Expressions of basic fibroblast growth factor and its receptors and their relationship to proliferation of human hepatocellular carcinoma cell lines

Expressions of Basic Fibroblast Growth Factor and Its Receptors and Their Relationship to Proliferation of Human Hepatocellular Carcinoma Cell Lines SACHIKO OGASAWARA, HIROHISA YANO, AKIHIRO IEMURA, TOHRU HISAKA,

On six human hepatocellular carcinoma (HCC) cell lines (KIM-1, KYN-1, KYN-2, KYN-3, HAK-1A, and HAK1B), we examined expressions and functions of the proteins and messenger RNAs (mRNAs) of basic fibroblast growth factor (bFGF) and its receptor, i.e., fibroblast growth factor receptor-1 (FGFR-1), as well as mRNA expressions of FGFR-2 Ç4. All six cell lines expressed the proteins and mRNAs of bFGF and FGFR-1, and at least one of FGFR-2 Ç4 mRNAs. Two of the six cell lines (KYN1 and KYN-3) presented significant release of bFGF in culture supernatant, while the release in the remaining four cell lines was quite small. Addition of anti-bFGF neutralizing antibody (1, 10, or 20 mg/mL) to culture medium resulted in marked suppression of cell proliferation in all cell lines except HAK-1A. On the other hand, addition of exogenous bFGF (0.1, 1, or 5 ng/mL) to culture medium stimulated cell proliferation except in KIM-1 and KYN-2. When KIM-1 was transplanted to nude mice and anti-bFGF antibody was injected subcutaneously to a space surrounding the developed tumor, tumor proliferation was significantly suppressed in nude mice that received anti-bFGF antibody than in control mice, but there were no histological differences between the groups, including blood space formation in the stroma. In conclusion, hepatocellular carcinoma (HCC) cells may possess a proliferation mechanism regulated by an autocrine mechanism, a paracrine mechanism, or both, which are mediated by bFGF/FGFR. (HEPATOLOGY 1996;24:198-205.) The fibroblast growth factor (FGF) family is a group of structurally related multifunctional mitogenic polypeptides, and its members possess heparin-binding property. At present, nine members, from FGF-1 to FGF-9, have been identified.1-9 The FGF family has been widely distributed in normal and/or tumor tissues, and they are known to take various important roles, e.g., in angiogenesis, tissue regeneration, wound healing, and embryonic development.10-17 Among the FGF family, basic fibroblast growth factor (bFGF, or FGF-2) acts as a potent mitogen and as a differentiation factor for various mesoderm- and neuroectoderm-derived cells.11 bFGF

Abbreviations: FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; FGFR, fibroblast growth factor receptor; HCC, hepatocellular carcinoma; mRNA, messenger RNA; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction. From the The First Department of Pathology, Kurume University School of Medicine, Kurume 830, Japan. Received January 2, 1996; accepted March 11, 1996. Supported in part by Sarah Cousins Memorial Fund, Boston, MA, and by a Grant-inAid for General Scientific Research (C) from The Ministry of Education, Science, Sports and Culture, Japan. Address reprint requests to: Sachiko Ogasawara, The First Department of Pathology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2401-0032$3.00/0

AND

MASAMICHI KOJIRO

is also known to possess the following biological effects: It affects vascular endothelial cells and stimulates angiogenesis, and it influences bFGF-responsive cells to change their morphology and growth pattern, and to increase their migratory activities.11,16,18-20 To date, bFGF has been identified in epithelial cells, endothelial cells, fibroblasts, macrophages, and extracellular matrix, in various organs in vivo,11-14 and its presence has also been confirmed in tumor tissues.11,12,21-23 Schlze-Osthoff et al.12 reported that tumors revealed a very heterogeneous staining pattern, e.g., bFGF can be expressed (1) only in tumor cells, (2) only on vascular endothelial cells, or (3) only on macrophages. Identification of bFGF in tumor tissues and in some cancerous cell lines allowed researchers to presume that bFGF is involved in the development and progression of tumors. It is possible that bFGF produced by tumor cells affects proliferation of the tumor cells themselves through an autocrine or intracrine mechanism; or, bFGF acts on endothelial cells through a paracrine mechanism and stimulates angiogenesis; or bFGF induces higher productions of plasminogen activators, various proteases, and collagenase, and contributes to infiltration and metastasis of tumor cells.11,16,19,22 As high-affinity cell surface receptors for the FGF family, five types (fibroblast growth factor receptor [FGFR]-1 Ç5) have been identified so far,24-27 and their expressions have been reported in various tumor tissues and cell lines, and in normal endothelial cells.14,17,28-30 Expression of bFGF in normal liver tissues has been a matter of controversy. Hughes and Hall14 conducted an immunohistochemical study and observed bFGF expressions at high levels in normal hepatocytes. However, other studies before them reported that normal hepatocytes did not express bFGF, or they only weakly expressed bFGF.12,13 On the other hand, Motoo et al.21 identified high bFGF expressions in the cytoplasm of hepatocellular carcinoma (HCC) cells of 16 of 56 patients (29%), and another study reported the cultured HCC cell line, SK Hep-1, can produce bFGF in vitro.31 Normal hepatocytes do not express the bFGF receptor, FGFR-1, while a hepatoblastoma cell line (HepG2) has been reported to express this receptor.14,32 Consequently, expressions of bFGF/ FGFR could be involved in the angiogenesis and proliferation of HCC cells, even though their precise mechanism has not been fully elucidated. In the present study, we examined (1) protein and messenger RNA (mRNA) expressions of bFGF and FGFR1 Ç4, and (2) proliferation effects of bFGF on HCC cells through an autocrine mechanism both in vitro and in nude mice. MATERIALS AND METHODS Cell Lines and Cell Culture. We used six human HCC cell lines that were originally established in our laboratory, i.e., KIM-1,33 KYN-1,34 KYN-2,35 KYN-3,36 HAK-1A,37 and HAK-1B.37 These cell lines were previously confirmed to retain morphological and functional characteristics of the original HCC. HAK-1A and HAK-1B were two clonally related HCC cell lines established from a single HCC nodule showing a three-layered structure with a different histological grade in each

