Inhibition of hormone-stimulated ornithine decarboxylase activity by lithium chloride

Life Sciences, Vol. 47, pp. 233-240 Printed in the U.S.A.

Pergamon Press

INHIBITION OF HORMONE-STIMULATED ORNITHINE DECARBOXYLASE

ACTIVITY BY LITHIUM CHLORIDE James F. Richards 1, Kelly Fox2, Ted Peng 1, Jerry Hsiao I and Peter W. Gout2 1Department of Biochemistry, The University of British Columbia, Vancouver, B.C., V6T 1W5 2Department of Cancer Endocrinology, Cancer Control Agency of British Columbia, Vancouver, B.C., V5Z 4E6, Canada (Received in final form May 15, 1990)

Summary Effects of Li + on hormone-stimulated ornithine decarboxylase (ODC) activity were determined in kidney and liver of rats treated with dexamethasone or prolactin (PRL) and also in cultured, PRL-stimulated Nb2 lymphoma cells. In both systems, LiC1 led to rapid and marked decreases in ODC activity. The inhibitory effect of Li+ in exponentially growing Nb2 lymphoma cell cultures, measured at 45 rain, was dosedependent, ranging from 10% at 0.1 mM LiC1 to 95% at 10 mM LiC1. Surprisingly, on continued incubation with 10 mM LiC1, the lymphoma cells partially overcame the inhibition, showing ODC activities which reached a maximal value of ca 50% of the control at 4.5 h. The inhibition by Li + could not be reduced by adding myo-inositol to the culture medium. LiC1 did not inhibit ODC activity when added to cell-free extracts of rat tissues and Nb2 lymphoma cells indicating it did not act directly on the enzyme; however, there is evidence that, in intact cells, Li + enhances the rate of inactivation of the enzyme. Ornithine decarboxylase (ODC: EC 4.1.1.17) is a key regulatory enzyme in the biosynthesis of polyamines in mammalian cells, and as such appears to play an important role in the control of a variety of biological processes, including cellular metabolism, differentiation, proliferation and membrane function (1-4). The activity of the enzyme is low in quiescent cells, and increases 5-500 fold in a tissue in response to an appropriate stimulus (1,3,5). Because polyamines are involved in such a wide variety of physiological processes, their metabolism, and particularly the activity of ODC, responds to a large number of stimuli, including such diverse compounds as peptide and steroid hormones, growth factors, amino acid supply, inorganic ions, tumor promoters, carcinogens and foreign organic chemicals (1,5). The chemical properties of these signals and their mechanisms of interaction with their target cells are quite varied, which would suggest that regulation of ODC activity might be imposed by several mechanisms. Changes in ODC activity have been linked at the molecular level to alterations in transcription a n d / o r translation (6-11). There is also evidence for control at the post-translational level by specific modifications of the protein. The activity of ODC may be determined not only by the amount of enzyme synthesized, but also by its rate of degradation and the form in which it occurs, properties which depend on interaction with a specific polypeptide inhibitor of ODC 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc

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(12-15) or phosphorylation of the protein (16-22). It is not yet known how these posttranslational mechanisms are activated by external signals, nor how they are integrated with the regulation of transcription or translation to produce the observed changes in ODC activity. Lithium has been shown to have several biochemical effects (23), including interference with adenyl cyclase (24), G-proteins (23) and the phosphoinositol system (25), components of signal transduction pathways of hormones and growth factors. It has also been shown to have an effect on ODC activity. The injection of LiC1 into mice severely decreased their renal, cardiac and splenic ODC activities (26). In another study, LiCl inhibited the small (2-3 fold) increase in ODC activity that occurred in T lymphocytes 5-10 minutes after treatment with Con A (27). It appeared from these studies that Li + could be a useful tool for studying the mechanisms involved in hormonal regulation of ODC. In the present investigation, we have examined the effects of LiC1 on hormone induced ODC in two experimental systems, i.e., in vivo, using rats injected with dexamethasone or prolactin, and in vitro, using a line of cultured PRL-dependent Nb2 lymphoma cells. The latter provides a less complex, better defined biological system, and is useful for investigating the regulation of ODC activity and growth by peptide hormones (28-30). Materials and Methods Animal studies Female Wistar rats (Animal Care Centre, The University of British Columbia) were used at a weight of 60-80 g. Groups of 3-5 animals were given i.p. injections of dexamethasone (200 ~g in 1 ml 10% ethanol-saline), bovine PRL (400 ~tg in 0.5 ml saline pH 8-8.5) or saline (controls). In addition, some of the animals were given i.p. injections of LiC1 in saline (1 ml at 5 ~tmole/g body weight), or saline. After 5 h, the rats were sacrificed, the kidneys and livers were pooled for each group and homogenized with a Potter-Elvehjem homogenizer in ice-cold buffer (50 mM Hepes, 3 mM dithiothreitol, 0.1 mM EDTA, pH 7.3-7.4). The tissue homogenates were then centrifuged at 20,000g for 20 min at 4°C and the supernatants used for assay of ODC activity. Rats treated with LiC1 at the above dose did not show any toxic effects (eg., in behaviour, gastro-intestinal function or weight) either in the first 5 h or over more extended periods (eg., 1 week). Studies involving cell cultures Lactogen-dependent Nb2 lymphoma cells were cultured using procedures already described (31). The cells were maintained in suspension culture in 80 cm 2 tissue culture flasks (50 ml portions) in Fischer's medium supplemented with fetal bovine serum (FBS; 10%) as a source of lactogens, lactogen-deficient horse (gelding) serum (10%), 2-mercaptoethanol (0.1 mM), penicillin (50 units/ml) and streptomycin (50 u g / m l ) at 37°C in an atmosphere of 5% CO2- 95% air. In some experiments, inositol-free medium was used, i.e. inositol-free Fischer's medium supplemented with 10% horse serum (dialyzed against PBS), 2-mercaptoethanol (0.1 mM), antibiotics and ovine PRL (10 ng/ml). Cell populations were determined using an electronic cell counter (Coulter Electronics, Hialeah, FL). LiC1, ovine PRL, myo-inositol, putrescine and spermidine were a d d e d to cultures as solutions in Fischer's medium supplemented with lactogen-deficient horse serum (10%). Quiescent Gl-arrested cells were obtained using procedures already described (30).

