Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Dec 1;115(5):1397–1408. doi: 10.1083/jcb.115.5.1397

Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells able to form functional bile canaliculi in vitro

PMCID: PMC2289240  PMID: 1955480

Abstract

A large number of hepatoma cell lines has been used to study expression and regulation of liver-specific function. However these cells, even the most differentiated, are morphologically far from hepatocytes. In no case is the typical hepatocyte cell polarity well maintained. Cell hybridization has been used as a potential means for turning on specific genes. From hybrids between well differentiated Fao rat hepatoma cells and WI 38 human fibroblasts, we have attempted to isolate segregated cells that are highly differentiated and polarized. Such cells, detected in aged cultures of only one hybrid (WIF12), were isolated by subcloning. One subclone, WIF12-1 was analyzed. Expression of liver-specific functions extinguished in the original hybrid is restored in all WIF12-1 cells at a very high level, similar to that of hepatocytes and 5-30 times higher that that of parental cells. Moreover human genes coding for liver-specific proteins (albumin, fibrinogen, and alcohol dehydrogenase) are actively expressed. WIF12-1 cells have acquired a polarized phenotype as attested by the presence of bile canaliculi between adjacent cells and by the asymmetrical localization of apical (Mg(2+)-ATPase, gamma-glutamyl transpeptidase) and basolateral membrane markers. The bile canaliculi formed are dynamic and functional structures, characterized by long periods of expansion followed by rapid contractions. The ability to polarize is a general and permanent property of WIF12-1 cells. These cells appear to constitute a valid model for the in vitro study of hepatocyte cell polarity, membrane domain formation and mechanisms of membrane protein sorting.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Auger G., Blanot D., van Heijenoort J., Nadal C., Gournay M. F., Winchenne J. J., Boffa G. A., Lambin P., Maes P., Tartar A. Purification and partial characterization of a hepatocyte antiproliferative glycopeptide. J Cell Biochem. 1989 Aug;40(4):439–451. doi: 10.1002/jcb.240400405. [DOI] [PubMed] [Google Scholar]
  2. Bartles J. R., Feracci H. M., Stieger B., Hubbard A. L. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation. J Cell Biol. 1987 Sep;105(3):1241–1251. doi: 10.1083/jcb.105.3.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartles J. R., Hubbard A. L. Plasma membrane protein sorting in epithelial cells: do secretory pathways hold the key? Trends Biochem Sci. 1988 May;13(5):181–184. doi: 10.1016/0968-0004(88)90147-8. [DOI] [PubMed] [Google Scholar]
  4. Bertolotti R. A selective system for hepatoma cells producing gluconeogenic enzymes. Somatic Cell Genet. 1977 Jul;3(4):365–380. doi: 10.1007/BF01542966. [DOI] [PubMed] [Google Scholar]
  5. Buys C. H., Aanstoot G. H., Nienhaus A. J. The Giemsa-11 technique for species-specific chromosome differentiation. A simple stain modification leading to dependable direct and sequential staining procedures. Histochemistry. 1984;81(5):465–468. doi: 10.1007/BF00489751. [DOI] [PubMed] [Google Scholar]
  6. Carlson J., Stenflo J. The biosynthesis of rat alpha 1-antitrypsin. J Biol Chem. 1982 Nov 10;257(21):12987–12994. [PubMed] [Google Scholar]
  7. Cassio D., Hassoux R., Dupiers M., Uriel J., Weiss M. C. Coordinate secretion of mouse alphafetoprotein, mouse albumin and rat albumin by mouse hepatoma-rat hepatoma hybrid cells. J Cell Physiol. 1980 Sep;104(3):295–308. doi: 10.1002/jcp.1041040304. [DOI] [PubMed] [Google Scholar]
  8. Cassio D., Rogier E., Feldmann G., Weiss M. C. Plasma-protein production by rat hepatoma cells in culture, their variants and revertants. Differentiation. 1986;30(3):220–228. doi: 10.1111/j.1432-0436.1986.tb00784.x. [DOI] [PubMed] [Google Scholar]
  9. Cassio D., Weiss M. C., Ott M. O., Sala-Trepat J. M., Friès J., Erdos T. Expression of the albumin gene in rat hepatoma cells and their dedifferentiated variants. Cell. 1981 Dec;27(2 Pt 1):351–358. doi: 10.1016/0092-8674(81)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Chin A. C., Fournier R. E. Tse-2: a trans-dominant extinguisher of albumin gene expression in hepatoma hybrid cells. Mol Cell Biol. 1989 Sep;9(9):3736–3743. doi: 10.1128/mcb.9.9.3736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chiu J. H., Hu C. P., Lui W. Y., Lo S. C., Chang C. M. The formation of bile canaliculi in human hepatoma cell lines. Hepatology. 1990 May;11(5):834–842. doi: 10.1002/hep.1840110519. [DOI] [PubMed] [Google Scholar]
