Polycomb dysregulation in gliomagenesis targets a Zfp423-dependent differentiation network.

Malignant gliomas constitute one of the most significant areas of unmet medical need, owing to the invariable failure of surgical eradication and their marked molecular heterogeneity. Accumulating evidence has revealed a critical contribution by the Polycomb axis of epigenetic repression. However, a coherent understanding of the regulatory networks affected by Polycomb during gliomagenesis is still lacking. Here we integrate transcriptomic and epigenomic analyses to define Polycomb-dependent networks that promote gliomagenesis, validating them both in two independent mouse models and in a large cohort of human samples. We find that Polycomb dysregulation in gliomagenesis affects transcriptional networks associated with invasiveness and de-differentiation. The dissection of these networks uncovers Zfp423 as a critical Polycomb-dependent transcription factor whose silencing negatively impacts survival. The anti-gliomagenic activity of Zfp423 requires interaction with the SMAD proteins within the BMP signalling pathway, pointing to a novel synergic circuit through which Polycomb inhibits BMP signalling.

The dual PI3K/mTOR inhibitor NVP-BEZ235 and chloroquine synergize to trigger apoptosis via mitochondrial-lysosomal cross-talk.

On the basis of our previous identification of aberrant phosphatidylinositol-3-kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235-induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235-induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase-dependent apoptosis. Lysosome-mediated apoptosis occurs in a ROS-dependent manner, as ROS scavengers significantly reduce BEZ235/CQ-induced loss of MMP, LMP and apoptosis. There is a mitochondrial-lysosomal cross-talk, since lysosomal enzyme inhibitors significantly decrease BEZ235- and CQ-induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome-mediated apoptosis via a mitochondrial-lysosomal cross-talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor-based combination therapies.

Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor α-induced necroptosis.

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

Comparative cellular and molecular analysis of cytotoxicity and apoptosis induction by doxorubicin and Baneh in human breast cancer T47D cells.

It is now widely accepted that dietary phytochemicals inhibit cancer progression and enhance the effects of conventional chemotherapy. In this report, we comparatively studied the cellular and molecular aspects of apoptosis induction by the methanolic extract of Baneh fruit skin in comparison to Doxorubicin (Dox), a well-known anticancer drug, in human breast cancer T47D cells. The MTT assay was used to determine the antiproliferative effects. The flow cytometric and microscopic analyses were done to evaluate the apoptosis induction. Furthermore, western blot analyses have been done to study the role of key molecular players of apoptosis including caspase 3 and PARP. The Baneh extract showed strong antiproliferative activity against T47D cells in a dose- and time-dependent manner that was comparable to and even stronger than Dox in certain concentrations. Analysis of Baneh-treated cells by flow cytometry and fluorescence microscopy indicated strong apoptosis induction and nuclear morphological alterations similar to or greater than Dox. Finally, molecular analysis of apoptosis by western blotting proved activation of caspase 3 followed by poly ADP ribose polymerase (PARP) cleavage more efficiently in Baneh than in Dox treated cancer cells. These findings indicate that Baneh extract contains phytochemicals which act as inhibitor of cell proliferation and inducer of apoptosis in human breast cancer T47D cells that makes it a potentially good candidate for new anticancer drug development.

NF-κB is required for Smac mimetic-mediated sensitization of glioblastoma cells for γ-irradiation-induced apoptosis.

Evasion of apoptosis contributes to radioresistance of glioblastoma, calling for novel strategies to overcome apoptosis resistance. In this study, we investigated the potential of the small molecule Smac mimetic BV6 to modulate radiosensitivity of glioblastoma cells. Here, we identify a novel proapoptotic function of NF-κB in γ-irradiation-induced apoptosis of glioblastoma cells by showing, for the first time, that NF-κB is critically required for Smac mimetic-mediated radiosensitization. BV6 significantly increases γ-irradiation-triggered apoptosis in several glioblastoma cell lines in a dose- and time-dependent manner. Calculation of combination index (CI) reveals that the interaction of BV6 and γ-irradiation is highly synergistic (CI < 0.3). Molecular studies show that BV6 stimulates NF-κB activation, which is critical for radiosensitization, because genetic inhibition of NF-κB by overexpression of the dominant-negative superrepressor IκBα-SR significantly decreases BV6- and γ-irradiation-induced apoptosis. Also, the BV6-mediated enhancement of γ-irradiation-triggered caspase activation, drop of mitochondrial membrane potential, and cytochrome c release is abolished in cells overexpressing IκBα-SR. Similarly, NF-κB inhibition by ectopic expression of a kinase dead mutant of IKKß prevents the BV6-mediated sensitization for γ-irradiation. The clinical relevance is underscored by experiments with primary tumor samples showing that BV6 sensitizes primary cultured glioma cells as well as glioblastoma-initiating cancer stem cells derived from surgical specimens for γ-irradiation. In conclusion, we identify NF-κB as a critical mediator of Smac mimetic-conferred radiosensitization of glioblastoma cells. These results have important implications for the development of Smac mimetic-based combination protocols for radiosensitization of glioblastoma.

