Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effect of steroid hormones on liver cells in culture M. Vetterlein & L. Desser‐Wiest To cite this article: M. Vetterlein & L. Desser‐Wiest (1979) Effect of steroid hormones on liver cells in culture, Journal of Toxicology and Environmental Health, 5:2-3, 561-564, DOI: 10.1080/15287397909529767 To link to this article: http://dx.doi.org/10.1080/15287397909529767
Published online: 20 Oct 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 06 November 2015, At: 05:51
EFFECT OF STEROID HORMONES ON LIVER CELLS IN CULTURE M. Vetterlein, L. Desser-Wiest
Downloaded by [University of Florida] at 05:51 06 November 2015
Institute for Cancer Research, University of Vienna, Vienna, Austria
A variety of liver and hepatoma cell lines were incubated in the presence of corticosterone. DNA synthesis was inhibited in some but not all cell lines. One of them, the Fu 5a clone of the Reuber hepatoma cell line, was subjected to corticosterone for 48 h. Incorporation of labeled thymidine into DNA was inhibited up to 10%. Afterward, corticosterone was removed. DNA synthesis then increased 16-fold relative to controls. It was concluded that corticosterone blocks these cells in late G, phase of the cell cycle and thus leads to synchronization.
INTRODUCTION Recent investigations have shown that the physiological regulators of liver cell proliferation, the steroid hormones, influence the origin and the development of liver tumors. In vivo experiments in rats demonstrated that corticosterone blocks liver cells in late Gj phase of the cell cycle and thus prevents the cells from entering the S phase. This observation and the fact that endogenous corticosterone is quickly catabolized by liver cells led to the conclusion that corticosterone, when injected in large quantities or stimulated by stress situations, synchronizes liver parenchymal cells. EFFECT OF CORTICOSTERONE ON CULTURED LIVER CELLS In order to evaluate the individual effect of corticosterone, we tested this hormone in a series of cultured liver and hepatoma cells. To exclude any other influence on the cells, we checked whether the addition of alcohol, in which the hormones are usually dissolved, has any effect on cell proliferation. A variety of cells were subjected to different concentrations of ethanol in 10 ml growth medium. Two of three cell lines tested showed a significantly decreased cell yield after 6 d of incubation compared with controls (Table 1). To avoid a possible inhibitory effect of ethanol on cell proliferation, we evaporated the ethanol before adding the medium. This method was used in the following experiment. Four cell lines derived from normal liver tissue and four cell lines derived from hepatomas were examined to determine their response to 561[391] Copyright © 1979 by Hemisphere Publishing Corporation
562[392]
M. VETTERLEIN AND L. DESSER-WIEST
TABLE 1. Influence of Ethanol Concentration on Cell Number Cell count for ethanol addition of Cell line
0
10 Ml
20 Ml
30 MI
40 Ml
8 0 A[I
Ml Fu5a DE-TA
8.0 + 0.5 1.98 + 0.13 2.37 ± 0.6
5.75 :t 1.7 1.21 i t 0.1 1.6 i t0.6
6.67 + 1.26 1.6 ±0.4 2.6 ± 0.6
4.5 ± 1 1.3 ±0.2 1.93 ±0.1
4.13 ± 0.75 1.525 ± 0.2 1.5 ± 0.4
4.13 ± 63 1.428 ± 0.3 1.01 ± 0.12
Downloaded by [University of Florida] at 05:51 06 November 2015
°2.5 X 10s cells were seeded in 10 ml medium in plastic petri dishes and counted after 6 d of incubation.
