Mitochondrial transfer between cells: Methodological constraints in cell culture and animal models.

Interest in the recently discovered phenomenon of mitochondrial transfer between mammalian cells has gained momentum since it was first described in cell culture systems more than a decade ago. Mitochondria-targeting fluorescent dyes have been repurposed and are now widely used in these studies and in acute disease models, sometimes without due consideration of their limitations, while vectors containing mitochondrially-imported fluorescent proteins have complemented the use of mitochondria-targeting dyes. Genetic approaches that use mitochondrial DNA polymorphisms have also been used in some in vitro studies and in tumor models and are particularly useful where mtDNA is damaged or deleted. These approaches can also be used to study the long-term consequences of mitochondrial transfer such as in bone marrow and organ transplantation and in tumour biology where inherent mitochondrial damage is often a key feature. As research on intercellular mitochondrial transfer moves from cell culture into animal models and human diseases it will be important to understand the limitations of the various techniques in order to apply appropriate methodologies to address physiological and pathophysiological conditions.

The Δ133p53 isoform and its mouse analogue Δ122p53 promote invasion and metastasis involving pro-inflammatory molecules interleukin-6 and CCL2.

A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.

The antiproliferative effects of phenoxodiol are associated with inhibition of plasma membrane electron transport in tumour cell lines and primary immune cells.

Although the redox-active synthetic isoflavene, phenoxodiol, is in Phase 3 clinical trials for drug-resistant ovarian cancer, and in early stage clinical trials for prostate and cervical cancer, its primary molecular target is unknown. Nevertheless, phenoxodiol inhibits proliferation of many cancer cell lines and induces apoptosis by disrupting FLICE-inhibitory protein, FLIP, expression and by caspase-dependent and -independent degradation of the X-linked inhibitor of apoptosis, XIAP. In addition, phenoxodiol sensitizes drug-resistant tumour cells to anticancer drugs including paclitaxel, carboplatin and gemcitabine. Here, we investigate the effects of phenoxodiol on plasma membrane electron transport (PMET) and cell proliferation in human leukemic HL60 cells and mitochondrial gene knockout HL60rho(o) cells that exhibit elevated PMET. Phenoxodiol inhibited PMET by both HL60 (IC(50) 32 microM) and HL60rho(o) (IC(50) 70 microM) cells, and this was associated with inhibition of cell proliferation (IC(50) of 2.8 and 6.7 microM, respectively), pan-caspase activation and apoptosis. Unexpectedly, phenoxodiol also inhibited PMET by activated murine splenic T cells (IC(50) of 29 microM) as well as T cell proliferation (IC(50) of 2.5 microM). In contrast, proliferation of WI-38 cells and HUVECs was only weakly affected by phenoxodiol. These results indicate that PMET may be a primary target for phenoxodiol in tumour cells and in activated T cells.

Peloruside A enhances apoptosis in H-ras-transformed cells and is cytotoxic to proliferating T cells.

Peloruside A (peloruside), a compound isolated from the marine sponge Mycale hentscheli , inhibits growth of human (HL-60) and mouse (32D-ras) myeloid leukemic cells, as well as non-transformed 32D cells. Using the MTT cell proliferation assay and trypan blue dye exclusion tests, little difference was seen in growth inhibition between 32D and 32D- ras cells; however, peloruside was more cytotoxic to the oncogene-transformed cells. Peloruside also blocked 32D- ras cells more readily in G2/M of the cell cycle, leading to apoptosis. Annexin-V/propidium iodide staining of 32D and 32D- ras cells showed that 1.6 microM peloruside induced significant cell death by 36 hours in 32D cells (16% survival), but to comparable levels as early as 14 hours in 32D- ras cells (11% survival). There was no evidence for activation of either of the initiator caspases-8 or -9 by 0.1 microM peloruside following 12 hours of exposure. Peloruside inhibited T cell proliferation and IL-2 and IFN gamma production in both the mixed lymphocyte reaction and following CD3 cross-linking, and this effect was shown to be a non-specific cytotoxic effect. It is concluded that peloruside preferentially targets oncogene-transformed cells over non-transformed cells by inducing transformed cells to undergo apoptosis.

Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine.

The marine sponge metabolites mycalamide A (mycalamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine-beta-D-arabinofuranoside (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.

The novel cytotoxic sponge metabolite peloruside A, structurally similar to bryostatin-1, has unique bioactivity independent of protein kinase C.

A novel secondary sponge metabolite, peloruside A (peloruside), isolated from the marine sponge Mycale sp. (New Zealand), was tested for its cytotoxic effects on mammalian cells in culture. The macrolide structure of peloruside is similar to that of the protein kinase C (PKC) activator, bryostatin-1 (bryostatin), both containing a pyranose ring adjacent to a gemdimethyl moiety. Peloruside is a potent inhibitor of cell proliferation. Treatment of different mammalian cell lines with peloruside for 48-96 h gave IC50 values ranging from 4 to 15 nM, using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium (MTT) cell proliferation assay. Peloruside was shown to be both cytostatic and cytotoxic by trypan blue dye exclusion tests. Peloruside induced apoptosis in a dose-dependent manner in murine (32D) and human (HL-60) myeloid cell lines, revealed by DNA laddering in agarose gels and flow cytometric analysis of annexin-V- and propidium iodide-stained cells. Treatment of HL-60 cells caused vacuolisation, partial substrate adherence, and the appearance of multi-lobed nuclei, suggesting the induction of a differentiation pathway. Vacuolisation was also observed in a human lung cancer cell line (H441). Opening of the pyranose ring of peloruside by sodium borohydride reduction increased the 48 h IC50 value by 26-fold in 32D cells, suggesting a similar active site to that proposed for bryostatin. However, unlike bryostatin, peloruside failed to bind to PKC in HL-60 cells and was unable to synergize with the calcium ionophore, ionomycin, or with interleukin-2, to activate T-lymphocytes in culture. In summary, although structurally similar to bryostatin, peloruside is a potent inhibitor of cell proliferation, has apoptosis-inducing properties and has a unique mode of action independent of PKC.

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.

High-capacity redox control at the plasma membrane of mammalian cells: trans-membrane, cell surface, and serum NADH-oxidases.

The high capacity of proliferating mammalian cells to transfer electrons from cytosolic NADH to extracellular electron acceptors like oxygen is poorly understood and not widely recognized. Nevertheless, trans-plasma membrane electron transport (plasma membrane redox control) probably ranks alongside the Na+/H+ antiport system (pH control) and glucose transport in facilitating cellular responses to physiological stimuli. These plasma membrane transport systems are acutely responsive to receptor ligation by growth factors, polypeptide hormones, and other cell activators. A novel tetrazolium-based cell proliferation assay that we have shown to measure an NADH-oxidoreductase component of the trans-plasma membrane electron transport system has allowed direct comparisons with NADH:ferricyanide-oxidoreductase and respiratory burst NADPH-oxidoreductase. In addition, an NAD(P)H-oxidase at the cell surface and an NADH-oxidase activity in body fluids can be measured by modifying the basic cell proliferation assay. As determined by reduction of the cell-impermeable tetrazolium reagent, WST-1, electron transfer across the plasma membrane of dividing cells can exceed that of fully activated human peripheral blood neutrophils. Cellular reduction of WST-1 is dependent on the presence of an intermediate electron acceptor and is inhibited by superoxide dismutase (SOD) and by oxygen, implying indirect involvement of superoxide in WST-1 reduction. Cell-surface NAD(P)H-oxidase and serum NADH-oxidase are shown to be distinct from trans-plasma membrane NADH-oxidoreductase by their differential sensitivity to capsaicin and pCMBS. The glycolytic metabolism of cancer cells may be linked to changes in trans-plasma membrane NADH:WST-1-oxidoreductase activity and to increased serum NADH-oxidase in cancer.

Cell-surface NAD(P)H-oxidase: relationship to trans-plasma membrane NADH-oxidoreductase and a potential source of circulating NADH-oxidase.

