[show abstract][hide abstract] ABSTRACT: It is increasingly appreciated that cancer cells must be endowed with specific metabolic adaptations
to support enhanced growth and to ensure survival under stressful conditions. On the other hand, many
oncogenic mutations of protooncogenes and tumor suppressor genes directly cause metabolic derangements
and, conversely,mutations of enzymes have been found to underlie several forms of cancer. Thus, cancer-specific
metabolic alterations are now considered among the hallmarks of malignant tumors. Most commonly,
cancer cells exhibit enhanced glycolysis under aerobic conditions (the Warburg effect) but alterations in the
metabolism of amino acids, such as glutamine, serine and proline are increasingly described as important
metabolic features of selected tumor types. In theory, all these deranged cancer-specific metabolic pathways
may constitute novel therapeutic targets, although the only “metabolic” drug in clinical use is still represented
by the enzyme L-asparaginase. However, the increasing amount of experimental evidence, as well as the
number of trials in progress, suggests that metabolic drugs will soon complement standard anti-cancer
chemotherapy and modern biological drugs.
L' Ateneo parmense. Sezione I: Acta bio-medica 12/2012; 83:168-176.
[show abstract][hide abstract] ABSTRACT: It is increasingly appreciated that cancer cells must be endowed with specific metabolic adaptations to support enhanced growth and to ensure survival under stressful conditions. On the other hand, many oncogenic mutations of protooncogenes and tumor suppressor genes directly cause metabolic derangements and, conversely, mutations of enzymes have been found to underlie several forms of cancer. Thus, cancer-specific metabolic alterations are now considered among the hallmarks of malignant tumors. Most commonly, cancer cells exhibit enhanced glycolysis under aerobic conditions (the Warburg effect) but alterations in the metabolism of amino acids, such as glutamine, serine and proline are increasingly described as important metabolic features of selected tumor types. In theory, all these deranged cancer-specific metabolic pathways may constitute novel therapeutic targets, although the only "metabolic" drug in clinical use is still represented by the enzyme L-asparaginase. However, the increasing amount of experimental evidence, as well as the number of trials in progress, suggests that metabolic drugs will soon complement standard anti-cancer chemotherapy and modern biological drugs.
[show abstract][hide abstract] ABSTRACT: This study reports the structure-activity relationship of a series of 8-hydroxoquinoline derivatives (8-HQs) and focuses on the citotoxic activity of 5-Cl-7-I-8-HQ (clioquinol, CQ) copper complex (Cu(CQ)). 8-HQs alone caused a dose-dependent loss of viability of the human tumor HeLa and PC3 cells, but the coadministration of copper increased the ligands effects, with extensive cell death occurring in both cell lines. Cytotoxic doses of Cu(CQ) promote intracellular copper accumulation and extensive endoplasmic reticulum vacuolization that precede a non-apoptotic (paraptotic) cell death. The cytotoxic effect of Cu(CQ) was reproduced in normal human endothelial cells (HUVEC) at concentrations double than those effective in tumor cells, pointing to a potential therapeutic window for Cu(CQ). Finally, the results showed that the paraptotic cell death induced by Cu(CQ) does not require nor involve caspases, giving an indication for the current clinical assessment of clioquinol as an antineoplastic agent.
Journal of Medicinal Chemistry 11/2012; · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 GSK3β/β-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.
[show abstract][hide abstract] ABSTRACT: L-Methionine sulfoximine (MSO) and DL-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators.
[show abstract][hide abstract] ABSTRACT: We report a quantitative structure-activity relationship study of a new class of pyrazole-pyridine copper complexes that establishes a clear correlation between the ability to promote copper accumulation and cytotoxicity. Intracellular metal accumulation is maximized when ligand lipophilicity allows the complex to rapidly cross the membrane. Copper and ligand follow different uptake kinetics and reach different intracellular equilibrium concentrations. These results support a model in which the ligand acts as an ionophore for the metal ion, cycling between intra- and extracellular compartments as dissociated or complexed entities. When treating cancer cells with structurally unrelated disulfiram and pyrazole-pyridine copper complexes, as well as with inorganic copper, the same morphological and molecular changes were reproduced, indicating that copper overload is responsible for the cytotoxic effects. Copper-based treatments drive sensitive cancer cells toward paraptotic cell death, a process hallmarked by endoplasmic reticulum stress and massive vacuolization in the absence of apoptotic features. A lack of caspase activation, as observed in copper-treated dying cells, is a consequence of metal-mediated inhibition of caspase-3. Thus, copper acts simultaneously as an endoplasmic reticulum (ER) stress inducer and a caspase-3 inhibitor, forcing the cell into caspase-independent paraptotic death. The establishment of a mechanism of action common to different copper binding agents provides a rationale for the exploitation of copper toxicity as an anticancer tool.
