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The role of calcium in the induction of ornithine decarboxylase in rat HTC cells
Life Sciences, Vol. 29, pp. 707-710 Printed in the U.S.A.
Pergamon Press
THE ROLE OF CALCIUM IN THE INDUCTION OF ORNITHINE DECARBOXYLASE IN RAT HTC CELLS
Zoe Nakos Canellakis #*, Theoharis C. Theoharides, and Efthalia T. Triarhos +.
Philip K. Bondy
Veterans Administration Medical Center, W~st Haven, CT 06516 and Department of Internal Medicine, and *Pharmacology Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510 (Received in final form June 8, 1981) Summary The possible role of calcium ions in the induction of ornithine decarboxylase (ODC) in rat hepatoma cells in culture (HTC) has been investigated by manipulating cellular calcium levels as follows: a) use of the calcium chelating agent EGTA to inhibit induction of ODC by dibutyryl cyclic AMP (cAMP), b) addition of Ca ++ to reverse the inhibition of cAMP induction of ODC by EGTA, c) use of a calcium ionophore in the presence of Ca ++ to induce ODC. In each case there was positive evidence for the participation of Ca ++ in the i~duction of ODC. Introduction Since Ca ++ has been implicated in the proliferative phase in many biological systems (I), we have attempted to evaluate its role in the induction of ODC, the first and rate limiting enzyme in po!yamine biosynthesis (2). Because of its well documented elevation in situations of rapid growth, ODC probably has a regulatory function in cellular metabolism (3,4). Experiments were therefore performed in hepatoma cells in culture to investigate the role of Ca ++ in ODC induction with the calcium chelator EGTA and with the calcium ionophore A23187. These studies support a role for Ca ++ in the induction of ODC. Methods HTC cells (Morris rat hepatoma 7288C) were maintained under standard conditions in suspension culture as reported earlier (5). Diluted cultures were permitted to grow for 16 hours with stirring at 37 ° to approximately 5xi05 per ml. After al~quoting volumes of 20 ml into 125 m] capped Erlenmeyer flasks, additions were made as indicated in the legends to the figures, in volumes not in excess of I% of the final volume, and the flasks incubated with shaking at 37 ° . Samples were taken for assay of ODC activity as described previously (6). Controls indicate ODC activity in non-induced cells. Results represent the mean ~ S.D. of at least two experiments with assays performed in triplicate.
+Present Address: Department of Biology, American University, #To whom reprint requests may be addressed. 0024-3205/81/070707-04502.00/0 Copyright (e) 1981 Pergamon Press Ltd.
Washington,D.C.
708
Ca++ and ODC in HTC Cells
Vol. 29, No. 7, 1981
The cation ionophore A23187, a gift from Dr. Robert J. Hosley (Eli Lilly), was dissolved in dimethyl suifoxide (DMSO; I mg/ml) and stored at -20 ° until used. Sodium salts of dibutyryl cAMP (Boehringer-Mannheim), ethyleneglycolbis(aminoethylether) tetraacetic acid (EGTA, Eastman)and ethylene diaminetetraacetic acid (EDTA, Fischer), were dissolved in water prior to use. Calcium chloride and magnesium chloride were the source of calcium and magnesium ions respectively. Results HTC cells growing in log phase suspension culture regularly responded to added 5xi0 -4 M cAMP with a maximum enhancement of ODC activity of approximately 5 fold at 2 hr. This level of induction corresponded to ODC activity of about 400 pmo!es CO2/hr/5xl05 cells. In early studies we observed that exposure of the cells to 10 -4 M EDTA for 30 min prior to the addition of cAMP caused approximately 80% inhibition of the cAMP induction of ODC. Since EGTA chelates Ca ++ preferentially, we studied the effect of 30 min preincubation with this chelator on ODC induction by cAMP. The dose-response curve shows complete inhibition of cAMP induction of ODC at 5xlO -4 M EGTA (Figure I) . Cells pretreated for 30 min with 5xlO-4M EGTA and then induced with 5xi0 -4 M cAMP in the presence of various concentrations of Ca ++ for 2 hours exhibited maximum enhancement of ODC activity at about 10-3 M Ca ++ (Figure 2). This peak effect of 10 -3 M Ca ++ in reversing the EGTA-induced inhibition of cAMP induction occurred at 2 hours of incubation, which coincided with the time peak in cAMP induction as well (unpublished data). Addition of Ca ++ in the absence of cAMP resulted in a small but reproducible increase of ODC activity in a dose-dependent manner.
