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

Abstract

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. Colchicine and nocodazole, at the doses used in this study, induced microtubule disassembly in the absence as well as in the pesence 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 [cyclic 8-bromoadenosine monophosphate]. 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, increased cAMP levels and a disrupted microtubule network regulate independent pathways involved in proliferative response.