Induction of catastrophic cell death in p53 siRNA-treated human fibroblast cells by hyperthermia

Abstract

Research has shown that high temperatures can damage and kill cancer cells, usually with minimal injury to normal tissue cells. However, the precise mechanism of hyperthermia-induced cell death is not yet fully under stood. CCD32SK diploid normal human fibroblasts were transiently transfected with short interfering RNA (siRNA) specific for human p53 (CCD/p53i) and negative siRNA (CCD/NEGi) for 24h. After p53 siRNA transfection (final concentration: 100 nM), CCD32SK cells were heated at 45°C for 0.5h. After the 45°C hyperthermia treatment, CCD/NEGi cells exhibited normal nuclear shapes, and almost all cells were dead at 120h. In contrast, CCD/p53i cells showed a marked increase in abnormal nuclear shapes after hyperthermia. While CCD/NEGi cells were arrested in G1 and G2 after heat shock, significant numbers of mitot ic cells with multiple poles appeared in CDD/p53i cells. Subsequently, cells with multiple micronuclei increased in number as tumor cells, with time, and after heat shock. We then investigated the induction of centrosome amplification by p53 siRNA transfection after hyperther mia. There was a small increase in the frequency of centrosome amplification in CCD/p53i cells (5.0%) without hyperthermia treatment. In contrast, at 48 h after hyperthermia treatment, CCD/p53i cells exhibited pronounced centrosome amplification (45%). In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, and,together, with DNA damage by hyperthermia, centrosome amplification and mitotic catastrophe is efficiently induced. However, these phenomena are not induced by either siRNA-mediated silencing of p53 or hyper thermia alone.