N-Acetylation of Arginine-Rich Hepatoma Histones1

[CANCER RESEARCH 31, 468-470, April 1971] N-Acetylation of Arginine-rich Hepatoma Histones1

Paul Byvoetand Harold P. Morris Cellular and Molecular Biology, Division of Biological Sciences, University of Florida, Gainesville, Florida Â¡P.B.Â¡,and Department of Biochemistry, Howard University College of Medicine. Washington, D. C. 20001 Â¡H.P.M./

SUMMARY represents a fraction of the total number of sites available for acetylation (2). Previous work in this laboratory has shown that decay rates On the other hand, Starbuck et al. (11) have purified of histones and DNA isolated from deoxyribonucleoprotein of histone fractions which were either 100% acetylated at the normal and neoplastic rat tissues were approximately equal, NH2-terminal end or not at all. In addition, when isolated indicating that not only DNA but the nucleohistone complex nuclei were incubated in the presence of radioactive acetate, as a whole is metabolically inert. In contrast, the vV-acetyl incorporation of label appeared to occur primarily in groups in the combined arginine and slightly lysine-rich e-jV-acetyllysine of histones (6, 12). Thus far, all evidence histones from these same tissues were found to turn over at would therefore lead to the conclusion that the e-jV-acetyl much faster rates than whole histones or DNA, with exception groups on the internal lysine residues are metabolically active, of the Novikoff hepatoma, in which this turnover was wheras the a-jV-acetyl groups at the NH2-terminal ends are remarkably slow, approaching that of the histones. Studies stable features of certain histone fractions, similar to those of were therefore carried out on a number of hepatomas with some other proteins, such as carbonic anhydrase (3). The varying growth rates, which indicated that the half-lives of previously mentioned in vitro experiments with isolated nuclei their histone W-acetyl groups were in general an order of also revealed that the incorporation of labeled acetate was magnitude smaller than that of the whole histones and similar most active in the f2al and ÃŸ fractions and was practically to that of most tissues studied thus far. The data derived from negligible in the lysine-rich histones (12). These data indicate very-slow-growing tumors would seem to indicate that the that the acetylation of internal lysine residues is not random histone jV-acetyl groups from hepatocyte histones exhibit a and definitely rule out the possibility of a spontaneous or rapid turnover similar to that in other tissues. artifactual process. In our previous studies, rats were given injections of alanine-14C to label the histones and tritiated acetate to label INTRODUCTION the histone TV-acetyl groups. At various time points after the injection, groups of animals were killed, and lysine-rich and It has been suggested that chemical modifications of arginine-rich (combined arginine- and slightly lysine-rich) histones introduce structural changes in the otherwise inert histone fractions isolated from various tissues and their deoxyribonucleoprotein complex which may influence the specific activities with respect to 14C and jV-acetyl-3H were flow of information from the DNA. One of these reactions determined (2). concerns the acetylation of amino groups present in the As shown in Table 1, the results of these studies indicated histone molecule. Evidence indicating the presence of 1 acetyl that uptake of acetate into ,/V-acetyl groups from lysine-rich group or less per molecule of histone and the finding that the histones was much less than that into the arginine-rich NH2-terminal amino groups of a number of more or less crude histones, which is to be expected in view of the absence of histone fractions were masked to varying degrees when e-./V-acetylgroups in the former (12). exposed to fluorodinitrobenzene, according to the method of The fact that some uptake of label occurs in the lysine-rich Sanger (9), lead to the general assumption that histones were histones is presumably due to incorporation of acetate in the acetylated only NH2-terminally. Gershey et al. (6) have a-yV-acetyl position of NH2-terminal residues. Phillips (9) has recently reported, however, that histones also contain established that the lysine-rich histone fraction is completely e-amino-acetyllysine. Experiments carried out in this acetylated at the NH2-terminal end. It is therefore not laboratory have shown that the W-acetyl groups in histones surprising that the specific activity of lysine-rich histones with turn over much faster than the histones themselves, and it respect to radioactive acetate decayed at a rate which seems therefore conceivable that the amount of ./V-acetyl approached that of the histones themselves. Accurate groups found in a pure histone species at any given time measurements of this turnover were, however, often hampered by the exceedingly low level of labeling (2). 'This research was supported in part by Grant CA-10472 to the As was already indicated, the turnover rate of Af-acetyl University of Florida, Grant CA-10729 to Howard University from the groups in the arginine-rich (combined slightly lysine-rich and National Cancer Institute, Grant P-439 from the American Cancer arginine-rich) histone fraction was found to be much faster Society, and a grant from the Florida Division of the American Cancer Society. than that of the histones (2). In view of the preceding Received May 25, 1970; accepted December 14, 1970. discussion, it seems that this rapid turnover must be attributed

