An analysis of changes in the expression of cyclins A and B1 by the cell array system during the cell cycle: Comparison between cell synchronization methods

Abstract

Background: A novel high-throughput analysis, cell array system, was developed for an extensive study of the expression of genes and/or the degradation of gene products at the cellular level. To exemplify the usefulness of this system, we showed the changes in the expression level of cyclin A and B1 during the cell cycle in a single experiment. Methods: We used the cell array system to chase the changes in cyclin A and B1 expression during the cell cycle in HeLa cells. Cells were synchronized by mitotic selection and thymidine-hydroxyurea methods. Cells were harvested at intervals of 1 h from 0 through 23 h. These 48 cell samples were spotted on a circle of the cell array glass slide. Cyclin A and B1 were immunologically stained with Alexa Fluor 488, and nuclear DNA was stained with propidium iodide. The amount of cyclins and nuclear DNA

Effects of hydroxyurea and aphidicolin on phosphorylation of ataxia telangiectasia mutated on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis

“…As mentioned earlier, both HU and APH are widely used to arrest cells in S-phase to obtain cell populations synchronized in the cell cycle for a variety of biochemical or molecular studies (2,3,13,14). The present data clearly demonstrate that the cells treated with these inhibitors have a multiplicity of DSBs.…”

“…Its mechanism of action involves inhibition of ribonucleotide reductase, the enzyme which, by converting ribonucleotide diphosphates to deoxyribonucleotide diphosphates, is essential for the de novo synthesis of all the DNA precursors (1). Because of its ability to reversibly inhibit DNA replication, HU is often used to arrest cells in S phase for the purpose of cell cycle synchronization (2,3). However, stalled DNA replication can cause DNA damage and there is evidence that exposure of cells to HU leads to DNA damage (4-7) including formation of DNA double-strand breaks (DSBs) (6).…”

Identification of G1-Regulated Genes in Normally Cycling Human Cells

“…For example, cells synchronized using aphidicolin or mimosine show highly elevated levels of checkpoint regulatory proteins, p53 and p21, and these elevated protein levels persist even after the cells are released from the block [44]. Also, cells synchronized by thymidine and/or hydroxyurea show higher expression levels of cyclins A and B1 in S and G2 phases than in mitotically selected cells progressing normally through the cell cycle [45]. The downstream effects of the elevated levels of important regulatory proteins on cellular signal pathways are likely to be significant.…”

Cytometry in cell necrobiology revisited. Recent advances and new vistas

“…By having many attributes of both flow cytometry and fluorescent imaging, LSC appears to be an optimal instrumentation for multiparameter cell necrobiology studies as described by us earlier (54). LSC has recently found interesting applications in clinical pathology, tissue microarrays, and in the analysis of living-cell microar-rays (55,56). Combination with the latter provides a budding outlook for a very high throughput and multidimensional analysis on rare populations of cells (57—59).…”