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.

Adaptive regulation of amino acid transport across the cell membrane in avian and mammalian tissues.

The regulation of amino acid transport across the cell membrane by adaptive mechanisms has been studied in a variety of mesenchymal and epithelial cells and tissues of avian and mammalian origin. Changes in transport activity as a function of time under various in vitro conditions (amino acid dependence, active and inhibited protein synthesis) have been evaluated by measurements of initial entry rates with representative amino acids. Results and conclusions based on the adopted experimental approach include the following. (1) An adaptive control mechanism for the transport of neutral amino acids corresponding to the typical substrates of the A mediation is operative in (a) mesenchymal cells (fibroblasts, chondroblasts, osteoblasts and myoblasts) from embryonic tissues of avian (chick embryo) origin and (b) mesenchymal cells from immature rat uterus (fibroblasts and smooth muscle cells) and other mammalian tissues (cardiac cells from newborn mouse and rat heart). (2) Adaptive regulation is restricted to a discrete subgroup of amino acids (L-proline, glycine and the analogue alpha-aminoisobutyric acid) in rat peritoneal macrophages and thymic lymphocytes. (3) Adaptive regulation is absent in erythroid cells (human erythrocytes, rabbit erythrocytes and reticulocytes, avian erythrocytes) which lack the A mediation and are incapable of active gene transcription. (4) Adaptive regulation is absent in the epithelial kidney cortex tissue and possibly absent in the epithelial component of liver tissue from adult rats; it is fully operative in the chick embryo crystalline lens, i.e. an epithelial preparation of embryonic origin. (5) These observations indicate that adaptive control mechanisms of amino acid transport across the cell membrane are quite common among tissues and species and emphasize their broad biological significance in eukaryotes.

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.

Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions.

Changes in the intracellular concentrations of Na+ and K+ of fetal human fibroblasts have been followed after replacement of serum-containing growth media with unsupplemented and serum-supplemented saline solution (Earle's balanced salt solution). Incubation in unsupplemented salt solution was followed by a progressive increase of the internal Na+ counterbalanced by a decrease of internal K+, without major alterations of the internal osmolarity. After 3 h incubation the intracellular Na+ and K+ concentrations were 120 mM and 50 mM, respectively. These intracellular ion derangements were not associated with a failure of the (Na+ + K+)-ATPase pump, whose activity actually increased with enhanced intracellular Na+ concentration. Ion changes did not take place when serum (in excess of 0.5%, final concentration) was present in the saline solution and a complete restoration to normal of the Na+ and K+ gradients occurred upon addition of serum to cells previously incubated in plain saline solution. The effects of serum were mimicked by furosemide, thus suggesting that channels sensitive to this diuretic are involved in the movement of Na+ and K+ following fibroblast incubation in unsupplemented saline solution.

Effect of insulin on the activity of amino acid transport systems in cultured human fibroblasts.

The regulation of amino acid transport by insulin has been studied in cultured human fibroblasts. Among the six amino acid transport systems operating in cultured human fibroblasts, two systems (A and X-C) are strongly stimulated by insulin and four (ASC, X-AG, y+ and L) are essentially not sensitive to the presence of the hormone in the incubation medium. The hormonal stimulation of system A and system X-C became significant after 3 h of incubation and increased up to 12 h. The stimulatory effect was related to insulin concentration, with a half-maximal stimulation at 10(-9) M hormone concentration. Insulin enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in Km values.

Effect of extracellular potassium on amino acid transport and membrane potential in fetal human fibroblasts.

The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K+. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), X-AG (for anionic amino acids), and y+ (for cationic amino acids). High doses (100 microM) of the K+-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y+. Transport systems L (for neutral amino acids) and x-C (for anionic amino acids) are insensitive to changes in extracellular K+ or to valinomycin. System X-AG is inhibited by the addition of 100 microM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x-C is potential-independent and (b) the activity of systems A, ASC, X-AG and y+ is sensitive to alterations of external [K+] associated to changes in membrane potential.

The regulation of sodium-dependent transport of anionic amino acids in cultured human fibroblasts.

In cultured human fibroblasts the transport of anionic amino acids through the sodium-dependent system X-AG is stimulated rapidly and transiently by phorbol 12,13-dibutyrate. Transport stimulation is consistent with an effect due to the activation of protein kinase C. Bradykinin (1 microM) and PDGF-AA (100 ng/ml) also stimulate the activity of system X-AG. The bradykinin effect appears to be fully dependent upon PKC activation whereas the stimulation of aspartate transport by PDGF-AA is also due to PKC-independent mechanisms.

