PT  - JOURNAL ARTICLE
AU  - Hélène Lemieux
AU  - Patrick Subarsky
AU  - Christine Doblander
AU  - Martin Wurm
AU  - Jakob Troppmair
AU  - Erich Gnaiger
TI  - Impairment of mitochondrial respiratory function as an early biomarker of apoptosis induced by growth factor removal
AID  - 10.1101/151480
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 151480
4099  - http://biorxiv.org/content/early/2017/06/18/151480.short
4100  - http://biorxiv.org/content/early/2017/06/18/151480.full
AB  - Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of IL-3, whereas succinate-supported respiration remained unchanged, consistent with control by Complex IV. The apparent Complex IV excess capacity was zero above NADH&succinate-pathway capacity reconstituting tricarboxylic acid cycle function. In comparison, electron flow reached only 60% when supported by succinate alone through Complexes II, III and IV, and was therefore relatively insensitive to Complex IV injuries up to a threshold of 40% inhibition. A slight increase in respiration following addition of cytochrome c, a marker of mitochondrial outer membrane leakage, was present after IL-3 depletion, indicating mitochondrial fragility. Our results highlight a novel link between the key mitogenic and survival kinase CRAF and mitochondrial energy homeostasis.