250
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150
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EGTA (rnM]
Figure I Effect of EGTA on the cAMP induction of ODC. HTC cells were exposed for 30 min to increasing concentrations of EGTA as indicated and then incubated with 5x10-4M cAMP for 2 hr.
0
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,
,,,,,I
O5
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I0
(mM]
Figure 2 Effect of Ca ++ restoration on the induction of ODC by EGTA. HTC cells wer~ exposed for 30 min to 5x10-4M EGTA, followed by incubation with 5x10-4M cAMP and increasing concentration of Ca ++ as indicated for 2 hr.
Vol. 29, No. 7, 1981
Ca ++ and ODC in HTC Cells
709
In order to investigate the possibility that Ca ++ may have a more specific role in inducing ODC activity, the calcium ionophore A23187 was tested in various concentrations in the presence of optimal levels of 10 -3 M Ca ++ in the absence of cAMP. The dose response curve for A23187 shows approximately a 6 fold induction at a level of O.1 ~g/m! ionophore (Figure 3). Dimetbyl sulfoxide, the diluent for the ionophore, had no effect on induction.
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A23187 (/,.glint)
Figure 3 Effect of A23187 and Ca ++ on ODC activity. HTC cells were incubated with 10-3M Ca ++ and ~ncreasing concentrations of the cation ionophore A23187 as indicated for 2 hrs. In the presence of 0.1 pg/ml A23187, I0-3M Mg ++ could not substitute for Ca ++ .
Discussion Early experiments had shown that cAMP could cause a 3 to 5 fold induction of ODC activity in HTC cells (7) which was largely due to de novo synthesis of enzyme protein (8). ODC activity has been routinely induced in HTC cells by dilution (9) and induction by cAMP has been documented in Reuber 35 hepatoma cells in culture (10). In HTC cells, in particular, cAMP has also been shown to induce tyroslne aminotransferase, another enzyme with a short half-life like ODC (11-12). We had postulated that Ca ++ , because of its known involvement in cell proliferation (I), might also have some role in the induction of ODC activity (13). Our present studies indicate that Ca ++ is required for cAMP-mediated induction of ODC in HTC cells and that Mg++ cannot serve as a substitute. We have also shown that C a + + , when added to HTC cells at extracellular concent,ations which correspond to physiological levels, can effect a significant induction of ODC activity. This effect was demonstrated either by pretreatment of stimulated HTC cells with the calcium chelator EGTA, followed by addition of Ca ++ , or by facilitating Ca ++ influx with the use of the calcium ionophore A23187 in otherwise unstimulated cells. A23187 appears to be effective over a short range of concentrations, which correspond roughly to the dose-response curve for A23187-mediated induction of ODC in guinea pig lymphocytes (14). A Ca ++ requirement for the induction of ODC has also been demonstrated recently in some other cell lines (15-17). In our studies, treatment with EGTA lowers
710
Ca ++ and ODC in HTC Cells
Vol. 29, No. 7, 198]
basal ODC activity (Figures I and 2), an effect which has also been reported in astrocytoma cells (17). Although the mechanism of the Ca ++ role in ODC induction remains to be elucidated, it may involve a uniq~le calcium-binding protein recently isolated from a Morris hepatoma (18), which appears to belong to the calmodulin-like Ca++-dependent cellular regulators (19).
Acknowledgement: Review Program.