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Cell-free extracts of Nb2 lymphoma cells were prepared for ODC assay using procedures alread~ described (14). Cells [(10-25) x 106] were collected from growing cultures (ca 5 x 10~ cells/ml) by centrifugation (350g, 5 rain) and then resuspended in ice-cold Hanks' balanced salt solution. This procedure was repeated twice and, after the final centrifugation, the cells were resuspended in 0.5 ml of the ice-cold buffer described above containing in addition 0.1% Triton X-100. The preparations were then centrifuged at 20,000 g for 20 min at 4°C and the supernatants (i.e. the cell-free extracts) used for assay of ODC activity. Assay of ornithine decarboxylase activity Tissue and cellular extracts were assayed for ODC activity in duplicate or triplicate by incubating aliquots of 100-250 ~tl with 260 ~tM ornithine (containing 0.25 ~tCi of L[1-14C]-ornithine), 200 ~tM pyridoxal phosphate, 4 mM dithiotreitol, and 100 ~tM EDTA in a final volume of 400 ~tl. The release of 14CO2 was measured as previously described (14). Materials Ovine PRL, dexamethasone, LiCI and other chemicals were obtained from the Sigma Chemical Co. (St. Louis, MO). L[1-14C]-ornithine-HC1 (50-60 mCi/mmole) was purchased from the Amersham Corporation (Oakville, Ontario, Canada). Bovine PRL was supplied by the National Hormone and Pituitary Program of the NIH. Results Effects of LiC1 on ODC activity of rat tissues When normal, non-hormonally treated rats were given i.p. injections of LiC1 (5 ~tmole/g body weight), the ODC activities of extracts of liver, kidney and thymus had decreased after 1 h by 70-85% (data not shown). Intraperitoneal injection of dexamethasone c(~ 3 ~tg/g body weight) or PRL c(~ 5 ~g/g body weight) into rats led to a marked rise in the ODC activities of liver and kidney extracts starting at 2-2.5 h, peaking at about 5 h and thereafter rapidly declining to levels of control animals. The effects of the hormones on ODC activity at h 5 are shown in Table 1. The effects of LiC1 on hormonally induced ODC activity in kidney and liver, measured at h 5, were determined under two different conditions by injecting LiC1 i) at the same time as the hormones (i.e., prior to the increase in ODC activity) or ii) 4 h after administration of the hormones (i.e., at a time when the hormonally induced ODC activity was reaching a maximum). The results in Table 1 show that in each case the ODC activities in the tissue extracts were lower than when only the hormones were given (controls). The magnitude of the inhibitory effect of LiC1 varied, however, with the time of its administration relative to that of the hormones. When LiC1 was given at the same time as the hormones, the ODC activity at 5 h was only marginally lower (10-18%) than that of the control; on the other hand, if LiC1 was injected 4 h after the hormones, ODC activity measured 1 h later was 38-96% lower than the controls. Incubation of extracts of liver and kidney from PRL- or dexamethasone-treated rats with LiC1 (5-50 mM) had no effect on the ODC activities (data not shown), indicating that the marked inhibition of tissue ODC activity by LiC1 in the animal was not due to a direct effect of Li+ on the enzyme.

