THAPSIGARGIN AND DI-TERT-BUTYLHYDROQUINONE INDUCE SYNERGISTIC STIMULATION OF DNA-SYNTHESIS WITH PHORBOL ESTER AND BOMBESIN IN SWISS 3T3 CELLS

The specific inhibitors of the endoplasmic reticulum Ca2+ pump, thapsigargin and 2,5-di-tert-butylhydroquinone (DBHQ), stimulated reinitiation of DNA synthesis in synergy with either phorbol 12,13-dibutyrate or bombesin in Swiss 3T3 cells. Maximum stimulation was achieved at 0.5 nM thapsigargin and 7.5 mu M DBHQ. Kinetics of [H-3]thymidine incorporation were consistent with exit from G(0) and entry into S phase. Autoradiography of labeled nuclei showed that the increase in [H-3]thymidine incorporation was due to an increase in the proportion of cells entering into DNA synthesis. Down-regulation or selective inhibition of protein kinase C abolished this synergistic stimulation of DNA synthesis. Thapsigargin and DBHQ and did not potentiate protein kinase C-mediated signals such as direct phosphorylation of myristoylated alanine-rich C-kinase substrate, activation of mitogen-activated protein kinase, and tyrosine phosphorylation of bands 110,000-130,000 and 70,000-80,000. Thapsigargin and DBHQ caused a marked reduction in the ability of bombesin to induce a rapid and transient increase in intracellular Ca2+ via depletion of total cellular Ca2+, measured by Ca-45(2+) content. The synergistic stimulation of DNA synthesis by DBHQ and phorbol 12,13-dibutyrate was dependent on a high concentration of extracellular Ca2+ (ED(50) = 410 mu M) and was preferentially inhibited by the inhibitor of Ca2+ influx econozole. This suggests a role for Ca2+ entry in growth control. This is the first time that either thapsigargin or DBHQ has been shown to stimulate the reinitiation of DNA synthesis in any target cell.