This research was supported by funds from the V.A. Merit
References I.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
J.F. WHITFIELD, A.L. BOYNTON, J.P. MacMANUS, R.H. RIXON, M.SIKORSKA, B. TSANG, P.R. WALKER and S.S.H. SWIERENGA, Ann. N.Y. Acad. Sci. 399, 216-240 (1980). C.W. TABOR and H. TABOR, Ann. Rev. Bioehem. 45, 285-306 (1976). H.G. WILLIAMS-ASHMAN ~nd Z.N. CANELLAKIS, Perspeet. Biol. Med. ~3, 421-453 (]979) D.H. RUSSELL, Pharmacology 20, 117-129 (1980). T. GEHLERTER, J.R. EMANUEL and C.J. SPENCER, J. Biol. Chem. 247, 6197-6203 (1972). Z.N. CANELLAKIS, Biochem. Biophys. Res. Commun. (In press) T.C. THEOHARIDES and Z.N. CANELLAKIS, Nature 255, 733-734 (1975). Z.N. CANELLAKIS and T.C. THEOHARIDES, J. Biol. Chem. 251, 4436-4441 1976. B.L.M. HOGAN, A. McILHINNEY and S. MURDEN, J. Cell. Physiol. 83, 353-357 ( 1973). C.V. BYUS, W.D. WICKS and D.H. RUSSELL, J. Cyclic Nucleotide Res. 2, 241-250 (1976). R.H. STELLWAGEN, K.K. KOHIL and R.D. SAILOR, J. Cellular Physiol. 91, 51-62 (1977). K.W. van de POLL, J.M. van AKEN and R. van WIJK, Cell Biol. Int. Rep. 3, 247-255 (1979). T.C. THEOHARIDES, Life Sei. 27, 703-713 (1980). S. OTANI, I. MATSUI, S. NAKAJIMA, M. MASUTANI, Y. MIZOGUCHI and S. MORISAWA, J. Biochem. 8__8, 77-85 (1980). M. COSTA and J.S. NYE, Biochem. Biophys. Res. Commun. 8__5_,1156-11~4 (1978). P.A. D'AMORE and D. SHEPRO, Life Sci. 22, 571-576 (1978). J.B. GIBBS, C-Y. HSU, W.L. TERASAKI and G. BROOKER, Proc. Natl. Acad. Sci. 7_7, 995-999 (1980). J.P. MacMANUS, Bioehim. Biophys. Acta 621, 296-304 (1980). A.R. MEANS and J.R. DEDMAN, Nature 285, 73-77 (1980).
Pergamon Press
THE ROLE OF CALCIUM IN THE INDUCTION OF ORNITHINE DECARBOXYLASE IN RAT HTC CELLS
Zoe Nakos Canellakis #*, Theoharis C. Theoharides, and Efthalia T. Triarhos +.
Philip K. Bondy
Veterans Administration Medical Center, W~st Haven, CT 06516 and Department of Internal Medicine, and *Pharmacology Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510 (Received in final form June 8, 1981) Summary The possible role of calcium ions in the induction of ornithine decarboxylase (ODC) in rat hepatoma cells in culture (HTC) has been investigated by manipulating cellular calcium levels as follows: a) use of the calcium chelating agent EGTA to inhibit induction of ODC by dibutyryl cyclic AMP (cAMP), b) addition of Ca ++ to reverse the inhibition of cAMP induction of ODC by EGTA, c) use of a calcium ionophore in the presence of Ca ++ to induce ODC. In each case there was positive evidence for the participation of Ca ++ in the i~duction of ODC. Introduction Since Ca ++ has been implicated in the proliferative phase in many biological systems (I), we have attempted to evaluate its role in the induction of ODC, the first and rate limiting enzyme in po!yamine biosynthesis (2). Because of its well documented elevation in situations of rapid growth, ODC probably has a regulatory function in cellular metabolism (3,4). Experiments were therefore performed in hepatoma cells in culture to investigate the role of Ca ++ in ODC induction with the calcium chelator EGTA and with the calcium ionophore A23187. These studies support a role for Ca ++ in the induction of ODC. Methods HTC cells (Morris rat hepatoma 7288C) were maintained under standard conditions in suspension culture as reported earlier (5). Diluted cultures were permitted to grow for 16 hours with stirring at 37 ° to approximately 5xi05 per ml. After al~quoting volumes of 20 ml into 125 m] capped Erlenmeyer flasks, additions were made as indicated in the legends to the figures, in volumes not in excess of I% of the final volume, and the flasks incubated with shaking at 37 ° . Samples were taken for assay of ODC activity as described previously (6). Controls indicate ODC activity in non-induced cells. Results represent the mean ~ S.D. of at least two experiments with assays performed in triplicate.
