169
Biochimic¢t et Bioph),~:i,.'a.4eta, 11195( 1991 ) 169-174
t13 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4~1~9/:~t/5113.511 ADONIS 016748809101}2792 BBAMCR 131}3¢)
Hepatocyte condi :'Jned medium modulates the response of primary rat hepatocyte cultures to epidermal growth factor G e o r g e G. Skouteris t and Matthew R. Kaser 2 t gh,llenic A~tlictlttt'er ln.~timte. Rt'search C't'trlt,r of ()lwoloj, 3' "(;. Papanikohum'..S~..S'aias ttovpmd. Athens ((,;rt't'c~')and " Department of Pt.diatri~v, thtrhor-U('l~[ Mcdh'al ('enter. Torram'c. (-t ( U.S.A, J
IReceivcd 8 April 19911
Key words: Hepatocytc: DNA synthesis: Conditioned medium: Growth factor: Secrelion
Primary hepatocytes may produce autocr]ne growth trigger(s) with or without a mitogenic stimulus. We explored the potential of hepatocyte conditioned medium - from untreated quiescent cultures - to modulate the DNA synthetic responses induced by EGF. The EGF-induced responses were similar when EGI~:was continuously or transiently (3 h) present. Conditioned medium (CM) from 48 h hepatocyte culture was the most effective in eliciting thymidine incorporation into hepatocyte DNA. At the same time the conditioned medium from hepatocyte cultures stimulated lymphocyte DNA synthesis at levels much lower than those observed using PHA, a specific lymphocyte inducer. The maximal EGF.binding by intact hepatocytes was also significantly increased in the presence of conditioned medium (48 h). We therefore suggest that hepatocytes produce autocrine growth trigger(s) which might be in part responsible for the regulation of the in vitro a n d / o r in vivo hepatocyte proiiferatton.
Introduction
Primary hepatocytes are not capable of undergoing multiple rounds of DNA synthesis in cuiture when stimulated with a variety of growth promoters [I-3]. Specific growth factors for primary hepatocytes have recently been described, however, a complete model of hepatocyte growth in vitro and in vivo has not yet been deeribed [4,5]. Primary hepatocytes in culture secrete a number of factors possessing growth-modulating properties on other cultured cells [6]. In the whole animal, the focus of attention has recently become the potential role of TGFa, which was shown to be a powerful stimulator of primary hepato~yte proliferation [6]. Several r,ormal and transformed cell lines secrete a number of polypeptides which are often correlated with either normal cell proliferation or tumorigenicity [7-9].
Abbrevialions: CM. conditioned medium: EGF, epidermal growth factor; MEM, minimal essential medium; TCA. trichloroacetic acid: PHA. phytohaemagglutinin; PBS, phosphate-buffered saline, Correspondence: G,G. Skouteris. Hellenic Anticancer Institute, Research Center of Ontology "G. Papanikolaou'. St. Savas Hospital. 171 Alexandras Avenue, 155 22 Athens, Greece.
We explored the capacity of conditioned medium from quiescent hepatocyte cultures, to modulate hepatocyte DNA synthesis and responsiveness to epidermal growth factor (EGF). Conditioned medium, was found to incease [3H]TdR incorporation into hepatocyte DNA and in parallel to upregulate the EGF-receptor number. We therefore suggest that primary hepatocytes secrete growth modulators whose activity is not restricted to these cells. Materials and Methods
Materials. Chemicals and culture media in powdered or liquid form were from Sigma, Surrey, U.K. Epidermal growth factor was from Collaborative Research, Lexington, MA, U.S.A. Radiochemical compounds were from Amersham International, U.K. Prostaglandins were a kind gift of Dr. J.E, Pike, The Upjohn Company, Kalamazoo, MI, U.S.A. Isolation end culture of Primary hepatocytes. Hepatocytes were isolated using a two-step collagenase procedure essentially as previously described [10], Hepatocytes were plated at 5- 105 cells/60 mm dish per 2 ml of minimum essential medium (MEM) containing Earle's salts and supplemented with 10% of I 0 " essential and non-essential amino acids, insulin {1o-7 M) and gentamycin (50/.tg/ml). Cultures were maintained
170 NaOH. The radioactivity was measured in a gammascintillation counter.
at 37 ° C, 5% CO t in a 95% humidified atmosphere. When various growth modulators were added for short periods, the culture media were replaced by ordinary MEM supplemented with insulin and the incubation continued. Conditioned media were collected after the appropriate incubation timcs (always 2 h after cell at,achment) and centrifuged at 3000 x g for 10 rain to remove contaminating cellular material. Material originating from cellular lysis did not interfere with the conditioned medium as shown from methionine synthetic labeling and absence of hepatocyte specific filamentous p~oteins.
Isolation of human peripheral lymphocytes and assay of thymidine incorporation by liquid scintillation. Human peripheral blood lymphocytes were isolated as previously described [13]. Briefly, lymphocytes from defibrihated human peripheral blood were separated using the above sedimentation method and cultured in bicarbonate-buffered RPMI 1640 medium. Cells were cultured at 5-40000 cells/weU and pulsed with l gCi/well of [3H]TdR (specific activity 2 Ci/mmol). The radioactivity was measured as previously described
Estimation of DNA synthesis in hepatocyte cultures.
[141.
