Summary
Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10−3 m) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7×10−6 m), Persantin (5×10−5 m) or RO-20-1724 (10−4 m), prevents cell replication induced by PMA or serum. In contrast, ouabain (10−4 m) and N,N′-dicyclohexylcarbodiimide (10−5 m), inhibitors of Na+−K+-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G1 phase at the time of reinitiation of the cell cycle from a stationary (G0) phase, a second is associated with the G1-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane ezymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner.
Similar content being viewed by others
References
Boutwell, R. K. 1974. The function and mechanism of promoters of carcinogenesis. CRC Crit. Rev. Toxicol. 2: 419–443.
O'Brien, T. G., R. C. Simsiman, and R. K. Boutwell. 1975. Induction of polyamine-biosynthetic enzymes in mouse epidermis by tumor-promoting agents. Cancer Res. 35: 1662–1670.
Lichti, U., and S. Yuspa. 1976. Ornithine decarboxylase (ODC) stimulation in mouse epidermis with tumor promotersin vitro andin vivo. Proc. Am. Assoc. Cancer Res. 17: 138.
Sivak, A., and B. L. Van Duuren. 1967. Phenotypic expression of transformation: induction in cell culture by a phorbol ester. Science 157: 1443–1444.
Sivak, A. 1972. Induction of cell division. Role of cell membrane sites. J. Cell. Physiol. 80: 167–174.
Sivak, A., and B. T. Mossman. 1973. Alteration of cell membrane enzymes in the stimulation of cell division. J. Cell Biol. 59: 323a.
Sivak, A., B. T. Mossman, and B. L. Van Duuren. 1972. Activation of cell membrane enzymes in the stimulation of cell division. Biochem. Biophys. Res. Commun. 46: 605–609.
Diamond, L., S. O'Brien, C. Donaldson, and Y. Shimizu. 1974. Growth stimulation of human diploid fibroblasts by the tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate. Int. J. Cancer 13: 721–730.
Estensen, R. D., J. W. Hadden, E. M. Hadden, F. Touraine, J-L. Touraine, M. K. Haddox, and N. D. Goldberg. 1974. Phorbol myristate acetate: effects of a tumor promoter on intracellular cyclic GMP in mouse fibroblasts and as a mitogen on human lymphocytes. In: B. Clarkson, and R. Baserga (Eds.),Control of Proliferation in Animal Cells. Vol. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 627–634.
Wang, J. L., D. A. McClain, and G. M. Edelman. 1975. Modulation of lymphocyte mitogenesis. Proc. Natl. Acad. Sci. U.S.A. 72: 1917–1921.
Estensen, R. D., H. R. Hill, P. G. Quie, N. Hogan, and N. D. Goldberg. 1973. Cyclic GMP and cell movement. Nature 245: 458–460.
Zucker, M.B., W. Troll, and S. Behman. 1974. The tumor promoter phorbol ester (12-0-tetradecanoyl-phorbol-13-acetate), a potent aggregating agent for blood platelets. J. Cell Biol. 60: 325–336.
Whitfield, J. F., J. P. MacManus, and D. J. Gillan. 1973. Calcium-dependent stimulation by a phorbol ester (PMA) of thymic lymphoblast DNA synthesis and proliferation. J. Cell. Physiol. 82: 151–156.
Clarkson, B., and R. Baserga (Eds.). 1974.Control of Proliferation in Animal Cells. Vol. 1. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.
Lelièvre, L., B. Prigent, and A. Paraf. 1971. Contact inhibition—plasma membranes enzymatic activities in cultured cell lines. Biochem. Biophys. Res. Commun. 45(3): 637–643.
Quastel, M. R., and J. G. Kaplan. 1970. Early stimulation of potassium uptake in lymphocytes treated with PHA. Exp. Cell Res. 63: 230–233.
Sivak, A. 1974. Phenotypic expression of transformation and cyclic AMP phosphodiesterase activity (abstr.) In Vitro 9: 358.
Van Duuren, B. L., A. Sivak, A. Segal, I. Seidman, and C. Katz. 1973. Dose response studies with a pure tumor-promoting agent: phorbol myristate acetate. Cancer Res. 33: 2166–2172.
Noonan, K. D., and M. M. Burger. 1973. Induction of 3T3 cell division at the monolayer stage. Exp. Cell Res. 80: 405–414.
Lamb, J. F., L. J. Boardman, J. P. Newton, and J. F. Aiton. 1973. Effect of calf serum on sodium pump density in HeLa cells. Nature [New Biol.] 242: 115–117.