corticosterone and the synthetic compound dexamethasone (Table 2). Clone Fu 5a of the Reuber hepatoma cell line was the most sensitive of the eight cell lines tested. Therefore the effect of corticosterone on these cells was examined in detail. The cells were seeded in plastic petri dishes in RPMI 1640 medium supplemented with 10% fetal calf serum and were incubated for 72 h at 37°C in a humidified atmosphere of 5% CO2 in air. After this incubation period the medium was removed and fresh medium supplemented with dialyzed fetal calf serum and containing corticosterone at a final concentration of 10~6 M was added. At 12, 24, and 48 h later the incorporation into DNA of labeled thymidine, added 1 h before the harvest, was measured by the method of Shatkin (1969). The incorporation decreased continuously, to 40% of control values at 24 h and 10% at 48 h (Fig. 1). The medium containing corticosterone was removed from the remaining cultures after 48 h of incubation; fresh corticosterone-free growth medium was added and DNA synthesis was measured at 4-h intervals (Fig. 2). Control cultures showed an expected increase of DNA synthesis following the change of medium. However, in cultures that had previously TABLE 2. Reactions of Different Cell Lines to Corticosterone and Dexamethasone
Cell line
Corticosterone (10-« M)
Chang liver Chimp liver
+ — — +
H4
+
Fu5a RH-A BP-RA M1
+ + — —
8PP RL
Dexamethasone (10-«/W)
+ + + + + + — +
563[393]
Downloaded by [University of Florida] at 05:51 06 November 2015
STEROID HORMONES AND LIVER CELLS IN CULTURE
12
24
36
48
h after adding corticosterone FIGURE 1. Incorporation of labeled thymidine into DNA after addition of corticosterone.
been incubated for 48 h in the presence of corticosterone, DNA synthesis was markedly delayed, probably because there was residual corticosterone in these cells. The first peak of DNA synthesis was reached 6 h later than in the controls. Afterward, DNA synthesis decreased rapidly. Between 16 and 28 h after medium change, DNA synthesis increased 16-fold. Since the duration of the second DNA synthesis phase
12
16
20
24
28
32
h after medium change FIGURE 2. Uptake of labeled thymidine (—) by cells that had been incubated for 48 h in the presence of corticosterone and (—) by control cultures without prcincubation in corticosterone.
564[394]
M. VETTERLEIN AND L. DESSER-WIEST
corresponds to the length of the S phase of these cells, we can assume synchronization during this period of the cell cycle. Autoradiographic studies are now in progress to prove these results.
Downloaded by [University of Florida] at 05:51 06 November 2015
CONCLUSIONS We conclude that corticosterone is a physiological agent that blocks liver cells in G1 phase and presumably also in late S phase, thus leading under certain conditions in vivo and in vitro to synchronization of liver cell populations. These findings have several implications. On the one hand, we know that certain carcinogens and viruses act on and transform cells only in certain phases of the cell cycle. On the other hand, synchronization of tumor cell populations has been widely used in cancer therapy. But most synchronizing agents, when effective, are highly toxic, and their application is limited. Therefore the search for physiological cell-proliferation regulators is important for a basic understanding of carcinogenesis as well as for the therapeutic management of tumor cell populations. REFERENCES Desser-Wiest, L. 1974. Stimulation of DNA synthesis in rat liver by adrenalectomy. J. Endocrinol. 60:315-319. Desser-Wiest, L. 1975. Autosynchronization of rat liver cells with endogeneous corticosterone after partial hepatectomy. Cell Tissue Kinet. 8:1-9. Klein, H., Lennartz, K. Y., and Gross, R. 1973. Pharmakologische Synchronisation von Tumorzellen und ihre Bedeutung für die zytostatische Behandlung. Aktuel. Probl. Ther. Maligner Tumoren. Int. Symp., Münster, 1972, ed. G. Wüst, pp. 270-273. Stuttgart: Thieme. Loeb, J. N., et al. 1973. Suppression of DNA synthesis in hepatoma cells exposed to glucocorticoid hormone in vitro. Proc. Natl. Acad. Sci. U.S.A. 70:3852-3856. Nitze, H. R., et al. 1973. Die Strahlenbehandlung menschlicher Tumoren nach partieller Synchronisation der Zellproliferation. Aktuel. Probl. Ther. Maligner Tumoren. Int. Symp., Münster, 1972, ed. G. Wüst, pp. 286-295. Stuttgart: Thieme. Raab, K. H. and Webb, T. E. 1969. Inhibition of DNA synthesis in regenerating rat liver by hydrocortisone. Experientia 25:1240-1243. Shatkin, Y. A. 1969. Colorimetric reactions for DNA, RNA, and protein determinations. In Fundamental Techniques in Virology, eds. K. Habel and N. P. Salzmann, pp. 231-237. New York: Academic. Zeuthen, E. 1964. Synchrony of Cell Division and Growth, pp. 1-630. New York: Interscience.