Hepatocyte culture~ were labeled with 5 tzCi of [methyl-3H]thymi~iine (specific activity > 95 Ci/mmol) for 2 h. The cells were washed five times with ice-cold phosphate-buffered saline (PBS) (pH 7.4), dissolved in 0.33 M NaOH and transferred into tubes kept on ice. 0.3 ml of 40% tr:.:hloroacetic acid (TCA) was subsequently added. The resulting pellets were dissolved in 0.33 M NaOH and aliquots were measured for radioactivity using Aquasol on a liquid scintillation spectrometer. The DNA content was estimated using a method employing diaminobenzoic acid [1 I]. EGF ce~uptor assay. These assays were performed on hepatocyteb ,:,t!tured onto 24-multi well plastic culture plates [12]. Isolated hepatocytes were plated in MEM, supplemented with gentamycin at 150000 cells per well. After attachment, the cultures were washed twice with binding buffer (containing MEM, 25 mM Hepes, 1 mg/ml bovine serum albumin pH 7.0). 1 ml of binding buffer containing [I~I]EGF (2 ng/ml, 100 mCi/mg) was added together with or without conditioned medium. Non-specific binding was less than 4% and it was determined in the presence of a 1000-fold excess of unlabeled EGF. The plates were incubated for 2 h at 5 °C and subsequently washed three times with binding buffer and dissolved in 1 ml of 1 M
L EAG~
Results
The continuous presence of EGF stimulates DNA synthesis and mitosis of hepatocytes in culture [1,15]. We performed experiments to determine the minimum time required for EGF to be present such that DNA synthesis was triggered (Fig. 1). EGF present for 3 or 6 h at low cell density hepatocyte culture (5- 105 cells/2 ml) induced the DNA synthetic peak to occur approx. 12 h earlier than if EGF was continuously present. Prostaglandin E2 added for 3 or 6 h followed by medium containing insulin alone did not induce early DNA synthesis; the DNA synthetic response in hepatocytes stimulated with PGFza was similar to that with EGF. Continuous stimulation with PGF2, delayed ihe peak of DNA synthesis by 25 h, compared with a shorter duration of exposure (Fig. 1). These results confirm EGF to be a competence faiztor for cultured hepatocytes, Although the peak in hepatocyte DNA synthesis after transient addition of EGF occurred at earlier times, the average duration of the G0-G1/S traverse did not vary significantly in comparison to cultures treated continuously with EGF. Previous studies have shown that prostaglandins stimulate hepato.
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Fig. I. Effects of the transient presence of EGF and prostag[andinsE2 and F2, upon hepalccyte entry, into S phase. Points represent mcans±S.E, from at [cast three indcpenden~experiments.(A) a, culturessupplementedwith insulinonly;b, EGF continmmslypresent in culture; c, EGF present in culture for the first 6 h anet attachment; and d, E G F present in culture for the first 3 h. (B and C) Conlinuous and
transientpresenceof PGE2('3)and PGF2~(C) at 6 #g/dish in hepatocyteeullmes,a-d, as for EGF-supplementedcultures.
171 cyte proliferation either directly or indirectly through an autocrine mechanism activated after EGF addition 115]. We next attempted to test the effects of conditioned medium/rom unstimulated hepatocyte cultures, upon the DNA synthesis of EGF-stimulated hepatoc'ytcs. Conditioned medium from hepatocyte cultures treated with insulin for 1, 6, 24, 36, 48, 72 and 96 h was collected and used to supplement EGF-stimulated cultures. EGF was added for 3 h together with 10% conditioned medium from hepatocytes cultured for different times. After the removal of EGF, the cultures were switched on conventional medium supplemented with 10% conditioned medium (CM). No significant difference in [3H]TdR incorporation was observed using CM from 1, 6 and 24 h cultures (Fig. 2A). However, the CM from 36 and 48 h culture ir~du,ed a dramatic increase in [3H]TdR incorporation into hepatocyt,: DNA t)bserved at 48 and 72 h (Fig. 2B). We further explored the effects of different concentrations of CM in modulating the EGF-promoted hepatocyte DNA synthesis. Thymidine incorporation observed using three different concentrations of CM (10, 15 and 20%) to supplement EGF-indueed hepatocyte cultures,
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Fig. 2. Effect of the conditioned medium from cultured hepatocytes, upon DNA synthesis of primary hepatocytes induced with EGF for 3 h. Points represent means+S.E, from at least three independent experiments. Hepatocyte cultures were supldemented with EGF' for 3 h and subsequentlyswitchedon conventionalmediumcontaining
10%CMfromculturesmaintainedin insulinonly,(A) a,'CMfrom 1 h; b, 6 h; e, 24 h; d, 36 h (B) e, 48 h; f, 72 h; 8. CM from 48 h culture with no cells (conlrol); h, 96 h.
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Fig. 3. Effecls of 10, 15 and 20% conditioned medium from 48 h hepatoc3'te culture up, ,n DNA synthesis of primary hepatoc~es. The points represent mea):~ : q.E. from z: lea~: three independent experiments. Primary hepato(yte cultures ,vere treated with EGF for 3 h and then supplemented with 10 (a), 15 (b) and 20% (b) CM from 48 h culture.