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TABLE I ODC Activities in Tissues of Rats Treated with Hormones: Effect of LiC1

Treatment of rats a

ODC activity at h 5b (pmoles CO2/30 m i n / m g protein) kidney

None

liver

758

15

dexamethasone dexamethasone + LiCI (at h 0) dexamethasone + LiC1 (at h 4)

3273 2829 1072

585 480 23

PRL PRL + LiC1 (at h 0) PRL + LiC1 (at h 4)

4089 3656 2531

671 581 110

aGroups of rats (3-5 animals per group) received hormone at h 0, and, where indicated, LiC1 at h 0 or 4. bCell-free extracts were assayed in duplicate or triplicate. The data are from a typical experiment. In a series of 6-8 experiments, the specific activity of ODC in tissues of animals treated with hormone and LiCI at h 0 was 82-90% of that in tissues of animals receiving only a hormone. Effects of LiC1 on ODC activity of Nb2 lymphoma cells The effects of LiC1 on the ODC activity of the lymphoma cells were determined in two different types of experiments. In the first type, LiC1 was added to cultures of cells growing exponentially in regular culture medium or in inositol-free medium. These cells were proliferating rapidly and had a high ODC activity. In the second type, the effect of LiCI was studied in cultures of quiescent (Gl-arrested) cells during response to stimulation by PRL. The cells in the quiescent cultures have barely detectable ODC activity; following the addition of PRL, there is an increase in ODC activity followed by DNA synthesis and subsequent cell division (30). When exponentially growing cultures of Nb2 lymphoma cells were incubated in regular culture medium with LiC1 at concentrations of 0.1, 1, 2, 5 and 10 mM, ODC activity had decreased after 45 min by 10, 18, 28, 65 and 95%, respectively (Fig. 1); similar results were obtained with cultures of Nb2 lymphoma cells growing in inositol-free medium. In contrast, incubation of cell-free extracts of Nb2 lymphoma cells with LiC1 (5mM) did not have any effect on ODC activity (data not shown), in spite of the marked inhibitory effect of 5 mM LiCl on ODC activity in intact cells (see Fig. 1). In view of the possibility that the LiCl-induced inhibition of ODC activity

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could be prevented by addition of myo-inositol, exponentially growing Nb2 lymphoma cell cultures were incubated with LiC1 (2 and 5 mM) in the presence and absence of added myo-inositol (0.1 and 1 mM). It was found that the inositol did not prevent the marked decrease in ODC activity induced by LiCI (Fig. 1). It was observed, too, that LiC1 interfered with the large increase in ODC activity which occurred in the quiescent cells treated only with PRL. While ODC activity increased from 3 pmoles C02/30 min/106 cells at the time of addition of PRL to 139.8 and 281.5 at 4 and 6 h, respectively, the corresponding values in cells treated with LiCI at the same time as PRL were 54.8 and 49.7.

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FIG. 1 Effect of LiC1 a n d / o r Myo-inositol on ODC Activity in Nb2 Lymphoma Cells LiC1 a n d / o r myo-inositol were added, at the concentrations shown, to exponentially growing Nb2 lymphoma cell cultures for 45 min at 37°C. Cellfree extracts were then prepared for ODC assay. Results are the means +/- SEM of values from 3-9 experiments. In spite of its dramatic effect on ODC activity, LiC1 (10 mM), when added to exponentially growing cells, only moderately inhibited the population growth of the Nb2 lymphoma cell cultures, reducing the growth rate by only about 30% at 24 and 48 h (leading to an increase of the culture doubling time from 12.7 to 16.4 h). The relatively modest inhibitory effect of LiC1 on population growth could not be prevented by the addition of putrescine (10-100 I~M) or spermidine (100 ~M), alone or in combination, or by myo-inositol (2 mM) either alone or in the presence of the polyamines. The possibility that the Nb2 lymphoma cells could overcome the inhibition of ODC activity by LiC1 was investigated by incubating cells with I0 mM LiCI for longer periods of time, and measuring their ODC activities at intervals. Figure 2 shows that the ODC activity in the LiCl-treated cells, after a very marked decline to c_~a1% percent of the controls at 90 rain, did indeed recover on continued incubation to a maximal value (at h 4.5) of about 50% of the controls; it was also 50% of the control values at 24 h. Myo-inositol had no effect on the recovery of ODC activity (Fig. 2).

