J. Bwchem., 81, 461^t65 (1977)
L Cells1 II.
Effects of Additions of Amino Acids and Serum Yasuhide YAMASAKI and Akira ICHIHARA Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770 Received for publication, July 8, 1976
Induction of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells by components of the culture medium was investigated. It was found that further addition of amino acid mixture, but not of calf serum, to confluent cells of 5 day culture induced ODC and that this induction was accelerated by actinomycin D. In salt solution, addition of either amino acids or serum alone did not cause full induction, but addition of both together did. This induction, in contrast, was inhibited by actinomycin D. Induction by insulin, but not by cyclic AMP, was enhanced by a higher concentration of amino acids. These results can be explained by supposing that in non-growing cells there is stable RNA which is involved in ODC induction, possibly mRNA of ODC, and that the observed induction is caused by inhibition of enzyme degradation and accelerated translation, while in growing cells this RNA is unstable and ODC induction is controlled at the level of transcription.
Polyamines are known to be involved in cell replication in normal tissues, tumors and cultured tissues {1-4) and ornithine decarboxylase [L-ornithine carboxylyase, EC 4.1.1.17] (ODC), the rate-limiting enzyme in polyamine synthesis, is markedly induced in growing tissues. It is well known that addition of serum or insulin to cultures stimulates cell growth with concomitant induction of ODC (4-10). It was reported that this ODC induction is due, at least in part, to inhibition of enzyme degradation (5,6). The mechanism of stimulation of cell replication by serum is still unclear. It has been 1
This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture of Japan and from the Princess Takamatsu Cancer Research Fund. Vol. 81, No. 2, 1977
461
thought that polypeptide factors in the serum may change the intracellular concentration of cyclic nucleotides or Ca ions and that this initiates DNA synthesis (11-13). However, there are several reports on the effect of amino acid supplementation on ODC induction (14,15), showing that amino acids inhibited enzyme degradation. In previous work in this laboratory on the induction of ODC in cultured mouse L cells, actinomycin D (Act-D) was also found to induce ODC in confluent cells, but not in growing cells (16). We also showed that the turnover rates of proteins in growing and nongrowing cells are different (17). This paper describes the effects of insulin, serum and amino acids on ODC induction in growing and non-growing mouse L cells.
Downloaded from https://academic.oup.com/jb/article-abstract/81/2/461/875796 by Western Sydney University Library user on 11 January 2019
Induction of Ornithine Decarboxylase in Cultured Mouse
Y. YAMASAKI and A. ICHIHARA
462
950
Materials—Earle's balanced salt mixture and the amino acid mixture for Eagle's minimal essential medium (MEM) were obtained from Nissui Chemical Co., Tokyo. Bovine serum albumin was obtained from Sigma Chemical Co., St Louis. All other materials were as described previously (16). Assay Methods—All methods were as described previously (16). Briefly, the procedure involved culture of mouse L cells as monolayers in MEM plus 10% calf serum, and cells in either the growing (2-day culture) or confluent (5-day culture) state were treated in various ways for 4 h as indicated. Then they were homogenized and centrifuged and the ODC activity in the supernatant was measured. The activity was expressed as 14COi formed from DL-[l-14C]ornithine/30 min/mg protein. Some results are expressed as percentages of the activity at 0 h. RESULTS AND DISCUSSION It was shown previously that L cells grow as monolayers in MEM with 10% calf serum, reaching a confluent state in 5 days. The cellular level of cyclic AMP(cAMP) increased at first and ODC was induced markedly when the cells started to grow, but both cAMP and ODC decreased to basal levels when the cells reached a confluent state (16). However, as shown in Fig. 1, ODC was again induced when fresh medium with serum was added to cells in the confluent state. ODC induction was greater in cells at low density (2 x 105 cells/ml) than in confluent cells (2.5x10' cells/ml), as reported by Hogan (18). Addition of conditioned medium from 5-day cultures did not induce ODC, even in cultures at low cell density, so the induction was due to the fresh medium or calf serum. Addition of calf serum alone to conditioned medium did not induce ODC (Table I), but addition of amino acid mixture alone did cause ODC induction. This induction by amino acids in conditioned medium was not increased by further addition of calf serum. Therefore, in conditioned medium, amino acids became rate-limiting for ODC induction and serum was still effective. The effect of the concentration of amino acids on induction in conditioned medium is shown in Fig. 2. The effective concentration was similar to that in MEM. However, amino
600 •
o 'o 350 -
100
2
4 Time (h)