468 CANCER RESEARCH VOL. 31

Table 1 feature of hepatomas in general, as the half-lives of histone Incorporation of acetate-3 H into N-acetyl groups of histories ./V-acetyl groups in all tumors studied appeared to be an order Specific activity due to /V-acety! (acetate-3 H) of magnitude smaller than those of the whole histones and similar to that of most tissues studied thus far (2). Tissue (cpm/mg histone) Because the liver consists of a variety of cell types (4, 5), histones"665a histones75 the half-life found for histones or DNA will differ depending on the time period during which measurement takes place. In Liver Â±161 Â±5.5 the "short-term" studies outlined above, the 1st group of Novikoff hepatoma"Arginine-rich71 Â±11Lysine-rich 34 Â±4.5 animals was killed 24 hr after injection of the radioactive "Averages of 3 experimental values Â±S.E.at peak activity 1 hr after amino acid. Such experiments will yield an experimental administration of radioactive acetate. half-life for DNA or histones of 100 to 200 hr. If, however, animals are killed at weekly intervals, after an incubation to the internal e-jV-acetyl groups and that the a-TV-acetyl period of 3 weeks following the administration of the tracers, groups on the NH2 -terminal residues turn over at the same rate much longer half-lives will be found, since during the long as the histones themselves. incubation period the DNA and histones of faster-turning-over The 2 arginine-rich histone fractions that are acetylated cell populations have lost their radioactivity. For ensurance of most actively incorporate radioactive acetate into the internal a reasonable uptake of radioactive precursors into DNA and e-/V-acetyl positions at approximately similar rates. Thus far, histones of hepatocytes, the animals used in such long-term no conditions have been reported that caused a differential experiments were 3 weeks old at the time of injection. The effect on the respective turnover rates. The near absence of decay of the radioactively labeled DNA and histones was turnover of TV-acetyl groups in Novikoff hepatoma histones therefore due only to dilution with newly synthesized encountered in our studies (2) would seem to indicate that in unlabeled material, since in this case no death or migration of this case both fractions were affected to the same extent. It cells occurs. The half-life obtained in such studies (Table 3) was this unexpected finding that prompted us to survey a agreed very well with the growth rate of the liver in rats of number of hepatoma lines with different growth rates to see that age (1), although it may be expected to become much longer when the animals reach adulthood.2 whether this slow turnover might be a general property of hepatomas. Because the liver cell population is varied, it was thus far In view of the similarity of jV-acetylation patterns of the not possible to decide which cell population is responsible for f2al and f3 fractions, the feasibility of the study, and the the rapid turnover of ./V-acetyl groups in liver. The present absence of e-TV-acetyl groups in lysine-rich histones, it was results indicate that the turnover of histone jV-acetyl groups in decided to carry out all experiments on the "arginine-rich" hepatomas with very slow growth rates R! and R7 appeared to histones without further fractionation. be very similar to that of normal liver. Since it can be assumed that these tumors consist of pure clones of "minimally" malignant hepatocytes, the rapid turnover of jV-acetyl groups MATERIALS AND METHODS in liver histones shown in Table 3 can in all probability be assigned to the hepatocytes, although it seems reasonable to The present studies were carried out as described previously assume that the histone W-acetyl groups in littoral cells of (1,2). Rats were given i.p. injections of 100 AiCi/kg normal liver turn over rapidly as well. This instance points out L-alanine-14C (uniformly labeled) to label the histones and 2 the experimental advantage of these slow-growing tumors, mCi/kg sodium acetate-3 H to label the histone TV-acetyl which consist of uniform populations of cells, very similar to groups. In general, 4 groups of 2 to 3 animals were killed after hepatocytes, in contrast to normal liver which, besides the injection of radioactive acetate and the arginine-rich hepatocytes, contains a multitude of other cell types. (combined slightly lysine-rich and arginine-rich) histone In order to explain the slow turnover of ./V-acetyl groups in fractions isolated from tumor and liver. All animals received Novikoff hepatoma, in vitro studies have been carried out on 14C-labeled alanine 24 hr prior to the injection of tritiated histone acetyltransferase activity in chromatin from rat liver acetate. The specific activities of the isolated histone fractions with respect to l4Cand Af-acetyl(acetate-3H) were determined and hepatoma (10), which indicated that although the activity in hepatoma chromatin was approximately 70% of that in liver as described previously (1,2). chromatin, this difference did not seem adequate to explain the almost complete absence of histone TV-acetyl turnover in this tumor. Meanwhile, in vitro studies by Libby (8) have RESULTS AND DISCUSSION revealed that the most plausible explanation for this The results of these experiments are summarized in Table 2. Decay rates were calculated from semilogarithmic plots of 2In view of low uptake of labeled precursors into histones and DNA, specific activity against time by the method of least squares. as well as the long half-lives, which generally exceeded the observation Each half-life reported was obtained from 9 to 12 period, some spread may be expected in these data. It is, however, obvious that the ratio of histone turnover to that of DNA was close to experimental points. unity in all cases, which would agree with the notion that histones are As can be seen, the near absence of turnover found in the metabolically inert and are conserved in a manner similar to that of case of the Novikoff hepatoma did not appear to be a specific DNA (1, 7).