did not vary significantly. This reflects the inability of hepatocytes to respond in a dose-dependent manner to the growth triggers included in the CM (Fig. 3). The effect of CM in stimulating DNA synthesis was appreciable using 36 and 48 h culture supernatant and not that from 72 h culture. Increased incorporation of [3H]TdR into hepatecyte DNA was not due to a feeding effect caused by the added medium as shown in control cultures; cultures were supplemented with conventional medium and incubated for the respective periods at 37°C with no cells present (2B, g). The conditioned medium was unable to induce DNA synthetic ~esponses when hepatoeyte cultures were not 'primed' with EGF for 3 h (results not shown). We furtr=er explored the potential of conditioned medium to modulate the responsiveness of cultured hepatoojtes to EGF. We carried out a receptor binding assay using [t~I]EGF and included 10 or 20% of CM from 48 h culture supernatant in the binding buffer (Fig. 4)..Ad. dition of control CM did not affect maximal EGF-binding, however, addition of 10% CM caused a 25% increase in EGF binding capacity at 20 ng/ml EGF. When EGF used was at 5 ng/ml, the EGF binding capacity was further increased to 32% compared to the control bind[ag capacity. Addition of 20% CM in the binding buffer caused a similar increa.~ to the EGFbinding capacity due to the presumably lower concentrations of free EGF (Fig. 4). These results showed that some f,,ctors produced by hepatocytes after 36 h in culture, increased thymidine incorporation and at the same time also increased the binding capacity of EGF on intact hepatocytes. C6-incoba.tionof EGF with con-
28/
172 ditioned medium at binding conditions did not reduce the counts bound, indicating that the growth trigger(s) contained in the medium were not competing EGF for its receptor (results not shown). Experiments with CM from 1 and 6 h hepatocyte cultures failed to show any increase or decrease in EGF-binding capacity (results not shown). Hepatocytes secrete a number of proteins which may have a paracrine role on parenehymal, non-parenchymal and on other heterologous cells. Some experiments were carried out to explore the potential of hepatocyte conditioned medium to induce a DNA synthetic response in cultured human peripheral blood lymphocytes. Lymphocytes plated at 540000 celIs/well were used to test the capacity of hepatocyte CM to induce thymidine incorporation. This incorporation was compared to phytohaemagglutinin (PHA)-stimutated lymphocyte cultures. Both agents were tested at different concentrations, however, 10% CM and 1 : 110 final dilution of unpurified PHA (Wellcome) were shown to produce representative [3H]TdR incorporation responses (Fig. 5). Previous studies have suggested that PHA induces lymphocyte proliferation after 24 h in cultures pulsed with thymidine (2 h). This was also observed in our system using 5-40000 cells/well (Fig. 5b). At day 3, addition of 10% conditioned medium from 48 h hepatocyte culture induced thymidine incorporation into lymphocyte DNA in a cell number-dependent manner. However, the levels of thymidine incorporation were far below those observed after PHA addition. Co-addition of conditioned medium and PHA, resulted a slightly elevated, but temporally similar response to that with PHA alone, suggesting that factors in the conditioned medium might enhance the mitogenic response of lymphocytes to PHA. In particular, this seemed to occur at intermediate cell densi,qes, when cell-cell contact is expected to be reduced (low) At Mgkc.r cell densities, the re-
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80
100
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Fig, 4. Maximal binding of EGF by intact hepatocytes supp|cmentcd with or without conditioned medium from hepatccyt¢ c-ltures. Binding conditlon~ were as described in Materials and Methods section and points represent means from two indcpc,'.d•=t cz~.':r!ments. (a) EGF only" (b) EGF+control CM; (c) E G F + 10% 48 h CM and (d) EGF÷ 20% 48 h CM.
24
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Fig. 5. Effects of hepatocyte conditioned medium upon lymphocyte DNA synthesis. Points represent means±S.E, from three independent experiments. Lymphocytes from human peripheral blood were plated in microp]ates as described in Materials and Methods, and stimulated with PHA. (a, c and e) Control cultures (unstimulated) at the day 1, 2 and 3. (b) PHA*sfimulaled |ymphocytes at day 3. (d)
Lymphocytesstimulatedwith t0% CM from48 h hepatocyteculture (day 3) and (D lymphocytes stimulated with PHA+ 10% CM from 48 h hepatocyl¢ culture (day 3).
spouse to PHA or to both adjuvants wa~ similm (Fig. 50. Discussion
in this study we explored the potential of EGF and prostaglandins to induce hepatocyte DNA synthesis if present in culture transiently. The results reported here have shown that growth promoters such as EGF and PGF2, induced intracellular changes !eadipg to initiation of a DNA synthetic response, by 3 h upon their addition, it has been reported that EGF and prostaglandins cooperate within an autocrine mechanism regulating hepatocyte proliferation [15-18]. However, prostaglandins were not secreted by primary hepatocytes in unstimulated cultures [15]. EGF and PGF2~, transiently in culture, were able to induce thymidine incorporation into hepatocyte DNA in a manner similar to that elicited under continuous presence of these growth factors. The minimum time required for these factors to induce a DNA synthetic response in hepatocytes was estimated to be approx. 3 h. However, the observed shifting of the respective thymidine incorporation curves may be correlated with a relative asynchrony of the hepato~te population, or may be related to the effects of EGF continuously in the culture medium. Although EGF was required for 3 h in hepatocyte cultures to trigger DNA synthesis, the length of the G0-G1/S traverse was not very different using the