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effect of steroid hormones on liver cells in culture M. Vetterlein & L. Desser‐Wiest To cite this article: M. Vetterlein & L. Desser‐Wiest (1979) Effect of steroid hormones on liver cells in culture, Journal of Toxicology and Environmental Health, 5:2-3, 561-564, DOI: 10.1080/15287397909529767 To link to this article: http://dx.doi.org/10.1080/15287397909529767
Published online: 20 Oct 2009.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 06 November 2015, At: 05:51
EFFECT OF STEROID HORMONES ON LIVER CELLS IN CULTURE M. Vetterlein, L. Desser-Wiest
Downloaded by [University of Florida] at 05:51 06 November 2015
Institute for Cancer Research, University of Vienna, Vienna, Austria
A variety of liver and hepatoma cell lines were incubated in the presence of corticosterone. DNA synthesis was inhibited in some but not all cell lines. One of them, the Fu 5a clone of the Reuber hepatoma cell line, was subjected to corticosterone for 48 h. Incorporation of labeled thymidine into DNA was inhibited up to 10%. Afterward, corticosterone was removed. DNA synthesis then increased 16-fold relative to controls. It was concluded that corticosterone blocks these cells in late G, phase of the cell cycle and thus leads to synchronization.
INTRODUCTION Recent investigations have shown that the physiological regulators of liver cell proliferation, the steroid hormones, influence the origin and the development of liver tumors. In vivo experiments in rats demonstrated that corticosterone blocks liver cells in late Gj phase of the cell cycle and thus prevents the cells from entering the S phase. This observation and the fact that endogenous corticosterone is quickly catabolized by liver cells led to the conclusion that corticosterone, when injected in large quantities or stimulated by stress situations, synchronizes liver parenchymal cells. EFFECT OF CORTICOSTERONE ON CULTURED LIVER CELLS In order to evaluate the individual effect of corticosterone, we tested this hormone in a series of cultured liver and hepatoma cells. To exclude any other influence on the cells, we checked whether the addition of alcohol, in which the hormones are usually dissolved, has any effect on cell proliferation. A variety of cells were subjected to different concentrations of ethanol in 10 ml growth medium. Two of three cell lines tested showed a significantly decreased cell yield after 6 d of incubation compared with controls (Table 1). To avoid a possible inhibitory effect of ethanol on cell proliferation, we evaporated the ethanol before adding the medium. This method was used in the following experiment. Four cell lines derived from normal liver tissue and four cell lines derived from hepatomas were examined to determine their response to 561[391] Copyright © 1979 by Hemisphere Publishing Corporation
562[392]
M. VETTERLEIN AND L. DESSER-WIEST
TABLE 1. Influence of Ethanol Concentration on Cell Number Cell count for ethanol addition of Cell line
0
10 Ml
20 Ml
30 MI
40 Ml
8 0 A[I
Ml Fu5a DE-TA
8.0 + 0.5 1.98 + 0.13 2.37 ± 0.6
5.75 :t 1.7 1.21 i t 0.1 1.6 i t0.6
6.67 + 1.26 1.6 ±0.4 2.6 ± 0.6
4.5 ± 1 1.3 ±0.2 1.93 ±0.1
4.13 ± 0.75 1.525 ± 0.2 1.5 ± 0.4
4.13 ± 63 1.428 ± 0.3 1.01 ± 0.12
Downloaded by [University of Florida] at 05:51 06 November 2015
°2.5 X 10s cells were seeded in 10 ml medium in plastic petri dishes and counted after 6 d of incubation.
