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A FIBROBLAST-DERIVED TUMORCYTOTOXIC FACTOR/F-TCF(llEPATOCYTE GROWTBFACTOR/IIGF) HAS MULTIPLE FUNCTIONSIN VITRO Nobuyuki Shima Yasuharu Itagaki, Masaya Nagao, Hisataka Yasuda, Tomonori Mdrinaga and Kanji lligashio l
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd. 519 Ishibashi-machi,Shimotsuga-gun, Tochigi 329-05, Japan. * To whom all
correspondence
should be addressed.
ABSTRACT We previously demonstrated that a tumor cytotoxic factor(F-TCF) purified from the culture broth of human embryonic lung diploid fibroblast, IMR-90 cells was one of the human hepatocyte growth In the present report, we demonstrate its factors(hllGFs). biological functions. F-TCF showed moderate cytotoxicity on human tumor cell lines KB, BG-1, MCF-7 and Hs913 T, and strong cytotoxity on mouse tumor cell lines Sarcoma 180, Meth A sarcoma and P388. On the contrary, F-TCF was a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, BUVECand human melanocytes. Moreover, F-TCF induced the differentiation of premyelocyte leukemia, BL-60 cells into morphologically granulocyte-like cells. These biological functions suggest that F-TCF is an effector molecule responsible for inflammation and repair in injured tissues including tumor and liver. INTRODUCTION studies have demonstrated that fibroblasts play an important role in inflammatory response not only producing matrix proteins but also mediating and amplifying locally the effects of inflammatory cytokines such as interleukin-l(IL-1) (Elias et al., 1989), interferon-P (IFN -B )(Kohase et a1.,1987), and colony stimulating factors(CSFs)(Zucali et a1.,1986). In we reported that human embryonic lung the previous study, diploid fibroblast, IMR-90 cells secreted a tumor cytotoxic factor (F-TCF) distinct from previously characterized cytotoxic cytokines. Amino acid sequence analysis of F-TCF(Bigashio et al., 1990) revealed that this protein was identical to one of the human hepatocyte growth factors(hHGFs), cDNAs of which were recently cloned independently by two groups(Nakamura et Indeed, F-TCF was found to be al., 1989; Miyazawa et a1.,1989). a potent mitogen for adult rat hepatocytes(Higashio et This finding indicates the possibility that F-TCF al., 1990). Recent
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may have a broad spectrum of activities in addition to cytotoxic activity. To clarify why fibroblasts widely distribute in various tissues secret F-TCF(hHGF), we investigated biological In the present report, we show functions of F-TCF in vitro. that F-TCF is cytotoxic to several human and mouse tumor cell 1 ines, whereas it has a strong mitogenic activity for human endothelial cells and melanocytes. Furthermore, we demonstrated that F-TCF induced the differentiation of premyelocyte leukemia, HL-60. MATER1 ALS AND METHODS Purification of F-TCF:F-TCF was purified from the conditioned medium of human embryonic lung diploid fibroblast, of UF concentration, CM IMR-90 cells by the combination sephadex C-50, Con A sepharose, Mono S, and heparin sepharose as previously described(Higashio et a1.,1990). Six human tumor cell lines, KB(Epidermoid Cell lines:HeLa(Epitheloid carcinoma), BG-l(Ovarian carcinoma), adenocarcinoma), MCF-7(Breast carcinoma), A549(Lung carcinoma ) and five mouse tumor cell lines, and Hs913T( Fibrosarcoma), P388(Lymphocytic leukemia ), L1210(Lymphocytic leukemia), Sarcoma 180, Meth A sarcoma and Colon 26(Adenocarcinoma) were used to analyse cytotoxic activity of F-TCF. BG-1 was kindly University School of provided by Dr. C.E.Wellander(Emory IMR-90 was used as a normal cell control. Human Medicine). umbilical vein endothelial cells, HUVEC(Kurabo Co., Japan), human melanocytes(Kurabo Co.), and a mouse melanoma cell line, B-lG(melanotic melanoma) were used to analyse mitogenic activity of F-TCF. K562(human chronic myelogenous leukemia) and HL-GO(human premyelocyte leukemia) were used to analyse differentiation inducing activity of F-TCF. KB, IleLa, IMR-90, lls913T, B-16, and Cell culture media:Sarcoma 180 were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10 % fetal bovine serum(FBS). L1210, P388, Meth A, Colon 26, HL-60, and K562 were cultured in RPM1 1640 with 10 % FBS. BC-1 was cultured in McCoy’s 5A with 10 % FBS. MCF-7 was cultured in Eagle’s minimal essential medium(MEM) with 10 % FBS, 0.1 mMnon-essential amino acids and 1 % pyruvic acid . A549 was cultured in llam F-12K with 15 % FBS . HUVECwas cultured in E-GM(Kurabo Co.), which is a modified MCDB 131 supplemented with 2 % FBS, 0.4 % bovine pituitary gland extracts(BPE), epidermal growth factor(EGF,lO rig/ml) and hydrocortisone(1 Y g/ml). Human melanocytes were cultured in MGM(Kurabo Co. ) , which is a modified MCDB 153 supplemented with 0.2 % BPE, recombinant basic fibroblast growth factor(r-bFGF, insulin(5 u g/ml), hydrocortisone(0.5 Y g/ml) and 1 rig/ml), phorbol myristate acetate(PMA, 10 rig/ml) .
