PubMed Health⌕ Search

Biomedical subjects

Michael V Berridge

Publications and source records attributed to Michael V Berridge.

12 recordsLinked to original sources

Plasma membrane electron transport: a new target for cancer drug development.

The view that mitochondrial electron transport is the only site of aerobic respiration and the primary bioenergetic pathway in mammalian cells is well established in the literature. Although this paradigm is widely accepted for most tissues, the situation is less clear for proliferating cells. Increasing evidence indicates that glycolytic ATP production contributes substantially to fulfilling the energy requirements of rapidly dividing somatic cells, many tumour cells, and self-renewing stem cells in hypoxic environments. Glycolytic cells have been shown to consume oxygen at the cell surface via plasma membrane electron transport (PMET), a process that oxidises intracellular NADH, supports glycolytic ATP production and may contribute to aerobic energy production. PMET, as determined by reduction of a cell-impermeable tetrazolium dye, is highly active in rapidly-dividing tumour cell lines, where it ameliorates intracellular reductive stress, originating from the mitochondrial TCA cycle. Thus, mitochondrial NADH production is linked to dye reduction outside the cell via the malate-aspartate shuttle. PMET activity increases several-fold under hypoxic conditions, consistent with the view that oxygen competes for electrons from this PMET system. In addition, rho(o) cells that lack mitochondrial electron transport are characterised by elevated PMET presumably to recycle NADH, a role traditionally assumed by lactate dehydrogenase. PMET presents an excellent target for developing novel anticancer drugs that exploit its unique plasma membrane localisation. We propose that PMET is a ubiquitous, high-capacity acute NADH redox-regulatory system responsible for maintaining the mitochondrial NADH/NAD+ ratio. Blocking this pathway compromises the viability of rapidly proliferating cells that rely on PMET.

Animals↗

Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases.

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

Antigens, Neoplasm↗

Mitochondrial gene knockout HL60rho0 cells show preferential differentiation into monocytes/macrophages.

This study compares the differentiation potential of mitochondrial gene knockout (rho0) and parental HL60 cells in response to 1.25% dimethylsulfoxide (DMSO) and 10nM phorbol myristate acetate (PMA). Compared to HL60 cells, undifferentiated HL60rho0 cells showed partial monocyte/macrophage differentiation, with increased CD11c and CD14 expression, decreased CD71 expression, and weak non-specific esterase staining. Differentiation along the monocyte/macrophage pathway (PMA) was more pronounced in HL60rho0 than parental HL60 cells with increased CD11c and CD14 expression and stronger non-specific esterase staining. DMSO-exposure resulted in a poorly differentiated nuclear morphology, small respiratory burst and marginal up-regulation of CD15 expression in HL60rho0 cells.

Antigens, CD↗

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.

Animals↗

Anti-inflammatory sesquiterpene-quinones from the New Zealand sponge Dysidea cf. cristagalli.

The inhibition of superoxide production by human neutrophils has been used to screen New Zealand's unique biota for anti-inflammatory natural products. Bioactivity-directed isolation on an extract of the sponge Dysidea cf. cristagalli led to a new sesquiterpene-quinone (4) with anti-inflammatory activity, plus acetylated hydroquinone (3). These compounds inhibited superoxide production in vitro with IC50's of 3 microM (3) and 11 microM (4).

Animals↗

Cell surface oxygen consumption by mitochondrial gene knockout cells.

Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.

Aerobiosis↗

Hemopoietic cell transformation is associated with failure to downregulate glucose uptake during the G2/M phase of the cell cycle.

Growth factors and cytokines initiate multiple signal transduction pathways that lead to cell survival, cell cycle progression or differentiation. A common feature of these pathways is increased cellular metabolism and glucose uptake. Furthermore, the energy requirements of many cancers and transformed cell lines are met by constitutive upregulation of glucose uptake. Relationships among transforming events, glucose uptake and cell cycle progression are not well understood. Here we investigated the regulation of glucose transport during the cell cycle of growth factor-dependent 32D cells, primary T-cells, src-transformed 32D cells and Jurkat cells. Cells were enriched in the G1, S and G2/M phases of the cell cycle, and glucose transporter expression and 2-deoxyglucose uptake were measured. Glucose transporter expression increased with cell volume as cells progressed through the cell cycle. Growth factor-dependent 32D cells and T-lymphocytes were characterised by increased 2-deoxyglucose uptake from G1 to S and reduced uptake at G2/M, with the highest specific activity of transporters in the S phase. In contrast, src-transformed 32D cells and Jurkat cells showed increased 2-deoxyglucose uptake from S to G2/M, with the highest glucose transporter specific activity in G2/M. Our results show that glucose transport is regulated in a cell cycle-dependent manner and suggest that this regulation may be altered in transformed cells.

Animals↗

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

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 CoQ(1)) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ(1), 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.

1-Octanol↗

Kottamides A-D: novel bioactive imidazolone-containing alkaloids from the New Zealand ascidian Pycnoclavella kottae.

Kottamides A-D (1-4), novel 2,2,5-trisubstituted imidazolone-containing alkaloids, were isolated from the New Zealand endemic ascidian Pycnoclavella kottae and structurally characterized using 15N natural abundance 2-D NMR in addition to standard spectroscopic methods. The kottamides exhibited anti-inflammatory and anti-metabolic activity as well as cytotoxicity toward tumor cell lines.

Alkaloids↗

Peloruside A, a novel antimitotic agent with paclitaxel-like microtubule- stabilizing activity.

Peloruside A is a novel secondary metabolite isolated from a New Zealand marine sponge, Mycale hentscheli, that has potent paclitaxel-like microtubule-stabilizing activity and is cytotoxic at nanomolar concentrations. Its 16-membered macrolide ring is similar to that of epothilone, a drug currently under clinical investigation as an anticancer agent. Like paclitaxel, peloruside A arrests cells in the G(2)-M phase of the cell cycle and induces apoptosis. The relatively simple structure of peloruside makes it suitable for the design and synthesis of analogues with improved tumor targeting and reduced tumor cross-resistance.

Actins↗

Oncogenes modulate cell sensitivity to apoptosis induced by glucose deprivation.

BACKGROUND: Increased glucose uptake is characteristic of many tumours and transformed cells and, consequently, glucose deprivation would be expected to differentially affect cancer cell survival and growth. In this study we investigated whether specific oncogenes sensitise cells to apoptosis induced by glucose deprivation. MATERIALS AND METHODS: Oncogene-transformed 32D cells were deprived of glucose with or without IL-3 and [3H]2-deoxyglucose uptake measured. Apoptosis was determined by AnnexinV/propidium iodide staining, cell cycle distribution was analysed and MYC expression determined by Western blotting. RESULTS: v-Hras and to some extent v-src and v-abl enhanced apoptosis induced by glucose deprivation in the presence of IL-3 but attenuated apoptosis in its absence. In contrast, bcr-abl was highly protective against glucose deprivation-induced apoptosis for 72 hours in the presence and absence of IL-3, while bcl-2 was mildly protective in the presence of IL-3. With strongly transforming oncogenes, c-MYC expression correlated with cell sensitivity to apoptosis induced by glucose deprivation. CONCLUSION: Oncogenes vary markedly in their ability to protect cells from apoptosis following glucose deprivation and, in some situations, promote apoptosis.

Animals↗