Fig. 1. Effect of change of medium on the induction of ODC. Confluent cells on the 5th day of culture (2.5 x 107 cells/ml) were incubated in fresh medium with 10% calf serum at the same cell density • , or diluted to a low cell density (2x 105 cells/ml) and incubated in fresh medium with 10% calf serum —O—, or in conditioned medium from a 5-day culture — O — . TABLE I. Effects of additions of amino acids and serum on the induction of ODC in confluent L cells. Cultures of confluent cells on the 5th day of culture (conditioned medium) were supplemented with either calf serum or amino acids at the same concentration as that in MEM without changing the medium and incubated for 4 h. Other cells were washed with Earle's salt medium and then incubated in salt medium with additions as indicated. Activity of ODC at 0 h was 880 cpm/30 min/mg protein. ODC activity (cpm/30 min/mg protein) Additions
Conditioned medium
None 997 10% Calf serum 782 1% Bovine serum albumin Amino acids 3,324 Ammo acids and 10% calf 3,513 serum Ammo acids and 1% bovine serum albumin
Salt medium 255 600 941 821 3,607 1,638
J. Biochem.
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MATERIALS AND METHODS
INDUCTION OF ORNTTHINE DECARBOXYLASE IN L CELLS. II
463
Medium Conditioned plus Act-D plus amino acids plus amino acids and Act-D Fresh and 10% calf serum plus Act-D 1
2
ODC activity (cpm/30 min/mg protein) 1,110 1,410 2,944 4,256 2,423 1,975
3
Amlno Adds (fold)
Fig. 2. Effect of the concentration of amino acids on ODC induction. Ammo acid mixture of the same concentration as that in MEM was added to confluent cells without change of medium. The concentration of amino acids is shown as a ratio to that in MEM.
acids alone were not sufficient to induce ODC in L cells suspended in salt solution, as shown in Table I. Similarly, neither 1 % bovine serum albumin nor 10 % calf serum alone caused full induction of ODC. The additions of amino acid mixture and 1 % bovine serum albumin had additive effects, but the sum of their effects was still only half that of amino acids and 10% calf serum. Recently, it was reported that albumin was the effective component in serum for the growth of lymphocytes (79), but under the present conditions the effect of serum did not seem to be attributable to its albumin content only, although 10% calf serum probably contains more albumin than 1 % bovine serum albumin. There are several reports describing the induction of ODC in cultured cells by amino acids or hormones without added serum (14, 20). Bachrach showed that cAMP or inducers of intracellular cAMP stimulated ODC synthesis much more without added serum than in conditioned medium. This suggested the accumulation of an inhibitor in conditioned medium (20). In this connection, Heller et al. recently reported the formation of a protein inhibitor of the enzyme in cells incubated with polyamines (21). The possible involvement of an inhibitor in the present experiments is still unclear. Vol. 81, No. 2, 1977
Table I also shows that, on incubation of cells in salt solution alone, their ODC activity decreased rapidly. This is because intracellular ODC is very unstable (3,5); we previously reported that the half-life of this enzyme in L cells is 81 min in the non-growing state and 112 min in the growing state (16). Previous work showed that Act-D alone induced ODC in non-growing cells and that the induction of ODC by cAMP or insulin can be explained by translational control of ODC synthesis (16). Table II shows that Act-D enhanced the induction of ODC by addition of amino acids in conditioned medium, but inhibited the induction by fresh medium with 10% calf serum. Therefore, the induction by fresh medium must involve synthesis of unstable RNA. Figure 3 shows that a concentration of about 5% calf serum caused maximal induction in cells at both low and high cell densities. It is also interesting that the level of ODC was high in cells at low density even without added serum. These results suggest that there may be two different mechanisms of ODC induction, one depending on cell replication and the other not. The former occurs not only in cells at low density, but also in confluent cells in complete medium for cell growth and is dependent on RNA synthesis, while the latter occurs on addition of amino acids or hormones in confluent cells. This explanation accords well with, recent reports showing biphasic induction of ODC in cultured cells, with one phase dependent on cell proliferation and the other not (9,10, 22). Simi-
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TABLE n. Effect of Act-D on the induction of ODC. Confluent cells on the 5th day of culture were either incubated in fresh medium with additions as indicated or in the same medium (conditioned medium) supplemented with the same concentration of amino acids as m MEM with or without Act-D at a concentration of 2
Y. YAMASAKI and A. ICHIHARA
464
600 o "o - Insulin
300 • Insulin ( 1 0 - ^ ) added o Control
4 10 Calf serum (%) Fig. 3. Effect of calf serum concentration on ODC induction. Confluent cells were incubated in fresh medium with various concentrations of calf serum • , or diluted to a low cell density and incubated in fresh medium with various concentrations of calf serum O.