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Assay for cell growth:The cells were seeded in 96-well flat bottomed plates(3072, Falcon) at 1 x lo3 cells per 50 u 1 per we1l(BG-1, MCF-7, A549, Sarcoma 180, Meth A, Colon 26, L1210 and P388) or at 5 x lo3 ccl 1s per 50 IL 1 per well( IMR-90, IIUVEC, KB, lls913T, B-16 and BeLa). To each test well, 50~ 1 of culture medium containing serially diluted F-TCF was added. The plates were incubated at 37 o C in a humidified atmosphere with 5 % CO 7 for three days for P388, L1210 and Meth A, for four days for IMR-90, KB, IleLa, lls913T, A549, BG-1, MCF-7, Sarcoma 180, B-16, Colon 26, and for six days for BUVEC. Viable cell numbers of anchorage-dependent cell lines were determined by dye extract method(Lee et a1.,1984) as follows. The well contents were removed and each well was gently washed with phosphate buffered sal ine(PBS). Viable cells in each well were fixed and stained with 0.5 % crystal violet in a mixture of methanol and water(l : 4) for 10 min. The crystal violet solution in each well was removed, each well was gently washed three times with water. After drying at room temperature, crystal violet in each well was extracted with Sorenson’s buffer(6.1 ml of 0.1 M disodium citrate, 3.9 ml of 0.1 N IlCl and 10 ml of 95 % ethanol) and the absorbance at 570 nrn(A57” ) was measured. Viable cell numbers of anchorage-independent cell lines(Ll210, P388 and Meth A) or the cell lines tend to detach easily from the bottom of the well(Sarcoma 180, BUVEC) were counted using a heamacytometer. Cytotoxicity or growth stimulating activity was calculated by the following equation: cytotoxicity(%)= [ Control sample(A6, 0 or viable cell numbers/ml) - Test sample(A5, D or viable cell numbers/ml)] / Control sample(A6, 0 or viable cell numbers/ml) x 100 or growth stimulating activity(%)= Test sample(A5, 0 or viable cell numbers/ml) / Control sample (As, n or viable cell numbers/ml) x 100. Assay for DNA synthesis in human melanocytes:Iluman melanocytes( six passages ) were suspended in M-GM and seeded in 24 well plates(3047, Falcon) at 2.5 x 10” cells per well. After 24 hrs of the incubation at 37°C in a humidified atmosphere with 5 % COz, the medium was replaced with M-GM containing F-TCF. The cells were subsequently incubated for 24 hrs under the conditions described above. Then the cells were incubated for 4 hrs in the medium containing Iv Ci/m1(86 Ci,/mmol) of [3111thymidine(TRK 686, Amersham). After the incubation, incorporation of [311]thymidine into DNA was measured by the method of Gohda et a1.(1986). Assay for differentiation inducing activity:K562 and HL-60 cells were seeded in 96-well flat bottomed plates at 3.5 x 10” cells per 100 ill 1 per well. To each test well, 100 ti 1 of culture medium containing F-TCF was added. The plates were incubated at 37 “C in a humidified atmosphere with 5 % COz for 3 of K562 and BL-60 cells were and 7 days. Differentiation monitored by o-dianisidine staining assay(Murata et a1.,1988) and nitroblue tetrazorium(NBT) reduction assay(Takeda et
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al., 1988),respectively. RESULTS Purification of F-TCF:- The purified F-TCF showed homogeneity on SDS-polyacrylamide gel electrophoresis under non-reducing conditions, when the gel was stained with silver(Fig. I). F-TCF had moderate Cytotoxic activity of F-TCF:cytotoxicity(more than 25 %, less than 50 % ) on human tumor cell lines, KB, BG-1, MCF-7 and Hs913T. However, the growth of human normal fibroblasts, IMR-90, and A549 were not affected(less than 15 %)(Fig. 2a). F-TCF was found to have strong cytotoxicity(more than 70 % ) on mouse tumor cell lines, Sarcoma 180, Meth A and P388. The amount of F-TCF that gave 50% cytotoxicity on sarcoma 180, Meth A and P388 was 6, 40 and 460 ng/ml,respectively(Fig. 2b). The changes in morphology were observed in KB,BG-l,A549 and B-16 cells by the addition of FTCF to the culture medium. The morphological change of KB from higher fibroblast-like to spherical was observed at F-TCF induced the dispersion of dosages(more than 500 rig/ml). BG-1, A549 and B-16 from cohesive colonies or tightly packed colonies(Fig. 3). Differentiation inducing activity of F-TCF:F-TCF did not induce the differentiation of K562 at 16 to 1000 rig/ml, but did induce the differentiation of HL-60 into morphologically granulocyte-like cells(Fig. 4). At a concentration of 250 Start MW Kd 94 67 43 31 21.5 14.4
Fig. 1: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining purified F-TCF. The gel was stained (1) F-TCF under non-reducing conditions, kit(Pharmacia Co.). (2) Molecular marker:;.
of
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O--’ oFv 0.5
1.0
1.0
F-TCF( ug/ml)
F-TCF(ug/ml)
(a)
(b)
Fig.2 : Cytotoxic activity of F-TCF for tumor cell . (a) Cytotoxity against human tumor cell lines. BG-l(a-,Ovarian adenocarcinoma),KB(-O-,Epidermoid carcinoma),MCF-7 (-&,Breast carcinoma),HeLa(-A-,Epitheloid carcinoma),A549(-•-,Lung carcinoma epithelial),Hs-913T(-¤-,Fibrosarcoma),IMR-90(-xNormal fibroblast). (b) Cytotoxity against mouse cell lines. karcoma 180(-•-),Meth A sarcoma(()-),P388(&,Lymphocytic Lymphoblast),Colon 26(- A-,Adenocarcinoma),Ll210(n leukemia ,Lymphocytic leukemia). All values are mean of duplicate experiments.
