In Vitro Cell. Dev. Biol. 2 8 A : 5 6 1 - 5 6 4 , September-October 1992 © 1992 Tissue Culture Association 0883-8364/92 $01,50+0.00
L e t t e r to t h e E d i t o r MODULATION OF THE GROWTH AND MORPHOLOGY OF A HUMAN NASOPttARYNGEAL CARCINOMA CELL LINE BY GROWTH FACTORS
mixture was overlayed on top of a solidified 0.9% agarose layer in a 35 mm dish. After the cell-agarose layer was gelled, 1 ml of basal medium containing appropriate amount of FBS and desired testing factors was added to each dish and the cultures were incubated for 7 days without a medium change. After incubation, the medium was removed and the colonies were fixed with 4% formaldehyde in phosphate buffered saline and the agar colonies stained with 0.01% crystal violet. Colonies with a diameter greater than 12 ~m were then scored under a dissecting microscope. Modulation of cell growth by growth factors. To examine the effect of growth factors on CG-1 cell growth, it was necessary to choose a serum concentration at which the cells are able to survive and the growth is minimal. For this purpose, the growth rate of the cells at various concentrations of FBS was therefore measured. Cells were plated at 2 5 0 / c m 2 (2000 cells per 35 mm dish) in basal medium containing indicated concentrations of FBS and cell number was counted every third day for up to 9 days. It was found that in 0.5% FBS, cells were able to survive and underwent a very slow cell cycling and doubled less than twice in 9 days. At 2% and 4% FBS, cell growth was pretty much the same indicating cell growth at this range of serum concentration was close to optimal. The clonal growth of cells in monolayer culture was examined by
Dear Editor: Nasopharyngeal carcinoma (NPC) is a human squamous cancer which is common in certain regions of Southeastern Asia and East and North Africa (3,17). Recent studies showed that 91% of the NPC biopsies from North Africa and China were positive with Epstein Barr virus (EBV) nuclear antigen 1 (5) and human nasopharyngeal epithelium was found to express functional EBV receptor (19,24). Furthermore, molecular biological studies have revealed the presence of EBV-DNA sequences in NPC cells (1,2). These findings suggest a possible link between EBV and the pathogenesis of NPC. Establishment of human NPC cell line from biopsy specimens has been cumbersome due to the presence of high proportions of lymphoid cells and fibroblasts in the biopsy and the difficulties in optimizing the culture conditions. Thus far, only a few NPC cell lines have been established (7,10,12,23). Recently, a NPC cell line, designated CG-1, was successfully established by one of us and has been characterized and was found to carry EBV-DNA sequences even though it has been carried in culture for more than 200 generations (1). In the present study, we examined CG-1 cell response to epithelial cell growth modulators including epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and transforming growth factor-/~ (TGF-/3) in 0 . 5 - 1 % FBS. We found that the anchorage-dependent growth of CG-1 cells was moderately stimulated by EGF, aFGF and TGF-~ and was inhibited by bFGF. In addition, EGF and bFGF induced a morphological transformation of CG-1 cells from a fight-packed, cobblestone-shaped epithelial morphology into a dispersed, fibroblastic morphology, while the morphological effect by aFGF was less obvious. Anchorage-independent growth of CG-1 cells was modulated by the aforementioned growth factors in a similar fashion. NPC CG-1 cell line were established from a WHO type II NPC tumor biopsy (1). This line has not been cloned and cells at Passages 30 to 40 were used in this study. Stock cultures were maintained in a basal medium containing 75% DMEM and 25% F-12 supplemented with 5% FBS in a humidified incubator at 37 ° C under 95% air/5% CO2, and were passed at a 1 to 3 split at confluence. Cell growth was measured by counting cell number at the end of experiments using a Coulter counter (model Zm). Cells were plated on type 1 collagen (10 gg/ml) coated dishes at 2 0 0 0 / 35 mm dish or 2 0 0 / 6 0 mm dish in basal medium containing 0.5% FBS and the indicated growth factors. At time points indicated, cells were harvested by trypsin (0.1%) treatment, suspended in 10 ml isoton (Coulter Electronics) and counted. For anchorage-independent growth assay, cells were mixed with 0.45% agarose in basal medium at 1000 cells per ml medium under 40 ° C and 2 ml of the
40
30
10
t
t
t
t
t
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Growth factor
ng/ml
Fla. 1. Anchorage-dependentclonalgrowthofCG-1 cells in low serum. Cells were plated at 200 per 60 mm collagen-coated dish in 3 ml of basal medium supplemented with 0.5% FBS and graded dose of various growth factors indicated. Cells were fixed on Day 10 and stained with crystal violet. Colonies with cell number greater than 64 were scored. Data are mean ± SD (n = 3). A, TGF-/~; A, aFGF; O, EGF; ©, bFGF.
