Potentiation by Retinoic Acid of Ornithine Decarboxylase Induction by Phytohemagglutinin or Phorbol 12-Myristate 13-Acetate in Guinea Pig Lymphocytes

J. Biochem. 99, 1789-1797 (1986) Potentiation by Retinoic Acid of Ornithine Decarboxylase Induction by or Phorbol 12-Myristate 13-Acetate in Guinea Pig Lymphocytes Shuzo OTANI, Isao MATSUI-YUASA, Yasuko MIMURA, and Seiji MORISAWA Department of Biochemistry, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka, Osaka 545 Received for publication, January 13, 1986 Retinoic acid potentiated the increases in ornithine decarboxylase (L-ornithine carboxy-lyase [EC 4.1.1.17]) activity, [ 3 H]difluoromethylornithine binding to ornithine decarboxylase, intracellular levels of polyamines and DNA synthesis in guinea pig lymphocytes stimulated with phytohemagglutinin. The stimulatory effect on the ornithine decarboxylase induction was dependent on the dose of retinoic acid and on the time of addition of the drug. Retinoic acid had to be added not later than 2 h after phytohemagglutinin to elicit the potentiation. Retinyl acetate also potentiated ornithine decarboxylase induction caused by phytohemagglutinin. Both of these retinoids augmented ornithine decarboxylase induction caused by phorbol 12-myristate 13-acetate. The half-life of ornithine decarboxylase activity estimated after addition of actinomycin D was longer in cells treated with phytohemagglutinin or phorbol 12-myristate 13-acetate together with retinoic acid than in cells treated with the mitogen alone. The half-life after addition of cycloheximide was not affected by retionic acid. These results suggest that the retinoids are stimulators rather than inhibitors of ornithine decarboxylase induction caused by phytohemagglutinin or phorbol 12-myristate 13-acetate in guinea pig lymphocytes and that retinoic acid potentiates the enzyme activity at the transcriptional or posttranscriptional, but not at the post-translational stage. Retinoic acid and certain of its analogues have lines, suggesting retinoids to be possible cancerbeen shown to affect the proliferation and differ- chemopreventive or therapeutic agents, although entiation of various cells (7). Many studies have some studies have shown that retinoids potentiate shown an inhibitory action of retinoids on cell mitogenic activity of various growth factors (2-7). growth of transformed and nontransformed cell Ornithine decarboxylase (L-ornithine carboxy-lyase [EC 4.1.1.17]) is the enzyme responsible for poly- Abbreviations: PMA, phorbol 12-myristate 13-acetate; amine formation. It has been shown to be im- DPO, 2,5-diphenyloxazole; POPOP, l,4-bis-[2-(5-phen- portant for cell growth and is induced by a wide yloxazolyl)]benzene. variety of hormones and growth factors (8). Reti- Vol. 99, No. 6, 1986 1789

1790 S. OTANI, I. MATSUI-YUASA, Y. MIMURA, and S. MORISAWA noids inhibit the induction of ornithine decarboxylase by tumor promoters in lymphocytes (9) and mouse epidermis (10-12), and by growth factors in rat kidney cells (13). Retinoids also reduce ornithine decarboxylase activity in glioma cells, neuroblastoma cells (14), Chinese hamster ovary cells (15), and tracheal epithelial cells (16). The inhibitory action of retinoids on cell growth may be based at least partially on the inhibition of ornithine decarboxylase activity by these agents, although the mechanism of the inhibition of the enzyme activity is not clear. In this paper we report that retinoic acid potentiates phytohemagglutinin- or phorbol 12- myristate 13-acetate (PMA)-induced ornithine decarboxylase activity, as well as the increases in intracellular levels of putrescine and spermidine and DNA synthesis