The role of Bcl-x on Apoptosis of p53 -/- Cells Following DNA Damaging Treatments By Ellen Bailey Mentor: Dr. Steven J. Weintraub Department of Internal Medicine and Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO My laboratory is exploring the steps involved in the response of cells to cancer therapy. Cancerous cells that have lost many of the checkpoints and pathways involved in regulating division and life characteristic of normal cells undergo apoptosis, or cellular suicide, more readily than healthy cells when exposed to radiation or toxins. The initial question presented was "how does pRb block apoptosis in p53 -/- cells?" The retinoblastoma protein (pRb) was initially discovered in cell cycle control pathways and additional research showed that it played a role in blocking apoptosis. p53 was one of the first pro-apoptotic proteins discovered that would, in response to DNA damage, either arrest the cell in order to repair the damage, or, if the damage was too severe, instigate a pathway cumulating in the death of the cell, or apoptosis. At least 50% of tumor cells contain a mutated form of p53, explaining their failure to apoptose despite genetic abnormalities, and many of the remaining carcinogenic cells probably contain a functionally inactive form of p53. Therefore, the study of p53 independent apoptosis became important to determine the mechanisms of apoptosis in cancer cells. The lab proceeded to grow a p53 -/- cell line of mouse embryo fibroblasts and induced active Rb in both the knockouts and tumor cells. Rb is usually also inactive when p53 is not present because it depends on an increased expression of p53 for its activating dephosphorylation. The retinoblastoma protein performed an anti-apoptotic role, inhibiting apoptotic pathways in both cell lines. These results suggest that Rb protects either directly or indirectly an anti-apoptotic protein downstream from DNA damage in the apoptotic pathway. Further research proved that this protein was Bcl-x, from the Bcl-2 family. Additional procedures, including techniques such as immunoprecipitations, western blots, and 2D gels, detected a modification (prevented by active Rb) in Bcl-x in p53 -/- cells that underwent treatment with chemotherapeutic drugs and radiation, both DNA damaging agents. On the initial western blots, two bands appeared above the usual band of Bcl-x observed in untreated cells and, the more severe the treatment, the less protein left in the initial band. In these treated cells, Bcl-x failed to protect from apoptosis as effectively. Our conclusion was that only the lowest band of Bcl-x, the one disappearing as DNA damage became more severe, is active in protecting the cells from apoptosis. When apoptosis becomes the only option for damaged cells, Bcl-x undergoes a modification (more specifically a deamidation) at two sites, rendering it inactive and unable to arrest apoptosis. My goal over the past six weeks was to establish the universal nature of the Bcl-x modification. My laboratory believes that this change must occur in every cell lacking p53 before apoptosis can take place. I was assigned the task of proving that the inactivating change in this key protein is the same in every cell without p53. I therefore treated Saos-2 (osteosarcoma) cells without p53 and p53 -/- MEFs with varying dosages of cisplatin, a common chemotherapy drug, and radiation, to determine whether the same modification occurs in both cell lines and also to a degree corresponding to the degree of the treatment. I then lysed the cells and performed an immunoprecipitation with Bcl-x antibodies to collect the protein. I ran these samples on a 2D gel, testing changes based upon both molecular weight and charge. My first set of data was inconclusive. The significance of my research is this: if a good majority of cancer cells lack an active form of p53 and if Bcl-x must be modified before these cells apoptose, then the effectiveness of new cancer treatments could be tested based on its ability to modify Bcl-x. If this pathway for apoptosis and this Bcl-x modification is indeed universal, treatments more specific to this Bcl-x change could be manufactured, making therapies more effective. Scholar Index Next Natural Sciences Learning Center Washington University - Biology All contents copyright © 2001 Email comments to nslc webmanager