We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227–2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24–72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution ( m 1) and 10% contained two 13C substitutions ( m 2). A similar m 1-to- m 2ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose.13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

The metabolic network of cancer cells confers adaptive mechanisms against many chemotherapeutic agents, but also presents critical constraints that make the cells vulnerable to perturbation of the network due to drug therapy. To identify these fragilities, combination therapies based on targeting the nucleic acid synthesis metabolic network at multiple points were tested. Results showed that cancer cells overcome single hit strategies through different metabolic network adaptations, demonstrating the robustness of cancer cell metabolism. Analysis of these adaptations also identified the maintenance of pentose phosphate cycle oxidative and nonoxidative balance to be critical for cancer cell survival and vulnerable to chemotherapeutic intervention. The vulnerability of cancer cells to the imbalance on pentose phosphate cycle was demonstrated by phenotypic phase plane analysis. ' 2006 Wiley-Liss, Inc.Key words: tumor growth inhibition; metabolic profiling; combined therapy; pentose phosphate pathway; phase plane analysis Recent molecular studies have revealed that several of the multiple genetic alterations that cause tumor development directly affect cellular energy metabolism through the glucose metabolic network.1 In particular, it has been demonstrated that most tumors produce high levels of lactate and fructose 1,6-bisphosphate, and have high rates of aerobic glycolysis, with correspondingly high rates of biosynthesis of both lipids and nucleic acids.2 This constellation of metabolic adaptations of cancer cells is crucial to their capacity for unregulated proliferation in a hypoxic tumor environment, and confers a strong robustness to cancer cells for the development of drug resistance. However, these adaptive mechanisms are constrained by sharing of precursor substrates and cofactors of other interconnecting pathways, which make cancer cells vulnerable to perturbation of the network. Such constraints imply the existence of rare points of fragility which would be logical new drug targets. Thus, it has been suggested that strategies designed to prevent metabolic network adaptation necessary for cancer cell proliferation would be particularly efficacious in the treatment of cancer drug resistance. 2-6Several antitumor therapies have been developed based on preventing nucleic acid synthesis. 7,8 More commonly, different chemotherapeutic agents currently in use are designed to inhibit the synthesis of the purine or pyrimidine ring of the nucleic acids, such as methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR).9 Recently, other targets of the synthetic pathways of ribose 5-phosphate, the sugar part of the nucleic acid molecule, have been proposed by Cascante et al. 4 Ribose 5-phosphate is synthesized from glucose or glycolytic intermediates through 2 pathways: the oxidative branch of the pentose phosphate pathway (PPP) (catalyzed by glucose 6-P dehydrogenase and 6-P-gluconate dehydrogenase) and the nonoxidative branch of the PPP (catalyzed by transketolase and transaldolase). In previous articles,...

Among K-ras mutations, codon 12 mutations have been identified as those conferring a more aggressive phenotype. This aggressiveness is primarily associated with slow proliferation but greatly increased resistance to apoptosis. Using transfected NIH3T3 fibroblasts with a mutated K-ras minigene either at codon 12 (K12) or at codon 13 (K13), and taking advantage of [1,2-13 C 2 ]glucose tracer labeling, we show that codon 12 mutant K-ras (K12)-transformed cells exhibit greatly increased glycolysis with only a slight increase in activity along pathways that produce nucleic acid and lipid synthesis precursors in the oxidative branch of the pentose phosphate pathway and via pyruvate dehydrogenase flux. K13 mutants display a modest increase in anaerobic glycolysis associated with a large increase in oxidative pentose phosphate pathway activity and pyruvate dehydrogenase flux. The distinctive differences in metabolic profiles of K12 and K13 codon mutated cells indicate that a strong correlation exists between the flow of glucose carbons towards either increased anaerobic glycolysis, and resistance to apoptosis (K12), or increased macromolecule synthesis, rapid proliferation, and increased sensitivity to apoptosis. (Cancer Res 2005; 65(13): 5512-5)