Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts.

BACKGROUND: A subset of human hepatocellular carcinomas (HCC) exhibit mutations of ß-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO). METHODS: Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1. RESULTS: Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro. CONCLUSIONS: The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

Valproic acid induces the glutamate transporter excitatory amino acid transporter-3 in human oligodendroglioma cells.

Glutamate transport in early, undifferentiated oligodendrocytic precursors has not been characterized thus far. Here we show that human oligodendroglioma Hs683 cells are not endowed with EAAT-dependent anionic amino acid transport. However, in these cells, but not in U373 human glioblastoma cells, valproic acid (VPA), an inhibitor of histone deacetylases, markedly induces SLC1A1 mRNA, which encodes for the glutamate transporter EAAT3. The effect is detectable after 8h and persists up to 120h of treatment. EAAT3 protein increase becomes detectable after 24h of treatment and reaches its maximum after 72-96h, when it is eightfold more abundant than control. The initial influx of d-aspartate increases in parallel, exhibiting the typical features of an EAAT3-mediated process. SLC1A1 mRNA induction is associated with the increased expression of PDGFRA mRNA (+150%), a marker of early oligodendrocyte precursor cells, while the expression of GFAP, CNP and TUBB3 remains unchanged. Short term experiments have indicated that the VPA effect is shared by trichostatin A, another inhibitor of histone deacetylases. On the contrary, EAAT3 induction is neither prevented by inhibitors of mitogen-activated protein kinases nor triggered by a prolonged incubation with lithium, thus excluding a role for the GSK3ß/ß-catenin pathway. Thus, the VPA-dependent induction of the glutamate transporter EAAT3 in human oligodendroglioma cells likely occurs through an epigenetic mechanism and may represent an early indicator of commitment to oligodendrocytic differentiation.

L-Asparaginase and inhibitors of glutamine synthetase disclose glutamine addiction of ß-catenin-mutated human hepatocellular carcinoma cells.

Selected oncogenic mutations support unregulated growth enhancing glutamine availability but increasing the dependence of tumor cells on the amino acid. Data from literature indicate that a subset of HepatoCellular Carcinomas (HCC) is characterized by mutations of ß-catenin and overexpression of Glutamine Synthetase (GS). To assess if this phenotype may constitute an example of glutamine addiction, we treated four human HCC lines with the enzyme L-Asparaginase (ASNase), a glutaminolytic drug. ASNase had a significant antiproliferative effect only in the ß-catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and α-ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2α phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the ß-catenin wild type cell line Huh-7. When used with ASNase, the GS inhibitor methionine sulfoximine (MSO) emptied cell glutamine pool, arresting proliferation in ASNase-insensitive Huh-7 cells and activating caspase-3 and apoptosis in HepG2 cells. Compared with Huh-7 cells, HepG2 cells accumulated much higher levels of glutamine and MSO, due to the higher expression and activity of SNAT2, a concentrative transporter for neutral amino acids, but were much more sensitive to glutamine withdrawal from the medium. In the presence of ASNase, MSO caused a paradoxical maintenance of rapamycin-sensitive mTOR activity in both HepG2 and Huh-7 cells. ß-catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another ß-catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, ß-catenin mutated HCC cells are more sensitive to glutamine depletion and accumulate higher levels of GS inhibitors. These results indicate that glutamine deprivation may constitute a targeted therapy for ß-catenin-mutated HCC cells addicted to the amino acid.

Analysis of living cells grown on different titanium surfaces by time-lapse confocal microscopy.

In this study we have combined fluorescence- and reflection-confocal laser scanning microscopy for the simultaneous visualization of living cells and surface topography beneath them. To this purpose we have designed a specific flow chamber and we have tested it with osteoblasts grown on an opaque, thick support, made of smooth or sandblasted titanium. Cells were loaded with Calcein-AM or tetramethylrhodamine methyl ester (TMRM), two probes employed as indicators of cell viability/morphology and mitochondrial membrane potential, respectively. Besides the acquisition of stacks of confocal sections, the system allowed also vertical views and faithful three-dimensional reconstruction of the samples. Confocal microscope implemented with our flow chamber proved to be a promising tool for time-lapse investigation of cell-biomaterial interactions.