198

AID

Hepa 0048

/

5p10$$$941

06-25-96 09:38:54

hpta

WBS: Hepatology

HEPATOLOGY Vol. 24, No. 1, 1996

OGASAWARA ET AL.

layer. HAK-1A is, morphologically, a well-differentiated HCC cell line, while HAK-1B is a poorly differentiated HCC cell line and biologically more malignant than HAK-1A. HAK-1B is presumed to be derived from HAK-1A through its clonal dedifferentiation.37 KIM-1 and KYN-1 were established from surgically resected moderately differentiated HCC nodules; KYN-2, from a resected moderately to poorly differentiated HCC nodule; and KYN-3, from peritoneal effusion of a HCC patient who had moderately to poorly differentiated HCC in his liver. Each cell line was grown in Dulbecco’s modified Eagle medium (Nissui Seiyaku Co., Japan) supplemented with 5% heat-inactivated (567C, 30 minutes) fetal bovine serum (Bioserum, Victoria, Australia), 100 U/mL penicillin, 100 mg/mL streptomycin (GIBCO BRL/ Life Technologies, Inc., Gaithersburg, MD), and 12 mmol/L sodium bicarbonate, in a humidified atmosphere in 5% CO2 in air at 377C. Immunohistochemistry. For immunohistochemistry, cells were cultured on Lab-Tek tissue culture chamber slides (Nunc, Inc., Roskilde, Denmark) for several days, then fixed with cold acetone for 20 minutes, air dried, and immunostained with Histofine streptoavidin-biotin peroxidase kits (SAB-PO[M], Nichirei, Tokyo, Japan). As primary antibodies, we used (1) immunoneutralizing monoclonal antibody against human bFGF (anti-bFGF antibody), i.e., designated 3H338 (immunoglobulin G1 , final dilution, 1/500; Wako Pure Chemical Industries, Inc., Tokyo, Japan), which does not cross-react to acidic FGF (aFGF), and (2) a monoclonal antibody that is specific to the human flg gene product, FGFR-1 (anti–FGFR-1 antibody) (immunoglobulin G1 , final dilution, 1/200; Upstate Biotechnology, Inc., NY). Peroxidase reaction was developed by using 3-amino-9ethylcarbazole, and the cells were counterstained with hematoxylin. Flow Cytometric Analysis. Cell suspension (4 1 105 cells per tube) was washed in a tube with 2 mL washing buffer (0.2% bovine serum albumin, 0.1% NaN3 /10 mmol/L phosphate-buffered saline, pH 7.4), centrifuged at 47C (300g, 5 minutes), and the supernatant was removed. The remaining cell pellet was fixed with 0.25% paraformaldehyde for at least 15 minutes in a dark room and at room temperature, then it was washed twice with 2 mL washing buffer, incubated for 1 hour at 47C with 70% cold methanol, and washed again. To examine expression of bFGF or FGFR-1, the cells were incubated for 1 hour at 47C with anti-bFGF antibody (final dilution, 1/100) or anti–FGFR1 antibody (final dilution, 1/100), washed twice, incubated for 30 minutes on ice in a dark room with 4 mL fluorescein isothiocyanate conjugated goat anti-mouse IgG (Becton Dickinson Immunocytometry System USA, San Jose, CA), washed twice, and suspended into 1 mL washing buffer for analyses using a FACScan (Becton Dickinson Immunocytometry System USA). Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and Western Blotting. Cultured cells were incubated in lysis buffer (10

mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L ethylenediametetraacetic acid, 1% Triton-X 100, 2 mmol/L phenymethylsulfonyl fluoride) on ice for 1 hour. The mixture of the cells and the lysis buffer was centrifuged for 15 minutes (15,000 rpm, 47C). The supernatant was mixed with Laemmli’s sample buffer to a final concentration of 1 1 104 cells/mL, boiled for 5 minutes, and then used as samples. To examine the expression of bFGF or FGFR-1 protein, a sample (20 mL per lane) was applied to a 12% or 7.5% sodium dodecyl sulfate–polyacrylamide slab gel, respectively, and electrophoresed. The proteins were transferred to nitrocellulose membranes by using the Transblot apparatus (Bio-Rad, Richmond, CA). The membranes were used for blotting with monoclonal antibody against human bFGF (immunoglobulin G1 , final dilution, 1/250; Transduction Laboratories, USA) or anti–FGFR-1 antibody (final dilution, 1/500), by using an Immunoblot detection kit (Wako Pure Chemical Industries, Tokyo, Japan). ELISA. Cultured cells (6 Ç25 1 104 cells per well) were seeded on 12-well plates (Nunc, Inc.), and the plates were cultured for 3 days. The bFGF amount in the supernatant was measured by using an enzyme-linked immunosorbent assay (ELISA) kit (FGF basic, human, ELISA system; Amersham International plc, Buckinghamshire, England). After counting the cells in a well by using the trypanblue dye exclusion method, bFGF amount per 1 1 104 cells was obtained. All experiments were quadruplicated. Analyses of bFGF and FGFR mRNAs with Reverse-Transcription PCR Method. Total RNA of cultured cells was extracted by using

RNAzol B (Biotecx Laboratories, Inc., Houston, TX). To prepare firststrand complementary DNAs, 4 mg of total RNA was added to 10 mL of reverse-transcriptase buffer (50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2 , 0.4 mmol/L deoxyribonucleoside