Journal of the American Chemical Society 03/2011; 133(16):6235-42. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: The copper(II) complex A0 induces a type of non-apoptotic cell death also known as paraptosis. Paraptosis involves extensive endoplasmic reticulum vacuolization in the absence of caspase activation. A wide panel of human cancer cell lines was used to demonstrate differences in cytotoxicity by the paraptosis-inducing drug A0 and the metal-based pro-apoptotic drug cisplatin. Gene expression profiling of the human fibrosarcoma HT1080 cells showed that, while cisplatin induced p53 targets, A0 up-regulated genes involved in the unfolded protein response (UPR) and response to heavy metals. The cytotoxic effects of A0 were associated with inhibition of the ubiquitin-proteasome system and accumulation of ubiquitinylated proteins, in a manner dependent on protein synthesis. Cycloheximide inhibited the accumulation of ubiquitinylated proteins and hampered A0-induced cell death process. The occurrence of the UPR during A0-induced death process was shown by the increased abundance of spliced XBP1 mRNA, transient eIF2alpha phosphorylation, and a series of downstream events, including attenuation of global protein synthesis and increased expression of ATF4, CHOP, BIP, and GADD34. Mouse embryonic fibroblasts expressing a mutant eIF2alpha, which could not be phosphorylated, were more resistant to A0 than wild type cells, pointing to a pro-death role of eIF2alpha phosphorylation. A0 may thus represent the prototypical member of a new class of compounds that cause paraptotic cell death via mechanisms involving eIF2alpha phosphorylation and the UPR.
Journal of Biological Chemistry 07/2009; 284(36):24306-19. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two ruthenium(III) complexes structurally similar to the anticancer compound NAMI were prepared: Na[RuCl4(DMSO)(L1)] ( 1) and Na[RuCl4(DMSO)(L2)] ( 2), where L1 and L2 are differently functionalised triazole-thiadiazine ligands. To facilitate the crystallisation of the complex anions, Na+ was substituted with the [bis(triphenylphosphoranylidene)ammonium] cation (PPN+), allowing the X-ray characterisation of PPN[RuCl4(DMSO)(L1)].2H2O ( 1a.2H2O) and PPN[RuCl4(DMSO)(L2)].3H2O ( 2a.3H2O), respectively. The two compounds undergo stepwise hydrolytic processes, as assessed by means of UV-vis and 1H NMR spectroscopy. The first hydrolytic step consists of the replacement of a chloride anion with a water molecule, with a half-life of 50 min ( 1) and 110 min ( 2), while the subsequent hydrolytic steps are more complicated to describe since more than one product is generated at the same time. The redox potential of the Ru(III)/Ru(II) couple (0.31 V for 1 and 0.28 V for 2) suggests that these complexes can be reduced in the intracellular environment, in agreement with the activation by reduction mechanism proposed for NAMI and NAMI-A. 1 and 2 were tested on a human cancer cell line derived from a fibrosarcoma (HT1080), and on non-cancerous primary human fibroblasts (HF), where they showed a modest inhibitory effect.
[show abstract][hide abstract] ABSTRACT: The copper(II) complex A0 induces non-apoptotic programmed cell death in human HT1080 fibrosarcoma cells but not in normal
fibroblasts (J Med Chem, 50(8):1916–1924, 2007). While typical apoptotic features, such as caspase-3 activation or nuclear fragmentation, are evident in
cisplatin-treated cells, they are absent in A0-dependent cell death. In contrast, the latter process is hallmarked by the
development of huge vacuoles originating from endoplasmic reticulum (Histochem Cell Biol 126(4):473–482, 2006), a feature consistent with the newly described type of cell death named paraptosis (PNAS 97(26):14376–14381, 2000). Consistently, in a panel of human cancer cells there is no correlation between the sensitivities to
A0 and cis-platin. In the same panel, paraptosis-like cell death is observed in all the A0-sensitive cell lines. Moreover,
the copper complex kills cisplatin-sensitive cells (HT1080 and ovarian carcinoma 2008) as well as their cisplatin-resistant
counterparts (C13* cells and the newly established HT1080PTR line) with comparable potencies. The different activity spectrum
between A0 and cisplatin suggests distinct mechanisms of action for the two drugs. In agreement with this hypothesis, a whole-genome
expression analysis, performed in HT1080 cells, showed that the transcriptional response evoked by the two drugs is poorly
overlapping. A0 induces genes involved in oxidative- and endoplasmic reticulum-stress (ER stress), while cisplatin increases
the expression of typical p53 targets. Moreover, A0 strongly induces metal responsive genes, as well as HSPs, chaperones and
other genes involved in the Unfolded Protein Response (UPR). The validation of the microarray results by qRT PCR and Western
Blot confirms that A0, but not cisplatin, activates two pathways of the UPR. In particular, IRE1 mRNA is up-regulated, resulting
in the increased abundance of the spliced form of XBP1 mRNA that encodes for the active transcription factor. Moreover, the
translation initiator complex subunit eIF2alpha is rapidly phosphorylated, with the consequent attenuation of protein synthesis
and the concomitant preferential translation of the pro-death ER stress responsive proteins ATF4, CHOP and GADD34. In conclusion,
A0 kills sensitive cancer cells through the triggering of ER stress, inducing a paraptotic process. Therefore, the copper
complex may constitute a novel device to overcome apoptosis resistance.
