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. 1996 Jan 1;313(Pt 1):65–70. doi: 10.1042/bj3130065

Inhibition of mitogen-induced DNA synthesis by bafilomycin A1 in Swiss 3T3 fibroblasts.

A J Saurin 1, J Hamlett 1, M J Clague 1, S R Pennington 1
PMCID: PMC1216910  PMID: 8546711

Abstract

Quiescent cells (in G0) can be stimulated to enter the cell cycle and proceed to DNA synthesis in S-phase by a wide range of growth factors and mitogens. Activation of cell-surface growth factor receptors with intrinsic protein tyrosine kinase activity initiates autophosphorylation of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced internalization to endosomes, which become acidified by the action of a vacuolar H(+)-ATPase (V-ATPase). The extent to which vesicular acidification plays a role in mitogenic signalling by receptors with intrinsic tyrosine kinase activity remains unknown. Here we have shown that bafilomycin A1, a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A1 at successively later times during G1 progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A1 was added at the onset of S-phase. Bafilomycin A1 also induced a dramatic but reversible change in the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was unaffected by bafilomycin A1. Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G1 phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoked signalling events that lead to c-fos mRNA expression.

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Selected References

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  1. Ashwell G., Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982;51:531–554. doi: 10.1146/annurev.bi.51.070182.002531. [DOI] [PubMed] [Google Scholar]
  2. Baldin V., Roman A. M., Bosc-Bierne I., Amalric F., Bouche G. Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells. EMBO J. 1990 May;9(5):1511–1517. doi: 10.1002/j.1460-2075.1990.tb08269.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumer K. J., Johnson G. L. Diversity in function and regulation of MAP kinase pathways. Trends Biochem Sci. 1994 Jun;19(6):236–240. doi: 10.1016/0968-0004(94)90147-3. [DOI] [PubMed] [Google Scholar]
  4. Bowman E. J., Siebers A., Altendorf K. Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7972–7976. doi: 10.1073/pnas.85.21.7972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Clague M. J., Urbé S., Aniento F., Gruenberg J. Vacuolar ATPase activity is required for endosomal carrier vesicle formation. J Biol Chem. 1994 Jan 7;269(1):21–24. [PubMed] [Google Scholar]
  7. Fantl W. J., Johnson D. E., Williams L. T. Signalling by receptor tyrosine kinases. Annu Rev Biochem. 1993;62:453–481. doi: 10.1146/annurev.bi.62.070193.002321. [DOI] [PubMed] [Google Scholar]
  8. Geisow M. J., Beaven G. H., Hart P. D., Young M. R. Site of action of a polyanion inhibitor of phagosome-lysosome fusion in cultured macrophages. Exp Cell Res. 1980 Mar;126(1):159–165. doi: 10.1016/0014-4827(80)90481-4. [DOI] [PubMed] [Google Scholar]
  9. Henomatsu N., Yoshimori T., Yamamoto A., Moriyama Y., Tashiro Y. Inhibition of intracellular transport of newly synthesized prolactin by bafilomycin A1 in a pituitary tumor cell line, GH3 cells. Eur J Cell Biol. 1993 Oct;62(1):127–139. [PubMed] [Google Scholar]
  10. Kimura H. Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2165–2169. doi: 10.1073/pnas.90.6.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. King A. C., Hernaez-Davis L., Cuatrecasas P. Lysosomotropic amines inhibit mitogenesis induced by growth factors. Proc Natl Acad Sci U S A. 1981 Feb;78(2):717–721. doi: 10.1073/pnas.78.2.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  13. Li C. Y., Watkins J. A., Glass J. The H(+)-ATPase from reticulocyte endosomes reconstituted into liposomes acts as an iron transporter. J Biol Chem. 1994 Apr 8;269(14):10242–10246. [PubMed] [Google Scholar]
