Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1979 Sep 1;82(3):644–653. doi: 10.1083/jcb.82.3.644

Endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture

PMCID: PMC2110496  PMID: 511930

Abstract

Variations of endocytic and of lysosomal functions during the cell cycle have been investigated in synchronized hepatoma cells (derived from Morris hepatoma 7288c) by following the cellular uptake of horseradish peroxidase, dextran (mol wt. 70,000), and chloroquine. Cell fractionation and cytochemistry show that in asynchronously growing cells exposed for 1 h to 5 mg/ml peroxidase, the bulk of the enzyme taken up by the cells is found in phagosomes. By using the same experimental system with synchronized HTC cells, large variations of endocytosis are observed during the cell cycle. Peroxidase uptake is lowest during mitosis, increases 5--10 times during G1 phase, reaches a plateau, and finally decreases at the end of S phase and during G2 phase. A similar evolution is observed for the uptake of dextran (0.5 or 1 mg/ml), but it is likely that a significant part of the polysaccharide is still associated with the pericellular surface after 1 h. Moreover, dextran is transferred more slowly than peroxidase to lysosomes. Cellular accumulation of chloroquine is related to intralysosomal pH or to the buffering capacity of lysosomes. Our results show that this drug is taken up more rapidly during G1 and S phases while the rate of accumulation is lowest in mitotic cells. The results are discussed in relation to the modifications of the physical properties of lysosomes during the cell cycle observed previously by cell fractionation and electron microsocopy, and to the possible role of lysosomes in the initiation of mitosis. Cyclic changes of endocytosis in actively dividing cells are demonstrated by our observations and may induce large differences in the uptake rate of extracellular substances.

Full Text

The Full Text of this article is available as a PDF (893.7 KB).