173
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Fig. 6. Effect of hepatocyte cell density upon the ability of It)% conditioned medium to induce DNA synthetic responses in primary hepatocytes. Points arc means_+S.E. from three independent experiments. (A) a, cultures supplemented with EGF (controD: b. EGF + CM from 48 h culture of I. 10~' cells/2 ml. (B) c, EGF+ CM from 36 h culture of 10~' cells/2 ml; and d, EGF+CM from 72 h culture of 10~' cells/2 mL
two ways of stimulating hepatocytes (continuous/ transient). It is currently believed that hepatocyees are capable of producing autocrine growth factors only after stimulation by external growth signals [16,17]. Furthermore, in our study conditioned medium from cultured hepatocytes contributed to a stimulation primarily induced by EGF. Regardless of its role in costimulating DNA synthesis, the conditioned medium was unable to elicit DNA synthetic responses if administered alone to the cultures. Thus, it malt be suggested that potential growth factors secreted in the conditioned medium may belong to the co-mitogenic growth factor class [19]. In addition, conditioned medium added in culture after removal of EGF did not affect thymidine incorporation into hepatocyte DNA. This is an additional evidence that hepatocyte culture supernatant contains growth triggers which are secreted in culture between 24 and up to 72 h ~fter cell attachment. These growth 'triggers' are able to cooperate with established growth factors when they deri,,e from hepatocytes cultured for up to 72 h (results not shown). The production of potential autocrine or paracrine growth triter(s), was found to be cell density-dependent (Fig. 6). Hepatocytes plated at lower d,~nsities (approx. 5" 10s cells/2 ml) seemed capable to p r o d u c e
the growth trigger(s), in contrast to those plated at higher densities (approx. (1-1.5)- 10~'cells/2 mI). Thus, we may suggest that the production of a potential growth trigger is related with mechanisms regulating celI-to-cell contact in this particular cell type. Although 'spatial distribution' signals do not ~em to coordinate directly hep~ttocytc proliferation in vitro, hepatocyte cell density was correlated with the appearance of growth triggcrs in the conditioned medium (Fig. 6). Potential growth triggers contained in the conditioned medium of cultured hepatocytes, seemed to increase EGF binding, especially when 48 h conditioned medium was used. tn order to explore further the ability of hepatocyte growth triggers to induce DNA synthetic responses in cells from different species, we have carried out experiments using peripheral blood lymphocytes (Fig. 5). Our data has shown that hepatocyte conditioned medium contains factors which may induce proliferation of beterologous cells such as lymphocytes. However, conditioned medium alone failed to produce a striking effect in eliciting lymphocyte DNA synthesis. Therefore, we may suggest that the conditioned medium contains factor(s) capable to super-induce or to 'cock the trigger' of the DNA wnthetic responses in primary cells, which may be secondary to the addition of a cell-specific growth inducer. TGFa expression increases approx. 6-8 ia after partial hepatectomy [3] and the peak in TGFa production coincided with the onset of DNA synthesis observed in vivo (approx. 24 h). However, the increase in TGFa production is correlated with a dramatic decline in EGF receptor number and mRNA [20]. Whether the secreted hepatocyte growth triggers share structural or functional similarities with known growth factor~ is a point of further investigation.
Acknowledgments We would like to thank Dr. M.G. Ord for constructive discussions regarding a part of experiments presented in this study. We also thank Peter R.F.D. Aylott for valuable and excellent assistance. Preliminary experiments presented here were performed by G.G.~. in the Department of Biochemistry, University of Oxford, U.K.
References I McGowan, J.A., Strain, A.L and Bucber, N LR. (1981)J. Ceil. Physiol. 180, 353-363. 2 Michalopoulns, G.. Jirtle. R.L, Kligerman. A.D., Cianciulli, D. and Novotny, A, (1982) Cancer Res. 42, 4673-468~ 3 Mead..J,E. and Fausto, N. {1989) ['roe. Natl. Acad. Sci. USA 86. 1558-1.'62. 4 Zarneg~r, R., Muga, S.. E~ghild. J. and Michalopoutos, G.K, (1989) Biochem. Biophys. Res. Commun. 163, 1370-1376.
I74 5 Nakamura, T.. Nishizawa. T.. Hagiya, M.. Seki. T.. Shimonishi. M.. Sugimura. A.. Tashiro, K. and Shimizu. S. 11989) Nature 342. 4411-443. 6 Luetleke, N.C., Michalopouh~s. G.K., Teixido. J. Gilmore. R.. Massague. J. and Lee, D.C. (1988) Biuchemislry 27, 6487-M94. "1 ':...r3~:.:k. R.. G~eddd. ~.V., Ullrich. A., Gulterman. J'.U., Williams, R.D., Bringman. T.S, and Berger. W.H. 11987) Cancer Res. 47, 71)7-712, 8 Scnger, D.R. and Peruzzi. C.A. 11985) Cancer Res. 45, ~818-5823. 0 Craig. A.M., Nemir. M, Mukherjee. Chambers, A.F. and Denhardt, D~T. (ItJ88) Bioehem. Biophys. Res. Commun. 157, 166173. II) Seglen. P.O. (tc,~76)Melhods Cell. Bio]. 13. 29-83. 11 Kapp. N.L. Brown, S.L and Levecz. R. ([~,74) Biochim. Biophys. Acla 361. 141)-143. 12 Cruise. J.L. Cotecchia. S. and Michalopt~ulos. G. (l~Sfi)J'. Cell. Physiol. 127. 39-44.
13 Boyum, A. (1%8) Scand. L Clin, Lab. Invest. 21, supp], 97, p. ")'7. 14 Hume. D,A. and Weidemann, MJ. (Iq~)) Mitogenic Lymphocyte Transh~rmation. Elsevier/North Holland. Amsterdam. 15 Skouteris. G.G.. Ord. M.G. and SIoeken, L.A. (I988,1 J. Cell. Physiol. 135. 516-520. 16 Whitfield, J.F. (Iqql)) Calcium, Cell Cycles and Cancer. pp. 92-98. CRC Press. Boca Raton. 17 Little, P.. Skouteris, G.G., Ord. M,G. and Sloeken, L.A. (|988)J. Cell Set. 91,549-553. 18 $kouteris. G.G.(Iq88) in Regulation of Liver Cell Growlh, Thesis l:k)clor of Philosophy. University of Oxford. Chapter 4. 19 Micbalopoulos. G.K, 11991))FASEB J. 4. 176-187. 211 Hsich. LL, Peraino, C- and Weinstein. I.B. (19~,8) Cancer Res. 48. 265-269.