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: Dispersion of BG-1 and B-16 cells. BC-1 cells were Fig.3 cultured for 4 days without(a) or with(b) 62.5 rig/ml of F-TCF. B-16 cells were cultured for 3 days without(c) or with(d) 62.5 were stained with crystal violet: x 90. rig/ml of F-TCF. Cells
rig/ml, NBT reduction was induced in 45.2 % of HL-60 cells after incubation at 37 “C for 7 days(Fig.5). At higher dosages(more than 250 rig/ml), NBT reducing ability decreased. F-TCF stimulated the growth of Mitogenic activity of F-TCF:human endothelial cells, HUVEC. The maximum growth stimulating F-TCF also activity was obtained at 125 ng/ml(Fig. 6a). stimulated DNA synthesis in human melanocytes in a dose range of 10 to 100 ng/ml(Fig. 6b). After 6 days of incubation with FTCF, cell number increased to 140 % at 10 rig/ml and to 220 % at 100 rig/ml, comparing to the control. F-TCF stimulated the growth of B-16 cells(mouse melanotic melanoma)(Fig. 6~). Interestingly, stimulation of growth caused inhibition of the melanin production which is a typical characteristic of B-16 ccl 1s. DISCUSSION Fibroblasts were known to secret various tumor cytotoxic factors(Kohase et a1.,1987; Kirk et a1.,1983; Imanishi et al., 1983). We previously reported (Higashio et a1.,1990) that a tumor cytotoxic factor (F-TCF) derived from human embryonic
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Fig.4 : Morphology of or with(b) 250ng/ml Wright-Giemsa; X 90.
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HL-60 cells of F-TCF.
cultured The cells
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7 days without(a) were stained with
ID--.-. 3”--.. 20-IO ll I 0
16
62
125
250
500
IO00
F-TCF(ng/ml) Fig. 5: NBT reducing activity of F-TCF for HL-60. HL-60 cells were cultured for 7 days with F-TCF. The percentage of cells containing blue-black formazan deposits was determined by counting at least 200 cells. Values are mean of duplicate experiments.
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Fig. 6: Mitogenic activity F-TCF. of ( a ) Ii II m a n umbilical vein endothelial cells(HUVEC)were cultured with Ffor 6 days TCF.Viable cell numbers counted were using a heamacytometer. Values are mean of t r i p 1 i c a t e experiments. Error bars represent SEM. (b)Human melanocytes were cultured for 211 hours with F-TCF. DNA synthesis was determined I 7n [ 3 H ] t h y m i d i n e by incorporation assay. Values Inn Ih 31 62 175 250 500 I”“0 R are the mean of triplicate experiments. Error bars F-TCF(ng/ml) represent SEM. (c)R-16 (cl cells(mouse m e I. a n 0 t i c melanoma) were cultured for 4 days with F-TCF. Viable cell numbers were determined by dye extract method descrived in materials and methods. Values are mean of duplicate experiments.
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f ibroblast, IMR-90 cells was identical to one of the human hepatocyte growth factors(hMGFs). This indicated that F-TCF was bifunctional. And so, we started investigating the detailed biological functions of F-TCF in vitro. F-TCF showed cytotoxicity on several human and mouse tumor cell lines. Therefore, F-TCF as well as TNF(Sugarman et al.,1985)does not have a species-specificity on its cytotoxic activity against tumor cells. TNF is known to be cytolytic against various tumor cell lines. In a preliminary experiment, we found that when Meth A was used as a target tiell, radioactivity was not released from the cells prelabeled with ’ ‘Cr, but uptake of [“Ii jthymidine by the cells was suppressed by F-TCF at 10 rig/ml. This result suggests that effect of F-TCF on tumor cell inhibition is probably cytostasis rather than cytolysis. F-TCF induced the dispersion of several tumor cell lines from cohesive colonies or tightly packed colonies. Similar motility factors, scatter factors(Stocker et a1.,1987; Gherardi et a1.,1989; Gherardi and Stocker, 1990; Rosen et al., 1990), migration stimulating factor(Grey et al., 1989) and autocrine motility factor(Kohn et al., 1990) have been purified in other laboratories. Among these motility factors, a scatter factor (Gherardi et al., 1989), which is derived from mouse fibroblasts and causes separation of contiguous epithelial cells, is similar to F-TCF in physicochemical properties. This protein is a heterodimer composed of a large subunit with Mw. about 57 kD and a small subunit with Mw. about 30 kD as similar to FTCF(lligashio et al., 1990). Both factors are basic, heat-labile and have affinity for heparin. Recently, this group reported that amino terminal sequence of mouse scatter fatter is highly related to that of human hepatocyte growth factor(Gherardi and Stocker,l990). These similarities suggest that F-TCF may belong to one of the scatter factors,and may act as one of the motility factors. F-TCF induced the differentiation of BL-60 ccl 1s. TNF(Takeda et al.,1986), IFN-7 (Takuma et al.,1987) and lymphotoxin(LT)(Hemmi et al., 1987) have been found to induce differentiation of leukemia cells besides tumor cytotoxity. FTCF may have various activities for immune cells as similar to these inflammatory cytokines. F-TCF is a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, IIUVEC, human melanocytes and mouse melanotic melanoma ccl Is, B-16. These results indicate that F-TCF is a multitarget growth factor as seen in many other growth factors. Angiogenesis is an essential component of many biological processes including embryogenesis, tissue regeneration and repair. FGF is a potent mitogen for endothelial cells and is known as an angiogenic factor in vivo(Gospodarowicz et a1.,1987) . We found that F-TCF showed a strong mitogenic activity for HUVECat 125 rig/ml (about 1.6 p mole/ml) even in the optimized F-TCF may act as an growth medium, E-GM for endothelial cells. angiogenic factor in vivo in the similar manner as FGF. F-TCF has marked affinity for heparin(fiigashio et a1.,1990) as FGF does. The glycosaminoglycan of heparin is closely related to
