DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to gamma radiation, the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei increases. The majority of the gamma-H2AX-positive micronuclei are centromere-negative. The number of gamma-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most gamma-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are gamma-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, gamma-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with gamma-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with gamma-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with gamma-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.

We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures. In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined. Dry cell culture vessels were preirradiated with 2 Gy of either alpha particles or X rays and immediately plated with nonirradiated cells. About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or alpha-particle radiation. Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells. In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments.

Background: Cell migration is one of the important processes, where cytoskeleton takes the main part. Vimentin intermediate filaments are also implicated in cell migration and adhesion, but their role is not clear yet. So, earlier we have investigated the influence of different vimentin forms (knocked out, with mutation that blocks filament formation on the stage of ULFs, and normal filaments) on the polarity determination in rat fibroblasts. Based on these results we have shown the principle role of vimentin even in the form of ULFs in determination of the cellular polarity and the directionality of cell migration. But ULFs are prone to aggregate, so in order to get their structure by electron microscopy we decided to investigate the influence of different polymers on prevention of ULFs aggregation. Methods: To investigate the aggregation of ULFs we used the method of dynamic light scattering. The polymers such as dextran sulfate and heparin were mixed with filaments in different concentrations to prevent the aggregation. Results: The ULFs assembly was conducted by adding of high salt concentration (160 mM NaCl). The process of aggregation was determined, mainly, at 37 0 C, and during various time periods-15 minutes and 1 hour. The results revealed the start of aggregation after 15 minutes of incubation. The hydrodynamic diameters of aggregated ULFs were from 120 nm till 350 nm depending on the incubation time, while the approximate hydrodynamic diameter of single ULF is 70-80 nm. Addition of polymers, such as dextran sulfate and heparin at different concentrations, maintained the ULFs in separate mode during one-hour incubation at 37 0 C with hydrodynamic diameter of 80 nm. Conclusion: Based on the obtained results we can conclude that both polymers, dextran sulfate and heparin, positively influence on ULFs separation and prevent their aggregation. Thus, this approach may help us for getting the single vimentin ULFs by electron microscopy in order to determine their structure.

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