Biochimic¢t et Bioph),~:i,.'a.4eta, 11195( 1991 ) 169-174
t13 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4~1~9/:~t/5113.511 ADONIS 016748809101}2792 BBAMCR 131}3¢)
Hepatocyte condi :'Jned medium modulates the response of primary rat hepatocyte cultures to epidermal growth factor G e o r g e G. Skouteris t and Matthew R. Kaser 2 t gh,llenic A~tlictlttt'er ln.~timte. Rt'search C't'trlt,r of ()lwoloj, 3' "(;. Papanikohum'..S~..S'aias ttovpmd. Athens ((,;rt't'c~')and " Department of Pt.diatri~v, thtrhor-U('l~[ Mcdh'al ('enter. Torram'c. (-t ( U.S.A, J
IReceivcd 8 April 19911
Key words: Hepatocytc: DNA synthesis: Conditioned medium: Growth factor: Secrelion
Primary hepatocytes may produce autocr]ne growth trigger(s) with or without a mitogenic stimulus. We explored the potential of hepatocyte conditioned medium - from untreated quiescent cultures - to modulate the DNA synthetic responses induced by EGF. The EGF-induced responses were similar when EGI~:was continuously or transiently (3 h) present. Conditioned medium (CM) from 48 h hepatocyte culture was the most effective in eliciting thymidine incorporation into hepatocyte DNA. At the same time the conditioned medium from hepatocyte cultures stimulated lymphocyte DNA synthesis at levels much lower than those observed using PHA, a specific lymphocyte inducer. The maximal EGF.binding by intact hepatocytes was also significantly increased in the presence of conditioned medium (48 h). We therefore suggest that hepatocytes produce autocrine growth trigger(s) which might be in part responsible for the regulation of the in vitro a n d / o r in vivo hepatocyte proiiferatton.
Introduction
Primary hepatocytes are not capable of undergoing multiple rounds of DNA synthesis in cuiture when stimulated with a variety of growth promoters [I-3]. Specific growth factors for primary hepatocytes have recently been described, however, a complete model of hepatocyte growth in vitro and in vivo has not yet been deeribed [4,5]. Primary hepatocytes in culture secrete a number of factors possessing growth-modulating properties on other cultured cells [6]. In the whole animal, the focus of attention has recently become the potential role of TGFa, which was shown to be a powerful stimulator of primary hepato~yte proliferation [6]. Several r,ormal and transformed cell lines secrete a number of polypeptides which are often correlated with either normal cell proliferation or tumorigenicity [7-9].
Abbrevialions: CM. conditioned medium: EGF, epidermal growth factor; MEM, minimal essential medium; TCA. trichloroacetic acid: PHA. phytohaemagglutinin; PBS, phosphate-buffered saline, Correspondence: G,G. Skouteris. Hellenic Anticancer Institute, Research Center of Ontology "G. Papanikolaou'. St. Savas Hospital. 171 Alexandras Avenue, 155 22 Athens, Greece.
We explored the capacity of conditioned medium from quiescent hepatocyte cultures, to modulate hepatocyte DNA synthesis and responsiveness to epidermal growth factor (EGF). Conditioned medium, was found to incease [3H]TdR incorporation into hepatocyte DNA and in parallel to upregulate the EGF-receptor number. We therefore suggest that primary hepatocytes secrete growth modulators whose activity is not restricted to these cells. Materials and Methods
Materials. Chemicals and culture media in powdered or liquid form were from Sigma, Surrey, U.K. Epidermal growth factor was from Collaborative Research, Lexington, MA, U.S.A. Radiochemical compounds were from Amersham International, U.K. Prostaglandins were a kind gift of Dr. J.E, Pike, The Upjohn Company, Kalamazoo, MI, U.S.A. Isolation end culture of Primary hepatocytes. Hepatocytes were isolated using a two-step collagenase procedure essentially as previously described [10], Hepatocytes were plated at 5- 105 cells/60 mm dish per 2 ml of minimum essential medium (MEM) containing Earle's salts and supplemented with 10% of I 0 " essential and non-essential amino acids, insulin {1o-7 M) and gentamycin (50/.tg/ml). Cultures were maintained
170 NaOH. The radioactivity was measured in a gammascintillation counter.
at 37 ° C, 5% CO t in a 95% humidified atmosphere. When various growth modulators were added for short periods, the culture media were replaced by ordinary MEM supplemented with insulin and the incubation continued. Conditioned media were collected after the appropriate incubation timcs (always 2 h after cell at,achment) and centrifuged at 3000 x g for 10 rain to remove contaminating cellular material. Material originating from cellular lysis did not interfere with the conditioned medium as shown from methionine synthetic labeling and absence of hepatocyte specific filamentous p~oteins.
Isolation of human peripheral lymphocytes and assay of thymidine incorporation by liquid scintillation. Human peripheral blood lymphocytes were isolated as previously described [13]. Briefly, lymphocytes from defibrihated human peripheral blood were separated using the above sedimentation method and cultured in bicarbonate-buffered RPMI 1640 medium. Cells were cultured at 5-40000 cells/weU and pulsed with l gCi/well of [3H]TdR (specific activity 2 Ci/mmol). The radioactivity was measured as previously described
Estimation of DNA synthesis in hepatocyte cultures.
[141.
