Tohoku
J. Exp.
Med., 1992,
168, 287-290
Heparin-Binding (Fibroblast) Growth Factors are Potential Autocrine Regulators of Esophageal Epithelial Cell Proliferation MASAFUMI KATAYAMA and MIKI0KAN* The Second Department of Surgery, Tohoku University School of Medicine,Sendai 980, and * W.Alton Jones Cell Science Center, Inc., Lake Placid, NY, USA KATAYAMA, M. and KAN, M. Heparin-Binding (Fibroblast) Growth Factors are Potential Autocrine Regulators of Esophageal Epithelial Cell Proliferation. Tohoku J. Exp. Med., 1992, 168 (2), 287-290 A serum-free culture system supplemented with neural tissue extract for normal human esophagus was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life-span, but retained morphology and other characteristics of normal epithelial cells. A screen for growth factors that stimulated growth of MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating, but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 cells had properties similar to HBGF-1 (acidic fibroblast growth factor). esophagus ; epithelial cells ; heparin-binding growth factors ; FGF ; autocrine mechanisms
We previously established a serum-free culture system for normal human esophageal epithelial cells (Katayama et al. 1986) and reported that crude bovine neural tissue extracts was an effective growth stimulant of normal human esophageal epithelial cells. The same culture system used for normal human esophageal cell culture was applied to establish an immortal non-malignant epithelial (MEE) cell line from normal esophageal epithelium from the BALB/c mouse. In the absence of neural extract, a malignant cloned cell line (MEE/C8) was selected from the MEE cells. Here we compare the growth kinetics of MEE, MEE/C8 and human esophageal cancer-derived cell lines and their response to EGF and heparin-binding growth factor-1(HBGF-1). We show that an HBGF-like activity may play a growth cycle-dependent autocrine role in non-malignant esoAddress
for reprints
: 1-1 Seiryomachi,
Aoba-ku, 287
Sendai
980, Japan.
288
M.Katayamaand M.Kan
phageal epithelial cell proliferation and a constitutive role in support of the malignant cell line. MATERIALS ANDMETHODS Methodsfor primaryculture of mouseesophagealepithelial(MEE) cells were a modificationof previouslydescribedprocedures(Katayamaet al. 1986). HeparinSepharosechromatography and reversed-phase high performance liquid chromatography (HPLC)werecarriedout as described(McKeehan and Crabb1987). RESULTS Establishment of mouse esophageal cell lines (MEE and MEE/C8). The previously reported serum-free culture system for normal human esophageal epithelial cells (Katayama et al. 1986) was applied to normal mouse esophageal epithelium. Rapidly growing colonies of cells (MEE) with apparent normal epithelial cell morphology were observed within several days and subcultured after 20 to 30 days. Bovine neural extract (BHE) was absolutely required for colony formation in the primary culture. MEE cells have been maintained for over one year (100 passages). To determine whether neural extract-independent cells could be selected from the MEE cultures, cells at passage 31 were inoculated in 96-well plates containing BHE-free medium. Only one colony of cells, MEE/ 08, could be expanded into monolayer serially passagedin the BHE-free medium. In the tumorigenicity studies, MEE/C8 cells formed invasive tumors in syngenic hosts. Responseof MEE cells,MEE/C8 cells and human esophagealcancercell lines to growthfactors. A preliminary screen of the activity of single members of major growth factor families on MEE cells in BHE-free medium indicated that EGF and
Fig. 1. Effects of HBGF-1 or EGF on growth of mouse esophageal (MEE) cells (.), MEE/C8 cells (o), TE9 (.) and TE11 (0). The cells were cultured in serum-free medium in the presence of the indicated amounts of HBGF-1 (A) or EGF (B).