larly, induction of hyaluronate synthetase in rat fibroblasts parallels cellular proliferation and is induced by serum and cAMP (23). It has also been found that there are two factors in serum, one for induction of the synthetase and the other for cell proliferation. Furthermore, Morley found that fetal bovine serum contains a factor that induces ODC in vivo, but he did not determine whether it was different from growth factor (24). It is known that serum, insulin and amino acids are pleiotypic effectors of cell growth (25) and these compounds all induce ODC. In cultured fetal rat hepatocytes arginine controls DNA synthesis, and it was reported that serum contains a factor stimulating the transport of arginine, whereas cAMP inhibits arginine transport (26). Insulin induced ODC in confluent L cells, as shown in Fig. 4, and this induction was enhanced by a higher concentration of amino acids, but induction by cAMP was not (Fig. 5). It has also been shown that induction by insulin only occurs in medium containing a high concentration of amino acids (8). Based on the present results and previous findings (16), the mechanisms of induction of ODC in
2
4
6
Time ( h )
Fig. 4. Induction of ODC by insulin. Insulin was added at a concentration of 1 x 10"* M to confluent cells without change of medium.
o
300 -
0
o
200 o 100
o •
o #
• 1.0 2.0 Amino acids Concentration (/old)
3.0
Fig. 5. Effects of insulin and cAMP on the induction of ODC by amino acids. Experimental conditions were as for Fig. 2, except that 1 x 10"* M insulin O, or 1 x 10"' M cAMP • was added. The activities with insulin or cAMP are expressed as percentages of those without insulin or cAMP.
/. Biochem.
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INDUCTION OF ORNTTfflNE DECARBOXYLASE IN L CELLS. H
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Vol. 81, No. 2, 1977
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Neoplastic Growth pp. 1-413, Raven Press, New York 3. Morns, D.R. & Fillingame, R.H. (1974) Ann. Rev. Biochem. 43, 303-325 4. Heby, O., Marton, L.J., Zardi, L., Russell, D.H., &. Baserga, R. (1975) Exp. Cell Res. 90, 8-14 5. Hogan, B.L.M., Mcllhinney, A., & Murden, S(1974) /. Cell. Physiol. 83, 353-358 6. Hogan, B., Shields, R., & Curtis, D. (1974) Cell 2_ 229-233 7. Lembach, K.J. (1974) Biochim. Biophys. Ada 354,. 88-100 8. Pariza, M.W., Becker, J.E., Yager, J.D , Jr., Bonney, R.J., & Potter, V.R. (1974) in Differentiation and Control of Malignancy of Tumor Cells (Nakahara, W., Ono, T., Sugimura, T., & Sugano, H., eds.) pp. 267-284, Univ. Tokyo Press, Tokyo 9. Oka, T. & Perry, J.W. (1976) /. Biol. Chem. 251,. 1738-1744 10. McCann, P.P., Tardif, C , Mamont, P.S., & Schuber^ F. (1975) Biochem. Biophys. Res. Commun. 64, 336341 11. Whitefield, J.F., MacManus, J.P., Rixon, R.H, Boynton, A.L , Youdale, T., & Swierenga, S. (1976> In Vitro 12, 1-18 12. Temin, H.M., Pierson, R.W., Jr , & Dulak, N.C. (1972) in Growth, Nutrition and Metabolism of Cellsm Culture (Rothblat, G.H. & Cristofalo, V.J., eds > Vol. 1, pp. 49-81, Academic Press, New York 13. Holley, R.W. (1975) Nature 258, 487^190 14. Hogan, B.L.M. & Murden, S. (1974) J. Cell. PhysioL 83, 345-352 15. Kay, J.E. & Lindsay, V.J. (1973) Exp. Cell Res. 77'„ 428-436 16. Yamasaki, Y. & Ichihara, A (1976) /. Biochem. 80,. 