Mechanism of Cancer Cell Death Induced by Depletion of an Essential Replication Regulator

Background: Depletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized. Methodology/Principal Findings: We have used the recently-developed cell cycle indicator, Fucci, to precisely characterize the cell death process induced by Cdc7 depletion. We have also generated and utilized similar fluorescent cell cycle indicators using fusion with other cell cycle regulators to analyze modes of cell death in live cells in both p53-positive and-negative backgrounds. We show that distinct cell-cycle responses are induced in p53-positive and-negative cells by Cdc7 depletion. p53-negative cells predominantly arrest temporally in G2-phase, accumulating CyclinB1 and other mitotic regulators. Prolonged arrest at G2-phase and abrupt entry into aberrant M-phase in the presence of accumulated CyclinB1 are followed by cell death at the post-mitotic state. Abrogation of cytoplasmic CyclinB1 accumulation partially decreases cell death. The ATR-MK2 pathway is responsible for sequestration of CyclinB1 with 14-3-3s protein. In contrast, p53-positive cancer cells do not accumulate CyclinB1, but appear to die mostly through entry into aberrant S-phase after Cdc7 depletion. The combination of Cdc7 inhibition with known anti-cancer agents significantly stimulates cell death effects in cancer cells in a genotype-dependent manner, providing a strategic basis for future combination therapies

The CyclinB1 protein level and Cdc2-CyclinB1 kinase activity decrease in p53-positive HCT116 cells after Cdc7 depletion.

<p>(<b>A</b>) p53-positive or -negative HCT116 cells were treated with control or Cdc7-D siRNA for 48 hrs, and whole cell extracts were analyzed by western blotting. (<b>B</b>) CSK-soluble extracts were prepared from the same cells as in (<b>A</b>) and immunoprecipitation was conducted with anti-CylinB1 antibody. Cdc2-CyclinB1 kinase activity was measured with Histone H1 as a substrate (upper panel), as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036372#s4" target="_blank">Materials and Methods</a>”. The graph below shows quantification of the level of phosphorylation. Lower panel, western blotting analyses of CyclinB1 proteins in the immunoprecipitates used for kinase assays. (<b>C</b>) p53-positive (left) or -negative (right) HCT116 cells expressing mKO2-CyclinB1 were treated with indicated siRNA and time lapse images were recorded. The time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signal and its translocation into the nucleus was measured in the time lapse images. The P-values of the two-tailed unpaired t-test was calculated by Prism software.</p

CyclinB1 does not accumulate in cytoplasm in Cdc7-depleted U2OS cells.

<p>(<b>A</b>) U2OS cells expressing mKO2-CycinB1 were treated with siRNAs and time lapse images were recorded by LCV100. The time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signal and its translocation into the nucleus was measured in the time lapse images of each cell population. In Cdc7-depleted U2OS, CyclinB1 does not accumulate in cytoplasm. However, co-depletion of Cdc7 and p53 caused CyclinB1 accumulation in cytoplasm for a longer period. The P-values of the two-tailed unpaired t-test were calculated by Prism software. (<b>B</b>) Western analysis of the whole cell extracts of U2OS cells treated with indicated siRNAs for 48 hrs. A phosgel was used for the detection of MK2. Other proteins were detected on a 4–12% gradient gel.</p

14-3-3σ sequesters CyclinB1 in the cytoplasm in Cdc7-depleted HeLa cells.

<p>(<b>A</b>) HeLa cells were treated with control or Cdc7-D siRNA for 24 hrs. Extracts were prepared and the immunoprecipitates with anti-CyclinB1 antibody (lanes 1 and 2) and input extracts (lanes 3 and 4; 20% of the extract used for immunoprecipitation) were analyzed by western blotting. (<b>B</b>) HeLa cells were treated with control or Cdc7-D siRNA, followed by transfection of a Flag-tagged 14-3-3σ-expressing plasmid. Extracts were prepared at 48 hrs after siRNA transfection and the immunoprecipitates with anti-Flag antibody (lanes 1 and 2) or normal (control) IgG (lanes 3 and 4) and input extracts (lanes 5 and 6; 17% of the extract used for immunoprecipitation) were analyzed by western blotting. The binding of Cdc2/CyclinB1 with 14-3-3σ increases after Cdc7 siRNA treatment, suggesting that 14-3-3σ retains CyclinB1 in the cytoplasm after Cdc7 depletion in HeLa cells. (<b>C</b> and <b>D</b>) HeLa cells were treated with Cdc7-D siRNA, 14-3-3σ and Cdc7-D siRNAs, 14-3-3σ siRNA and control siRNA for 48 hrs. (<b>C</b>) Western blot analysis of the whole cell extracts. (<b>D</b>) DNA contents of the cells in <b>C</b> were analyzed by FACS (10,000 cells for each) and the fraction of the cells in each cell cycle stage is presented. (<b>E</b>) HeLa cells expressing mKO2-CyclinB1 were treated with Cdc7-D and/or 14-3-3σ siRNA as indicated. The time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signals and its translocation into the nucleus was measured using the time lapse images, which started at 24 hrs after siRNA transfection. The P-value of the two-tailed unpaired t-test was calculated by Prism software. Co-depletion of 14-3-3σ led to a decrease in the overall CyclinB1 protein level (<b>C</b>), reduced cell death (<b>D</b>), and reduced the duration of its cytoplasmic retention (<b>E</b>).</p