Chemosensitization of glioblastoma cells by the histone deacetylase inhibitor MS275.

Glioblastoma is the most common primary brain tumor with a dismal prognosis, highlighting the need for novel treatment strategies. Here, we provide the first evidence that the histone deacetylase inhibitor, MS275, sensitizes glioblastoma cells for chemotherapy-induced apoptosis. Pretreatment of glioblastoma cells with MS275 causes acetylation of histone H3 protein and significantly enhances doxorubicin-induced apoptosis. Calculation of combination index showed that MS275 and doxorubicin acted in a synergistic manner to trigger apoptosis. Furthermore, pre-exposure to MS275 significantly increases apoptosis in response to temozolomide, etoposide, and cisplatin. In contrast, treatment with MS275 before the addition of vincristine and taxol significantly reduces the induction of apoptosis. Analysis of cell cycle alterations showed that treatment with MS275 triggers G1 cell cycle arrest, which in turn renders cells less sensitive to the cytotoxic effects of mitotic inhibitors, such as vincristine and taxol. Thus, these findings show for the first time that the histone deacetylase inhibitor, MS275, represents a promising strategy to prime glioblastoma cells for chemotherapy-induced apoptosis in a drug-specific manner.

Bortezomib primes glioblastoma, including glioblastoma stem cells, for TRAIL by increasing tBid stability and mitochondrial apoptosis.

PURPOSE: Searching for novel approaches to sensitize glioblastoma for cell death, we investigated the proteasome inhibitor bortezomib. EXPERIMENTAL DESIGN: The effect of bortezomib on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures, and in an in vivo model. RESULTS: Bortezomib and TRAIL synergistically trigger cell death and reduce colony formation of glioblastoma cells (combination index < 0.1). Investigations into the underlying molecular mechanisms reveal that bortezomib and TRAIL act in concert to cause accumulation of tBid, the active cleavage product of Bid. Also, the stability of TRAIL-derived tBid markedly increases on proteasome inhibition. Notably, knockdown of Bid significantly decreases bortezomib- and TRAIL-mediated cell death. By comparison, silencing of Noxa, which is also upregulated by bortezomib, does not confer protection. Coinciding with tBid accumulation, the activation of Bax/Bak and loss of mitochondrial membrane potential are strongly increased in cotreated cells. Overexpression of Bcl-2 significantly reduces mitochondrial perturbations and cell death, underscoring the functional relevance of the mitochondrial pathway. In addition, bortezomib cooperates with TRAIL to reduce colony formation of glioblastoma cells, showing an effect on long-term survival. Of note, bortezomib profoundly enhances TRAIL-triggered cell death in primary cultured glioblastoma cells and in patient-derived glioblastoma stem cells, underlining the clinical relevance. Importantly, bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo glioblastoma model. CONCLUSION: These findings provide compelling evidence that the combination of bortezomib and TRAIL presents a promising novel strategy to trigger cell death in glioblastoma, including glioblastoma stem cells, which warrants further investigation.

Requirement of nuclear factor κB for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis.

