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Life Soioaoes Vol. 17, pp . 1769-1776 Printed in the II .S .A.

RELATIONSHIP OF ORNTTHINE DECARBOXYLASE TO RNA POLYMERASE T ACTTVITY Carol-Ann Manen and Diane H. Russell Department of Pharnincology University of Artzone Medical Center Tucson, Artzone 85724 (Received in final form October 28, 1975)

SUMMARY Ornithine decarboxylase activity can be rapidly elevated 50- to 100-fold by the administration of methylxanthtne derivatives such as methyllsobutylxanthine . This elevation occurs just prior to the increase in RNA polymerise I activity . Inhibitors of RNA synthesis and of protein synthesis suggest that arty alteration in the ornithine decarboxylase response results in a similar alteration in the level of a-amanitln-insensitive RNA polymerise . Addition of a partially purified .ornithine decarboxylase preparation to the RNA polymerise assay increased both the initial rate of aH-UTP incorporation and the length of time that the polymerise assay was linear . It is suggested that ornithine decarboxylase is the labile protein that modulates the level of RNA polymerise I . Studies of bacterial and mammalian systems suggest that poi amines might be growth factors (1-5) by regulating RNA synthesis (4,6,7 . There are, for example, indications of a correlation between the amount of spermtdine in a cell and the amount of rRNA that can be accumulated (7,8) . However, this relationship would have to result from earlier biosynthetic events since both ornìthine decarboxylase, the initial enzyme of olyamine biosynthesis, and RNA polymerise I increase in activity within 4 ~ of partial hepatectomy in the rat (5,9), and significant changes in the accumulation of spermidine and rRNA occur much later. The link between polyamines and RNA polymerise I is strengthened by the discovery that a short-lived protein is required for a nonual level of transcription of nucleolar genes (10,11), and thus to regulate the activity of the enzyme . As amino acids stimulate the synthesis, or slow the degradation of this proteln(s) (11), as they do ornithine decarboxylase (12,13), and because orntthine decerboxylase is very labile, with a half-life of 10-20 minutes (14,15), we suggest that orntthlne decarboXylase might regulate the activity of RNA polymerise I . By evaluating the activity of ornithine decarboxylase and of RNA polymerise I after the administration of methylisobutylxanthine (MIX), we have 1769

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Ornithine Dacarboxylase and RNA Polymeraee I

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shown : (1) a 50- to 100-fold increase in ornithine decarboxylase activity within 4~ h after MIX administration, followed by more than a 2-fold increase in RNA polymerase I activity within 5 h of MIX administration ; and (2) administration of inhibitors which alter the extent of the increase in ornithine decarboxylase activity results in a similar attenuation of the increase in RNA polymerase I activity . In addition, we have shown a direct effect of the addition of a partially purified ornithine decarboxylase preparation on RNA polymerase I activity . MATERIALS AND METHODS Male Spra ue-Dawley rats (90-125g) were given 3-isobutyl, 1-methylxanthine (MIX)(10 mg/kg, i .p .) in ethanol/0 .9% saline (20% v/v) . Controls received an equivalent amount of ethanol/0 .9% saline (20% v/v) . Cyclo heximide (50 mg/kg, i .p .) or cordycepin (30 mg/kg, i .p .) in 0 .9% saline was given at the appropriate times . The rats were sacrificed and the livers removed and assayed for enzyme activity . Ornithine decarboxylase activity was determined by measuring the release of 14C02 from D,L-[1-14C]- ornithine (7 .7 mCi/~, New England Nuclear, Boston, Mass .) as previously described (16) . Ornithine decarboxylase was isolated and partially purified from the livers of rats treated with MIX 4 h before sacrifice. The purification procedure employed was that of Jänne and Williams-Ashman (17) with the exception that the buffers used were Na-K phosphate containing 1 mM phenylmethylsulfonylfluoride and 5 mM NaF. After DEAE-cellulose chromatography, ornithine decarboxylase was reprecipitated with ammonium sulfate (55% of saturation) . The resulting pellet was resuspended in the homogenizing buffer containing 0 .1 mM ornithine and dialyzed for 1 h against the homogenization buffer . The preparation had an activity of approximately 126 .5 uUnits/mg protein, a 40-fold purification, and was added to the RNA polymerase I assay as the source of a partially purified ornithine decarboxylase preparation . Using a purified nuclear preparation as the enzyme source (18), RNA polymerase I activity was determined by measuring the incorporation of uridine 5'-triphosphate [5-aH] (18.0 Ci/mM, Schwarz/Mann, Yan Nuys, CA) into RNA in the presence and absence of 1 uM a-amaniti n, according to the method of Roeder and Rutter (19) . RESULTS AND DISCUSSION Figure 1 shows that both ornithine decarboxylase and RNA polymerase I activities increased after the administration of MIX . Other known inhibitors of phosphodiesterases, such as theophy111ne and aminophylline, were reported to increase ornithine decarboxylase activity in various rat tissues (20) . Ornithine decarboxylase activity was substantially increased within 3 h, and by 4~ h reached a level 80 times greater than control . RNA polymerase I activity was increased within 4 h and reached a level more than twice the control level within 5 h . A high dose of a-amanitin (0 .8 mM) has been reported to inhibit both RNA polymerase II and III activities (21) . Using this criterion, RNA polymerase III activity remained constant after the administration of MIX and the increased incorporation of aH-UTP into RNA was attributable to an increase in RNA polymerase I activity . Inhibitors that altered the extent of the increase in ornithine decarboxylase activity also attenuated the increase in RNA polymerise I activity (Table 1) . Cycloheximide, administered simultaneously with MIX, completely inhibited the increases in both ornithine decarboxylase and RNA polymerise