  14. Manabe T., Yoshimori T., Henomatsu N., Tashiro Y. Inhibitors of vacuolar-type H(+)-ATPase suppresses proliferation of cultured cells. J Cell Physiol. 1993 Dec;157(3):445–452. doi: 10.1002/jcp.1041570303. [DOI] [PubMed] [Google Scholar]
  15. Martinez-Zaguilan R., Lynch R. M., Martinez G. M., Gillies R. J. Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells. Am J Physiol. 1993 Oct;265(4 Pt 1):C1015–C1029. doi: 10.1152/ajpcell.1993.265.4.C1015. [DOI] [PubMed] [Google Scholar]
  16. Mellman I., Fuchs R., Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986;55:663–700. doi: 10.1146/annurev.bi.55.070186.003311. [DOI] [PubMed] [Google Scholar]
  17. Muroi M., Shiragami N., Takatsuki A. Destruxin B, a specific and readily reversible inhibitor of vacuolar-type H(+)-translocating ATPase. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1358–1365. doi: 10.1006/bbrc.1994.2815. [DOI] [PubMed] [Google Scholar]
  18. Nanda A., Grinstein S. Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10816–10820. doi: 10.1073/pnas.88.23.10816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ohkuma S., Poole B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3327–3331. doi: 10.1073/pnas.75.7.3327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ohkuma S., Shimizu S., Noto M., Sai Y., Kinoshita K., Tamura H. Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H(+)-ATPase. In Vitro Cell Dev Biol Anim. 1993 Nov;29A(11):862–866. doi: 10.1007/BF02631364. [DOI] [PubMed] [Google Scholar]
  21. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  22. Pawson T., Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. doi: 10.1016/0960-9822(93)90350-w. [DOI] [PubMed] [Google Scholar]
  23. Pennington S. R., Moore J. P., Evan G. I., Hesketh T. R., Metcalfe J. C. GTP gamma S inhibits early c-myc protein accumulation but not DNA synthesis in Swiss 3T3 fibroblasts. FEBS Lett. 1990 Oct 29;273(1-2):243–247. doi: 10.1016/0014-5793(90)81095-6. [DOI] [PubMed] [Google Scholar]
  24. Poole B., Ohkuma S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol. 1981 Sep;90(3):665–669. doi: 10.1083/jcb.90.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rakowicz-Szulczynska E. M., Rodeck U., Herlyn M., Koprowski H. Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3728–3732. doi: 10.1073/pnas.83.11.3728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reaves B., Banting G. Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma membrane. FEBS Lett. 1994 May 23;345(1):61–66. doi: 10.1016/0014-5793(94)00437-4. [DOI] [PubMed] [Google Scholar]
  27. Seger R., Krebs E. G. The MAPK signaling cascade. FASEB J. 1995 Jun;9(9):726–735. [PubMed] [Google Scholar]
  28. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  29. Werner G., Hagenmaier H., Drautz H., Baumgartner A., Zähner H. Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics. Production, isolation, chemical structure and biological activity. J Antibiot (Tokyo) 1984 Feb;37(2):110–117. doi: 10.7164/antibiotics.37.110. [DOI] [PubMed] [Google Scholar]
  30. Xu H., Shields D. Prosomatostatin processing in permeabilized cells. Endoproteolytic cleavage is mediated by a vacuolar ATPase that generates an acidic pH in the trans-Golgi network. J Biol Chem. 1994 Sep 9;269(36):22875–22881. [PubMed] [Google Scholar]
  31. Yilla M., Tan A., Ito K., Miwa K., Ploegh H. L. Involvement of the vacuolar H(+)-ATPases in the secretory pathway of HepG2 cells. J Biol Chem. 1993 Sep 5;268(25):19092–19100. [PubMed] [Google Scholar]
  32. Yoshimori T., Yamamoto A., Moriyama Y., Futai M., Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991 Sep 15;266(26):17707–17712. [PubMed] [Google Scholar]
  33. Zen K., Biwersi J., Periasamy N., Verkman A. S. Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. J Cell Biol. 1992 Oct;119(1):99–110. doi: 10.1083/jcb.119.1.99. [DOI] [PMC free article] [PubMed] [Google Scholar]

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