: 2-FAA, Af-2-fluorenylacetamide; PCMB, p-chloromercuribenzoate. Even with the dialyzed soluble fractions and nuclear Received September 7, 1971; accepted January 14, 1972. extracts from normal rat liver and hepatoma, the joining of

886 CANCER RESEARCH VOL. 32

Table 1 Polynucleotide ligase activity from normal and neoplastic rat liver Conditions of the standard assay were used. 32P resistant to phosphatase (mjumoles/mg of protein) in following cell fractions

Normal Neoplastic

ComponentsComplete extract0.450.04<0.010.020.020.030.250.150.46<0.01Soluble0.2600.006

systemMinus Mg2+Minus enzymeMinus 0.0010.0040.0020.0050.0300.0220.050<0.001Nuclear0.0010.0100.0020.0090.1200.1010.250<0.001Nuclear ATPMinus Mn2*MinusMg2+,plus NAD*MinusATP, plus mercaptoethanolPlus mM)PlusPCMB (0.5 plusmercaptoethanolPCMB (0.5 mM), mM)Single (16 DNASoluble0.0490.005<

Table 2 Comparison of polynucleotide ligase activity from normal and neoplastic livers Conditions of the standard assay were used. 32P resistant to phosphatase (m^moles/nig of protein) in following cell fractions lissuesNormal g extract0.48 supernatant0.046

liver (5f Â±0.05b1.40+0.20 Â±0.268 Neoplastic liver (4) Hepatoma area Â± Nonhepatoma areaNuclear 0.60 Â±0.06105,OOOX0.063 Â±0.0050.0300.006 " No. of determinations. IO 20 6 Mean Â±S.D. TIME IN MMUTES Chart l. Rates of polynucleotide ligase activity by soluble fractions DNA is dependent on the addition of ATP, Mg2*, from normal rat livers and hepatoma. The standard assay conditions and described in "Materials and Methods" were used with 80 fig of enzyme mercaptoethanol, as shown in Table 1. The ATP requirement could not be replaced by NAD+ in preparations. Â»,hepatoma; o, normal rat liver. both normal rat liver and hepatoma. Mn2+ (5 X 10~3 M) could not replace Mg2*. In the absence of mercaptoethanol, 5 X degraded by the nuclear extract. Mixtures of soluble fractions 1CT4 M PCMB inactivated the ligase activity about 50%, and or nuclear extracts from normal livers and hepatomas provided this inactivation was overcome by adding 1.6 X IGT2 M the degradation of nicked 5'-phosphoryl-DNA-32P at less than mercaptoethanol. As noted in Tables 1 and 2 and Chart 1, the 5% of the calculated values. Endonuclease activity was activity of the polynucleotide ligase in the soluble fraction determined by measuring the amount of 10 ^g of native from rat hepatoma was about 5 times greater than that in DNA-14C (10,000 cpm) that became acid insoluble after incubation at 37Â°with the same assay procedure as described normal rat liver. As shown in Tables 1 and 2, the ligase activity in the nuclear extract from rat hepatoma was about 3 times in Chart 2 in the presence of soluble fractions or nuclear greater than that in normal rat liver. It is possible that these extracts from both normal liver and hepatoma, and the changes were caused by differences in the contamination of substrate was not degraded by the preparations. It is nucleases in the enzyme preparations. For examination of this concluded that the increased activity of the ligase from rat possibility, the substrate nicked DNA-32P was incubated with hepatoma is not due to the change of the activity of nuclease. both soluble fractions and nuclear extracts. As shown in Chart The results were obtained with the same procedures (9) as 2, the degradation of nicked 5'-phosphoryl-DNA-32P by the described previously to show that this enzyme catalyzed the formation of 3' ,5'-phosphodiester bond at the interrupted site. soluble enzyme preparations proceeded at almost the same rate with different concentrations of the extracts from both After incubation with the single-strand breaks in DNA and the normal liver and hepatoma, and the substrate was not ligase in nuclear extracts, the sedimentation coefficient in

MAY 1972 887

cofactor. These results suggest that ligase activity is correlated with increased DNA synthesis.

