Itor applied in this experiment showed delayed S and G2/M phase progression and accumulated CyclinB1 in HeLa cells (Fig. S4). We noted that each etoposide and 5FU augmented the cell death effect of Cdc7 inhibition in p53-positive HCT116 but not in p53-negative cells (Fig. 9). It is speculated that cell death for the duration of S phase in Cdc7-inhibited p53-positive HCT116 is additional stimulated by the inhibition of DNA chain elongation through etoposide or 5FU. Meanwhile, in p53-negative HCT116 cells, cell death, induced largely by aberrant M phase progression from G2arrest, will not be affected considerably by the added S phase inhibitions. Equivalent impact of etoposide on cancer cell death induced by Cdc7 depletion was previously reported [41]. These final results suggest potentially effective cancer therapy techniques based on the genotype of tumors. In p53-positive cancer cells, a mixture of inhibitors of DNA replication initiation and genotoxic agents interfering the DNA chain elongation method may very well be an efficient measure for cell death induction, whereas mixture of Cdc7 inhibition with genotoxic agents targeting G2-M phase progression might be an efficient measure in p53negative cancer cells. The latter possibility is now getting tested. In summary, we show that distinctive cell death pathways are induced in cancer cells by inhibition of Cdc7 kinase, depending onthe p53 status (Fig. 10). Cdc7 depletion would induce “defective initiation” which may possibly send checkpoint signals straight to ATM/ ATR or via DNA damages caused by aberrant initiation of DNA replication in the absence of Cdc7. Within the absence of p53, aberrant S phase may well proceed to completion but the Pralidoxime Activator activated checkpoint could induce G2 elongation via MK2, ultimately top to post-mitotic cell death. Inside the presence of p53, the initiation defect triggered by Cdc7 inhibition could predominantly trigger transient G1 or S phase arrest. Aberrant progression into S phase and generation of pathological stalled fork structures under these circumstances may possibly bring about collapsed replication forks and produce lethal DNA damages, leading to cell death in S phase. A p53-induced pro-apoptotic factor might also contribute to cell death. In normal cells with wild-type p53 and all other checkpoint machinery functioning, a defect in initiation would be proficiently detected and stalled ahead of entering abortive S phase, hence permitting the cells to escape from cell death [16,42].Components and Solutions Cell lines and the cells expressing fluorescence-tagged proteinsAll cells including HeLa, U2OS, HCT116 (p53-positive), NHDF and 293T cells were obtained from ATCC, and were maintained as described previously [5,15,19]. Lentiviruses forPLoS A single | plosone.orgCancer Cell Death Induced by Replication Defectexpressing fluorescence-tagged proteins have been generated as described previously [18]. mKO2-CyclinB1 and mKO2-AuroraA expressing plasmids have been constructed by replacing the Cdt1 part of the mKO2-Cdt1 vector with the full-length CyclinB1 and AuroraA, respectively. p53-negative HCT116 cells were obtained from Dr. B. Vogelstein.phosphorylated proteins in accordance with the manufacture’s instruction.Supporting InformationFigure S1 Cdc7 depletion in cancer and normal cells. (A) FACS analyses of HeLa or U2OS cells (10,000 cells for every) treated with manage (green) or Cdc7-D (red) siRNA for times indicated. Sub-G1 population elevated immediately after Cdc7 depletion in each cell lines. (B) FACS analyses of NHDF cells (10,000 cells for every) treat.