221

Biochimica et Biophysica Acta, 1052 (1990) 221-228

Elsevier BBAMCR 12662

Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin Oystein Spydevold

a,

Hilde Sorensen a, Ole Petter Clausen b and Kaare M. Gautvik a

a Institute of Medical Biochemistry and b Institute of Pathology, University of Oslo, Oslo (Norway)

(Received 30 June 1989) (Revised manuscript received 31 October 1989)

Key words: Cell cycle; DNA synthesis; Dexamethasone; Cell proliferation; (Rat hepatoma cell)

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 f m o l / m g cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyI-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methylthymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.

Introduction Considerable effort has been directed towards identification of biochemical events involved in changes of cell proliferation rates. Studies on regenerating liver following hepatectomy and on cells in culture have shown that proliferation of eukaryotic cells are regulated by a series of humoral factors including hormones [1-6]. Glucocorticoids and insulin have been shown to regulate proliferation as well as synthesis of cell components and products [1,2,7]. The effects of dexamethasone on the growth of cells in culture seems to be complex and cell-type specific. Both inhibitory as well as stimulating effects have been observed. In melanoma Abbreviations: [3H]TdR, [3H]methylthymidine; PBS, phosphatebuffered saline. Correspondence: O. Spydevold, Institute of Medical Biochemistry, P.O. Box 11120 Blindern, Oslo 3, Norway.

cells and in mouse epidermal cells the proliferation is inhibited by glucocorticosteroid hormones [8,9] and lymphoid cells are irreversibly arrested in the Gl-phase of the cell cyle [10]. In contrast to this action of dexamethasone, enhanced DNA synthesis in rat hepatocytes in culture has been reported [6,7] as well as stimulation of growth responses in fibroblasts [11,12] and in HeLa cells [13]. Furthermore, dexamethasone promotes differentiation of hepatocytes from suckling rats in culture [14]. Insulin, which is a vital hormone in metabolism, is also an important growth factor in many tissues [1-3] and seems to be necessary for the initiation of the DNA synthesis and proliferation of fiver after partial hepatectomy [1]. A clonal strain, 7800 C1 cells, derived from Morris hepatoma by Richardson et al. [15] is a potential model for growing liver cells, since it is a minimum deviation tumour which has retained several properties characteristic for normal liver cells. In this study we describe the action of dexamethasone on the growth rate characteristics of the 7800 C1 cells which contain receptors for

0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

222 corticosteroids. The results show that the cell proliferation is inhibited by low concentrations of dexamethasone, and this effect is reversed by insulin. The actions of these hormones are reflected in changes in the cell cycle distribution and mitotic rates. Materials and Methods Materials. Ham’s FlO medium, horse serum and calf serum were from Flow Laboratories (Irvine, U.K.) Anti PPLO, Fungizone, penicillin and streptomycin were from Gibco (Grand Island, New York, U.S.A.). [ 3H]Methylthymidine ([3H]TdR) was from New England Nuclear (Boston, MA, U.S.A.). RNase was from Boehringer (Mannheim, F.R.G.). Other chemicals were obtained from Sigma (St. Louis, MO, U.S.A.) Cell culture. The establishment, cloning and cell propagation of the 7800 Cl Morris hepatoma cells have been described by Richardson et al. [15]. The cells were cultivated as monolayers in 60 x 15 mm culture dishes or in 24-well dishes (Costar). The cells were grown in Ham’s F 10 medium supplemented with 10% horse serum and 3% calf serum and incubated at 37’ C in a humidified atmosphere of 5% CO, and 95% air. The growth medium was supplemented with penicillin (50 U/ml), streptomycin (50 fig/ml), fungizone (2.5 pg/ml) and anti I-PPLO (50 pi/ml) and was changed every 48 h. The cells were plated at 2-4 . lo5 cells per dish from cultures in plateau phase of growth. Cell growth. Cell growth was determined both by counting the number of cells and by measuring the increase in cellular DNA according to Labarca and Paigen [16]. Cell treatment. Dexamethasone and insulin were added to cultures in concentrations from 0.1 nM to 250 nM and from 10 nM to 400 nM, respectively, for the time periods indicated. The physiological concentrations of insulin and cortisol in serum are: 0.035-0.700 nM and 250-750 nM, respectively. The cells were harvested by washing three times in ice-cold 50 mM Tris buffer (pH 7.4). The cells were then scraped off the dishes and used for DNA estimations and enzyme analysis. [-‘H]Dexamethasone binding to intact 7800 Cl cells at different concentrations. The uptake of [3H]dexamethasone to the hepatoma cells at 37” C can be used to study the interaction between the steroid and its intracellular receptors [17]. In these experiments, cell suspensions made freshly from the stock monolayer cultures after treatment with trypsin for 1 min were used. The Trypan-blue test indicated that the cells were not damaged by the trypsin treatment. After two washes with 0.9% NaCl (5 ml), and centrifugation at 1200 rpm (Sorwall GLC 2B), the cells were resuspended in Ham’s F 10 medium supplemented with 20 mM Hepes (pH = 7.4). The binding reaction was started when cell suspen-