Hepatocyte culture~ were labeled with 5 tzCi of [methyl-3H]thymi~iine (specific activity > 95 Ci/mmol) for 2 h. The cells were washed five times with ice-cold phosphate-buffered saline (PBS) (pH 7.4), dissolved in 0.33 M NaOH and transferred into tubes kept on ice. 0.3 ml of 40% tr:.:hloroacetic acid (TCA) was subsequently added. The resulting pellets were dissolved in 0.33 M NaOH and aliquots were measured for radioactivity using Aquasol on a liquid scintillation spectrometer. The DNA content was estimated using a method employing diaminobenzoic acid [1 I]. EGF ce~uptor assay. These assays were performed on hepatocyteb ,:,t!tured onto 24-multi well plastic culture plates [12]. Isolated hepatocytes were plated in MEM, supplemented with gentamycin at 150000 cells per well. After attachment, the cultures were washed twice with binding buffer (containing MEM, 25 mM Hepes, 1 mg/ml bovine serum albumin pH 7.0). 1 ml of binding buffer containing [I~I]EGF (2 ng/ml, 100 mCi/mg) was added together with or without conditioned medium. Non-specific binding was less than 4% and it was determined in the presence of a 1000-fold excess of unlabeled EGF. The plates were incubated for 2 h at 5 °C and subsequently washed three times with binding buffer and dissolved in 1 ml of 1 M
L EAG~
Results
The continuous presence of EGF stimulates DNA synthesis and mitosis of hepatocytes in culture [1,15]. We performed experiments to determine the minimum time required for EGF to be present such that DNA synthesis was triggered (Fig. 1). EGF present for 3 or 6 h at low cell density hepatocyte culture (5- 105 cells/2 ml) induced the DNA synthetic peak to occur approx. 12 h earlier than if EGF was continuously present. Prostaglandin E2 added for 3 or 6 h followed by medium containing insulin alone did not induce early DNA synthesis; the DNA synthetic response in hepatocytes stimulated with PGFza was similar to that with EGF. Continuous stimulation with PGF2, delayed ihe peak of DNA synthesis by 25 h, compared with a shorter duration of exposure (Fig. 1). These results confirm EGF to be a competence faiztor for cultured hepatocytes, Although the peak in hepatocyte DNA synthesis after transient addition of EGF occurred at earlier times, the average duration of the G0-G1/S traverse did not vary significantly in comparison to cultures treated continuously with EGF. Previous studies have shown that prostaglandins stimulate hepato.
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Fig. I. Effects of the transient presence of EGF and prostag[andinsE2 and F2, upon hepalccyte entry, into S phase. Points represent mcans±S.E, from at [cast three indcpenden~experiments.(A) a, culturessupplementedwith insulinonly;b, EGF continmmslypresent in culture; c, EGF present in culture for the first 6 h anet attachment; and d, E G F present in culture for the first 3 h. (B and C) Conlinuous and
transientpresenceof PGE2('3)and PGF2~(C) at 6 #g/dish in hepatocyteeullmes,a-d, as for EGF-supplementedcultures.
171 cyte proliferation either directly or indirectly through an autocrine mechanism activated after EGF addition 115]. We next attempted to test the effects of conditioned medium/rom unstimulated hepatocyte cultures, upon the DNA synthesis of EGF-stimulated hepatoc'ytcs. Conditioned medium from hepatocyte cultures treated with insulin for 1, 6, 24, 36, 48, 72 and 96 h was collected and used to supplement EGF-stimulated cultures. EGF was added for 3 h together with 10% conditioned medium from hepatocytes cultured for different times. After the removal of EGF, the cultures were switched on conventional medium supplemented with 10% conditioned medium (CM). No significant difference in [3H]TdR incorporation was observed using CM from 1, 6 and 24 h cultures (Fig. 2A). However, the CM from 36 and 48 h culture ir~du,ed a dramatic increase in [3H]TdR incorporation into hepatocyt,: DNA t)bserved at 48 and 72 h (Fig. 2B). We further explored the effects of different concentrations of CM in modulating the EGF-promoted hepatocyte DNA synthesis. Thymidine incorporation observed using three different concentrations of CM (10, 15 and 20%) to supplement EGF-indueed hepatocyte cultures,
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Fig. 2. Effect of the conditioned medium from cultured hepatocytes, upon DNA synthesis of primary hepatocytes induced with EGF for 3 h. Points represent means+S.E, from at least three independent experiments. Hepatocyte cultures were supldemented with EGF' for 3 h and subsequentlyswitchedon conventionalmediumcontaining
10%CMfromculturesmaintainedin insulinonly,(A) a,'CMfrom 1 h; b, 6 h; e, 24 h; d, 36 h (B) e, 48 h; f, 72 h; 8. CM from 48 h culture with no cells (conlrol); h, 96 h.
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Fig. 3. Effecls of 10, 15 and 20% conditioned medium from 48 h hepatoc3'te culture up, ,n DNA synthesis of primary hepatoc~es. The points represent mea):~ : q.E. from z: lea~: three independent experiments. Primary hepato(yte cultures ,vere treated with EGF for 3 h and then supplemented with 10 (a), 15 (b) and 20% (b) CM from 48 h culture.