557-562 17. Tanaka, K. & Ichihara, A. (1976) Exp. Cell Res. 99, 1-6 18. Hogan, B.L.M. (1971) Biochem. Biophys. Res. Commun. 45, 301-307 19. Spieker-Polet, H & Polet, H. (1976) J. Biol. Chem251, 987-992 20. Bachrach, U (1975) Proc. Natl. Acad Sa. U.S. 11^ 3087-3091 21. Heller, J.S., Fong, W.F , & Canellakis, E.S. (1976> Proc. Natl. Acad. Sa U.S 73, 1858-1862 22. Heby, O., Gray, J.W., Lindl, P A., Marton, L.J., &Wilson, C B. (1976) Biochem. Biophys Res. Commun. 71,99-105 23. Tomida, M , Koyama, H , & Ono, T. (1975) J CelL Physiol. 86, 121-130 24. Morley, C G D . (1972) Biochem. Biophys. ResCommun. 49, 1530-1535 25. Hershko, A., Mamont, P., Shields, R., & Tomkins,. G.M. (1971) Nature New Biol. 232, 206-211 26. Paul, D. & Walter, S. (1975) J. Cell. Physiol. 85,113124 27. van Rijn, H , Bevers, M.M., van Wijk, R., & Wicks, REFERENCES W.D. (1974) J. Cell Biol. 60, 181-191 28. Elsas, L.J., Wheeler, F.B., Danner, D.J., & Dehaan,. 1. Inoue, H., Kato, Y., Takigawa, M., Adachi, K., & L. (1975) / . Biol. Chem. 250, 9381-9390 Takeda, Y. (1975) /. Biochem. 77, 879-893 29. Fulks, R.M., Li, J.B., & Goldberg, A.L. (1975) /_ 2. Russell, D.H. (ed.) (1973) Polyamines in Normal and Biol. Chem. 250, 290-298
growing and non-growing cells can be tentatively suggested to be as follows: in cells just before or during growth, such as those in fresh medium with serum, change of cyclic nucleotide concentration causes synthesis of mRNA for ODC, followed by an increase in ODC and hence in polyamine concentration, and this stimulates DNA synthesis. However, it is still uncertain whether decrease in cAMP or increase in cGMP is a prerequisite for cell growth {13). The mRNA in growing cells is unstable and hence Act-D, which inhibits the formation of mRNA, inhibits ODC synthesis in cells in fresh medium, although it may also inhibit the degradation of mRNA or ODC. Added cAMP was reported to inhibit DNA synthesis and cell growth (27), and we found that it inhibits both ODC induction and cell growth (16). It may inhibit ODC induction by inhibiting transcription of mRNA in growing cells. In non-growing cells mRNA is stable, and added cAMP stimulates the translation of mRNA. Increase of polyamine concentration cannot stimulate DNA synthesis, because this has been inhibited by cAMP or an inhibitor in the conditioned medium. In nongrowing cells, degradation of ODC is accelerated (16). ODC degradation in cultured cells is inhibited by amino acids in the medium (14,15), although this could not be demonstrated in cultured hepatocytes (8). Figure 5 shows that insulin stimulated ODC induction more when the amino acid concentration was higher, and this is compatible with the report that insulin stimulates amino acid transport (28). It was reported that insulin and amino acids inhibited protein degradation in tissue slices as well as stimulating protein synthesis (29). In non-growing cells Act-D inhibits only the degradation of ODC, because mRNA is stable. Hence Act-D alone causes induction of ODC in conditioned medium, but not in fresh medium, and it also stimulates the induction of ODC by amino acids. However, the exact mechanism of ODC induction requires further studies, because there are no reports on mRNA for ODC or the processes of its transcription and translation in cell-free systems.
L Cells1 II.