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heparan sulfate which is a structural component of extracellular matrix. It has been known that extracellular matrix components contain FGF(Rogelj et a1.,1989). Therefore, extracellular matrix components may bind F-TCF and modulate its activity in vivo. Chemotaxis and proliferation of fibroblasts are essential processes in normal inflammatory response. Proliferated fibroblasts secret various inflammatory cytokines. Our studies revealed that F-TCF derived from human fibroblasts had multiple functions such as inhibition of tumor cell growth, angiogenesis, mitogenesis on hepatocytes and melanocytes, and differentiation of leukemia cells. These results suggest that F-TCF is an effector molecule responsible for inflammation and repair, and that fibloblasts may contribute to repair the injured tissues by secreting F-TCF. REFFERENCES Elias, J. A., Reynolds, M. M., Kotloff, R. M., and Kern J. A. (1989). Fibroblast interleukin 1P : Synergistic stimulation by recombinant interleukin 1 and tumor necrosis factor and posttranscriptional regulation. Proc. Natl. Acad. Sci. USA 86, 6171 - 6175. Cherardi, E., Gray, J., Stoker, M., Perryman, M., and Furlong, R. (1989). Purification of scatter factor,a fibroblastderived basic protein that modurates epithelial interactions and movement. Proc. Natl. Acad. Sci. 86, 5844 - 5848. Gherard i , E. and Stoker, M. (1990). llepatocytes and scatter Nature 346, 228. factor. Gohda, E., Tsubouchi,. , Nakayama, 1~., flirono, S., Takahashi, K. , Koura, M., llashimoto, S., and Daikuhara, Y. (1986). fluman hepatocyte growth factor in plasma from patients with fulminant hepatic failure. Exp. Cell Res. 166, 139 - 150. Gospodarowicz, D., Ferrara, N., Schweigerer, L. (1987). Structural characterization and biological functions of 95 - 114. fibroblast growth factor. Endocrine Reviews 8, Grey, A. M., Schor, A. M., Rushton, G., El1 is, I., and Schor, S. L. (1989). Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc. Natl. Acad. Sci. USA 86, 2438 -2442. Hemmi, fl., Nakamura, T., Tamura, K., Shimizu, Y., Kato, S., Miki, T., Takahashi, N., Muramatsu, Y., Numao, N., and Induction of terminal Sugamura, K. (1987). Lymphotoxin: differentiation of the human myeloid leukemia cell lines HL-60 and THP-1. J. Immunol. 138, 664 - 666. Y., Nagao, M., fligashio, K., Shima, N., Goto, M., Itagaki, of a tumor Yasuda, H., and Morint)ga, T. (1990). Identity cytotxic factor from human fibroblasts and hepatocyte growth factor. Biochem. Biophys. Res. Commun. 170, 397 404.
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Imanishi, J., Hoshino, S., Matsuoka, H., Uemura, Il., Imanishi, T . , Tanaka, A., Nishino, Il., and Kishida, T. (1983). Tumor degeneration by human embryonic fibroblasts and its enhancement by interferon. 43, 4323 Cancer Research 4326. Kirk, D. , Kagawa, S. , and Verner, G. (1983). Comparable growth regulation of five human tumor cell lines by neonatal human lung fibroblasts in semisolid culture media. Cancer 3753 -3758. Research 2, Kohase, M., May, L. T., Tamm, I., Vilcek, J., and Sehgal, P. B. (1987). A cytokine network in human diploid fibroblasts: Interactions of P -interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1. Mol. Cell. Biol. 1, 273 - 280. Kohn, E. C., Liotta, L. A., and Schiffmann, E. (1990). Autocrine motility factor stimulates a three-fold increase in inositol triphosphate in human melanoma cells. Biochem. Biophis. Res. Commun. 166 , 757 -764. Lee, S. Il., Aggarwal, B. B., Rinderknecht, E., Assisi, F., and Chiu, H. (1984). The synergistic anti-proliferative effect of r-interferon and human lymphotoxin. J. Immunol. 133, 1083 - 1089. Miyazawa, K., Tsubouchi, II., Naka, D., Takahashi, K., Okigaki, M Arakaki, N., Nakayama, Il., Hirono, S., Sakiyama, 0.) Takahashi, K., Gohda, E. , Dai kuhara, Y. , and Hi tamura, N. (1989). Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 163, 967 - 973. Murata, M., Eto, Y., Shibai, Il., Sakai, M., and Muramatsu, M. (1988). Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin P A chain. Proc. Natl. Acad. Sci. USA 85, 2434 - 2438. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth Nature 342, 440 - 443. factor. Rogel j, S., Klagsbrun, M., Atzmon, R., Kurokawa, M., Haimovitz, A Fuks, Z., and Vlodavsky, 1. (1989). Basic fibroblast growth factor is an exracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 eels. J. Cell Biol. 109, 823 - 831. Rosen, E. M., Meromsky, I,. , Romero, R., Setter, E., and Goldberg, I. (1990). Human placenta contains an epithelial scatter protein. Biochem. Biophys. Res. Commun. 168 , 1082 - 1088. Stocker, M., Gherardi, E., Pennyman, M., and Gray, J. (1987). Scatter factor is a fibroblast-derived modulator of epithelial sell mobility. Nature 327, 239 - 242.
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Sugarman, P., Aggarwal, B. B., Bass, P. E., Figari, I. S., Palladino, Jr., M. A., and Shepard, II. M. (1985). Recombinant human tumor necrosis factora : Effect on proliferation of normal and transformed cells in vitro. 943 -945. Science 230, Takeda, K., I wamoto, T. , Sugimoto, II. , Takuma, T. , Kawatan i , N. , Noda, M., Masaki, A., Morise, II., Arimura, II., and Sugamura, K. (1986). Identity of differentiation inducing factor and tumor nezrosis factor. Nature 323, 338 - 340. Takeda, K. , flosoi , T. , Noda, M. , Ar imura, II. , and Konno, K. (1988). Effect of fibroblast-derived differentiation inducing factor on the differentiation of human monocytoid and myeloid leukemia cell lines. Biochem. Biophys. Res. 24 - 31. Commun. 155, Takuma, T., Takeda, K., and Konno, K. (1987). Synegism of tumor necrosis factor and interferon-r in induction of differentiation of human myeloblastic leukemic ML-l cells. Biochem. Biophys. Res. Commun. 145, 514 - 521. Zucali, J.R., Dinarello, C.A., Oblon, D. J., Gross, M.A., Anderson,L., and Weiner, R.S. (1986). Interleukin 1 stimulates fibroblasts to produce granulocyte-macrophage colony stimulating activity and prostaglandin Ez.
