Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34^cdc2/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 μM in cells synchronized in S phase versus 1.64 ± 0.12 μM in asynchronized cells; drug exposure, 1 hr). Treatment of synchronized cells in early S phase with 1 μM Dox (2 × 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 p34^cdc2/cyclin B₁ complex because it plays a key role in regulating G2/M phase transition. In untreated control P388 cells, p34^cdc2 kinase localizes in the nucleus and cytoplasm, particularly in the centrosomes, and p34^cdc2 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 μM Dox for 1 hr did not affect the pattern of subcellular distribution of the enzyme but completely abrogated its function for ≥10 hr. In a cell-free system, Dox did not inhibit p34^cdc2 kinase activity, indicating that it has no direct effect on the enzyme function. In whole cells, Dox treatment prevented p34^cdc2 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 induce 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 p34^cdc2 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 p34^cdc2/cyclin B₁ complex, thus leading to G2/M phase arrest.