Effects of Additions of Amino Acids and Serum Yasuhide YAMASAKI and Akira ICHIHARA Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770 Received for publication, July 8, 1976
Induction of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells by components of the culture medium was investigated. It was found that further addition of amino acid mixture, but not of calf serum, to confluent cells of 5 day culture induced ODC and that this induction was accelerated by actinomycin D. In salt solution, addition of either amino acids or serum alone did not cause full induction, but addition of both together did. This induction, in contrast, was inhibited by actinomycin D. Induction by insulin, but not by cyclic AMP, was enhanced by a higher concentration of amino acids. These results can be explained by supposing that in non-growing cells there is stable RNA which is involved in ODC induction, possibly mRNA of ODC, and that the observed induction is caused by inhibition of enzyme degradation and accelerated translation, while in growing cells this RNA is unstable and ODC induction is controlled at the level of transcription.
Polyamines are known to be involved in cell replication in normal tissues, tumors and cultured tissues {1-4) and ornithine decarboxylase [L-ornithine carboxylyase, EC 4.1.1.17] (ODC), the rate-limiting enzyme in polyamine synthesis, is markedly induced in growing tissues. It is well known that addition of serum or insulin to cultures stimulates cell growth with concomitant induction of ODC (4-10). It was reported that this ODC induction is due, at least in part, to inhibition of enzyme degradation (5,6). The mechanism of stimulation of cell replication by serum is still unclear. It has been 1
This work was supported in part by grants for cancer research from the Ministry of Education, Science and Culture of Japan and from the Princess Takamatsu Cancer Research Fund. Vol. 81, No. 2, 1977
461
thought that polypeptide factors in the serum may change the intracellular concentration of cyclic nucleotides or Ca ions and that this initiates DNA synthesis (11-13). However, there are several reports on the effect of amino acid supplementation on ODC induction (14,15), showing that amino acids inhibited enzyme degradation. In previous work in this laboratory on the induction of ODC in cultured mouse L cells, actinomycin D (Act-D) was also found to induce ODC in confluent cells, but not in growing cells (16). We also showed that the turnover rates of proteins in growing and nongrowing cells are different (17). This paper describes the effects of insulin, serum and amino acids on ODC induction in growing and non-growing mouse L cells.
Downloaded from https://academic.oup.com/jb/article-abstract/81/2/461/875796 by Western Sydney University Library user on 11 January 2019
Induction of Ornithine Decarboxylase in Cultured Mouse
Y. YAMASAKI and A. ICHIHARA
462
950
Materials—Earle's balanced salt mixture and the amino acid mixture for Eagle's minimal essential medium (MEM) were obtained from Nissui Chemical Co., Tokyo. Bovine serum albumin was obtained from Sigma Chemical Co., St Louis. All other materials were as described previously (16). Assay Methods—All methods were as described previously (16). Briefly, the procedure involved culture of mouse L cells as monolayers in MEM plus 10% calf serum, and cells in either the growing (2-day culture) or confluent (5-day culture) state were treated in various ways for 4 h as indicated. Then they were homogenized and centrifuged and the ODC activity in the supernatant was measured. The activity was expressed as 14COi formed from DL-[l-14C]ornithine/30 min/mg protein. Some results are expressed as percentages of the activity at 0 h. RESULTS AND DISCUSSION It was shown previously that L cells grow as monolayers in MEM with 10% calf serum, reaching a confluent state in 5 days. The cellular level of cyclic AMP(cAMP) increased at first and ODC was induced markedly when the cells started to grow, but both cAMP and ODC decreased to basal levels when the cells reached a confluent state (16). However, as shown in Fig. 1, ODC was again induced when fresh medium with serum was added to cells in the confluent state. ODC induction was greater in cells at low density (2 x 105 cells/ml) than in confluent cells (2.5x10' cells/ml), as reported by Hogan (18). Addition of conditioned medium from 5-day cultures did not induce ODC, even in cultures at low cell density, so the induction was due to the fresh medium or calf serum. Addition of calf serum alone to conditioned medium did not induce ODC (Table I), but addition of amino acid mixture alone did cause ODC induction. This induction by amino acids in conditioned medium was not increased by further addition of calf serum. Therefore, in conditioned medium, amino acids became rate-limiting for ODC induction and serum was still effective. The effect of the concentration of amino acids on induction in conditioned medium is shown in Fig. 2. The effective concentration was similar to that in MEM. However, amino
600 •
o 'o 350 -
100
2
4 Time (h)
Fig. 1. Effect of change of medium on the induction of ODC. Confluent cells on the 5th day of culture (2.5 x 107 cells/ml) were incubated in fresh medium with 10% calf serum at the same cell density • , or diluted to a low cell density (2x 105 cells/ml) and incubated in fresh medium with 10% calf serum —O—, or in conditioned medium from a 5-day culture — O — . TABLE I. Effects of additions of amino acids and serum on the induction of ODC in confluent L cells. Cultures of confluent cells on the 5th day of culture (conditioned medium) were supplemented with either calf serum or amino acids at the same concentration as that in MEM without changing the medium and incubated for 4 h. Other cells were washed with Earle's salt medium and then incubated in salt medium with additions as indicated. Activity of ODC at 0 h was 880 cpm/30 min/mg protein. ODC activity (cpm/30 min/mg protein) Additions
Conditioned medium
None 997 10% Calf serum 782 1% Bovine serum albumin Amino acids 3,324 Ammo acids and 10% calf 3,513 serum Ammo acids and 1% bovine serum albumin