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LiCI Inhibits Hormone-Stimulated

ODC

Vol. 47, No. 3, 1990

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FIG. 2 Effects of Prolonged Incubation with LiC1 on ODC Activity of Nb2 Cell Cultures The results show the mean and the range of values obtained from duplicate cultures assayed at least in duplicate. The half-life of ODC in cultures of exponentially growing Nb2 lymphoma cells was found to be 26.5 min using cycloheximide at 50 ~g/ml to block protein synthesis. The activity of the enzyme declined even more rapidly in cultures incubated with 10 mM LiC1. Thus, whereas ODC activity in cultures treated with cycloheximide decreased by only 29% and 53% at 15 min and 30 min, respectively, the activity of the enzyme in the LiC1 treated cultures decreased by 63% and 92% at the same times. The half-life of ODC activity in the LiC1 containing cultures was calculated to be approximately 9.5 min. Discussion Studies by Adlercreutz et al (26) have shown that treatment of NMRI mice with LiC1 can cause a major decrease in the ODC activities of kidney, heart and spleen (26). In the present investigation, we have observed a similar decrease in the ODC activities of the liver and kidney of Li+-treated Wistar rats. The study has been extended to include the effects of LiC1 on the greatly increased ODC activity of these tissues in rats treated with dexamethasone or PRL, and also in cultured PRLdependent Nb2 lymphoma cells which, in the presence of PRL, have high levels of ODC activity (30). In both the animal (Table 1) and the cultured cells (Figs 1, 2), Li + caused a marked and rapid decrease in the hormonally induced ODC activity. In view of the rapid and almost complete inhibition of ODC activity by 10 mM LiCI at 90 min (Fig. 2), it was unexpected to find that, on continued incubation with LiC1, a partial recovery of ODC activity occurred in the Nb2 lymphoma cells (Fig. 2). While the mechanism of this recovery is not understood, it provides a plausible explanation for the observation that LiCI at 10 mM only moderately reduced the proliferation rate of the cells even after 48 hours. The much lower levels of tissue ODC activity in

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hormonally treated rats after a 1 h treatment with LiC1 than after a 5 h treatment (Table 1), may be due not only to a lowering of LiCI levels by excretion, but also to the ability of the tissues to overcome the Li+-induced inhibition of ODC activity. The mechanism by which Li + reduces the activity of ODC in cells and tissues is not known. However, it does not involve direct inhibition of the enzyme per se but appears to be mediated by processes occurring in intact cells, as indicated by the findings that the ODC activities of cell-free extracts of the hormonally stimulated tissues and Nb2 lymphoma cells were not affected by incubation with LiC1. Similar findings have been reported by others (26). The marked effect of LiC1 on the half-life of ODC in the intact Nb2 lymphoma cells raises the possibility that the effects of Li + on the levels of ODC activity may be due, at least in part, to an enhancement of the rate of inactivation of the enzyme. Li + (e.g., 10 raM) has been shown to lead to a deficiency in free intracellular inositol and thereby to interfere with the formation of inositol phospholipids essential for the effects of certain mitogens on their target cells (23). For example, LiCI inhibits the rapid increase in ODC activity in T lymphocytes, which occurs within 10 min of stimulation with the mitogen Con A; this inhibition can be completely prevented by the addition of exogenous inositol (27). In the present study, the addition of exogenous myo-inositol to Nb2 lymphoma cell cultures did not prevent the inhibition of ODC activity by 2-10 mM LiC1 (Fig. 1), nor did it alter the recovery of ODC activity (Fig. 2) or the rate of growth of LiCl-containing cultures. These findings indicate that the inhibition of ODC activity by LiC1 in the Nb2 lymphoma cells cannot be attributed merely to an interference with phosphoinositide metabolism produced by an intracellular deficiency of inositol, unlike the inhibition in the LiCl-treated mitogen-stimulated T lymphocytes (27). Furthermore, the Nb2 lymphoma cells grow quite readily in the presence of 10 mM LiCI suggesting that phosphoinositide metabolism may not be very important in the mitogenic stimulation of the cells by PRL. This suggestion is consistent with observations that the mitogenic stimulation of the Nb2 lymphoma cells by human growth hormone (also a lactogen) is not mediated by an immediate acceleration of phosphoinositide metabolism (32). PRL and dexamethasone can both induce large increases in the ODC activities of their target cells. The levels of ODC activity in hormone-stimulated systems are dependent on a number of factors, including ODC gene activation by the hormones, synthesis of ODC and degradation a n d / o r inactivation of the enzyme. The marked and rapid inhibition by Li + of the ODC activities induced by dexamethasone and PRL suggests that this agent may be a useful tool for identifying and investigating the mechanisms which are important for achieving and maintaining high levels of the enzyme in hormone-stimulated cells. Acknowledgements This work was supported by grants from the Medical Research Council of Canada (J.F.R.) and the National Cancer Institute of Canada (P.W.G.). Dr. Salvatore Raiti of the National Institute of Diabetes and Digestive and Kidney Diseases is thanked for a generous gift of bovine PRL. References 1. 2. 3. 4.

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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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