Analysis of 13 C and 14 C labeling in pyruvate and lactate in tumor and blood of lymphoma-bearing mice injected with 13 C- and 14 C-labeled pyruvate.

Measurements of hyperpolarized 13 C label exchange between injected [1-13 C]pyruvate and the endogenous tumor lactate pool can give an apparent first-order rate constant for the exchange. The determination of the isotope flux, however, requires an estimate of the labeled pyruvate concentration in the tumor. This was achieved here by measurement of the tumor uptake of [1-14 C]pyruvate, which showed that <2% of the injected pyruvate reached the tumor site. Multiplication of this estimated labeled pyruvate concentration in the tumor with the apparent first-order rate constant for hyperpolarized 13 C label exchange gave an isotope flux that showed good agreement with a flux determined directly by the injection of non-polarized [3-13 C]pyruvate, rapid excision of the tumor after 30 s and measurement of 13 C-labeled lactate concentrations in tumor extracts. The distribution of labeled lactate between intra- and extracellular compartments and the blood pool was investigated by imaging, by measurement of the labeled lactate concentration in blood and tumor, and by examination of the effects of a gadolinium contrast agent and a lactate transport inhibitor on the intensity of the hyperpolarized [1-13 C]lactate signal. These measurements showed that there was significant export of labeled lactate from the tumor, but that labeled lactate in the blood pool produced by the injection of hyperpolarized [1-13 C]pyruvate showed only relatively low levels of polarization. This study shows that measurements of hyperpolarized 13 C label exchange between pyruvate and lactate in a murine tumor model can provide an estimate of the true isotope flux if the concentration of labeled pyruvate that reaches the tumor can be determined.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 is essential for p53-null cancer cells.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) controls metabolic flux through allosteric regulation of glycolysis. Here we show that p53 regulates the expression of PFKFB4 and that p53-deficient cancer cells are highly dependent on the function of this enzyme. We found that p53 downregulates PFKFB4 expression by binding to its promoter and mediating transcriptional repression via histone deacetylases. Depletion of PFKFB4 from p53-deficient cancer cells increased levels of the allosteric regulator fructose-2,6-bisphosphate, leading to increased glycolytic activity but decreased routing of metabolites through the oxidative arm of the pentose-phosphate pathway. PFKFB4 was also required to support the synthesis and regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) in p53-deficient cancer cells. Moreover, depletion of PFKFB4-attenuated cellular biosynthetic activity and resulted in the accumulation of reactive oxygen species and cell death in the absence of p53. Finally, silencing of PFKFB4-induced apoptosis in p53-deficient cancer cells in vivo and interfered with tumour growth. These results demonstrate that PFKFB4 is essential to support anabolic metabolism in p53-deficient cancer cells and suggest that inhibition of PFKFB4 could be an effective strategy for cancer treatment.

Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK).

Towards a successful clinical implementation of fluorescence-guided surgery.

During the European Molecular Imaging Meeting (EMIM) 2013, the fluorescence-guided surgery study group held its inaugural session to discuss the clinical implementation of fluorescence-guided surgery. The general aim of this study group is to discuss and identify the steps required to successfully and safely bring intraoperative fluorescence imaging to the clinics. The focus group intends to use synergies between interested groups as a tool to address regulatory and implementation hurdles in Europe and operates within the intraoperative focus group of the World Molecular Imaging Society (WMIS) that promotes the same interests at the WMIS level. The major topics on the critical path of implementation identified within the study group were quality controls and standards for ensuring accurate imaging and the ability to compare results from different studies, regulatory affairs, and strategies to increase awareness among physicians, regulators, insurance companies, and a broader audience. These hurdles, and the possible actions discussed to overcome them, are summarized in this report. Furthermore, a number of recommendations for the future shape of the fluorescence-guided study group are discussed. A main driving conclusion remains that intraoperative imaging has great clinical potential and that many of the solutions required are best addressed with the community working together to optimally promote and accelerate the clinical implementation of fluorescence imaging towards improving surgical procedures.

Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation.

A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid ß-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species.

Detecting treatment response in a model of human breast adenocarcinoma using hyperpolarised [1-13C]pyruvate and [1,4-13C2]fumarate.