MK2 is activated in Cdc7-depleted HeLa cells and is required for cytoplasmic accumulation of CyclinB1.

<p>(<b>A</b>) HeLa (lanes 1, 2, 7 and 8), U2OS (lanes 3 and 4) and NHDF (lanes 5 and 6) cells were treated with control or Cdc7 siRNA and the whole cell extracts were run on a phosgel and analyzed by western blotting. Lanes 7 and 8, extracts from Cdc7 siRNA-treated HeLa cells were non-treated (−) or treated with λ-phosphatase (+). Arrowheads indicate the phosphorylated MK2 band. (<b>B</b>) HeLa cells were treated with control siRNA (lanes 1 and 5), Cdc7 siRNA (lanes 2 and 6), Cdc7 and MK2 siRNAs (lanes 3 and 7) and MK2 siRNA (lanes 4 and 8) for 48 hrs, and CSK-soluble (lanes 1–4; Sup) and -insoluble (lanes 5–8; Ppt) proteins were analyzed by western blotting. Tubulin and LaminB are shown as loading controls. (<b>C</b>) Glutathion Sepharose 4B beads carrying GST-14-3-3σ protein was incubated for 1 hr at 4°C with CSK-soluble extracts of HeLa cells treated with siRNA, as shown. Bound proteins were examined by Western blotting. “Input” represents only the extracts without added GST-14-3-3σ protein. Cdc7 and MK2 co-depletion reduced the binding between 14-3-3σ and Cdc2/CyclinB1. (<b>D</b>) HeLa cells expressing mKO2-CyclinB1 were treated with indicated siRNAs. The time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signal and its translocation into the nucleus was measured in the time lapse images. Co-depletion of MK2, p38 (upstream kinase of MK2) or ATR reduced cytoplasmic retention of CyclinB1 (G2 elongation) was observed in Cdc7-depleted HeLa cells. The P-values of the two-tailed unpaired t-test were calculated by Prism software. Cdc7-D siRNA was used in all the experiments.</p

Cdc7 depletion in HeLa cells leads to accumulation of cytoplasmic CyclinB1.

<p>(<b>A</b>) HeLa cells were cultured on cover glasses, transfected with control or Cdc7-D siRNA for 48 hrs, fixed with 4% paraformaldehyde and stained by anti-CyclinB1 antibody followed by FITC-conjugated anti-mouse IgG and Hoechst33342. Left, Cdc7 siRNA; right, control siRNA. Green, CyclinB1; blue, DNA. Photos were taken by FSX100 Olympus microscopy. Bar, 16 µm. (<b>B</b>) More than 3,000 cells were examined and cells with nuclear CyclinB1 signals were scored and the fractions are presented. “n” represents the numbers of independent experiments conducted. (<b>C</b>) HeLa cells expressing mKO2-CyclinB1 were treated with Cdc7-D siRNA or control siRNA. Time lapse images were recorded by Olympus LCV100 (<b>movies S3 and S4</b>). Images taken from the time lapse data at the times indicated are presented. Upper, control siRNA; lower, Cdc7 siRNA. Red signals show mKO2-CyclinB1. The control siRNA-treated cells indicate those undergoing periodic cytoplasmic appearance, nuclear transfer, and degradation (upper panel), whereas the Cdc7 siRNA-treated cells show persistent strong cytoplamic signals for a long period (lower panel). Numbers in each panel show time (hrs) after siRNA treatment. Bar: 20 µm. (<b>D</b>) The time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signal and its translocation into the nucleus was measured in the time lapse images of control or Cdc7-D siRNA treated HeLa cells. The P-value of the two-tailed unpaired t-test was calculated by Prism software.</p

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.</p

Coadministration of conventional anti-cancer drugs enhances cell death in a p53-dependent manner.