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A FIBROBLAST-DERIVED TUMORCYTOTOXIC FACTOR/F-TCF(llEPATOCYTE GROWTBFACTOR/IIGF) HAS MULTIPLE FUNCTIONSIN VITRO Nobuyuki Shima Yasuharu Itagaki, Masaya Nagao, Hisataka Yasuda, Tomonori Mdrinaga and Kanji lligashio l
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd. 519 Ishibashi-machi,Shimotsuga-gun, Tochigi 329-05, Japan. * To whom all
correspondence
should be addressed.
ABSTRACT We previously demonstrated that a tumor cytotoxic factor(F-TCF) purified from the culture broth of human embryonic lung diploid fibroblast, IMR-90 cells was one of the human hepatocyte growth In the present report, we demonstrate its factors(hllGFs). biological functions. F-TCF showed moderate cytotoxicity on human tumor cell lines KB, BG-1, MCF-7 and Hs913 T, and strong cytotoxity on mouse tumor cell lines Sarcoma 180, Meth A sarcoma and P388. On the contrary, F-TCF was a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, BUVECand human melanocytes. Moreover, F-TCF induced the differentiation of premyelocyte leukemia, BL-60 cells into morphologically granulocyte-like cells. These biological functions suggest that F-TCF is an effector molecule responsible for inflammation and repair in injured tissues including tumor and liver. INTRODUCTION studies have demonstrated that fibroblasts play an important role in inflammatory response not only producing matrix proteins but also mediating and amplifying locally the effects of inflammatory cytokines such as interleukin-l(IL-1) (Elias et al., 1989), interferon-P (IFN -B )(Kohase et a1.,1987), and colony stimulating factors(CSFs)(Zucali et a1.,1986). In we reported that human embryonic lung the previous study, diploid fibroblast, IMR-90 cells secreted a tumor cytotoxic factor (F-TCF) distinct from previously characterized cytotoxic cytokines. Amino acid sequence analysis of F-TCF(Bigashio et al., 1990) revealed that this protein was identical to one of the human hepatocyte growth factors(hHGFs), cDNAs of which were recently cloned independently by two groups(Nakamura et Indeed, F-TCF was found to be al., 1989; Miyazawa et a1.,1989). a potent mitogen for adult rat hepatocytes(Higashio et This finding indicates the possibility that F-TCF al., 1990). Recent
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may have a broad spectrum of activities in addition to cytotoxic activity. To clarify why fibroblasts widely distribute in various tissues secret F-TCF(hHGF), we investigated biological In the present report, we show functions of F-TCF in vitro. that F-TCF is cytotoxic to several human and mouse tumor cell 1 ines, whereas it has a strong mitogenic activity for human endothelial cells and melanocytes. Furthermore, we demonstrated that F-TCF induced the differentiation of premyelocyte leukemia, HL-60. MATER1 ALS AND METHODS Purification of F-TCF:F-TCF was purified from the conditioned medium of human embryonic lung diploid fibroblast, of UF concentration, CM IMR-90 cells by the combination sephadex C-50, Con A sepharose, Mono S, and heparin sepharose as previously described(Higashio et a1.,1990). Six human tumor cell lines, KB(Epidermoid Cell lines:HeLa(Epitheloid carcinoma), BG-l(Ovarian carcinoma), adenocarcinoma), MCF-7(Breast carcinoma), A549(Lung carcinoma ) and five mouse tumor cell lines, and Hs913T( Fibrosarcoma), P388(Lymphocytic leukemia ), L1210(Lymphocytic leukemia), Sarcoma 180, Meth A sarcoma and Colon 26(Adenocarcinoma) were used to analyse cytotoxic activity of F-TCF. BG-1 was kindly University School of provided by Dr. C.E.Wellander(Emory IMR-90 was used as a normal cell control. Human Medicine). umbilical vein endothelial cells, HUVEC(Kurabo Co., Japan), human melanocytes(Kurabo Co.), and a mouse melanoma cell line, B-lG(melanotic melanoma) were used to analyse mitogenic activity of F-TCF. K562(human chronic myelogenous leukemia) and HL-GO(human premyelocyte leukemia) were used to analyse differentiation inducing activity of F-TCF. KB, IleLa, IMR-90, lls913T, B-16, and Cell culture media:Sarcoma 180 were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10 % fetal bovine serum(FBS). L1210, P388, Meth A, Colon 26, HL-60, and K562 were cultured in RPM1 1640 with 10 % FBS. BC-1 was cultured in McCoy’s 5A with 10 % FBS. MCF-7 was cultured in Eagle’s minimal essential medium(MEM) with 10 % FBS, 0.1 mMnon-essential amino acids and 1 % pyruvic acid . A549 was cultured in llam F-12K with 15 % FBS . HUVECwas cultured in E-GM(Kurabo Co.), which is a modified MCDB 131 supplemented with 2 % FBS, 0.4 % bovine pituitary gland extracts(BPE), epidermal growth factor(EGF,lO rig/ml) and hydrocortisone(1 Y g/ml). Human melanocytes were cultured in MGM(Kurabo Co. ) , which is a modified MCDB 153 supplemented with 0.2 % BPE, recombinant basic fibroblast growth factor(r-bFGF, insulin(5 u g/ml), hydrocortisone(0.5 Y g/ml) and 1 rig/ml), phorbol myristate acetate(PMA, 10 rig/ml) .
