Nitrobenzylthioinosine-insensitive uridine transport in human lymphoblastoid and murine leukemia cells

Both human lymphoblastoid (RPMI 6410) and murine leukemia (L1210) cells were found to have a component of uridine transport which is insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In both cell lines NBMPR-insensitive uridine transport is inhibited by other nucleosides and by the sulfhydryl reagent p-chloromercuribenzenesulfonate. In RPMI 6410 cells NBMPR-insensitive transport accounts for only 2% of the initial rate of uridine transport. In contrast, 20% of the initial rate of transport of L1210 cells is insensitive to NBMPR, and uridine uptake over longer periods (10 min) is completely insensitive to NBMPR.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Calcium sensitivity of dicarboxylate transport in cultured proximal tubule cells

Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na(+)-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation.     

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.     

Mouse very long-chain Acyl-CoA synthetase 3/fatty acid transport protein 3 catalyzes fatty acid activation but not fatty acid transport in MA-10 cells

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.     

A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells

In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. Deficiencies in the apoptotic pathways may lead to chemoresistance. Here we report that glutathione (GSH) plays a critical role in activation of apoptosis pathways by CD95 (APO-1/Fas) or anticancer drugs. Upon treatment with anticancer drugs or CD95 triggering, CD95-resistant or Bcl-x(L) overexpressing CEM cells were deficient in activation of apoptosis pathways. CD95-resistant and Bcl-x(L) overexpressing CEM cells exhibited higher intracellular GSH levels in comparison to parental cells. Downregulation of GSH by L-buthionine-(S,R)-sulfoxime (BSO), a specific inhibitor of glutathione synthesis, reversed deficiencies in activation of apoptosis pathways by anticancer drugs or CD95. Interestingly, downregulation of GSH by BSO increased CD95 DISC formation in type I cells. In hybrids of CD95-resistant cells with sensitive cells and hybrids of overexpressing Bcl-x(L) cells with sensitive cells, the phenotype of apoptosis resistance was dominant. Also, in these hybrids, downregulation of GSH reversed CD95- and chemoresistance. We conclude that dominant apoptosis resistance depends, at least in part, on intracellular GSH levels, which may affect apoptosis signaling at different compartments, for example, the death receptor or mitochondria.     

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.     

Effect of plasma membrane cholesterol depletion on glucose transport regulation in leukemia cells

GLUT1 is the predominant glucose transporter in leukemia cells, and the modulation of glucose transport activity by cytokines, oncogenes or metabolic stresses is essential for their survival and proliferation. However, the molecular mechanisms allowing to control GLUT1 trafficking and degradation are still under debate. In this study we investigated whether plasma membrane cholesterol depletion plays a role in glucose transport activity in M07e cells, a human megakaryocytic leukemia line. To this purpose, the effect of cholesterol depletion by methyl-β-cyclodextrin (MBCD) on both GLUT1 activity and trafficking was compared to that of the cytokine Stem Cell Factor (SCF). Results show that, like SCF, MBCD led to an increased glucose transport rate and caused a subcellular redistribution of GLUT1, recruiting intracellular transporter molecules to the plasma membrane. Due to the role of caveolae/lipid rafts in GLUT1 stimulation in response to many stimuli, we have also investigated the GLUT1 distribution along the fractions obtained after non ionic detergent treatment and density gradient centrifugation, which was only slightly changed upon MBCD treatment. The data suggest that MBCD exerts its action via a cholesterol-dependent mechanism that ultimately results in augmented GLUT1 translocation. Moreover, cholesterol depletion triggers GLUT1 translocation without the involvement of c-kit signalling pathway, in fact MBCD effect does not involve Akt and PLCγ phosphorylation. These data, together with the observation that the combined MBCD/SCF cell treatment caused an additive effect on glucose uptake, suggest that the action of SCF and MBCD may proceed through two distinct mechanisms, the former following a signalling pathway, and the latter possibly involving a novel cholesterol dependent mechanism.     

Adenosine transport and metabolism in mouse leukemia cells and in canine thymocytes and peripheral blood leukocytes.

The intracellular accumulation of free [³H] adenosine was measured by rapid kinetic techniques in P388 murine leukemia cells in which adenosine metabolism (phosphorylation and deamination) was completely prevented by depletion of cellular ATP and by treatment with deoxycoformycin. Nonlinear regression of integrated rate equations on the data demonstrate that the time courses of labeled adenosine accumulation at various extracellular adenosine concentrations in zero-trans and equilibrium exchange protocols are well described by a simple, completely symmetrical, transport model with a carrier:substrate affinity constant of about 150 μM. Adenosine transport was not affected by 1 mM deoxycoformycin indicating that this analog has a low affinity for the nucleoside transport system. The transport capacity of dog thymocytes and peripheral leukocytes was similar to that of P388 cells. Transport was not inhibited by deoxycoformycin and remained constant during the first two hours after mitogenic stimulation with concanavalin A. In untreated, metabolizing P388 cells transport was found to be the major determinant of the rate of intracellular metabolism, regardless of the extracellular adenosine concentration (up to at least 160 μM), but the long-term accumulation (longer than 30–60 seconds) of radioactivity from extracellular adenosine strictly reflected the rate of formation of nucleotides (mainly ATP). The metabolism of adenosine by whole cells was entirely consistent with the kinetic properties of the transport system and those of the metabolic enzymes. At low exogenous adenosine concentrations (1 μM and below) transport was slow enough to allow direct phosphorylation of most of the entering adenosine. The remainder was deaminated and rapidly converted to nucleotides via inosine, hypoxanthine, and IMP. At concentrations of 100 μM or higher, on the other hand, influx exceeded the maximum velocity of adenosine kinase about 100 times so that most of the entering adenosine was deaminated. But since the maximum velocity of adenosine deaminase exceeded those of nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase about 5 and 100 times, respectively, hypoxanthine and inosine rapidly exited from the cells and accumulated in the medium. A 98% reduction of adenosine transport (at 100 μM), caused by the transport inhibitor Persantin, inhibited adenosine deamination by whole cells to about the same extent as transport, whereas adenosine phosphorylation was relatively little affected; thus in the presence of Persantin, transport and metabolism resembled that occurring at the low adenosine concentration. These and other results indicate that adenosine deamination is an event distinct from transport, which occurs only subsequent to adenosine's transport into the cell.     