induced by phytohemagglutinin. MATERIALS AND METHODS Materials DL-[l- 14 C]Ornithine (57 mci/mmol) and [methyl- 3 H]thymidine (20 Ci/mmol) were obtained from Amersham International, Amersham, UK. DL-a-[3,4-3 H]Difiuoromethylornithine (33.3 Ci/mmol) was purchased from New England Nuclear, Boston, MA. All-fraiw-retinoic acid, retinyl acetate, actinomycin D and cycloheximide were the products of Sigma, St. Louis, MO. Phorbol 12-myristate 13-acetate (PMA) was the product of LC Services Corp., Woburn, MA. -p was from Difco Laboratories. Horse serum was purchased from Commonwealth Serum Laboratories, Victoria, Australia. Other reagents used were special grade commercial products. Cell Culture Guinea pigs weighing about 300 g were sensitized by subcutaneous injections of purified protein derivatives of tubercle bacilli emulsified in Freund's adjuvant to obtain large amounts of lymphocytes from lymph nodes. One month after the injection, the animals were killed and lymphocytes were obtained from cervical, inguinal and axillary lymph nodes as described previously (17), and were separated from other cells by. the method of Boyum (18). The cells were suspended in Eagle's minimum essential medium containing 5% horse serum and 2'rnM glutamine at!a concentration of 1 x 10 7 cells/ml. One ml of the cell suspension was poured into a flat-bottomed glass tube with a glass cover and kept in a CO 2 - incubator with aeration by 5 % CO 2 in air at 37 C. Assay of Ornithine Decarboxylase Activity The enzyme activity was measured by estimation of the release of [ 14 C]CO 2 from DL-[l- 14 C]ornithine. A crude enzyme extract was prepared from cells (1 x 10 7 ), which were suspended in 0.1 ml. of 50 mm Tris (ph 7.4)/200 /IM pyridoxal phosphate/0.1 ITIM EDTA/2 ITIM dithiothreitol. The cells were disrupted by freeze-thawing three times and then centrifuged at 30,000 x g for 30 min. The supernatant (0.1 ml) was reacted with DL-[l- 14 C]ornithine (0.25 /uci) and L-ornithine (40 nmol). [ 14 C]CO 2 released was measured as described previously (17). Assay of [ 3 H]'Difluoromethylomithine Binding to Ornithine Decarboxylase Crude enzyme extracts were prepared as described above. The assay mixture contained 50 mm Tris-HCl (ph 7.4)/ 200 /.«M pyridoxal phosphate/0.1 mm EDTA/2 mm dithiothreitol/0.02% Brij 35/0.6 /IM [^difluoromethylomithine (2 /*Ci)/30,000 x gr supernatant of cell extract (100 /xg protein) in a final volume of 50 /A. After incubation at 37 C for 2.5 h, [ 3 H]- difluoromethylornithine-bound ornithine decarboxylase was precipitated by addition of 100 /il of cold 1 M perchloric acid. The precipitate was washed four times with cold 0.5 M perchloric acid and once with 100% ethanol. Then it was dissolved in 0.1 N NaOH and the radioactivity was measured using a toluene-based scintillator containing 0.4% DPO, 0.01% POPOP, and 33% Triton X-100. Incorporation of [ z H]Leucine into Acid-Insoluble Fraction After incubation of cells in Eagle's minimum essential medium containing 5% horse serum and 2 mm glutamine for 18 h, the medium was replaced by fresh medium from which leucine was depleted. The cells were incubated with phytohemagglutinin and retinoic acid for 6 h and then labeled with [ 3 H]leucine (1 /tci/ml) for the last 1.5 h. The labeled cells were centrifuged at 700xg for 10min and then suspended in 10% trichloroacetic acid. After standing in ice for 30 min, the precipitates were collected on glass microfiber filters (Whatman GF/B). The filters were washed with 5% trichloroacetic acid, dried and placed in counting vials containing 5 ml of a toluene-based scintiliator (0.4% DPO, 0.01% POPOP) and radioactivity was measured using a J. Biochem.