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Assay for cell growth:The cells were seeded in 96-well flat bottomed plates(3072, Falcon) at 1 x lo3 cells per 50 u 1 per we1l(BG-1, MCF-7, A549, Sarcoma 180, Meth A, Colon 26, L1210 and P388) or at 5 x lo3 ccl 1s per 50 IL 1 per well( IMR-90, IIUVEC, KB, lls913T, B-16 and BeLa). To each test well, 50~ 1 of culture medium containing serially diluted F-TCF was added. The plates were incubated at 37 o C in a humidified atmosphere with 5 % CO 7 for three days for P388, L1210 and Meth A, for four days for IMR-90, KB, IleLa, lls913T, A549, BG-1, MCF-7, Sarcoma 180, B-16, Colon 26, and for six days for BUVEC. Viable cell numbers of anchorage-dependent cell lines were determined by dye extract method(Lee et a1.,1984) as follows. The well contents were removed and each well was gently washed with phosphate buffered sal ine(PBS). Viable cells in each well were fixed and stained with 0.5 % crystal violet in a mixture of methanol and water(l : 4) for 10 min. The crystal violet solution in each well was removed, each well was gently washed three times with water. After drying at room temperature, crystal violet in each well was extracted with Sorenson’s buffer(6.1 ml of 0.1 M disodium citrate, 3.9 ml of 0.1 N IlCl and 10 ml of 95 % ethanol) and the absorbance at 570 nrn(A57” ) was measured. Viable cell numbers of anchorage-independent cell lines(Ll210, P388 and Meth A) or the cell lines tend to detach easily from the bottom of the well(Sarcoma 180, BUVEC) were counted using a heamacytometer. Cytotoxicity or growth stimulating activity was calculated by the following equation: cytotoxicity(%)= [ Control sample(A6, 0 or viable cell numbers/ml) - Test sample(A5, D or viable cell numbers/ml)] / Control sample(A6, 0 or viable cell numbers/ml) x 100 or growth stimulating activity(%)= Test sample(A5, 0 or viable cell numbers/ml) / Control sample (As, n or viable cell numbers/ml) x 100. Assay for DNA synthesis in human melanocytes:Iluman melanocytes( six passages ) were suspended in M-GM and seeded in 24 well plates(3047, Falcon) at 2.5 x 10” cells per well. After 24 hrs of the incubation at 37°C in a humidified atmosphere with 5 % COz, the medium was replaced with M-GM containing F-TCF. The cells were subsequently incubated for 24 hrs under the conditions described above. Then the cells were incubated for 4 hrs in the medium containing Iv Ci/m1(86 Ci,/mmol) of [3111thymidine(TRK 686, Amersham). After the incubation, incorporation of [311]thymidine into DNA was measured by the method of Gohda et a1.(1986). Assay for differentiation inducing activity:K562 and HL-60 cells were seeded in 96-well flat bottomed plates at 3.5 x 10” cells per 100 ill 1 per well. To each test well, 100 ti 1 of culture medium containing F-TCF was added. The plates were incubated at 37 “C in a humidified atmosphere with 5 % COz for 3 of K562 and BL-60 cells were and 7 days. Differentiation monitored by o-dianisidine staining assay(Murata et a1.,1988) and nitroblue tetrazorium(NBT) reduction assay(Takeda et
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al., 1988),respectively. RESULTS Purification of F-TCF:- The purified F-TCF showed homogeneity on SDS-polyacrylamide gel electrophoresis under non-reducing conditions, when the gel was stained with silver(Fig. I). F-TCF had moderate Cytotoxic activity of F-TCF:cytotoxicity(more than 25 %, less than 50 % ) on human tumor cell lines, KB, BG-1, MCF-7 and Hs913T. However, the growth of human normal fibroblasts, IMR-90, and A549 were not affected(less than 15 %)(Fig. 2a). F-TCF was found to have strong cytotoxicity(more than 70 % ) on mouse tumor cell lines, Sarcoma 180, Meth A and P388. The amount of F-TCF that gave 50% cytotoxicity on sarcoma 180, Meth A and P388 was 6, 40 and 460 ng/ml,respectively(Fig. 2b). The changes in morphology were observed in KB,BG-l,A549 and B-16 cells by the addition of FTCF to the culture medium. The morphological change of KB from higher fibroblast-like to spherical was observed at F-TCF induced the dispersion of dosages(more than 500 rig/ml). BG-1, A549 and B-16 from cohesive colonies or tightly packed colonies(Fig. 3). Differentiation inducing activity of F-TCF:F-TCF did not induce the differentiation of K562 at 16 to 1000 rig/ml, but did induce the differentiation of HL-60 into morphologically granulocyte-like cells(Fig. 4). At a concentration of 250 Start MW Kd 94 67 43 31 21.5 14.4
Fig. 1: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining purified F-TCF. The gel was stained (1) F-TCF under non-reducing conditions, kit(Pharmacia Co.). (2) Molecular marker:;.
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O--’ oFv 0.5
1.0
1.0
F-TCF( ug/ml)
F-TCF(ug/ml)
(a)
(b)
Fig.2 : Cytotoxic activity of F-TCF for tumor cell . (a) Cytotoxity against human tumor cell lines. BG-l(a-,Ovarian adenocarcinoma),KB(-O-,Epidermoid carcinoma),MCF-7 (-&,Breast carcinoma),HeLa(-A-,Epitheloid carcinoma),A549(-•-,Lung carcinoma epithelial),Hs-913T(-¤-,Fibrosarcoma),IMR-90(-xNormal fibroblast). (b) Cytotoxity against mouse cell lines. karcoma 180(-•-),Meth A sarcoma(()-),P388(&,Lymphocytic Lymphoblast),Colon 26(- A-,Adenocarcinoma),Ll210(n leukemia ,Lymphocytic leukemia). All values are mean of duplicate experiments.