Selected References

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

  1. ALLISON A. C., MALLUCCI L. LYSOSOMES IN DIVIDING CELLS, WITH SPECIAL REFERENCE TO LYMPHOCYTES. Lancet. 1964 Dec 26;2(7374):1371–1373. doi: 10.1016/s0140-6736(64)91162-6. [DOI] [PubMed] [Google Scholar]
  2. Barlogie B., Drewinko B., Johnston D. A., Freireich E. J. The effect of adriamycin on the cell cycle traverse of a human lymphoid cell line. Cancer Res. 1976 Jun;36(6):1975–1979. [PubMed] [Google Scholar]
  3. Bartholeyns J., Quintart J., Baudhuin P. Inhibition of the discharge of endocytosed protein from phagosomes into lysosomes in hepatoma cells exposed to dimerized ribonuclease A. Biochem J. 1979 Feb 15;178(2):433–442. doi: 10.1042/bj1780433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baudhuin P., Leroy-Houyet M. A., Quintart J., Berthet P. Application of cluster analysis for characterization of spatial distribution of particles by stereological methods. J Microsc. 1979 Jan;115(1):1–17. doi: 10.1111/j.1365-2818.1979.tb00148.x. [DOI] [PubMed] [Google Scholar]
  5. Baudhuin P. Morphometry of subcellular fractions. Methods Enzymol. 1974;32:3–20. doi: 10.1016/0076-6879(74)32004-6. [DOI] [PubMed] [Google Scholar]
  6. Berlin R. D., Oliver J. M., Walter R. J. Surface functions during Mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A. Cell. 1978 Oct;15(2):327–341. doi: 10.1016/0092-8674(78)90002-8. [DOI] [PubMed] [Google Scholar]
  7. COHEN S. N., YIELDING K. L. SPECTROPHOTOMETRIC STUDIES OF THE INTERACTION OF CHLOROQUINE WITH DEOXYRIBONUCLEIC ACID. J Biol Chem. 1965 Jul;240:3123–3131. [PubMed] [Google Scholar]
  8. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donato S. D., Wiesmann U. N., Herschkowitz N. Membrane adsorption and internalization of (14C)chloroquine by cultured human fibroblasts. Biochem Pharmacol. 1977 Jan 1;26(1):7–10. doi: 10.1016/0006-2952(77)90122-8. [DOI] [PubMed] [Google Scholar]
  10. Doyle D., Baumann H., England B., Friedman E., Hou E., Tweto J. Biogenesis of plasma membrane glycoproteins in hepatoma tissue culture cells. J Biol Chem. 1978 Feb 10;253(3):965–973. [PubMed] [Google Scholar]
  11. Fedorko M. E., Hirsch J. G., Cohn Z. A. Autophagic vacuoles produced in vitro. II. Studies on the mechanism of formation of autophagic vacuoles produced by chloroquine. J Cell Biol. 1968 Aug;38(2):392–402. doi: 10.1083/jcb.38.2.392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein J. L., Brown M. S. The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem. 1977;46:897–930. doi: 10.1146/annurev.bi.46.070177.004341. [DOI] [PubMed] [Google Scholar]
  13. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  14. Henning R. pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium. Biochim Biophys Acta. 1975 Aug 20;401(2):307–316. doi: 10.1016/0005-2736(75)90314-4. [DOI] [PubMed] [Google Scholar]
  15. Herzog V., Farquhar M. G. Luminal membrane retrieved after exocytosis reaches most golgi cisternae in secretory cells. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5073–5077. doi: 10.1073/pnas.74.11.5073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. KELLENBERGER E., RYTER A., SECHAUD J. Electron microscope study of DNA-containing plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J Biophys Biochem Cytol. 1958 Nov 25;4(6):671–678. doi: 10.1083/jcb.4.6.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kaplan J. Cell contact induces an increase in pinocytotic rate in cultured epithelial cells. Nature. 1976 Oct 14;263(5578):596–597. doi: 10.1038/263596a0. [DOI] [PubMed] [Google Scholar]
  18. Knutton S., Sumner M. C., Pasternak C. A. Role of microvilli in surface changes of synchronized P815Y mastocytoma cells. J Cell Biol. 1975 Sep;66(3):568–576. doi: 10.1083/jcb.66.3.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O'Brien R. L., Allison J. L., Hahn F. E. Evidence for intercalation of chloroquine into DNA. Biochim Biophys Acta. 1966 Dec 21;129(3):622–624. doi: 10.1016/0005-2787(66)90078-5. [DOI] [PubMed] [Google Scholar]
  20. Porter K., Prescott D., Frye J. Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J Cell Biol. 1973 Jun;57(3):815–836. doi: 10.1083/jcb.57.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Quintart J., Baudhuin P. Accumulation of chloroquine by hepatoma cells as a function of the cell cycle [proceedings]. Arch Int Physiol Biochim. 1978 Oct;86(4):881–882. [PubMed] [Google Scholar]
  22. Quintart J., Baudhuin P. Isopycnic equilibration of hepatoma-cell homogenates at different times during the cell cycle [proceedings]. Biochem Soc Trans. 1977;5(4):1174–1176. doi: 10.1042/bst0051174. [DOI] [PubMed] [Google Scholar]
  23. Quintart J., Baudhuin P. Uptake of horseradish peroxidase during the cell cycle of hepatoma cells in culture. Arch Int Physiol Biochim. 1976 Apr;84(2):409–410. [PubMed] [Google Scholar]
  24. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reijngoud D., Tager J. M. Effect of ionophores and temperature on intralysosomal pH. FEBS Lett. 1975 Jun 1;54(1):76–79. doi: 10.1016/0014-5793(75)81072-6. [DOI] [PubMed] [Google Scholar]
  26. STRAUS W. Colorimetric analysis with N, N-dimethyl-p-phenylenediamine of the uptake of intravenously injected horseradish peroxidase by various tissues of the rat. J Biophys Biochem Cytol. 1958 Sep 25;4(5):541–550. doi: 10.1083/jcb.4.5.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schneider Y. J., Tulkens P., Trouet A. Recycling of fibroblast plasma-membrane antigens internalized during endocytosis [proceedings]. Biochem Soc Trans. 1977;5(4):1164–1167. doi: 10.1042/bst0051164. [DOI] [PubMed] [Google Scholar]
  28. Sellers L., Granner D. Regulation of tyrosine aminotrasferase activity in two liver-derived permanent cell lines. J Cell Biol. 1974 Feb;60(2):337–345. doi: 10.1083/jcb.60.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  30. Steinman R. M., Brodie S. E., Cohn Z. A. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976 Mar;68(3):665–687. doi: 10.1083/jcb.68.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steinman R. M., Silver J. M., Cohn Z. A. Pinocytosis in fibroblasts. Quantitative studies in vitro. J Cell Biol. 1974 Dec;63(3):949–969. doi: 10.1083/jcb.63.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Trouet A., Deprez-de Campeneere D., De Duve C. Chemotherapy through lysosomes with a DNA-daunorubicin complex. Nat New Biol. 1972 Sep 27;239(91):110–112. doi: 10.1038/newbio239110a0. [DOI] [PubMed] [Google Scholar]
  33. Trouet A. Perspectives in cancer research. Increased selectivity of drugs by linking to carriers. Eur J Cancer. 1978 Feb;14(2):105–111. doi: 10.1016/0014-2964(78)90167-6. [DOI] [PubMed] [Google Scholar]
  34. Wibo M., Poole B. Protein degradation in cultured cells. II. The uptake of chloroquine by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B1. J Cell Biol. 1974 Nov;63(2 Pt 1):430–440. doi: 10.1083/jcb.63.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Youngdahl-Turner P., Rosenberg L. E., Allen R. H. Binding and uptake of transcobalamin II by human fibroblasts. J Clin Invest. 1978 Jan;61(1):133–141. doi: 10.1172/JCI108911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Duve C., de Barsy T., Poole B., Trouet A., Tulkens P., Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. doi: 10.1016/0006-2952(74)90174-9. [DOI] [PubMed] [Google Scholar]
  37. de Laat S. W., van der Saag P. T., Shinitzky M. Microviscosity modulation during the cell cycle of neuroblastoma cells. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4458–4461. doi: 10.1073/pnas.74.10.4458. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES