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Because heat shock factor 1 (HSF1) phosphorylation by Plk1 has been previously reported to be involved in mitotic regulation and p53 function may be involved in this mitotic regulation, we have further examined HSF1 functions in mitotic regulation according to p53 status. Nocodazole-mediated aneuploidy was increased in p53-defective (p53Mut) cells; however, it was not increased in p53 wild-type (p53WT) cells. Phosphorylation of HSF1 at Ser216 was increased in p53Mut cells with increased stability of securin and cyclin B1 in mitosis compared with p53WT cells. The interaction of p53 with Plk1 that was shown in p53WT cells and that induced normal mitotic checkpoint function was not observed in p53Mut cells; instead, the binding of HSF1 with Plk1 and HSF1 phosphorylation at Ser216 were seen in p53Mut cells, which resulted in increased aneuploidy production. Moreover, the interaction affinity of Cdc20 with Mad2 was inhibited in p53Mut cells, whereas the interaction between Cdc20 and HSF1 was increased. From the data, it was suggested that HSF1-mediated aneuploidy was more facilitated in p53-defective cells, indicating the importance of novel mechanisms for p53 function in HSF1-mediated mitotic regulation and genomic instability. [Cancer Res 2009;69(24):9404–12]

Although mechanisms of arsenic trioxide (As2O3)-induced cell death have been studied extensively in hematologic cancers, those in solid cancers have yet to be clearly defined. In this study, we showed that the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for As2O3-induced cell death in human cervical cancer cells. We also showed that reactive oxygen species (ROS)-mediated poly(ADP-ribose) polymerase-1 (PARP-1) activation is necessary for AIF release from mitochondria. The treatment of human cervical cancer cells with As2O3 induces dissipation of mitochondrial membrane potential (Δψm), translocation of AIF from mitochondria to the nucleus, and subsequent cell death. Small interfering RNA targeting of AIF effectively protects cervical cancer cells against As2O3-induced cell death. As2O3 also induces an increase of intracellular ROS level and a marked activation of PARP-1. N-acetyl-l-cystein, a thiol-containing antioxidant, completely blocks As2O3-induced PARP-1 activation, Δψm loss, nuclear translocation of AIF from mitochondria, and the consequent cell death. Furthermore, pretreatment of 1,5-dihydroxyisoquinoline or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, PARP-1 inhibitors, effectively attenuates the loss of Δψm, AIF release, and cell death. These data support a notion that ROS-mediated PARP-1 activation signals AIF release from mitochondria, resulting in activation of a caspase-independent pathway of cell death in solid tumor cells by As2O3 treatment.

Previously, heat shock factor 1 (HSF1) had been reported to induce genomic instability and aneuploidy by interaction with Cdc20. Here, we have further examined the functions of HSF1 in the regulation of mitosis. A null mutant or knockdown of HSF1 caused defective mitotic progression. By monitoring chromosomes in living cells, we determined that HSF1 was localized to the centrosome in mitosis and especially to the spindle poles in metaphase. HSF1 was phosphorylated by Plk1 at Ser216 of the DSGXXS motif during the timing of mitosis and a phospho-defective mutant form of HSF1 inhibited mitotic progression. Phosphorylated HSF1 during spindle pole localization underwent ubiquitin degradation through the SCFβ-TrCP pathway. However, binding of HSF1 with Cdc20 stabilized the phosphorylation of HSF1. Moreover, SCFβ-TrCP–mediated degradation only occurred when phosphorylated HSF1 was released from Cdc20. HSF1 phosphorylation at Ser216 occurred in the early mitotic period with simultaneous binding of Cdc20. The interaction of HSF1 with SCFβ-TrCP was followed and then the interaction of APC/Cdc20 was subsequently observed. From these findings, it was shown that Plk1 phosphorylates HSF1 in early mitosis and that the binding of phosphorylated HSF1 with Cdc20 and ubiquitin degradation by SCFβ-TrCP regulates mitotic progression. [Cancer Res 2008;68(18):7550–60]

Although mechanisms of arsenic trioxide (As(2)O(3))-induced cell death have been studied extensively in hematologic cancers, those in solid cancers have yet to be clearly defined. In this study, we showed that the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for As(2)O(3)-induced cell death in human cervical cancer cells. We also showed that reactive oxygen species (ROS)-mediated poly(ADP-ribose) polymerase-1 (PARP-1) activation is necessary for AIF release from mitochondria. The treatment of human cervical cancer cells with As(2)O(3) induces dissipation of mitochondrial membrane potential (Deltapsi(m)), translocation of AIF from mitochondria to the nucleus, and subsequent cell death. Small interfering RNA targeting of AIF effectively protects cervical cancer cells against As(2)O(3)-induced cell death. As(2)O(3) also induces an increase of intracellular ROS level and a marked activation of PARP-1. N-acetyl-l-cystein, a thiol-containing antioxidant, completely blocks As(2)O(3)-induced PARP-1 activation, Deltapsi(m) loss, nuclear translocation of AIF from mitochondria, and the consequent cell death. Furthermore, pretreatment of 1,5-dihydroxyisoquinoline or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone, PARP-1 inhibitors, effectively attenuates the loss of Deltapsi(m), AIF release, and cell death. These data support a notion that ROS-mediated PARP-1 activation signals AIF release from mitochondria, resulting in activation of a caspase-independent pathway of cell death in solid tumor cells by As(2)O(3) treatment.