Figure 1.
HCT116 clones vary in sensitivity to cisplatin.
A) HCT116 cells were plated at single cell density and ten individual clones (designated D1–D10) were isolated and expanded. The clones were exposed to 15 µM Cisplatin for 72 hrs, and apoptosis determined by the percentage of cells with decreased mitochondrial membrane potential (% low ΛΨm), sub-G1 DNA content, or Annexin-V positive staining. (H) refers to the parental HCT116 cell population. Data represent the average of 3 experiments +/− s.e.m. B) Clones D3 and D6 were treated with 5 or 15 µM cisplatin for 24 hrs, followed by cisplatin removal. Percent colony formation was determined 2 weeks after cisplatin removal. Plotted is the average of 3 separate experiments +/− s.e.m.
Figure 2.
Cisplatin resistant HCT116 clones display a prolonged 4N arrest in response to cisplatin.
A) HCT116 clones that are relatively cisplatin sensitive (D3, D8) or relatively cisplatin resistant (D6, D7) were untreated (NT) or treated with 15 µM cisplatin for 1–3 days. Cell cycle profiles were determined by flow cytometry. B) The percentage of sub-G1, 2N, S-phase, 4N, or greater than 4N cells was determined at the indicated time points in cisplatin treated cells. Numbers represent the average of 3 separate experiments +/− s.e.m.
Figure 3.
UCN01 abrogates CP-induced G2 arrest and enhances killing of CP-resistant HCT116.
The CP resistant clone D6 was untreated (NT) or treated with 15 µM cisplatin for 6 hrs followed by cisplatin removal for 18 hrs (CP). The cisplatin was then removed by media change (−CP), and the cells were either untreated, treated with UCN01 alone, or treated with UCN01 plus colcemid for 6, 24, or 48 hrs. A) Cell cycle profiles were examined at the indicated time points. B) The percentage of sub-G1, 2N, S-phase, 4N, or greater than 4N cells was determined and is graphed.
Figure 4.
UCN01 sensitizes CP-resistant HCT116 clones to CP-induced killing.
HCT116 clones D6 and D7 were treated with either CP alone for 72 hrs, UCN01 alone for 72 hrs, or treated with CP alone for 24 hrs and then treated with UCN01 in the continued presence of CP for an additional 48 hrs. A) Cell cycle analysis was determined at the 72 hr time point. Representative cell cycle profiles are shown. The number in the upper right corner is the percentage of sub-G1 cells. B) Plotted is the percentage of sub-G1 (apoptotic) cells at the 72 hr time point. The data represent the average of 3 experiments +/− s.e.m. C) Clones D6 and D7 were either mock-transfected (MT) or transfected with control siRNA (siC) or siRNA against Chk1 and/or Chk2. Immunoblotting was performed 24 hrs after transfection. D) Clones D6 and D7 were transfected with the indicated siRNA for 24 hrs, followed by cisplatin (15 µM) treatment for an additional 72 hrs. Representative cell cycle profiles are shown. The number in the upper right corner is the percentage of sub-G1 (apoptotic) cells.
Figure 5.
Cisplatin induces a tetraploid G1 arrest that is p53 and p21 dependent.
A) HCT116 clones D6 and D7 were treated with 15 µM cisplatin (CP) for 24 or 48 hrs, followed by immunoblotting for the indicated proteins. Actin levels were used as a loading control. B) HCT116 clones D6 and D7 mock transfected (MT) or transfected with control siRNA or siRNA targeting p53 or p21. Twenty four hrs after transfection, the cells were treated with cisplatin (15 µM) for an additional 48 hrs. Protein lysates examined by immunoblotting.
Figure 6.
P21 knockdown abrogates the tetraploid G1 arrest induced by cisplatin and sensitizes cells to p53-dependent killing.
A) HCT116 clones were either untreated (NT) or treated with 15 µM cisplatin. At the 24 hr time point after cisplatin treatment, cells were either mock transfected (MT) or transfected with the indicated siRNAs, and cultured in the continued presence of cisplatin for an additional 48 hrs. Representative cell cycle profiles are shown. The number in the upper right corner is the percentage of sub-G1 cells. B) Plotted is the percentage of sub-G1 (apoptotic) cells from the experiments in A). The data represent the average of 3 experiments +/− s.e.m.