561
562
CHEN ET AL.
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0.6
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.4
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0.2 0.0
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FIG. 2. Effect of growth factors on the anchorage-independent growth of CG-1 cells in low serum. Cells (2000 per 2 ml agarose solution per 35 mm dish) were cultured in soft agar in basal medium supplemented with 0.5% FBS and indicated amounts of growth factors. On Day 7, colonies were fixed and stained with 0.01% crystal violet. Colonies with diameter greater than 12 ttm were scored and data are expressed as mean + SD (n = 4). A, TGF-fl; A, aFGF; O, EGF; ©, bFGF.
plating cells at 8 / c m z (200 cells per 60 mm dish) in basal medium supplemented with 0.5% FBS and graded doses of EGF (Mouse, receptor grade, Collaborative Research), aFGF (human, recombinant, Boehringer Mannheim) bFGF (bovine, recombinant, Boehringer Mannheim) or TGF-/~ (type I, Collaborative Research). Ceils were fixed on day 10 and stained with 0.1% crystal violet and cell colonies were scored. Fig. 1 shows that colony formation was stimulated by EGF, aFGF and TGF-fl and was inhibited by bFGF at the same dose range as that of aFGF. Of the factors tested, TGF-fl appeared to be the most growth promoting. At an initial plating density of 250 cells/cm 2 and 0.5% FBS, cell growth was also promoted, though to a lesser extent, by EGF, aFGF and TGF-fl, and again, bFGF appeared to be inhibitory at dose range between 1 - 1 0 ng/ml (not shown). In this study, two lots of bFGF were used (as described by the manufacturer, both lots contained no carrier proteins), they were both growth stimulatory to human umbilical cord vein endothelial cells and skin fibroblasts indicating that the bFGF used were not cytotoxic. Fig. 2. shows that soft agar growth of CG-1 cells in 0.5% FBS was also promoted by either EGF, aFGF or TGF-/~ and was somewhat inhibited by bFGF. The stimulatory effect of EGF and TGF-fl was slightly enhanced by retinoic acid (not shown), Morphological effect of the growth factors. CG-1 cells are of epithelial origin, and exhibit epithelial characteristics in culture
FIG. 3. Effect of growth factors on the monolayer colony morphology of CG-I cells. Cells were plated at 200 per 35 mm collagencoated dish in basal medium containing 1% FBS. One h after plating, the cultures were either unsupplemented (A) or were supplemented with EGF (B), aFGF (C} or bFGF (D) at 5 ng/ml. The cultures were incubated for 10 days with one medium change on day 5 and were fixed with 4% formaldehyde and stained with crystal violet. Photos were taken under a phase contrast microscope at a magnification of X60.
