A growth inhibitor of rat hepatocytes synthesized by a rat hepatoma cell line, FF101

ELSEVIER

International Hepatology Communications 2 (1994) 333-340

A growth inhibitor of rat hepatocytes synthesized by a rat hepatoma cell line, FF 10 1 Hirofumi Kitaharaa,*, Hiroko Matsudab, Masahiro Matsumotob, Tsunehisa Kawasakia, Koichi Kanaib “The Second Department of Internal Medicine, Hamamatsu University School of Medicine. Handa-cho 3600, Hamamatsu, Shizuoka 431-31, Japan “Department of Gastroenterology, Toshiba Hospital, 6-3-22, Higashiohi, Shinagawa, Tokyo 140, Japan

(Received 4 February 1994; accepted 30 March 1994)

Abstract

A rat hepatoma cell line, FFlOl, established in serum-free, protein-free medium synthesizes a growth inhibitor of adult rat hepatocytes in primary culture. Gel filtration of FFlOl-conditioned medium disclosed two peaks of growth inhibitory activity with the molecular sizes over 200 kDa and smaller than 10 kDa. The growth inhibitor of smaller molecular size was tentatively designated as FF-GI. The DNA synthesis of rat hepatocytes was inhibited by FF-GI. The inhibitory activity of FF-GI was abolished by treatments with trypsin, dithiothreitol and heating at 100°C for 30 min, although it was stable against acid treatment and heating at 100°C for 5 min. These results indicate that FF-GI may be a novel growth inhibitor of adult rat hepatocytes. Key words: Growth inhibitor; Hepatocyte; Hepatoma cell line; Primary culture

1. Introduction

Although the presence of growth factors [1,2] and inhibitors [3,4] in both normal and neoplastic growth have been reported, their roles in the regulation of cell growth have not been elucidated in detail. Cultured tumor cells are known to synthesize and secrete several factors with biological activities. Previously, we reported the establishment of a rat hepatoma cell line, FFlOl, which proliferates in serum-free, protein-free medium [5], and the purification of a novel growth factor, FF-GF, synthesized by FFlOl cells [6]. FF-GF (molecular mass 70 kDa) has very little specificity since it

* Corresponding author. 0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved 0928-4346(94)00027-3

SSDI

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H. Kitahara et al. Ilnt. Hepatol. Commun. 2 (1994) 333-340

stimulated several kinds of cells from different species and different organs, whereas it exerted no effect on the proliferation of adult rat hepatocytes. In this paper, we describe the presence of growth inhibitory activity against rat hepatocytes in FFlOlconditioned medium and the partial purification and characterization of this growth inhibitor synthesized by FFlOl cells.

2. Materials and methods Cell line

A rat hepatoma cell line, FFlOl, established and maintained in serum-free, proteinfree RPM1 1640 medium (Nissui Pharmaceutical Co., Tokyo) as described previously [5], was used for the experiments. Preparation of conditioned medium

Conditioned medium from 2 days of FFlOl cell culture was centrifuged at 250 x g for 5 min and the supernatant was passed through a 0.22 pm membrane filter (MillexGV, Millipore Co., Tokyo). The filtrate was concentrated lOOO-fold using a membrane filter with a molecular cutoff of 2000 (high-performance ultrafiltration cells; Amicon Corp., Lexington, MA), filter-sterilized (Millex), and stored at -20°C. Geljltration

chromatography

Two hundred microliters of lOOO-fold concentrated conditioned medium was applied to a Superose-12 column (1 x 30 cm, Pharmacia AB Laboratory, Uppsala, Sweden). Elution was carried out with phosphate-buffered saline (PBS) at a flow rate of 0.7 ml/min. Four hundred microliter fractions were collected, concentrated 6-fold using a membrane filter (M, cutoff 2000, Amicon Corp), and filter sterilized (0.22 ,um; Millex-GV). Primary culture of hepatocytes

