ADF Cells Research Articles

Pathways of 4-Hydroxy-2-Nonenal Detoxification in a Human Astrocytoma Cell Line.

One of the consequences of the increased level of oxidative stress that often characterizes the cancer cell environment is the abnormal generation of lipid peroxidation products, above all 4-hydroxynonenal. The contribution of this aldehyde to the pathogenesis of several diseases is well known. In this study, we characterized the ADF astrocytoma cell line both in terms of its pattern of enzymatic activities devoted to 4-hydroxynonenal removal and its resistance to oxidative stress induced by exposure to hydrogen peroxide. A comparison with lens cell lines, which, due to the ocular function, are normally exposed to oxidative conditions is reported. Our results show that, overall, ADF cells counteract oxidative stress conditions better than normal cells, thus confirming the redox adaptation demonstrated for several cancer cells. In addition, the markedly high level of NADP+-dependent dehydrogenase activity acting on the glutahionyl-hydroxynonanal adduct detected in ADF cells may promote, at the same time, the detoxification and recovery of cell-reducing power in these cells.

The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems.

In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-β1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100 nM–100 μM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM–100 μM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-β1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.

Anxiolytic properties of a 2-phenylindolglyoxylamide TSPO ligand: Stimulation of in vitro neurosteroid production affecting GABA A receptor activity

A number of neurosteroids have been demonstrated to exert anxiolytic properties by means of a positive modulation of inhibitory GABAergic neurotransmission. The observation that neurosteroid synthesis can be pharmacologically regulated by ligands to the mitochondrial translocator protein (TSPO) has prompted the search for new, more selective TSPO ligands able to stimulate steroidogenesis with great efficacy. In the present study, the potential anxiolytic activity of a selective TSPO ligand, N,N-di-n-propyl-2-(4-methylphenyl)indol-3-ylglyoxylamide (MPIGA), was tested by means of the elevated plus maze paradigm. Moreover, the in vitro effects on synaptoneurosomal GABA(A) receptor (GABA(A)R) activity exerted by the conditioned salt medium from MPIGA-treated ADF human glial cells were investigated. MPIGA (30mg/kg) was found to affect rats' performance in the elevated plus maze test significantly, leading to an increase in both entries and time spent in the open arms. This same dose of MPIGA had no effect on rats' spontaneous exploratory activity. The conditioned salt medium from MPIGA-treated ADF cells potentiated the (36)Cl(-) uptake into cerebral cortical synaptoneurosomes. The exposure of ADF cells to MPIGA stimulated the production of pregnelonone derivatives including allopregnanolone, one of the major positive GABA(A)R allosteric modulator. In conclusion, the TSPO ligand MPIGA is a promising anxiolytic drug. The mechanism of action by which MPIGA exerts its anxiolytic activity was identified in the stimulation of endogenous neurosteroid production, which in turn determined a positive modulation of GABA(A)R activity, thus opening the way to the potential use of this TSPO ligand in anxiety and depressive disorders.

The silencing of adenine nucleotide translocase isoform 1 induces oxidative stress and programmed cell death in ADF human glioblastoma cells

Adenine nucleotide translocases (ANTs) are multitask proteins involved in several aspects of cell metabolism, as well as in the regulation of cell death/survival processes. We investigated the role played by ANT isoforms 1 and 2 in the growth of a human glioblastoma cell line (ADF cells). The silencing of ANT2 isoform, by small interfering RNA, did not produce significant changes in ADF cell viability. By contrast, the silencing of ANT1 isoform strongly reduced ADF cell viability by inducing a non-apoptotic cell death process resembling paraptosis. We demonstrated that cell death induced by ANT1 depletion cannot be ascribed to the loss of the ATP/ADP exchange function of this protein. By contrast, our findings indicate that ANT1-silenced cells experience oxidative stress, thus allowing us to hypothesize that the effect of ANT1-silencing on ADF is mediated by the loss of the ANT1 uncoupling function. Several studies ascribe a pro-apoptotic role to ANT1 as a result of the observation that ANT1 overexpression sensitizes cells to mitochondrial depolarization or to apoptotic stimuli. In the present study, we demonstrate that, despite its pro-apoptotic function at a high expression level, the reduction of ANT1 density below a physiological baseline impairs fundamental functions of this protein in ADF cells, leading them to undertake a cell death process.