REGULATION OF CARCINOMA CELL GROWTH AND MORPHOLOGY (Fig. 3 A). In the presence of EGF or bFGF, CG-1 cells were induced to lose their cell-cell adhesiveness and to acquire morphological properties typical of mesenchymal cells (Fig. 3 B,D). The morphological effect by aFGF was not as striking in the same dose range ( 1 - 1 0 ng/ml) in that only the cells at the peripheral region of a colony assumed a motile, fibroblastic morphology (Fig. 3 C). The opposite effects of aFGF and bFGF on CG-1 cell growth are interesting and deserve further study. Acidic FGF and bFGF are structurally related molecules, they both stimulate the growth of a variety of ceil types and have been shown to interact with the same cell surface receptors (16). Recent FGF receptor cDNA sequence studies by Hou et al. (9) predicted the existence of a minimum of 6 possible receptor phenotypes and Kan et al. (11) found that aFGF and bFGF affected liver cell growth in a different manner and they did not compete with each other for the binding to hepatocytes. These findings suggested that aFGF and bFGF may bind to different cell surface FGF receptor subtypes and modulate cell growth differently. Thus, it is tempting to speculate that the opposite effects of aFGF and bFGF on CG-1 cells may be mediated through distinct FGF receptor subtypes. TGF-/~ is a potent growth inhibitor in many primary and secondary ceil cultures, particularly epithelial cells (14,15,18). The fact that TGF-/3 stimulates CG-1 cell growth indicates that the growth inhibitory signal conveyed by TGF-/~-receptor complex in normal epithelial ceils was diverted into a signaling pathway leading to growth stimulation due perhaps, to neoplastic transformation. The maintenance of the state of an epithelium is very much dependent on the intercellular adhesive forces (4,21) and cell interactions with the extracellular matrix (8), how these interactions may be regulated by soluble factors have not been fully elucidated. Recently a scatter factor produced by fetal fibroblasts was shown to disorganize cultures of normal epithelial cells (6,20), and a motility factor produced by several tumor ceil hnes that affect the cohesion of cancer cells was also reported (13). These factors appeared to be different from other polypeptide growth factors. More recently, aFGF, but not bFGF, was reported to modulate epithelial plasticity in a rat bladder carcinoma cell line (22). In the present study, we showed that EGF and bFGF stimulated CG-1 cell scattering and motility, while the effect by aFGF was not as obvious. In our hands, EGF was a growth and scattering stimulator and bFGF was a growth inhibitor and a scattering stimulator. Thus, the regulation of cell growth and scattering were not coupled in CG-1 ceils. Our results with CG-1 ceils, which are induced by EGF, bFGF, and to a lesser extent, by aFGF, to change from an epithelial state to a dispersed, motile one, are of cell biological importance because similar transitions are seen in wound repair and perhaps, cancer metastasis. Further studies are needed to elucidate the cellular mechanisms by which EGF and bFGF induce CG-1 ceil scattering and to examine why aFGF and bFGF, two structurally related mitogens, influence CG-1 ceil growth and morphology in a different manner.
ACKNOWLEDGEMENTS This study was supported by grants from National Science Council, Taiwan (NSC 80-0412-B18231) and Chang Gung Memorial Hospital (CMRP 291).
563