Heparin-Binding GrowthFactorsin EsophagealEpithelialCell
2$9
HBGF (HBGF-1, HBGF-2) were most active. HBGF-1 exhibited about a 2-fold higher specific activity than that of HBGF-2, therefore HBGF-1 was utilized for further study. Dose-responsesof MEE cells, MEE/C8 cells, and human esophageal cancer cell lines, TE9 and TEIl, to HBGF-1 and EGF are compared in Fig. 1. HBGF-1 or EGF significantly stimulated growth of MEE cells, but had no effect or a slight inhibitory effect on MEE/C8, TE9 or TE11cells. Partial characterization of the growthpromoting activity in extracts of the tumorigenic MEE/C8 cell line. Crude cell extracts from MEE cells, MEE/C8, TE9 and TE11 cells were tested for effect on DNA synthesis of quiescent, confluent MEE cells. Activity of crude extracts from confluent MEE/C8 cells, TE9 or TE11 on DNA synthesis of resting MEE cells was significant. Although the activity in extracts of growing, subconfluent MEE cells was significant, extracts of quiescent, confluent MEE cells exhibited no detectable effects. Because of the ease of scale-up, rapid growth rates, and constitutive expression of activity at high cell densities, we first performed a partial characterization of the activity in extracts of MEE/C8 cells. Crude cell extracts were applied to heparin-agarose, fractions were eluted at different NaCI concentrations, and tested for mitogenic activity on quiescent, confluent MEE cells (Fig. 2A). Fractions which eluted from the column between 0.7 and 1.0 M NaCI yielded significant growthpromoting activity. No activity was detected in the unretarded fractions or fractions which eluted between 1 to 2 M NaCI. The fractions from the MEE/C8 cells which were eluted between 0.6 M and 1.0 M NaCI from heparin-agarose chromatography was further purified by HPLC (Mckeehan and Crabb 1987).
Fig. 2. Partial purification of the growth-promoting activity in extracts of MEE/ C8 cells.
290
M. Katayama
and M. Kan
The major growth-promoting activity eluted between 36 and 38% acetonitrile (Fig. 2B). The partially purified activity from heparin-agarose was completely destroyed by heat (100°C, 10min) and trypsin (0.5 mg/ml trypsin, 37°C, 1 hr). Exposure to acid (pH 2.0 in 20% acetonitrile and 0.1% TFA) for 8 hr at 4°C resulted in only a 25% decline in activity. The biological activity was potentiated by the addition of heparin in the growth assay and masked by the presence of purified bovine brain HBGF-1. (A) Crude extracts of approximately 2 X 108 MEE/C8 cells was prepared and applied to a heparin-agarose column. The column was washed with 20 ml of 25 mM Tris-HC1(pH 7.1),1 mM EDTA and 0.5 M NaCI, and eluted stepwise with 3 ml of 25 mM Tris-HC1(pH 7.1) and 1 mM EDTA containing 0.6, 0.7, 0.8, 0.9, 1.0, 2.0 M NaCI (fractions 1-6). Eluents were dialyzed against water, concentrated by freeze-drying, and reconstituted with 0.5 ml of water. Aliquots (20 pl) of eluted fractions were added to quiescent, confluent MEE cells and DNA synthesis was determined. (B) The cell extract from approximately 1 X 109MEE/C8 cells was applied to heparin-agarose column and the material retained between 0.6 M and 1.0M NaCI was collected, dialyzed, reconstituted with 10% acetonitrile and 0.1% TFA and applied to a reversed-phase C4 silica column. Gradient elution was performed, and 1 ml fractions were collected. Aliquotes (10 p l) of each fraction was added to quiescent, confluent MEE cells and DNA synthesis was determined. References 1) Katayama, M., Akaishi, T., Nishihira, T., Kasai, M., Kan, M. & Yamane, I. (1986) Primary cultures and serial passages of normal human esophageal epithelial cells in a serum-free medium. In : Esophageal Cancer (Proceedings of the International Symposium on Cancer of the Esophagus, Sendai, October 27-29, 1985; current clinical practice series No. 40) edited by M. Kasai, Excerpta Medica, Amsterdam-Princeton-NJ, Geneva-Tokyo, pp. 31-34. 2) McKeehan, W.L. & Crabb, J.W. (1987) Isolation and characterization of different molecular and chromatographic forms of heparin-binding growth factor 1 from bovine brain. Anal. Biochem., 164, 563-569.
