Breast cancer is the most common form of cancer women are afflicted with, and about one in every eight women will be diagnosed with it in their lifetime. It can affect anybody from your neighbor, to your teacher, and even your own mother. It is for these people that we love so dearly that many scientists are searching for a cure. Breast cancer is a complex disease, but that is my favorite part of our research. My faculty partner and I are looking to explore the significance in the interaction between a tumor suppressor called Retinoblastoma (Rb), and the pro-apoptotic protein Bax in cell culture systems and in three dimensional cultures of breast cancer cells.
Rb is a tumor suppressor protein which prevents excessive proliferation in cells depending on its state of phosphorylation. Phosphorylation is a chemical modification of the protein that turns it on and off. When Rb is highly modified by phosphorylation, cancers such as breast, ovarian, and colon develop. Our research focuses on the phosphorylation of Rb, which has 15 potential sites. In previous work, it has been shown that modification of some sites gives cells the ability to evade apoptosis (cell death). Therefore Rb controls life and death in the cell. This project will test the hypothesis that certain phosphorylation sites regulate Rb binding to other proteins, such as the pro-apoptotic protein Bax. Association of Bax with Rb is a new finding that we plan to further investigate in these studies.
In order to find out what sites of Rb phosphorylation are involved in association with Bax, we will use plasmids that express mutant Rb in breast cancer cells. Following this we will determine the association between Rb and Bax. These studies require a great deal of laboratory work. Cell stocks must be grown under aseptic conditions, in complex media, for weeks at a time. Experiments take 4-5 days, starting with setting up on Mondays or Tuesdays followed by analysis of the experiments on Thursdays or Fridays. To analyze, we use laboratory procedures such as Immunoprecipitation and Western blotting. Luckily, I learned these procedures this past summer while training in Dr. Krucher’s lab. These experiments determine the absence or presence of proteins, as well as binding of specific proteins. Because the experiments are complex and challenging, and one or other small steps often go wrong, we need to troubleshoot each week until we obtain results that we believe are correct and are reproducible. Therefore it will likely take almost the full academic year to find our answer.
For the sake of our loved ones, it is vital for people like my faculty partner and I to be dedicated to our research. It takes an enormous amount of determination and brain power to conduct these experiments, but once you’re in it – you’re hooked. I believe we can make a difference in the future of breast cancer and hopefully through these experiments, all of us will be able to see it unfold.