REFERENCES 1. Chang, Y-S.; Lin, S-Y.; Lee, P-F., et al. Establishment and characterization of tumor cell line from human nasopharyngeal carcinoma tissue. Cancer Res. 49:6752-6757; 1989. 2. Chen, J-Y.; Ou, Y-H.; Hsu, T-Y., et al. Detection of EBV-DNAin nude mice passaged nasopharyngeal carcinoma (NPC) tissue. In: Levine, P. H.; Ablashi, D. V.; Nonoyama, M., et al., eds. Epstein-Barr virus and human disease, vol. 1. Clifton, NJ: The Humana Press, Inc.; 1987:183-187. 3. Desgranges, C.; Wolf, H.; de-The, G., et al. Nasopharyngeal carcinoma. X. Presence of Epstein-Barr genomes in epithelial cells of tumors from high and medium risk areas. Int. J. Cancer. 16:7-15; 1975. 4. Edelman, G. M. Morphoregulatory molecules. Biochemistry 27: 3533-3543; 1988. 5. Fahraeus, R.; Fu, H. L.; Emberg, l., et at. Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. Int. J. Cancer 42:329-338; 1988. 6. Gherardi, E.; Gray, J.; Stoker, M., et at. Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interactions and movement. Proc. Natl. Acad. Sci. USA 86:5844-5848; 1989. 7. Gu, S. U.; Tann, B. F.; Zeng, Y., et al. Establishment of an epithelial cell line (CNE-2) from an NPC patient with poorly differentiated squamous cell carcinoma. Chinese J. Cancer 2:70-72; 1983. 8. Hay, E. D. Collagen and embryonic development, chapter 12. In: Hay, E. D., ed. Cell biology of the extracellular matrix. New York, NY: Plenum Press; 1981:379-409. 9. Hou, J.: Kan, M.; McKeehan, K., et al. Fibroblast growth factor receptors from liver vary in three structural domains. Science 251:665668; 1991. 10. Huang, D. P.; Ho, J. H. C.; Poon, Y. F., et at. Establishment of a cell line (NPC/HK) from a differentiated squamous carcinoma of the nasopharynx. Int. J. Cancer 26:127-132; 1980. 11. Kan, M.; Shi, E.; McKeehan, W. L. Different effects of heparin binding (fibroblast) growth factor type 1 (aFGF) and type 2 (bFGF) on primary cultured rat hepatocytes. In Vitro Cell. Dev. Biol. 27A (part II):496; 1991. 12. Lin, C-T.; Wong, C-I.; Chan, W-Y., et al. Establishment mad characterization of two nasopharyngeal carcinoma cell lines. Lab. Invest. 62:713-724; 1990. 13. Liotta, L. A.; Mandler, R.; Murano, G., et at. Tumor cell autocrine motility factor. Proc. Natl. Acad. Sci. USA 83:3302-3306; 1986. 14. Masui, T.; Wakefield, L. M.; Lechner, J. F., et al. Type ~ transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 83:2438-2442; 1986. 15. Moses, H. L.; Tucker, R. F.; Leof, E. B., et at. Type-/~ transforming growth factor is a growth stimulator and a growth inhibitor. In: Feramisco, J.; Ozanne, B.; Stiles, C., eds. Cancer cells, growth factors and transformation, vol. 3. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1985:65-71. 16. Neufeld, G.; Gospodarowicz, D. Basic and acidic fibroblast growth factors interact with the same cell surface receptors. J. Biol. Chem. 261:5631-5637; 1986. 17. Pagano, J. S.; Huang, C. H.; Klein, G., et al. Homology of EpsteinBarr virus DNA in nasopharyngeal carcinoma from Kenya, Taiwan, Singapore and Tunisia. In: de-Thee, G.; Epstein, M. A.; zur Hansen, H., eds. Oncogenesis and herpesviruses, Vol. 2. Lyon: International Agency for Research on Cancer; 1975:179-190. 18. Shipley, G. D.; Pittelkow, M. R.; Wille, J. J., Jr., et al. Reversible inhibition of normal human prokeratinocyte proliferation by type-/~ transforming growth factor-growth inhibition in serum-free medium. Cancer Res. 46:2068-2071; 1986. 19. Sixbey, J. W.; Davis, D. S.; Young, L. S., et at. Human epithelial cell expression of an Epstein-Barr virus receptor. J. Gen. Virol. 68:805-811; 1987. 20. Stoker, M.; Gherardi, E.; Perryman, M., et al. Scatter factor is a fibro-
564
21. 22. 23.
24.
CHEN ET AL. blast-derived modulator of epithelial cell mobility. Nature (London) 327:239-242; 1987. Takeichi, M. The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development 102:639-655; 1988. Valles, A. M.; Boyer, B.; Badet, J., et al. Acidic fibroblast growth factor is a modulator of epithelial plasticity in a rat bladder carcinoma cell line. Proc. Natl. Acad. Sci. USA 87:1124-1128; 1990. Yao, K.; Zhang, H-Y.; Zhu, H-C., et al. Establishment and characterization of two epithelial tumor cell lines (HNE-1 and HONE-l) latently infected with Epstein-Barr virus and derived from nasopharyngeal carcinomas. Int. J. Cancer 45:83-89; 1990. Young, I. S.; Clark, D.; Sixbey, J. W., et al. Epstein=Barrvirus receptors on human pharyngeal epithelia. Lancet 1:240-242; 1986.