Hepatocytes were isolated from young adult male Donryu rats (150-200 g), by in situ perfusion with collagenase (collagenase S-l, Nitta Gelatin Inc., Osaka) as described by Tanaka et al. [7]. The cells were seeded at a density of 1.4 x lo5 cells/well on 24-well dishes (Corning 25820; Corning, NY) which had been precoated with rat tail collagen. The cells were cultured in 0.5 ml of Williams’ medium E (ICN Biomedical Inc., Costa Mesa, CA) supplemented with 5% FBS (GIBCO, Grand Island, NY), 10m9M dexamethasone (Tokyo Chemical Industry Co., Ltd. Tokyo) and lo-* M insulin (Sigma Chemical Co., St. Louis, MS) under 5% CO, in air at 37°C. Three hours after seeding, the medium was changed to serum-free, hormone-free Williams’ medium E. Assay of DNA synthesis low7 M insulin, 10 ng/ml human epidermal growth factor (EGF: Wakunaga Phar-

maceutical Co., Hiroshima) and test samples were added to the culture dishes 24 h after plating, and 24 h later 1 pCi/ml [3H]thymidine (New England Nuclear, Boston,

H. Kitahara et al. IInt. Hepatol. Commun. 2 (1994) 333-340

335

MA) was added. After a 4-h labelling period, the radioactivity in the hot trichloroacetic acid soluble fraction was measured using a liquid scintillation counter [Xl. Protein measurement was done by the method of Bradford [9]. Results were expressed as dpm per h per mg cellular protein. In order to determine the phases of the cell cycle where the growth inhibitor exerts its effect, the test sample or TGF-j? (1 ng/ml: King Brewing Co., Kakogawa, Japan) as control, was added to the medium after various periods of incubation. Assay of protein synthesis

lo-’ M insulin, 10 nglml EGF, test samples and 1 pCi/ml [3H]leucine (ICN Biomedicals, Inc., CA) were added to the culture dishes 24 h after plating. Culture was continued for another 24 h and the medium was removed for assay of extracellular protein synthesis, as described by Tanaka et al. [7]. The cells were solubilized and radioactivity in the hot trichloroacetic acid insoluble fraction was measured using a liquid scintillation counter to determine intracellular protein synthesis. Analysis of amino acid composition in culture medium

Williams’ medium E was incubated with test samples at 37°C for 24 h and the amino acid composition before and after incubation was analyzed on an amino acid analyzer (Hitachi 835-200; Hitachi Co., Ltd., Tokyo). Physicochemical treatments

To test trypsin sensitivity, the sample was incubated for 2 h at 37°C with 100 pg/ml trypsin (GIBCO, Grand Island, NY) and then 200 &ml soybean trypsin inhibitor (Sigma Chemical Co., St. Louis, MI) was added for 30 min at 37°C. Another sample was exposed to 65 mM dithiothreitol plus 100 mM NaHCO, for 1 h at room temperature and dialyzed extensively in Spectra/Par 7 tubing (Mr 2000 cutoff; Spectrum, TX) against PBS (20 h at 4°C two changes of 1000 x volume). In addition, the conditioned medium was heated at 100°C for periods ranging from 5 min to 30 min. In order to test acid resistance, the sample was incubated for 20 h at 4°C with 1 N acetic acid and then dialyzed against PBS. Influence on proliferation of other cell lines

The fraction with growth inhibitory activity against rat hepatocytes eluted by gel filtration chromatography was added to cultures of two other cell lines (rat hepatoma cell, AH66 [lo], human erythroleukemia cell, K562 [l 11). AH66 cells and K562 cells were maintained in RPM1 1640 containing 10% FBS. For assays, they were suspended in RPM1 1640 by pipetting, washed three times with RPM1 1640, and then plated in microplates (Corning 25860: 96-well with lid flat bottom) containing RPM1 1640 with 1% FBS (AH66, 5 x 103/well; K562, 3 x 103/well). Five microcuries of [3H]thymidine was added to each culture well 24 h after addition of the sample, and culture was continued for another 6 h. The medium was then aspirated and the cells were washed with cold 5% trichloroacetic acid. Individual wells were harvested by the addition of 150 ,~l of 0.25 M sodium hydroxide. Radioactivity was determined using a liquid scintillation counter.

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3. Results Inhibitory effects of FFlOI conditioned medium In hepatocyte cultures, insulin, EGF and insulin plus EGF increased the incorporation of [3H]thymidine into rat hepatocytes by about 3-, 5- and lo-fold, respectively. The addition of FFlOl conditioned medium inhibited the incorporation of [3H]thymidine into rat hepatocytes induced by insulin and/or EGF in a dose-dependent manner (Fig. 1). A 50% inhibition of DNA synthesis occurred when 0.5 ~1 of IOOOfold conditioned medium was present in incubation medium (500,~l). Maximal inhibition was observed at 2.5 ,ull500 ~1. PartialpuriJication of a growth inhibitor synthesized by FFlOl When lOOO-fold concentrates of FFlOl-conditioned medium was fractionated by gel titration chromatography on a Superose-12 column, two peaks with inhibitory activity on the incorporation of t3H]thymidine into hepatocytes were observed (Fig. 2). One inhibitory peak was eluted at fractions 17 to 19 and the molecular mass of this peak was estimated to be larger than 200 kDa. The other was eluted at fraction 37 to 40 and its molecular mass was estimated to be smaller than 10 kDa. The former was

