Inhibition of MCL-1 by obatoclax sensitizes Sp2/0-Ag14 hybridoma cells to glutamine deprivation-induced apoptosis.

For several cancer cell types, the lack of an adequate supply of the amino acidl-glutamine (Gln) triggers apoptosis, a phenomenon termed Gln addiction. In this report, we examined the role of the anti-apoptotic proteins of the B-cell lymphoma 2 (BCL-2) protein family in the survival of Sp2/0-Ag14 (Sp2/0) mouse hybridoma cells, a cell line that undergoes apoptosis within minutes of Gln deprivation. Western blot analysis revealed that myeloid cell leukaemia 1 (MCL-1) was expressed at much higher levels than BCL-2, B-cell lymphoma extra-large and BCL-2-like protein 2 making it the prominent pro-survival BCL-2 family member in this hybridoma. Gln deprivation triggered a progressive decrease in MCL-1 protein levels, which coincided with the decrease in Sp2/0 cell survival. Moreover, Sp2/0 cells were much more sensitive to the broad Bcl-2 homology domain-3 (BH3) mimetic obatoclax (which targets MCL-1) than to the more selective drug ABT-737 (which does not target MCL-1). Finally, we show that obatoclax sensitizes Sp2/0 cells to apoptosis following Gln starvation. All together, the data presented here reveal that modulation of the pro-survival protein MCL-1 is an important step in the sequence of events leading to the initiation of apoptosis in Gln-starved Sp2/0 cells. Cancer cells require an adequate supply ofl-glutamine for their survival. Using a mouse hybridoma cell line that is exquisitely sensitive to glutamine starvation, we show that the levels of the pro-survival BCL-2 family protein MCL-1 decrease upon glutamine starvation in a manner that correlates with the loss of cell viability. Moreover, inhibiting MCL-1 with the drug obatoclax sensitizes hybridoma cells to glutamine starvation. Thus, in some cancer cells, glutamine starvation triggers the inactivation of pro-survival proteins. Our data suggest that the combined inhibition of glutamine biosynthesis pathways and BCL-2 proteins may prove effective against some cancers.

Ammonium ions improve the survival of glutamine-starved hybridoma cells.

BACKGROUND: As a consequence of a reprogrammed metabolism, cancer cells are dependent on the amino acid l-glutamine for their survival, a phenomenon that currently forms the basis for the generation of new, cancer-specific therapies. In this paper, we report on the role which ammonium ions, a product of glutaminolysis, play on the survival of l-glutamine-deprived Sp2/0-Ag14 mouse hybridoma cells. RESULTS: The supplementation of l-glutamine-starved Sp2/0-Ag14 cell cultures with either ammonium acetate or ammonium chloride resulted in a significant increase in viability. This effect did not depend on the ability of cells to synthesize l-glutamine, and was not affected by the co-supplementation with α-ketoglutarate. When we examined the effect of ammonium acetate and ammonium chloride on the induction of apoptosis by glutamine deprivation, we found that ammonium salts did not prevent caspase-3 activation or cytochrome c leakage, indicating that they did not act by modulating core apoptotic processes. However, both ammonium acetate and ammonium chloride caused a significant reduction in the number of l-glutamine-starved cells exhibiting apoptotic nuclear fragmentation and/or condensation. CONCLUSION: All together, our results show that ammonium ions promote the survival of l-glutamine-deprived Sp2/0-Ag14 cells and modulate late-apoptotic events. These findings highlight the complexity of the modulation of cell survival by l-glutamine, and suggest that targeting survival-signaling pathways modulated by ammonium ions should be examined as a potential anti-cancer strategy.