Jan-Kan Chen Yu-Sun Chang
Song-Shu Lin Hsiou-Hsien Chao
Department of Physiology (J-K. C., S-S. L., H-H. C.) and Department of Immunology and Microbiology (Y-S. C.) Chang Gung Medical College Taoyuan 333 Taiwan, Republic of China (Received 21 January 1992)
L e t t e r to t h e E d i t o r MODULATION OF THE GROWTH AND MORPHOLOGY OF A HUMAN NASOPttARYNGEAL CARCINOMA CELL LINE BY GROWTH FACTORS
mixture was overlayed on top of a solidified 0.9% agarose layer in a 35 mm dish. After the cell-agarose layer was gelled, 1 ml of basal medium containing appropriate amount of FBS and desired testing factors was added to each dish and the cultures were incubated for 7 days without a medium change. After incubation, the medium was removed and the colonies were fixed with 4% formaldehyde in phosphate buffered saline and the agar colonies stained with 0.01% crystal violet. Colonies with a diameter greater than 12 ~m were then scored under a dissecting microscope. Modulation of cell growth by growth factors. To examine the effect of growth factors on CG-1 cell growth, it was necessary to choose a serum concentration at which the cells are able to survive and the growth is minimal. For this purpose, the growth rate of the cells at various concentrations of FBS was therefore measured. Cells were plated at 2 5 0 / c m 2 (2000 cells per 35 mm dish) in basal medium containing indicated concentrations of FBS and cell number was counted every third day for up to 9 days. It was found that in 0.5% FBS, cells were able to survive and underwent a very slow cell cycling and doubled less than twice in 9 days. At 2% and 4% FBS, cell growth was pretty much the same indicating cell growth at this range of serum concentration was close to optimal. The clonal growth of cells in monolayer culture was examined by
Dear Editor: Nasopharyngeal carcinoma (NPC) is a human squamous cancer which is common in certain regions of Southeastern Asia and East and North Africa (3,17). Recent studies showed that 91% of the NPC biopsies from North Africa and China were positive with Epstein Barr virus (EBV) nuclear antigen 1 (5) and human nasopharyngeal epithelium was found to express functional EBV receptor (19,24). Furthermore, molecular biological studies have revealed the presence of EBV-DNA sequences in NPC cells (1,2). These findings suggest a possible link between EBV and the pathogenesis of NPC. Establishment of human NPC cell line from biopsy specimens has been cumbersome due to the presence of high proportions of lymphoid cells and fibroblasts in the biopsy and the difficulties in optimizing the culture conditions. Thus far, only a few NPC cell lines have been established (7,10,12,23). Recently, a NPC cell line, designated CG-1, was successfully established by one of us and has been characterized and was found to carry EBV-DNA sequences even though it has been carried in culture for more than 200 generations (1). In the present study, we examined CG-1 cell response to epithelial cell growth modulators including epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and transforming growth factor-/~ (TGF-/3) in 0 . 5 - 1 % FBS. We found that the anchorage-dependent growth of CG-1 cells was moderately stimulated by EGF, aFGF and TGF-~ and was inhibited by bFGF. In addition, EGF and bFGF induced a morphological transformation of CG-1 cells from a fight-packed, cobblestone-shaped epithelial morphology into a dispersed, fibroblastic morphology, while the morphological effect by aFGF was less obvious. Anchorage-independent growth of CG-1 cells was modulated by the aforementioned growth factors in a similar fashion. NPC CG-1 cell line were established from a WHO type II NPC tumor biopsy (1). This line has not been cloned and cells at Passages 30 to 40 were used in this study. Stock cultures were maintained in a basal medium containing 75% DMEM and 25% F-12 supplemented with 5% FBS in a humidified incubator at 37 ° C under 95% air/5% CO2, and were passed at a 1 to 3 split at confluence. Cell growth was measured by counting cell number at the end of experiments using a Coulter counter (model Zm). Cells were plated on type 1 collagen (10 gg/ml) coated dishes at 2 0 0 0 / 35 mm dish or 2 0 0 / 6 0 mm dish in basal medium containing 0.5% FBS and the indicated growth factors. At time points indicated, cells were harvested by trypsin (0.1%) treatment, suspended in 10 ml isoton (Coulter Electronics) and counted. For anchorage-independent growth assay, cells were mixed with 0.45% agarose in basal medium at 1000 cells per ml medium under 40 ° C and 2 ml of the
40
30
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t
t
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2
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Growth factor
ng/ml
Fla. 1. Anchorage-dependentclonalgrowthofCG-1 cells in low serum. Cells were plated at 200 per 60 mm collagen-coated dish in 3 ml of basal medium supplemented with 0.5% FBS and graded dose of various growth factors indicated. Cells were fixed on Day 10 and stained with crystal violet. Colonies with cell number greater than 64 were scored. Data are mean ± SD (n = 3). A, TGF-/~; A, aFGF; O, EGF; ©, bFGF.