+Present Address: Department of Biology, American University, #To whom reprint requests may be addressed. 0024-3205/81/070707-04502.00/0 Copyright (e) 1981 Pergamon Press Ltd.
Washington,D.C.
708
Ca++ and ODC in HTC Cells
Vol. 29, No. 7, 1981
The cation ionophore A23187, a gift from Dr. Robert J. Hosley (Eli Lilly), was dissolved in dimethyl suifoxide (DMSO; I mg/ml) and stored at -20 ° until used. Sodium salts of dibutyryl cAMP (Boehringer-Mannheim), ethyleneglycolbis(aminoethylether) tetraacetic acid (EGTA, Eastman)and ethylene diaminetetraacetic acid (EDTA, Fischer), were dissolved in water prior to use. Calcium chloride and magnesium chloride were the source of calcium and magnesium ions respectively. Results HTC cells growing in log phase suspension culture regularly responded to added 5xi0 -4 M cAMP with a maximum enhancement of ODC activity of approximately 5 fold at 2 hr. This level of induction corresponded to ODC activity of about 400 pmo!es CO2/hr/5xl05 cells. In early studies we observed that exposure of the cells to 10 -4 M EDTA for 30 min prior to the addition of cAMP caused approximately 80% inhibition of the cAMP induction of ODC. Since EGTA chelates Ca ++ preferentially, we studied the effect of 30 min preincubation with this chelator on ODC induction by cAMP. The dose-response curve shows complete inhibition of cAMP induction of ODC at 5xlO -4 M EGTA (Figure I) . Cells pretreated for 30 min with 5xlO-4M EGTA and then induced with 5xi0 -4 M cAMP in the presence of various concentrations of Ca ++ for 2 hours exhibited maximum enhancement of ODC activity at about 10-3 M Ca ++ (Figure 2). This peak effect of 10 -3 M Ca ++ in reversing the EGTA-induced inhibition of cAMP induction occurred at 2 hours of incubation, which coincided with the time peak in cAMP induction as well (unpublished data). Addition of Ca ++ in the absence of cAMP resulted in a small but reproducible increase of ODC activity in a dose-dependent manner.
250
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400
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200
150
-
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50
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0
#
,
,
OJ
,
,
, I , l l L
05
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EGTA (rnM]
Figure I Effect of EGTA on the cAMP induction of ODC. HTC cells were exposed for 30 min to increasing concentrations of EGTA as indicated and then incubated with 5x10-4M cAMP for 2 hr.
0
,
,
,,,,,I
O5
Ol C01oum
I0
(mM]
Figure 2 Effect of Ca ++ restoration on the induction of ODC by EGTA. HTC cells wer~ exposed for 30 min to 5x10-4M EGTA, followed by incubation with 5x10-4M cAMP and increasing concentration of Ca ++ as indicated for 2 hr.
Vol. 29, No. 7, 1981
Ca ++ and ODC in HTC Cells
709
In order to investigate the possibility that Ca ++ may have a more specific role in inducing ODC activity, the calcium ionophore A23187 was tested in various concentrations in the presence of optimal levels of 10 -3 M Ca ++ in the absence of cAMP. The dose response curve for A23187 shows approximately a 6 fold induction at a level of O.1 ~g/m! ionophore (Figure 3). Dimetbyl sulfoxide, the diluent for the ionophore, had no effect on induction.
600
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400
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. . . . . . . . .
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005
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. . . . . .
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A23187 (/,.glint)
Figure 3 Effect of A23187 and Ca ++ on ODC activity. HTC cells were incubated with 10-3M Ca ++ and ~ncreasing concentrations of the cation ionophore A23187 as indicated for 2 hrs. In the presence of 0.1 pg/ml A23187, I0-3M Mg ++ could not substitute for Ca ++ .