REFERENCES 1. Burton, K. The Relationship between the Synthesis of Deoxyribonucleic Acid and the Synthesis of Protein in the Multiplication of Bacteriophage T2. Biochem. J., 61: 473-483, 1955. 2. Cozzarelli, N. R., Melechen, N. R., Jovin, J. M., and Kornberg, A. Polynucleotide Cellulose as a Substrate for a Polynucleotide Ligase Induced by Phage T4. Biochem. Biophys. Res. Commun., 28: 578-586, 1967. 3. Gefter, M. L., Becker, A., and Hurwitz, J. The Enzymatic Repair of Â¿G OF PROTEIN DNA, I. Formation of Circular DNA. Proc. Nati. Acad. Sei. U. S., Chart 2. Degradation of nicked 5'-phosphoryl-DNA-32P by soluble 58: 240-247, 1967. 4. Geliert, M. Formation of Covalent Circles of Lambda DNA by E. fractions from normal rat livers and hepatoma. The reaction mixture (0.2 ml) contained 10 jugof nicked 5'-phosphoryl-DNA-"P, 10 jumÃ³les coli Extracts. Proc. Nati. Acad. Sei. U. S., 57: 148-155, 1967. of Tris-HCl buffer (pH 8.0), 2 jumÃ³lesof MgCl2, 2 jumÃ³lesof 5. Hoagland, M. B., Stephenson, M. L., Scott, J. F., Hecht, L. L, and Zamecnik, P. C. A Soluble Ribonucleic Acid Intermediate in mercaptoethanol, and enzyme preparations as indicated. After incubation at 37Â°for 20 min, the reaction was stopped by adding 0.2 Protein Synthesis. J. Biol. Chem., 231: 241-257, 1958. 6. Ichimura, M., and Tsukada, K. Polynucleotide Kinase from Rat ml of 0.1 M sodium pyrophosphate, followed by the procedures described in "Materials and Methods" to count in a Packard Tri-Carb Liver Nuclear Extracts. J. Biochem., 69: 823-828, 1971. 7. Lea, M. A., Morris, H. P., and Weber, G. Comparative Biochemistry liquid scintillation spectrometer. Â»,soluble fraction from hepatoma; o, of Hepatoma Growth Rate. Cancer Res., 26: 465-469, 1966. soluble fraction from normal liver. 8. Lea, M. A., Morris, H. P., and Weber, G. DNA Metabolism in Liver and Kidney Tumors of Different Growth Rates. Cancer Res., 28: alkali increased from about 4 S to 6 to 7 S in normal liver and 71-74, 1968. from about 4 S to 7 to 8 S in hepatoma. 9. Lindahl, T., and Edelman, G. M. Polynucleotide Ligase from In normal liver about 50% of the ligase activity was Myeloid and Lymphoid Tissues. Proc. Nati. Acad. Sei. U. S., 61: localized in the nuclear fraction and 40% in cytoplasma, while 680-687, 1968. in hepatoma about 30% of the ligase activity was localized in 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein Measurement with the Folin Phenol Reagent. J. Biol. the nuclear fraction and 60% in cytoplasma. This suggests that Chem., Â¡93:265-275,1951. in hepatoma some of ligase activity in the nuclear fraction is 11. Marugami, M., Ito, N., Konishi, Y., Hiasa, Y., and Farber, E. more readily extractable compared with normal liver. Influence of 3-Methylcholanthrene on Liver Carcinogenesis in Rats Ingesting DL-Ethionine, 3'-Methyl-4-dimethylaminoazobenzene and A'-2-Fluorenylacetamie. Cancer Res., 27: 2011-2019, 1967. DISCUSSION 12. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., and Sugino, A. Mechanism on DNA Chain Growth, I. Possible Discontinuity and Unusual Secondary Structure of Newly Synthesized Chains. Proc. The polynucleotide ligase in mammalian cells was first Nati. Acad. Sei. U. S.,59. 598-605, 1968. described by Lindahl and Edelman (9) in rabbit bone marrow. 13. Olivera, B. M., and Lehman, I. R. Linkage of Polynucleotides They have shown that the enzyme utilizes ATP as a cofactor. through Phosphodiester Bonds by an Enzyme from Escherichia The activity of the ligase in regenerating rat liver was about 4 coli. Proc. Nati. Acad. Sei. U. S., 57: 1426-1433, 1967. times greater in both soluble fractions and nuclear extracts 14. Sugimoto, K., Okazaki, T., and Okazaki, R. Mechanism of DNA Chain Growth, II. Accumulation of Newly Synthesized Short than in normal rat liver (15). Chains in E. Coli Infected with Ligase-defective T4 Phages. Proc. Okazaki et al. (12) and Sugimoto et al. (14) reported that Nati. Acad. Sei. U. S., 60: 1356-1362,1968. nascent DNA is synthesized in a discontinuous manner in 15. Tsukada, K., and Ichimura, M. Polynucleotide Ligase from Rat bacteria and that the polynucleotides are subsequently joined Liver after Partial Hepatectomy. Biochem. Biophys. Res. to growing DNA strands by polynucleotide ligase. We have Commun., 42: 1156-1161,1971. confirmed their observations in regenerating rat liver (15, 16). 16. Tsukada, K., Moriyama, T., Lynch, W. E., and Lieberman, I. It is known that incorporation of labeled thymidine into Polydeoxynucleotide Intermediates in DNA Replication in DNA is low in normal livers; however, incorporation was Regenerating Liver. Nature, 220: 162-164, 1968. markedly increased in the regenerating livers and in all 17. Weiss, B., and Richardson, C. C. Enzymatic Breakage and Joining of Deoxyribonucleic Acid, I. Repair of Single-Strand Breaks in hepatomas (7), and thymidine incorporation into DNA and DNA by an Enzyme System from Escherichia coli Infected with T4 growth rate of hepatoma showed a good correlation (8). In Bacteriophage. Proc. Nati. Acad. Sei. U. S., 57: 1021-1028, 1967. this communication, we have shown that ligase activity in 18. Zimmerman, S. B., Little, J. W., Oshinsky, C. K., and Geliert, M. hepatoma induced by 2-FAA is about 5 times greater in the Enzymatic Joining of DNA Strands: A Novel Reaction of soluble fraction and about 3 times greater in the nuclear Diphosphopyridine Nucleotide. Proc. Nati. Acad. Sei. U. S., 57: fraction than those in normal rat liver and required ATP as a 1841-1848, 1967.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1972 American Association for Cancer Research. Increased Activity of Polynucleotide Ligase from Rat Hepatoma Induced by N-2-Fluorenylacetamide

Kinji Tsukada, Shigeru Hokari, Nobuko Hayasaki, et al.

Cancer Res 1972;32:886-888.

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