561
562
CHEN ET AL.
0.8 •~
0.6
~
.4
~
0.2 0.0
I
I
0
1
I
2.5
Growth factor
I
5
I
10
ng/ml
FIG. 2. Effect of growth factors on the anchorage-independent growth of CG-1 cells in low serum. Cells (2000 per 2 ml agarose solution per 35 mm dish) were cultured in soft agar in basal medium supplemented with 0.5% FBS and indicated amounts of growth factors. On Day 7, colonies were fixed and stained with 0.01% crystal violet. Colonies with diameter greater than 12 ttm were scored and data are expressed as mean + SD (n = 4). A, TGF-fl; A, aFGF; O, EGF; ©, bFGF.
plating cells at 8 / c m z (200 cells per 60 mm dish) in basal medium supplemented with 0.5% FBS and graded doses of EGF (Mouse, receptor grade, Collaborative Research), aFGF (human, recombinant, Boehringer Mannheim) bFGF (bovine, recombinant, Boehringer Mannheim) or TGF-/~ (type I, Collaborative Research). Ceils were fixed on day 10 and stained with 0.1% crystal violet and cell colonies were scored. Fig. 1 shows that colony formation was stimulated by EGF, aFGF and TGF-fl and was inhibited by bFGF at the same dose range as that of aFGF. Of the factors tested, TGF-fl appeared to be the most growth promoting. At an initial plating density of 250 cells/cm 2 and 0.5% FBS, cell growth was also promoted, though to a lesser extent, by EGF, aFGF and TGF-fl, and again, bFGF appeared to be inhibitory at dose range between 1 - 1 0 ng/ml (not shown). In this study, two lots of bFGF were used (as described by the manufacturer, both lots contained no carrier proteins), they were both growth stimulatory to human umbilical cord vein endothelial cells and skin fibroblasts indicating that the bFGF used were not cytotoxic. Fig. 2. shows that soft agar growth of CG-1 cells in 0.5% FBS was also promoted by either EGF, aFGF or TGF-/~ and was somewhat inhibited by bFGF. The stimulatory effect of EGF and TGF-fl was slightly enhanced by retinoic acid (not shown), Morphological effect of the growth factors. CG-1 cells are of epithelial origin, and exhibit epithelial characteristics in culture
FIG. 3. Effect of growth factors on the monolayer colony morphology of CG-I cells. Cells were plated at 200 per 35 mm collagencoated dish in basal medium containing 1% FBS. One h after plating, the cultures were either unsupplemented (A) or were supplemented with EGF (B), aFGF (C} or bFGF (D) at 5 ng/ml. The cultures were incubated for 10 days with one medium change on day 5 and were fixed with 4% formaldehyde and stained with crystal violet. Photos were taken under a phase contrast microscope at a magnification of X60.
