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Growth factor regulation of proliferation in primary cultures of small intestinal epithelium

  • Growth, Differentiation, And Senescence
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Summary

Although the intestinal epithelium is one of the most rapidly renewing tissues, little is known about the major growth factors that control the rate of cell replacement and migration. Recently, a primary culture model has been described for the developing rat small intestinal epithelium, which permits epithelial growth while maintaining interactions with associated stromal cells, thereby possessing several contextual advantages over established cell lines (Evans et al., 1992). We have used this model to begin to determine the factors that may be involved in controlling intestinal epithelial cell proliferation. Under the conditions examined, no single growth factor promoted exclusive proliferation of epithelial cells; stromal cell proliferation was also apparent. The most potent stimulators of epithelial proliferation were insulin and insulin-like growth factor 1 (IGF-1). These factors also appeared to inhibit migration of the epithelial cells. 5–10 ng/ml EGF, 5–20 ng/ml TGFα, and 10–20 ng/ml PDGF also slightly increased epithelial cell numbers. Cell proliferation was inhibited by 0.1 ng/ml TGFβ-1. In Dulbecco’s modified Eagle’s medium (DMEM) containing 0.25 IU/ml insulin, glucose levels of 2–3 g/liter permitted epithelial growth with limited expansion of the stromal cell population. Higher levels of glucose further stimulated the nonepithelial cell types. Transferrin was also a potent stimulator of both cell types.

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References

  • Aisen, P.; Listowsky, I. Iron transport and storage proteins. Annu. Rev. Biochem. 49:357–393; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Al-Nafussi, A. I.; Wright, N. A. The effect of epidermal growth factor (EGF) on cell proliferation of the gastric mucosa in rats. Virchows Arch. B Cell Pathol. 40:63–69; 1982.

    CAS  Google Scholar 

  • Anderson, G. J.; Walsh, M. D.; Powell, L. W., et al. Intestinal transferrin receptors and iron absorption in the neonatal rat. Br. J. Haematol. 77:229–239; 1991.

    PubMed  CAS  Google Scholar 

  • Avery, A.; Pareskava, C.; Hall, P., et al. TGFβ expression in the human colon: differential immunostaining along the crypt epithelium. Br. J. Cancer 68:137–139; 1993.

    PubMed  CAS  Google Scholar 

  • Baliga, B. S.; Borowitz, S. M.; Barnard, J. A. Effects of EGF and PMA on the growth and proliferation of IEC6 cells. Biochem. Int. 19:1045–1056; 1989.

    PubMed  CAS  Google Scholar 

  • Beach, R. L.; Popiela, H.; Festof, B. W. The identification of a neurotrophic factor as transferrin. FEBS Lett. 156:151–156; 1983.

    Article  PubMed  CAS  Google Scholar 

  • Bennet, K. L.; Plowman, G. D.; Buckley, S. D., et al. Regulation of amphiregulin mRNA by TGF-β in the human lung adenocarcinoma cell line A549. Growth Factors 7:207–213; 1992.

    Google Scholar 

  • Bissel, M. J.; Hall, G.; Parry, G. How does the extracellular matrix direct gene expression? J. Theor. Biol. 99:31–68; 1982.

    Article  Google Scholar 

  • Blay, J.; Brown, K. D. Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells. J. Cell. Physiol. 124:107–112; 1985.

    Article  PubMed  CAS  Google Scholar 

  • Boyd, D.; Florent, G.; Chakrabarty, S., et al. Alterations of the biological characteristics of a colon carcinoma cell line by colon derived substrata material. Cancer Res. 48:2825–2831; 1988.

    PubMed  CAS  Google Scholar 

  • Boyd, F. T.; Massague, J. Transforming growth factorβ inhibition of epithelial cell proliferation linked to the expression of a 53 kDa membrane receptor. J. Biol. Chem. 264:2272–2278; 1989.

    PubMed  CAS  Google Scholar 

  • Brasaemle; Attie. Microelisa reader quantification of fixed, stained, solubilised cells in microfibre dishes. Biotechniques 6:418; 1988.

    PubMed  CAS  Google Scholar 

  • Brattain, M.; Levine, A.; Chakrabarty, S., et al. Heterogeneity of human colon carcinomas. Cancer Metastasis Rev. 3:177–191; 1984.

    Article  PubMed  CAS  Google Scholar 

  • Cartlidge, S. A.; Elder, J. B. Transforming growth factorα and epidermal growth factor levels in normal human gastrointestinal mucosa. Br. J. Cancer 60:657–660; 1989.

