Grant: Master's Award: Canada Graduate Scholarships

The focus of my research is understanding structure, function and regulation of membrane proteins. I am currently studying the regulation of the equilibrative nucleoside transporter, ENT1, which is responsible for the flux of nucleosides and nucleoside analog drugs, used in anti-cancer treatments, into and out of cells. ENT1 is sufficient and necessary for the effective uptake of nucleosides and nucleoside analog drugs but the contribution of substrate binding/translocation to subsequent drug uptake and the potential for optimizing nucleoside analog therapies in the presence of low levels of ENT1 are not well understood. The first hypothesis I will test is that binding of substrate to ENT1 leads to endocytosis. This process is likely to affect overall efficacy of analog drugs as transporters are taken “out of service” before being recycled back to the membrane. To test this hypothesis, I will transiently transfect GFP-h(human)ENT1 into RPE (retinal pigment epithelium) cells and measure the change in localization of the fluorescent label before and after treating the cells with the high-affinity, tight-binding non-transportable nucleoside analog NBTI (nitrobenzylthioinosine). NBTI binds specifically to ENT1 and does not generate receptor-dependent or other effects that may occur if natural substrates (such as adenosine) are used. Treated RPE cells will be analyzed using confocal microscopy (fixed and live cell imaging with TIRF). I predict that substrate binding leads to an increase in intracellular fluoresence (which is confirmed by preliminary data). I propose that the underlying mechanism is clathrin-dependent endocytosis of ENT1, because the cytoplasmic loop of ENT1 has an AP2 (a clathrin adaptor) binding site, YQQL. Clathrin-dependence will be confirmed using the clathrin inhibitor, Pitstop, in the presence and absence of substrate. Inhibition of endocytosis following substrate binding will prevent localization of fluorescence inside the cell confirming a role for clathrin in ENT1 endocytosis. Endocytosis of ENT1 is predicted to lead to a decrease in uptake of nucleoside, which would correlate with a decrease in cytotoxicity of a nucleoside analog drug such as gemcitabine (a cytidine analog used as a front-line treatment for bladder and pancreatic cancer). Therefore, there may be an optimal interval between doses of nucleoside analog drugs which result in maximal efficacy and these data will provide an insight in the duration of drug transporters being “out of action.” My second hypothesis is that nucleoside analog drug efficacy can be enhanced using ultrasound and lipid stabilized microbubbles (LSM) in the presence of low levels of ENT1. Combination therapies are increasingly important as disease progresses and metastasis occurs. However, low or decreased expression of ENT1 occurs in certain individuals and can lead some cancers to become non-responsive to gemcitabine. I hypothesize that gemcitabine delivery and cytotoxicity can be enhanced in cells expressing low levels of ENT1 using ultrasound and lipid stabilized microbubbles (LSM). I will test the efficacy of gemcitabine using cytotoxicity and MTT assays in cell lines expressing low levels of ENT1 (determined by qRT-PCR and SDS-PAGE/immunoblotting), such as MDA-MB-231 (breast cancer cells), in the presence and absence of LSM. Gemcitabine released from LSM can enter the cells through the transient holes, circumventing the decreased levels of ENT1. Successful gemcitabine delivery will result in increased cytotoxicity compared to untreated cells. These data will provide insights into innovative combination therapies for nucleoside analog drugs.