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

Abstract

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.

Figure 1

Western blotting analysis of CCD32SK cells following irradiation (IR). Whole‐cell lysates (10 µg) were loaded into each lane. Actin was used as the loading control. siRNA, small interfering RNA.

Figure 2

Radiation‐induced abnormal nuclear shapes in p53 short interfering RNA (siRNA)‐treated CCD32SK cells. (a) Propidium iodide (PI) staining (×400, scale bar = 10 µm). (b) DNA histogram produced using laser scanning cytometry. IR, irradiation; siRNA, small interfering RNA.

Figure 3

Radiation‐induced abnormal amplification of centrosomes in p53 short interfering RNA (siRNA)‐treated CCD32SK cells. (□), n = 1; (▒), n = 2; (▪), n ≥ 3. DAPI, 4′‐diamidino 2‐phenylindole; IR, irradiation; PI, propidium iodide.

Figure 4

Representative γ‐tubulin immunostaining images of radiation‐induced abnormal amplification of centrosomes in p53 short interfering RNA (siRNA)‐treated CCD32SK cells (scale bar = 10 µm).

Figure 5

Cell cycle analysis of short interfering RNA (siRNA)‐treated CCD32SK cells. (▪), M phase; (▒), G₂ phase; (▓), interphase (G₁ + S); (□), post‐mitotic cells. IR, irradiation.

Figure 6

Centrosome duplication throughout the cell cycle determined by laser scanning cytometry in p53 short interfering RNA‐treated CCD32SK cells without irradiation. (a) G₁ phase; (b) S phase; (c) G₂ phase; (d) M phase. Numbers (%) in the figure represent the incidence of abnormal amplification of centrosomes (AAC) for each cell‐cycle stage (original magnification ×600). PI, propidium iodide.

Figure 7

Centrosome duplication throughout the cell cycle determined by laser scanning cytometry in p53 short interfering RNA‐treated CCD32SK cells at 48 h after 5‐Gy irradiation. (a) Post‐mitotic cell; (b) sub‐G₁ phase; (c) G₁ phase; (d) S phase; (e) G₂ phase; (f) prometa phase; (g) meta phase. Numbers (%) in the figure represent the incidence of abnormal amplification of centrosomes (AAC) for each cell cycle stage (original magnification ×600). PI, propidium iodide.