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IsoTopics™ – November 2015

Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation.

Metabolic isotopic analysis (MIA) utilizes stable isotope-enriched substrates and mass spectrometry to study metabolism in living cells and tissue. Because isotope-labeled substrates make the determination of metabolic pathways and flux in living cells or tissue possible, MIA has become an integrated platform in oncology research.
This application note presents an example of MIA for studying metabolism in mice and cultured human cells used in oncology research by summarizing research conducted by Nicolay et al. Previously these researchers used MIA to study the inactivation of the pRB tumor suppressor in the fly model. In this presented work, Nicolay and coworkers found that loss of pRB promotes altered glutamine metabolism in mice. A follow-up study demonstrated that the acute loss of pRB in adult mouse tissues led to rapid changes in glucose oxidation of which are independent of the proliferation status of the tissue (Nicolay et al. 2015). This subsequent study shows that MIA of glucose-derived intermediates in the TCA cycle for mitochondria in pRB-/- tissue are less functional than the pRB+/+ tissue. These effects are mimicked in human cell culture models of pRB loss. This work also showed that glutamine oxidation is reduced in human cells upon loss of pRB and that these alterations in mitochondria oxidation capacity led to pRB-/- cells being more sensitive to mitochondrial poisons.


The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.
© 2015 Nicolay et al.; Published by Cold Spring Harbor Laboratory Press.
Reference: Genes Dev. 2015 Sep 1;29(17):1875-89. doi: 10.1101/gad.264127.115. Epub 2015 Aug 27.
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