The effect of glutamine absence on the viability of endothelial cells with mTOR knockdown genes
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BACKGROUND: Endothelial cells line the inner vessels of all organ systems. Endothelial cells share many similarities with cancer cells from their preference for glucose consumption even in the presence of oxygen availability (Warburg effect) to their incredibly fast proliferation through the mTOR pathway. Both cell types also rely heavily on glutamine, in addition to glucose, to maintain their metabolic activities. Glutamine is the most abundant free amino acid in the body, making it readily available to both endothelial and tumor cells as a source of carbons for the synthesis of biomass. Due to these overlapping similarities, there has become an interest in understanding how mutations of cells can allow them to survive in the absence of glutamine. A greater understanding of the mechanism of action from down regulation of specific genes can lead to many clinical applications. OBJECTIVE: To understand if down regulation of specific mTOR genes can affect the viability and proliferation of endothelial cells in glutamine deficient and glutamine supplemented media, respectively. METHODS: siRNA transfection was used as a secondary method to knock down specific genes in endothelial cell lines (ECFC and HUVEC). QPCR was used to validate knockdown efficiency. Cell progression was documented both visually and through cell counting over a long period of time. Cells were placed in both glutamine deficient and glutamine supplemented media to monitor mTOR activity and viability. RESULTS: Of all the genes tested, endothelial cells with CS, SLC7A11, WDR59, and WDR24 knockdowns were shown to have greater viability in glutamine deficient media than the controls. They were also shown to have suppressed proliferative behavior in complete media. CONCLUSIONS: Despite initial CRISPR screening results, not all of the knockdown genes selected from the list were able to survive in glutamine deficient conditions. However, we were able to show that knockdown of CS and SLC7A11 demonstrate similar viability in endothelial cells as previously published studies in cancer cells in glutamine deficient media. Interestingly, both GATOR2 complex genes (WDR59 and WDR24), which act as positive and negative regulators of the mTORC1 pathway via amino acid sensing, were able to survive without glutamine. Future studies could be done to understand the mechanisms behind their survival.