Salt medium 255 600 941 821 3,607 1,638
J. Biochem.
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MATERIALS AND METHODS
INDUCTION OF ORNTTHINE DECARBOXYLASE IN L CELLS. II
463
Medium Conditioned plus Act-D plus amino acids plus amino acids and Act-D Fresh and 10% calf serum plus Act-D 1
2
ODC activity (cpm/30 min/mg protein) 1,110 1,410 2,944 4,256 2,423 1,975
3
Amlno Adds (fold)
Fig. 2. Effect of the concentration of amino acids on ODC induction. Ammo acid mixture of the same concentration as that in MEM was added to confluent cells without change of medium. The concentration of amino acids is shown as a ratio to that in MEM.
acids alone were not sufficient to induce ODC in L cells suspended in salt solution, as shown in Table I. Similarly, neither 1 % bovine serum albumin nor 10 % calf serum alone caused full induction of ODC. The additions of amino acid mixture and 1 % bovine serum albumin had additive effects, but the sum of their effects was still only half that of amino acids and 10% calf serum. Recently, it was reported that albumin was the effective component in serum for the growth of lymphocytes (79), but under the present conditions the effect of serum did not seem to be attributable to its albumin content only, although 10% calf serum probably contains more albumin than 1 % bovine serum albumin. There are several reports describing the induction of ODC in cultured cells by amino acids or hormones without added serum (14, 20). Bachrach showed that cAMP or inducers of intracellular cAMP stimulated ODC synthesis much more without added serum than in conditioned medium. This suggested the accumulation of an inhibitor in conditioned medium (20). In this connection, Heller et al. recently reported the formation of a protein inhibitor of the enzyme in cells incubated with polyamines (21). The possible involvement of an inhibitor in the present experiments is still unclear. Vol. 81, No. 2, 1977
Table I also shows that, on incubation of cells in salt solution alone, their ODC activity decreased rapidly. This is because intracellular ODC is very unstable (3,5); we previously reported that the half-life of this enzyme in L cells is 81 min in the non-growing state and 112 min in the growing state (16). Previous work showed that Act-D alone induced ODC in non-growing cells and that the induction of ODC by cAMP or insulin can be explained by translational control of ODC synthesis (16). Table II shows that Act-D enhanced the induction of ODC by addition of amino acids in conditioned medium, but inhibited the induction by fresh medium with 10% calf serum. Therefore, the induction by fresh medium must involve synthesis of unstable RNA. Figure 3 shows that a concentration of about 5% calf serum caused maximal induction in cells at both low and high cell densities. It is also interesting that the level of ODC was high in cells at low density even without added serum. These results suggest that there may be two different mechanisms of ODC induction, one depending on cell replication and the other not. The former occurs not only in cells at low density, but also in confluent cells in complete medium for cell growth and is dependent on RNA synthesis, while the latter occurs on addition of amino acids or hormones in confluent cells. This explanation accords well with, recent reports showing biphasic induction of ODC in cultured cells, with one phase dependent on cell proliferation and the other not (9,10, 22). Simi-
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TABLE n. Effect of Act-D on the induction of ODC. Confluent cells on the 5th day of culture were either incubated in fresh medium with additions as indicated or in the same medium (conditioned medium) supplemented with the same concentration of amino acids as m MEM with or without Act-D at a concentration of 2
Y. YAMASAKI and A. ICHIHARA
464
600 o "o - Insulin
300 • Insulin ( 1 0 - ^ ) added o Control
4 10 Calf serum (%) Fig. 3. Effect of calf serum concentration on ODC induction. Confluent cells were incubated in fresh medium with various concentrations of calf serum • , or diluted to a low cell density and incubated in fresh medium with various concentrations of calf serum O.