POTENTIATION BY RETINOIC ACID OF ORNITHINE DECARBOXYLASE INDUCTION 1791 Packard Tri-Carb liquid scintillation counter. Incorporation of [ z H]Thymidine into Acid- Insoluble Fractions Cells suspended in Eagle's minimum essential medium containing 5% horse serum and 2 mm glutamine were incubated with phytohemagglutinin and retinoic acid for 48 h. f H]Thymidine (1 /ici/ml) was added at 47 h after the start of the incubation and the incorporation of [ 3 H]thymidine into the acid-insoluble fraction was measured as described for the incorporation of [ 3 H]leucine. Estimation of Intracellular Polyamine Levels Cells were harvested by centrifugation at 700 x g for 5 min and washed once with phosphate-buffered saline. The cells (2 x 10 7 ) were suspended in 0.4 N perchloric acid (100 fii) and disrupted by freezethawing three times. After centrifugation at 30,000 x g for 30 min, portions (20 /A) of the supernatant were analyzed for polyamines with a Shimadzu LC-3A liquid chromatograph using a cation-exchange column (ISC-05). The columii was eluted with 0.2 N sodium citrate containing 2.5 M NaCl. Polyamines separated by the column were reacted with O-phthalaldehyde and the fluorescence was measured. RESULTS Effects of Retinoic Acid on Omithine Decarboxylase Induction Caused by When cells were incubated with phytohemagglutinin together with various concentrations of retinoic acid, retinoic acid stimulated the ornithine decarboxylase activity in a dose-dependent manner (Table I). The extent of the stimulation by retinoic acid was highest when the cells were stimulated with phytohemagglutinin at the concentration which caused maximum induction of ornithine decarboxylase (Fig. 1). Retinoic acid did not 0.25 0.5 1.0 2.0 3.8 (/ig/m0 7.5 Fig. 1. Effect of retinoic acid on ornithine decarboxylase induction caused by various concentrations of phytohemagglutinin. Cells were incubated with various concentrations of phytohemagglutinin together with ( ) or without (O) retinoic acid (20 im) for 6 h and then ornithine decarboxylase activity was measured. Each point is the mean of duplicate experiments. TABLE I. Effect of concentration of retinoic acid on ornithine decarboxylase induction. Cells were incubated with phytohemagglutinin (7.5 /«g/ml) in the presence of various concentrations of retinoic acid for 6 h and then ornithine decarboxylase activity was measured as described in " MATERIALS AND METHODS." Each value is the mean±s.e. of triplicate experiments. Mitogen Additions Concentration of retinoic acid (JIM) Ornithine decarboxylase activity (nmol CO 2 /h/10 7 cells) None 0 0 0.05 0.5 5.0 50.0 0.021+0.001 0.219+0.007 0.253±0.011 0.287+0.006 0. 384±0.017 0.457 ±0.012 Vol. 99, No. 6, 1986

1792 S. OTANI, I. MATSUI-YUASA, Y. MIMURA, and S. MORISAWA affect the ornithine decarboxylase activity of cells incubated in the absence of phytohemagglutinin, indicating that retinoic acid itself could not induce the enzyme activity, but potentiated the enzymeinducing ability of phytohemagglutinin. Time Course of Ornithine Decarboxylase Induction Caused by in the Presence or Absence of Retinoic Acid In order to know whether retinoic acid affects the time course of ornithine decarboxylase induction, the time course in the presence or absence of retinoic acid was studied. The time course of the enzyme induction in the presence of retinoic acid was similar to that in the absence of the drug. The peak of the induction was 6-7 h after addition of phytohemagglutinin (Fig. 2). Effects of Addition Time of Retinoic Acid on Ornithine Decarboxylase Induction We examined the effect of time of addition of retinoic acid on the enzyme induction. Figure 3 shows that the stimulatory effect was marked when retinoic acid was added not later than 2 h after phytohemagglutinin. Effects of Retinoic Acid and Retinyl Acetate on Ornithine Decarboxylase Induction Caused by or PMA To determine whether retinoic acid potentiation of ornithine decarboxylase induction is specific for the induction 11 ">> o0.4 x o o t- 2 \ Q> \ 0.3 w o time ol C s 1 o 0.5 0.2 0.1 0 A / Jo / /\.// / / \ c / / / > *?? 