BACKGROUND: The recent introduction of a dynamic nuclear polarisation technique has permitted noninvasive imaging of tumour cell metabolism in vivo following intravenous administration of (13)C-labelled cell substrates. METHODS: Changes in hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate metabolism were evaluated in both MDA-MB-231 cells and in implanted MDA-MB-231 tumours following doxorubicin treatment. RESULTS: Treatment of MDA-MB-231 cells resulted in the induction of apoptosis, which was accompanied by a decrease in hyperpolarised (13)C label flux between [1-(13)C]pyruvate and lactate, which was correlated with a decrease in the cellular NAD(H) coenzyme pool. There was also an increase in the rate of fumarate conversion to malate, which accompanied the onset of cellular necrosis. In vivo, the decrease in (13)C label exchange between pyruvate and lactate and the increased flux between fumarate and malate, following drug treatment, were shown to occur in the absence of any detectable change in tumour size. CONCLUSION: We show here that the early responses of a human breast adenocarcinoma tumour model to drug treatment can be followed by administration of both hyperpolarised [1-(13)C]pyruvate and [1,4-(13)C(2)]fumarate. These techniques could be used, therefore, in the clinic to detect the early responses of breast tumours to treatment.

The assessment of antiangiogenic and antivascular therapies in early-stage clinical trials using magnetic resonance imaging: issues and recommendations.

Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used.

Early detection of tumour immune-rejection using magnetic resonance imaging.

Dynamic contrast agent-enhanced magnetic resonance imaging measurements of the perfusion of an immunogenic murine tumour showed that immune rejection was preceded by an increase in the apparent vascular volume of the tumour. This increase in vascularity, which has been observed previously in other tumours undergoing immune rejection, was confirmed by histological analysis of tumour sections obtained postmortem. Magnetic resonance imaging measurements similar to this could be used in the clinic to monitor the early responses of tumours to immunotherapy, before there is any change in tumour growth rate or volume.

Molecular imaging using magnetic resonance: new tools for the development of tumour therapy.

Molecular imaging - the exploitation of specific molecules as the source of image contrast - promises new insights into disease processes in the laboratory and since the imaging modalities employed are applicable clinically, can be used to translate this knowledge into new diagnostics and treatments in the clinic. This brief review focuses on the use of MR-based molecular imaging techniques for developing tumour therapy. As examples, methods for detecting drug-induced tumour cell apoptosis; the response of tumours and their susceptibilities to an antivascular drug; early signs of tumour immune rejection and methods for detecting immune cell infiltration of tumours are described.

Measurement of bioreactor perfusion using dynamic contrast agent-enhanced magnetic resonance imaging.

Dynamic magnetic resonance imaging was used to monitor solute diffusion through aggregates of Chinese hamster ovary cells growing on macroporous carriers in a fixed-bed bioreactor. Diffusion-weighted (1)H magnetic resonance imaging (MRI) and scanning electron microscopy demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the periphery of the carriers. T(1)-weighted magnetic resonance imaging measurements of the inflow of a commonly used magnetic resonance contrast agent, gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA), showed that migration of the agent through the peripheral cell masses could be explained by diffusion. However, appearance of the contrast agent in the center of the carriers was too fast to be explained by simple diffusion and indicated that these regions were perfused by convective flow. The average diffusivity of Gd-DTPA through the cell mass was found to be (2.4 +/- 0.2) x 10(-10) m(2) sec(-) (mean +/- SEM). This technique will be useful in the characterization and development of high-cell-density bioreactor systems, in which solute transport plays a critical role in cell growth and physiology.

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.

The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs.

Potent anti-metastatic activity of combretastatin-A4.

The requirement for tumour vascularisation to permit the expansion of solid tumours beyond a threshold size of approximately 1 mm diameter has focussed attention on anti-vascular and anti-angiogenic agents for cancer therapy. Combretastatin-A4 (cis CA-4P) is a tubulin-binding agent that is cytotoxic for proliferating endothelial cells in vitro and causes anti-vascular effects in the established tumour vessels of some primary tumours. Preliminary data from Phase I clinical trials indicate that cis CA-4 may also be effective in targeting the vasculature of human tumours. As metastatic disease is the principal cause of mortality in cancer, we have investigated the effects of cis CA-4 on metastatic development using an in vivo model. We show that bolus or continuous administration of cis CA-4P results in potent inhibition of metastases derived from ectopic primary Lewis lung carcinomas in mice whereas the trans CA-4 isomer is without effect. These data further characterise the activity of CA-4 in vivo and suggest that the drug should be evaluated clinically as an anti-metastatic agent.

The susceptibility of tumors to the antivascular drug combretastatin A4 phosphate correlates with vascular permeability.

