| Chemical Induction of Apoptosis - 1 May 2001 |
| p53, p21WAF1, Myc, Bcl-2, Bax, Bcl-x and bak are among the proteins involved in the regulation of apoptosis. The agents and doses listed in the table below have all been used to induce apoptosis by scientists at Oncogene Research Products. Not every agent will induce apoptosis in every cell type. Depending on the agent chosen and the concentrations used, maximal induction of a particular protein may occur at any time between 8 and 72 hours post-treatment. We have found that not all proteins are effected by each of these reagents in a particular cell line, and even proteins involved in preventing apoptosis, such as Bcl-2, may be induced by these treatments (but with a unique time course). AgentDoseStock SolventCat. No.Actinomycin D0.5 μg/mLMethanol114666Aphidicolin2 μg/mLDMSO178273A2318710 mMDMSO100105Caffeine16 mMBoiling H2O205548Camptothecin4 μg/mLDMSO208925Cycloheximide100 μg/mLH2O239763Dexamethasone1 μMEthanol265005Doxorubicin (Adriamycin)0.2 μg/mLH2O3243805-Fluorouracil25 μg/mLDMSO343922Hydroxyurea2.5 mMPBS400046Staurosporine500 nMDMSO569397Taxol (Paclitaxel)100 nM/580nMDMSO580555Thymidine2 mMPBS6060Vinblastine60 nMMethanol677175 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 or 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 buffer to the uninduced control. Day 3: Check cells; may begin to see some degree of cell death. 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 under "Cell Lysate Preparation." 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 4 or 5: Resolve proteins on sodium dodecylsulfate-polyacrylamide gels. Visualize the protein of interest from total lysates by Western blotting using chemiluminescent detection as described in the accompanying protocols. Visualize immunoprecipitates by either autoradiography of metabolically labeled proteins or by western blotting. Always compare levels of p53 or p21WAF1 from treated cells with those from untreated controls to confirm induction. For γ-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. |
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