sions (100 ~1) were incubated with increasing concentrations of [3H]dexamethasone in the absence (total binding) or presence of a lOOO-fold molar excess of unlabelled dexamethasone (nonspecific binding). The specific binding was calculated as the difference between total and nonspecific binding. After 4 h the reaction was stopped by transferring 100 ~1 of the reaction suspension to Eppendorf tubes containing 1 ml ice-cold 0.9% NaCl. Cell-bound and free [‘Hldexamethasone were separated by pelleting the cells by spinning for 3-5 s (Eppendorf centrifuge 5414). The cell pellet was washed three times with 0.9% NaCl and dissolved in 500 ~1 0.1 M NaOH. 100 ~1 was used for protein measurements and 300 ~1 for measurement of the radioactivity (Packard Tri Carb 3255 liquid scintillation spectrometer). Micro-flow fluorometry. Cells were removed from the (60 X 15 mm) culture dishes by trypsinization, and centrifugation (5 min at 500 X g). After washing, the cells were resuspended in PBS to give single-cell suspension (controlled by microscopical examination). The cells were fixed in 96% ethanol and stored at a final concentration of 70% at 4 o C until further treatment. The cells were then centrifuged and washed in saline before incubation in RNase (1% aqueous solution) at 37 o C for 1 h followed by incubation in 0.5% pepsin at 37 o C for 15 min before staining with ethidium bromide as described by Clausen et al. [18]. After 30 min at room temperature, the cells were measured in a Scatron argus high-performance flow cytometer (Scatron, Lier, Norway) giving the DNA histograms and cell cycle distributions. The proportions of diploid, tetraploid and S phase cells were scored at intervals after the start of treatment for 2 weeks and are illustrated in Fig. 4. The mitotic rate. Preliminary results showed that 3 . lop6 M colcemide represented an optimal concentration for accumulating metaphases in this cell strain. Control dishes were incubated with this concentration of colcemide during exponential growth in parallel with dishes treated with dexamethasone and with dexamethasone plus insulin for the times indicated. Four dishes in each treatment group were harvested after 3 h of colcemide incubation, whereas control dishes were harvested at 1, 2, 3 and 4 h following colcemide incubation. The number of accumulated metaphases per 1000 cells were scored per h in the controls and per 3 h in treatment groups. Continuous [“HI TdR labelling. Cultures incubated with 250 nM dexamethasone, 250 nM dexamethasone plus 400 nM insulin and control cultures received 0.1 PCi [3H]TdR (spec. act. 6.7 Ci/mmol, 1.0 mCi/ml) for 3 days during the period of exponential growth. Dishes were harvested at day 1, 2 and 3, washed in saline, fixed in ethanol and processed for autoradiography as described by Clausen et al. [18]. The dishes were dipped in Kodak NTB 2 emulsion diluted 1 : 1 with distilled water

223 and exposed for 4 weeks and stained with haematoxylin. The portion of labelled cells was scored among 300 cells for each dish at each time point, and cells with 5 grains or more, depending on the background, were considered labelled. Measurements of enzyme actioities and protein. Lactate and glucose-6-phosphate dehydrogenases were assayed as described by Bergmeyer [19] and tyrosine aminotransferase was assayed according to Granner and Tomkins [20]. The peroxisomal acyl-CoA oxidase was measured as described by Mannaerts et al. [21]. Protein was measured by the method of Lowry et al. [22]. Statistical calculation. All the experiments were carried out 3 times with quadruplicate dishes at each time-point. The results are given as mean values ± S.E. and the level of significance calculated by the Fischers t-test. Results

