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Preneoplastic rat liver cells in vitro: Slow progression without promoters, hormones, or growth factors

  • Original Papers
  • Experimental Oncology
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Summary

In order to determine early changes in liver cells during carcinogenesis and to compare them with normal or neoplastic hepatocytes, an experimental model was established which allowed enrichment of this population at early stages of carcinogenesis and provided sufficient viable material for biochemical and cytogenetic analysis. This paper describes a method that allows in vitro selection and propagation of hepatocytes after in vivo initiation by alkylating agents, without the use of hormones, growth factors, or promoters which might affect their progression. From 6 different rat livers (5 initiated by continuous diethylnitrosamine feeding, 1 by a single exposure to N-methyl-N-nitrosourea) we established slow-growing lines, each of which had its own typical characteristics of growth behavior, morphology, and chromosome number. One of these lines (CL 38) transformed spontaneously after 8 weeks in primary culture, with an abrupt change to typical tumor cell behavior such as focal growth, anchorage independence, cloning ability in soft agar, and tumorigenicity in nude mice and newborn rats. In none of the other lines (now in culture for 11–15 months) has a similar abrupt change yet been observed, but all of them show a steady, albeit slow progression towards the properties of neoplastic liver cells, together with a reduction in chromosoome number.

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References

  • Armato U, Romano F, Andreis PG, Paccagnella L, Marchesini C (1986) Growth stimulation and apoptosis induced in cultures of neonatal rat liver cells by repeated exposures to EGF/urogastrone with or without associated pancreatic hormones. Cell Tissue Res 245:471–480

    Google Scholar 

  • Bannasch P, Hacker HJ, Klimek F, Mayer D (1984) Hepatcellular glucogenesis and related pattern of enzyme changes during hepatocarcinogenesis. Adv Enzyme Regul 22:97–121

    Google Scholar 

  • Cifone MA (1981) Correlation between bizarre colony morphology and metastatic potential of tumor cells. Exp Cell Res 131:435–441

    Google Scholar 

  • Dougherty KK, Spilman SD, Green CE, Steward AR, Byard JL (1980) Primary cultures of adult mouse and rat hepatocytes for studying the metabolism of foreign chemicals. Biochem Pharmacol 29:2117–2124

    Google Scholar 

  • Emmelot P, Scherer E (1980) The first relevant cell stage in rat liver carcinogenesis. A quantitative approach. Biochim Biophys Acta 605:247–304

    Google Scholar 

  • Evarts RP, Marsden E, Hanna P, Wirth PJ, Thorgeirsson SS (1984) Isolation of preneoplastic liver cells by centrifugal elutriation and binding to asialofetuin. Cancer Res 44:5718–5724

    Google Scholar 

  • Farber E (1980) The sequential analysis of liver cancer induction. Biochem Biophys Acta 605:149–166

    Google Scholar 

  • Farber E (1984) Chemical carcinogenesis: a current biological perspective. Carcinogenesis 5:1–5

    Google Scholar 

  • Grabske RJ (1978) Separating cell populations by elutriation. Fractions No. 1 (Beckman Instruments): 1–8

  • Hanigan HM, Pitot HC (1982) Isolation of GGT positive hepatocytes during the early stages of hepatocarcinogenesis. Carcinogenesis 3:1349–1354

    Google Scholar 

  • Holecek B, Rabes HM (1986) Cytogenetic analysis of normal and diethylnitrosamine-initiated preneoplastic hepatocytes. J Cancer Res Clin Oncol 111: S 95

  • Isom HC, Secott T, Georgoff I, Woodworth C, Mummaw J (1985) Maintenance of differentiated rat hepatocytes in primary culture. Proc Natl Acad Sci USA 82:3252–3256

    Google Scholar 

  • Kaufmann WK, Tsao MS, Novicki DL (1986) In vitro colonization ability appears soon after innitiation of hepatocarcinogenesis in the rat. Carcinogenesis 7:669–671

    Google Scholar 

  • Kerler R, Rabes HM (1984) Separation of subpopulations from the Zajdela hepatoma by elutriation centrifugation. Eur J Cell Biol (Suppl) 5 33:18

    Google Scholar 

  • Kerler R, Rabes HM (1986) In vitro propagation of preneoplastic hepatocytes initiated in vivo by DEN. J Cancer Res Clin Oncol 111:S 46

    Google Scholar 

  • LaBrecque DR, Bachur NR (1982) Hepatic stimulator substance: physiochemical characteristics and specificity. Am J Physiol 242:G281-G288

    Google Scholar 

  • LaBrecque DR, Wilson M, Fogerty S (1984) Stimulation of HTC hepatoma cell growth in vitro by hepatic stimulator substance (HSS). Exp Cell Res 150:419–429

    Google Scholar 

  • Luetteke NC, Michalopoulos GK (1985) Partial purification and characterization of a hepatocyte growth factor, produced by rat hepatocellular hepatoma cells. Cancer Res 45:6331–6337

    Google Scholar 

  • Lewis JG, Swenberg JA (1980) Differential repair of O6-methylguanine in DNA of rat hepatocytes and non parenchymal cells. Nature 288:185–187

    Google Scholar 

  • Lin KH, Leach MF, Winters AL, Lindahl R (1986) Characteristics and ALDH activity of four rat hepatoma cell lines produced by DEN-PB treatment. In Vitro Cell Dev Biol 22:263–272