Prasad, K. N., and J. R. Sheppard. 1972. Inhibitors of cyclic-nucleotide phosphodiesterase induce morphological differentiation of mouse neuroblastoma cell culture. Exp. Cell Res. 73: 436–440.
Wahrmann, S. P., R. Winand and D. Luzzati. 1973. Effect of cyclic AMP on growth and morphological differentiation of an established myogenic cell line. Nature [New Biol.] 245: 112–113.
Lazarides, E., and K. Weber. 1974. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc. Natl. Acad. Sci. U.S.A. 71(6): 2268–2272.
Puszkin, E. A., and M. B. Zucker. 1973. Enhancement of platelet and muscle actomyosin ATPase activity and superprecipitation by the tumour-promoter 12-0-tetradecanoyl-phorbol-13-acetate (PMA). Nature [New Biol.] 245: 277–280.
Hsie, A. W., K. Kawashima, J. P. O'Neill, and C. H. Schröder 1975. Possible role of adenosine cyclic 3′:5′-monophosphate phosphodiesterase in the morphological transformation of Chinese hamster ovary cells mediated by N6, O2-dibutyryl adenosine cyclic 3′:5′-monophosphate. J. Biol. Chem. 250: 984–989.
Plagemann, P. G. W. 1973. Deoxyglucose transport by uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells. J. Cell. Physiol. 82: 421–434.
Pledger, W. J., W. J. Thompson, and S. J. Strada. 1975. Mediation of serum-induced changes in cyclic nucleotide levels in cultured fibroblasts by cyclic nucleotide phosphodiesterase. Nature 256: 729–731.
Grimm, J., and W. Frank. 1972. Stimulierung von Kulturen embryonaler Rattenzellen durch Kälberserum. II. Veränderung der ATP- und cAMP-Spiegel; aktivierung einer cAMP-spaltenden phosphodiesterase. Z. Naturforsch. 27b: 562–566.
Whitfield, J. F., J. P. MacManus, D. J. Franks, D. J. Gillan, and T. Youdale. 1971. The possible mediation by cyclic AMP of the stimulation of thymocyte proliferation by cyclic GMP (35598). Proc. Soc. Exp. Biol. Med. 137: 453–457.
Pardee, A. B., L. Jiménez de Azúa, and E. Rozengurt. 1974. Functional membrane changes and cell growth: significance and mechanism. In: B. Clarkson, and R. Baserga (Eds.),Control of Proliferation in Animal Cells. Vol. 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 547–561.
Rubin, C. S., R. D. Rosenfeld, and O. M. Rosen, 1973. Studies on the orientation of cyclic AMP-dependent protein kinase in human erythrocyte membranes. Proc. Natl. Acad. Sci. U.S.A. 70: 3735–3738.
Swislocki, N. I., and J. Tierney. 1973. Solubilization, stabilization and partial purification of brain adenylate cyclase from rat. Biochemistry 12(10): 1862–1866.
Stein, G. S., and T. W. Borun. 1972. The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. I. The accelerated accumulation of acidic residual nuclear protein before the initiation of DNA replication. J. Cell Biol. 52: 292–307.
Johnson, L. D., and J. W. Hadden. 1975. Cyclic GMP and lymphocyte proliferation: effects on DNA-dependent RNA polymerase I and II activities. Biochem. Biophys. Res. Comm. 66: 1498–1505.
Sivak, A., and B. T. Mossman. 1975. Plasma membrane and tumor-promoter effects on 3T3 cell RNA synthesis. Proc. Amer. Assoc. Cancer Res. 16: 104.
Boynton, A. L., J. F. Whitfield, and R. J. Isaacs. 1976. Calcium-dependent stimulation of BALB/c-3T3 mouse cell DNA synthesis by a tumor-producing phorbol ester (PMA). J. Cell. Physiol. 87: 25–32.
Author information
Authors and Affiliations
Additional information
This work was supported by a USPHS Research Grant CA12503, and a Center Grant ES-00260 awarded to the Institute of Environmental Medicine. Mrs. Susan Kulina provided the consistent and excellent technical aid necessary to perform this work.
Note added in proof: During the preparation and review of this paper, Boynton reported that PMA appears to sensitize BALB/c-3T3 cells to calcium ion which may play a critical role in the regulation of the DNA synthesis (36).
Rights and permissions
About this article
Cite this article
Sivak, A. Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: Effects of inhibitors of cyclic AMP phosphodiesterase and Na+−K+-ATPase. In Vitro 13, 337–343 (1977). https://doi.org/10.1007/BF02615093
Issue Date:
DOI: https://doi.org/10.1007/BF02615093