APRIL 1971 469

Table 2 Half-lives of arginine-rich histories and their N-acetyl groups from hepatomas and normal liver

(hr) (hr) TissueNovikoff Af-acetyl histone(L-alanine-'4C)41 generation250, rateFastFastFastIntermediateIntermediateIntermediateSlowSlow/VÂ»(acetate-3H)21

hepatoma"Morris (0.28)025 Â±(0.10)52 (0.39)1.5 Â±(0.32)N.D.d56 hepatomas3924AC1111e9121e7800e9098eR,cR,cNormal 2615017,1928,29432816,1710, (0.20)3.3(0.13)2.4(0.15)4.1 (0.34)37(0.13)34

(0.41)3.6 (0.24)39(0.16)43(0.21)56 (0.36)6.4 (0.32)4.1 (0.10)2.5 (0.69)N.D.168 (0.34)3.2 12Growth (0.16)2.3 (0.28) liverAGIBuffalo9121 (0.28)1.9 (0.24)1.9(0.06)2.1 (AGI)Sprague-DawleyTransplant (0.22)2.2(0.15)/Vi

" Sprague-Dawley rats. 0 Ratio S.E. of slope/absolute value of slope (S.E./n,i). S.E. derived according to:

S.E. = where

The ii/2 for acetate-H3 of all hepatomas differed significantly from that of Novikoff hepatoma at p = 0.05 for tin <4 hr and p = 0.10 for tin >4 hr. c ACI rats. d N.D., not determined. * Buffalo rats.

Table 3 Anhydrase from Dog Kidney and Dog Erythrocytes. Mol. Half-lives in days of histories and DNA in rat liver Pharmacol., 3: 142-152, 1967. 4. Edwards, J. L., and Klein, R. E. Cell Renewal in Adult Mouse Experiment 1 Average Tissues. Am. J. Pathol., 38: 437-452, 1961. 5. Edwards, J. L., and Koch, A. Parenchyma! and Littoral Cell DNALysine-rich Proliferation during Liver Regeneration. Lab. Invest., 13: 32-43, histonesOther 1964. histones21.621.723.326.722.217.028.717.614.225.620.518.1 6. Gershey, E. L., Vidali, G., and Allfrey, V. G. Chemical Studies of Histone Acetylation. The Occurrence of e-TV-Acetyllysine in the Pal Histone. J. Biol. Chem., 243: 5018-5022, 1968. phenomenon must be sought in the very low content of a 7. Hancock, R. Conservation of Histones in Chromatin during nuclear histone deacetylase in Novikoff hepatoma. In view of Growth and Mitosis in Vitro. J. Mol. Biol., 40: 457-466, 1969. these findings, it should be interesting to study the in vitro 8. Libby, P. R. Activity of Histone Deacetylase in Rat Liver and histone acetyltransferase and deacetylase activities of the Novikoff Hepatoma. Biochim. Biophys. Acta, 213: 234-236, various hepatoma lines, in order to see whether any 1970. correlations may exist with growth rates or other biochemical 9. Phillips, D. M. P. The Presence of Acetyl Groups in Histones. Biochem. Â¡.,87: 258-263, 1963. properties. 10. Racey, L. A., and Byvoet, P. In Vitro Acetylation of Histones. Proc. Am. Assoc. Cancer Res., 10: 71, 1969. 11. Starbuck, W. C., Mauritzen, C. M., Taylor, C. W., Soraja, I. S., and REFERENCES Busch, H. A. Large Scale Procedure for Isolation of the Glycine-rich, Arginine-rich Histone and the Arginine-rich, 1. Byvoet, P. Metabolic Integrity of Deoxyribonucleohistones. J. Mol. Lysine-rich Histone in a Highly Purified Form. J. Biol. Chem., 243: Biol., 77: 311-318, 1966. 2038-2047, 1968. 2. Byvoet, P. Differences in Turnover between Histones and Their 12. Vidali, G., Gershey, E. L., and Allfrey, V. G. Chemical Studies of JV-Acetyl Groups. Biochim. Biophys. Acta, 160: 217-223, 1968. Histone Acetylation. The Distribution of e-W-Acetyllysine in Calf 3. Byvoet, P., and Gotti, A. Isolation and Purification of Carbonic Thymus Histones. J. Biol. Chem., 243: 6361-6366, 1968.

470 CANCER RESEARCH VOL. 31

Paul Byvoet and Harold P. Morris

Cancer Res 1971;31:468-470.

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