    Google Scholar 

  • McMillan TI, Rao J, Hart IR (1986) Enhancement of experimental metastasis by pretreatment of tumor cells with hydroxyurea. Int J Cancer 38:61–65

    Google Scholar 

  • Miyazaki M, Wahid S, Miyano K, Yabe T, Sato J (1983) Expression of GGT in cultures of spontaneously and chemically transformed rat liver cells. Int J Cancer 32:373–377

    Google Scholar 

  • Mori H, Mu B, Williams GM (1982) Isolation and enrichment of cells resistant to iron accumulation from carcinogen-treated rat liver altered foo. Exp Mol Pathol 37:101–110

    Google Scholar 

  • Pierschbacher MD, Ruoslahti E (1984) Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309:30–33

    Google Scholar 

  • Quastler H, Sherman FG (1959) Cell population kinetics in the intestinal epithelium of the mouse. Exp Cell Res 17:420–438

    Google Scholar 

  • Rabes HM (1983) Development and growth of early preneoplastic lesions induced in the liver by chemical carcinogens. J Cancer Res Clin Oncol 196:85–92

    Google Scholar 

  • Rabes HM, Szymkowiak W (1979) Cell kinetics of hepatocytes during the preneoplastic period of DEN-induced liver carcinogenesis. Cancer Res 39:1298–1304

    Google Scholar 

  • Rabes HM, Scholze P, Jantsch B (1972) Growth kinetics of diethylnitrosamine-induced, enzyme-deficient preneoplastic liver cell populations in vivo and in vitro. Cancer Res 32:2577–2586

    Google Scholar 

  • Rabes HM, Bücher T, Hartmann A, Linke I, Dünnwald M (1982) Clonal growth of carcinogen-induced enzyme-deficient preneoplastic cell populations in mouse liver. Cancer Res 42:3220–3227

    Google Scholar 

  • Rabes HM, Müller L, Hartmann A, Kerler R, Schuster C (1986) Cell cycle-dependent initiation of ATPase-deficient populations in adult rat liver by a single dose of MNU. Cancer Res 46:645–650

    Google Scholar 

  • Rutenberg AM, Kim H, Fischbein J, Hauker JS, Wasserkrug HC, Seligman R (1968) Histochemical and ultrastructural demonstration of GGT activity. J Histochem Cytochem 17:517–526

    Google Scholar 

  • Sarafoff M, Rabes HM, Dörmer P (1986) Correlations between ploidy and initiation probability determined by DNA cytophotometry in individual altered hepatic foci. Carcinogenesis 7:1191–1196

    Google Scholar 

  • Schwarze PE, Pettersen EO, Seglen PO (1986a) Characterization of hepatocytes from carcinogen-treated rats by two-parametric flow cytometry. Carcinogenesis 7:171–173

    Google Scholar 

  • Schwarze PE, Pettersen EO, Tolleshaug H, Seglen PO (1986b) Isolation of carcinogen-induced diploid rat hepatocytes by centrifugal elutriation. Cancer Res 46:4732–4737

    Google Scholar 

  • Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83

    Google Scholar 

  • Sporn MB, Todaro GJ (1980) Autocrine secretion and malignant transformation of cells. New Engl J Med 303:878–880

    Google Scholar 

  • Swenberg JA, Bedell MA, Billings KC, Umbenhauer DR, Pegg AE (1982) Cell-specific differences in O6-alkylguanine DNA repair activity during continuous exposure to carcinogen. Proc Natl Acad Sci USA 79:5499–5502

    Google Scholar 

  • Tsao MS, Grisham JW, Nelson KG, Smith JD (1985) Phenotypic and karyothypic changes induced in cultured rat hepatic epithelial cells that express the oval cell phenotype by exposure to MNNG. Am J Pathol 118:306–315

    Google Scholar 

  • Wachstein M, Meisel B (1959) Enzyme histochemistry of ethionine induced liver cirrhosis and hepatoma. Histochem Cytochem 7:189–201

    Google Scholar 

  • Wanson JC, Bernaert D, Penasse W, Mosselmans R, Bannasch P (1980) Separation of distinct subpopulations by elutriation of liver cells following exposure of rats to NNM. Cancer Res 40:459–471

    Google Scholar 

  • Williams GM (1976) Primary and long-term culture of adult rat liver epithelial cells. In: Prescott P (ed) Methods in cell biology, vol XIV. Academic Press, New York San Francisco London pp 357–364

    Google Scholar 

  • Williams GM (1980) Pathogenesis of rat liver cancer caused by chemical carcinogens. Biochim Biophys Acta 605:167–189

    Google Scholar 

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Authors and Affiliations

  1. Institute of Pathology, University of Munich, Thalkirchnerstrasse 36, D-8000, Munich 2, Federal Republic of Germany

    R. Kerler & H. M. Rabes

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  1. R. Kerler
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  2. H. M. Rabes
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Kerler, R., Rabes, H.M. Preneoplastic rat liver cells in vitro: Slow progression without promoters, hormones, or growth factors. J Cancer Res Clin Oncol 114, 113–123 (1988). https://doi.org/10.1007/BF00417823

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  • DOI: https://doi.org/10.1007/BF00417823

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