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: Dispersion of BG-1 and B-16 cells. BC-1 cells were Fig.3 cultured for 4 days without(a) or with(b) 62.5 rig/ml of F-TCF. B-16 cells were cultured for 3 days without(c) or with(d) 62.5 were stained with crystal violet: x 90. rig/ml of F-TCF. Cells
rig/ml, NBT reduction was induced in 45.2 % of HL-60 cells after incubation at 37 “C for 7 days(Fig.5). At higher dosages(more than 250 rig/ml), NBT reducing ability decreased. F-TCF stimulated the growth of Mitogenic activity of F-TCF:human endothelial cells, HUVEC. The maximum growth stimulating F-TCF also activity was obtained at 125 ng/ml(Fig. 6a). stimulated DNA synthesis in human melanocytes in a dose range of 10 to 100 ng/ml(Fig. 6b). After 6 days of incubation with FTCF, cell number increased to 140 % at 10 rig/ml and to 220 % at 100 rig/ml, comparing to the control. F-TCF stimulated the growth of B-16 cells(mouse melanotic melanoma)(Fig. 6~). Interestingly, stimulation of growth caused inhibition of the melanin production which is a typical characteristic of B-16 ccl 1s. DISCUSSION Fibroblasts were known to secret various tumor cytotoxic factors(Kohase et a1.,1987; Kirk et a1.,1983; Imanishi et al., 1983). We previously reported (Higashio et a1.,1990) that a tumor cytotoxic factor (F-TCF) derived from human embryonic
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Fig.4 : Morphology of or with(b) 250ng/ml Wright-Giemsa; X 90.
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HL-60 cells of F-TCF.
cultured The cells
for
7 days without(a) were stained with
ID--.-. 3”--.. 20-IO ll I 0
16
62
125
250
500
IO00
F-TCF(ng/ml) Fig. 5: NBT reducing activity of F-TCF for HL-60. HL-60 cells were cultured for 7 days with F-TCF. The percentage of cells containing blue-black formazan deposits was determined by counting at least 200 cells. Values are mean of duplicate experiments.
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F-TCF(ng/ml)
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Fig. 6: Mitogenic activity F-TCF. of ( a ) Ii II m a n umbilical vein endothelial cells(HUVEC)were cultured with Ffor 6 days TCF.Viable cell numbers counted were using a heamacytometer. Values are mean of t r i p 1 i c a t e experiments. Error bars represent SEM. (b)Human melanocytes were cultured for 211 hours with F-TCF. DNA synthesis was determined I 7n [ 3 H ] t h y m i d i n e by incorporation assay. Values Inn Ih 31 62 175 250 500 I”“0 R are the mean of triplicate experiments. Error bars F-TCF(ng/ml) represent SEM. (c)R-16 (cl cells(mouse m e I. a n 0 t i c melanoma) were cultured for 4 days with F-TCF. Viable cell numbers were determined by dye extract method descrived in materials and methods. Values are mean of duplicate experiments.
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f ibroblast, IMR-90 cells was identical to one of the human hepatocyte growth factors(hMGFs). This indicated that F-TCF was bifunctional. And so, we started investigating the detailed biological functions of F-TCF in vitro. F-TCF showed cytotoxicity on several human and mouse tumor cell lines. Therefore, F-TCF as well as TNF(Sugarman et al.,1985)does not have a species-specificity on its cytotoxic activity against tumor cells. TNF is known to be cytolytic against various tumor cell lines. In a preliminary experiment, we found that when Meth A was used as a target tiell, radioactivity was not released from the cells prelabeled with ’ ‘Cr, but uptake of [“Ii jthymidine by the cells was suppressed by F-TCF at 10 rig/ml. This result suggests that effect of F-TCF on tumor cell inhibition is probably cytostasis rather than cytolysis. F-TCF induced the dispersion of several tumor cell lines from cohesive colonies or tightly packed colonies. Similar motility factors, scatter factors(Stocker et a1.,1987; Gherardi et a1.,1989; Gherardi and Stocker, 1990; Rosen et al., 1990), migration stimulating factor(Grey et al., 1989) and autocrine motility factor(Kohn et al., 1990) have been purified in other laboratories. Among these motility factors, a scatter factor (Gherardi et al., 1989), which is derived from mouse fibroblasts and causes separation of contiguous epithelial cells, is similar to F-TCF in physicochemical properties. This protein is a heterodimer composed of a large subunit with Mw. about 57 kD and a small subunit with Mw. about 30 kD as similar to FTCF(lligashio et al., 1990). Both factors are basic, heat-labile and have affinity for heparin. Recently, this group reported that amino terminal sequence of mouse scatter fatter is highly related to that of human hepatocyte growth factor(Gherardi and Stocker,l990). These similarities suggest that F-TCF may belong to one of the scatter factors,and may act as one of the motility factors. F-TCF induced the differentiation of BL-60 ccl 1s. TNF(Takeda et al.,1986), IFN-7 (Takuma et al.,1987) and lymphotoxin(LT)(Hemmi et al., 1987) have been found to induce differentiation of leukemia cells besides tumor cytotoxity. FTCF may have various activities for immune cells as similar to these inflammatory cytokines. F-TCF is a potent mitogen not only for adult rat hepatocytes, but also for human endothelial cells, IIUVEC, human melanocytes and mouse melanotic melanoma ccl Is, B-16. These results indicate that F-TCF is a multitarget growth factor as seen in many other growth factors. Angiogenesis is an essential component of many biological processes including embryogenesis, tissue regeneration and repair. FGF is a potent mitogen for endothelial cells and is known as an angiogenic factor in vivo(Gospodarowicz et a1.,1987) . We found that F-TCF showed a strong mitogenic activity for HUVECat 125 rig/ml (about 1.6 p mole/ml) even in the optimized F-TCF may act as an growth medium, E-GM for endothelial cells. angiogenic factor in vivo in the similar manner as FGF. F-TCF has marked affinity for heparin(fiigashio et a1.,1990) as FGF does. The glycosaminoglycan of heparin is closely related to
