Cdc7 depletion in cancer cells induces cell death: effect on Cdc2-CyclinB1 and mitosis.

<p>(<b>A and B</b>) HeLa (<b>A</b>) or U2OS (<b>B</b>) cells expressing Fucci were treated with control or Cdc7-D siRNA, and time lapse image was recorded with Olympus LCV100 (<b>movies S1 and S2</b>). Images taken from the time lapse data at the times indicated are presented. The uppermost panels (control siRNA) indicate cells undergoing normal cell division. Numbers in each panel show time (hrs) after siRNA transfection. Lower two panels (<b>a</b> and <b>b</b>) show Cdc7 siRNA treated cells. Some cells died in red color (G1 phase, <b>a</b>), and other cells died in green (S/G2/M phase, <b>b</b>). Lengths of cell cycle stages are indicated in the panels (G1, arrowed broken lines; S/G2/M, arrowed solid lines). Bar, 20 µm. (<b>C</b>) Dead cells in Cdc7 siRNA-treated HeLa-Fucci (left, 324 cells) or U2OS-Fucci (right, 180 cells) were counted from the time lapse data to determine the fractions of the dead cells in red and in green. Cell death occurs at both G1 and S/G2/M phases in Cdc7 siRNA treated cancer cells. (<b>D, E and F</b>) HeLa cells were transfected with control or Cdc7-D siRNA and were harvested at 48 hrs (<b>D</b>) or at the times indicated (<b>E and F</b>). The whole cell extracts (<b>D</b>) or CSK-soluble extracts (<b>E</b>) were analyzed by western blotting using the antibodies indicated. (<b>F</b>) Cdc2-CyclinB1 kinase activity was measured using the CSK-soluble extracts. The immunoprecipitates (IP) used for the assays and the input extracts were analyzed by western blotting. The extent of Cdc7 depletion was similar between HeLa and U2OS. Cdc7 was not detectable by western after siRNA treatment in both cells. (<b>G</b>) HeLa cells were treated with control or Cdc7-D siRNA for indicated times, collected, washed with PBS, swollen in 75 mM KCl for 20 min at 37°C, and fixed with glacial acetic acid/methanol (1∶3) solution three times. Fixed chromosomes were dropped on a slide glass, air dried and stained with 5% Giemsa's solution in 1/15 M PBS. Spread chromosomes were observed under All-in-One microscopy (Keyence). The mitotic cells with aberrantly condensed chromosomes were counted and the fractions are presented. The insets show representative images of aberrantly condensed chromosomes observed in a Cdc7 siRNA treated HeLa cell (left) and properly condensed chromosomes observed in a control cell (right). Bar, 50 µm. (<b>H</b>) HeLa cells were treated with control or Cdc7-D siRNA for 48 hrs, washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature and then stained with Hoechst 33342. Cells were examined under confocal microscopy LSM510 (1427 cells [Cdc7] and 1023 cells [control]), and the cells in M phase stages were scored. The fractions of cells in each mitotic stage are presented. (<b>I</b>) Spread and fixed chromosomes prepared in U2OS as described above were observed by FSX100 Olympus microscopy. No significant difference was observed in mitotic cells after Cdc7 depletion. However, the numbers of apoptotic cells increased in Cdc7-depleted U2OS cells. Bar, 32 µm. In C, G and H, “n” represents the numbers of independent experiments conducted.