Drugs targeting the mitochondrial pore act as citotoxic and cytostatic agents in temozolomide-resistant glioma cells

BackgroundHigh grade gliomas are one of the most difficult cancers to treat and despite surgery, radiotherapy and temozolomide-based chemotherapy, the prognosis of glioma patients is poor. Resistance to temozolomide is the major barrier to effective therapy. Alternative therapeutic approaches have been shown to be ineffective for the treatment of genetically unselected glioma patients. Thus, novel therapies are needed. Mitochondria-directed chemotherapy is an emerging tool to combat cancer, and inner mitochondrial permeability transition (MPT) represents a target for the development of cytotoxic drugs. A number of agents are able to induce MPT and some of them target MPT-pore (MPTP) components that are selectively up-regulated in cancer, making these agents putative cancer cell-specific drugs.ObjectiveThe aim of this paper is to report a comprehensive analysis of the effects produced by selected MPT-inducing drugs (Betulinic Acid, Lonidamine, CD437) in a temozolomide-resistant glioblastoma cell line (ADF cells).MethodsEGFRvIII expression has been assayed by RT-PCR. EGFR amplification and PTEN deletion have been assayed by differential-PCR. Drugs effect on cell viability has been tested by crystal violet assay. MPT has been tested by JC1 staining. Drug cytostatic effect has been tested by mitotic index analysis. Drug cytotoxic effect has been tested by calcein AM staining. Apoptosis has been assayed by Hoechst incorporation and Annexine V binding assay. Authophagy has been tested by acridine orange staining.ResultsWe performed a molecular and genetic characterization of ADF cells and demonstrated that this line does not express the EGFRvIII and does not show EGFR amplification. ADF cells do not show PTEN mutation but differential PCR data indicate a hemizygous deletion of PTEN gene. We analyzed the response of ADF cells to Betulinic Acid, Lonidamine, and CD437. Our data demonstrate that MPT-inducing agents produce concentration-dependent cytostatic and cytotoxic effects in parallel with MPT induction triggered through MPTP. CD437, Lonidamine and Betulinic acid trigger apoptosis as principal death modality.ConclusionThe obtained data suggest that these pharmacological agents could be selected as adjuvant drugs for the treatment of high grade astrocytomas that resist conventional therapies or that do not show any peculiar genetic alteration that can be targeted by specific drugs.

Knockdown of cytosolic 5′-nucleotidase II (cN-II) reveals that its activity is essential for survival in astrocytoma cells

IMP preferring cytosolic 5′-nucleotidase (cN-II) is an ubiquitous nucleotide hydrolysing enzyme. The enzyme is widely distributed and its amino acid sequence is highly conserved among vertebrates. Fluctuations of cN-II activity have been associated with the pathogenesis of neurological disorders. The enzyme appears to be involved in the regulation of the intracellular availability of the purine precursor IMP and also of GMP and AMP, but the contribution of this activity and of its regulation to cell metabolism and to CNS cell functions remains uncertain. To address this issue, we used a vector based short hairpin RNA (shRNA) strategy to knockdown cN-II activity in human astrocytoma cells. Our results demonstrated that 53 h after transduction, cN-II mRNA was reduced to 17.9 ± 0.03% of control cells. 19 h later enzyme activity was decreased from 0.7 ± 0.026 mU/mg in control ADF cells to 0.45 ± 0.046 mU/mg, while cell viability (evaluated by the MTT reduction assay) decreased up to 0.59 ± 0.01 (fold vs control) and caspase 3 activity increased from 136 ± 5.8 pmol min − 1 mg − 1 in control cells to 639 ± 37.5 pmol min − 1 mg − 1 in silenced cells, thus demonstrating that cN-II is essential for cell survival. The decrease of enzyme activity causes apoptosis of the cultured cells without altering intracellular nucleotide and nucleoside concentration or energy charge. Since cN-II is highly expressed in tumour cells, our finding offers a new possible therapeutical approach especially against primary brain tumours such as glioblastoma, and to ameliorate chemotherapy against leukemia.

Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

Proteasome Inhibitors Potentiate Etoposide-Induced Cell Death in Human Astrocytoma Cells Bearing a Mutated p53 Isoform

Resistance to anticancer agents is often due to defects of intracellular pathways of cell death. Thus, the identification of the apoptotic pathways that can still be recruited by chemotherapeutic agents in cancerous cells can disclose new opportunities to treat malignancies. Here we show that human astrocytoma ADF cells (which are resistant to "mitochondriotropic" agents as well as to the antineoplastic drug etoposide and to proteasome inhibitors when used alone) undergo dramatic apoptotic death when exposed to a combination protocol based on the use of etoposide in the presence of proteasome inhibitors. Sensitization to cell death involved an autoamplifying loop of caspase activation, where the "executioner" phase of apoptosis was sustained by cooperation of caspase-2, -9, -8, and -3. We also show that sensitization of cells to the combination protocol involved the nuclear relocalization of p53, despite the presence of a polymorphism in its DNA-binding domain, suggesting the likely induction of p53-dependent proapoptotic genes. Conversely, p53 phosphorylation on Ser-15 did not play any role in apoptosis. In conclusion, use of etoposide in combination with proteasome inhibitors may represent an effective strategy to restore sensitivity to apoptosis in human astrocytoma cells bearing multiple defects of intracellular apoptotic pathways.

Human glioblastoma ADF cells express tyrosinase, L‐tyrosine hydroxylase and melanosomes and are sensitive to L‐tyrosine and phenylthiourea

Melanocytes and neuroblasts share the property of transforming L-tyrosine through two distinct metabolic pathways leading to melanogenesis and catecholamine synthesis, respectively. While tyrosinase (TYR) activity has been shown to be expressed by neuroblastoma it remains to be established as to whether also glioblastomas cells are endowed with this property. We have addressed this issue using the human continuous glioblastoma cell line ADF. We demonstrated that these cells possess tyrosinase as well as L-tyrosine hydroxylase (TH) activity and synthesize melanosomes. Because the two pathways are potentially cyto-genotoxic due to production of quinones, semiquinones, and reactive oxygen species (ROS), we have also investigated the expression of the peroxisomal proliferators activated receptor alpha (PPARalpha) and nuclear factor-kB (NFkB) transcription factor as well the effect of L-tyrosine concentration on cell survival. We report that L-tyrosine down-regulates PPARalpha expression in ADF cells but not neuroblastoma and that this aminoacid and phenylthiourea (PTU) induces apoptosis in glioblastoma and neuroblastoma.

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

The A3 adenosine receptor induces cytoskeleton rearrangement in human astrocytoma cells via a specific action on Rho proteins.

In previous studies, we have demonstrated that exposure of astroglial cells to A3 adenosine receptor agonists results in dual actions on cell survival, with "trophic" and antiapoptotic effects at nanomolar concentrations and induction of cell death at micromolar agonist concentrations. The protective actions of A3 agonists have been associated with a reinforcement of the actin cytoskeleton, which likely results in increased resistance of cells to cytotoxic stimuli. The molecular mechanisms at the basis of this effect and the signalling pathway(s) linking the A3 receptor to the actin cytoskeleton have never been elucidated. Based on previous literature data suggesting that the actin cytoskeleton is controlled by small GTP-binding proteins of the Rho family, in the study reported here we investigated the involvement of these proteins in the effects induced by A3 agonists on human astrocytoma ADF cells. The presence of the A3 adenosine receptor in these cells has been confirmed by immunoblotting analysis. As expected, exposure of human astrocytoma ADF cells to nanomolar concentrations of the selective A3 agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CI-IB-MECA) resulted in formation of thick actin positive stress fibers. Preexposure of cells to the C3B toxin that inactivates Rho-proteins completely prevented the actin changes induced by CI-IB-MECA. Exposure to the A3 agonist also resulted in significant reduction of Rho-GDI, an inhibitory protein known to maintain Rho proteins in their inactive state, suggesting a potentiation of Rho-mediated effects. This effect was fully counteracted by the concomitant exposure to the selective A3 receptor antagonist MRS1191. These results suggest that the reinforcement of the actin cytoskeleton induced by A3 receptor agonists is mediated by an interference with the activation/inactivation cycle of Rho proteins, which may, therefore, represent a biological target for the identification of novel neuroprotective strategies.