Effects of heat deproteinate bone and polynucleotides on bone regeneration: an experimental study on rat.

This experimental study evaluated the effects of polynucleotides on bone regeneration on rats. Defects with a diameter of 2mm were prepared in the thickness of cortical bone of 32 rat tibiae and filled with different compounds: polynucleotide gel (PDRN), deproteinated porcine cortical bone (HDB) obtained by high temperature heating in the form of granules and a paste made of HDB granules and PDRN gel. Bone regeneration of the gaps was histologically analysed after a treatment time ranging from 1 to 12 weeks. Both PDRN and HDB stimulated bone growth and repair, but the paste prepared combining HDB granules and PDRN showed the best performance with faster filling, better osteconductive and biocompatible properties and easier handling. This study suggests that the paste prepared combining HDB and PDRN gel induces rapid bone regeneration in different clinical situations.

The inhibition of glutamine synthetase sensitizes human sarcoma cells to L-asparaginase.

PURPOSE: To evaluate the activity of the antitumor enzyme L: -asparaginase (ASNase) on tumor cells of mesenchymal origin and the contribution of glutamine synthetase (GS) to the adaptation to the metabolic stress caused by the anti-tumor enzyme. METHODS: We studied the effects of ASNase in six human sarcoma cell lines: HT1080 (fibrosarcoma); RD (rhabdomyosarcoma); SW872 (liposarcoma); HOS, SAOS-2, and U2OS (osteosarcoma) in the absence or in the presence of the GS inhibitor methionine L: -sulfoximine (MSO). RESULTS: HT1080 and SW872 cells were highly sensitive to ASNase-dependent cytotoxicity. In contrast, RD, SAOS-2, HOS, and U2OS cells exhibited only a partial growth suppression upon treatment with the anti-tumor enzyme. In these cell lines ASNase treatment was associated with increased levels of GS. When ASNase was used together with MSO, the proliferation of the poorly sensitive cell lines was completely blocked and a significant decrease in the IC(50) for ASNase was observed. Moreover, when ASNase treatment was carried on in the presence of MSO, HOS and U2OS osteosarcoma cells exhibited a marked cytotoxicity, with increased apoptosis. CONCLUSIONS: In human sarcoma cells (1) GS markedly contributes to the metabolic adaptation of tumor cells to ASNase and (2) the inhibition of GS activity enhances the antiproliferative and cytotoxic effects of ASNase. The two-step interference with glutamine metabolism, obtained through the combined treatment with ASNase and MSO, may provide a novel therapeutic approach that should be further investigated in human tumors of mesenchymal origin.

Non-apoptotic programmed cell death induced by a copper(II) complex in human fibrosarcoma cells.

A0, a Cu(II) thioxotriazole complex, produces severe cytotoxic effects on HT1080 human fibrosarcoma cells with a potency comparable to that exhibited by cisplatin. A0 induced a characteristic series of changes, hallmarked by the formation of eosin- and Sudan Black-B-negative vacuoles. No evidence of nuclear fragmentation or caspase-3 activation was detected in cells treated with A0 which, rather, inhibited cisplatin-stimulated caspase-3 activity. Membrane functional integrity, assessed with calcein and propidium iodide, was spared until the late stages of the death process induced by the copper complex. Vacuoles were negative to the autophagy marker monodansylcadaverine and their formation was not blocked by 3-methyladenine, an inhibitor of autophagic processes. Negativity to the extracellular marker pyranine excluded vacuole derivation from the extracellular fluid. Ultrastructural analysis indicated that A0 caused the appearance of many electronlight cytoplasmic vesicles, possibly related to the endoplasmic reticulum, which progressively enlarge and coalesce to form large vacuolar structures that eventually fill the cytoplasm. It is concluded that A0 triggers a non-apoptotic, type 3B programmed cell death (Clarke in Anat Embryol (Berl) 181:195-213, 1990), characterized by an extensive cytoplasmic vacuolization. This peculiar cytotoxicity pattern may render the employment of A0 to be of particular interest in apoptosis-resistant cell models.