KeywordsCopper complex–Non-apoptotic cell death–Paraptosis–Vacuolization–Endoplasmic reticulum stress
[show abstract][hide abstract] ABSTRACT: 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.
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).
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.
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.
Cancer Chemotherapy and Pharmacology 11/2007; 60(5):751-8. · 2.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: The thioamido function of [CuCl2(1H)]Cl (2) (1=4-amino-1,4-dihydro-3-(2-pyridyl)-5-thioxo-1,2,4-triazole), a cytotoxic copper complex, was converted into thioether moieties, leading to the synthesis of [CuCl2(3)]2 (4) and [CuCl2(5)] (6) (3=6-methyl-3-pyridin-2-yl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine; 5=4-amino-5-ethylthio-3-(2-pyridyl)-1,2,4-triazole). These complexes were structurally characterized, and their stability constants, along with their biological activity, were determined. 4 and 6 were slightly less stable and significantly less active than 2. However, as 2, both complexes induced nonapoptotic vacuolar cell death. Copper uptake, investigated in both 2-sensitive and -insensitive cell types, was markedly higher in sensitive cells where it was associated with an increase in oxidized glutathione. These data suggest that the thioamido function enhances the cytotoxicity of copper complexes in cancer cells promoting the accumulation of the metal and its interaction with cell thiols.
Journal of Medicinal Chemistry 05/2007; 50(8):1916-24. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Nutritional stress caused by amino acid starvation involves a coordinated cellular response that includes the global decrease of protein synthesis and the increased production of cell defense proteins. Part of this response is the induction of transport system A for neutral amino acids that leads to the recovery of cell volume and amino acid levels once extracellular amino acid availability is restored. Hypertonic stress also increases system A activity as a mechanism to promote a rapid recovery of cell volume. Both a starvation-dependent and a hypertonic increase of system A transport activity are due to the induction of SNAT2, the ubiquitous member of SLC38 family. The molecular mechanisms underlying SNAT2 induction were investigated in tissue culture cells. We show that the increase in system A transport activity and SNAT2 mRNA levels upon amino acid starvation were blunted in cells with a mutant eIF2alpha that cannot be phosphorylated. In contrast, the induction of system A activity and SNAT2 mRNA levels by hypertonic stress were independent of eIF2alpha phosphorylation. The translational control of the SNAT2 mRNA during amino acid starvation was also investigated. It is shown that the 5'-untranslated region contains an internal ribosome entry site that is constitutively active in amino acid-fed and -deficient cells and in a cell-free system. We also show that amino acid starvation caused a 2.5-fold increase in mRNA and protein expression from a reporter construct containing both the SNAT2 intronic amino acid response element and the SNAT2-untranslated region. We conclude that the adaptive response of system A activity to amino acid starvation requires eukaryotic initiation factor 2alpha phosphorylation, increased gene transcription, and internal ribosome entry site-mediated translation. In contrast, the response to hypertonic stress does not involve eukaryotic initiation factor 2alpha phosphorylation, suggesting that SNAT2 expression can be modulated by specific signaling pathways in response to different stresses.
