RESUMO
Recent observations on cancer cell metabolism indicate increased serine synthesis from glucose as a marker of poor prognosis. We have predicted that a fraction of the synthesized serine is routed to a pathway for ATP production. The pathway is composed by reactions from serine synthesis, one-carbon (folate) metabolism and the glycine cleavage system (SOG pathway). Here we show that the SOG pathway is upregulated at the level of gene expression in a subset of human tumors and that its level of expression correlates with gene signatures of cell proliferation and Myc target activation. We have also estimated the SOG pathway metabolic flux in the NCI60 tumor-derived cell lines, using previously reported exchange fluxes and a personalized model of cell metabolism. We find that the estimated rates of reactions in the SOG pathway are highly correlated with the proliferation rates of these cell lines. We also observe that the SOG pathway contributes significantly to the energy requirements of biosynthesis, to the NADPH requirement for fatty acid synthesis and to the synthesis of purines. Finally, when the PC-3 prostate cancer cell line is treated with the antifolate methotrexate, we observe a decrease in the ATP levels, AMP kinase activation and a decrease in ribonucleotides and fatty acids synthesized from [1,2-(13)C2]-D-glucose as the single tracer. Taken together our results indicate that the SOG pathway activity increases with the rate of cell proliferation and it contributes to the biosynthetic requirements of purines, ATP and NADPH of cancer cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Ácido Fólico/metabolismo , Glicina/metabolismo , NADP/metabolismo , Neoplasias/metabolismo , Purinas/metabolismo , Serina/metabolismo , Aminoácido Oxirredutases/genética , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Metotrexato/farmacologia , Camundongos , Complexos Multienzimáticos/genética , Neoplasias/genética , Biossíntese de Proteínas , Transferases/genéticaRESUMO
Nanosecond fluorescence decay characteristics of the calcium-binding probe Quin2 and two of its cation complexes were examined by time-resolved fluorescence spectroscopy. Binding of Ca2+ and Cd2+ resulted in fluorescence lifetime enhancements as compared to that of free Quin2 ('tau' = 0.9 ns). The Quin2-Ca2+ complex displays a monoexponential decay of tau = 7.4 ns, while the cadmium complex gives an average decay time of ca. 4 ns. Lifetime measurements made on heterogeneous cationic solutions demonstrate that decay times for individual complexes can be retrieved. Time-resolved measurements were used to monitor the kinetics of ionomycin-mediated calcium and cadmium transport across artificial membranes. Fluorescence decays, collected on the time-scale of second, were sufficient to measure individual ion fluxes or those of mixtures into liposomes. The combination of steady-state and time-resolved fluorescence techniques offers the unique advantage of simultaneously detecting other cations in the presence of calcium.
Assuntos
Cádmio/química , Cálcio/química , Corantes Fluorescentes/química , Lipossomos/química , Aminoquinolinas/química , Ionomicina/química , Ionóforos/química , Quelantes de Ferro/química , Cinética , Espectrometria de FluorescênciaRESUMO
The binding of 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl] 6-methoxy-8-bis[carboxymethyl] aminoquinoline, the fluorescent calcium probe Quin2, to serum albumin and several other proteins has been investigated. Changes in fluorescence emission spectra and fluorescence anisotropy revealed interactions between Quin2 and several proteins including human serum albumin, bovine serum albumin, aldolase, phosphoglucose isomerase, glyceraldehyde-3-phosphate dehydrogenase, and alkaline phosphatase. Protein-probe interactions were inhibited by the presence of calcium. Binding was also measured by resonance energy transfer and gel permeation chromatography. Equilibrium binding constants for Quin2 were quantitated by the application of the recently-developed "SPECTRABIND' program to spectroscopic data (D. Toptygin and L. Brand, Anal. Biochem., 224 (1995) 330-338). Binding of Quin2 to human serum albumin is discussed in terms of the published X-ray crystal structure of human serum albumin (X.M. He and D.C. Carter, Nature, 358 (1992) 209-215).
Assuntos
Proteínas de Ligação ao Cálcio/química , Aminoquinolinas , Anisotropia , Quelantes , Cromatografia em Gel , Transferência de Energia , Fluorescência , Corantes Fluorescentes , Humanos , Ligantes , Ligação Proteica , Albumina Sérica/química , Software , Espectrometria de FluorescênciaRESUMO
A fluorescence technique for characterizing ligand binding is evaluated. This technique uses the combination of steady-state and time-resolved methods to recover molar concentrations and overcomes errors inherent in the use of either method alone. The technique is applicable to time-resolved measurements made either with time-domain or frequency-domain instrumentation. A straightforward single-frequency phase/modulation approach is presented to determine whether an experimental system can be described by a two-state system. The approach is based on a nonlinear transformation of the phase/modulation data that results in a linear model function. Here, the theory is applied to the fluorescent calcium-binding probes Quin-2 and Calcium Green, but is relevant to studies involving other interacting systems. The technique described is used to assess the fraction of bound ligand (Ca2+) and binding constants for these probes.