REGULATION OF CARCINOMA CELL GROWTH AND MORPHOLOGY (Fig. 3 A). In the presence of EGF or bFGF, CG-1 cells were induced to lose their cell-cell adhesiveness and to acquire morphological properties typical of mesenchymal cells (Fig. 3 B,D). The morphological effect by aFGF was not as striking in the same dose range ( 1 - 1 0 ng/ml) in that only the cells at the peripheral region of a colony assumed a motile, fibroblastic morphology (Fig. 3 C). The opposite effects of aFGF and bFGF on CG-1 cell growth are interesting and deserve further study. Acidic FGF and bFGF are structurally related molecules, they both stimulate the growth of a variety of ceil types and have been shown to interact with the same cell surface receptors (16). Recent FGF receptor cDNA sequence studies by Hou et al. (9) predicted the existence of a minimum of 6 possible receptor phenotypes and Kan et al. (11) found that aFGF and bFGF affected liver cell growth in a different manner and they did not compete with each other for the binding to hepatocytes. These findings suggested that aFGF and bFGF may bind to different cell surface FGF receptor subtypes and modulate cell growth differently. Thus, it is tempting to speculate that the opposite effects of aFGF and bFGF on CG-1 cells may be mediated through distinct FGF receptor subtypes. TGF-/~ is a potent growth inhibitor in many primary and secondary ceil cultures, particularly epithelial cells (14,15,18). The fact that TGF-/3 stimulates CG-1 cell growth indicates that the growth inhibitory signal conveyed by TGF-/~-receptor complex in normal epithelial ceils was diverted into a signaling pathway leading to growth stimulation due perhaps, to neoplastic transformation. The maintenance of the state of an epithelium is very much dependent on the intercellular adhesive forces (4,21) and cell interactions with the extracellular matrix (8), how these interactions may be regulated by soluble factors have not been fully elucidated. Recently a scatter factor produced by fetal fibroblasts was shown to disorganize cultures of normal epithelial cells (6,20), and a motility factor produced by several tumor ceil hnes that affect the cohesion of cancer cells was also reported (13). These factors appeared to be different from other polypeptide growth factors. More recently, aFGF, but not bFGF, was reported to modulate epithelial plasticity in a rat bladder carcinoma cell line (22). In the present study, we showed that EGF and bFGF stimulated CG-1 cell scattering and motility, while the effect by aFGF was not as obvious. In our hands, EGF was a growth and scattering stimulator and bFGF was a growth inhibitor and a scattering stimulator. Thus, the regulation of cell growth and scattering were not coupled in CG-1 ceils. Our results with CG-1 ceils, which are induced by EGF, bFGF, and to a lesser extent, by aFGF, to change from an epithelial state to a dispersed, motile one, are of cell biological importance because similar transitions are seen in wound repair and perhaps, cancer metastasis. Further studies are needed to elucidate the cellular mechanisms by which EGF and bFGF induce CG-1 ceil scattering and to examine why aFGF and bFGF, two structurally related mitogens, influence CG-1 ceil growth and morphology in a different manner.
ACKNOWLEDGEMENTS This study was supported by grants from National Science Council, Taiwan (NSC 80-0412-B18231) and Chang Gung Memorial Hospital (CMRP 291).
