Daniel Sharp and Dr. Kim O’Neill, MMBio
Immunotherapy is a developing field in cancer treatment that relies upon the bodies natural defenses to target and eliminate cancerous cells. In order for the immune system to differentiate cancerous tissue from normal tissue there must be some mechanism for targeting only cancerous cells. Without a targeting mechanism, stimulation of the immune system can lead to attack of normal tissue. The use of antibodies to cancer-specific antigens is a viable option to solve this targeting issue. If a cancerous cell specifically expresses a protein on its surface, an antibody to that protein could direct immune cells to the cancerous cell, allowing them to attack and kill it without damaging non-cancerous cells.
The role of Thymidine kinase 1 (TK1) in cancer has been extensively studied and researched 1-3. TK1 is a salvage pathway enzyme (SPE), a protein important in the recycling of used DNA molecules. It has been shown to be useful as a diagnostic and prognostic marker in several types of cancers. Previous research in Dr. Kim O’Neill’s lab at BYU has shown that TK1 is localized to the surface of the plasma membrane of many cancerous cells. I wanted to know if other SPEs might also be up-regulated on the surface of cancer cells.
Along with TK1, I decided to study the expression of three other SPEs, one for each DNA nucleotide: Adenine phosphoribosyltransferase (APRT), Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and Deoxycytidine kinase (dCK). After obtaining polyclonal rabbit anti-human antibodies to each of these four SPEs, I began to test their expression on several cancer cell lines using flow cytometry (FCM). Over the course of my experiments I tested the expression of all four SPEs on 18 different cancer cell lines. These cell lines originated from many different types of cancer including breast, lung, colon, cervical and skin cancers. All were adherent cell lines, meaning that they bound to the wall of the flask as they grew. Non-cancerous 293-T kidney epithelial cells were used as a negative control to compare to.
The culturing of these cell lines required a lot of time and effort. In order to avoid error, I followed the correct protocol for culturing each cell line and always took samples to test 24 hours after feeding them. I experimented with a few different methods for removing the cells from the wall of the flask before settling on a non-enzymatic solution that helped weaken their hold without compromising the cells’ exterior proteins.
Once I had the cells in suspension, I followed our standard lab protocols for FCM on all samples. This entails thorough washings before and after binding with a primary antibody to each SPE. I then used a secondary antibody, FITC-conjugated goat antirabbit, before taking them to the flow cytometer at the RIC Facility to run. The flow cytometer can measure the amount of green fluorescent light emitted by the FITC molecules and determine if any cell passing through it is above threshold. Propidium iodide, a chemical that binds to DNA and RNA, was used to select only for nonpermeabilized cells. The FITC-conjugated secondary antibody was used as a negative control to set the threshold. I recorded the results of my experiments as the percent of cells above threshold (see Figure 1). I also took pictures of the stained cells using fluorescent microscopy to provide a visualization of the location of staining (see Figure 2).
On all the cell lines I tested, I found that all the SPEs were expressed at a significantly higher level than control (p<0.05). TK1 and APRT were the highest, with an average of 70.3% events above threshold, with dCK at 41.8% and HGPRT at 22.3%. If my research is correct, these SPEs offer interesting options for immunotherapy targets, because they are expressed on the surface of many different cancer cell lines. Further work needs to be done to elucidate the mechanism by which these enzymes are located to and remain on the surface of the plasma membrane. Experimentation should also be done with cancerous tissue removed from patients’ tumors instead of only with in-vitro cell lines. Regardless, the results are promising.
Special thanks to Robert Whitehurst, Dagoberto Estevez, Melissa Alegre and Dr. Kim O’Neill for helping me with my project.
References
- He E, et al. Thymidine kinase 1 is a potential marker for prognosis and monitoring the response to treatment of patients with breast, lung, and esophageal cancer and non-Hodgkin’s lymphoma. Nucleosides Nucleotides Nucleic Acids [Internet]. 2010 Jun [cited 28 Oct 2010]; 29(4-6): 352-8. Available from: http://www.ncbi.nlm.nih.gov/pubmed
- Konopley SN, et al. High serum thymidine kinase 1 level predicts poorer survival in patients with chronic lymphocytic leukemia. American Journal of Clinical Pathology [Internet]. 2010 Sep [cited 28 Oct 2010]; 134(3): 472-7. Available from: http://www.ncbi.nlm.nih.gov/pubmed
- Zhang F, et al. Thymidine kinase 1 immunoassay: a potential marker for breast cancer. Cancer Detect Prev. [Internet]. 2001 [cited 28 Oct 2010]; 25(1): 8-15. Available from: http://www.ncbi.nlm.nih.gov/pubmed