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heparan sulfate which is a structural component of extracellular matrix. It has been known that extracellular matrix components contain FGF(Rogelj et a1.,1989). Therefore, extracellular matrix components may bind F-TCF and modulate its activity in vivo. Chemotaxis and proliferation of fibroblasts are essential processes in normal inflammatory response. Proliferated fibroblasts secret various inflammatory cytokines. Our studies revealed that F-TCF derived from human fibroblasts had multiple functions such as inhibition of tumor cell growth, angiogenesis, mitogenesis on hepatocytes and melanocytes, and differentiation of leukemia cells. These results suggest that F-TCF is an effector molecule responsible for inflammation and repair, and that fibloblasts may contribute to repair the injured tissues by secreting F-TCF. REFFERENCES Elias, J. A., Reynolds, M. M., Kotloff, R. M., and Kern J. A. (1989). Fibroblast interleukin 1P : Synergistic stimulation by recombinant interleukin 1 and tumor necrosis factor and posttranscriptional regulation. Proc. Natl. Acad. Sci. USA 86, 6171 - 6175. Cherardi, E., Gray, J., Stoker, M., Perryman, M., and Furlong, R. (1989). Purification of scatter factor,a fibroblastderived basic protein that modurates epithelial interactions and movement. Proc. Natl. Acad. Sci. 86, 5844 - 5848. Gherard i , E. and Stoker, M. (1990). llepatocytes and scatter Nature 346, 228. factor. Gohda, E., Tsubouchi,. , Nakayama, 1~., flirono, S., Takahashi, K. , Koura, M., llashimoto, S., and Daikuhara, Y. (1986). fluman hepatocyte growth factor in plasma from patients with fulminant hepatic failure. Exp. Cell Res. 166, 139 - 150. Gospodarowicz, D., Ferrara, N., Schweigerer, L. (1987). Structural characterization and biological functions of 95 - 114. fibroblast growth factor. Endocrine Reviews 8, Grey, A. M., Schor, A. M., Rushton, G., El1 is, I., and Schor, S. L. (1989). Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc. Natl. Acad. Sci. USA 86, 2438 -2442. Hemmi, fl., Nakamura, T., Tamura, K., Shimizu, Y., Kato, S., Miki, T., Takahashi, N., Muramatsu, Y., Numao, N., and Induction of terminal Sugamura, K. (1987). Lymphotoxin: differentiation of the human myeloid leukemia cell lines HL-60 and THP-1. J. Immunol. 138, 664 - 666. Y., Nagao, M., fligashio, K., Shima, N., Goto, M., Itagaki, of a tumor Yasuda, H., and Morint)ga, T. (1990). Identity cytotxic factor from human fibroblasts and hepatocyte growth factor. Biochem. Biophys. Res. Commun. 170, 397 404.
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Imanishi, J., Hoshino, S., Matsuoka, H., Uemura, Il., Imanishi, T . , Tanaka, A., Nishino, Il., and Kishida, T. (1983). Tumor degeneration by human embryonic fibroblasts and its enhancement by interferon. 43, 4323 Cancer Research 4326. Kirk, D. , Kagawa, S. , and Verner, G. (1983). Comparable growth regulation of five human tumor cell lines by neonatal human lung fibroblasts in semisolid culture media. Cancer 3753 -3758. Research 2, Kohase, M., May, L. T., Tamm, I., Vilcek, J., and Sehgal, P. B. (1987). A cytokine network in human diploid fibroblasts: Interactions of P -interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1. Mol. Cell. Biol. 1, 273 - 280. Kohn, E. C., Liotta, L. A., and Schiffmann, E. (1990). Autocrine motility factor stimulates a three-fold increase in inositol triphosphate in human melanoma cells. Biochem. Biophis. Res. Commun. 166 , 757 -764. Lee, S. Il., Aggarwal, B. B., Rinderknecht, E., Assisi, F., and Chiu, H. (1984). The synergistic anti-proliferative effect of r-interferon and human lymphotoxin. J. Immunol. 133, 1083 - 1089. Miyazawa, K., Tsubouchi, II., Naka, D., Takahashi, K., Okigaki, M Arakaki, N., Nakayama, Il., Hirono, S., Sakiyama, 0.) Takahashi, K., Gohda, E. , Dai kuhara, Y. , and Hi tamura, N. (1989). Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 163, 967 - 973. Murata, M., Eto, Y., Shibai, Il., Sakai, M., and Muramatsu, M. (1988). Erythroid differentiation factor is encoded by the same mRNA as that of the inhibin P A chain. Proc. Natl. Acad. Sci. USA 85, 2434 - 2438. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M Sugimura, A., Tashiro, K., and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth Nature 342, 440 - 443. factor. Rogel j, S., Klagsbrun, M., Atzmon, R., Kurokawa, M., Haimovitz, A Fuks, Z., and Vlodavsky, 1. (1989). Basic fibroblast growth factor is an exracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 eels. J. Cell Biol. 109, 823 - 831. Rosen, E. M., Meromsky, I,. , Romero, R., Setter, E., and Goldberg, I. (1990). Human placenta contains an epithelial scatter protein. Biochem. Biophys. Res. Commun. 168 , 1082 - 1088. Stocker, M., Gherardi, E., Pennyman, M., and Gray, J. (1987). Scatter factor is a fibroblast-derived modulator of epithelial sell mobility. Nature 327, 239 - 242.
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