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. 1983 Jun 1;96(6):1743–1750. doi: 10.1083/jcb.96.6.1743

Interplay of cyclic AMP and microtubules in modulating the initiation of DNA synthesis in 3T3 cells

PMCID: PMC2112466  PMID: 6189842

Abstract

The results presented here show that disruption of the microtubule network acts synergistically with cAMP-elevating agents to stimulate the entry into DNA synthesis of 3T3 cells. Antimicrotubule agents and increased cAMP levels require an additional growth-promoting factor for inducing initiation of DNA synthesis; such requirement can be furnished by insulin, vasopressin, epidermal growth factor, platelet-derived growth factor, or fibroblast-derived growth factor. The involvement of the microtubules is indicated by the fact that enhancement of the DNA synthetic response was demonstrated with the chemically diverse agents colchicine, nocodazole, vinblastine, or demecolcine, all of which elicited the response in a dose-dependent manner. We verified that colchicine and nocodazole, at the doses used in this study, induced microtubule disassembly in the absence as well as in the presence of cAMP-elevating agents as judged by measurement of [3H]colchicine binding of total and pelletable tubulin. The involvement of cAMP was revealed by increasing its endogenous production by cholera toxin or by treatment with 8BrcAMP. The enhancing effects of antimicrotubule drugs and cAMP-elevating agents could be demonstrated by incorporation of [3H]thymidine into acid-insoluble material, autoradiography of labeled nuclei, or flow cytofluorometric analysis. The addition of antimicrotubule drugs does not increase the intracellular level of cAMP nor does addition of cAMP-elevating agents promote disassembly of microtubules (as judged by measuring [3H]colchicine binding of total and pelletable tubulin) in 3T3 cells. In view of these findings and the striking synergistic effects between these agents in stimulating DNA synthesis in the presence of a peptide growth factor, we conclude that increased cAMP levels and a disrupted microtubule network regulate independent pathways involved in proliferative response.

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Selected References

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  1. Borisy G. G. A rapid method for quantitative determination of microtubule protein using DEAE-cellulose filters. Anal Biochem. 1972 Dec;50(2):373–385. doi: 10.1016/0003-2697(72)90046-2. [DOI] [PubMed] [Google Scholar]
  2. Bourne H. R., Rozengurt E. An 18,000 molecular weight polypeptide induces early events and stimulates DNA synthesis in cultured cells. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4555–4559. doi: 10.1073/pnas.73.12.4555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brinkley B. R., Fuller E. M., Highfield D. P. Cytoplasmic microtubules in normal and transformed cells in culture: analysis by tubulin antibody immunofluorescence. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4981–4985. doi: 10.1073/pnas.72.12.4981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brooker G., Harper J. F., Terasaki W. L., Moylan R. D. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv Cyclic Nucleotide Res. 1979;10:1–33. [PubMed] [Google Scholar]
  5. Crossin K. L., Carney D. H. Microtubule stabilization by taxol inhibits initiation of DNA synthesis by thrombin and by epidermal growth factor. Cell. 1981 Dec;27(2 Pt 1):341–350. doi: 10.1016/0092-8674(81)90417-7. [DOI] [PubMed] [Google Scholar]
  6. Deuel T. F., Huang J. S., Proffitt R. T., Baenziger J. U., Chang D., Kennedy B. B. Human platelet-derived growth factor. Purification and resolution into two active protein fractions. J Biol Chem. 1981 Sep 10;256(17):8896–8899. [PubMed] [Google Scholar]
  7. DiPasquale A. M., McGuire J., Moellmann G., Wasserman S. J. Microtubule assembly in cultivated Greene melanoma cells is stimulated by dibutyryl adenosine 3':5'-cyclic monophosphate or cholera toxin. J Cell Biol. 1976 Dec;71(3):735–748. doi: 10.1083/jcb.71.3.735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dicker P., Pohjanpelto P., Pettican P., Rozengurt E. Similarities between fibroblast-derived growth factor and platelet-derived growth factor. Exp Cell Res. 1981 Sep;135(1):221–227. doi: 10.1016/0014-4827(81)90314-1. [DOI] [PubMed] [Google Scholar]
  9. Eichhorn J. H., Peterkofsky B. Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents. J Cell Biol. 1979 Apr;81(1):26–42. doi: 10.1083/jcb.81.1.26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eichhorn J. H., Peterkofsky B. Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells. J Cell Biol. 1979 Aug;82(2):572–576. doi: 10.1083/jcb.82.2.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Friedkin M., Legg A., Rozengurt E. Antitubulin agents enhance the stimulation of DNA synthesis by polypeptide growth factors in 3T3 mouse fibroblasts. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3909–3912. doi: 10.1073/pnas.76.8.3909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedkin M., Legg A., Rozengurt E. Enhancement of DNA synthesis by colchicine in 3T3 mouse fibroblasts stimulated with growth factors. Exp Cell Res. 1980 Sep;129(1):23–30. doi: 10.1016/0014-4827(80)90327-4. [DOI] [PubMed] [Google Scholar]
  13. Gibbs J. B., Hsu C. Y., Terasaki W. L., Brooker G. Calcium and microtubule dependence for increased ornithine decarboxylase activity stimulated by beta-adrenergic agonists, dibutyryl cyclic AMP, or serum in a rat astrocytoma cell line. Proc Natl Acad Sci U S A. 1980 Feb;77(2):995–999. doi: 10.1073/pnas.77.2.995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hsie A. W., Puck T. T. Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3':5'-monophosphate and testosterone. Proc Natl Acad Sci U S A. 1971 Feb;68(2):358–361. doi: 10.1073/pnas.68.2.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Insel P. A., Kennedy M. S. Colchicine potentiates beta-adrenoreceptor-stimulated cyclic AMP in lymphoma cells by an action distal to the receptor. Nature. 1978 Jun 8;273(5662):471–473. doi: 10.1038/273471a0. [DOI] [PubMed] [Google Scholar]
  16. Jameson L., Caplow M. Modification of microtubule steady-state dynamics by phosphorylation of the microtubule-associated proteins. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3413–3417. doi: 10.1073/pnas.78.6.3413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnson G. S., Friedman R. M., Pastan I. Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3':5'-cyclic monphosphate and its derivatives. Proc Natl Acad Sci U S A. 1971 Feb;68(2):425–429. doi: 10.1073/pnas.68.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Malawista S. E., Oliver J. M., Rudolph S. A. Microtubules and cyclic AMP in human leukocytes: on the order of things. J Cell Biol. 1978 Jun;77(3):881–886. doi: 10.1083/jcb.77.3.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McClain D. A., Edelman G. M. Density-dependent stimulation and inhibition of cell growth by agents that disrupt microtubules. Proc Natl Acad Sci U S A. 1980 May;77(5):2748–2752. doi: 10.1073/pnas.77.5.2748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Otto A. M., Zumbé A., Gibson L., Kubler A. M., Jimenez de Asua L. Cytoskeleton-disrupting drugs enhance effect of growth factors and hormones on initiation of DNA synthesis. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6435–6438. doi: 10.1073/pnas.76.12.6435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Porter K. R., Puck T. T., Hsie A. W., Kelley D. An electron microscopy study of the effects on dibutyryl cyclic AMP on Chinese hamster ovary cells. Cell. 1974 Jul;2(3):145–162. doi: 10.1016/0092-8674(74)90089-0. [DOI] [PubMed] [Google Scholar]
  22. Prentki M., Crettaz M., Jeanrenaud B. Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. J Biol Chem. 1981 May 10;256(9):4336–4340. [PubMed] [Google Scholar]
  23. Puck T. T. Cyclic AMP, the microtubule-microfilament system, and cancer. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4491–4495. doi: 10.1073/pnas.74.10.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rasenick M. M., Stein P. J., Bitensky M. W. The regulatory subunit of adenylate cyclase interacts with cytoskeletal components. Nature. 1981 Dec 10;294(5841):560–562. doi: 10.1038/294560a0. [DOI] [PubMed] [Google Scholar]
  25. Rozengurt E. Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division. Exp Cell Res. 1982 May;139(1):71–78. doi: 10.1016/0014-4827(82)90319-6. [DOI] [PubMed] [Google Scholar]
  26. Rozengurt E. Cyclic AMP: a growth-promoting signal for mouse 3T3 cells. Adv Cyclic Nucleotide Res. 1981;14:429–442. [PubMed] [Google Scholar]
  27. Rozengurt E., Legg A., Strang G., Courtenay-Luck N. Cyclic AMP: a mitogenic signal for Swiss 3T3 cells. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4392–4396. doi: 10.1073/pnas.78.7.4392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rozengurt E. Stimulation of Na influx, Na-K pump activity and DNA synthesis in quiescent cultured cells. Adv Enzyme Regul. 1980;19:61–85. doi: 10.1016/0065-2571(81)90009-1. [DOI] [PubMed] [Google Scholar]
  29. Rozengurt E. Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells. J Cell Physiol. 1982 Aug;112(2):243–250. doi: 10.1002/jcp.1041120213. [DOI] [PubMed] [Google Scholar]
  30. Rudolph S. A., Greengard P., Malawista S. E. Effects of colchicine on cyclic AMP levels in human leukocytes. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3404–3408. doi: 10.1073/pnas.74.8.3404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Somers K. D., Weberg A. D., Steiner S. Cyclic AMP-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus. Expression of transformation-associated markers. J Cell Biol. 1977 Sep;74(3):707–716. doi: 10.1083/jcb.74.3.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. TODARO G. J., GREEN H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963 May;17:299–313. doi: 10.1083/jcb.17.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Teng M. H., Bartholomew J. C., Bissell M. J. Synergism between anti-microtubule agents and growth stimulants in enhancement of cell cycle traverse. Nature. 1977 Aug 25;268(5622):739–741. doi: 10.1038/268739a0. [DOI] [PubMed] [Google Scholar]
  34. Tobey R. A., Crissman H. A. Unique techniques for cell analysis utilizing mithramycin and flow microfluorometry. Exp Cell Res. 1975 Jun;93(1):235–239. doi: 10.1016/0014-4827(75)90445-0. [DOI] [PubMed] [Google Scholar]

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