Ethanol increases the paracellular permeability of monolayers of CAPAN-1 pancreatic duct cells.

When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.

Identification of immunodominant hepatitis C virus (HCV)-specific cytotoxic T-cell epitopes by stimulation with endogenously synthesized HCV antigens.

Hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTL) are believed to play an important role in the pathogenesis of liver cell injury and viral clearance in HCV infection. Because HCV does not efficiently infect human cells in vitro and primary infected hepatocytes cannot be used as stimulator/target cells for CTL analysis, development of efficient systems to activate and expand CTL in vitro, reproducing antigen presentation to CTL occurring during natural infection, is mandatory to study CTL activity and to define the hierarchy of immunodominance of CTL epitopes. To achieve this goal, 5 different defective adenoviruses carrying structural and nonstructural HCV genes (core, core-E1-E2, E2, NS3-NS4A, NS3-NS5A) were used to induce the endogenous synthesis of HCV proteins in human adherent mononuclear cells in vitro and to allow their entry into the HLA class I cytosolic pathway of antigen processing. The cytolytic activity of peripheral blood lympho-mononuclear cells (PBMC) from HLA-A2(+) HCV-infected patients stimulated with recombinant adenovirus-infected cells was tested against target cells either pulsed with a panel of synthetic peptides containing the HLA-A2 binding motif or infected with recombinant vaccinia viruses carrying HCV genes. Our study defines a reproducible system to stimulate and expand HCV-specific CTL in vitro that mimics the conditions of antigen encounter in vivo. By this approach, we have identified several HLA-A2-restricted epitopes that should correspond to immunodominant HCV sequences recognized by CTL during natural infection. Therefore, these amino acid sequences represent ideal candidates for the design of therapeutic vaccines for chronic HCV infection.

CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained.

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

The stimulation of Na,K,Cl cotransport and of system A for neutral amino acid transport is a mechanism for cell volume increase during the cell cycle.

It has been known for several years that the triggering of cell proliferation is associated with an increase of the activity of Na,K,Cl cotransport and of transport system A for neutral amino acids. These systems are also enhanced during the volume recovery of hypertonically shrunk cells. We demonstrate here that during the cell cycle of NIH3T3 cells, an increase in cell volume is associated with an enhanced cell content of potassium and amino acids. Bumetanide delays cell cycle progression and hampers volume increase. The nonmetabolizable analog 2-methylamino-isobutyric acid, a specific substrate of system A, can partially substitute natural amino acids accumulated during the cell cycle as intracellular osmolytes. It is therefore proposed that the stimulation of Na,K,Cl cotransport and of system A, observed in proliferating cells, causes an expansion of cell volume through an enhanced intracellular accumulation of both inorganic and organic osmolytes and the concurrent, osmotically obliged uptake of water.

Emerging roles for sodium dependent amino acid transport in mesenchymal cells.

The functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X(-) AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.

Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells.