corticosterone and the synthetic compound dexamethasone (Table 2). Clone Fu 5a of the Reuber hepatoma cell line was the most sensitive of the eight cell lines tested. Therefore the effect of corticosterone on these cells was examined in detail. The cells were seeded in plastic petri dishes in RPMI 1640 medium supplemented with 10% fetal calf serum and were incubated for 72 h at 37°C in a humidified atmosphere of 5% CO2 in air. After this incubation period the medium was removed and fresh medium supplemented with dialyzed fetal calf serum and containing corticosterone at a final concentration of 10~6 M was added. At 12, 24, and 48 h later the incorporation into DNA of labeled thymidine, added 1 h before the harvest, was measured by the method of Shatkin (1969). The incorporation decreased continuously, to 40% of control values at 24 h and 10% at 48 h (Fig. 1). The medium containing corticosterone was removed from the remaining cultures after 48 h of incubation; fresh corticosterone-free growth medium was added and DNA synthesis was measured at 4-h intervals (Fig. 2). Control cultures showed an expected increase of DNA synthesis following the change of medium. However, in cultures that had previously TABLE 2. Reactions of Different Cell Lines to Corticosterone and Dexamethasone
Cell line
Corticosterone (10-« M)
Chang liver Chimp liver
+ — — +
H4
+
Fu5a RH-A BP-RA M1
+ + — —
8PP RL
Dexamethasone (10-«/W)
+ + + + + + — +
563[393]
Downloaded by [University of Florida] at 05:51 06 November 2015
STEROID HORMONES AND LIVER CELLS IN CULTURE
12
24
36
48
h after adding corticosterone FIGURE 1. Incorporation of labeled thymidine into DNA after addition of corticosterone.
been incubated for 48 h in the presence of corticosterone, DNA synthesis was markedly delayed, probably because there was residual corticosterone in these cells. The first peak of DNA synthesis was reached 6 h later than in the controls. Afterward, DNA synthesis decreased rapidly. Between 16 and 28 h after medium change, DNA synthesis increased 16-fold. Since the duration of the second DNA synthesis phase
12
16
20
24
28
32
h after medium change FIGURE 2. Uptake of labeled thymidine (—) by cells that had been incubated for 48 h in the presence of corticosterone and (—) by control cultures without prcincubation in corticosterone.
564[394]
M. VETTERLEIN AND L. DESSER-WIEST
corresponds to the length of the S phase of these cells, we can assume synchronization during this period of the cell cycle. Autoradiographic studies are now in progress to prove these results.
Downloaded by [University of Florida] at 05:51 06 November 2015
CONCLUSIONS We conclude that corticosterone is a physiological agent that blocks liver cells in G1 phase and presumably also in late S phase, thus leading under certain conditions in vivo and in vitro to synchronization of liver cell populations. These findings have several implications. On the one hand, we know that certain carcinogens and viruses act on and transform cells only in certain phases of the cell cycle. On the other hand, synchronization of tumor cell populations has been widely used in cancer therapy. But most synchronizing agents, when effective, are highly toxic, and their application is limited. Therefore the search for physiological cell-proliferation regulators is important for a basic understanding of carcinogenesis as well as for the therapeutic management of tumor cell populations. REFERENCES Desser-Wiest, L. 1974. Stimulation of DNA synthesis in rat liver by adrenalectomy. J. Endocrinol. 60:315-319. Desser-Wiest, L. 1975. Autosynchronization of rat liver cells with endogeneous corticosterone after partial hepatectomy. Cell Tissue Kinet. 8:1-9. Klein, H., Lennartz, K. Y., and Gross, R. 1973. Pharmakologische Synchronisation von Tumorzellen und ihre Bedeutung für die zytostatische Behandlung. Aktuel. Probl. Ther. Maligner Tumoren. Int. Symp., Münster, 1972, ed. G. Wüst, pp. 270-273. Stuttgart: Thieme. Loeb, J. N., et al. 1973. Suppression of DNA synthesis in hepatoma cells exposed to glucocorticoid hormone in vitro. Proc. Natl. Acad. Sci. U.S.A. 70:3852-3856. Nitze, H. R., et al. 1973. Die Strahlenbehandlung menschlicher Tumoren nach partieller Synchronisation der Zellproliferation. Aktuel. Probl. Ther. Maligner Tumoren. Int. Symp., Münster, 1972, ed. G. Wüst, pp. 286-295. Stuttgart: Thieme. Raab, K. H. and Webb, T. E. 1969. Inhibition of DNA synthesis in regenerating rat liver by hydrocortisone. Experientia 25:1240-1243. Shatkin, Y. A. 1969. Colorimetric reactions for DNA, RNA, and protein determinations. In Fundamental Techniques in Virology, eds. K. Habel and N. P. Salzmann, pp. 231-237. New York: Academic. Zeuthen, E. 1964. Synchrony of Cell Division and Growth, pp. 1-630. New York: Interscience.