The surface of mammalian cells faces an oxidizing environment that has the potential to damage proteins, lipids, and carbohydrates to which it is exposed. In contrast, the cytoplasm is reducing and its redox state is tightly regulated. Trans-plasma membrane oxidoreductases that shift electrons from cytosolic NADH to external electron acceptors such as oxygen are widely involved in cellular redox control. They reduce oxygen to water and may generate reactive oxygen species such as superoxide and hydrogen peroxide. In addition, external NAD(P)H-oxidases have been demonstrated on intact cells and as eluted proteins, but the relationship between trans-plasma membrane NADH-oxidoreductases and cell-surface NAD(P)H-oxidases is not known. To investigate further the relationship between plasma membrane NAD(P)H-oxidoreductases, and to gain insight into the physiological functions of these redox active membrane proteins, we have adapted a simple colorimetric assay for measuring the trans-plasma membrane NADH-oxidoreductase activity of viable cells to measure NAD(P)H-oxidase at the cell surface in real time. Using the cell-impermeable tetrazolium salt WST-1 in the presence of NADH or NADPH, but in the absence of an intermediate electron acceptor, we show that cell-surface NAD(P)H-oxidase is widely expressed on mammalian cells, being more abundant on rapidly proliferating cells than on resting neutrophils and spleen cells. The ratio of cofactor dependence of NAD(P)H-oxidase (NADH:NADPH) varied widely between different cells (0.7-5.2), suggesting a family of cell surface oxidases or that the activity of these enzymes may be modulated in various ways. Comparison of NAD(P)H-oxidase on the surface of viable cells with trans-membrane NADH-oxidoreductase, measured with WST-1 in the presence of 1-methoxy PMS, showed that cell-surface NAD(P)H-oxidase was differentially inhibited by the cell-impermeable thiol-blocking agent pCMBS, but was unaffected or stimulated by other thiol blocking agents. Capsaicin, which inhibits trans-plasma membrane NADH-oxidoreductase activity, stimulated surface NAD(P)H-oxidase. Metabolic inhibitors had little effect on surface NAD(P)H-oxidase activity but inhibited trans-plasma membrane activity. These results do not support the view the surface NAD(P)H-oxidase is a terminal oxidase for trans-plasma membrane NADH-oxidoreductase.

Ceramides that mediate apoptosis reduce glucose uptake and transporter affinity for glucose in human leukaemic cell lines but not in neutrophils.

We have demonstrated that CD95-induced apoptosis in a human leukaemic T-cell line resulted in loss of glucose transporter function (Berridge et al. 1996). To determine whether ceramide, a mediator of CD95 and tumour necrosis factor-alpha-induced apoptosis, has similar effects on glucose transport, the human leukaemic cell lines, Jurkat and U937, and human peripheral blood neutrophils were treated with ceramide or sphingomyelinase and the effects on glucose transport determined by measuring [3H]-2-deoxyglucose uptake. We show that in U937 and Jurkat cells, the cell permeable ceramides, C2 (N-acetylsphingosine) and C6 (N-hexanoylsphingosine) inhibit glucose uptake within minutes of initiating ceramide treatment, 60-70% inhibition being observed within 2 hr. Loss of glucose transport correlated with loss of proliferative response, but metabolic activity as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction, was affected to a much lesser extent. With Jurkat and U937 cells, the inhibitory effects of ceramides on glucose transport were associated with reduced affinity of glucose transporters for glucose (Km). Similar effects were observed with sphingomyelinase. With human peripheral blood neutrophils, C2 and C6-ceramides inhibited glucose uptake by 70-80% within 30 min. without affecting transporter affinity for glucose, but the maximum velocity of uptake (Vmax) was reduced. These results show that acute regulation of glucose transport is an early effector mechanism of cell death induced by ceramides in human leukaemic cell lines and peripheral blood neutrophils. This is the first study which describes ceramide-induced early physiological/biochemical events leading to cell death in human cells.

N-glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells.

