Ling Y H, el-Naggar A K, Priebe W, Perez-Soler R
Department of Thoracic@Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Mol Pharmacol. 1996 May;49(5):832-41.
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.
我们研究了阿霉素（Dox）对p53突变型P388细胞的细胞周期进程的影响及其与DNA损伤和细胞毒性的相关性。与处于G1期的细胞相比，同步于S期和G2/M期的P388细胞对Dox的敏感性高3倍以上（同步于S期的细胞中Dox的半数抑制浓度[ID50]为0.50±0.16微摩尔，而异步细胞中为1.64±0.12微摩尔；药物暴露1小时）。用1微摩尔Dox（2倍ID50）处理早期S期同步化的细胞1小时，在药物孵育后6至12小时诱导细胞在G2/M期显著停滞。然后我们研究了Dox对p34cdc2/细胞周期蛋白B1复合物的影响，因为它在调节G2/M期转换中起关键作用。在未处理的对照P388细胞中，p34cdc2激酶定位于细胞核和细胞质中，特别是在中心体中，并且p34cdc2激酶活性依赖于细胞周期进程，该酶活性从G1/S期到G2/M期稳步增加，此后显著下降。用1微摩尔Dox处理早期S期同步化的P388细胞1小时，不影响该酶的亚细胞分布模式，但使其功能完全丧失≥10小时。在无细胞系统中，Dox不抑制p34cdc2激酶活性，表明其对该酶功能无直接影响。在完整细胞中，Dox处理可防止p34cdc2激酶去磷酸化而不改变其合成，并且这种作用既不是由于cdc25C的下调也不是由于蛋白酪氨酸磷酸酶活性的抑制。相反，发现Dox处理通过刺激细胞周期蛋白B1的合成并抑制其降解而诱导其积累。发现DNA双链断裂程度与p34cdc2激酶活性抑制之间存在良好的相关性。我们的结果表明，蒽环类药物诱导的细胞毒性是细胞周期依赖性的，并且至少部分是由p34cdc2/细胞周期蛋白B1复合物调节紊乱介导的，从而导致G2/M期停滞。
