Cell cycle-dependent potentiation of cisplatin by UCN-01 in non-small-cell lung carcinoma

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Abstract
Purpose We evaluated the combination of UCN-01 plus cisplatin and sought to determine how the cell cycle effects of each agent affected the combined response. Cisplatin-induced DNA damage results in cell cycle arrest, primarily at the S and G2 checkpoints, providing the opportunity for DNA damage repair prior to mitosis. Thus, strategies to enhance cisplatin cytotoxicity include attenuation of DNA damage-induced checkpoints. The cyclin-dependent kinase inhibitor 7-hydroxystaurosporine (UCN-01) can potentiate cisplatin activity, likely via abrogation of the S and G2 checkpoints. UCN-01 has additional effects on cell cycling, including induction of an RB-associated G1 arrest. Methods NSCLC cell lines A549 (wt p53, wt RB), Calu1 (p53-null, wt RB) and H596 (mt p53, RB-null) were treated with UCN-01 and/or cisplatin with two-drug treatments delivered in alternate sequences. Effects of drug treatment on cell growth, cell cycling, apoptosis and levels and phosphorylation of cell cycle-associated proteins were evaluated. The interaction between the two drugs was assessed using median effect analysis. Results When UCN-01 preceded cisplatin, growth inhibition was additive or less than additive, as assessed by median effect analysis. In contrast, when NSCLC cells were treated with cisplatin followed by UCN-01, the combination was synergistic. In this treatment sequence, a decrease in the proportion of cells at the G2 checkpoint was confirmed by reduced expression of cyclins A and B and activation of Cdk1. Abrogation of the G2 DNA damage checkpoint and apoptosis were prevalent only in cell populations treated with cisplatin followed by UCN-01 and was markedly enhanced in the cell lines with disrupted p53. Conclusions These studies indicate that timing of drug administration strongly influences response to cisplatin plus UCN-01 in NSCLC cells, and this is related to the cell cycle-modulatory effects of these agents. Furthermore, this sequence combination was more effective in cell lines with dysfunctional p53. These findings support the hypothesis that checkpoint abrogation is the major mechanism of UCN-01-mediated potentiation of cisplatin cytotoxicity.