Bioscience Reports, Vol. 12, No. 3, 1992

Decreased Protein Kinase C Activity is Associated with Programmed Cell Death (Apoptosis) in Freshly Isolated Rat Hepatocytes Victor Sanchez, ~ Miguel Lucas, 1'2 Aureo Sanz, 1 and Raimundo Goberna ~ Received: December 12, 1991; revised April 14, 1992 Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporin. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporin and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival. KEY WORDS: Nucleosome; DNA; Protein-kinase C; apoptosis; hepatocyte.

INTRODUCTION Apoptosis (programmed cell death) is characterized by chromatin condensation, fragmentation of D N A into oligonucleosome-sized pieces, cell shrinkage and progressive cell degradation (1, 2). It occurs during embryo morphogenesis, the development of immune tolerance and tissue degeneration (3, 4). In liver, apoptosis has been found to be responsible for the regression of liver hyperplasia (5) caused by the withdrawal of the stimuli, and the regulation of cell turnover in the alteration of liver mass in tumour-bearing rats (6). Possible intracellular signaling mechanisms in the initiation of apoptosis include influx of calcium (7), altered expression of oncogenes c-los and c-myc (8) 1 Departamento de Bioquimica M6dica y Biologia Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Avda S~inchez Pizjuan 4, 41009 Sevilla. Spain. 2 To whom correspondence should be addressed. 199 0144-8463/92/0600-0199506.50/0~ 1992PlenumPublishingCorporation


Sanchez, Lucas, Sanz and Goberna

and of protein kinase C (9, 10). To gain insight into the possible factors involved in the initiation of the apoptotic process in hepatocytes, we have studied the effect of serum and two well-known inhibitors of protein kinase C (polymyxine B and staurosporin) on the production of apoptosis in cultured rat hepatocytes. Changes in the activity of protein kinase C were measured in parallel with DNA analysis.


Agarose was from BRL; Z-DNA/HindlII digested molecular weights markers, proteinase K, ribonuclease A and collagenase were from BoehringerMannheim; bleomycin was from Almirall (Spain); o~-L-phosphatidyl serine, 1-3-diolein, lysine-rich histone, phorbol myristate acetate (PMA), polymyxin B, N-laurylsarcosine and ethidium bromide from Sigma; RPMI medium, fetal bovine serum, streptomycin and penicillin from Flow. [y32p]-ATP was from Amersham. Animals

Male Wistar rats weighing 180-250 g were used. The animals were fed a standard diet and libitum and were exposed to an automatically regulated light-dark cycle of 14: 10 hours at a controlled temperature of 23~ Isolation of Hepatocytes

Hepatocytes were prepared by perfusion of the liver with collagenase as described by Krebs et al. (11) and modified by Hems et al. (12). The isolated cells were resuspended in Krebs-Ringer bicarbonate medium containing 20mM glucose, 0.1%(w/v) bovine serum albumin and 2.5mMCaC12. Hepatocyte viability was evaluated by the trypan blue test. The stain was excluded by 90-95% of cells. Incubation of Hepatocytes

Hepatocytes (8 x 106/mol) were suspended in RPMI medium containing 20mM hepes buffer pH7.3, supplemented with L-glutamine (20mM), streptomycin (100 #g/ml), penicillin (100 IU/ml), and 10% fetal bovine serum, except the experiment lacking serum. Incubations were at 37~ in an atmosphere of 95% air and 5% CO2. Experiments were carried out in 50 ml tubes with shaking (60 cycles/min). Effectors were added as indicated under Results. Samples for the studies of DNA fragmentation and protein kinase C activity were taken at different time-points as indicated in the text.