0 2 4 6 8 Time (h ) Fig. 2. Time course of ornithine decarboxylase induction. Cells were incubated with phytohemagglutinin (7.5 /ig/ml) in the presence ( ) or absence (O) of retinoic acid (20 /IM), and harvested at the times indicated. The ornithine decarboxylase activity was measured. Each point is the mean of duplicate experiments. caused by phytohemagglutinin and whether a retinoid other than retinoic acid could also potentiate the induction, we examined the effects of retinoic acid and retinyl acetate on phytohemagglutininor PMA-induced ornithine decarboxylase activity. Retinyl acetate as well as retinoic acid augmented both phytohemagglutinin- and PMA-induced ornithine decarboxylase activity (Table II). Effect of Retinoic Acid on [ Z H]Difluoromethylornithine Binding to Ornithine Decarboxylase To determine if the increase in ornithine decarboxylase activity caused by retinoic acid results from an increase in the amount of the enzyme protein, the amount of the enzyme protein was measured by estimation of the binding of [ 3 H]difiuoromethylornithine to ornithine decarboxylase (19). Table III shows that [ 3 H]difluoromethylornithine binding to soluble protein extracted from cells treated with phytohemagglutinin or PMA was potentiated by treatment of the cells with retinoic acid. Ornithine decarboxylase activity paralleled [ 3 H]difluoromethylornithine binding, suggesting that retinoic acid -18-1 0 1 2 3 4 5 6 Time of addition of retinoic acid (h ) Fig. 3. Effect of time of addition of retinoic acid on ornithine decarboxylase induction caused by phytohemagglutinin. Retinoic acid (20 /IM) was added at the times indicated before or after phytohemagglutinin (7.5 /ig/ml) addition. Cells were harvested at 6 h and then ornithine decarboxylase activity was measured. The results are expressed as percent stimulation with respect to ornithine decarboxylase activity of cells treated with phytohemagglutinin alone (286±12pmol CO 2 /h/10 7 cells). Each point and vertical bar is the mean of triplicate experiments with the standard error. /. Biochem.

POTENTIATION BY RETINOIC ACID OF ORNITHINE DECARBOXYLASE INDUCTION 1793 TABLE n. Effects of retinoids on omithine decarboxylase induction caused by phytohemagglutinin or PMA. Cells were incubated with phytohemagglutinin (7.5 /'g/ml) or PMA (100 ng/ml) in the presence or absence of retinoid for 6 h and the omithine decarboxylase activity of the cells was measured. The concentrations of retinyl acetate and retinoic acid were 5 /IM in Experiment 1 and 10 //M in Experiment 2. Each value is the mean+s.e. of quadruplicate experiments. Mitogen Retinoid Omithine decarboxylase activity (nmol CO 2 /h/10 7 cells) Experiment 1 PMA PMA PMA Experiment 2 None Retinoic acid Retinyl acetate None Retinyl acetate 1.021+0.044 2.300+0.060 2.304+0.039 0. 191 ±0.022 0.334±0.014 TABLE III. Binding of [ 3 H]difluoromethylornithine to guinea pig lymphocyte omithine decarboxylase. Cells were treated with phytohemagglutinin (7.5 /*g/ml) or PMA (100 ng/ml) for 6 h in the presence or absence of retinoic acid (20 /<M). Crude enzyme extracts of these cells were prepared and omithine decarboxylase activity and [ 3 H]difluoromethylornithine binding to omithine decarboxylase were measured as described in " MATERIALS AND METH- ODS." Each value is the mean of duplicate experiments. Similar results were obtained from three independent experiments. Treatment of cells None +Retinoic acid PMA PMA+Retinoic acid potentiation of omithine decarboxylase activity is due to an increase in the amount of the enzyme protein, not to activation of the enzyme. Since it is possible that the omithine level in crude enzyme extract affects [ 3 H]difiuoromethylornithine binding, we tested the effect of dialysis of the crude enzyme extract on the binding. Removal of amino acids from the crude extract did not affect the binding (data not shown). Effect of Retinoic Acid on [ 3 H]Leucine Incorporation into Acid-Insoluble Fraction It is possible that the potentiation of omithine decarboxylase induction by retinoic acid is ascribable to a nonspecific enhancement of protein biosynthesis. To test this, we studied the effect of retinoic acid on Omithine decarboxylase activity (nmol CO 2 /h/mg protein) 0.3 8.0 14.3 21.3 51.4 [ 3 H]Difluoromethylornithine bound (fmol/mg protein) 5.9 172.6 265.6 382.9 1,147.9 [ 3 H]leucine incorporation into acid-insoluble fraction. The amount of [ 3 H]leucine incorporated into acid-insoluble fractions of unstimulated, phytohemagglutinin-stimulated and phytohemagglutinin plus retinoic acid-treated cells were 52,191 ±841 cpm/10' cells, 79,583 ±6,501 cpm/10 7 cells and 78,095 ± 4,628 cpm/10' cells, respectively. stimulated the incorporation. The incorporation of cells treated with phytohemagglutinin together with retinoic acid was not more than that of cells treated with phytohemagglutinin alone, suggesting that retinoic acid potentiation of omithine decarboxylase induction is not due to a nonspecific stimulation of general protein synthesis. Effects of Retinoic Acid on Decrease in Orni- Vol. 99, No. 6, 1986

