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 radiationinduced 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.

Objective: In order to elucidate the effects of radiation on centrosome hyperamplification (CH), we examined the centrosome duplication cycle in KK47 bladder cancer cells following irradiation. Methods: KK47 cells were irradiated with various doses of radiation and were examined for CH immunostaining for γ-tubulin. Results: Nearly all control cells contained one or two centrosomes, and mitotic cells displayed typical bipolar spindles. The centrosome replication cycle is well regulated in KK47. Twenty-four hours after 5-Gy irradiation, ∼80% of irradiated cells were arrested in G2 phase, and at 48 h after irradiation, 56.9% of cells contained more than two centrosomes. Laser scanning cytometry performed 48 h after irradiation showed the following two pathways: (1) unequal distribution of chromosomes to daughter cells, or (2) failure to undergo cytokinesis, resulting in polyploidy. With mitotic collection, M-phase cells with CH could be divided into G1 cells with micronuclei and polyploidal cells. Fluorescence in situ hybridization analysis showed clear signs of chromosomal instability (CIN) at 48 h after irradiation. The present study had two major findings: (1) continual duplication of centrosomes occurred in the cell cycle-arrested cells upon irradiation, leading to centrosome amplification; (2) cytokinesis failure was due to aberrant mitotic spindle formation caused by the presence of amplified centrosomes. Abnormal mitosis with amplified centrosomes was detected in the accumulating G2/M population after irradiation, showing that this amplification of centrosomes was not caused by failure to undergo cytokinesis, but rather that abnormal mitosis resulting from amplification of centrosomes leads to cytokinesis block. Conclusion: These results suggest that CH is a critical event leading to CIN following exposure to radiation.

It has been suggested that renal tubular cell damage induced by oxalic acid, one of the components of urinary calculi, may be involved in a variety of ways in the development of urolithiasis. During our study on a calculus related protein, renal prothrombin fragment-1 (RPTF-1), we noted that this is an inflammation related substance that mediates an acute inflammatory reaction, one of the original roles of prothrombin. RPTF-1 is a part of prothrombin that is a coagulation factor known to be expressed in the renal tubule. We examined whether oxalic acid may cause cytotoxic effects on tubular epithelial cells and whether such chemical stimulation may promote the translation of RPTF-1 mRNA into RPTF-1 proteins. We used Madin-Darby canine kidney (MDCK) cells derived from the distal tubule of a dog kidney. In this study, the effects of oxalic acid in culture solution at different concentrations on cytotoxicity were assessed using a MTT assay. The location of active oxygen species was identified using dichlorofluorescein diacetate. After the prothrombin sequence of RPTF-1 was confirmed in MDCK cells, RPTF-1 mRNA expression was determined by RT-PCR. The gene sequence of the same promoter area was ligated, and a luciferase sequence was inserted downstream of the vector. The target sequence was transfected into MDCK cells and the relation between oxalic acid and prothrombin promoter was examined. In addition, the variable expression of RPTF-1 mRNA was quantitatively compared depending on oxalic acid concentrations using real-time PCR. When cytotoxicity was investigated, cells were not damaged but, by contrast, were stimulated and activated under oxalic acid below a certain concentration. The relation between cytotoxicity on the cultured MDCK cell membrane and active oxygen species was confirmed. Luminescence in MDCK cells containing the luciferase gene was detected by the addition of oxalic acid, which activated the prothrombin promoter. A part of the prothrombin gene sequence in the MDCK cells was detected and an increase in the expression of RPTF-1 mRNA in MDCK cells by the addition of oxalic acid was confirmed using real-time PCR. Increased expression of prothrombin by adding oxalic acid has already been demonstrated in previous studies. In this study, however, RPTF-1 mRNA was promoted by oxalic acid and a direct association between oxalic acid and RPTF-1 is indicated. This finding shows that increased oxalic acid in urine induces the expression of RPTF-1 in tubular epithelial cells and thereby causes the generation of active oxygen species.