    PubMed  CAS  Google Scholar 

  • Clemmons, D. R.; Shaw, D. S. Variables controlling somatomedin production by cultured human fibroblasts. J. Cell. Physiol. 115:127–; 1983.

    Google Scholar 

  • Conteas, C. N.; Adhip, P.; Majumdar, N. The effect of gastrin, epidermal growth factor and somatostatin on DNA synthesis in a small intestinal cell line (IEC6). Proc. Soc. Exp. Biol. Med. 184:307–311; 1987.

    PubMed  CAS  Google Scholar 

  • Conteas, C. N.; McMonov, B.; Luck, G. D. Modulation of epidermal growth factor induced cell proliferation and receptor binding by insulin in cultured intestinal epithelial cells. Biochem. Biophys. Res. Commun. 161:414–419; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Conteas, C. N.; Mujumdar, N. The effects of gastrin epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6). Proc. Soc. Exp. Biol. (NY) 184:307–311; 1986.

    Google Scholar 

  • Culouscou, J.-M.; Remade-Bonnet, M.; Conton, G. W., et al. Colorectum cell derived growth factor (CRDGF) is homologous to amphiregulin, a member of the epidermal growth factor family. Growth Factors 7:195–205; 1992.

    PubMed  CAS  Google Scholar 

  • DeLeon, D. D.; Wilson, D. M.; Powers, M., et al. Effects of insulin-like growth factors (IGF’s) and IGF receptor antibodies on the proliferation of human breast cancer cells. Growth Factors 6:327–336; 1992.

    CAS  Google Scholar 

  • Derynck, R. Transforming growth factor alpha. Cell 54:593–595; 1988.

    Article  PubMed  CAS  Google Scholar 

  • East, J. A.; Langdon, S. P.; Townsend, K. M. S., et al. The influence of type I collagen on the growth and differentiation of the human colonic adenocarcinoma cell line HT29 in vitro. Differentiation 50:179–188; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Evans, G. S.; Flint, N.; Somers, A. S., et al. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J. Cell Sci. 101:219–231; 1992.

    PubMed  Google Scholar 

  • Finch, P. W.; Rubin, J. S.; Miki, J., et al. Human KGF is FGF related with properties of a paracrine effector of epithelial cell growth. Science 245:752–755; 1990.

    Article  Google Scholar 

  • Flint, N.; Cove, F. L.; Evans, G. S. Heparin stimulates the proliferation of intestinal epithelial cells in primary culture. J. Cell Sci. 107:401–411; 1994.

    PubMed  CAS  Google Scholar 

  • Fonti, R.; Latella, G.; Bises, G., et al. Human colonocytes in primary culture: a model to study epithelial growth, metabolism and differentiation. Int. J. Colorectal Dis. 9:13–22; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Fukamachi, H. Proliferation and differentiation of foetal rat intestinal epithelial cells in primary serum-free culture. J. Cell Sci. 103:511–519; 1992.

    PubMed  Google Scholar 

  • Goodlad, R. A.; Wilson, T. J. G.; Lenton, W., et al. Intravenous but not intragastric urogastrone-EGF is trophic to the intestine of parentally fed rats. Gut 28:573–582; 1987.

    PubMed  CAS  Google Scholar 

  • Goyette, M. C.; Cho, K.; Fasching, C. L., et al. Progression of colorectal cancer is associated with multiple tumour suppression gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol. Cell. Biol. 12:1387–1395; 1992.

    PubMed  CAS  Google Scholar 

  • Heinz-Erian, P.; Kessler, U.; Funk, B., et al. Identification and in-situ localisation of the insulin-like growth factor-II/mannose-6-phosphate (IGFII/M6P) receptor in the rat gastrointestinal tract: comparison with the IGF-I receptor. Endocrinology 129:1769–1778; 1991.

    PubMed  CAS  Google Scholar 

  • Humble, R. E. Insulin-like growth factors I and II. Eur. J. Biochem. 190:445–462; 1990.

    Article  Google Scholar 

  • Ito, M.; Yasui, W.; Nakayama, H., et al. Reduced levels of transforming growth factorβ type 1 receptor in human gastric carcinoma. Jpn. J. Cancer Res. 83:86–92; 1992.

    PubMed  CAS  Google Scholar 

  • Kedinger, M.; Simon-Assmann, P. M.; Lacroix, B., et al. Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev. Biol. 113:474–483; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Kimchi, A.; Wang, X. F.; Weinberg, R. A., et al. Absence of TGFβ receptors and growth inhibition responses in retinoblastoma cells. Science 240:196–199; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Kitada, S.; Hays, E. F. Transferrin-like activity produced by murine malignant T lymphoma cell lines. Cancer Res. 45:3537–3540; 1985.