Cell growth Cell growth was initially estimated both by cell counting and by measurement of cellular DNA. The curves obtained by the two methods were in excellent agreement (not shown). Since D N A measurement was the most convenient method, this was used for routine purposes. Fig. 1 shows that the cells growed exponentially for 7 days with a doubling time of approx. 36 h and the cells then reached an early plateau phase from about 9 days. The presence of dexamethasone (250 nM) significantly reduced the growth rate. The combined treatment of dexamethasone (250 nM) and insulin (400

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Fig. 1. Growth curves of 7800 C1 hepatoma cells. (©) control; (zx) 250 nM dexamethasone; (Ill) 400 mM insulin; ([2) 250 nM dexamethasone plus 400 nM insulin. Treatment was given from day 1. The results are shown as means values + S.E. The experiment was carried out 4 times and a representative experiment is shown, where each point represents 4 replicates.

TABLE I

Effect of different concentrations of dexamethasone alone and in combintion with insulin on the growth of 7800 C1 hepatoma cells The hormone additions were started at the second day of plating. The values are given as means 5- S.E. There were four observations in each group. Hormone additions to the culture media at day 1

Days in culture

~t D N A / well

None None Dexamethasone Dexamethasone Dexamethasone Insulin Dexamethasone + Insulin Dexamethasone + Insulin Dexamethasone + Insulin

2 14 14 14 14 14

0.55-0.01 8.2 5-0.56 7.2 5-0.60 3.25-0.52 2.0 5-0.40 7.9 5-0.62

14

8.0 5-1.00

14

7.8+1.00

14

4.0 + 0.64

1 10 250 400 250 400 250 50 250 10

nM nM nM nM nM nM nM nM nM nM

nM) gave rise to a growth curve closely paralleling the control curve. The growth of cells observed with insulin alone was however, not different from the control. In contrast to insulin E G F (10 -8 M and 10 -7 M) did not affect the growth rate, whether dexamethasone was present in the medium or not (data not shown). Table I shows the effect of different concentrations of dexamethasone and insulin on the growth of cells during 12 days of treatment. No significant growth inhibition was obtained with 1 nM dexamethasone. At 10 nM and 250 mM dexamethasone, the inhibitory effect was about 60 and more than 80%, respectively. When the cell growth was inhibited with 250 nM dexamethasone, insulin at both 400 and 50 nM almost completely restored the growth rate, while 10 nM of insulin had a minimal antagonistic effect. Fig. 2 shows that when dexamethasone was removed from the medium after different treatment intervals a vigorous resumption of growth started without delay and the cell mass reached the level of the control group after plateau phase had been obtained.

Binding of dexamethasone to intact 7800 C1 hepatoma cells In order to determine the binding of dexamethasone to functional receptors in the hepatoma cells, the binding of the hormone at equilibrium was studied both in control cells and in cells treated with 400 mM insulin for 7 days. Almost identical binding data were obtained from the two groups of cells, showing that insulin did not modify that dexamethasone effects by changing its binding to the cells. Only the data obtained from experiments with control cells are therefore presented. The total binding showed half-maximal saturation at about 13 nM (Fig. 3A). The maximal binding of the

steroid hormone was attained at approx. 300 nM. When the specific binding was plotted according to Scatchard [23], a linear relationship was obtained indicating one class of receptor molecules (Fig. 3B). The total binding capacity obtained from the intercept with the x-axis was found to be 24 nmol/mg cell protein and from the slope of the curve the K, was calculated to be 0.24 nM [23,24].

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Fig. 2. Reversal of the effect of 250 nM of dexamethasone on the growth of the 7800 Cl cells. When present, dexamethasone was added at day 1 of plating and the interval of treatment given for each curve given below. (0) control; (v) dexamethasone: day 1-3; (v) dexamethasone: day l-5; (A) dexamethasone: day 1-9; (A) dexamethasone: day l-19. When changed to dexamethasone-free medium, the cells were washed four times with medium to remove the hormone. The values are means f SE. of four observations.