To elucidate the role of N-glycosylation in the functional activity of the universal glucose transporter, Glut-1, we investigated effects of the N-glycosylation inhibitor, tunicamycin, on glucose transport by human leukemic cell lines K562, U937 and HL60. Treatment with tunicamycin produced a 40-50% inhibition of 2-deoxyglucose uptake and this was associated with a 2-2.5-fold decrease in transporter affinity for glucose (Km) without a change in Vmax. Leukemic K562, U937 and HL60 cells expressed Glut-1 transporter protein. With K562 cells Glut-1 appeared as a broad band of 50-60 kDa, whereas with U937 and HL60 cells a diffuse band was observed at approximately 55 kDa. Treatment of K562 cells with tunicamycin for 18 h, resulted in extensive loss of the 50-60 kDa glycoprotein, appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With U937 cells, tunicamycin treatment resulted in the appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With HL60 cells loss of the 55 kDa Glut-1 band was observed and a band of 45 kDa appeared. Tunicamycin-treatment resulted in 75-90% inhibition in [3H]mannose incorporation but only 20-25% inhibition in [3H]thymidine and [3H]leucine incorporation. In contrast, tunicamycin had little effect on the viability and MTT responses of the cells used. These results suggest that in leukemic cells N-glycosylation of Glut-1 plays an important role in maintaining its structure and functional integration.

Distinct regulation of glucose transport by interleukin-3 and oncogenes in a murine bone marrow-derived cell line.

Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dc13 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dc13 cells by 40-50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72-112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases.

IL-3 induces apoptosis in a ras-transformed myeloid cell line.

Growth factors promote cell survival and proliferation. Homeostasis is maintained by programmed cell death which occurs when the growth stimulus is withdrawn, in response to negative growth regulators such as interferons, TNF-alpha and CD95 ligand, or following differentiation. Although acutely-transforming oncogenes often overcome the need for growth factors, growth regulatory cytokines can influence proliferative responses of transformed cells. In this study we investigated the effects of IL-3 on the proliferative responses of parental bone marrow-derived 32D cells and cells transformed with ras and abl oncogenes. We show that treatment of ras-transformed 32D cells with IL-3 reduced proliferative responses and decreased colony-forming ability. These effects were exacerbated in the absence of serum and associated with inhibition of tyrosine kinase activity, down-regulation of RAS and MYC expression, and induction of apoptosis as indicated by DNA fragmentation. In contrast, treatment of parental 32D cells with IL-3, which is obligatory for cell survival and proliferation, increased tyrosine kinase activity, upregulated MYC and RAS expression and maintained DNA integrity. With abl-transformed cells, proliferation and colony-forming ability were also inhibited by IL-3. Tyrosine kinase activity and MYC expression were reduced, but early apoptosis was not evident. Calcium uptake however, was stimulated by IL-3 in both parental and oncogene-transformed cells. These results suggest that threshold levels of tyrosine kinase activity are necessary for cell survival and proliferation and that with ras-transformed cells, IL-3 treatment may result in this threshold being breached. We conclude that in some situations, growth-promoting cytokines can inhibit proliferation of transformed cells and induce cell death by apoptosis.

Transforming oncogenes regulate glucose transport by increasing transporter affinity for glucose: contrasting effects of oncogenes and heat stress in a murine marrow-derived cell line.

Transforming oncogenes often overcome the growth factor requirements of cells by activating growth factor signal transduction pathways. Increased energy utilization by transformed cells is a well known phenomenon, but whether glucose uptake is regulated at the level of the glucose transporter has not been clearly established. To determine whether cell transformation by specific oncogenes like, v-H-ras and v-abl affects the activation state of glucose transporters, bone marrow-derived IL-3-dependent 32D (clone3) cells transfected with temperature-sensitive ras and abl oncogenes were used to compare proliferative responses and glucose transporting ability of these cells with the parental cell line at permissive (32 degrees C) and non-permissive (40 degrees C) temperatures. Transformed cells showed elevated incorporation of [3H]thymidine and enhanced tyrosine kinase activity, both of which were abrogated in temperature-sensitive mutants maintained at the non-permissive temperature. Compared with control cells, 2-deoxy-D-[1-(3)H]glucose (2-DOG) uptake was not significantly different in transformed cells at the permissive temperature. However, transformation was associated with a 2-2.5-fold greater affinity of glucose transporters for glucose (Km) and this was reversed following treatment with tyrosine kinase inhibitor, genistein. Maximum velocity of glucose transport (Vmax) and membrane expression of transporters were reduced in oncogene-transformed cells. At the non-permissive temperature, glucose uptake was elevated in both control and oncogene-transformed cells. This increase in glucose transport was not associated with a change in transporter affinity for glucose, but increased Glut-1 expression was observed indicating a 'heat stress' effect that overrode the effects attributable to oncogene loss. The 'heat stress' effect was inhibited by protein synthesis inhibitor cycloheximide. These results provide evidence for intrinsic activation of glucose transporters by the transforming oncogenes ras and abl, and indicate that oncogenes and 'heat stress' regulate glucose transport by different mechanisms.