<p>(<b>A</b>) p53-positive (upper) or -negative (lower) HCT116 cells were treated with Cdc7-D or control siRNA for 24 hrs, followed by treatment with DMSO or 10 µM etoposide (Wako) for 32–40 hrs. (<b>B</b>) p53-positive (upper) or -negative (lower) HCT116 cells were treated with indicated chemicals (10 µM etoposide or 5FU (Wako) and 1 µM Cdc7 inhibitor (Calbiochem)) for 16 hrs. In A and B, DNA contents were analyzed by FACS (10,000 cells for each) and the fractions of sub-G1 population were calculated and presented. Synergistic effect of etoposide and 5FU on cell death induced by Cdc7 inhibition was observed in a p53-positive HCT116 but not in p53-negative HCT116. (<b>C</b>) Colony formation assays were conducted in p53-positive HCT116 cells. Cells treated with drugs for 16 hrs were collected and 500 cells were seeded in a 3.5 cm plate and cultured. The numbers of colonies were counted after 7 days. “n” represents the numbers of independent experiments conducted.</p

FoxM1 mRNA level increases after Cdc7 depletion in HeLa and p53-negative HCT116.

<p>(<b>A</b>) HeLa cells were treated with indicated siRNAs for 24 hrs. FoxM1 (left), Plk1 (middle) and CyclinB1 (right) mRNA levels are presented. (<b>B</b>) Western analysis of the whole cell extracts of HeLa cells treated with indicated siRNAs for 48 hrs. A phosgel was used for the detection of MK2. Other proteins were separated on a 4–12% gradient gel. (<b>C</b>) The FoxM1 mRNA levels of HCT116 (p53-positive and -negative) cells treated with control or Cdc7 siRNA for 24 hrs. In A and C, mRNA levels were quantified by real time-PCR and the relative values normalized by the level of GAPDH mRNA are presented. (<b>D</b>) HeLa cells treated with indicated siRNAs for 48 hrs were fixed with 4% paraformaldehyde for 10 min and stained with anti-CyclinB1 antibody. Fractions of the cells showing nuclear localization of CyclinB1 are shown. Cdc7-D siRNA was used in these experiments.</p

Expression of nuclear localization signal-targeted CyclinB1 partially reduces cell death in Cdc7-depleted HeLa cells.

<p>(<b>A</b>) HeLa cells expressing mKO2-NLS-CyclinB1 (left) or mKO2-CyclinB1 (right) were treated with control or Cdc7-D siRNA and were monitored by Olympus LCV100. Dead cells were counted in the time lapse images at the times indicated. Cell death was suppressed in mKO2-NLS-CyclinB1 expressing HeLa cells up to 72 hrs (at which half of the Cdc7-depleted HeLa cells are usually dead). mKO2-NLS-CyclinB1 plasmid was constructed by inserting the NLS sequence (PPKKKRKVEDP) from the SV40 large T antigen into the mKO2-CyclinB1 plasmid between mKO2 and CyclinB1. About 200 or 60 cells expressing mKO2-NLS-CyclinB1 or mKO2-CyclinB1, respectively, were counted. “n” represents the numbers of independent experiments conducted. (<b>B</b>) HeLa cells expressing mKO2-CyclinB1 (WT) or mKO2-NLS-CyclinB1 (NLS) were treated with control or Cdc7-D siRNA and the duration of CyclinB1 expression before entry into M phase was measured in the time lapse images. Upon Cdc7 depletion, NLS cells did not accumulate the tagged CyclinB1 in cytoplasm, and continued through the cell cycle more or less normally. (<b>C</b>) HeLa cells were treated with indicated siRNAs for 48 hrs. DNA contents were analyzed by FACS (10,000 cells for each) and the fractions of sub-G1 population were calculated and presented. Co-depletion of CyclinB1 reduced the cell death induced by Cdc7-D siRNA in HeLa cells. (<b>D</b>) HeLa cells expressing mKO2-CyclinB1 (WT) were treated with Cdc7-D or Cdc7-D+CyclinB1 siRNA and the time (hr) between the first appearance of cytosolic mKO2-CyclinB1 signal and its translocation into the nucleus was measured in the time lapse images of each cell population. Down-regulation of CyclinB1 expression shortened the G2 arrest induced by Cdc7 depletion. In (B) and (D), the P-values of the two-tailed unpaired t-test were calculated by Prism software.</p