Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. p53?/? cells showed DNA damage and a strong capability 1009298-59-2 IC50 of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition. Keywords: p53, BI 2536, BI 6727, Poloxin, monastrol Introduction Polo-like kinase 1 (Plk1), the best-characterized member of the Plk family, is crucial for the cell cycle and exerts multiple functions throughout mitosis.1-3 Overexpression FGF2 of Plk1 enables cells to override control checkpoints and to promote transformation of mammalian cells.1,4 In line with these observations, numerous studies reveal that elevated Plk1 in tumor tissues is well correlated with a poor prognosis of tumor patients.4,5 Moreover, Plk1 has been identified as the only kinase selectively required for the viability of Ras cancer cells in a genome-wide RNA interference screening.6 Thus, Plk1 has been widely considered as one of the most promising targets for molecular intervention. In fact, multiple small-molecule inhibitors targeting the enzymatic kinase domain and the regulatory Polo-box binding domain (PBD) have been recently developed and characterized.1,7-19 In particular, BI 2536 and BI 6727 are the most intensively investigated Plk1 inhibitors.20-25 Poloxin, the first reported non-peptidic inhibitor 1009298-59-2 IC50 of the PBD of Plk1, shows its specificity and anti-proliferative activity in vitro as well as in vivo.15-17 While the preclinical data of Plk1 inhibitors are encouraging, the clinical results are rather less inspiring, showing limited anticancer activity.20,23,26,27 It is of importance to identify the molecules and mechanisms responsible for the sensitivity of Plk1 inhibitors. It has been reported that the cytotoxicity resulting from Plk1 inhibition is elevated in cancer cells with defective p53,28-30 leading to the hypothesis that the inactive p53 might be a predictive marker for sensitive response of Plk1 inhibition. However, in our previous work based on various cancer cell lines with or without functional p53, we demonstrated that inactive p53 is clearly not a predictor for the sensitive response to Plk1 inhibition.15 In contrast, cancer cells with wild type p53 responded more strongly in apoptosis induction than cancer cells without p53.15 We could not exclude the possibility that other circumstances, such as mitotic stress or DNA damage, could render cancer cells with inactive p53 more susceptible to Plk1 inhibitors. In the current work, we have systematically addressed whether mitotic stress, which is very often observed in cancer cells, could affect the efficiency of Plk1 inhibitors in cancer cells with or without functional p53. Results Plk1 inhibitors trigger more apoptosis in HCT116 p53+/+ cells than in HCT116 p53?/? cells under mitotic 1009298-59-2 IC50 stress To address the impact of mitotic stress on the efficiency of Plk1 inhibitors in context of the p53 status of cancer cells, we have chosen the isogenic HCT116 p53+/+ and HCT116 p53?/? cell lines, as they comprise comparable cellular context with the exception of the p53 status and are very well 1009298-59-2 IC50 characterized.31 Microtubule destabilizer nocodazole and vincristine, microtubule stabilizer paclitaxel and the kinesin Eg5 inhibitor monastrol were chosen as mitotic stress inducers for pretreatment. As indicated in the figure legend, all mitotic stress inducers were used in a low dose after performing dose-kinetics, so that they induce mitotic stress but.