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 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 cytoplasm, 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 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 p34cdc2 kinase activity, indicating that it 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 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 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.
|Original language||English (US)|
|Number of pages||10|
|State||Published - May 1 1996|
ASJC Scopus subject areas
- Molecular Medicine