563
REFERENCES 1. Chang, Y-S.; Lin, S-Y.; Lee, P-F., et al. Establishment and characterization of tumor cell line from human nasopharyngeal carcinoma tissue. Cancer Res. 49:6752-6757; 1989. 2. Chen, J-Y.; Ou, Y-H.; Hsu, T-Y., et al. Detection of EBV-DNAin nude mice passaged nasopharyngeal carcinoma (NPC) tissue. In: Levine, P. H.; Ablashi, D. V.; Nonoyama, M., et al., eds. Epstein-Barr virus and human disease, vol. 1. Clifton, NJ: The Humana Press, Inc.; 1987:183-187. 3. Desgranges, C.; Wolf, H.; de-The, G., et al. Nasopharyngeal carcinoma. X. Presence of Epstein-Barr genomes in epithelial cells of tumors from high and medium risk areas. Int. J. Cancer. 16:7-15; 1975. 4. Edelman, G. M. Morphoregulatory molecules. Biochemistry 27: 3533-3543; 1988. 5. Fahraeus, R.; Fu, H. L.; Emberg, l., et at. Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma. Int. J. Cancer 42:329-338; 1988. 6. Gherardi, E.; Gray, J.; Stoker, M., et at. Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interactions and movement. Proc. Natl. Acad. Sci. USA 86:5844-5848; 1989. 7. Gu, S. U.; Tann, B. F.; Zeng, Y., et al. Establishment of an epithelial cell line (CNE-2) from an NPC patient with poorly differentiated squamous cell carcinoma. Chinese J. Cancer 2:70-72; 1983. 8. Hay, E. D. Collagen and embryonic development, chapter 12. In: Hay, E. D., ed. Cell biology of the extracellular matrix. New York, NY: Plenum Press; 1981:379-409. 9. Hou, J.: Kan, M.; McKeehan, K., et al. Fibroblast growth factor receptors from liver vary in three structural domains. Science 251:665668; 1991. 10. Huang, D. P.; Ho, J. H. C.; Poon, Y. F., et at. Establishment of a cell line (NPC/HK) from a differentiated squamous carcinoma of the nasopharynx. Int. J. Cancer 26:127-132; 1980. 11. Kan, M.; Shi, E.; McKeehan, W. L. Different effects of heparin binding (fibroblast) growth factor type 1 (aFGF) and type 2 (bFGF) on primary cultured rat hepatocytes. In Vitro Cell. Dev. Biol. 27A (part II):496; 1991. 12. Lin, C-T.; Wong, C-I.; Chan, W-Y., et al. Establishment mad characterization of two nasopharyngeal carcinoma cell lines. Lab. Invest. 62:713-724; 1990. 13. Liotta, L. A.; Mandler, R.; Murano, G., et at. Tumor cell autocrine motility factor. Proc. Natl. Acad. Sci. USA 83:3302-3306; 1986. 14. Masui, T.; Wakefield, L. M.; Lechner, J. F., et al. Type ~ transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells. Proc. Natl. Acad. Sci. USA 83:2438-2442; 1986. 15. Moses, H. L.; Tucker, R. F.; Leof, E. B., et at. Type-/~ transforming growth factor is a growth stimulator and a growth inhibitor. In: Feramisco, J.; Ozanne, B.; Stiles, C., eds. Cancer cells, growth factors and transformation, vol. 3. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1985:65-71. 16. Neufeld, G.; Gospodarowicz, D. Basic and acidic fibroblast growth factors interact with the same cell surface receptors. J. Biol. Chem. 261:5631-5637; 1986. 17. Pagano, J. S.; Huang, C. H.; Klein, G., et al. Homology of EpsteinBarr virus DNA in nasopharyngeal carcinoma from Kenya, Taiwan, Singapore and Tunisia. In: de-Thee, G.; Epstein, M. A.; zur Hansen, H., eds. Oncogenesis and herpesviruses, Vol. 2. Lyon: International Agency for Research on Cancer; 1975:179-190. 18. Shipley, G. D.; Pittelkow, M. R.; Wille, J. J., Jr., et al. Reversible inhibition of normal human prokeratinocyte proliferation by type-/~ transforming growth factor-growth inhibition in serum-free medium. Cancer Res. 46:2068-2071; 1986. 19. Sixbey, J. W.; Davis, D. S.; Young, L. S., et at. Human epithelial cell expression of an Epstein-Barr virus receptor. J. Gen. Virol. 68:805-811; 1987. 20. Stoker, M.; Gherardi, E.; Perryman, M., et al. Scatter factor is a fibro-
564
21. 22. 23.
24.
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Jan-Kan Chen Yu-Sun Chang
Song-Shu Lin Hsiou-Hsien Chao
Department of Physiology (J-K. C., S-S. L., H-H. C.) and Department of Immunology and Microbiology (Y-S. C.) Chang Gung Medical College Taoyuan 333 Taiwan, Republic of China (Received 21 January 1992)
