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 microM DBHQ. Kinetics of [3H]thymidine incorporation were consistent with exit from G0 and entry into S phase. Autoradiography of labeled nuclei showed that the increase in [3H]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 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 45Ca2+ content. The synergistic stimulation of DNA synthesis by DBHQ and phorbol 12,13-dibutyrate was dependent on a high concentration of extracellular Ca2+ (ED50 = 410 microM) 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.
Charlesworth
A
A
Imperial Cancer Research Fund, London, United Kingdom.
Rozengurt
E
E
D016475:3T3 Cells; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001839:Bombesin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004261:DNA Replication / Q000187:drug effects*; D004357:Drug Synergism; D006873:Hydroquinones / Q000494:pharmacology*; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D000975:Antioxidants; D006873:Hydroquinones; D008934:Mitogens; D013729:Terpenes; C019359:2,5-di-tert-butylhydroquinone; D015240:Phorbol 12,13-Dibutyrate; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D001839:Bombesin; D002118:Calcium