AID

Hepa 0048

/

5p10$$$942

06-25-96 09:38:54

199

triphosphates), which contained 100 U of RNase H-reverse transcriptase (Superscript II, GIBCO BRL/Life Technologies, Inc.), 50 pmol random primer, and 20 U of RNAsin (Promega Corp., Madison, WI). Reverse-transcription reaction progressed for 45 minutes at 377C, and then was stopped by boiling the solution for 5 minutes at 957C. One fourth of this complementary DNA solution was used as a template for DNA synthesis, and polymerase chain reaction (PCR) reaction was made with a primer specific either to bFGF, FGFR-1 Ç4, or b-actin (control).39 The PCR reaction solution consisted of 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2 , 50 mmol/L KCl, 0.01% gelatin, 0.1 mmol/L deoxyribonucleoside triphosphate, 0.25 mg appropriate 3* and 5* primers, and 2.5 U Amplitaq DNA Polymerase (Perkin Elmer, Branchburg, NJ). PCR reaction was repeated for 40 cycles by using a Thermocycler (Perkin-Elmer Cetus Corp., Norwalk, CT), and one cycle consisted of denaturation (947C, 30 seconds), annealing (627C, 30 seconds), and extension (727C, 30 seconds). PCR product (5 mL) was electrophorased with a 2% NuSieve agarose gel (FMC Bioproducts, Rockland, ME), which contained 0.5% ethidium bromide, and specific DNA bands were examined under an ultraviolet transilluminator. Effect of Anti-bFGF Antibody on HCC Cell Proliferation. Effect of anti-bFGF neutralizing antibody on cultured cell proliferation was investigated by using colorimetric assays with 3-(4,5-dimethylthiazol-2yl-yl)-2, 5-diphenyl tetrazolium bromide cell growth assay kits (Chemicon, Temecula, CA). Cultured cells (0.25 Ç1 1 104 cells per well) were seeded on 96-well plates (Falcon, Becton Dickinson Labware, Tokyo, Japan), cultured for 24 hours, and then the medium was exchanged to fresh 100 mL medium containing anti-bFGF antibody (1, 10, or 20 mg/mL) or control antibody at the same levels (mouse immunoglobulin G1 ; Chemicon). After 3 days, 100 mL of 3(4,5-dimethylthiazol-2yl-yl-2, 5-diphenyl tetrazolium bromide, 160 mg/mL, was added to each well, cultured for 4 hours, the supernatant was removed, and 100 mL of 40 mmol/L HCl/dimethylsulfoxide was added to each well. Viable cell numbers were estimated by measuring the absorbance with an Easy Reader EAR 400 (SLT Lab Instruments, Salzburg, Austria) by setting a test wavelength at 570 nm and a reference wavelength at 630 nm. The absorbance (viable cell number) for the anti-bFGF antibody–treated group and the control antibody group were obtained in quadruplicated examinations, and the absorbance was comparatively examined. To confirm reproducibility of the test results, the same examination was repeated twice more. Effect of Exogenous bFGF on HCC Cell Proliferation. Cultured cells (1 Ç4 1 104 cells per well) were seeded on 12-well plates. After a 24-hour culture, culture medium was changed to the medium with bFGF (0.1, 1, or 5 ng/mL; Genzyme, Cambridge, USA) or without bFGF. The plates were cultured for 72 hours, then viable cells were counted using the trypan-blue dye exclusion method (n Å 6), and the effect of exogenous bFGF on cell proliferation was examined. To prevent nonspecific adherence of bFGF, the medium was supplemented with 0.001% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid. To monitor morphological changes of the cells, cultured cells were seeded on Lab-Tek tissue culture chamber slides, cultured with or without bFGF at the same concentrations as shown above, and then stained with hematoxylin-eosin. Effect of Anti-bFGF Antibody on HCC Cell Proliferation in Nude Mice. KIM-1 cells (8 1 106 cells per mouse) were transplanted subcu-

taneously to 5-week-old female BALB/c athymic nude mice (Clea Japan, Inc., Osaka, Japan). After tumor formation was confirmed on day 3, either an anti-bFGF neutralizing antibody (n Å 10, 40 mg per mouse per day) or control antibody (n Å 8, 40 mg per mouse per day) was injected subcutaneously for 5 days to a space surrounding the tumor. Tumor measurements were made in two directions using calipers, and tumor volume (mm3) was estimated by using ‘‘length 1 (width)2 1 0.5.’’ The solid tumor in each mouse was resected on day 31 of transplantation, fixed into formalin, prepared into paraffin sections, and stained with hematoxylin-eosin and elastica van Gieson for histological examinations. Animals were treated in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals’’ (National Institutes of Health publication no. 86-23, revised 1985). RESULTS Expressions of bFGF and FGFR-1 Proteins in HCC Cell Lines. In immunohistochemical staining, expression of bFGF

proteins was confirmed as positive granular pictures in the cytoplasm, at different intensities, in all cell lines except HAK-1A (Fig. 1A). FGFR-1 protein expression was diffusely observed on the cell membrane and in the cytoplasm of all

hpta

WBS: Hepatology

200

OGASAWARA ET AL.