Micro-flow jluorometry Fig. 4 shows typical DNA distributions of control cultures (top) and dexamethasone treated cultures (bottom) during exponential growth. All cultures showed one major diploid DNA stem line and a second minor tetraploid DNA stem line. Less than 10% of all cells had a DNA content higher than tetraploid. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. The tetraploid population represents G, + M cells of the diploid stem line and G, cells of the tetraploid stemline. Fig. 5A shows the changes in the proportions of cells in S phase with time after start of culture. The treatments were given from the day of plating. It is clearly shown that the proportion of cells in S phase was reduced during dexamethasone treatment, and that ad-

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Fig. 3. A. Binding of [3H]dexamethasone to 7800 Cl hepatoma cells at 37OC during addition of increasing concentrations. Monolayer cultures M) of [3H]dexamethasone for 4 h (total binding, #) or in the presence of were incubated with increasing concentrations (5. lo-” - 1 .lO-’ lOOO-fold molar excess of unlabelkd examethasone (nonspecific binding, 0). B. Scatchard analysis [23] of the specific binding (7). From the cell protein and from the slope of the curve R, was calculated to intercept with the x-axis, the binding capacity is obtained (I?,,, = 24 fmol/mg 0.24 nM. The values representing the binding are the means of four observations f S.E.

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Fig. 4. DNA distribution histograms of control cells (top) and cells treated with dexamethasone (250 mM) for 6 days (bottom). The first peak of the histograms represent the diploid state ((3]) cells) and the second peak the tetraploid cells. The area between these two peaks represents cells with S phase DNA content. The pulses to the right of the tetraploid peak represent S and G 2 phase cells of a small tetraploid DNA stemline. Note the reduced fraction of cells with an S phase DNA content in the dexamethasone treated culture.

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Fig. 6. The mitotic rate (%/h) was determined in colcemide-treated cultures. (© ©) control; (a) dexamethasone (250 mM); (C2) dexamethasone (250 mM) plus insulin (400 mM). Each value is the mean of four observations + S.E. Asterix indicates P < 0.01 compared to control.

dition of insulin to the dishes only reversed this effect at day 2, otherwise the action dexamethasone was dominating Fig. 5B shows that dexamethasone did also

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Fig. 5. The calculated cell cycle distribution data. A, percentage cells with S phase DNA content. B, percentage cells with tetraploid DNA content. (©) control, (zx) dexamethasone (250 raM), (D) dexamethasone (250 mM) Plus insulin (400 nM). Each value is the mean of four observations + S.E, Asterices indicate P < 0.01 compared to control.

226 reduce the proportion of tetraploid cells from day 3 and for the rest of the experiment, but that addition of insulin prevented this reduction completely. The mitotic rate The results are illustrated in Fig. 6. The accumulation of metaphases was linear during the first three hours after colcemide incubation of control dishes during exponential growth at concentration of 3 . lop6 M. The results show that the mitotic rate was significantly reduced during dexamethasone treatment, whereas addition of insulin was again able to almost completely reverse this effect. Continuous [3H] TdR labelling The continuous [3H]TdR labelling shows that close to 100% of cells in all treatment groups were labelled after 3 days, indicating a growth fraction of unity

TABLE

II

Effect of dexamethasone and insulin on enzyme activities in the 7800 Cl hepatoma cells The hormone additions were started after 7 days of growth. When added, 250 nM dexamethasone and 400 nM insulin were used. The medium was changed every day. After 3 days, the medium was removed and 50 mM Tris (pH 7.4) was added. The cells were then scraped off and sonicated for 10 s. The enzymes were measured as described in Materials and Methods. The values are given as means + SE. There were four observations in each group.

(Data not shown). A transient delay in labelling in the dexamethasone-treated group.

is seen

Hormonal effects on the activities of LDH, peroxisomal acyl-CoA oxidase, glucose-6-phosphate dehydrogenase and tyrosine aminotransferase. Lack of hormonal induction of albumin

synthesis

Table II shows that at 250 nM, dexamethasone induced an increase in the peroxisomal acyl-CoA oxidase of about 608, but did not affect the activity of lactate or glucose-6-phosphate dehydrogenases. At 400 nM, insulin caused a small reduction in basal as well in the dexamethasone-induced activities of the peroxisomal acyl-CoA oxidase. Tyrosine aminotransferase was increased about 15-times by dexamethasone treatment. Insulin alone did not change the activity of the enzyme but the hormone almost abolished the inductive effect of dexamethasone. No albumin production could be detected when the 7800 Cl cells were grown in the serum-free medium (Neumann-Tytell) and neither dexamethasone (250 nM) nor insulin (400 nM) induced any production (data not shown). Insulin and dexamethasone at these high concentrations therefore probably do not bring about general and unphysiological changes, but do show differentiated effects on diverse cellular processes such as cell proliferation and enzyme induction. In both instances insulin inhibited the actions of dexamethasone. Discussion

Hormone

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(nmol/ag DNA per min) None Dexamethasone Insulin None Dexamethasone Insulin Dexamethasone + Insulin None Dexamethasone Insulin None Dexamethasone Insulin Dexamethasone + Insulin

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* P < 0.01 vs. control.