1794 S. OTANI, I. MATSUI-YUASA, Y. MIMURA, and S. MORISAWA thine Decarboxylase Activity after Addition of Actinomycin D or Cycloheximide The ornithine decarboxylase activity of cells treated with phytohemagglutinin for 6 h in the absence of retinoic acid decreased rapidly after addition of actinomycin D (Fig. 4A). In contrast, cells treated with phytohemagglutinin for 6 h in the presence of retinoic acid retained the enzyme activity at a high level for 60 min after actinomycin D addition and then the enzyme activity decreased. However, the enzyme activity of phytohemagglutinin plus retinoic acid-treated cells decreased rapidly, like that of cells treated with phytohemagglutinin alone after addition of cycloheximide (Fig. 4B). To know whether 30 60 90 120 Time ( min ) 30 60 90 Time (min) Fig. 4. Effects of retinoic acid on the decrease of ornithine decarboxylase activity after actinomycin D (A) or cycloheximide (B) addition. Cells were preincubated for 6h with phytohemagglutinin (7.5^g/ml) or PMA (100 ng/ml) in the presence or absence of retinoic acid (20 //M). After addition of actinomycin D (2.5 /<g/ml) or cycloheximide (20 ^g/ml), cells were harvested at the times indicated, and the ornithine decarboxylase activity of the cells was measured. The results are expressed as percentages of ornithine decarboxylase activities at zero time. The activities at zero time were as follows; A, 481 ±15 pmol CO 2 /h/10 7 cells (phytohemagglutinin alone), 730±2 pmol CO 2 /h/10' cells (phytohemagglutinin plus retinoic acid), 891 ±70 pmol CO 2 /h/10 7 cells (PMA alone), l,458±50pmol CO 2 /h/10' cells (PMA plus retinoic acid); B, 218±12pmol CO 2 /h/10 7 cells (phytohemagglutinin alone), 349 ±14 pmol CO 2 /h/10 7 cells (phytohemagglutinin plus retinoic acid), 536± 30 pmol CO 2 /h/10 7 cells (PMA alone), 878±21 pmol CO 2 /h/10 7 cells (PMA plus retinoic acid). Each point is the mean of triplicate experiments. (O), phytohemagglutinin; ( ), phytohemagglutinin plus retinoic acid; (A), PMA; (A), PMA plus retinoic acid. the stabilization of ornithine decarboxylase activity by retinoic acid after actinomycin D addition is specific to phytohemagglutinin-treated cells, we tested the effect of retinoic acid on PMA-treated cells. Retinoic acid, stabilized ornithine decarboxylase activity after actinomycin D addition in PMA-treated cells, as observed in phytohemagglutinin-treated cells (Fig. 4A). The PMA-induced increase in ornithine decarboxylase activity was not prolonged by retinoic acid after cycloheximide addition (Fig. 4B). Effects of Retinoic Acid on Polyamine Levels of Lymphocytes Since the results described above show that retinoic acid potentiated ornithine decarboxylase induction, we tested the effect of retinoic acid on intracellular levels of -polyamines (Fig. 5). Putrescine level was higher in phytohemagglutinin-stimulated cells than in unstimulated cells. Retinoic acid treatment augmented the phytohemagglutinin-induced increase in putrescine level as well as ornithine decarboxylase activity. The spermidine level of phytohemagglutinin-stimulated cells was also higher than that of unstimulated cells and was potentiated by retinoic acid, although Fig. 5. Effects of retinoic acid on intracellular levels of putrescine (A) and spermidine (B). Cells were incubated with phytohemagglutinin (7.5 ^g/ml) with or without retinoic acid (20 /*M) and harvested at the times indicated. Polyamines were extracted with 0.4 N perchloric acid. Concentrations of polyamines were determined by liquid chromatography as described in " MATERIALS AND METHODS." Each point and vertical bar is the mean of triplicate experiments with the standard error. (O), none; ( ), phytohemagglutinin; (A), phytohemagglutinin plus retinoic acid. J.Biochem.

