Technical Tips: Protocols for Apoptosis Research - Induction of Apoptosis
 |
1. Chemical Induction of Apoptosis Top
Proteins such as p53, p21WAF1, Myc, Bcl-2, Bax, and Bak are involved in the regulation of apoptosis. The agents and doses listed in the table below can be used to induce apoptosis. Not every agent will induce apoptosis in every cell type; indeed, dexamethasone will actually stimulate growth of some cells. Depending on the agent selected and the concentrations used, maximal induction of a particular protein may occur within 8 to 72 hours post-treatment. We have found that not all proteins are affected by each of these reagents in a particular cell line. Even proteins involved in preventing apoptosis, such as Bcl-2, may induce apoptosis by these treatments (but with a different time course).
Agent | Dose | Stock Solvent | Cat. No. |
|---|
| Actinomycin D | 500 ng/ml | Methanol | | | Aphidicolin | 2 mg/ml | DMSO | | | A23187 | 10 mM | DMSO | | | Caffeine | 16 mM | Boiling H2O | | | Camptothecin | 4 mg/mL | DMSO | | | Cycloheximide | 100 mg/mL | H2O | | | Dexamethasone | 1 mM | Ethanol | | | Doxorubicin (Adriamycin) | 0.2 mg/mL | H2O | | | 5-Fluorouracil | 25 mg/mL | DMSO , Hot H2O | | | Hydroxyurea | 500 nM | H2O | | | Staurosporine | 500 nM | DMSO | | | Paclitaxel (TAXOL®) | 100-580nM | DMSO | | | Thymidine | 2 mM | PBS | | | Vinblastine | 60 nM | Methanol | |
48 hour protocol for DNA Damage-Induced Apoptosis:
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 each of 2 or more 10 cm tissue culture dishes for adherent cells or T-75 flasks for non-adherent cells with approximately 1 x 106 cells. One dish or flask will be used as 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 to the indicated final concentration. Add appropriate volume of bufferor solvent (i.e. DMSO) to the uninduced control.
Day 3: Check cells to determine if cells have begun to die. Harvest cells if greater than 75% of the cells appear to have died upon visual inspection.
Day 4: Harvest cells and prepare lysates for either Western blotting or immunoprecipitation as described in the accompanying protocols. 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. For the examination of apoptotic proteins, dead cells should also be collected.
Day 5: Resolve proteins on SDS-PAGE. Visualize the protein of interest from total lysates by Western blotting using chemiluminescent detection as described in the accompanying protocols.
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 Research 54, 1169-1174 or Deng, et al. 1995, Cell 82, 675-684.
|