HEPATOLOGY July 1996

FIG. 1. Immunohistochemical staining of (A) bFGF and (B) FGFR-1 using the streptoavidin-biotin method. (A) Intensive cytoplasmic staining for bFGF was observed in KYN-3. (B) KYN-3 showed weak cell surface and cytoplasmic immunoreactivity (Hematoxylin, original magnification 2001).

cell lines, but the expression was generally weak (Fig. 1B). In flow cytometry, bFGF and FGFR-1 expressions were observed in all cell lines, and their intensity was in agreement with the results of immunohistochemical staining. The positive cell rate to bFGF was the highest in KYN-3 (98.3%) and the lowest in HAK-1A (31.8%). The rate to FGFR-1 was the highest in KYN-2 (62.0%) and the lowest in HAK-1A (22.9%) (Fig. 2A and 2B). In Western blot analyses, bFGF expression

was the most intensive in KYN-3, as observed in the other assays, and expression of four isoforms (18, 22, 24, and 28 kd) were identified. In the other cell lines except HAK-1A, expression of three isoforms (18, 22, and 28 kd) were identified (Fig. 3A). For FGFR-1, all cell lines presented a band around 120 kd (Fig. 3B). Detection of bFGF in Spent Media by Using ELISA. A relatively high amount of bFGF protein was detected in the su-

FIG. 2. Flow cytometric analysis of (A) bFGF and (B) FGFR-1, which were expressed on the HCC cell lines. Staining with specific antibodies or control antibody is shown by solid or dotted line, respectively. KYN-3 expressed bFGF at a high level with an average positive cell rate of 98.3%. KYN-2 expressed FGFR at relatively high levels with an average positive cell rate of 62.0%.

AID

Hepa 0048

/

5p10$$$942

06-25-96 09:38:54

hpta

WBS: Hepatology

HEPATOLOGY Vol. 24, No. 1, 1996

OGASAWARA ET AL.

201

FIG. 3. Western blot analysis of (A) bFGF and (B) FGFR-1 on the HCC cell lines. Twenty microliters (104 cells/mL) of lysate obtained from each cell line (lane 1, KIM-1; lane 2, KYN-1; lane 3, KYN-2; lane 4, KYN-3; lane 5, HAK-1A; lane 6, HAK-1B) was applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a (A) 12% or (B) 7.5% slab gel. Molecular weights of bFGF are 18, 22, 24, and 28 kd, and FGFR is approximately 120 kd.

pernatant of KYN-3 (1.97 pg per 104 cells) and KYN-1 (1.39 pg per 104 cells), whereas the amount was relatively low for KYN-2 (0.33 pg per 104 cells) and HAK-1B (0.13 pg per 104 cells), and quite low for KIM-1 and HAK-1A. Table 1 summarizes expressions of bFGF and FGFR-1 proteins in each cell line and in its culture supernatants. Expression of bFGF and FGFR mRNAs in HCC Cell Lines.

tion of bFGF to culture medium stimulated cell proliferation in KYN-1, KYN-3, HAK-1A, and HAK-1B. In cultures with the highest bFGF level (5 ng/mL), the cell counts for KYN1, KYN-3, HAK-1A, and HAK-1B increased to 138.1% (P ° .001), 131.5% (P õ .01), 150.1% (P ° .001), and 159.6% (P ° .001), respectively, of the cultures that did not have bFGF (Fig. 6). KYN-3 cells usually proliferate in the form of paving stones on the bottom of a flask, but addition of 5 ng/mL bFGF made them grow in a spindle form or pile up (Fig. 7).

All cell lines presented a 235-bp band that corresponds to a PCR product of bFGF. The PCR products of FGFR-1, -2, -3, and -4 are expected to be expressed as bands at 425, 743, 295, and 560 bp, respectively. In the present study, each cell line expressed at least two of these four bands. FGFR-1 expression was detected in all cell lines, whereas FGFR-2 was not expressed in KYN-1 and KYN-3; FGFR-3 was not expressed in KYN-3; and FGFR-4 was not expressed in HAK1A (Fig. 4). PCR using b-actin specific primers demonstrated a 931-bp band in all cell lines, suggesting that an almost equal amount of complementary DNA has been used and similarly amplified.

KIM-1 cells to nude mice, the estimated tumor volume did not differ statistically significantly between the anti–bFGFtreated group and the control group. However, after day 13, tumor development in the anti–bFGF-treated group was significantly suppressed in contrast to the control (P õ .05, by ANOVA) (Fig. 8). There were no morphological differences, such as blood space formation in the stroma, between the two groups.

Effect of Anti-bFGF Neutralizing Antibody on HCC Cell Proliferation. Addition of anti-bFGF neutralizing antibody to

DISCUSSION

culture medium suppressed cell proliferation in all cell lines, except HAK-1A. Fig. 5 shows a representative case in each experiment. In cultures with the highest anti-bFGF antibody level (20 mg/mL), cell counts for KIM-1, KYN-1, KYN-2, KYN3, and HAK-1B were suppressed to 49.8% (P ° .001), 55% (P ° .001), 47.4% (P ° .001), 60.4% (P ° .001), and 86.5% (P õ .05), respectively, of the counts in the cultures with control antibody. The same experiments were repeated three times, and similar results were obtained. Effect of Exogenous bFGF on HCC Cell Proliferation. Addi-

Effects of Anti-bFGF on HCC Cell Proliferation in Nude Mice. Until day 10 after subcutaneous transplantation of

The present immunohistochemistry, flow cytometry, Western blot analysis, and reverse-transcription PCR assay showed all six HCC cell lines express proteins and mRNAs of bFGF and its receptor, FGFR-1. Except HAK-1A, which expresses bFGF at quite low levels detectable only by flow cytometry, proliferation of the other five cell lines was statistically significantly suppressed by the addition of immunoneutralizing monoclonal antibody against human bFGF to the cultures. This strongly indicates the presence of autono-

TABLE 1. Summary of Protein Assay Results for bFGF and FGFR-1 Expressions in the Cell Lines bFGF

FGFR-1

Cell Line

IH* Positivity

FCM† (%)

Western‡ Positivity

ELISA (pg/104 cells)

IH* Positivity

FCM† (%)

Western‡ Positivity

KIM-1 KYN-1 KYN-2 KYN-3 HAK-1A HAK-1B

/ // / // 0 /

51.2 75.2 40.9 98.3 31.8 53.0

{ { { // 0 {

0.03 1.39 0.33 1.97 0.01 0.13

/ / / / / /

29.9 39.4 62.0 45.3 22.9 43.5

/ / / / / /

Abbreviations: IH, immunohistochemistry; FCM, flow cytometry; Western, Western blotting. * Immunostaining intensity was classified as follows: 0, negative; /, positive; //, strongly positive. † Figures show positive cell rate. ‡ Western blotting intensity was classified as follows: 0, negative; {, weakly positive; /, positive; //, strongly positive.

