An S. Tan

Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.

Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

Metabolic flexibility and cell hierarchy in metastatic cancer.

Cancer is characterized by disturbed homeostasis of self-renewing cell populations, and their ability to seed and grow in multiple microenvironments. This overarching cellular property of metastatic cancer emerges from the contentious cancer stem cell hypothesis that underpins the more generic hallmarks of cancer (Hanahan and Weinberg, 2000) and its subsequent add-ons. An additional characteristic, metabolic flexibility, is related to concepts developed by Warburg and to subsequent work by mid 20th century biochemists who elucidated the bioenergetic workings of mitochondria. Metabolic flexibility may circumvent limitations inherent in the increasingly popular but erroneous view that aerobic glycolysis is a universal property of cancer cells. Cancer research in the second half of the 20th century was largely the domain of geneticists and molecular biologists using reductionist approaches. Integrated approaches that address cancer cell hierarchy and complexity, and how cancer cells adapt their metabolism according to their changing environment are now beginning to emerge, and these approaches promise to address the poor mortality statistics of metastatic cancer.

Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents

Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 microg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 microM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay sensitivity. Addition of the intermediate electron acceptor, 1-methoxy phenazine methosulfate, increased the background of the neutrophil assay but did not affect the overall magnitude of the response. We have used the WST-1 assay to assess human neutrophil dysfunction and to compare anti-inflammatory activity.

Mitochondrial Genome Acquisition Restores Respiratory Function and Tumorigenic Potential of Cancer Cells without Mitochondrial DNA

We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer. DOI: http://dx.doi.org/10.7554/eLife.22187.001

The Hemopoietic Growth Factor, Interleukin-3, Promotes Glucose Transport by Increasing the Specific Activity and Maintaining the Affinity for Glucose of Plasma Membrane Glucose Transporters

Most mammalian cells rely on an external supply of glucose for survival, proliferation, and function. Glucose enters cells through specific transporter molecules at the plasma membrane by a facilitative process that does not expend energy. Regulation of glucose transport into cells is thought to occur largely through transporter expression at the cell surface, but the extent to which the intrinsic properties of glucose transporters are regulated is at present controversial. Using a bone marrow-derived cell line that responds to the hemopoietic growth factor, interleukin-3 (IL-3), we investigated IL-3 regulation of glucose transport. IL-3 significantly increased 2-deoxyglucose (2-DOG) uptake within 1 h (26 ± 8.0%, n = 11) with a maximum 73% increase after 6 h. Withdrawal of IL-3 resulted in decreased uptake within 1 h and this continued to decline to 43% of initial uptake by 16 h. To determine whether these changes in 2-DOG uptake were associated with corresponding changes in glucose transporter expression, subtype-specific antisera against Glut-1 and Glut-3 were used. Little change in membrane expression of these transporters was observed prior to 16 h. Fractionation of cell membranes on Nycodenz gradients showed that the majority of each transporter subtype was associated with the plasma membrane (63–93%) and that transporter distribution did not change markedly in response to addition or withdrawal of IL-3. These results demonstrate that IL-3 regulates glucose uptake by modulating the intrinsic transporting ability of glucose transporters. Decreased transporter affinity for 2-DOG and 3-O-methylglucose was observed following IL-3 withdrawal. Similar affinity changes were observed with 2-DOG following exposure of IL-3-stimulated cells to the protein kinase inhibitors, genistein and staurosporine. In contrast, the tyrosine phosphatase inhibitor, vanadate, acted like IL-3 to increase transporter affinity for glucose. Together these results demonstrate that IL-3 acts to maintain the intrinsic transport properties of glucose transporters without markedly affecting their expression or translocation.

The role of mitochondrial electron transport in tumorigenesis and metastasis.

BACKGROUND Tumor formation and spread via the circulatory and lymphatic drainage systems is associated with metabolic reprogramming that often includes increased glycolytic metabolism relative to mitochondrial energy production. However, cells within a tumor are not identical due to genetic change, clonal evolution and layers of epigenetic reprogramming. In addition, cell hierarchy impinges on metabolic status while tumor cell phenotype and metabolic status will be influenced by the local microenvironment including stromal cells, developing blood and lymphatic vessels and innate and adaptive immune cells. Mitochondrial mutations and changes in mitochondrial electron transport contribute to metabolic remodeling in cancer in ways that are poorly understood. SCOPE OF REVIEW This review concerns the role of mitochondria, mitochondrial mutations and mitochondrial electron transport function in tumorigenesis and metastasis. MAJOR CONCLUSIONS It is concluded that mitochondrial electron transport is required for tumor initiation, growth and metastasis. Nevertheless, defects in mitochondrial electron transport that compromise mitochondrial energy metabolism can contribute to tumor formation and spread. These apparently contradictory phenomena can be reconciled by cells in individual tumors in a particular environment adapting dynamically to optimally balance mitochondrial genome changes and bioenergetic status. GENERAL SIGNIFICANCE Tumors are complex evolving biological systems characterized by genetic and adaptive epigenetic changes. Understanding the complexity of these changes in terms of bioenergetics and metabolic changes will permit the development of better combination anticancer therapies. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Evidence for NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling via plasma membrane electron transport: A sensitive cellular assay for NQO1

2,3-Dimethoxy 1,4-naphthoquinone (DMNQ), which redox cycles via two-electron reduction, mediates reduction of the cell-impermeative tetrazolium dye WST-1 in kidney epithelial cells (MDCK), which express high levels of NQO1, but not in HL60 or CHO cells, which are NQO1 deficient. DMNQ-dependent WST-1 reduction by MDCK cells was strongly inhibited by low concentrations of the NQO1 inhibitor dicoumarol and was also inhibited by diphenyleneiodonium, capsaicin, and superoxide dismutase (SOD), but not by the uncoupler FCCP or the complex IV inhibitor cyanide. This suggests that DMNQ-dependent WST-1 reduction by MDCK cells is catalyzed by NQO1 via redox cycling and plasma membrane electron transport (PMET). Interestingly, we observed an association between DMNQ/WST-1 reduction and extracellular H(2)O(2) production as determined by Amplex red. Exposure of MDCK cells to DMNQ for 48 h caused cellular toxicity that was extensively reversed by co-incubation with dicoumarol or exogenous SOD, catalase, or N-acetylcysteine. No effects were observed in NQO1-deficient CHO and HL60 cells. In conclusion, we have developed a simple real-time cellular assay for NQO1 and show that PMET plays a significant role in DMNQ redox cycling via NQO1, leading to cellular toxicity in cells with high NQO1 levels.

Effects of Mitochondrial Gene Deletion on Tumorigenicity of Metastatic Melanoma: Reassessing the Warburg Effect

Metabolic flexibility is a hallmark of cancer. Although many tumors preferentially use glycolysis in the presence of oxygen for bioenergetic purposes (Warburg effect), the effects of glycolytic metabolism on tumor metastasis have not been investigated. We have employed an extreme model of glycolytic metabolism to investigate the ability of metastatic B16 mouse melanoma cells to grow as primary subcutaneous tumors and to form lung tumors when injected intravenously into syngeneic and immunocompromised mice. Mitochondrial gene-knockout B16rho degrees cells showed delayed subcutaneous tumor growth and, surprisingly, failed to form lung tumors. The results suggest that mitochondrial reactive oxygen species (ROS) may be required for tumor metastasis.

Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells.

Abstract Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ1) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ1, whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40–60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.