015 Volume

1:O

of FFlOl

1:5

210

conditioned

215 medium

(PC)

Fig. 1.Dose-response curve of FFlOl conditioned medium on DNA synthesis of adult rat hepatocytes in primary culture. 1.4 x 10’ hepatocytes were incubated with insulin (lo-’ M) plus EGF (10 ngknl) (0) or EGF (A) or insulin (H) or no addition (0) after 24 h of incubation, 0 to 2.5 ~1 of lOOO-foldconcentrated conditioned medium were added to incubation medium (500 ~1). Triplicate cultures were harvested after labeling with [‘E[lthymidine for 4 h (48-52 h) and were processed for DNA synthesis assay.

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situated in the position of the void volume of the column and therefore, we chose the latter, especially fraction number 38, for further experiments and tentatively designated it as FF-GI. Effect of FF-GI on protein synthesis of rat hepatocytes Although FF-GI remarkably inhibited DNA synthesis of hepatocytes and this inhibitory effect could not be overcome by increasing the concentration of insulin and EGF, FF-GI did not exert any influence on either intracellular or extracellular protein synthesis of rat hepatocytes or the morphological appearance of cultured hepatocytes (data not shown). Inhibition of DNA synthesis by FF-GI relative to the cell cycle To determine the critical phase of the cell cycle where FF-GI exerts its biological action, rat hepatocytes were exposed to FF-GI for different periods after the addition of insulin and EGF, as shown in Table 1. The inhibitory activity of FF-GI was strongest when it was present in medium at 12 h to 18 h of incubation. In contrast, TGF-/I inhibited DNA synthesis of hepatocytes almost completely when present at any time during the first 18 h of incubation. DNA synthesis was not inhibited when FF-GI was present in culture medium between 24 h to 28 h of incubation. Infruence of FF-GI on the amino acid composition of culture medium FF-GI did not affect the amino acid composition of the culture medium (data not shown).

.l .o 7 .0.8 _,_

.0.6

d

~0.4 ; ~_ FL 0.2 $ 0

2

fraction number

Fig. 2. Fraction of FFlOl conditioned medium by Superose 12 gel filtration chromatography. 200 ~1 of lOOO-foldconcentrates of FFlOl conditioned medium were layered on a Superose 12 column (1 x 30 cm). Elution was done using PBS at an elution speed of 0.7 ml/min. Four hundred microliters fractions were collected and their inhibitory effects on [‘Hhhymidine incorporation into adult rat hepatocytes stimulated by insulin (lo-’ M) plus EGF (10 @ml) were determined. Catalase (M, 232 000), aldolase (M, 158 000), bovine serum albumin (M, 67 000), ovalbumin (i+f, 43 000), chymotrypsinogen A (M, 25 000), and cytochrome (M, 12 500) were used as markers.

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H. Kitahara et al. lint. Hepatol. Commun. 2 (1994) 333-340

Table 1 Inhibitory activity of FF-GI and TGF-/3 on DNA synthesis of adult rat hepatocytes in different phases of the cell cycle Addition period (h)

None O-28 t&6 612 12-18 18-24 24-28

Inhibition (%) FF-GI

TGF-,I3

0 100 5 30 86 33 1

0 100 100 98 88 34 2

Insulin and EGF were added to cultures at time 0 (24 h after plating). Hepatocytes were treated with FF-GI (25 ~llwell) or TGF-B (1 ng/ml) for the indicated periods. rH]Thymidine was added to the culture medium 24 h after addition of insulin plus EGF, and DNA synthesis were measured 28 h after addition of the hormones.