AID

Hepa 0048

/

5p10$$$942

06-25-96 09:38:54

hpta

WBS: Hepatology

202

OGASAWARA ET AL.

HEPATOLOGY July 1996

FIG. 4. Expression of mRNAs for bFGF, FGFR-1, -2, -3, and -4 in the HCC cell lines using reverse-transcription PCR. The PCR products (lane 1, KIM-1; lane 2, KYN-1; lane 3, KYN-2; lane 4, KYN-3; lane 5, HAK-1A; lane 6, HAK1B; lane 7, negative control) were electrophoresed in a 2% NuSieve agarose gel and stained with ethidium bromide. Lane M shows the DNA molecularweight markers. PCR reaction using b-actin specific primers demonstrated that equal amounts of complementary DNA had been used and similarly amplified.

mous cell proliferation mechanisms, such as autocrine, mediated by bFGF/FGFR, at least, in the five cell lines. Several studies have reported that some bFGF-producing cell lines release bFGF into conditioned media,18,40-43 and in these cells, the presence of an autocrine growth mechanism has been proposed, i.e., the released bFGF could bind to FGFR on the surface of the releasing cell and could act to transfer

FIG. 6. Effect of bFGF on the growth of HCC cell lines. Cells (1 Ç4 1 104 cells per well) were seeded on 12-well plates and incubated in medium with or without bFGF (h, 0.1; Ω, 1; j, 5 ng/mL) for 3 days. The relative viable cell number was calculated as a percentage of the viable cell number in an experimental well to the number in a corresponding control culture. Significant differences between values for experimental wells and their corresponding control wells are: *P õ .01, **P ° .001. Error bar represents SD.

growth signals.44 However, among the FGF family, bFGF and acidic FGF genes do not encode conventional signal peptide sequences for extracellular FGF release.3,4,11,22 Consequently, the following three mechanisms of extracellular bFGF release have been proposed: (1) It is released through cytolysis or cell leakage. (2) It is released from cells in association with extracellular matrix components and becomes an integral part of the insoluble extracellular matrix. Their release and activation could occur when specific enzymes, such as heparinase-like

FIG. 5. Cells (0.25 Ç1 1 104 cells per well) were seeded on 96-well plates and incubated in medium with an antibFGF antibody (Ω; 1, 10, 20 mg/mL) or control antibody (h, 1, 10, 20 mg/mL) for 3 days. The cell numbers of HCC cell lines were assessed by 3-(4,5-dimethylthiazol-2yl-yl-2, 5-diphenyl tetrazolium bromide assay. Significant differences between values for cells with anti-bFGF antibody and for cells with control antibody are: *P õ .05, **P õ .01, ***P ° .001. Error bar represents SD.

AID

Hepa 0048

/

5p10$$$942

06-25-96 09:38:54

hpta

WBS: Hepatology

HEPATOLOGY Vol. 24, No. 1, 1996

OGASAWARA ET AL.

203

FIG. 7. Photomicrograph of KYN-3 cultured in (A) the presence (bFGF) or (B) absence (control) of 5 ng/mL bFGF. bFGF induced morphological alterations in cell and spindle-shaped cells and pile-up of cells were observed (Hematoxylin-eosin; original magnification 1001.)

enzymes, degrade the extracellular structure. (3) It is released through unknown mechanisms.11,16,40,45,46 Among the five cell lines, which excludes HAK-1A, in which only a low amount of bFGF is produced within the cell, KYN-1 and KYN-3 released bFGF into culture supernatants at relatively high levels compared with the other three cell lines. In addition, proliferation of KYN-1 and KYN-3 was apparently suppressed when an antibFGF neutralizing antibody was added to the cultures, while it was stimulated when exogenous bFGF was added. Therefore, these two cell lines almost certainly possess an autocrine growth mechanism mediated by bFGF/FGFR. The amount of released bFGF into the culture supernatant in KIM-1, KYN-2, and HAK-1B was lower than KYN-1 and KYN-3. Specifically, KIM-1 released quite a small amount of bFGF into the supernatant. This questions the presence of an autocrine growth mechanism in which released bFGF

FIG. 8. KIM-1 cells (8 1 106 cells) were transplanted subcutaneously into BALB/c nude mice on day 0. Anti-bFGF antibody (40 mg per mouse per day) or control antibody was injected into the subcutaneous space surrounding the tumor mass daily for 5 days, starting on day 3. The tumor volume was significantly different (P õ .05, by ANOVA) between the anti–bFGF-treated group and the control group after day 13. Error bar represents SD. Arrows, date of antibody administration. s, control group that received injections of control antibody. ●, treated group that received injections of anti-bFGF antibody.

