Induction of centrosome amplification in p53 siRNA-treated human fibroblast cells by radiation exposure.

Centrosome amplification can be detected in the tissues of p53(-/-) mice. In contrast, loss of p53 does not induce centrosome amplification in cultured human cells. However, examination of human cancer tissues and cultured cells has revealed a significant correlation between loss or mutational inactivation of p53 and occurrence of centrosome amplification, supporting the notion that p53 mutation alone is insufficient to induce centrosome amplification in human cells, and that additional regulatory mechanisms are involved. It has recently been shown that gamma irradiation of tumor cells induces centrosome amplification. However, the precise mechanism of radiation-induced centrosome amplification is not fully understood. In the present study, CCD32SK diploid normal human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human p53 (CCD/p53i). There was a small increase in the frequency of centrosome amplification in CCD/p53i cells (4.0%) without irradiation. In contrast, CCD/p53i cells after 5-Gy irradiation showed a marked increase in abnormal nuclear shapes and pronounced amplification of centrosomes (46.0%). At 12 h after irradiation, irradiated CCD/p53i cells were arrested in G(2) phase. By laser scanning cytometry, abnormal mitosis with amplified centrosomes was observed frequently in the accumulating G(2)/M population at 48 h after irradiation. In the present study, we found that siRNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by irradiation, efficiently induced centrosome amplification and nuclear fragmentation, but these phenomena were not observed with either siRNA-mediated silencing of p53 or irradiation alone.