Serine, but not glycine, supports one-carbon metabolism and proliferation of cancer cells

Previous work has shown that some cancer cells are highly dependent on serine/glycine uptake for proliferation. Although serine and glycine can be interconverted and either might be used for nucleotide synthesis and one-carbon metabolism, we show that exogenous glycine cannot replace serine to support cancer cell proliferation. Cancer cells selectively consumed exogenous serine, which was converted to intracellular glycine and one-carbon units for building nucleotides. Restriction of exogenous glycine or depletion of the glycine cleavage system did not impede proliferation. In the absence of serine, uptake of exogenous glycine was unable to support nucleotide synthesis. Indeed, higher concentrations of glycine inhibited proliferation. Under these conditions, glycine was converted to serine, a reaction that would deplete the one-carbon pool. Providing one-carbon units by adding formate rescued nucleotide synthesis and growth of glycine-fed cells. We conclude that nucleotide synthesis and cancer cell proliferation are supported by serine—rather than glycine—consumption

Localization of NADPH production: a wheel within a wheel

In this issue of Molecular Cell, Lewis et al. (2014) describe a new method to determine where in the cell NADPH is produced, contributing to a growing appreciation that the THF cycle is an important source of mitochondrial NADPH

Localization of NADPH production: a wheel within a wheel

In this issue of Molecular Cell, Lewis et al. (2014) describe a new method to determine where in the cell NADPH is produced, contributing to a growing appreciation that the THF cycle is an important source of mitochondrial NADPH

Direct estimation of metabolic flux by heavy isotope labeling simultaneous with pathway inhibition: metabolic flux inhibition assay

Heavy isotope labeled metabolites are readily detected by mass spectrometry and are commonly used to analyze the rates of metabolic reactions in cultured cells. The ability to detect labeled metabolites—and infer fluxes—is influenced by a number of factors that can confound simplistic comparative assays. The accumulation of labeled metabolites is strongly influenced by the pool size of the metabolite of interest and also by changes in downstream reactions, which are not always fully perceived. Here, we describe a method that overcomes some of these limitations and allows simple calculation of reaction rates under low nutrient, rapid reaction rate conditions. Acutely increasing the pool of the metabolite of interest (by adding a pulse of excess unlabeled nutrient to the cells) rapidly increases accumulation of labeled metabolite, facilitating a more accurate assessment of reaction rate

Hoehenberechnungen von Heegnerpunkten auf Drinfeldschen Modulkurven

Available from TIB Hannover: RR 1608(1996,10) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Localization of NADPH Production: A Wheel within a Wheel

In this issue of Molecular Cell, Lewis et al. (2014) describe a new method to determine where in the cell NADPH is produced, contributing to a growing appreciation that the THF cycle is an important source of mitochondrial NADPH

A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis

<div><p>Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells. This approach is rapid and highly sensitive, allowing for the absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells as well as damage originating from multiple ROS sources. Furthermore, differences in the endogenous cellular redox state induced by transcriptional regulation of ROS scavenging enzymes were detected by following this protocol. Finally we showed that the F2-isoprostane 5-iPF_2α-VI is a metabolically stable end product, which is excreted from cells. Overall, this protocol enables accurate, specific and sensitive quantification of endogenous lipid peroxidation in cellular systems.</p> </div

F2-isoprostanes are formed in response to different ROS sources.

<p>(A) F2-isoprostane levels increased in response to PQ treatment. HepG2 cells were treated with increasing concentrations of PQ for 24 hours. 5-iPF2α-VI levels were measured by UPLC-MS/MS. (B) Exogenous H₂O₂ exposure of HepG2 cells resulted in a 3-fold increase in levels of 5-iPF2α-VI. HepG2 cells were incubated in the presence of GOX for 2 hours and 5-iPF2α-VI levels were analyzed every 20 minutes. (C) Rotenone increased lipid peroxidation in HepG2 cells in a dose-dependent manner. HepG2 cells were treated with an increasing concentration of rotenone and F2-isoprostanes were measured after 24 hours. (D) Lipid peroxidation induced by the peroxyl radical generator, AAPH, is quenched by the antioxidant Trolox. Cells were treated with either Trolox, AAPH or both, for 24 hours. Data are shown as mean fold increase ± S.D over untreated control cells (** p < 0.01, *** p < 0.001).</p

The F2-isoprostane approach allows for analysis of FOXO transcription factor control of endogenous oxidative stress.

<p>(A) GOX-induced lipid peroxidation is significantly decreased when FOXO3a is activated. DLD-1 and DL-23 cells were treated with 4-OHT to activate FOXO3a for 16 hours before H₂O₂ exposure by GOX treatment for 2 hours. F2-isoprostane levels were analyzed by UPLC-MS/MS and are shown as mean fold increase ± S.D (* p < 0.05) (B) Prolonged FOXO3a activation increased the protection against endogenously formed ROS damage in colon carcinoma cells. DLD-1 and DL-23 cells were incubated with 4-OHT to activate FOXO3a for 24 hours, before treatment with PQ for another 8 hours. 5-iPF2α-VI levels were analyzed and data are represented as mean fold increase ± S.E.M (*p < 0.05, *** p < 0.001).</p