Vol . 17, No . 12

Oraithiae Dacarboxylase aad HNA Polymeraea I

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Time after Methylisobutylxarlthine Administration (hr) Changes in liver ornithine decarboxylase and RNA polymerise I activities after the administration of MIX . Male SpragueDawley rats (90-125 )were iven 10 mg/kg, i .p . MIX in ethanol/0 .9% saline ~20% v/v~ . Ornithine decarboxylase activity was determined by measuring the release of 14 C02 from D,L-[1-14 C]-ornithine (16) . Using a purified nuclear preparation (18) as the enzyme source, RNA polymerise I activity was determined by measuring the lncorporation of 3H-UTP into RNA in the presence and absence of 1 uM a-amanitin, according to the method of Roeder and Rutter (19) . Each point represents the mean t S.E .M . in Units/mg protein of at least 4 different rats with triplicate assays of each liver . I. When this inhibitor was given 4 and 4~ h after MIX, at times when these enzyme activities were increasing, both elizymes were markedly inhibited . Ornithine decarboxylase activity was inhibited 75% and RNA polymerise I activity about 45%. The difference in inhibition of ornithine decarboxylase activity and RNA polymerise I activity after cycloheximide can be explained on the basis of the latency time for inhibition of RNA polymerise I activity (Table 1, Fig 2) . That is, we suggest that ornithine decarboxylase activity has to decrease significantly prior to a change in the activation pattern of RNA polymerise I . When the decreases in the activities of both enzymes were plotted on a semi-log scale at various times after the addition of cyctoheximtde so that half-lives could be calculated, both were about 15 min . There was, however, an initial lag of 15 min in the case of RNA polymerise I in which activity did not decrease after the addition of cycloheximide ; and thereafter 1t declined with a half-life of approximately 15 min . Since in the initial 15 min, half the ornithine decarboxylase activity has turned over, this could account for the lag time for the decreased activity of RNA polymerise I . Ornithine decarboxylase may have to decline below a certain level in order for RNA polymerise I to return to control

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Ornithine Decarboaylase and RNA Polymeraea I

Vol . 17, No . 12

Figure 2

Semi-logarithmic plot of the decline of ornithine decarboxylase and RNA polymerase I activities after cycloheximide administration . Male Sprague-Dawley rats (90-125g) were given 10 mg/kg, i .p . MIX in ethanol/0 .9% saline (20% v/v) . Cycloheximide, 50 mg/kg, i .p . was given 4 h after MIX . The rats were killed at times indicated and the livers removed and assayed for enzyme activity . Ornithine decarboxylase activity was determined by measuring the release of iaC02 from D,L-[1-14 C]-ornithine as previously described (16) . Using a purified nuclear preparation (18) as an enzyme source, RNA polymerase I activity was determined by measuring the incorporation of 3H-UTP into RNA in the presence and absence of 1 yM a-amanitin, according to the method of Rceder and Rutter (19) . Each point represents the mean of at least 5 determinations and the data are expressed in Units/mg protein . level . Cordycepin (30 mg/kg), an antibiotic which inhibits mRNA synthesis through its effect on the synthesis of poly(A) chains, inhibited both ornithine decarboxylase and RNA polymerase I about 15% (Table 1) . Since an increase in the putrescine concentration in response to an elevation of ornithine decarboxylase activity could be an alternative explanation to the regulation of RNA polymerase I by a short-lived protein (10,11), we assessed the effects of isolating nuclei (18) in concentrations of putrescine from 1 uM to 20 mM and were unable to show .any increased activity of RNA polymerase I (unpublished observations) . The addition to nuclear preparations of a given amount of ornithine decarboxylase activity (50 uUnits) rapidly altered the measurable activity of RNA polymerise I . The addition changed the initial rate of the RNA polymerise I reaction, as well as increased the time for which the enzyme assay was linear (Fig . 3) . RNA polymerise II activity was not affected by the addition of an enriched ornithine decarboxylase preparation . In order for ornithine decarboxylase to affect directly RNA polymerise I activity, it would have to be present in the nucleus . There is evidence to indicate that ornithine decarboxylase may be a nuclear enzyme when non-

