Induction of DNA Synthesis in Isolated Nuclei by Cytoplasmic Factors: Inhibition by Protease Inhibitors

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 1; pp. 241 - 245
• Main Authors: Wong, Robert L., Gutowski, Janice K., Katz, Michael, Goldfarb, Ronald H., Cohen, Stanley
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01-01-1987; National Acad Sciences
• Subjects: Adsorption; Animals; B lymphocytes; Biological and medical sciences; Cell growth; Cell Line; Cell nucleus; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Cytoplasm - physiology; DNA; DNA Replication - drug effects; Elution; Endopeptidases - metabolism; Enzymes; Fundamental and applied biological sciences. Psychology; Humans; Interleukin-2 - pharmacology; Kinetics; Leukemia; Lymphocytes; Molecular and cellular biology; Molecular genetics; Protease inhibitors; Protease Inhibitors - pharmacology; Replication; Serine Endopeptidases; Splenocytes; Xenopus; Human; Lymphoid cell; Regulation(control); Cell nucleus; Proteinase; Isolation; Replication; DNA synthesis; Nucleocytoplasmic interaction
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.1.241

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-α -tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotininconjugated 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.