Thus far in our research, Dr. Krucher and I have made progress with determining whether or not certain resistance pathways in breast cancer cells remain inactive in response to reducing PNUTS levels or PNUTS Knockdown. One of the resistant pathways we have discovered as remaining inactive when PNUTS levels were reduced is AKT. In order to come to these results I would perform an experiment utilizing a series of laboratory techniques including but not limited to performing cell lysis protocols, protein concentration assays, western blotting, and running gel electrophoresis.
One of the challenges that I had with regard to our research was figuring out why some of the samples I had made were not easily visible or clean when it came time to view the Western blots via the Bio-Rad Imager. This problem lead to communication between Dr. Krucher and I in an attempt to determine why this was happening in my samples. After careful consideration and discussion with Dr. Krucher we came to the conclusion that the problem may have been a result of not always keeping the cell pellets on ice before performing the lysis protocol. This would potentially allow proteases within the cells to kill off the proteins that we want to isolate which would have accounted for the problems I had viewing the blots in the Bio-Rad Imager.
Through this project I have learned something new with each passing day. From learning laboratory techniques, to better understanding Cancer cells and learning more about the pathways that lead to their resistance, this research has allowed me to learn immensely and I am looking forward to continuing to learn throughout the duration of my time doing research with Dr. Krucher.
The goal of the research I have been doing this year is to activate the tumor suppressor Rb, in breast cancer cells. To do that we activate an enzyme called PP1, which activates Rb, by a gene silencing technique called PNUTS knockdown. So far in this research I have learned many of the techniques necessary for setting up PNUTS Knockdown. I have learned how to efficiently count and plate MCF7 breast cancer cells, as well as learned how to trypsinize cells off of plates in order to remove the cancer cells and centrifuge them to collect them.
Throughout the semester I have also learned more about the intricacies of the PNUTS KD For example, I learned that using specific antibodies we tag the proteins that are on the nitrocellulose paper and after viewing them via specific computer software (Bio-rad image software) we determine whether or not PNUTS was knocked down by comparing a portion of cancer cells that didn’t receive any treatment (controls) that knocked down PNUTS to a portion of cancer cells that did have PNUTS knocked down and if the software shows the band where PNUTS is to be lighter or less prominent then we know the knockdown was a success. Furthermore, we also look to see if the band where Actin is, is more prominent or less prominent or the same. Ideally it is the same, meaning the same amount of protein was loaded in each cell. Therefore, expression of actin serves as our loading-control.
So far we don’t have any confirmed data because the results we do have need to be replicated several times to have confidence in the accuracy of the results. However, we do have general results. For example, so far the experiments that have shown promising results regard the phosphorylation of AKT detected by the antibody AKT-473. The way we know this antibody has shown promising results is by viewing the band on the nitrocellulose paper as mentioned before and determining whether or not the band is more prominent with the treatment or less prominent and in the experiments we have carried out so far the bands for this antibody have been less prominent after the PNUTS KD and this experiment will be carried out again a number of times to ensure the results are accurate.
The title of the research project that Dr. Krucher and I are working on is Investigation of Combination Treatments in Breast Cancer in Attempt to Overcome Drug Resistance. The reason we are investigating combination treatments is because in breast cancer, like in many other cancers, the cancer has the ability to develop resistance to the drugs that are used to attack and kill the cancer cells. This means that after a period of time the drug that was used to treat the cancer is rendered ineffective due to the cancers ability to activate specific resistance pathways and continue proliferating. Therefore, through this research project Dr. Krucher and I plan on determining whether or not combination treatments would be a more effective means at combating this resistance in cancer. From this project I personally expect to learn in a variety of different ways. For example, I plan on learning new laboratory skills and techniques or developing the techniques that I have been taught so far like running a Gel Electrophoresis or Culturing cells or performing Protein Concentration Assays. Furthermore, I also plan on learning how to properly go about conducting research in a laboratory setting.
Our target in this research is the Retinoblastoma (Rb) protein, which is inactivated in almost all cancer types. In cancer cells Rb is highly phosphorylated which inactivates its ability to suppress unwarranted proliferation. The way that Dr. Krucher and I plan on answering these questions is by utilizing a method known as PNUTS (Phosphatase Nuclear Targeting Subunit) knockdown where we reduce PNUTS, a PP1 interacting protein, in cancer cells. PNUTS depletion leads to an activation of PP1 activity which leads to dephosphorylation of the Rb protein, and its activation. We will determine whether PNUTS depletion is a method to suppress cancer drug resistance. We plan on answering this question by continuing to utilize Gel Electrophoresis in order to separate proteins and Western Blot them in order to image them and determine whether or not PNUTS was knocked down. In future experiments we are interested to see if PNUTS Knockdown coupled with other drugs will aid in combating any resistance pathways that certain cancer cells develop.