J. Exp.
Med., 1992,
168, 287-290
Heparin-Binding (Fibroblast) Growth Factors are Potential Autocrine Regulators of Esophageal Epithelial Cell Proliferation MASAFUMI KATAYAMA and MIKI0KAN* The Second Department of Surgery, Tohoku University School of Medicine,Sendai 980, and * W.Alton Jones Cell Science Center, Inc., Lake Placid, NY, USA KATAYAMA, M. and KAN, M. Heparin-Binding (Fibroblast) Growth Factors are Potential Autocrine Regulators of Esophageal Epithelial Cell Proliferation. Tohoku J. Exp. Med., 1992, 168 (2), 287-290 A serum-free culture system supplemented with neural tissue extract for normal human esophagus was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life-span, but retained morphology and other characteristics of normal epithelial cells. A screen for growth factors that stimulated growth of MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating, but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 cells had properties similar to HBGF-1 (acidic fibroblast growth factor). esophagus ; epithelial cells ; heparin-binding growth factors ; FGF ; autocrine mechanisms
We previously established a serum-free culture system for normal human esophageal epithelial cells (Katayama et al. 1986) and reported that crude bovine neural tissue extracts was an effective growth stimulant of normal human esophageal epithelial cells. The same culture system used for normal human esophageal cell culture was applied to establish an immortal non-malignant epithelial (MEE) cell line from normal esophageal epithelium from the BALB/c mouse. In the absence of neural extract, a malignant cloned cell line (MEE/C8) was selected from the MEE cells. Here we compare the growth kinetics of MEE, MEE/C8 and human esophageal cancer-derived cell lines and their response to EGF and heparin-binding growth factor-1(HBGF-1). We show that an HBGF-like activity may play a growth cycle-dependent autocrine role in non-malignant esoAddress
for reprints
: 1-1 Seiryomachi,
Aoba-ku, 287
Sendai
980, Japan.
288
M.Katayamaand M.Kan
phageal epithelial cell proliferation and a constitutive role in support of the malignant cell line. MATERIALS ANDMETHODS Methodsfor primaryculture of mouseesophagealepithelial(MEE) cells were a modificationof previouslydescribedprocedures(Katayamaet al. 1986). HeparinSepharosechromatography and reversed-phase high performance liquid chromatography (HPLC)werecarriedout as described(McKeehan and Crabb1987). RESULTS Establishment of mouse esophageal cell lines (MEE and MEE/C8). The previously reported serum-free culture system for normal human esophageal epithelial cells (Katayama et al. 1986) was applied to normal mouse esophageal epithelium. Rapidly growing colonies of cells (MEE) with apparent normal epithelial cell morphology were observed within several days and subcultured after 20 to 30 days. Bovine neural extract (BHE) was absolutely required for colony formation in the primary culture. MEE cells have been maintained for over one year (100 passages). To determine whether neural extract-independent cells could be selected from the MEE cultures, cells at passage 31 were inoculated in 96-well plates containing BHE-free medium. Only one colony of cells, MEE/ 08, could be expanded into monolayer serially passagedin the BHE-free medium. In the tumorigenicity studies, MEE/C8 cells formed invasive tumors in syngenic hosts. Responseof MEE cells,MEE/C8 cells and human esophagealcancercell lines to growthfactors. A preliminary screen of the activity of single members of major growth factor families on MEE cells in BHE-free medium indicated that EGF and
Fig. 1. Effects of HBGF-1 or EGF on growth of mouse esophageal (MEE) cells (.), MEE/C8 cells (o), TE9 (.) and TE11 (0). The cells were cultured in serum-free medium in the presence of the indicated amounts of HBGF-1 (A) or EGF (B).