Defects in apoptosis contribute to treatment resistance and poor outcome of pancreatic cancer, calling for novel therapeutic strategies. Here, we provide the first evidence that nuclear factor (NF) κB is required for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis. The Smac mimetic BV6 cooperates with gemcitabine to reduce cell viability and to induce apoptosis. In addition, BV6 significantly enhances the cytotoxicity of several anticancer drugs against pancreatic carcinoma cells, including doxorubicin, cisplatin, and 5-fluorouracil. Molecular studies reveal that BV6 stimulates NF-κB activation, which is further increased in the presence of gemcitabine. Importantly, inhibition of NF-κB by overexpression of the dominant-negative IκBα superrepressor significantly decreases BV6- and gemcitabine-induced apoptosis, demonstrating that NF-κB exerts a proapoptotic function in this model of apoptosis. In support of this notion, inhibition of tumor necrosis factor α (TNFα) by the TNFα blocking antibody Enbrel reduces BV6- and gemcitabine-induced activation of caspase 8 and 3, loss of mitochondrial membrane potential, and apoptosis. By demonstrating that BV6 and gemcitabine trigger a NF-κB-dependent, TNFα-mediated loop to activate apoptosis signaling pathways and caspase-dependent apoptotic cell death, our findings have important implications for the development of Smac mimetic-based combination protocols in the treatment of pancreatic cancer.

Intracellular prooxidant activity of melatonin induces a survival pathway involving NF-kappaB activation.

We have shown that melatonin exerts a prooxidant activity in U937 cells, a tumor human promonocytic cell line. (1) Here we show that melatonin induces a strong canonical activation of NF-kappaB, inducing IkappaBalpha degradation and the consequential nuclear translocation of p50/p65 subunits. The timing of NF-kappaB activation overlaps with the timing of reactive oxygen species (ROS) production due to melatonin. Overexpression of dominant-negative IkappaB, which prevents a possible NF-kappaB activation, transformed melatonin in a proapoptotic molecule. These data indicate for the first time that melatonin can trigger NF-kappaB activation and might suggest a possible role for ROS induced by melatonin. Results indicate a possible involvement in the survival pathway of melatonin-generated ROS as secondary messengers.

Subapoptogenic oxidative stress strongly increases the activity of the glycolytic key enzyme glyceraldehyde 3-phosphate dehydrogenase.

We have previously shown that oxidative stress induced by an apoptogenic dose of H(2)O(2) leads to a temporary block of glycolytic flux via inactivation of the glycolytic key enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in U937 cells. This corresponds to the activation of a cell defense pathway that is triggered to repair stress-induced damage and to rescue cells from death. Here, we show that subapoptogenic doses of H(2)O(2) affect GAPDH activity in an opposite way, leading to strong hyperactivation. This phenomenon is related to milder oxidative stress because induction of a moderate oxidative stress with an alternative approach (i.e., by decreasing glutathione content in the cells with buthionine sulphoximine) gives similar results. U937 cells hyperactivate GAPDH with the same timing observed for GAPDH alterations from apoptogenic doses of H(2)O(2). Additionally, the prevention of the glycolytic flux sensitizes stressed cells to apoptosis. This suggests that GAPDH hyperactivity might also be an active cell response to stress, thus depicting multiple roles for glycolytic flux in different prosurvival pathways where activation depends on the strength of the oxidative stress.

The inhibition of TNF-alpha-induced NF-kappaB activation by marine natural products.

The deregulated activation of NF-kappaB is associated with cancer development and inflammatory diseases. With an aim to find new NF-kappaB inhibitors, we purified and characterized compounds from extracts of the Fijian sponge Rhabdastrella globostellata, the crinoid Comanthus parvicirrus, the soft corals Sarcophyton sp. nov. and Sinularia sp., and the gorgonian Subergorgia sp. after an initial screening of 266 extracts from different marine origins. Results obtained show that selected purified compounds had a cytotoxic effect on the human leukaemia cell line K562, inhibited both TNF-alpha-induced NF-kappaB-DNA binding as well as TNF-alpha-induced IkappaBalpha degradation and nuclear translocation of p50/p65. Furthermore, we observed the inhibition of NF-kappaB activation induced by an overexpression of IKKbeta. Interestingly, natural products inhibited IKKbeta kinase as well as the 26S proteasome proteolytic activity.

Cell cycle arrest in early mitosis and induction of caspase-dependent apoptosis in U937 cells by diallyltetrasulfide (Al2S4).