    PubMed  CAS  Google Scholar 

  • Koyama, S.; Podolsky, D. Differential expression of transforming growth factorα andβ in rat intestinal epithelial cells. J. Clin. Invest. 83:1768–1773; 1989.

    PubMed  CAS  Google Scholar 

  • Laburthe, M.; Rouyer-Fessard, C.; Gammeltoft, S. Receptors for insulin-like growth factors I and II in rat gastrointestinal epithelium. Am. J. Physiol. 254:G457-G462; 1988.

    PubMed  CAS  Google Scholar 

  • Lichtenberger, L. M.; Lechago, J. M.; Miller, T. A. Cell culture of human intestinal mucosae. Gastroenterology 77:1291–1300; 1979.

    PubMed  CAS  Google Scholar 

  • Malden, L. T.; Novak, U.; Burgess, A. W. Expression of transforming growth factor alpha messenger RNA in the normal and neoplastic gastrointestinal tract. Int. J. Cancer 43:380–384; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Malo, C.; Menard, D. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology 83:28–35; 1982.

    PubMed  CAS  Google Scholar 

  • Manning, A. M.; Williams, A. C.; Game, S. M., et al. Differential sensitivity of human colonic adenoma and carcinoma cells to transforming growth factor beta: conversion of an adenoma cell line to a tumorigenic phenotype is accompanied by a reduced response to the inhibitory effects of TGF-beta. Oncogene 6:1471–1476; 1991.

    PubMed  CAS  Google Scholar 

  • May, W. S.; Cuatrecasas, P. Transferrin receptor: its biological significance. J. Memb. Biol. 88:205–215; 1985.

    Article  CAS  Google Scholar 

  • Migdalska, A.; Molineux, G.; Demuynck, H., et al. Growth inhibitory effects of transforming growth factor-β1 in vivo. Growth Factors 4:239–245; 1991.

    PubMed  CAS  Google Scholar 

  • Minniti, C. P.; Maggi, M.; Helman, L. J. Suramin inhibits the growth of human rhabdomyosarcoma by interrupting the insulin-like growth factor II autocrine growth loop. Cancer Res. 52:1830–1835; 1992.

    PubMed  CAS  Google Scholar 

  • Moyer, M. P. Culture of human gastrointestinal epithelial cells. Proc. Soc. Exp. Biol. Med. 174:12–15; 1983.

    PubMed  CAS  Google Scholar 

  • Neilson, F. C. The molecular and cellular biology of insulin-like growth factor II. Prog. Growth Factor Res. 4:257–290; 1992.

    Article  Google Scholar 

  • Park, J. H.; McCusker, R. H.; Van Derhoof, J. A., et al. Secretion of insulin like growth factor II (IGFII) and IGF binding protein 2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation. Endocrinology 131:1359–1368; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Park, J. H.; Vanderhoof, J. A.; Blackwood, D., et al. Characterisation of type I and type II insulin-like growth factor receptor in an intestinal epithelial cell line. Endocrinology 126:2998–3005; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Pelton, R. W.; Saxena, B.; Jones, M., et al. Immunohistochemical localisation of TGFβ-1, TGFβ-2 and TGFβ-3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development J. Cell Biol. 115:1091–1101; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Perez-Tomas, R.; Cullere, X.; Diaz, C. Immunohistochemical localisation of transforming growth factorα in the developing rat colon. Gastroenterology 104:789–795; 1993.

    PubMed  CAS  Google Scholar 

  • Pommier, G. J.; Garrouste, F. L.; El Atiq, F., et al. Potential autocrine role of insulin-like growth factor II during suramin induced differentiation of HT29-D4 human colonic adenocarcinoma line. Cancer Res. 52:3182–3188; 1992.

    PubMed  CAS  Google Scholar 

  • Potten, C. S.; Loeffler, M. Stem cells: attributes, cycles, spirals, uncertainties and pitfalls: lessons for and from the crypt. Development 110:1001–1019; 1990.

    PubMed  CAS  Google Scholar 

  • Potten, C. S.; Booth, C.; Chadwick, C. A., et al. A potent stimulator of small intestinal cell proliferation extracted by simple diffusion from intact irradiated intestine: in vitro studies. Growth Factors 10:53–61; 1993.

    Google Scholar 

  • Potten, C. S.; Chadwick, C. A. A potent stimulator of small intestinal cell proliferation extracted by simple diffusion from intact irradiated intestine: in vivo studies. Growth Factors 10:63–75; 1993.