POTENTIATION BY RETINOIC ACED OF ORNITHINE DECARBOXYLASE INDUCTION 1795 the extent of the potentiation was less than that of putrescine level. The concentration of spermine was higher than those of putrescine and spermidine and was not influenced by phytohemagglutinin and retinoic acid by 8 h after addition of the mitogen (data not shown). Effect of Retinoic Acid on [ 3 H]Thymidine Incorporation into Acid-Insoluble Fraction The results described above show that retinoic acid stimulated phytohemagglutinin-induced ornithine decarboxylase activity and accumulation of polyamines. Since we observed that the stimulation of ornithine decarboxylase activity paralleled that of DNA synthesis (77), we tested the effect of retinoic acid on [ 3 H]thymidine incorporation into DNA. Retinoic acid at the concentrations which potentiated ornithine decarboxylase activity enhanced the incorporation of [ 3 H]thymidine into acid-insoluble fraction (Fig. 6). However, retinoic acid at the concentration of 100 /im inhibited DNA synthesis. Judging from the results of the trypan blue exclusion test, the viability of cells treated 1 10 20 30 50 100 Retinoic acid (fm ) Fig. 6. Effect of retinoic acid on DNA synthesis. Cells were incubated with phytohemagglutinin (7.5 /*g/ml) and various concentrations of retinoic acid for 48 h. [ 3 H]- Thymidine (1 /<Ci/ml) was added at 47 h. The cells were labeled for 1 h and then incorporation of radioactivity into acid-insoluble fraction was measured as described in "MATERIALS AND METHODS." Each point and vertical bar is the mean of triplicate experiments with the standard error. with retinoic acid at 100 /<M for 48 h was low, suggesting that the inhibition of DNA synthesis was due to cytotoxicity of retinoic acid (data not shown). DISCUSSION Previous papers have shown that retinoic acid inhibited ornithine decarboxylase induction in various experimental systems. PMA-induced ornithine decarboxylase activity in whole mouse skin (10, 12) and cultured mouse epithelial cells (11) was inhibited by retinoic acid. Ornithine decarboxylase induction in rat kidney cells by various growth factors was also inhibited by retinoic acid (13). In contrast to these results, our results presented here show that retinoic acid potentiated phytohemagglutinin- or PMA-induced ornithine decarboxylase activity in guinea pig lymphocytes. Kensler et al. (9) reported that retinoic acid inhibited synergistic induction of ornithine decarboxylase by PMA in phytohemagglutinin-treated lymphocytes and this inhibition required administration of retinoic acid to the lectin-activated lymphocytes 1 h prior to PMA. Figure 3 shows that addition of retinoic acid 1 h prior to phytohemagglutinin did not inhibit, but rather stimulated the enzyme induction. Schroder et al. (20) showed that treatment of murine 3T3 cells with retinoic acid for less than 24 h potentiated the mitogenic response of the cells to PMA, but longer treatment inhibited the cell growth. Kensler et al. (9) measured the ornithine decarboxylase activity of bovine lymphocytes after incubation of the cells with phytohemagglutinin, PMA and retinoic acid for 18 h. The enzyme activity in guinea pig lymphocytes activated by phytohemagglutinin peaked at 6 h, at which time we measured the activity to test the effect of retinoic acid on it. Therefore, it is possible that the exposure period to retinoic acid in our experiments is not sufficient to inhibit the enzyme induction. However, this seems unlikely, because retinoic acid added 18 h prior to phytohemagglutinin also potentiated phytohemaglutinin-induced enzyme activity (Fig. 3). Recently, Chaproniere and Weber (21) demonstrated that retinyl acetate has biphasic effects on epidermal growth factor or insulin-induced proliferation of prostatic epithelium. Retinyl acetate at 3 x 10-' M inhibited the proliferation stimulated by epidermal Vol. 99, No. 6, 1986