Changes in neutral amino acid transport activity in myeloid leukemia cells differentiated by lipopolysaccharide

M1 cells derived from mouse myeloid leukemia have been reported to differentiate to macrophage-like cells upon treatment with substances such as lipopolysaccharide. Previously we found that in mouse peritoneal macrophages most of the neutral amino acids were taken up through a unique Na+-independent system. In this paper we have investigated the neutral amino acid transport in M1 cells and in those treated with lipopolysaccharide. In M1 cells serine, alanine and proline were taken up mainly by Na+-dependent transport systems, and leucine was largely transported by a Na+-independent system. By treating the cells with lipopolysaccharide, the activities of the Na+-dependent systems markedly decreased, whereas the activity of the Na+-independent system was little affected. The amino acid concentrations in the cells and the culture medium were measured. As a whole, the intracellular to extracellular distribution ratios for neutral amino acids that are preferred substrates for Na+-dependent systems were decreased on lipopolysaccharide treatment, whereas those for amino acids that are mainly transported by a Na+-independent system were slightly increased. From these results we conclude that M1 cells treated with lipopolysaccharide tend to differentiate to macrophage-like cells with respect to the neutral amino acid transport.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI⁺D⁺, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI⁺D^?, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI⁺D^? compared to the MGI⁺D⁺ clones. The MGI⁺D^? clones produced an unusual polyunsaturated C_20:5 fatty acid at 28°C, whereas the MGI⁺D⁺ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI⁺D^? clones than of the MGI⁺D⁺ clones. The cells of MGI⁺D⁺ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

Adriamycin and daunomycin induce interstrand DNA crosslinks in HeLa S3 cells

By using a new mild procedure for detecting DNA crosslinks it has been shown that adriamycin and daunomycin are able to form interstrand DNA crosslinks in HeLa cells. This effect seems to be preceded by transformation of the parent antibiotics in the cell to active forms. In addition, interstrand DNA crosslinks formed by adriamycin and daunomycin were found to be temperature- and alkali-labile.     

Regulation of fatty acid transport

PURPOSE OF REVIEW: The aim of the present review is to summarize recent developments in the area of regulation of fatty acid transport. RECENT FINDINGS: While controversy still exists regarding the contribution of passive diffusion versus protein-mediated fatty acid transport, both processes are now widely accepted. With the recent identification of an increasing number of putative fatty acid transporters, emphasis has been placed on regulation including fatty acid transport function of the protein, and also possible associated functions (acylCoA synthase activity and vectorial channelling to intracellular processing). Deciphering these issues has been facilitated through the use of loss-of-function (such as knockout) and gain-of-function (cell transfectants and transgenic mice) models. SUMMARY: It is likely that our concept of fatty acid transport will continue to converge, incorporating the individual functions of the wide variety of fatty acid transporters into an integrated physiologic framework with relevance to a number of diseases.     

Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells

In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs.     

The fatty acid transport function of fatty acid-binding proteins

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.     

Hypoxanthine transport in mammalian cells: Cell type-specific differences in sensitivity to inhibition by dipyridamole and uridine

Summary We have measured by rapid kinetic techniques the zero-trans influx of hypoxanthine in various cell lines and its sensitivity to inhibition by uridine, dipyridamole, nitrobenzylthioinosine and nitrobenzylthiopurine. The results and those reported earlier divided the cells into two distinct groups. In mouse P388, L1210 and L929 cells uridine and hypoxanthine had little effect on the transport of each other, supporting the view that nucleosides and hypoxanthine are transported by different carriers. In these cells, hypoxanthine transport was also uniquely resistant to inhibition by dipyridamole (IC₅₀ (50% inhibition dose) >30M). In Novikoff and HTC rat hepatoma, Chinese hamster ovary and Ehrlich ascites tumor cells, on the other hand, hypoxanthine and uridine inhibited the transport of each other about 50% at a concentration corresponding to the Michaelis-Menten constant of their transport, and hypoxanthine transport was strongly inhibited by dipyridamole (IC₅₀=100 to 400nM). Although these results are compatible with the view that nucleosides and hypoxanthine are transported by a common carrier in these cells, this conclusion is not supported by the finding that uridine transport is strongly inhibited in some of these cell lines, as in first group of cells, by nitrobenzylthioinsine, whereas hypoxanthine transport is highly resistant in all cell lines tested. In contrast, the transport of both substrates is highly resistant to inhibition by nitrobenzylthiopurine. The Michaelis-Menten constants for uridine transport are about the same in all cell lines. The Michaelis-Menten constants for hypoxanthine transport are similar to those for uridine transport in some cell lines, but are much higher in others. This difference is unrelated to the sensitivity of uridine and hypoxanthine transport to inhibition by each other or dipyridamole.