Acute regulation of glucose transport after activation of human peripheral blood neutrophils by phorbol myristate acetate, fMLP, and granulocyte-macrophage colony-stimulating factor.

Activation of human peripheral blood neutrophils by pathogens or by phorbol myristate acetate (PMA), fMLP, or myeloid growth factors generates a respiratory burst in which superoxide production plays an important role in killing invading microorganisms. Although the increased energy demands of activated neutrophils would be expected to be associated with increased glucose uptake and utilization, previous studies have shown that PMA inhibits 2-deoxyglucose (2-DOG) uptake. In this study, we show that PMA activation of neutrophils, isolated by methods not involving hypotonic lysis, increases the rate of 2-DOG uptake and results in a 1.6-fold to 2.1-fold increase in transporter affinity for glucose without changing Vmax. Increased transporter affinity in response to PMA was also observed with 3-O-methyglucose, which is not phosphorylated, and inclusion of glucose in the activation medium further increased respiratory burst activity. Increased 2-DOG uptake and increased transporter affinity for glucose were also observed with the peptide activator, fMLP, and with granulocyte-macrophage colony-stimulating factor (GM-CSF). The protein kinase C (PKC) inhibitor, calphostin C, and the tyrosine kinase inhibitor, genistein, inhibited both PMA- and fMLP-stimulated 2-DOG uptake. In contrast, genistein inhibited fMLP-induced superoxide production, but had little effect on the PMA-induced response, while staurosporine differentially inhibited PMA-induced superoxide production. These results show that neutrophil activation involves increased glucose transport and intrinsic activation of glucose transporter molecules. Both tyrosine kinases and PKC are implicated in the activation process.

Acute regulation of glucose transport in a monocyte-macrophage cell line: Glut-3 affinity for glucose is enhanced during the respiratory burst.

Activation of the respiratory burst imposes acute metabolic demands on phagocytic cells. These are met by mobilizing internal energy stores and by increasing the utilization of exogenous energy, including glucose in the circulation. To determine whether the increased glucose uptake that is known to be associated with the respiratory burst involves the regulation of glucose transporter molecules, the intrinsic transport properties of glucose transporters on the macrophage cell line RAW 264.7 were determined after activation with PMA, N-formyl-methionine-leucine-phenylalanine (fMLP) and the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3). Treatment with PMA resulted in a 2-fold increase in respiratory burst activity within 10 min; this was associated with a 30-50% increase in 2-deoxyglucose uptake and a 4-fold increase in transporter affinity for glucose. Similarly, fMLP, GM-CSF and IL-3 treatments stimulated 2-deoxyglucose uptake that was associated with a 3-4-fold increase in transporter affinity for glucose. To determine whether the changes observed in 2-deoxyglucose uptake in response to PMA, fMLP and growth factors were influenced by phosphorylation of the sugar, 3-O-methylglucose, which is not phosphorylated, was used. Increased 3-O-methylglucose uptake and increased transporter affinity for glucose were also observed after PMA, fMLP and GM-CSF treatments. Whereas both fMLP and GM-CSF stimulated superoxide production, IL-3 failed to activate respiratory burst activity. The protein kinase inhibitors genistein and staurosporine inhibited the increase in 2-deoxyglucose uptake observed with fMLP and GM-CSF, and partly reversed the affinity increase towards that of untreated control cells. In contrast, the phosphatidylinositol 3-kinase inhibitor wortmannin had little effect on 2-deoxyglucose uptake in response to these activators. Western blotting with subtype-specific antisera showed that Glut-3 was the predominant transporter on RAW 264.7 cells. These studies demonstrate that acute regulation of glucose transporters occurs in response to activators that promote respiratory burst activity, and show that this regulation involves both tyrosine kinases and protein kinase C activity.

