I. Induction of Apoptosis Protocol for DNA Damage-Induced Apoptosis (48 h) The following protocol is based on p53-dependent G1-arrest that occurs in response to DNA damage by chemical agents such as doxorubicin, 5-fluorouracil, paclitaxel, and vinblastine. A typical time course for p53 and p21WAF1 induction is 40 to 48 hours treatment with a DNA-damaging agent. Other proteins involved in apoptosis are also induced (although not all proteins involved in apoptosis will be induced by a particular agent in a given cell type). We recommend taking several time points (i.e., 24, 48, and 72 hours). Maximal induction of p21WAF1 requires wild-type p53 activity. In the absence of wild-type p53, p21WAF1 can also be induced by serum stimulation of G1-arrested cells or by treatment with agents such as dexamethasone, albeit at significantly lower levels than that seen upon p53-dependent induction.
Day 1: Inoculate 2 or more 10-cm tissue culture dishes for adherent cells 1 x 106 cells/dish or T-75 flasks for non-adherent cells with approximately (1-5 x 105 cells/ml). One dish or flask will be used as a negative control for uninduced or basal level expression.
Day 2: Confirm that cells are growing by visual inspection of tissue culture dishes or by viable cell counts on non-adherent cells in T-75 flasks. Add DNA damaging agents at the indicated final concentration. Add appropriate volume of buffer or solvent to the uninduced control.
Day 3: Check cells to determine if cells have begun to die. If too few cells are dead, incubate for additional time and check again. Harvest cells if greater than 75% of the cells appear to have died.
Day 4: Harvest cells and prepare lysates for either immunoblotting or immunoprecipitation. For any agent used, a time course of induction can be performed by inoculating additional dishes or flasks and harvesting at various times after addition of the DNA damaging agent.
Day 5: Resolve proteins on SDS-PAGE. Visualize the protein of interest from total lysates by immunoblotting using chemiluminescent detection.
Always compare levels of p53 or p21WAF1 from treated cells with those from untreated controls to confirm induction. For g irradiation treatment to induce p53 and p21WAF1. (See El-Deiry, et al. 1994. Cancer Res. 54, 1169 or Deng, et al. 1995. Cell82, 675.)
