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Supplementary Figures 1-6 from Heat Shock Factor 1–Mediated Aneuploidy Requires a Defective Function of p53

Supplementary Figures 1-6 from HSF1 as a Mitotic Regulator: Phosphorylation of HSF1 by Plk1 Is Essential for Mitotic Progression

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]

Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase Cδ (PKCδ)-V5 region with ensuing inhibition of PKCδ activity and PKCδ-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKCδ activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKCδ-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27. [Cancer Res 2007;67(13):6333–9]

Supplementary Methods and Figure Legends 1-6 from Heat Shock Factor 1–Mediated Aneuploidy Requires a Defective Function of p53

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]

Supplementary Figure Legends 1-6 from HSF1 as a Mitotic Regulator: Phosphorylation of HSF1 by Plk1 Is Essential for Mitotic Progression

Supplementary Figures 1-7 from Inhibition of Heat Shock Protein 27–Mediated Resistance to DNA Damaging Agents by a Novel PKCδ-V5 Heptapeptide