    Google Scholar 

  • Quaroni, A.; May, R. J. Establishment and characterisation of intestinal epithelial cell cultures. Methods Cell Biol. 21B:403–427; 1980.

    Article  PubMed  CAS  Google Scholar 

  • Ross, R.; Raines, E. W.; Bowen-Pope, D. F. The biology of platelet-derived growth factor. Cell 46:155–169; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Rouyer-Fessard, C.; Gammeltoft, S.; Laburthe, M. Expression of two types of receptor for insulin-like growth factors in human colonic epithelium. Gastroenterology 98:703–707; 1990.

    PubMed  CAS  Google Scholar 

  • Sato, Y.; Rifkin, D. B. Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis and DNA synthesis. J. Cell Biol. 107:1199–1205; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Shoyab, M.; McDonald, V. L.; Bradley, J. G., et al. Amphiregulin: a bifunctional growth modulating glycoprotein produced by phorbol-12-myristate 13-acetate treated human breast adenocarcinoma cell line MCF-7. PNAS 85:6528–6532; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Shoyab, M.; Plowman, G. D.; McDonald, V. L., et al. Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science 243:1074–1076; 1989.

    Article  PubMed  CAS  Google Scholar 

  • Simon-Assmann, P.; Bouziges, F.; Arnold, C., et al. Epithelial-mesenchyme interactions in the production of basement membrane components in the gut. Development 102:339–347; 1988.

    PubMed  CAS  Google Scholar 

  • Sutherland, R.; Delia, D.; Schneider, C., et al. Ubiquitous cell-surface glycoprotein on tumour cells is proliferation associated receptor for transferrin. P. N. A. S. 78:4515–4519; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Tait, I. S.; Evans, G. S.; Kedinger, M., et al. Progressive morphogenesis in vivo after transplantation of cultured small bowel epithelium. Cell Transplantation 3:33–40; 1994a.

    PubMed  CAS  Google Scholar 

  • Tait, I. S.; Flint, N.; Campbell, F. C., et al. Generation of neomucosa in vivo by transplantation of dissociated rat postnatal small intestinal epithelium. Differentiation 56:91–100; 1994b.

    PubMed  CAS  Google Scholar 

  • Tricoli, J. V.; Rall, L. B.; Kjarakousis, C. P., et al. Enhanced levels of insulin-like growth factor messenger RNA in human colon carcinomas and liposarcomas. Cancer Res. 46:6169–6173; 1986.

    PubMed  CAS  Google Scholar 

  • Wakefield, L. M.; Thompson, N. L.; Flanders, K. C., et al. Transforming growth factorβ: multifunctional regulator of cell growth and phenotype. Ann. NY Acad. Sci. 551:290–298; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Walling, J. M.; Blackmore, M.; Hickman, J. A., et al. Role of the extracellular matrix on the growth and differentiated phenotype of murine colonic adenocarcinoma cells in vitro. Int. J. Cancer 47:776–788; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Whitehead, R. H.; Brown, A.; Bhathal, P. S. A method for the isolation and culture of human colonic crypts in collagen gels. In Vitro Cell. Dev. Biol. 23:436–442; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Whitehead, R. H.; Nice, E. C.; Lloyd, C. J., et al. Detection of colonic growth factors using a human colonic carcinoma cell LIM1215. Int. J. Cancer 46:858–863; 1990.

    Article  CAS  Google Scholar 

  • Wright, N. A.; Alison, M. R. (1984). Kinetic parameters in the gastrointestinal mucosa. The biology of epithelial cell populations. Vol. 2 Clarendon Press, Oxford, UK.

    Google Scholar 

  • Wright, N. A.; Pike, C.; Elia, G. Induction of a novel epidermal growth factor secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature 343:82–85; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Young, G. P.; Taranto, T. M.; Jonas, H. A., et al. Insulin-like growth factors and the developing and mature rat small intestine: receptors and biological actions. Digestion 46(2):240–252; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Yoshinouchi, M.; Baserga, R. The role of the IGF-1 receptor in the stimulation of cells by short pulses of growth factors. Cell Proliferation 26:139–146; 1993.

    PubMed  CAS  Google Scholar 

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  1. Cancer Research Campaign, Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Wilmslow Road, M20 9BX, Manchester, United Kingdom

    C. Booth, G. S. Evans & C. S. Potten

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  1. C. Booth
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  2. G. S. Evans
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  3. C. S. Potten
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Booth, C., Evans, G.S. & Potten, C.S. Growth factor regulation of proliferation in primary cultures of small intestinal epithelium. In Vitro Cell Dev Biol - Animal 31, 234–243 (1995). https://doi.org/10.1007/BF02639439

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