A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes

In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog α,βmethyleneATP (α,βmeATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. α,βmeATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X 7 receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both α,βmeATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

Apoptosis induced by 2-chloro-adenosine and 2-chloro-2?-deoxy-adenosine in a human astrocytoma cell line: Differential mechanisms and possible clinical relevance

We have previously demonstrated that 2-chloro-adenosine (2-CA) can induce apoptosis of rat astroglial cells (Abbracchio et al. [1995] Biochem. Biophys. Res. Commun. 213:908-915). In the present study, we have characterized, for the first time, the effects induced on a human astrocytoma cell line (ADF cells) by both 2-CA and its related analog 2-chloro-2'-deoxy-adenosine (2-CdA, that is employed as anti-cancer agent in chronic lymphoid malignancies). Exposure of these cells to either adenosine analog resulted in time- and concentration-dependent apoptosis. Experiments with pharmacological agents known to interfere with adenosine receptors, its membrane transporter, and intracellular nucleoside kinases showed that: (i) cell death induced by either adenosine analog did not depend on extracellular adenosine receptors, but on a direct intracellular action; however, only in the case of 2-CA, was entry into cells mediated by the specific nitrobenzyl-tioinosine-sensitive transporter; (ii) for both adenosine analogs, induction of apoptosis required the phosphorylation/activation by specific intracellular nucleoside kinases, i.e., adenosine kinase for 2-CA, and deoxycytidine kinase for 2-CdA. In addition, only in the case of 2-CdA, was induction of apoptosis preceded by a block of cells at the G2/M phase of the cell cycle. Finally, at concentrations of either analog that killed about 80-90% of astrocytoma cells, a significantly lower effect on the viability of primary cortical neurons was observed. In conclusion, both adenosine analogs can trigger apoptosis of human astrocytoma cells, albeit with different mechanisms. This effect together with the relative sparing of neuronal cells, may have potential clinical implications for the therapy of tumors of glial origin.

Increase of BCNU sensitivity by wt-p53 gene therapy in glioblastoma lines depends on the administration schedule.

In this article, we investigated the effect induced by the reintroduction of wild-type p53 (wt-p53) protein on BCNU sensitivity in the ADF glioblastoma line. Using a wt-p53 recombinant adenovirus (Ad-p53), we demonstrated that exogenous wt-p53 expression was able to increase the sensitivity to BCNU in ADF cells. Interestingly, this effect was more evident when Ad-p53 infection was performed after BCNU treatment compared with the opposite sequence. To understand the biological basis of these different behaviors, we analyzed the cell cycle of the differently treated cells. We found that Ad-p53 infection induced a persistent accumulation of cells in the G0/G1 phase while, as expected, BCNU induced a block in the G2-M phase. Ad-p53-->BCNU sequence did not significantly modify the cell cycle profile in respect of Ad-p53 infected cells. In contrast, BCNU-->Ad-p53 sequence provoked G2-M arrest similar to that observed after treatment with BCNU alone, but prevented the later recovery of the cells through the cell cycle, by driving the cells to apoptotic death. These results demonstrate that the administration sequence is important to increase drug sensitivity. To generalize the phenomenon observed on ADF line, the antiproliferative effect of the two different schedules was analyzed on other glioblastoma lines (A172, CRS-A2, U373MG) with different BCNU sensitivity and p53 status. The data obtained confirm that the wt-p53 gene transfer enhances BCNU sensitivity in glioblastoma cells depending on the administration sequence.