AID

Hepa 0048

/

5p10$$$942

06-25-96 09:38:54

binds to FGFR and transfers growth signals. On the other hand, there is a proliferation mechanism, termed intracrine and proposed by Logan,47 in which there is a growth factor acting within a cell, and this factor is not necessarily secreted by a cell and does not require receptors on the cell surface to stimulate growth. For example, Bouche et al.48 reported that bFGF would act directly on the nuclei of endothelial cells to regulate ribosomal gene transcriptions. Therefore, bFGF could stimulate cell proliferation through the intracrine mechanism within a cell, and this could be applicable to KIM1. At present, we cannot conclude whether the intracrine mechanism presents not only in KIM-1 but also in KYN-2 and HAK-1B, because, in this study, addition of anti-bFGF neutralizing antibody suppressed cell proliferation of these cells, even though a previous study reported that anti-bFGF neutralizing antibody did not suppress cell proliferation mediated by the intracrine mechanism via bFGF.49 Therefore, we suspect that the following mechanism is more likely present, rather than the intracrine mechanism: bFGF produced within a cell is brought to the cell surface, and strongly and efficiently binds to FGFR soon after the release, or even without being released, and transfers growth signals. The lack of growth stimulation by added exogenous bFGF in KIM-1 and KYN-2 in this study might be the result of rapid binding of endogenous bFGF to FGFR on the cell surface, as Neufeld et al.49 reported. Another possible explanation is that there could be optimal concentrations of bFGF required to stimulate proliferation. In the present study, stepwise proliferation stimulation with increasing bFGF concentrations was observed only for HAK-1B, and the level of stimulation varied widely among the cell lines, e.g., proliferation stimulation was weakened at 5 ng/mL in KYN-3, while in the other cells, effects of 1 and 5 ng/mL were almost equivalent. Because similar variations have been reported for other non-HCC cell lines,18,30,43 these are not specific to HCC cells. These differences would be related to the expressions of FGF family members, other than bFGF, in the cells and the types of FGFR expressed on the cell surface. Among the four receptors of the FGF family that were examined in this study, FGFR-1, -2, and -3 function as common receptors for acidic FGF and bFGF, whereas FGFR-4 has been reported to be specific to acidic FGF.25-27 Expressions of mRNAs of FGFR-2, -3, and -4 have been reported in human fetal and adult liver tissues,32 but the expressions of FGFR1 protein and mRNA in hepatocytes have not been observed.14,32 In addition, Korhonen et al.32 detected the expressions of FGFR-1 and -3 in HCC tissues of a patient, as well as the expressions of FGFR-4 in addition to FGFR-1 and -3

hpta

WBS: Hepatology

204

OGASAWARA ET AL.

HEPATOLOGY July 1996

in HepG2, a human hepatoblastoma cell line; however, they reported that FGFR-2 was barely detectable in these cells. In our study, all six HCC cell lines expressed FGFR-1 protein and mRNA. This indicates that liver tissues start to express FGFR-1 at high levels along with the progression of transformation, and furthermore, increased FGFR-1 expression would be related to the proliferation of cancerous cells. In the same way, neoexpression of FGFR-1 along with transformation has been reported in glioblastoma.28 Besides FGFR1 mRNA, expressions of FGFR-2, -3, and -4 mRNAs were observed irregularly, and no specific tendency was observed. In addition to the cell proliferation stimulation effect, bFGF is reported to induce morphological and motility changes in some cells, e.g., endothelial cells, fibroblast cells, and cancer cells.11,18-20 In this study, only KYN-3 among the six cell lines presented morphological changes when cultured with 5 ng/mL bFGF, i.e., spindle shape of proliferated cells and their piling up. This indicates that bFGF not only stimulates proliferation but also affects morphology in some HCC cells. Furthermore, bFGF has been reported to stimulate productions of plasminogen activator and collagenase, and to increase migratory activity of cells.19 Therefore, it should be investigated in future studies whether bFGF presents such infiltration- and metastasis-related effects on HCC cells. bFGF is well known as a mitogen to cells and as an important angiogenesis factor.16 Hori et al.38 transplanted K1000 cells (a BALB/c 3T3 transformant in which the leader sequence-fused bFGF gene was transfected) to nude mice and injected anti-bFGF neutralizing antibody subcutaneously to a space surrounding the tumor; they observed that this antibody suppressed angiogenesis in the subcutaneous tumor and resulted in tumor growth suppression. KIM-1 is a moderately differentiated HCC cell line, and the tumor, which is developed by a subcutaneous injection of KIM-1 cells, presents histological features of typical HCC, e.g., polygonal cells in a trabecular pattern associated with blood space, and an endothelial layer that can be identified around the trabeculae.33 KIM-1 rarely releases bFGF into the culture supernatant, and this indicates a weak relationship between the angiogenesis and the bFGF release from HCC cells. In our study, when an anti-bFGF neutralizing antibody was injected subcutaneously around the tumor of nude mice, cell proliferation in the tumor was suppressed, but there were no histological differences. Therefore, it is unlikely that bFGF produced in KIM-1 in vivo is released extracellularly, acts on the endothelial cells through a paracrine mechanism, and is involved in the formation of the characteristic blood space and the endothelial layer in HCC. It is surmised that other factors would be involved in the angiogenesis in HCC. Matsuzaki et al.50 transplanted hybridoma cells, which produce neutralizing antibodies to bFGF, to nude mice subcutaneously, observed the formation of a highly vascularized solid tumor, and suggested that bFGF would not be an essential autocrine or paracrine growth factor for angiogenesis in vivo. On the other hand, among the six HCC cell lines, only HAK-1A produces and releases a very low amount of bFGF and does not form tumors after transplantation to nude mice. This would support the findings of Rogelj et al.,51 who reported a relationship between bFGF production and tumorigenicity. In the proliferation of HCC cells, there could be an autocrine or paracrine mechanism mediated by bFGF/FGFR. Cells like HAK-1A, which rarely express bFGF but produce FGFR, would be stimulated to proliferate through a paracrine mechanism when bFGF is released from the surrounding noncancerous cells. Among the cell lines that react to the anti-bFGF neutralizing antibody, KIM-1 and KYN-2, which express bFGF and FGFR and did not react to exogenous bFGF, would possess only an autocrine mechanism mediated by bFGF/FGFR, whereas the other three cell lines

