Effect of glutamine limitation on the behavior of Sp2/0-Ag14 mouse hybridoma cells.

Cancer cells often display a dependence toward the amino acid L-glutamine for their survival, a phenomenon termed glutamine addiction. The mouse hybridoma Sp2/0-Ag14 (Sp2/0) undergoes rapid apoptotic cell death upon glutamine deprivation, making it a useful model for uncovering the molecular and cellular processes through which glutamine controls cell survival. This work was aimed at gaining a better understanding of the molecular and cellular events triggered when Sp2/0 cells are exposed to limiting amounts of glutamine. First, the effect of glutamine limitation on Sp2/0 cell behavior was investigated. We found that a threshold concentration of 100 μM glutamine exists where Sp2/0 cell density does not increase, but cells remain viable. Under this threshold, Sp2/0 cells underwent apoptosis, but in a more protracted fashion than under conditions of acute glutamine deprivation. Unexpectedly, I found that exposure of Sp2/0 cells to 25μM glutamine triggered a biphasic activation of caspase-3. Interestingly, glutamine limitation, but not acute glutamine deprivation, was sufficient to maintain intact mitochondria for several hours and to trigger the expression of the stress-related transcription factor GADD-153. My results raise the possibility that glutamine limitation triggers a stress response which could enable Sp2/0 cells to adapt to its environment. Using microscopic and biochemical techniques, I also provided evidence for a reduction in autophagic processes in Sp2/0 cells exposed to glutamine-limiting conditions. Chemical inhibitors of autophagy caused Sp2/0 cell death even in the presence of adequate supply of glutamine. On the other hand, rapamycin, a known activator of autophagy, improved Sp2/0 viability under glutamine limitation conditions. Therefore, the loss of Sp2/0 cell viability when exposed to limiting amounts of glutamine could be the result, at least in part, of a reduction in the cell’s autophagic capabilities. Finally, I explored the effect of ammonium ions, a product of glutamine metabolism, on the behavior of Sp2/0 cells exposed to limiting amounts of glutamine. Ammonium ions treatment rescued Sp2/0 cell viability and proliferation in Sp2/0 cells cultured in glutamine-limiting conditions. Interestingly, ammonium acetate, but not ammonium chloride, caused a reduction in caspase-3 activity in Sp2/0 cells maintained under limiting glutamine conditions. Finally, my data suggest that ammonium salts led to a partial restoration of the autophagy process in Sp2/0 cells exposed to limiting amounts of glutamine, providing a potential explanation for the beneficial effect of ammonium ions on cell viability. All together, the results obtained in the course my studies argue in favor of a mechanistic link between autophagy and ammonium ions in the modulation of the viability of a Sp2/0 cells exposed to glutamine-limiting conditions.Doctor of Philosophy (PhD) in Biomolecular Science