did not vary significantly. This reflects the inability of hepatocytes to respond in a dose-dependent manner to the growth triggers included in the CM (Fig. 3). The effect of CM in stimulating DNA synthesis was appreciable using 36 and 48 h culture supernatant and not that from 72 h culture. Increased incorporation of [3H]TdR into hepatecyte DNA was not due to a feeding effect caused by the added medium as shown in control cultures; cultures were supplemented with conventional medium and incubated for the respective periods at 37°C with no cells present (2B, g). The conditioned medium was unable to induce DNA synthetic ~esponses when hepatoeyte cultures were not 'primed' with EGF for 3 h (results not shown). We furtr=er explored the potential of conditioned medium to modulate the responsiveness of cultured hepatoojtes to EGF. We carried out a receptor binding assay using [t~I]EGF and included 10 or 20% of CM from 48 h culture supernatant in the binding buffer (Fig. 4)..Ad. dition of control CM did not affect maximal EGF-binding, however, addition of 10% CM caused a 25% increase in EGF binding capacity at 20 ng/ml EGF. When EGF used was at 5 ng/ml, the EGF binding capacity was further increased to 32% compared to the control bind[ag capacity. Addition of 20% CM in the binding buffer caused a similar increa.~ to the EGFbinding capacity due to the presumably lower concentrations of free EGF (Fig. 4). These results showed that some f,,ctors produced by hepatocytes after 36 h in culture, increased thymidine incorporation and at the same time also increased the binding capacity of EGF on intact hepatocytes. C6-incoba.tionof EGF with con-
28/
172 ditioned medium at binding conditions did not reduce the counts bound, indicating that the growth trigger(s) contained in the medium were not competing EGF for its receptor (results not shown). Experiments with CM from 1 and 6 h hepatocyte cultures failed to show any increase or decrease in EGF-binding capacity (results not shown). Hepatocytes secrete a number of proteins which may have a paracrine role on parenehymal, non-parenchymal and on other heterologous cells. Some experiments were carried out to explore the potential of hepatocyte conditioned medium to induce a DNA synthetic response in cultured human peripheral blood lymphocytes. Lymphocytes plated at 540000 celIs/well were used to test the capacity of hepatocyte CM to induce thymidine incorporation. This incorporation was compared to phytohaemagglutinin (PHA)-stimutated lymphocyte cultures. Both agents were tested at different concentrations, however, 10% CM and 1 : 110 final dilution of unpurified PHA (Wellcome) were shown to produce representative [3H]TdR incorporation responses (Fig. 5). Previous studies have suggested that PHA induces lymphocyte proliferation after 24 h in cultures pulsed with thymidine (2 h). This was also observed in our system using 5-40000 cells/well (Fig. 5b). At day 3, addition of 10% conditioned medium from 48 h hepatocyte culture induced thymidine incorporation into lymphocyte DNA in a cell number-dependent manner. However, the levels of thymidine incorporation were far below those observed after PHA addition. Co-addition of conditioned medium and PHA, resulted a slightly elevated, but temporally similar response to that with PHA alone, suggesting that factors in the conditioned medium might enhance the mitogenic response of lymphocytes to PHA. In particular, this seemed to occur at intermediate cell densi,qes, when cell-cell contact is expected to be reduced (low) At Mgkc.r cell densities, the re-
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80
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Fig, 4. Maximal binding of EGF by intact hepatocytes supp|cmentcd with or without conditioned medium from hepatccyt¢ c-ltures. Binding conditlon~ were as described in Materials and Methods section and points represent means from two indcpc,'.d•=t cz~.':r!ments. (a) EGF only" (b) EGF+control CM; (c) E G F + 10% 48 h CM and (d) EGF÷ 20% 48 h CM.
24
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Fig. 5. Effects of hepatocyte conditioned medium upon lymphocyte DNA synthesis. Points represent means±S.E, from three independent experiments. Lymphocytes from human peripheral blood were plated in microp]ates as described in Materials and Methods, and stimulated with PHA. (a, c and e) Control cultures (unstimulated) at the day 1, 2 and 3. (b) PHA*sfimulaled |ymphocytes at day 3. (d)
Lymphocytesstimulatedwith t0% CM from48 h hepatocyteculture (day 3) and (D lymphocytes stimulated with PHA+ 10% CM from 48 h hepatocyl¢ culture (day 3).
spouse to PHA or to both adjuvants wa~ similm (Fig. 50. Discussion
in this study we explored the potential of EGF and prostaglandins to induce hepatocyte DNA synthesis if present in culture transiently. The results reported here have shown that growth promoters such as EGF and PGF2, induced intracellular changes !eadipg to initiation of a DNA synthetic response, by 3 h upon their addition, it has been reported that EGF and prostaglandins cooperate within an autocrine mechanism regulating hepatocyte proliferation [15-18]. However, prostaglandins were not secreted by primary hepatocytes in unstimulated cultures [15]. EGF and PGF2~, transiently in culture, were able to induce thymidine incorporation into hepatocyte DNA in a manner similar to that elicited under continuous presence of these growth factors. The minimum time required for these factors to induce a DNA synthetic response in hepatocytes was estimated to be approx. 3 h. However, the observed shifting of the respective thymidine incorporation curves may be correlated with a relative asynchrony of the hepato~te population, or may be related to the effects of EGF continuously in the culture medium. Although EGF was required for 3 h in hepatocyte cultures to trigger DNA synthesis, the length of the G0-G1/S traverse was not very different using the