AID

Hepa 0048

/

5p10$$$942

06-25-96 09:38:54

(KYN-1, KYN-3, HAK-1B) would possess both autocrine and paracrine mechanisms for proliferation. The six HCC cell lines used in this study did not show a marked relationship between bFGF/FGFR expressions and histological differentiation levels. HAK-1A and HAK-1B are derived from the same clone cells but have different differentiation levels. HAK-1B is a morphologically poorly differentiated cell line, has higher malignancy, and is presumed to be derived from HAK-1A through its clonal dedifferentiation.37 Between HAK-1A and HAK-1B, expressions of bFGF and FGFR were different. This strongly indicates that cancer cells acquire a more efficient cell proliferation mechanism mediated by bFGF/FGFR in their dedifferentiation process. The block of the autocrine and paracrine mechanisms of cancer cell proliferation mediated by bFGF is a promising, new approach in HCC treatment. Acknowledgment: We gratefully acknowledge Kazuo Takashima for photomicrographs and Akemi Fujiyoshi for her excellent technical assistance. REFERENCES 1. Tanaka A, Miyamoto K, Minamino N, Takeda M, Sato B, Matsuo H, Matsumoto K. Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells. Proc Natl Acad Sci U S A 1992;89:8928-8932. 2. Miyamoto M, Naruo K-I, Seko C, Matsumoto S, Kondo T, Kurokawa T. Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property. Mol Cell Biol 1993;13:4251-4259. 3. Jaye M, Howk R, Burgess W, Ricca GA, Chiu IM, Ravera MW, O’Brien SJ, et al. Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization. Science 1986;233:541-545. 4. Abraham JA, Whang JL, Tumolo A, Mergia A, Friedman J, Gospodarowicz D, Fiddes JC. Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J 1986;5:2523-2528. 5. Moore R, Casey G, Brookes S, Dixon M, Peters G, Dickson C. Sequence, topography and protein coding potential of mouse int-2: a putative oncogene activated by mouse mammary tumor virus. EMBO J 1986;5:919-924. 6. Delli Bovi P, Curatola AM, Kern FG, Greco A, Ittmann M, Basilico C. An oncogene isolated by transfection of Kaposi’s sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell 1987;50:729-737. 7. Zhan X, Bates B, Hu X, Goldfarb M. The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol Cell Biol 1988;8: 3487-3495. 8. Marics J, Adelaide J, Raybaud F, Mattei MG, Coulier F, Planche J, Lapeyriere O, et al. Characterization of the HST-related FGF 6 gene, a new member of the fibroblast growth factor gene family. Oncogene 1989;4:335340. 9. Finch PW, Rubin JS, Miki T, Ron D, Aaronson SA. Human KGF is FGFrelated with properties of a paracrine effector of epithelial cell growth. Science 1989;245:752-755. 10. Ohtani H, Nakamura S, Watanabe Y, Mlzoi T, Saku T, Nagura H. Immunocytochemical localization of basic fibroblast growth factor in carcinomas and inflammatory lesions of the human digestive tract. Lab Invest 1993; 68:520-527. 11. Gospodarowicz D, Neufeld G, Schweigerer L. Fibroblast growth factor: structural and biological properties. J Cell Physiol 1987;5(suppl):15-26. 12. Schulze-Osthoff K, Risau W, Vollmer E, Sorg C. In situ detection of basic fibroblast growth factor by highly specific antibodies. Am J Pathol 1990; 137:85-92. 13. Cordon-Cardo C, Vlodavsky I, Haimovitz-Friedman A, Hicklin D, Fuks Z. Expression of basic fibroblast growth factor in normal human tissues. Lab Invest 1990;63:832-840. 14. Hughes SE, Hall PA. Immunolocalization of fibroblast growth factor receptor 1 and its ligands in human tissues. Lab Invest 1993;69:173-182. 15. Burgess WH, Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem 1989;58:575-606. 16. Folkman J, Klagsbrun M. Angiogenic factor. Science 1987;235:442-447. 17. Baird A, Klagsbrun M. The fibroblast growth factor family. Ann N Y Acad Sci 1991;638:403-477. 18. Schofield PN, Granerus M, Lee A, Ektro¨m TJ, Engstro¨m W. Concentrationdependent modulation of basic fibroblast growth factor action on multiplication and locomotion of human teratocarcinoma cells. FEBS Lett 1992; 298:154-158. 19. Presta M, Moscatelli D, Joseph-Silverstein J, Rifkin DB. Purification from a human hepatoma cell line of a basic fibroblast growth factor–like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration. Mol Cell Biol 1986;6:4060-4066. 20. Gospodarowicz D, Morran J. Effect of a fibroblast growth factor, insulin, dexamethasone, and serum on morphology of BALB/c 3T3 cells. Proc Natl Acad Sci U S A 1974;71:4648-4652.

hpta

WBS: Hepatology

HEPATOLOGY Vol. 24, No. 1, 1996

OGASAWARA ET AL.

