Cancer Biology

Like all animals, Drosophila shows robust fat (triglyceride) turnover, i.e., they synthesize, store and utilize triglyceride for their daily metabolic needs. The protocol describes a simple assay to measure this turnover of triglycerides in Drosophila.

Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by measuring γ-glutamylhydroxamate synthesized from glutamine and hydroxylamine, and the chromatographic separation of the reaction product from the reactants (Deuel et al., 1978). An overview of the GS glutamyl transferase reaction can be found in Figure 1. GS activity was measured by a spectrophotometric assay at a specific wavelength of 560 nm using a microplate reader. The method is simple, and has a comparable sensitivity with those methods applying radioactively labelled substrates. This modified procedure has been applied to assay/determine GS activity in cultured cell lines including the human mammary epithelial MCF10A cells and the murine pre-B FL5.12 cells, and could be used to measure GS activity in other cell lines.


Figure 1. An overview of the GS glutamyl transferase reaction

Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of ⁵⁹Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.

Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al., 1980; Kranendijk et al., 2012). More recently, investigators have shown that D2HG is produced by mutant isocitrate dehydrogenase enzymes associated with a variety of human malignancies, such as acute myeloid leukemia, glioblastoma multiforme, and cholangiocarcinoma (Cairns and Mak, 2013; Dang et al., 2009; Ward et al., 2010). By contrast, we and others have shown that L2HG accumulates in response to cellular reductive stressors like hypoxia, activation of hypoxia inducible factors, and mitochondrial electron transport chain defects (Oldham et al., 2015; Reinecke et al., 2011; Intlekofer et al., 2015; Mullen et al., 2015). Each enantiomer is produced and metabolized in independent biochemical pathways in reactions catalyzed by separate enzymes and utilizing different cofactors with presumably different consequences for cellular metabolism (Kranendijk et al., 2012). Therefore, as research into the roles of D2HG and L2HG in human metabolism continues, it becomes increasingly important for investigators to consider each enantiomer independently (Struys, 2013). Several methods for quantification of biochemically relevant enantiomers in general have been developed and typically include enzymatic assays using enzymes specific for one enantiomeric species or the other, the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy, or the use of chiral derivatization reagents to convert a mixture of enantiomers to diastereomers with differing physical and chemical properties facilitating their chromatographic separation. In this protocol, we report the adaptation of a previously published derivatization method using diacetyl-L-tartaric anhydride (DATAN) for the quantification of 2HG enantiomers (Figure 1) (Oldham et al., 2015; Struys et al., 2004).


Figure 1. Reaction scheme for the derivatization protocol

Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.

2-Methylthio-N⁶-isopentenyladenosine (ms²i⁶A) is an evolutionally conserved posttranscriptional modification found at position 37 of four mammalian mitochondrial tRNAs, mt-tRNA^Ser(UCN), mt-tRNA^Trp, mt-tRNA^Phe and mt-tRNA^Tyr. The ms² modification in ms²i⁶A strengthens codon-anticodon interaction and contributes to accurate and efficient decoding. Deficiency of ms² modifications impairs mitochondrial protein synthesis, which ultimately leads to the development of myopathy in mice and patients having mitochondrial diseases. Therefore, the level of ms² could be utilized as an indicator that reflects the status of mitochondrial protein synthesis. Here, we describe a simple and fast quantitative PCR-based method to measure the ms² level in total RNA sample.

LDH (Lactate dehydrogenase) enzyme catalyzes the reversible conversion of pyruvate to lactate using NAD+ as a cofactor. Although the physiological significance of lactate accumulation in tumor cells, a dead-end product in cellular metabolism, is currently a topic of debate, it has long been known that many tumor cells express a high level of LDH-A (Koukourakis et al., 2003; Koukourakis et al., 2006; Koukourakis et al., 2009). So detection of its enzyme activity in vitro is important for researching on LDH-A. Recently, it has been reported that Lys-5 acetylation could decrease LDH-A enzyme activity (Zhao et al., 2013).