The present study demonstrates that the growth rate of 7800 Cl hepatoma cells is more than 80% reduced by 250 nM dexamethasone, and significant effect is observed in concentrations as low as 10 nM. This substantial retardation of growth is in fact normalized by insulin. The results suggest that, even if the hormones have distinct modes of action, their effects appear to converge towards the same phase in the cell cycle traverse, but act in opposite directions. The effects of dexamethasone clearly are not due to general pharmacological or toxicological actions, since they are reversible and several enzymes are unaffected by the hormone, except for an observed increase in the peroxisomal acyl-CoA oxidase and tyrosine aminotransferase activity (Table II). Moreover, glucocorticoid receptors are present in 7800 Cl cells, and their specific characteristics have been studied in detail recently (Sorensen, Norrheim, Spydevold and Gautvik, unpublished data). Our data show that 2, 10 and 100% of dexamethasone receptors in the 7800 Cl cells are occupied when the cells are incubated with 1 nM, 7 nM and 250 nM of the hormone, respectively. The receptor occupation parallels the degree of growth reduction caused by the various concentrations of the hormone (Table I). The existence of

227 only one class of dexamethasone binding sites with properties known to be characteristic for glucocorticoid receptors [25] suggests that the dexamethasone dependent growth inhibition of the 7800 C1 cells is a physiological effect mediated by the hormone-receptor interaction. The stimulating effect of insulin on the cell growth is probably a specific insulin effect, since growth retardation caused by dexamethasone was not counteracted by EGF. Insulin did not counteract dexamethasone effects by altering dexamethasone receptor affinity or concentration, as determined by Scatchard analysis of the dexamethasone binding. The antagonistic effects of insulin and dexamethasone on the 7800 C1 cells were demonstrated both on cell growth and on the induction of the peroxisomal acyl-CoA oxidase and on tyrosine aminotransferase. The antagonism between the hormones on tyrosine aminotransferase is in fine with the observations obtained on fetal hepatocytes [26] and on isolated rat hepatocytes [27]. The results of dexamethasone and insulin on cell growth reported here are in keeping with the effects of the hormones on human breast cancer cells reported by Osborne et al. [28]. These workers also found that dexamethasone inhibited the thymidine kinase activity and the nudeoside uptake into the cells. To which extent these effects contributed to the observed growth inhibition was not studied. Cook et al. [29] have recently reported that dexamethasone and insulin have similar effects on the growth also of Fu5 rat hepatoma cells. Furthermore, the growth inhibitory effect of dexamethasone reported here is also consistent with the effect of glucocorticoid on fibroblast [30] and on lymphoma cells [31]. In many other cells, dexamethasone has a stimulating effect on growth [1]. It is interesting that Bagnarelli et al. [32] found that in a continuous line of human hepatoma ceUs, hydrocortisone stimulated the growth at 10-0.1 nM, but was inhibitory at higher concentrations. The effect of dexamethasone on hepatoma cell growth presented in this report is apparently also in contrast with the stimulating effect on the DNA synthesis in hepatocytes from normal rat liver in culture [6,7]. Sand et al. [6] reported that dexamethasone was not inhibitory at any concentration, and had a maximal stimulating effect on DNA synthesis in hepatocytes at 400 nM. In line with its general growth-stimulating properties, insulin counteracted the effect of dexamethasone, but was alone without influence on the growth of the 7800 C1 cells. One explanation is that the growth in serum-supplemented medium provides sufficient insulin for the cells to secure maximal growth, so that a further stimulation by insulin is not possible. This could explain why Bagnarelli et al. [32] and Testa et al. [33] found that

