Centrosome hyperamplification and chromosomal damage after exposure to radiation.

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 gamma-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, approximately 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.

[The development of continuous crystallizer and observation of formation and growth of calcium oxalate crystals].

A continuous flow crystallizer system of calcium oxalate has been developed. Crystal nucleation rate (B0) and growth rate (G) were calculated by the formula by Randolph and Larson. Nucleation rate, growth rate and crystal mass concentration were decreased with increase of residence time. A linear relationship was observed between growth rate and nucleation rate (B0 = 1.58 x 10(4)G0.924, r = 0.981). Crystals formed in the solution were composed of calcium oxalate dihydrate by infrared spectroscopic analysis. Growth rate and crystal mass did not change between pH 5.0 and 6.5 but increased above pH 7.0. CG-120 and sodium pentosan polysulfate inhibited the nucleation rate, crystal mass concentration but did not influence the growth rate of the crystals.

[Determination of urinary oxalate using a Sigma kit].

Many methods for measuring urinary oxalate have been reported. However, most of them are unsuitable for clinical use because of complexity and difficulty. We tested the value of the Sigma kit for measuring urinary oxalate. In principle, after extraction from urine, oxalate is oxidized to hydrogen peroxide in the presence of oxalate oxidase and the hydrogen peroxide reacts with 3-methyl-2 benzothiozolinone hydrazone and 3-(dimethylamino) benzoic acid in the presence of peroxidase to yield an indamine dye with a maximum absorbance at 590 nm. The linearity of the standard curve and reproducibility of this method were measured and good results were obtained. (Linearity: r = 0.998, within assay: C.V. = 1.5 to 5.0%, between assays: C.V. = 4.0 to 11.6%). The correlation between this method and modified Hodgkinson and Williams' method or ion chromatographic method were 0.922 and 0.796, respectively. The overestimation effect of added ascorbic acid was completely inhibited by FeCl3 which had been added beforehand.