Decreased Protein Kinase C Activity


D N A Electrophoresis

DNA was extracted from hepatocytes as previously described for lymphocytes (10) with minor modifications. Hepatocytes, 2 x 106 were resuspended in 0.3 ml Tris-EDTA buffer (50 mM Tris, 10 mM EDTA pH 8.0) and supplemented with 0.5% (w-v) N-laurylsulphate and 0.5 mg/ml proteinase-K. After 1 hour at 50~ the incubation medium was supplemented with 0.5 mg/ml deoxyribonuclease-free ribonuclease-A, and further incubated at 50~ for 1 hour. DNA was precipitated with ethanol at -60~ and dissolved in Tris-EDTA buffer. Samples were heated at 65~ and supplemented with loading buffer (10raM EDTA, pH 8, containing 0.25% Bromophenol blue, 1% low-gelling-temperature agarose) at a 1 : 5 (v/v) ratio. Electrophoresis was carried out in a 2% agarose gel, and the buffer used was 80 mM Tris/20 mM phosphate/2 mM EDTA, pH 8.

Protein Kinase C Assay

Protein kinase C activity was assayed as phospholipid-sensitive, Ca 2+dependent phosphorylation of lysine-rich histone according to the procedure described in (13) with minor modifications. In brief, hepatocytes (4 • 106) were sonicated for 15 seconds at 4~ in calcium- and magnesium-free Hanks balanced salt solution. A crude homogenate was prepared by centrifugation at 15,000 • g for 10 min to remove debris, nuclei and mitochondria. Protein kinase C activity was assayed in a medium containing 25 mM Tris-HCl buffer pH 8, 10 mM MgCI2, 0.1 mM CaC12, 0.25 mg/ml o~-L-phosphatidyl serine, 0.6 mg/ml 1-3-diolein, 1 mg/ml lysine-rich histone, and 10 #M [32p]-ATP. Reaction was started by addition of 20/~1 of the homogenate to 180 #1 of the reaction mixture, giving a final concentration of 1 mg protein/ml. After 15 min incubation at 37~ reaction was stopped by pipetting 75 ~1 aliquots of the assay mixture on to Whatman 3MM filters. Thereafter the filters were washed 3 times with an excess of 10% trichloroacetic acid. Radioactivity was determined in a fl-counter. Specific radioactivity was calculated from an unwashed filter spotted with 10/~1 of the incubation mixture.

RESULTS Effect of Serum-Free Conditions and Inhibitors of Protein Kinase C on D N A Breakdown in Hepatocytes

Hepatocytes incubated in medium lacking in fetal bovine serum underwent a process of DNA degradation which could be detected within 5-8 hours of incubation. A characteristic pattern of oligonucleosome-sized fragments was observed on electrophoresis of DNA. Under these conditions, 200/~M polymyxin B and 10/tM staurosporin failed to modify the pattern of D N A breakdown produced by the deprivation of serum (see Fig. 1).


Sanchez, Lucas, Sanz and Goberna

Fig. 1. Induction of apoptosis in serum-free medium. Freshly isolated rat hepatocytes were cultured for either 5 hours (lane 1-3) or 8 hours (lane 4-6). Lane 1 and 4 were control tubes; lane 2 and 5 refer to the presence of 200/tM polymyxin B; lane 3 and 6 refer to the presence of 10/~M staurosporin. Experiments were performed in RPMI medium lacking bovine fetal serum.

Time-Course of Apoptosis after Addition of Bleomycin or Protein Kinase C Inhibitors. Effect of PMA Fetal b o v i n e s e r u m p r e v e n t e d the f r a g m e n t a t i o n of D N A in h e p a t o c y t e s i n c u b a t e d for up to 12 hours. T h e s e c o n d i t i o n s allowed us to study the effects of a n u m b e r of agents o n D N A of h e p a t o c y t e s which w e r e f o u n d to be as follows: a) T h e a d d i t i o n of b l e o m y c i n ( 0 . 6 m g / m l ) to h e p a t o c y t e s c u l t u r e d in c o m p l e t e m e d i u m , i.e. c o n t a i n i n g fetal b o v i n e s e r u m , c a u s e d a rapid f r a g m e n t a tion of D N A within 30 m i n i n c u b a t i o n (Fig. 2).