human hepatoma cells cultured in serum deficient medium were stimulated by insulin, but not if serumsupplemented medium was used. In hepatocytes, insulin has been able to stimulate DNA synthesis [6,7], but was not able to stimulate an increase in the number of hepatocytes. This is in contrast to the results with the 7800 C1 cells where insulin stimulated the DNA synthesis as well as the increase in cell number after prior suppression with dexamethasone. The results from the mitotic rate measurements confirmed the growth curves showing reduced cell division activity during dexamethasone treatment. This effect of dexamethasone was, however, reversed by insulin. A reduced growth of cells due to glucocorticoid has been shown earlier [18] and was assumed to reflect a partially reduced transition of cells from the G 1 compartment to the S phase. The present results demonstrate a reduced fraction of cells in S and G 2 phase, and accordingly leave support to the presence of increased G 1 fraction after dexamethasone treatment. The present data, however, do not exclude the possibility that dexamethasone has some effect also in other parts of the cell cycle. It is interesting that Fanger et al. [34] observed that glucocorticoids increase the length of the G 2 and M phases of the HeLa cell cycle. Furthermore, Osman et al. [8] showed that in a human hepatoma cell line, dexamethasone resulted in a complete block in the G1/S transition and also arrested the cells in G 1 phase and retarded progression through G 2. The continuous labelling with [3H]TdR shows that dexamethasone does not reduce the growth fraction, and therefore must induce an increased cycle time. If the cell cycle time is increased, however, and insulin is reversing the inhibition of cell growth, one would expect that the S phase fraction also would approach control values. In other cells lines, insulin has been shown to induce a transition from G 0 / G 1 phase through S phase [35]. Since insulin did not increase the size of the S phase fraction, it is likely that insulin in addition to stimulate G 1 to S transition also stimulates other parts of the cell cycle. It is possible, e.g., that the expected inverse increase in cells in S phase after insulin is counteracted by an increased rate of DNA synthesis. The normalized number of tetraploid cells after insulin treatment can be explained by an increased rate of DNA synthesis. Otto et al. [36] showed that in Swiss mouse 3T3 cells the DNA polymerase-ct was stimulated by insulin. In those cells, however, the increased rate of DNA synthesis correlated with the percentage of cells in S phase. Even if insulin probably stimulates other parts of the cell cycle in addition to the G 1 to S transition, e.g., the G 1 phase traverse, it does not increase the growth rate unless the G1 to S transition is retarded by dexamethasone, suggesting that this part of the cycle is rate limiting in the 7800 C1 cells.

228 Acknowledgements

Financial support was received from the Anders Jahre Foundation for the Promotion of Science, Norway, The Insulin Fond, Copenhagen, Denmark, the Norwegian Cancer Society and The Norwegian Research Council for Science and Humanities. We thank Drs. I. Richardson and A.H. Tashjian, Jr. for supplying the 7800 Cl hepatoma cells. Excellent technical assistance by Mette 0stby is greatly appreciated. References 1 Hepatotrophic factors (1978) Ciba Foundation Symposium 55 (new series), Elsevier/Excerpta Medica, Amsterdam. 2 Gospodarowicz, D. and Moran, J.S. (1976) Annu. Rev. Biochem. 45, 531-558. Biophys. 3 Rudland, P.S. and Jimenez de Asua, L. (1979) B&him. Acta 560, 91-134. 4 Bronstad, G. and Christoffersen, T. (1980) FEBS Lett. 120, 89-93. T. (1983) B&him. 5 Bronstad, G., Sand, T.-E. and Christoffersen, Biophys. Acta 763, 58-63. 6 Sand, T.-E., Branstad, G., Digemes, V., Killi, A., Amara, W., Refsnes, M. and Christoffersen, T. (1985) Acta Endocrinol. 109, 369-377. 1 Richman, R.A., Claus, T.H., Pilkis, S.J. and Friedman, D.L. (1976) Proc. Natl. Acad. Sci. USA 73, 3589-3593. C. 8 Osman, A.-M., Jansen, P.W.M., Smets, L.A. and Benckhuijsen, (1985) J. Cell. Physiol. 125, 306-312. 8, 265-274. 9 Bullough, W.S. (1952), J. Endocrinol. E.B. 10 Harmon, J.M., Norman, ,M.R, Fowlkes, B.J. and Thompson, (1979) J. Cell. Physiol. 98, 267-218. 11 Smith, B.T., Torday, J.S. and Giroud, C.J.(1974) J. Clin. Invest. 53, 1518-1526. V.J. (1987) Exp. Cell. Res. 168, 12 Finlay, C.O. and Christofalo, 191-203. R.E. and Griffin, M.J. (1972) Biochim. Bio13 Tu, S.H., Nordquist, phys. Acta 290, 92-109. , J. (1985) Can. J. 14 Marceau, N., Baribault, H. and Leroux-Nicollet, Biochem. 63,448-457. U.I., Snodgrass, P.J., Nuzum, C.T. and Tashjian, 15 Richardson, A.H., Jr. (1973) J. Cell Physiol. 83, 141-150.

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Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin.

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obta...
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