Murine pre-B-cells grown in the presence of lower (1 microM) or higher (5 microM) concentration of cadmium chloride were separated into 13 fractions by centrifugal elutriation. The rate of DNA synthesis after cadmium treatment determined in permeable cells was dependent on cell culture density during cadmium treatment. Cell cycle analysis revealed a shift in the profile of DNA synthesis from replicative to repair DNA synthesis upon cadmium treatment. The study of the relationship between cell culture density and cell diameter at lower and higher cell densities in the presence of 1 microM cadmium chloride concentration showed that a. at 5 x 10(5) cell/ml or lower densities cells were shrinking indicating apoptotic changes, b. at higher cell culture densities the average cell size increased, c. the treatment of cells with low CdCl(2) concentration (1 microM) at higher cell culture density (>5 x 10(5) cell/ml) did not change significantly the average cell diameter. At 5 microM cadmium concentration and higher cell culture densities (>5 x 10(5) cell/ml) the average cell size decreased in each elutriated fraction. Most significant inhibition of cell growth took place in early S phase (2.0-2.5 C value). Apoptotic chromatin changes in chromatin structure after cadmium treatment were seen as large extensive disruptions, holes in the nuclear membrane and stickiness of incompletely folded chromosomes.
Exponentially growing human erythroleukemia K562 cells were permeabilized and the dose dependent decrease of DNA synthesis rate was measured after ultraviolet (UV B, 290 nm) irradiation. Cells were able to overcome 2 and 5 J/m2 UV doses, partial recovery was observed at 15 J/m2, while at high (25 J/m2) UV dose replicative DNA synthesis remained suppressed. K562 cells were subjected to synchronization prior to and after UV irradiation (24 J/m2) and 18 fractions were collected by centrifugal elutriation. Cell cycle analysis by flow cytometry did not show early apoptotic cells after UV irradiation. The gradual increase in DNA content typical for non-irradiated cells was contrasted by an early S phase block between 2.2 and 2.4 C-values after UV irradiation. Cell cycle dependent chromatin changes after ultraviolet irradiation were seen as a fine fibrillary network covering the mainly fibrous chromatin structures and incompletely folded primitive chromosomes. Based on observations after UV irradiation and on earlier results with cadmium treatment and gamma irradiation, we confirm that typical chromatin changes characteristic to genotoxic agents can be recognized and classified.
Earlier, the interphase chromatin structures could not be visualized due to the stickiness of the nuclear material. We have reduced stickiness by the reversal of permeabilization allowing the isolation and microscopic imaging of interphase chromatin structures. By using a high resolution of synchronization, collecting 36 elutriation fractions, we show that major intermediates of chromatin condensation include: (a) decondensed veillike chromatin at the unset of the S phase (2.0-2.2 C-value), (b) polarization of veiled chromatin (2.2-2.6 C), (c) fibrous chromatin (2.6-3.0 C), chromatin bodies (3.0-3.3 C), early precondensed chromosomes (3.3-3.6). The compaction of Drosophila chromosomes did not reach that of the mammalian cells in the final stage of condensation (3.6-4.0 C). Drosophila chromosomes consist of smaller units called rodlets. Results demonstrate that nucleosomal chromatin ("beads on string") does not form a solenoid structure; rather, the topological arrangement consists of meandering and plectonemic loops.
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