Fig. 2. Induction of DNA breakdown by bleomycin. Experiments were performed in complete medium, i.e. RPMI containing 10% bovine fetal serum. Hepatocytes were incubated for 30 min in medium supplemented as follow: none, lane 2; 0.6 mg/ml bleomycin, lane 3. DNA molecular weight marker (LambdaDNA/HindlII digested) was run in lane 1. Kbp values are given on the left of the gel.

Decreased Protein Kinase C Activity


Fig. 3. Time-dependent apoptosis of hepatocytes. Hepatocytes were incubated with a number of agents as follow: none, lane 1; 0.6mg/ml bleomycin, lane 2; 200~M polymyxin B, lane 3; 10 ttM staurosporin, lane 4; 200/tM polymyxin B plus 200 nM PMA, lane 5; 10#M staurosporin plus 200nMPMA, lane 6; 200 nM PMA, lane 7. Hepatocytes were incubated in complete medium (RPMI containing 10% fetal bovine serum) for 3 hours (figure 3a) and 8 hours (figure 3b). b) The protein kinase C inhibitors staurosporin and polymyxin B, at 200 ~ M and 10 # M respectively, were able to initiate the apoptotic process after 3 hours incubation. The effect of staurosporin was hindered by the presence of 200 nM P M A (Fig 3a). After 8 hours incubation, P M A failed to prevent apoptosis caused by the protein kinase C inhibitors, and P M A by itself was able to produce apoptotic D N A degradation (see Fig. 3b).

Protein Kinase C Activity in Hepatocytes Freshly isolated hepatocytes were incubated in the absence and in the presence of fetal bovine serum together with a n u m b e r of agents. Protein kinase

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4. Protein kinase C activity of hepatocytes. The crude homogenates, prepared as Fig.

described in methods, were obtained from hepatocytes incubated for 3 0 m i n as follows: none, control; 200 n M P M A ; 10/~M staurosporin; 200/~M polymyxin B ; 2 0 0 n M P M A plus 1 0 / ~ M staurosporin; 200 n M P M A plus 200 # M polymyxin B. All experiments used R P M I medium and contained 10% fetal bovine serum, except the serum free control.

C activity of hepatocytes incubated under serum-free conditions was much lower, nearly 60%, than in control hepatocytes incubated in the presence of serum. When hepatocytes were incubated in the presence of fetal bovine serum, the activity of protein kinase C was modified as follows: a) PMA, 200 nM final concentration, strongly stimulated the activity of protein kinase C; b) 10/~M staurosporin and 200/~M polymyxin B inhibited protein kinase by nearly 60% and 40% respectively; C) PMA partially counteracted the inhibition produced by the above agents (see Fig. 4).

DISCUSSION The promotion of cell survival by suppression of the process of apoptosis may be crucial for cell differentation and tumorigenesis (14). The protective effect of PMA inhibiting the induction of DNA breakdown has been shown in fibroblasts and mature lymphocytes (9, 10). An increase in intracellular calcium is sufficient to induce apoptosis in immature thymocytes and PMA has no detectable effect (15). In the liver, apoptosis has been shown to occur during atrophy following ligation of the hepatic portal vein (16) and during regression of adaptative liver growth induced by mitogens and during involution of lead-induced liver hyperplasia (17). However, little is known about the mechanisms underlying this process.