  12. Clayton D. F., Weiss M., Darnell J. E., Jr Liver-specific RNA metabolism in hepatoma cells: variations in transcription rates and mRNA levels. Mol Cell Biol. 1985 Oct;5(10):2633–2641. doi: 10.1128/mcb.5.10.2633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coon H. G., Weiss M. C. A quantitative comparison of formation of spontaneous and virus-produced viable hybrids. Proc Natl Acad Sci U S A. 1969 Mar;62(3):852–859. doi: 10.1073/pnas.62.3.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Coquil J. F., Berthon B., Chomiki N., Combettes L., Jourdon P., Schteingart C., Erlinger S., Claret M. Effects of taurolithocholate, a Ca2(+)-mobilizing agent, on cell Ca2(+) in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line. Biochem J. 1991 Jan 1;273(Pt 1):153–160. doi: 10.1042/bj2730153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Deschatrette J., Moore E. E., Dubois M., Weiss M. C. Dedifferentiated variants of a rat hepatoma:reversion analysis. Cell. 1980 Apr;19(4):1043–1051. doi: 10.1016/0092-8674(80)90095-1. [DOI] [PubMed] [Google Scholar]
  16. Gallimore P. H., Richardson C. R. An improved banding technique exemplified in the karyotype analysis of two strains of rat. Chromosoma. 1973;41(3):259–263. doi: 10.1007/BF00344020. [DOI] [PubMed] [Google Scholar]
  17. Gautam A., Ng O. C., Boyer J. L. Isolated rat hepatocyte couplets in short-term culture: structural characteristics and plasma membrane reorganization. Hepatology. 1987 Mar-Apr;7(2):216–223. doi: 10.1002/hep.1840070203. [DOI] [PubMed] [Google Scholar]
  18. Gauthier F., Genell S., Mouray H., Ohlsson K. Interactions in vitro and in vivo between rat serum protease inhibitors and anodal and cathodal rat trypsin and chymotrypsin. Biochim Biophys Acta. 1979 Jan 12;566(1):200–210. doi: 10.1016/0005-2744(79)90262-6. [DOI] [PubMed] [Google Scholar]
  19. Gebhardt R. Disappearance of visible bile canaliculi caused by vinblastine in primary cultures of rat hepatocytes. Exp Cell Res. 1983 Mar;144(1):218–223. doi: 10.1016/0014-4827(83)90457-3. [DOI] [PubMed] [Google Scholar]
  20. Graessmann M., Graessmann A. Microinjection of tissue culture cells. Methods Enzymol. 1983;101:482–492. doi: 10.1016/0076-6879(83)01033-2. [DOI] [PubMed] [Google Scholar]
  21. Graf J., Gautam A., Boyer J. L. Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6516–6520. doi: 10.1073/pnas.81.20.6516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hubbard A. L., Bartles J. R., Braiterman L. T. Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies. J Cell Biol. 1985 Apr;100(4):1115–1125. doi: 10.1083/jcb.100.4.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Johnson P. F. Transcriptional activators in hepatocytes. Cell Growth Differ. 1990 Jan;1(1):47–52. [PubMed] [Google Scholar]
  24. Jung W., Gebhardt R., Mecke D. Alterations in activity and ultrastructural localization of several phosphatases on the surface of adult rat hepatocytes in primary monolayer culture. Eur J Cell Biol. 1982 Jun;27(2):230–241. [PubMed] [Google Scholar]
  25. Kao F. T., Puck T. T. Genetics of somatic mammalian cells: demonstration of a human esterase activator gene linked to the adeB gene. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3273–3277. doi: 10.1073/pnas.69.11.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kawahara H., French S. W. Role of cytoskeleton in canalicular contraction in cultured differentiated hepatocytes. Am J Pathol. 1990 Mar;136(3):521–532. [PMC free article] [PubMed] [Google Scholar]
  27. Kielty C. M., Povey S., Hopkinson D. A. Regulation of expression of liver-specific enzymes. III. Further analysis of a series of rat hepatoma X human somatic cell hybrids. Ann Hum Genet. 1982 Oct;46(Pt 4):307–327. doi: 10.1111/j.1469-1809.1982.tb01582.x. [DOI] [PubMed] [Google Scholar]
  28. Killary A. M., Fournier R. E. A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells. Cell. 1984 Sep;38(2):523–534. doi: 10.1016/0092-8674(84)90507-5. [DOI] [PubMed] [Google Scholar]
  29. Klee W. A., Nirenberg M. A neuroblastoma times glioma hybrid cell line with morphine receptors. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3474–3477. doi: 10.1073/pnas.71.9.3474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laurell C. B. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem. 1966 Apr;15(1):45–52. doi: 10.1016/0003-2697(66)90246-6. [DOI] [PubMed] [Google Scholar]
  31. Malawista S. E., Weiss M. C. Expression of differentiated functions in hepatoma cell hybrids: high frequency of induction of mouse albumin production in rat hepatoma-mouse lymphoblast hybrids. Proc Natl Acad Sci U S A. 1974 Mar;71(3):927–931. doi: 10.1073/pnas.71.3.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mancini G., Carbonara A. O., Heremans J. F. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry. 1965 Sep;2(3):235–254. doi: 10.1016/0019-2791(65)90004-2. [DOI] [PubMed] [Google Scholar]