21. Motoo Y, Sawabe N, Nakanuma Y. Expression of epidermal growth factor and fibroblast growth factor in human hepatocellular carcinoma: an immunohistochemical study. Liver 1991;11:272-277. 22. Takahashi JA, Mori H, Fukumoto M, Igarashi K, Jaye M, Oda Y, Kikuchi H, et al. Gene expression of fibroblast growth factors in human gliomas and meningiomas: demonstration of cellular source of basic fibroblast growth factor mRNA and peptide in tumor tissues. Proc Natl Acad Sci U S A 1990;87:5710-5714. 23. Takahashi JA, Suzuki H, Yasuda Y, Ito N, Ohta M, Jaye M, Fukumoto M, et al. Gene expression of fibroblast growth factor receptors in the tissues of human gliomas and meningiomas. Biochem Biophys Res Commun 1991; 177:1-7. 24. Avivi A, Zimmer Y, Yayon A, Yarden Y, Givol D. Flg-2, a new member of the family of fibroblast growth factor receptors. Oncogene 1991;6:10891092. 25. Dionne CA, Crumley G, Bellot F, Kaplow JM, Searfoss G, Ruta M, Burgess WH, et al. Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors. EMBO J 1990;9:2685-2692. 26. Keegan K, Johnson DE, Williams LT, Hayman MJ. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci U S A 1991;88:1095-1099. 27. Partanen J, Ma¨kela¨ TP, Eerola E, Korhonen J, Hirvonen H, ClaessonWelsh L, Alitalo K. FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern. EMBO J 1991;10:1347-1354. 28. Morrison RS, Yamaguchi F, Bruner JM, Tang M, McKeehan W, Berger MS. Fibroblast growth factor receptor gene expression and immunoreactivity are elevated in human glioblastoma multiforme. Cancer Res 1994;54: 2794-2799. 29. McLeskey SW, Ding IYF, Lippman ME, Kern FG. MDA-MB-134 brease carcinoma cells overexpress fibroblast growth factor (FGF) receptors and are growth-inhibited by FGF ligands. Cancer Res 1994;54:523-530. 30. Leung HY, Gullick WJ, Lemoine NR. Expression and functional activity of fibroblast growth factors and their receptors in human pancreatic cancer. Int J Cancer 1994;59:667-675. 31. Klagsbrun M, Sasse J, Sullivan R, Smith JA. Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor. Proc Natl Acad Sci U S A 1986;83:2448-2452. 32. Korhonen J, Partanen J, Eerola E, Vainikka S, Ilvesma¨ki V, Voutilainen R, Julkunen M, et al. Novel human FGF receptors with distinct expression patterns. Ann N Y Acad Sci 1991;638:403-405. 33. Murakami T. Establishment and characterization of human hepatoma cell line (KIM-1). Acta Hepatol Jpn 1984;25:532-539. 34. Yano H, Kojiro M, Nakashima T. A new human hepatocellular carcinoma cell line (KYN-1) with a transformation to adenocarcinoma. In Vitro Cell Dev Biol 1986;22:637-646. 35. Yano H, Maruiwa M, Murakami T, Fukuda K, Ito Y, Sugihara S, Kojiro M. A new human pleomorphic hepatocellular carcinoma cell line, KYN-2. Acta Pathol Jpn 1988;38:953-966. 36. Murakami T, Maruiwa M, Fukuda K, Kojiro M, Tanaka M, Tanikawa K. Characterization of a new human hepatoma cell line (KYN-3) derived from the ascites of the hepatoma patient [Abstract]. Proceeding of the 47th annual meeting. Jpn J Cancer Res 1988;292.

AID

Hepa 0048

/

5p10$$$943

06-25-96 09:38:54

205

37. Yano H, Iemura A, Fukuda K, Mizoguchi A, Haramaki M, Kojiro M. Establishment of two distinct human hepatocellular carcinoma cell lines from a single nodule showing clonal dedifferentiation of cancer cells. HEPATOLOGY 1993;18:320-327. 38. Hori A, Sasada R, Matsutani E, Naito K, Sakura Y, Fujita T, Kozai Y. Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor. Cancer Res 1991; 51:6180-6184. 39. Brogi E, Winkles JA, Underwood R, Clinton SK, Alberts GF, Libby P. Distinct patterns of expression of fibroblast growth factors and their receptors in human atheroma and nonatherosclerotic arteries. J Clin Invest 1993;92:2408-2418. 40. Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaeli R, Sasse J, Klagsburn M. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci U S A 1987;84:2292-2296. 41. Sato Y, Murphy PR, Sato R, Friesen HG. Fibroblast growth factor release by bovine endothelial cells and human astrocytoma cells in culture is density dependent. Mol Endocrinol 1989;3:744-748. 42. van Veggel JH, van Oostwaard TMJ, de Laat SW, van Zoelen EJJ. PC13 embryonal carcinoma cells produce a heparin-binding growth factor. Exp Cell Res 1987;169:280-286. 43. Sasada R, Kurokawa T, Iwane M, Igarashi K. Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA. Mol Cell Biol 1988;8:588-594. 44. Takahashi JA, Fukumoto M, Kozai Y, Ito N, Oda Y, Kikuchi H, Hatanaka M. Inhibition of cell growth and tumorigenesis of human glioblastoma cells by a neutralizing antibody against human basic fibroblast growth factor. FEBS Lett 1991;288:65-71. 45. Bashkin P, Doctrow S, Klagsbrun M, Svahn CM, Folkmann J, Vlodavsky I. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry 1989;28:1737-1743. 46. Baird A, Ling N. Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: implication for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 1987;142:428-435. 47. Logan A. Intracrine regulation at the nucleus—a further mechanism of growth factor activity? J Endocrinol 1990;125:339-343. 48. Bouche G, Gas N, Prats H, Baldin V, Tauber JP, Iberg N. Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing G0 -G1 transition. Proc Natl Acad Sci U S A 1987;84:6770-6774. 49. Neufeld G, Mitchell R, Ponte P, Gospodarowicz D. Expression of human basic fibroblast growth factor cDNA in baby hamster kidney–derived cells results in autonomous cell growth. J Cell Biol 1988;106:1385-1394. 50. Matsuzaki K, Yoshitake Y, Matuo Y, Sasaki H, Nishikawa K. Monoclonal antibodies against heparin-binding growth factor II/basic fibroblast growth factor that block its biological activity: invalidity of the antibodies for tumor angiogenesis. Proc Natl Acad Sci U S A 1989;86:9911-9915. 51. Rogelj S, Weinberg RA, Fanning P, Klagsbrun M. Basic fibroblast growth factor fused to a signal peptide transforms cells. Nature 1988;331:173175.

hpta

WBS: Hepatology