Discussion Early experiments had shown that cAMP could cause a 3 to 5 fold induction of ODC activity in HTC cells (7) which was largely due to de novo synthesis of enzyme protein (8). ODC activity has been routinely induced in HTC cells by dilution (9) and induction by cAMP has been documented in Reuber 35 hepatoma cells in culture (10). In HTC cells, in particular, cAMP has also been shown to induce tyroslne aminotransferase, another enzyme with a short half-life like ODC (11-12). We had postulated that Ca ++ , because of its known involvement in cell proliferation (I), might also have some role in the induction of ODC activity (13). Our present studies indicate that Ca ++ is required for cAMP-mediated induction of ODC in HTC cells and that Mg++ cannot serve as a substitute. We have also shown that C a + + , when added to HTC cells at extracellular concent,ations which correspond to physiological levels, can effect a significant induction of ODC activity. This effect was demonstrated either by pretreatment of stimulated HTC cells with the calcium chelator EGTA, followed by addition of Ca ++ , or by facilitating Ca ++ influx with the use of the calcium ionophore A23187 in otherwise unstimulated cells. A23187 appears to be effective over a short range of concentrations, which correspond roughly to the dose-response curve for A23187-mediated induction of ODC in guinea pig lymphocytes (14). A Ca ++ requirement for the induction of ODC has also been demonstrated recently in some other cell lines (15-17). In our studies, treatment with EGTA lowers
710
Ca ++ and ODC in HTC Cells
Vol. 29, No. 7, 198]
basal ODC activity (Figures I and 2), an effect which has also been reported in astrocytoma cells (17). Although the mechanism of the Ca ++ role in ODC induction remains to be elucidated, it may involve a uniq~le calcium-binding protein recently isolated from a Morris hepatoma (18), which appears to belong to the calmodulin-like Ca++-dependent cellular regulators (19).
Acknowledgement: Review Program.
This research was supported by funds from the V.A. Merit
References I.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
J.F. WHITFIELD, A.L. BOYNTON, J.P. MacMANUS, R.H. RIXON, M.SIKORSKA, B. TSANG, P.R. WALKER and S.S.H. SWIERENGA, Ann. N.Y. Acad. Sci. 399, 216-240 (1980). C.W. TABOR and H. TABOR, Ann. Rev. Bioehem. 45, 285-306 (1976). H.G. WILLIAMS-ASHMAN ~nd Z.N. CANELLAKIS, Perspeet. Biol. Med. ~3, 421-453 (]979) D.H. RUSSELL, Pharmacology 20, 117-129 (1980). T. GEHLERTER, J.R. EMANUEL and C.J. SPENCER, J. Biol. Chem. 247, 6197-6203 (1972). Z.N. CANELLAKIS, Biochem. Biophys. Res. Commun. (In press) T.C. THEOHARIDES and Z.N. CANELLAKIS, Nature 255, 733-734 (1975). Z.N. CANELLAKIS and T.C. THEOHARIDES, J. Biol. Chem. 251, 4436-4441 1976. B.L.M. HOGAN, A. McILHINNEY and S. MURDEN, J. Cell. Physiol. 83, 353-357 ( 1973). C.V. BYUS, W.D. WICKS and D.H. RUSSELL, J. Cyclic Nucleotide Res. 2, 241-250 (1976). R.H. STELLWAGEN, K.K. KOHIL and R.D. SAILOR, J. Cellular Physiol. 91, 51-62 (1977). K.W. van de POLL, J.M. van AKEN and R. van WIJK, Cell Biol. Int. Rep. 3, 247-255 (1979). T.C. THEOHARIDES, Life Sei. 27, 703-713 (1980). S. OTANI, I. MATSUI, S. NAKAJIMA, M. MASUTANI, Y. MIZOGUCHI and S. MORISAWA, J. Biochem. 8__8, 77-85 (1980). M. COSTA and J.S. NYE, Biochem. Biophys. Res. Commun. 8__5_,1156-11~4 (1978). P.A. D'AMORE and D. SHEPRO, Life Sci. 22, 571-576 (1978). J.B. GIBBS, C-Y. HSU, W.L. TERASAKI and G. BROOKER, Proc. Natl. Acad. Sci. 7_7, 995-999 (1980). J.P. MacMANUS, Bioehim. Biophys. Acta 621, 296-304 (1980). A.R. MEANS and J.R. DEDMAN, Nature 285, 73-77 (1980).