173
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Fig. 6. Effect of hepatocyte cell density upon the ability of It)% conditioned medium to induce DNA synthetic responses in primary hepatocytes. Points arc means_+S.E. from three independent experiments. (A) a, cultures supplemented with EGF (controD: b. EGF + CM from 48 h culture of I. 10~' cells/2 ml. (B) c, EGF+ CM from 36 h culture of 10~' cells/2 ml; and d, EGF+CM from 72 h culture of 10~' cells/2 mL
two ways of stimulating hepatocytes (continuous/ transient). It is currently believed that hepatocyees are capable of producing autocrine growth factors only after stimulation by external growth signals [16,17]. Furthermore, in our study conditioned medium from cultured hepatocytes contributed to a stimulation primarily induced by EGF. Regardless of its role in costimulating DNA synthesis, the conditioned medium was unable to elicit DNA synthetic responses if administered alone to the cultures. Thus, it malt be suggested that potential growth factors secreted in the conditioned medium may belong to the co-mitogenic growth factor class [19]. In addition, conditioned medium added in culture after removal of EGF did not affect thymidine incorporation into hepatocyte DNA. This is an additional evidence that hepatocyte culture supernatant contains growth triggers which are secreted in culture between 24 and up to 72 h ~fter cell attachment. These growth 'triggers' are able to cooperate with established growth factors when they deri,,e from hepatocytes cultured for up to 72 h (results not shown). The production of potential autocrine or paracrine growth triter(s), was found to be cell density-dependent (Fig. 6). Hepatocytes plated at lower d,~nsities (approx. 5" 10s cells/2 ml) seemed capable to p r o d u c e
the growth trigger(s), in contrast to those plated at higher densities (approx. (1-1.5)- 10~'cells/2 mI). Thus, we may suggest that the production of a potential growth trigger is related with mechanisms regulating celI-to-cell contact in this particular cell type. Although 'spatial distribution' signals do not ~em to coordinate directly hep~ttocytc proliferation in vitro, hepatocyte cell density was correlated with the appearance of growth triggcrs in the conditioned medium (Fig. 6). Potential growth triggers contained in the conditioned medium of cultured hepatocytes, seemed to increase EGF binding, especially when 48 h conditioned medium was used. tn order to explore further the ability of hepatocyte growth triggers to induce DNA synthetic responses in cells from different species, we have carried out experiments using peripheral blood lymphocytes (Fig. 5). Our data has shown that hepatocyte conditioned medium contains factors which may induce proliferation of beterologous cells such as lymphocytes. However, conditioned medium alone failed to produce a striking effect in eliciting lymphocyte DNA synthesis. Therefore, we may suggest that the conditioned medium contains factor(s) capable to super-induce or to 'cock the trigger' of the DNA wnthetic responses in primary cells, which may be secondary to the addition of a cell-specific growth inducer. TGFa expression increases approx. 6-8 ia after partial hepatectomy [3] and the peak in TGFa production coincided with the onset of DNA synthesis observed in vivo (approx. 24 h). However, the increase in TGFa production is correlated with a dramatic decline in EGF receptor number and mRNA [20]. Whether the secreted hepatocyte growth triggers share structural or functional similarities with known growth factor~ is a point of further investigation.
Acknowledgments We would like to thank Dr. M.G. Ord for constructive discussions regarding a part of experiments presented in this study. We also thank Peter R.F.D. Aylott for valuable and excellent assistance. Preliminary experiments presented here were performed by G.G.~. in the Department of Biochemistry, University of Oxford, U.K.
References I McGowan, J.A., Strain, A.L and Bucber, N LR. (1981)J. Ceil. Physiol. 180, 353-363. 2 Michalopoulns, G.. Jirtle. R.L, Kligerman. A.D., Cianciulli, D. and Novotny, A, (1982) Cancer Res. 42, 4673-468~ 3 Mead..J,E. and Fausto, N. {1989) ['roe. Natl. Acad. Sci. USA 86. 1558-1.'62. 4 Zarneg~r, R., Muga, S.. E~ghild. J. and Michalopoutos, G.K, (1989) Biochem. Biophys. Res. Commun. 163, 1370-1376.
I74 5 Nakamura, T.. Nishizawa. T.. Hagiya, M.. Seki. T.. Shimonishi. M.. Sugimura. A.. Tashiro, K. and Shimizu. S. 11989) Nature 342. 4411-443. 6 Luetleke, N.C., Michalopouh~s. G.K., Teixido. J. Gilmore. R.. Massague. J. and Lee, D.C. (1988) Biuchemislry 27, 6487-M94. "1 ':...r3~:.:k. R.. G~eddd. ~.V., Ullrich. A., Gulterman. J'.U., Williams, R.D., Bringman. T.S, and Berger. W.H. 11987) Cancer Res. 47, 71)7-712, 8 Scnger, D.R. and Peruzzi. C.A. 11985) Cancer Res. 45, ~818-5823. 0 Craig. A.M., Nemir. M, Mukherjee. Chambers, A.F. and Denhardt, D~T. (ItJ88) Bioehem. Biophys. Res. Commun. 157, 166173. II) Seglen. P.O. (tc,~76)Melhods Cell. Bio]. 13. 29-83. 11 Kapp. N.L. Brown, S.L and Levecz. R. ([~,74) Biochim. Biophys. Acla 361. 141)-143. 12 Cruise. J.L. Cotecchia. S. and Michalopt~ulos. G. (l~Sfi)J'. Cell. Physiol. 127. 39-44.
13 Boyum, A. (1%8) Scand. L Clin, Lab. Invest. 21, supp], 97, p. ")'7. 14 Hume. D,A. and Weidemann, MJ. (Iq~)) Mitogenic Lymphocyte Transh~rmation. Elsevier/North Holland. Amsterdam. 15 Skouteris. G.G.. Ord. M.G. and SIoeken, L.A. (I988,1 J. Cell. Physiol. 135. 516-520. 16 Whitfield, J.F. (Iqql)) Calcium, Cell Cycles and Cancer. pp. 92-98. CRC Press. Boca Raton. 17 Little, P.. Skouteris, G.G., Ord. M,G. and Sloeken, L.A. (|988)J. Cell Set. 91,549-553. 18 $kouteris. G.G.(Iq88) in Regulation of Liver Cell Growlh, Thesis l:k)clor of Philosophy. University of Oxford. Chapter 4. 19 Micbalopoulos. G.K, 11991))FASEB J. 4. 176-187. 211 Hsich. LL, Peraino, C- and Weinstein. I.B. (19~,8) Cancer Res. 48. 265-269.