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.

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.

The role of the neutral amino acid transporter SNAT2 in cell volume regulation.

Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.

[Regulation of amino acid transport in avian fibroblasts from growing and quiescent cell cultures (author's transl)]. / Regolazione del trasporto degli aminoacidi in fibroblasti da colture in crescita e quiescenti

The regulation of amino acid transport across the cell membrane by adaptive mechanisms has been studied in chick embryo fibroblasts obtained from growing and quiescent cell cultures. Changes in transport activity as a function of time under various in vitro conditions (amino acid dependence, active and inhibited protein synthesis) have been evaluated by measurements of initial entry rates with representative amino acids.

Adaptive increase of amino acid transport system A requires ERK1/2 activation.

Amino acid starvation markedly stimulates the activity of system A, a widely distributed transport route for neutral amino acids. The involvement of MAPK (mitogen-activated protein kinase) pathways in this adaptive increase of transport activity was studied in cultured human fibroblasts. In these cells, a 3-fold stimulation of system A transport activity required a 6-h amino acid-free incubation. However, a rapid tyrosine phosphorylation of ERK (extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium with amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced after a 15-min amino acid-free incubation and maintained at stimulated values thereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivation. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaffected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the presence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimulation of ERK1/2 was observed. To identify the stimulus that maintains MAPK activation, cell volume was monitored during amino acid-free incubation. It was found that amino acid deprivation caused a progressive cell shrinkage (30% after a 6-h starvation). If proline was added to amino acid-starved, shrunken cells, normal values of cell volume were rapidly restored. However, proline-dependent volume rescue was hampered if cells were pretreated with PD98059. It is concluded that (a) the triggering of adaptive increase of system A activity requires a prolonged activation of ERK1 and 2 and that (b) cell volume changes, caused by the depletion of intracellular amino acid pool, may underlie the activation of MAPKs.

Involvement of protein kinase Cepsilon in the stimulation of anionic amino acid transport in cultured human fibroblasts.

Protein kinase C (PKC) activation stimulates transport system X-AG for anionic amino acids in cultured human fibroblasts (Franchi-Gazzola, R., Visigalli, R., Bussolati, O., and Gazzola, G. C. (1994) FEBS Lett. 352, 109-112). To identify which PKC isoform is responsible for this effect, aspartate transport through system X-AG, PKC activity, and the subcellular distribution of PKC isoforms have been studied before and after treatment with phorbol 12, 13-dibutyrate (PDBu) in fibroblasts maintained at low serum for 1 (control cells) or 7 days (quiescent cells). In control cells aspartate transport and PKC activity in the particulate fraction were stimulated by short term PDBu treatment; both stimulatory effects were down-regulated by a prolonged exposure to the phorbol. In contrast, in quiescent cells aspartate transport and particulate PKC activity were higher than control under basal conditions, unaffected by a short term PDBu treatment, and lowered by a prolonged incubation with the phorbol. In both control and quiescent cells a short term PDBu treatment modified PKCalpha distribution, increasing its membrane-associated fraction. PKCdelta was mostly in the soluble fraction and scarcely sensitive to PDBu. A brief exposure to PDBu increased membrane-associated PKCepsilon in control but not in quiescent cells. In these cells epsilon isoform was found exclusively in the particulate fraction even in PDBu-untreated cells. A prolonged PDBu treatment caused a partial down-regulation of membrane-associated PKCepsilon in control cells and its marked decrease in quiescent cells. It is concluded that PKC-dependent changes in system X-AG activity parallel the behavior of PKCepsilon, thus suggesting a specific role for this isoform in system X-AG regulation.

The transport of alanine, serine, and cysteine in cultured human fibroblasts.

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.

Glycine transport by cultured human fibroblasts.

The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na+-dependent and Na+-independent mechanisms. Na+-independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na+-dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.

Post-translational control by carrier availability of amino acid transport in fetal human fibroblasts.

The action of serum on the expression of the starvation-enhanced amino acid transport by System A (as a part of the adaptive regulation mechanism) has been studied in cultured fetal human fibroblasts. Serum enhanced L-proline uptake of cells starved in serum-free medium. This effect was rapid, proportional to the amount of pre-existing transporters, insensitive to cycloheximide and kinetically characterized by an increase of transport Vmax. These results can be interpreted to indicate that serum is essential for a vectorial post-translational event leading to insertion of transport proteins into the cell membrane.