Physicochemical characterization of FF-GI FF-GI was subjected to several physicochemical treatments and subsequently assayed for its ability to inhibit DNA synthesis in primary rat hepatocyte cultures. FF-GI was inactivated by treatment with trypsin or dithiothreitol. The activity was partially lost after treatment at 100°C for 15 min, and completely lost at 100°C for 30 min. Treatment with 1 N acetic acid also partially inactivated the inhibitory activity of FF-GI. Results of physicochemical treatments are summarized in Table 2. Growth inhibitory activity of FF-GI on various cell lines FF-GI exerted no effect on [3H]thymidine incorporation K562 cells (data not shown).

in either AH66 cells or

Table 2 Effect of different physicochemical treatments on the inhibitory activity of FF-GI on the DNA synthesis of rat hepatocytes in primary culture Treatment

DNA synthesis (dpm/h/mg protein x 10m4)

Inhibition (%)

Ins. + EGF Ins. + EGF + FF-GI Heat 100°C 5 min 15 min 30 min Trypsin (100 &ml, 2 h) Dithiothreitol(65 mM, 1 h) Acetic acid (1 N, 20 h)

10.0 f 2.1 rt 2.0 + 4.3 + 9.6 f 10.0 f 9.3 f 4.2 f

100 100 72 5 0 9 16

0.8 0.1 0.1 0.2 0.4 0.4 0.2 0.3

0

Hepatocytes were exposed to aliquots of FF-GI after each treatment. The DNA synthesis was determined by the incorporation of [‘Hlthymidine into cultured hepatocytes as described in Materials and methods.

H. Kitahara et al. IInt. Hepatol. Commun. 2 (1994) 333-340

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4. Discussion

Various factors with biological activities are known to be synthesized and secreted by cultured tumor cells. Among them, growth factors have been extensively studied and it has been indicated that these factors play important roles in making a favorable microenvironment for tumor cells inducing autocrine proliferation [2,12], angiogenesis [2,13], extracellular matrix synthesis [14], and sometimes inhibition of normal cell growth [3,1.5].On the other hand, little is known about the possibility that tumor cells might synthesize factors which inhibit normal cell growth surrounding the tumor. Previously we have reported the establishment of a rat hepatoma cell line FFlOl, that proliferates in serum-free, protein-free RPM1 1640 medium [5] and the purification of a novel growth factor (FF-GF) from the culture medium of FFlOl cells, with intense growth promoting activity on several tumor cell lines [6]. In this report, we have demonstrated that FFlOl conditioned medium inhibits the incorporation of [3H]thymidine into DNA of cultured rat hepatocytes. Therefore, it appears that FFlOl cells synthesize a factor(s) that inhibits DNA synthesis of rat hepatocytes, in addition to FF-GF. Two peaks (molecular mass > 200 kDa and molecular mass < 10 kDa) with inhibitory activity on DNA synthesis were observed by gel filtration chromatography of FFlOl conditioned medium. Since the fraction of large molecular weight was situated in the position of the void volume of the column, we decided to concentrate on the fraction with small molecular size, especially fraction number 38, designated tentatively as FF-GI. FF-GI exerted remarkable inhibitory effect on cultured rat hepatocytes in a dose-dependent manner. The growth inhibition caused by FF-GI was not due to cytotoxic effect or changes of the amino acid in incubation medium, because FF-GI affected neither protein synthesis of hepatocytes nor amino acid concentrations of the culture medium. TGF-P is one of the most potent inhibitors on the proliferation of hepatocytes and acts on the late Gl phase of the cell cycle [16]. In our study, Ff-GI exerted its inhibitory activity at a later phase of the cycle indicating that FF-GI is different from TGF-p. When physicochemical and biological characteristics of FF-GI were compared with that of other hepatocyte growth inhibitors [l&19], the molecular size of FF-GI (less than 10 kDa) determined by gel filtration was smaller than many other hepatocyte growth inhibitors (IL-6; 26 kDa, TGFB; 25 kDa, IFN-y; 20 kDa, IL-l/I; 14 kDa). The inhibitory activity of FF-GI was abolished by treatment with trypsin or dithiothreitol, suggesting that a polypeptide is an essential structural element of FF- GI. FF-GI is relatively heat and acid stable, while some hepatocyte growth inhibitors, such as IL-lp and IFN-y, are heat labile. IL-6 is relatively heat stable but does not completely inhibit DNA synthesis of rat hepatocytes stimulated by insulin plus EGF [ 171. These results suggest that FF-GI is a novel hepatocyte growth inhibitor and further purification and characterization is warranted. FFlOl appears to be a unique cell line, because it synthesizes not only a novel autocrine growth factor, FF-GF, but also produces a potent inhibitor of the proliferation of normal rat hepatocytes from which FFlOl cells were originated. FFlOl may be a suitable model for the study of growth regulation of the tumor cells and to examine the relationship between tumor cells and surrounding normal cells.

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