Adenosine A3 Receptors and Viability of Astrocytes.

We investigated the role of the A3 adenosine receptor in cells of the astroglial lineage (both rat primary astrocytes and human astrocytoma ADF cells) by means of the selective A3 agonists N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and CI-IB-MECA, and by utilizing the selective A3 receptor antagonist MRS1191. Exposure of ADF cells to μM concentrations of either agonist resulted in reduction of cell number, likely due to cell death. In both rat astrocytes and human astrocytoma cells, at concentrations 2-3 orders of magnitude lower (which were not associated with cytotoxicity), these same agonists induced a marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions. Functionally, these morphological changes were associated with cell protection, as demonstrated by a significant reduction of spontaneous apoptosis in A3 agonist-treated cells. To confirm a role for the A3 receptor in this effect, MRS1191 completely counteracted CI-IB-MECA-induced reduction of spontaneous apoptosis. In ADF cells, A3 agonists also induced changes in the intracellular distribution of the anti-apoptotic protein Bcl-XL, which became localized in cell protrusions. Also, this effect was specifically antagonized by MRS1191. These dual actions of A3 agonists in vitro may have important in vivo implications. For example, a robust and acute activation of the A3 receptor following massive adenosine release during ischemia may contribute to brain cell death; conversely, a subthreshold activation of this receptor prior to ischemia may trigger protective mechanisms (i.e., induction of stress fibers and of a Bcl-XL-dependent reorganization of cytoskeleton) making the brain more resistant to subsequent insults ("ischemic tolerance").

The A3Adenosine Receptor Mediates Cell Spreading, Reorganization of Actin Cytoskeleton, and Distribution of Bcl-xL: Studies in Human Astroglioma Cells

The pathophysiological role of the adenosine A3receptor in the central nervous system is largely unknown. We have investigated the effects of the selective A3receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine, Cl-IB-MECA, in cells of the astroglial lineage (human astrocytoma ADF cells). A marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions, was found following exposure of cells to low (nM) concentrations of Cl-IB-MECA. These “trophic” effects were accompanied by induction of the expression of Rho, a small GTP-binding protein, which was virtually absent in control cells, and by changes of the intracellular distribution of the antiapoptotic protein Bcl-xL, that, in agonist-exposed cells, became specifically associated to cell protrusions. This is the first demonstration that the intracellular organization of Bcl-xLcan be modulated by the activation of a G-protein-coupled membrane receptor, such as the A3adenosine receptor. Moreover, modulation of the astrocytic cytoskeleton by adenosine may have intriguing implications in both nervous system development and in the response of the brain to trauma and ischemia.

Let the facts speak for themselves

Resistance to anticancer agents is often due to defects of intracellular pathways of cell death. Thus, the identification of the apoptotic pathways that can still be recruited by chemotherapeutic agents in cancerous cells can disclose new opportunities to treat malignancies. Here we show that human astrocytoma ADF cells (which are resistant to “mitochondriotropic” agents as well as to the antineoplastic drug etoposide and to proteasome inhibitors when used alone) undergo dramatic apoptotic death when exposed to a combination protocol based on the use of etoposide in the presence of proteasome inhibitors. Sensitization to cell death involved an autoamplifying loop of caspase activation, where the “executioner” phase of apoptosis was sustained by cooperation of caspase-2, -9, -8, and -3. We also show that sensitization of cells to the combination protocol involved the nuclear relocalization of p53, despite the presence of a polymorphism in its DNA-binding domain, suggesting the likely induction of p53-dependent proapoptotic genes. Conversely, p53 phosphorylation on Ser-15 did not play any role in apoptosis. In conclusion, use of etoposide in combination with proteasome inhibitors may represent an effective strategy to restore sensitivity to apoptosis in human astrocytoma cells bearing multiple defects of intracellular apoptotic pathways.