larly, induction of hyaluronate synthetase in rat fibroblasts parallels cellular proliferation and is induced by serum and cAMP (23). It has also been found that there are two factors in serum, one for induction of the synthetase and the other for cell proliferation. Furthermore, Morley found that fetal bovine serum contains a factor that induces ODC in vivo, but he did not determine whether it was different from growth factor (24). It is known that serum, insulin and amino acids are pleiotypic effectors of cell growth (25) and these compounds all induce ODC. In cultured fetal rat hepatocytes arginine controls DNA synthesis, and it was reported that serum contains a factor stimulating the transport of arginine, whereas cAMP inhibits arginine transport (26). Insulin induced ODC in confluent L cells, as shown in Fig. 4, and this induction was enhanced by a higher concentration of amino acids, but induction by cAMP was not (Fig. 5). It has also been shown that induction by insulin only occurs in medium containing a high concentration of amino acids (8). Based on the present results and previous findings (16), the mechanisms of induction of ODC in
2
4
6
Time ( h )
Fig. 4. Induction of ODC by insulin. Insulin was added at a concentration of 1 x 10"* M to confluent cells without change of medium.
o
300 -
0
o
200 o 100
o •
o #
• 1.0 2.0 Amino acids Concentration (/old)
3.0
Fig. 5. Effects of insulin and cAMP on the induction of ODC by amino acids. Experimental conditions were as for Fig. 2, except that 1 x 10"* M insulin O, or 1 x 10"' M cAMP • was added. The activities with insulin or cAMP are expressed as percentages of those without insulin or cAMP.
/. Biochem.
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900 •
INDUCTION OF ORNTTfflNE DECARBOXYLASE IN L CELLS. H
465
Vol. 81, No. 2, 1977
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growing and non-growing cells can be tentatively suggested to be as follows: in cells just before or during growth, such as those in fresh medium with serum, change of cyclic nucleotide concentration causes synthesis of mRNA for ODC, followed by an increase in ODC and hence in polyamine concentration, and this stimulates DNA synthesis. However, it is still uncertain whether decrease in cAMP or increase in cGMP is a prerequisite for cell growth {13). The mRNA in growing cells is unstable and hence Act-D, which inhibits the formation of mRNA, inhibits ODC synthesis in cells in fresh medium, although it may also inhibit the degradation of mRNA or ODC. Added cAMP was reported to inhibit DNA synthesis and cell growth (27), and we found that it inhibits both ODC induction and cell growth (16). It may inhibit ODC induction by inhibiting transcription of mRNA in growing cells. In non-growing cells mRNA is stable, and added cAMP stimulates the translation of mRNA. Increase of polyamine concentration cannot stimulate DNA synthesis, because this has been inhibited by cAMP or an inhibitor in the conditioned medium. In nongrowing cells, degradation of ODC is accelerated (16). ODC degradation in cultured cells is inhibited by amino acids in the medium (14,15), although this could not be demonstrated in cultured hepatocytes (8). Figure 5 shows that insulin stimulated ODC induction more when the amino acid concentration was higher, and this is compatible with the report that insulin stimulates amino acid transport (28). It was reported that insulin and amino acids inhibited protein degradation in tissue slices as well as stimulating protein synthesis (29). In non-growing cells Act-D inhibits only the degradation of ODC, because mRNA is stable. Hence Act-D alone causes induction of ODC in conditioned medium, but not in fresh medium, and it also stimulates the induction of ODC by amino acids. However, the exact mechanism of ODC induction requires further studies, because there are no reports on mRNA for ODC or the processes of its transcription and translation in cell-free systems.