The treatment of NIH3T3 cells with L-asparaginase causes a complete and reversible growth arrest with a decrease of cell number in the first 2 days. The enzyme induces impressive morphological changes that have been studied exploiting eosin in fixed cells and calcein in intact cells as sources of fluorescence for confocal microscopy. The first changes are observed after 12 h of treatment and the process is complete after 48 h. Both nucleus and cytoplasm shrink, while cells round and lose processes. Eventually most cells break; several debris include strongly hematoxylinic bodies negative for eosin fluorescence. Some cells neither round nor break in fragments. Throughout the process cells and fragments retain calcein fluorescence, thus indicating the integrity of the cell membrane. A rapid depletion of the intracellular pools of both glutamine and glutamate occurs in treated cells, followed by a decrease in DNA and protein syntheses, while the cell content of ATP, the transmembrane gradient of sodium, and the active transport of amino acids are scarcely affected. It is concluded that (i) L-asparaginase induces an apoptotic process in NIH3T3 cells that is forerun by a marked intracellular depletion of glutamate and glutamine; and (ii) although the enzyme completely suppresses cell proliferation, only a subset of cells undergoes apoptosis upon treatment. These findings provide a model for the characterization of factors that determine cell sensitivity to the effects of L-asparaginase.

Suppression of anionic amino acid transport impairs the maintenance of intracellular glutamate in Ha-ras-expressing cells.

When the expression of a Ha-ras oncogene is triggered in NIH3T3 cells, a progressive inhibition of sodium dependent transport of anionic amino acids through system X-AG is observed. After 48 h of ras expression the transport activity of system X-AG is almost abolished, while other transport systems involved in anionic amino acid transport are unaffected or even stimulated. In the presence of high extracellular concentrations of glutamine, the intracellular concentration of glutamate is comparable in ras expressing and non-expressing cells. On the contrary, when the extracellular pool of glutamine is depleted by the enzyme L-asparaginase, intracellular glutamate decreases at a much faster rate in ras expressing, low-transport cells. These results suggest that transport system X-AG significantly contributes to the homeostasis of intracellular glutamate under conditions of glutamine deprivation.

The relationship between sodium-dependent transport of anionic amino acids and cell proliferation.

The relationship between the transport of anionic amino acids and the proliferative status of the cell population has been studied in NIH-3T3 cells. Proliferative quiescence, verified by determinations of growth-rate quotient and incorporation of thymidine, is associated with a marked increase of the influx of L-aspartate. After 7-10 days of serum starvation, the initial influx of L-aspartate increases by 8-10-times with respect to the transport activity determined in growing cells. The operational properties of the influx of L-aspartate are similar in growing and quiescent cells; in particular, the influx of the anionic amino acid is mostly Na(+)-dependent and completely suppressed by an excess of L-glutamate and D-aspartate, but not of D-glutamate. These features suggest that, in both cases, aspartate uptake occurs through system X(-)AG. The quiescence-related increase in aspartate transport is gradual, sensitive to the inhibition of protein synthesis and referable to the enhanced maximal capacity of transport system X(-)AG. Restoration of serum concentration in the culture medium of serum-starved cells causes a decrease in aspartate transport that is maximal in correspondence to late G1/S phases. It is concluded that the X(-)AG system for anionic amino-acid uptake is sensitive to the proliferative status of the cell population and that, in particular, its transport activity is stimulated by the establishment of proliferative quiescence.

Anionic amino acid transport in ras- transformed fibroblasts.

The sodium-dependent transport of anionic amino acids is suppressed in NIH3T3 cells that constitutively express ras oncogenes. In a model of NIH3T3 cells in which ras expression is triggered in the presence of dexamethasone, aspartate transport decreases gradually upon dexamethasone treatment and is almost completely suppressed after two days of incubation in the presence of the steroid. In the same cell model, lovastatin, an inhibitor of beta hydroxy-beta methyl-glutaryl-CoA-reductase and, hence, of farnesylation of p21ras, partially protects aspartate transport from the inhibition observed upon steroid treatment. Determinations of cell glutamate in ras-expressing and non expressing cells indicate that in both cell models glutamate decreases when extracellular medium is depleted of glutamine. However, this decrease is much faster in cells expressing ras (either constitutively or conditionally). It is proposed i) that cell production of oncogenic p21ras hinders sodium-dependent transport of anionic amino acids and ii) that the transport alteration impairs the maintenance of cell levels of glutamate in ras-expressing cells.