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Ornithine Dacarboxylase and RNA Polymerase I

Vol. 17, No . 12

Figure 3

Omithine Decarboxylase Activity (NUrits of Enzyme Activity) RNA polymerase I activity in isolated rat liver nuclear preparations with and without the addition of ornithine decarboxylase activity . Fifty uUnits of an ornithine decarboxylase preparation ( ) purified through DEAE-cellulose chromatography (see Methods) was added to a purified nuclear preparation (17) and the incorporation of 3 HUTP into RNA in the presence and absence of 1 .8 ug/ml of a-amanitin was measured as previously described (19) . Controls (----) received an equivalent amount of protein in the same buffer . Each point represents the mean of 3 or more determinations in duplicate . aqueous extraction techniques are employed (22) . Since ornithine decarboxylase is a low molecular weight enzyme (70-90,000), it probably becomes cytoplasmic when cell fractions are obtained via aqueous sucrose gradient fractionation techniques . A serious consideration of the temporal relationship between the induction of ornithine decarboxylase and a subsequent modulation of RNA polymerase I activity which occurs in every system studied to date (4,9, 23,24) strongly suggests that ornithiné decarboxylase is the labile protein which modulates the level of RNA polymerase I activity thus controlling, to a great extent, the accumulation pattern of rRNA and the level of protein synthesis in tissues . ACKNOWLEDGEMENTS This work was supported by USPHS Grants CA-14783 and CA-17094 from the National Cancer Institute and American Cancer Society Grant IN-110 . Dr . Manen is the recipient of PHS Research Fellowship HD-00871 from the National Institute of Child Health and Development .

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Ornithine Decarboxylasa and RNA Polymerase I

Dr . Russell is the recipient of National Cancer Institute Research Career Development Award CA-00072 . 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 .

REFERENCES

E .J . Herbst and E .E . Snell, J . Biol . Chem . 181 : 47-54 (1949) . R .G . Ham, Biochem. Bio h s . es . ommun . 4~4-38 (1964) . W .K . Maas, Z . Le fer, and J . Poindexter, Ann . N .Y . Acad . Sci . _171 : 957-967 (1970) . D.H . Russell, Proc . Nat . Acad . Sci . USA 68 : 523-527 (1971) . D. Russell and S .H . ny er, Proc . at . Acâd . Sci . USA 60 : 1420-1427 (1968) . S .S . Cohen, Introduction to the Pol amines, pp . 44-47, Prentice-Hall, New Jersey (T37T . D.H . Russell and T .A . McVicker, Biochem. J . 130: 71-76 (1972) . N . Seiler, in Pol amines in Norma aT~n Néo ,lastic Growth (ed ., D .H . 973 . Russell) pp . 137-156, Raven Press, New York T. Lindell, F. Weinberg and W.J . Rutter, Fed . Proc . 29 : 669 (1970) . J . iTo . Biol . _53 : M. Muramatsu, N. Shimada and T . Higashina aga~a, 91-106 (1970) . M. Franze-Fernandez and A. Fontanive-Sengüesa, Biochim . Biophys . Acta 331 : 71-80 (1973) . Q.T.M . Hogan, S. Morden and A. Blackledge, in Pol amines in Normal , aven Press, and Neo lastic Growth (ed., D .H . Russell) pp . New Yorc 9 3 . B.L .M . Hogan, A. McIlhenney, S . Morden, J . Cell Physiol . _83 : 353-359 (1974) . D.H . Russell and S .H . Snyder, Mol . Pharmacol . 5 : 253-262 (1969) . D .H . Russell, S .H . Snyder, V .J . e na, En ocr~nology _86 : 1414-1419 (1970) . D .H . Russell and S .H . Snyder, ~Endocrinolo~ 84 : 223-229 (1968) . . 216 : 1725-1732 (1971) . J. Jänne, H .G . Williams-Ashman,J Bio1 . Chem . . T.S . Ro and H. Busch, Cancer Res2630-~637~T964) R .G . Roeder and W .J . Rutter, aT~turé224 : 234-237 (1969) . C .V . Byus and D .H . Russell, L~~i~5 : 1991-1997 (1974) . L .B . Schwartz, V .E .F . Sklar, J~aehn~ng, R . Weinmann and R .G . Roeder, J . Biol . Chem . 249 : 5889-5897 (1974) . S .H . Sny er, D .S . Kreuz,P.J . Medina and D .H . Russell, Ann. N .Y . Acad . Sci . 171 : 749-771 (1970) . D .H . tt~ell,Life Sci . 13 : 1635-1647 (1973) . .l .~ussêfl, Fed . Proc . 34 : 586 (1975) . C .A . Manen and~

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