Heparin-Binding GrowthFactorsin EsophagealEpithelialCell
2$9
HBGF (HBGF-1, HBGF-2) were most active. HBGF-1 exhibited about a 2-fold higher specific activity than that of HBGF-2, therefore HBGF-1 was utilized for further study. Dose-responsesof MEE cells, MEE/C8 cells, and human esophageal cancer cell lines, TE9 and TEIl, to HBGF-1 and EGF are compared in Fig. 1. HBGF-1 or EGF significantly stimulated growth of MEE cells, but had no effect or a slight inhibitory effect on MEE/C8, TE9 or TE11cells. Partial characterization of the growthpromoting activity in extracts of the tumorigenic MEE/C8 cell line. Crude cell extracts from MEE cells, MEE/C8, TE9 and TE11 cells were tested for effect on DNA synthesis of quiescent, confluent MEE cells. Activity of crude extracts from confluent MEE/C8 cells, TE9 or TE11 on DNA synthesis of resting MEE cells was significant. Although the activity in extracts of growing, subconfluent MEE cells was significant, extracts of quiescent, confluent MEE cells exhibited no detectable effects. Because of the ease of scale-up, rapid growth rates, and constitutive expression of activity at high cell densities, we first performed a partial characterization of the activity in extracts of MEE/C8 cells. Crude cell extracts were applied to heparin-agarose, fractions were eluted at different NaCI concentrations, and tested for mitogenic activity on quiescent, confluent MEE cells (Fig. 2A). Fractions which eluted from the column between 0.7 and 1.0 M NaCI yielded significant growthpromoting activity. No activity was detected in the unretarded fractions or fractions which eluted between 1 to 2 M NaCI. The fractions from the MEE/C8 cells which were eluted between 0.6 M and 1.0 M NaCI from heparin-agarose chromatography was further purified by HPLC (Mckeehan and Crabb 1987).
Fig. 2. Partial purification of the growth-promoting activity in extracts of MEE/ C8 cells.
290
M. Katayama
and M. Kan
The major growth-promoting activity eluted between 36 and 38% acetonitrile (Fig. 2B). The partially purified activity from heparin-agarose was completely destroyed by heat (100°C, 10min) and trypsin (0.5 mg/ml trypsin, 37°C, 1 hr). Exposure to acid (pH 2.0 in 20% acetonitrile and 0.1% TFA) for 8 hr at 4°C resulted in only a 25% decline in activity. The biological activity was potentiated by the addition of heparin in the growth assay and masked by the presence of purified bovine brain HBGF-1. (A) Crude extracts of approximately 2 X 108 MEE/C8 cells was prepared and applied to a heparin-agarose column. The column was washed with 20 ml of 25 mM Tris-HC1(pH 7.1),1 mM EDTA and 0.5 M NaCI, and eluted stepwise with 3 ml of 25 mM Tris-HC1(pH 7.1) and 1 mM EDTA containing 0.6, 0.7, 0.8, 0.9, 1.0, 2.0 M NaCI (fractions 1-6). Eluents were dialyzed against water, concentrated by freeze-drying, and reconstituted with 0.5 ml of water. Aliquots (20 pl) of eluted fractions were added to quiescent, confluent MEE cells and DNA synthesis was determined. (B) The cell extract from approximately 1 X 109MEE/C8 cells was applied to heparin-agarose column and the material retained between 0.6 M and 1.0M NaCI was collected, dialyzed, reconstituted with 10% acetonitrile and 0.1% TFA and applied to a reversed-phase C4 silica column. Gradient elution was performed, and 1 ml fractions were collected. Aliquotes (10 p l) of each fraction was added to quiescent, confluent MEE cells and DNA synthesis was determined. References 1) Katayama, M., Akaishi, T., Nishihira, T., Kasai, M., Kan, M. & Yamane, I. (1986) Primary cultures and serial passages of normal human esophageal epithelial cells in a serum-free medium. In : Esophageal Cancer (Proceedings of the International Symposium on Cancer of the Esophagus, Sendai, October 27-29, 1985; current clinical practice series No. 40) edited by M. Kasai, Excerpta Medica, Amsterdam-Princeton-NJ, Geneva-Tokyo, pp. 31-34. 2) McKeehan, W.L. & Crabb, J.W. (1987) Isolation and characterization of different molecular and chromatographic forms of heparin-binding growth factor 1 from bovine brain. Anal. Biochem., 164, 563-569.
