Biology subjects

Kuskovsky, R.

Publications and source records attributed to Kuskovsky, R..

2 recordsLinked to original sources

bioRxiv2018

Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry

Quantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.

biochemistry↗

bioRxiv2018

Suppression of p16 increases nucleotide synthesis via mTORC1

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurred via activation of mTORC1 signaling, which directly mediated increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlated with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibited proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.\n\nHighlightsO_LImTORC1 is activated by p16 knockdown to increase nucleotide synthesis and bypass senescence\nC_LIO_LImTORC1 directly increases translation RPIA to increase ribose-5-phosphate\nC_LIO_LIActivation of mTORC1 pathway downstream of p16 suppression is independent of RB\nC_LIO_LIRPIA suppression induces senescence only in cells and tumors with low p16\nC_LI

cancer biology↗