1796 S. OTANI, I. MATSUI-YUASA, Y. MIMURA, and S. MORISAWA growth factor. However, higher concentrations of retinyl acetate enhanced the mitogenic activity of epidermal growth factor. We tested the effect of retinoic acid at concentrations of 10~ 8 10~ 10 M on ornithine decarboxylase induction caused by phytohemagglutinin, but could not observe any inhibition of the induction (data not shown). As shown in Fig. 4A, retinoic acid-treated cells retained ornithine decarboxylase activity at a high level for longer periods after actinomycin D addition than did control cells. This suggests that retinoic acid potentiates phytohemagglutinin- or PMA-induced ornithine decarboxylase activity through a post-transcriptional mechanism. However, we cannot exclude the possibility that retinoic acid potentiates phytohemagglutinin- or PMAinduced transcription of ornithine decarboxylase gene and the resultant accumulation of ornithine decarboxylase mrna in retinoic acid-treated cells causes the prolongation of the enzyme activity after actinomycin D addition. It has been demonstrated that retinoids bind to a specific cytosolic protein receptor, migrate into the nucleus (22, 23) and influence gene expression (24). Therefore, it is possible that retinoic acid stimulates phytohemagglutinin- or PMA-induced gene expression, resulting in the potentiation of ornithine decarboxylase activity at the transcriptional level. There have been some reports showing that ornithine decarboxylase activity is regulated at the posttranslational level; retinoic acid induces transglutaminase, which inhibits ornithine decarboxylase activity through post-translational modification of the enzyme protein (25); further, antizyme, a specific inhibitor protein, binds to ornithine decarboxylase, resulting in a loss of the enzyme activity (26). However, it is unlikely that the retinoic acid potentiation of ornithine decarboxylase activity observed in our experiments is due to a posttranslational mechanism, because the half-life of the enzyme activity of retinoic acid-treated cells after cycloheximide addition was the same as that of control cells (Fig. 4B). Retinoic acid added 3 h after phytohemagglutinin addition did not potentiate ornithine decarboxylase induction caused by phytohemagglutinin (Fig. 3), and the retinoid added at the same time as actinomycin D did not prevent the decrease in ornithine decarboxylase activity (data not shown), suggesting that some metabolite derived from retinoic acid or some biochemical change induced by incubation of the cells with retinoic acid is involved in the stimulatory effect on ornithine decarboxylase activity. We reported previously that ornithine decarboxylase activity was inhibited when dibutyryl cyclic AMP and 3-isobutyl-l-methylxanthine were added simultaneously with phytohemagglutinin, but the decay of the enzyme activity after actinomycin D addition in the cells treated with phytohemagglutinin plus dibutyryl cyclic AMP and 3-isobutyl-l-methylxanthine was significantly slower than that in cells treated with phytohemagglutinin alone (27). The difference in the decay of ornithine decarboxylase activity was not observed when cycloheximide instead of actinomycin D was added. These results are similar to those shown in Fig. 4. Therefore, it is possible that the decrease in the decay of ornithine decarboxylase activity caused by treatment of cells with retinoic acid occurs through a cyclic AMP-dependent mechanism. This is consistent with the previous findings that retinoic acid potentiates prostaglandin production in dog kidney (MDCK) cells (28) and cyclic AMP-dependent protein kinase activity in mouse melanoma cells (29). However, the potentiation of ornithine decarboxylase activity by retinoic acid was not inhibited by indomethacin (data not shown). Further, although the cyclic AMP level of cells treated with phytohemagglutinin plus retinoic acid for 6 h was 1.2-fold higher than that of cells treated with phytohemagglutinin alone (data not shown), it is not clear now whether the increase in cyclic AMP level is responsible for the decrease in the decay of ornithine decarboxylase activity after actinomycin D addition. Further studies are necessary to elucidate in more detail the mechanism of the potentiation of ornithine decarboxylase activity by retinoic acid. REFERENCES 1. Lotan, R. (1980) Biochim. Biophys. Ada 605, 33-91 2. Dicker, P. & Rozengurt, E. (1979) Biochem. Biophys. Res. Commun. 91, 1203-1210 3. Dicker, P. & Rozengurt, E. (1980) Nature 289, 607-612 4. Schroder, E., Chou, L., & Black, P. (1980) Cancer Res. 40, 3038-3094 5. Jetten, A.M. (1984) Fed. Proc. 43, 134-139 6. Harper, R. & Savage, C.R. (1980) Endocrinology 107,2113-2114 /. Biochem.

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