Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells.
We studied the effect of doxorubicin (Dox) on cell cycle progression and its correlation with DNA damage and cytotoxicity in p53-mutant P388 cells. P388 cells synchronized in S and G2/M phases were > 3-fold more sensitive to Dox than were cells in G1 phase (Dox ID50 = 0.50 +/- 0.16 microM in cells synchronized in S phase versus 1.64 +/- 0.12 microM in asynchronized cells; drug exposure, 1 hr). Treatment of synchronized cells in early S phase with 1 microM Dox (2 x ID50) for 1 hr induced a marked cell arrest at G2/M phase at 6-12 hr after drug incubation. We then studied the effect of Dox on the p34cdc2/cyclin B1 complex because it plays a key role in regulating G2/M phase transition. In untreated control P388 cells, p34cdc2 kinase localizes in the nucleus and cytoplasms, particularly in the centrosomes, and p34cdc2 kinase activity is dependent on cell cycle progression, with the enzyme activity increasing steadily from G1/S to G2/M and markedly declining thereafter. Treatment of synchronized P388 cells in early S phase with 1 microM Dox for 1 hr did not affect the pattern of subcellular distribution of the enzyme but completely abrogated its function for > or = 10 hr. In a cell-free system, Dox did not inhibit p34cdc2 kinase activity, indicating that is has no direct effect on the enzyme function. In whole cells, Dox treatment prevented p34cdc2 kinase dephosphorylation without altering its synthesis, and this effect was due to neither down-regulation of cdc25C nor inhibition of protein-tyrosine phosphatase activity. In contrast, Dox treatment was found to induced cyclin B1 accumulation as a result of stimulating its synthesis and inhibiting its degradation. A good correlation was found between extent of DNA double-strand breaks and p34cdc2 kinase activity inhibition. Our results suggest that anthracycline-induced cytotoxicity is cell cycle dependent and is mediated, at least in part, by disturbance of the regulation of p34cdc2/cyclin B1 complex, thus leading to G2/M phase arrest.
Ling
Y H
YH
Department of Thoracic@Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. yihevling@isqm.mda.uth.tmc.edu
el-Naggar
A K
AK
Priebe
W
W
Perez-Soler
R
R
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002451:Cell Compartmentation / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D007941:Leukemia P388 / Q000503:physiopathology*; D051379:Mice; D010766:Phosphorylation
D000903:Antibiotics, Antineoplastic; C534700:Ccnb1 protein, mouse; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D004317:Doxorubicin; D016203:CDC2 Protein Kinase