Journal of Biological Chemistry 07/2006; 281(26):17929-40. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of the present study was the synthesis, the determination of formation constants, and the evaluation of the antiproliferative activity of two copper(II) complexes formed with triazole-type ligands. The synthesis of the unsymmetrical triazole ligand 4-amino-3-aminomethyl-5-methyl-1,2,4-triazole (L1), and its copper(II) complex is reported. The ligand was prepared by functionalization of the carboxylate function of tert-butyloxycarbonyl (BOC) protected glycine O-methyl ester. All intermediates and final products were isolated and characterized with IR, 1H NMR, and elemental analysis. X-ray structures of the ligand as a sulfate salt ((H2L1)2SO4.H2O) and the copper(II) complex [CuCl2(L1)(2)] are described. The ligand forms a (N,N) bidentate chelate with the amino group and one triazole nitrogen atom. The tetragonally distorted octahedral coordination of Cu(II) results from two axially coordinated chloride ions. Protonation constants for L1 and speciation of the Cu(II)/L1 system were determined in 0.1 M aqueous KCl solution at 25 degrees C. Complexes formed in solution were also characterized by visible spectrophotometry. Ligand substitution competition between L1 and glycine has also been studied using potentiometric titrations. Antiproliferative activities of ([CuCl2(L1)2]) and [CuCl2(H2L2)]Cl, where HL2 is the 5-thioxo analog of L1, against human tumor cell lines HT1080 and HT29 as well as normal human fibroblasts (HF) are presented along with the antiproliferative activities of L1, CuCl2, and cisplatin. Activity of these two complexes are discussed and compared with the activity of analogous compounds reported in the literature which contain pyridyl groups in place of the aminomethyl group. In particular, it is suggested that a lypophilic residue such as a pyridyl group is important for antiproliferative activity of this class of compounds.
Journal of Inorganic Biochemistry 09/2005; 99(8):1573-84. · 3.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.
[show abstract][hide abstract] ABSTRACT: In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.
Biochimica et Biophysica Acta 01/2005; 1667(2):157-66. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Preparations of copper(II) and palladium(II) complexes of 4-amino-5-methylthio-3-(2-pyridyl)-1,2,4-triazole (L(1)) and the copper(II) complex of 1,4-dihydro-4-amino-3-(2-pyridyl)-5-thioxo-1,2,4-triazole (HL) are described. These complexes have been characterized by means of spectroscopy and microanalysis. Molecular structures of HL (1), [CuCl(2)(H(2)L)]Cl.2H(2)O (2a), cis-[CuCl(2)(L(1))] (3), and cis-[PdCl(2)(L(1))] (4) have been determined by single-crystal X-ray diffraction. The HL ligand acts as a N,S bidentate through the thioxo moiety and the exo-amino group whilst the ligand L(1) forms N,N coordination complexes through the pyridine and triazole nitrogen atoms. Speciation in solution of the systems Cu/HL and Cu/L(1) have been determined by means of potentiometry and spectrophotometry as well as for the Cu/L(1)/A (HA=glycine) system in order to determine species present at physiological pH. Antiproliferative activity of these complexes and their ligands was evaluated, using the AlamarBlue Assay, on normal human fibroblasts (HF) and human fibrosarcoma tumor (HT1080) cells. The copper compounds cis-[CuCl(2)(H(2)L)]Cl and cis-[CuCl(2)(L(1))] exerted significant antiproliferative activity of both normal and neoplastic cells; although dose-response experiments revealed that the HT1080 cell line was more sensitive to the tested drugs than normal fibroblasts.
Journal of Inorganic Biochemistry 12/2002; 92(2):95-104. · 3.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: The expression of the osmosensitive sodium/myo-inositol cotransporter (SMIT) is regulated by multiple tonicity-responsive enhancers (TonEs) in the 5'-flanking region of the gene. In response to hypertonicity, the nuclear abundance of the transcription factor TonE-binding protein (TonEBP) is increased, and the transcription of the SMIT gene is induced. Transport system A for neutral amino acids, another osmosensitive mechanism, is progressively stimulated if amino acid substrates are not present in the extracellular compartment. Under this condition, as in hypertonicity, cells shrink and mitogen-activated protein kinases are activated. We demonstrate here that a clear-cut nuclear redistribution of TonEBP, followed by SMIT expression increase and inositol transport activation, is observed after incubation of cultured human fibroblasts in Earle's balanced salts (EBSS), an isotonic, amino acid-free saline. EBSS-induced SMIT stimulation is prevented by substrates of system A, although these compounds do not compete with inositol for transport through SMIT. We conclude that the incubation in isotonic, amino acid-free saline triggers an osmotic stimulus and elicits TonEBP-dependent responses like hypertonic treatment.
[show abstract][hide abstract] ABSTRACT: System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.