The acute effects of the antivascular drug, combretastatin A4 phosphate, on tumor energy status and perfusion were assessed using magnetic resonance imaging (MRI) and spectroscopy. Localized (31)P magnetic resonance spectroscopy showed that LoVo and RIF-1 tumors responded well to drug treatment, with significant increases in the P(i)/nucleoside triphosphate ratio within 3 h, whereas SaS, SaF, and HT29 tumors did not respond to the same extent. This variable response was also seen in MRI experiments in which tumor perfusion was assessed by monitoring the kinetics of inflow of the contrast agent, gadolinium diethylenetriaminepentaacetate. These data were analyzed to give the initial rate and time constant for inflow of contrast agent and the integral under the inflow curve. The differential susceptibility of the tumors to combretastatin A4 phosphate showed a positive correlation with prior MRI measurements of tumor vascular permeability, which was determined by measuring the inflow of a macromolecular contrast agent, BSA-gadolinium diethylenetriaminepentaacetate.

A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast.

Uncoupling protein 1 (UCP1) from mouse was expressed in yeast and the specific (GDP-inhibitable) and artifactual (GDP-insensitive) effects on mitochondrial uncoupling were assessed. UCP1 provides a GDP-inhibitable model system to help interpret the uncoupling effects of high expression in yeast of other members of the mitochondrial carrier protein family, such as the UCP1 homologues UCP2 and UCP3. Yeast expressing UCP1 at modest levels (approx. 1 microg/mg of mitochondrial protein) showed no growth defect, normal rates of chemically uncoupled respiration and an increased non-phosphorylating proton conductance that was completely GDP-sensitive. The catalytic-centre activity of UCP1 in these yeast mitochondria was similar to that in mammalian brown-adipose-tissue mitochondria. However, yeast expressing UCP1 at higher levels (approx. 11 microg/mg of mitochondrial protein) showed a growth defect. Their mitochondria had depressed chemically uncoupled respiration rates and an increased proton conductance that was partly GDP-insensitive. Thus, although UCP1 shows native behaviour at modest levels of expression in yeast, higher levels (or rates) of expression can lead to an uncoupling that is not a physiological property of the native protein and is therefore artifactual. This observation might be important in the interpretation of results from experiments in which the functions of UCP1 homologues are verified by their ability to uncouple yeast mitochondria.

Combretastatin-A4 disrupts neovascular development in non-neoplastic tissue.

Combretastatin-A4 phosphate (cis-CA-4) is a tubulin-binding agent currently undergoing clinical trials as an anti-tumour drug. We have investigated whether CA-4 functions as a tumour-specific anti-vascular agent using the hyperplastic thyroid as a novel in vivo model of neovascularization. CA-4 elicited pathological changes in normal tissue, manifested as the induction of multiple, discrete intravascular thrombi. These vascular-damaging effects indicate that CA-4P does not function as a tumour-specific agent but targets neovasculature irrespective of the primary angiogenic stimulus.

Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria.

We assessed the ability of human uncoupling protein 2 (UCP2) to uncouple mitochondrial oxidative phosphorylation when expressed in yeast at physiological and supraphysiological levels. We used three different inducible UCP2 expression constructs to achieve mitochondrial UCP2 expression levels in yeast of 33, 283, and 4100 ng of UCP2/mg of mitochondrial protein. Yeast mitochondria expressing UCP2 at 33 or 283 ng/mg showed no increase in proton conductance, even in the presence of various putative effectors, including palmitate and all-trans-retinoic acid. Only when UCP2 expression in yeast mitochondria was increased to 4 microg/mg, more than an order of magnitude greater than the highest known physiological concentration, was proton conductance increased. This increased proton conductance was not abolished by GDP. At this high level of UCP2 expression, an inhibition of substrate oxidation was observed, which cannot be readily explained by an uncoupling activity of UCP2. Quantitatively, even the uncoupling seen at 4 microgram/mg was insufficient to account for the basal proton conductance of mammalian mitochondria. These observations suggest that uncoupling of yeast mitochondria by UCP2 is an overexpression artifact leading to compromised mitochondrial integrity.

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations.

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line.

Overexpression of the liver subunit of 6-phosphofructo-1-kinase in Chinese hamster ovary K1 cells was shown to increase the steady-state level of the enzyme's product, fructose 1, 6-bisphosphate, and to produce a small but significant decrease in the concentration of fructose 2,6-bisphosphate, which is an allosteric activator of the enzyme. However, overexpression of the enzyme had no effect on glycolytic flux under a variety of different substrate conditions. This latter observation is consistent with similar studies in fungi and in potato tubers which indicate that 6-phosphofructo-1-kinase has very little control over flux in glycolysis.