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.

Regulation of glucose transport by interleukin-3 in growth factor-dependent and oncogene-transformed bone marrow-derived cell lines.

Growth factors maintain cell viability and promote cell growth by stimulating glucose transport into cells and by progressing cells through the cell cycle. In the short term, effects on glucose transport involve transporter activation, while in the longer term increased gene expression is involved. This study aimed to investigate growth factor regulation of glucose transport in an interleukin (IL)-3-dependent bone marrow-derived cell line and its oncogene-transformed counterparts. 32D clone 3 (32Dcl3) cells and cells transfected with temperature-sensitive (ts) ras and abl oncogenes, were treated with and without IL-3 and their ability to take up 2-deoxy-D-glucose compared. Transformed cells, which are not dependent on IL-3 for growth at the permissive temperature of 32 degrees C, exhibited a two- to six-fold higher proliferative response, enhanced tyrosine kinase activity and c-myc expression than control cells optimally stimulated with IL-3. Compared with control 32Dcl3 cells, 2-deoxy-D-glucose uptake was also 36-76% higher in transformed cells. The increased glucose uptake in transformed cells was consistent with 2.5-fold higher affinity of the glucose transporters for glucose. IL-3 stimulated glucose uptake in both control and oncogene-transformed cells. With control and ras-transformed cells, enhanced glucose uptake in response to IL-3 was associated with increased affinity of glucose transporters for glucose but with abl-transformed cells, no significant affinity changes were observed. IL-3 also increased glucose transporter expression in both control and oncogene-transformed cells, suggesting that increased transporter expression as well as changes in transporter affinity for glucose can affect glucose uptake.

CD95 (Fas/Apo-1)-induced apoptosis results in loss of glucose transporter function.

Treatment of activated human T cells with CD95 (Fas/Apo-1) ligand or Abs against CD95 results in apoptotic cell death. Although cellular responses to CD95 ligation have been described in some detail, the early molecular events that result in T cell death are only now beginning to be elucidated. Using Jurkat cells as a model of activated human T cells, we have investigated the effects of CD95 ligation on glucose transport and on glucose transporter function. We show that within minutes of CD95 activation, the ability to transport glucose across the plasma membrane is compromised and that transient exposure to Abs against CD95 for as little as 3 min results in reduced glucose transport and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) responses measured at 16 h. The effects of CD95 ligation on glucose transport are shown to be associated with loss of affinity of glucose transporters for glucose without altered maximum velocity and without changes in the cell surface expression of Glut 1, the predominant glucose transporter isotype on Jurkat cells. These results support a model of CD95 induced cell death that, at least in its early stages, does not depend on signaling to the nucleus or on macromolecular synthesis. Acute regulation of glucose transport is proposed to be an early effector mechanism in CD95-induced apoptotic cell death.

Evidence that cell survival is controlled by interleukin-3 independently of cell proliferation.

Hemopoietic cell proliferation is controlled by a set of polypeptide growth factors and regulatory molecules that bind to cell surface receptors inducing cellular responses. Maintenance of a viable state, cell growth, DNA synthesis and mitosis are basic properties of proliferating cells, but links between growth factor receptors and each of these cellular outcomes are poorly understood. Most studies have monitored DNA synthesis as a measure of progression through the cell cycle or directly measured viable cell numbers, but cell survival per se as an output of receptor activation by ligand, has received little attention. In this study we have used a bone marrow-derived murine cell line that is dependent on interleukin-3 for growth, to investigate the relationship between DNA synthesis and a biochemical marker of cell survival, reduction of the tetrazolium salt, MTT. We show that at times up to 6 hr, continued DNA synthesis, RNA synthesis, protein synthesis, and mitochondrial respiration are not necessary for background or IL-3-stimulated MTT reduction. Furthermore, dibutyryl cyclic AMP promoted background and IL-3-dependent MTT reduction while simultaneously inhibiting DNA synthesis. These results provide evidence that IL-3 controls events involved in MTT reduction and cell survival independently of DNA synthesis.