Decreased Protein Kinase C Activity


In the present work, we describe a number of experimental conditions leading to the apoptosis of rat hepatocytes. Bleomycin is an antitumor antibiotic that elicits its chemotherapeutic effects by degrading cellular DNA (18, 19) in a reaction that is dependent on metal ions and molecular oxygen. With bleomycin as experimental control for DNA breakdown to oligonucleosome-sized fragments, we could detect the apoptosis of freshly isolated hepatocytes by either the omission of fetal bovine serum or the addition of protein kinase C inhibitors. Since staurosporine and polymyxin B are not specific inhibitors of protein kinase C, the results should be viewed cautiously with regard to the mechanism involved in the apoptosis triggered by the above indicated compounds. In spite of the widely documented inhibitory effect of staurosporine and polymyxin B, the mechanism is poorly understood, although staurosporine appears to interact directly with the catalytic domain of protein kinase C to exert its effect (see 20 for a review). In contrast to the effect of the protein-kinase C inhibitors, we found a dual effect of the protein-kinase C agonist PMA, a functional analogue of the physiological modulator diacylglycerol. In fact, short-term incubation of hepatocytes with PMA produced a protective effect on staurosporin-induced apoptosis, which is in agreement with data previously reported (9, 10). On the other hand, long-term incubation of hepatocytes with PMA, eight hours in our experiments, produced apoptosis by itself. The protective effect may be explained by a mechanism involving the stimulation of protein-kinase C, see Fig. 4, whereas the apoptosis could be explained by depletion of protein-kinase C in a similar fashion to the reported down regulation following incubation of cells with high concentrations of PMA (21). Moreover, the fact that incubation of hepatocytes in serum-free conditions produced apoptosis and a decrease in protein-kinase C activity further suggest the involvement of protein-kinase C in the apoptosis of hepatocytes described in the present work.


Supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social, Ministerio de Sanidad y Consumo, (92/0390; 92/0399).

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Wyllie, A. H., Kerr, J. F. R. and Currie, A. R., (1980) Int. Rev. Cytol. 68:251-356. McConkey, D. J., Orrenius, S. and Jondal, M. (1990) Immunology Today 11:120-121. Kerr, J. F. R., Wyllie, A. H. and Currie, A. R. (1972) Br. J. Cancer 26:239-257. Wyllie, A. H. (1980) Nature 284:555-556. Columbano, A., Ledda-Columbano, G. M., Coni, P. P., Faa, G., Liguori, C., Santa Cruz, G. and Pani, P. (1985) Lab. Invest. 6:670-675. 6. Tessitori, L., Valente, G., Bonelli, P., Carteli, P. and Baccino, F. M. (1989) Int. J. Cancer 44: 697-700. 7. McConkey, D. J., Nicotera, P., Hartzell, P., Bolloma, G., Wyilie, A. H. and Orrenius, S. (1989) Arch. Biochem. Biophys. 269:365-370.


Sanchez, Lucas, Sanz and Goberna

8. Buttyan, R., Zakeri, Z., Lockshin, R. and Wogelmuth, D. (1988) Mol. Endocrinol. 2:650-657. 9. Tomei, D. L., Kanter, P. and Wenner, C. E. (1988) Biochem. Biophys. Res. Commun. 155: 324-331. 10. Lucas, M., Solano, F. and Sanz, A. (1991) FEBS Lett 1:19-20. 11. Krebs, H. A., Cornell, N. W., Lund, P. and Hems, R. (1974) Regulation of hepatic metabolism (eds. Lundquist F and Tygstrup N), Munksgaard, Copenhagen. pp. 726-750. 12. Hems, D. A., Rodrigues, L. M. and Whitton, P. D. (1978) Biochem. J. 172:311-317. 13. Wright, C. D., Hoffman, M. D., Thueson, D. O. and Conroy, M. C. (1987) Biochem. Biophys. Res. Commun. 148:1110-1117. 14. Williams, G. T., Smith, C. A., Spooncer, E., Dexter, T. M. and Taylor, D. R. (1990) Nature 343: 76-79. 15. Smith, C. A., Williams, G. T., Kingston, R., Jenkinson, E. J. and Owen, J. J. T. (1989) Nature 337:181-184. 16. Kerr, J. F. R. (1971) J. Path. 105:13-18. 17. Bursch, W., Lauer, B., Timmermann-Trossiener, L., Barthel, G., Schuppler, J. and SchulicHermann, R. (1984) Carcinogenesis 5:453-458. 18. Kuo, M. T. and Hus, T. C. (1978) Nature 271-'83-84. 19. Hertzberg, R. P., Caranfa, M. J. and Hecht, S. M. (1988) Biochemistry 27:3164-3174. 20. Huang, K. P. (1989) Trends Neurosci. 11:425-432. 21. Ballester, R. and Rosen, O. M. (1985) J. Biol. Chem. 260:15194-15199.

Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes.

Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protei...
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