  33. Maurice M., Durand-Schneider A. M., Garbarz M., Feldmann G. Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies. Eur J Cell Biol. 1985 Nov;39(1):122–129. [PubMed] [Google Scholar]
  34. Maurice M., Rogier E., Cassio D., Feldmann G. Formation of plasma membrane domains in rat hepatocytes and hepatoma cell lines in culture. J Cell Sci. 1988 May;90(Pt 1):79–92. doi: 10.1242/jcs.90.1.79. [DOI] [PubMed] [Google Scholar]
  35. McMillan P. N., Hixson D. C., Hevey K. A., Naik S., Jauregui H. O. Hepatocyte cell surface polarity as demonstrated by lectin binding. J Histochem Cytochem. 1988 Dec;36(12):1561–1571. doi: 10.1177/36.12.2848070. [DOI] [PubMed] [Google Scholar]
  36. Miyairi M., Oshio C., Watanabe S., Smith C. R., Yousef I. M., Phillips M. J. Taurocholate accelerates bile canalicular contractions in isolated rat hepatocytes. Gastroenterology. 1984 Oct;87(4):788–792. [PubMed] [Google Scholar]
  37. Mével-Ninio M., Weiss M. C. Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids. J Cell Biol. 1981 Aug;90(2):339–350. doi: 10.1083/jcb.90.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oshio C., Phillips M. J. Contractility of bile canaliculi: implications for liver function. Science. 1981 May 29;212(4498):1041–1042. doi: 10.1126/science.7015506. [DOI] [PubMed] [Google Scholar]
  39. PITOT H. C., PERAINO C., MORSE P. A., Jr, POTTER V. R. HEPATOMAS IN TISSUE CULTURE COMPARED WITH ADAPTING LIVER IN VIVO. Natl Cancer Inst Monogr. 1964 Apr;13:229–245. [PubMed] [Google Scholar]
  40. Pepperkok R., Zanetti M., King R., Delia D., Ansorge W., Philipson L., Schneider C. Automatic microinjection system facilitates detection of growth inhibitory mRNA. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6748–6752. doi: 10.1073/pnas.85.18.6748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Petit C., Levilliers J., Ott M. O., Weiss M. C. Tissue-specific expression of the rat albumin gene: genetic control of its extinction in microcell hybrids. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2561–2565. doi: 10.1073/pnas.83.8.2561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Polokoff M. A., Everson G. T. Hepatocyte-hepatoma cell hybrids. Characterization and demonstration of bile acid synthesis. J Biol Chem. 1986 Mar 25;261(9):4085–4089. [PubMed] [Google Scholar]
  43. REUBER M. D. A transplantable bile-secreting hepatocellular carcinoma in the rat. J Natl Cancer Inst. 1961 Apr;26:891–899. [PubMed] [Google Scholar]
  44. Rodriguez-Boulan E., Nelson W. J. Morphogenesis of the polarized epithelial cell phenotype. Science. 1989 Aug 18;245(4919):718–725. doi: 10.1126/science.2672330. [DOI] [PubMed] [Google Scholar]
  45. Rogier E., Cassio D., Weiss M. C., Feldmann G. An ultrastructural study of rat hepatoma cells in culture, their variants and revertants. Differentiation. 1986;30(3):229–236. doi: 10.1111/j.1432-0436.1986.tb00785.x. [DOI] [PubMed] [Google Scholar]
  46. Rutenburg A. M., Kim H., Fischbein J. W., Hanker J. S., Wasserkrug H. L., Seligman A. M. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J Histochem Cytochem. 1969 Aug;17(8):517–526. doi: 10.1177/17.8.517. [DOI] [PubMed] [Google Scholar]
  47. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  48. Sellem C. H., Weiss M. C., Cassio D. Activation of a silent gene is accompanied by its demethylation. J Mol Biol. 1985 Feb 5;181(3):363–371. doi: 10.1016/0022-2836(85)90225-6. [DOI] [PubMed] [Google Scholar]
  49. Sellem C., Cassio D., Weiss M. C. No extinction of tyrosine aminotransferase inducibility in rat hepatoma-human fibroblast hybrids containing the human X chromosome. Cytogenet Cell Genet. 1981;30(1):47–49. doi: 10.1159/000131587. [DOI] [PubMed] [Google Scholar]
  50. Simons K., Fuller S. D. Cell surface polarity in epithelia. Annu Rev Cell Biol. 1985;1:243–288. doi: 10.1146/annurev.cb.01.110185.001331. [DOI] [PubMed] [Google Scholar]
  51. Weiss M. C., Cassio D., Sellem C. H. Cell differentiation: contributions of somatic cell genetics. Cancer Surv. 1988;7(2):295–302. [PubMed] [Google Scholar]
  52. Worton R. G., Duff C. Karyotyping. Methods Enzymol. 1979;58:322–344. doi: 10.1016/s0076-6879(79)58148-8. [DOI] [PubMed] [Google Scholar]
  53. Zweibaum A., Pinto M., Chevalier G., Dussaulx E., Triadou N., Lacroix B., Haffen K., Brun J. L., Rousset M. Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose. J Cell Physiol. 1985 Jan;122(1):21–29. doi: 10.1002/jcp.1041220105. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES