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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Jan;84(1):241–245. doi: 10.1073/pnas.84.1.241

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

R L Wong, J K Gutowski, M Katz, R H Goldfarb, S Cohen
PMCID: PMC304179  PMID: 3540956

Abstract

Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-alpha-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself.

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Selected References

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

  1. Baugh R. J., Travis J. Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry. 1976 Feb 24;15(4):836–841. doi: 10.1021/bi00649a017. [DOI] [PubMed] [Google Scholar]
  2. Benbow R. M., Ford C. C. Cytoplasmic control of nuclear DNA synthesis during early development of Xenopus laevis: a cell-free assay. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2437–2441. doi: 10.1073/pnas.72.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benz W. C., Strominger J. L. Viral and cellular DNA synthesis in nuclei from human lymphocytes transformed by Epstein-Barr virus. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2413–2417. doi: 10.1073/pnas.72.6.2413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown R. L., Clark R. W., Chiu J. F., Stubblefield E. Protease activation of G1 nuclei isolated from Chinese hamster fibroblasts. Exp Cell Res. 1977 Jan;104(1):207–213. doi: 10.1016/0014-4827(77)90083-0. [DOI] [PubMed] [Google Scholar]
  5. Brown R. L., Stubblefield E. Enhancement of DNA synthesis in a mammalian cell-free system by trypsin treatment. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2432–2434. doi: 10.1073/pnas.71.6.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carney D. H., Glenn K. C., Cunningham D. D. Conditions which affect initiation of animal cell division by trypsin and thrombin. J Cell Physiol. 1978 Apr;95(1):13–22. doi: 10.1002/jcp.1040950103. [DOI] [PubMed] [Google Scholar]
  7. Chen L. B., Buchanan J. M. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc Natl Acad Sci U S A. 1975 Jan;72(1):131–135. doi: 10.1073/pnas.72.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chou I. N., Black P. H., Roblin R. O. Non-selective inhibition of transformed cell growth by a protease inhibitor. Proc Natl Acad Sci U S A. 1974 May;71(5):1748–1752. doi: 10.1073/pnas.71.5.1748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Das M. Mitogenic hormone-induced intracellular message: assay and partial characterization of an activator of DNA replication induced by epidermal growth factor. Proc Natl Acad Sci U S A. 1980 Jan;77(1):112–116. doi: 10.1073/pnas.77.1.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Floros J., Chang H., Baserga R. Stimulated DNA synthesis in frog nuclei by cytoplasmic extracts of temperature-sensitive mammalian cells. Science. 1978 Aug 18;201(4356):651–652. doi: 10.1126/science.675253. [DOI] [PubMed] [Google Scholar]
  11. Goldfarb R. H., Quigley J. P. Purification of plasminogen activator from Rous sarcoma virus transformed chick embryo fibroblasts treated with the tumor promoter phorbol 12-myristate 13-acetate. Biochemistry. 1980 Nov 25;19(24):5463–5471. doi: 10.1021/bi00565a001. [DOI] [PubMed] [Google Scholar]
  12. Goldfarb R. H., Timonen T., Herberman R. B. Production of plasminogen activator by human natural killer cells. Large granular lymphocytes. J Exp Med. 1984 Mar 1;159(3):935–951. doi: 10.1084/jem.159.3.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grayzel A. I., Hatcher V. B., Lazarus G. S. Protease activity of normal and PHA stimulated human lymphocytes. Cell Immunol. 1975 Jul;18(1):210–219. doi: 10.1016/0008-8749(75)90049-0. [DOI] [PubMed] [Google Scholar]
  14. Gutowski J. K., Cohen S. Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells. Cell Immunol. 1983 Feb 1;75(2):300–311. doi: 10.1016/0008-8749(83)90328-3. [DOI] [PubMed] [Google Scholar]
  15. Gutowski J. K., Mukherji B., Cohen S. The role of cytoplasmic intermediates in IL 2-induced T cell growth. J Immunol. 1984 Dec;133(6):3068–3074. [PubMed] [Google Scholar]
  16. Hirschhorn R., Grossman J., Troll W., Weissmann G. The effect of epsilon amino caproic acid and other inhibitors of proteolysis upon the response of human peripheral blood lymphocytes to phytohemagglutinin. J Clin Invest. 1971 Jun;50(6):1206–1217. doi: 10.1172/JCI106598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jazwinski S. M., Edelman G. M. Acitivity of yeast extracts in cell-free stimulation of DNA replication. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3933–3936. doi: 10.1073/pnas.73.11.3933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jazwinski S. M., Wang J. L., Edelman G. M. Initiation of replication in chromosomal DNA induced by extracts from proliferating cells. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2231–2235. doi: 10.1073/pnas.73.7.2231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Johnson E. M., Hadden J. W. Phosphorylation of lymphocyte nuclear acidic proteins: regulation by cyclic nucleotides. Science. 1975 Mar 28;187(4182):1198–1200. doi: 10.1126/science.163491. [DOI] [PubMed] [Google Scholar]
  20. Kishimoto T., Kikutani H., Nishizawa Y., Sakaguchi N., Yamamura Y. Involvement of anti-Ig-activated serine protease in the generation of cytoplasmic factor(s) that are responsible for the transmission of Ig-receptor-mediated signals. J Immunol. 1979 Oct;123(4):1504–1510. [PubMed] [Google Scholar]
  21. Ku G. S., Quigley J. P., Sultzer B. M. The inhibition of the mitogenic stimulation of B lymphocytes by a serine protease inhibitor: commitment to proliferation correlates with an enhanced expression of a cell-associated arginine-specific serine enzyme. J Immunol. 1983 Nov;131(5):2494–2499. [PubMed] [Google Scholar]
  22. Kupfer A., Gani V., Jiménez J. S., Shaltiel S. Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3073–3077. doi: 10.1073/pnas.76.7.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. MOSESSON M. W. The preparation of human fibrinogen free of plasminogen. Biochim Biophys Acta. 1962 Feb 26;57:204–213. doi: 10.1016/0006-3002(62)91112-5. [DOI] [PubMed] [Google Scholar]
  24. Nishizawa Y., Kishimoto T., Kikutani H., Yamamura Y. Induction and properties of cytoplasmic factor(s) which enhance nuclear nonhistone protein phosphorylation in lymphocytes stimulated by anti-Ig. J Exp Med. 1977 Sep 1;146(3):653–664. doi: 10.1084/jem.146.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pogo B. G., Katz J. R. Early events in lymphocyte transformation by phytohaemagglutinin. 2. Synthesis and phosphorylation of nuclear proteins. Differentiation. 1974 Apr;2(2):119–124. doi: 10.1111/j.1432-0436.1974.tb00343.x. [DOI] [PubMed] [Google Scholar]
  26. Richert N., Davies P. J., Jay G., Pastan I. Inhibition of the transformation-specific kinase in ASV-transformed cells by N-alpha-tosyl-L-lysyl chloromethyl ketone. Cell. 1979 Oct;18(2):369–374. doi: 10.1016/0092-8674(79)90056-4. [DOI] [PubMed] [Google Scholar]
  27. Saito M., Yoshizawa T., Aoyagi T., Nagai Y. Involvement of proteolytic acivity in early events in lymphocyte transformation by phytohemagglutinin. Biochem Biophys Res Commun. 1973 May 15;52(2):569–575. doi: 10.1016/0006-291x(73)90750-x. [DOI] [PubMed] [Google Scholar]
  28. Schnebli H. P., Burger M. M. Selective inhibition of growth of transformed cells by protease inhibitors. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3825–3827. doi: 10.1073/pnas.69.12.3825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zyznar E. S. A rationale for the application of trasylol as a protease inhibitor in radioimmunoassay. Life Sci. 1981 Apr 27;28(17):1861–1866. doi: 10.1016/0024-3205(81)90291-5. [DOI] [PubMed] [Google Scholar]

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