Naturally occurring organic sulfur compounds (OSCs), such as linear allylsulfides from Allium species, are attracting attention in cancer research, since several OSCs were shown to act beneficially both in chemoprevention and in chemotherapy, while hardly exerting any harmful side effects. Hence, we investigated the possible role of different OSCs in the treatment of leukemia. Thereby, we found that the compounds tested in this study induced apoptosis in U937 cells, with an efficiency depending on the number of sulfides, and selected the most promising candidate, diallyltetrasulfide (Al2S4), for detailed mechanistic studies. Here we show that Al2S4 induced an accumulation of cells in early mitosis (G2/M phase), followed by the activation of caspase-dependent apoptosis. The compound counteracted different anti-apoptotic Bcl-2 family members (Bcl-xL, phospho-Bad and Bcl-2), promoted activation of Bax and Bak and induced the release of cytochrome c into the cytoplasm. Treatment by Al2S4 let to the identification of early apoptotic events including Bcl-xL degradation, Bak activation and release of cytochrome c followed by late events including Bcl-2 proteolysis, Bax activation, Bad dephosphorylation, caspase activation, nuclear fragmentation and phosphatidylserine exposure.

The inhibitory effect of the proinflammatory cytokine TNFalpha on erythroid differentiation involves erythroid transcription factor modulation.

The hematopoietic transcription factor GATA-1 regulates the expression of several genes associated with differentiation of erythroid cells. We show here the inhibitory effect of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, on hemoglobinization and erythroid transcription factor GATA-1 expression in erythroleukemia (HEL) as well as in chronic myelogenous leukemia (K562) cells, which were induced to differentiate towards the erythroid lineage after aclacinomycin (Acla), doxorubicin (Dox) or hemin (HM) treatment. As a result, we observed i) a decreased expression of Friend of GATA-1 (FOG-1), an essential cofactor of GATA-1 transcription factor, ii) a downregulation of GATA-1 by proteasomal degradation and iii) a reduced acetylation level of GATA-1 in HM-induced K562 cells after TNF treatment. As a result, these modifications i) decreased the level of GATA-1/FOG-1 complex, ii) unsettled the GATA-1/GATA-2 balance, iii) reduced GATA-1 transcriptional activity and iv) inhibited erythroid marker gene expression (glycophorin A, erythropoietin receptor, gamma-globin) independently of the cell line or the inducer used. These data provided new insights into the role of GATA-1 regulation in TNFalpha-mediated inhibition of erythroid differentiation in erythroleukemia.

Oxidative, multistep activation of the noncanonical NF-kappaB pathway via disulfide Bcl-3/p50 complex.

Buthionine sulfoximine (BSO) is a well-known inhibitor of glutathione synthesis, producing slow glutathione (GSH) depletion and oxidative stress; some "responder" cells avoid BSO-induced death by trans-activating the prosurvival protein Bcl-2. Here we show that BSO activates a noncanonical, inhibitory NF-kappaB- and p65-independent NF-kappaB pathway via a multistep process leading to the up-regulation of Bcl-2. The slow BSO-induced GSH depletion allows separation of two redox-related phases, namely, early thiol disequilibrium and late frank oxidative stress; each phase contributes to the progressive activation of a p50-p50 homodimer. The early phase, coinciding with substantial thiol depletion, produces a cytosolic preparative complex, consisting of p50 and its interactor Bcl-3 linked by interprotein disulfide bridges. The late phase, coinciding with reactive oxygen species production, is responsible, probably via p38 activation, for nuclear targeting of the complex and trans-activation of Bcl-2.

Tumor necrosis factor alpha induces gamma-glutamyltransferase expression via nuclear factor-kappaB in cooperation with Sp1.

Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

Tumor necrosis factor alpha inhibits erythroid differentiation in human erythropoietin-dependent cells involving p38 MAPK pathway, GATA-1 and FOG-1 downregulation and GATA-2 upregulation.

The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) has been linked to inflammation- and cancer-related anemia, which reduces both quality of life and prognosis of patients. The aim of this study was to reveal molecular mechanisms linked to the inhibition of erythroid differentiation by TNFalpha. In this study, we showed that the inhibition of erythropoietin (Epo)-mediated differentiation by TNFalpha lead to a downregulation of hemoglobin synthesis and was correlated to a modulation of key erythroid transcription factors. Thus, a reverse of the transcription factor GATA-1/GATA-2 balance normally present during erythropoiesis, as well as a downregulation of the cofactor of GATA-1, friend of GATA-1 (FOG-1), and the coregulating transcription factor nuclear factor erythroid 2 (NF-E2) was observed after TNFalpha treatment. Moreover, we showed a reduction of GATA-1/FOG-1 interaction due to a reduced transcription of GATA-1 and a proteasome-dependent FOG-1 degradation after TNFalpha treatment. These changes led to an inhibition of erythroid gene expression including Epo receptor (EpoR), alpha- and gamma-globin, erythroid-associated factor (ERAF), hydroxymethylbilane synthetase (HMBS), and glycophorin A (GPA). An analysis of distinct signaling pathway activations then revealed an activation of p38 by TNF, as well as a corresponding involvement of this mitogen-activated protein kinase (MAPK) in the cytokine-dependent inhibition of erythroid differentiation. Indeed the p38 inhibitor, SB203580, abrogated the inhibitory effect of TNFalpha on the major erythroid transcription factor GATA-1 as well as erythroid marker expression in Epo-induced TF-1 cells. Overall, these data contribute to a better understanding of cytokine-dependent anemia, by giving first hints about key erythroid transcription factor modulations after TNFalpha treatment as well as an involvement of p38 in the inhibition of erythroid differentiation.

Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2.

We have recently shown that melatonin antagonizes damage-induced apoptosis by interaction with the MT-1/MT-2 plasma membrane receptors. Here, we show that melatonin interferes with the intrinsic pathway of apoptosis at the mitochondrial level. In response to an apoptogenic stimulus, melatonin allows mitochondrial translocation of the pro-apoptotic protein Bax, but it impairs its activation/dimerization The downstream apoptotic events, i.e. cytochrome c release, caspase 9 and 3 activation and nuclear vesiculation are equally impaired, indicating that melatonin interferes with Bax activation within mitochondria. Interestingly, we found that melatonin induces a strong re-localization of Bcl-2, the main Bax antagonist to mitochondria, suggesting that Bax activation may in fact be antagonized by Bcl-2 at the mitochondrial level. Indeed, we inhibit the melatonin anti-apoptotic effect (i) by silencing Bcl-2 with small interfering RNAs, or with small-molecular inhibitors targeted at the BH3 binding pocket in Bcl-2 (i.e. the one interacting with Bax); and (ii) by inhibiting melatonin-induced Bcl-2 mitochondrial re-localization with the MT1/MT2 receptor antagonist luzindole. This evidence provides a mechanism that may explain how melatonin through interaction with the MT1/MT2 receptors, elicits a pathway that interferes with the Bcl-2 family, thus modulating the cell life/death balance.

RETRACTED: Glutathione as a mediator of apoptotic cell signaling pathways.

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Oxidative upregulation of Bcl-2 in healthy lymphocytes.

In many cell systems, pharmacological glutathione (GSH) depletion with the GSH neosynthesis inhibitor buthionine sulfoximine (BSO) leads to cell death and highly sensitizes tumor cells to apoptosis induced by standard chemotherapeutic agents. However, some tumor cells upregulate Bcl-2 in response to BSO, thus surviving the treatment and failing to be chemosensitized. Cell lines of monocytic and lymphocytic origins respond to BSO treatment in an opposite way, lymphocytes being chemosensitized and unable to transactivate Bcl-2. In this article we investigate the response to BSO of lymphocytes freshly isolated from peripheral blood of healthy donors. After ensuring that standard separation procedures do not alter per se lymphocytes redox equilibrium nor Bcl-2 levels in the first 24 h of culture, we show that BSO treatment promotes the upregulation of Bcl-2, with a mechanism involving the increased radical production consequent to GSH depletion. Thus, BSO treatment may increase the differential cytocidal effect of cytotoxic drugs in tumor versus normal lymphocytes.

Glutathione depletion up-regulates Bcl-2 in BSO-resistant cells.

Glutathione depletion by inhibition of its synthesis with buthionine sulfoximine (BSO) is a focus of the current research in antitumor therapy, BSO being used as chemosensitizer. We had previously shown that two human tumor cell lines (U937 and HepG2) survive to treatment with BSO: BSO can elicit an apoptotic response, but the apoptotic process is aborted after cytochrome c release and before caspase activation, suggesting the development of an adaptive response (FASEB J., 1999, 13, 2031-2036). Here, we investigate the mechanisms of such an adaptation. We found that following BSO, U937 up-regulate Bcl-2 mRNA and protein levels, by a mechanism possibly involving NF-kappaB transcription factor; the increase in protein level is limited by a rapid decay of Bcl-2 in BSO-treated cells, suggesting that redox imbalance speeds up Bcl-2 turnover. BSO-dependent Bcl-2 up-regulation is associated with the ability to survive to BSO. Indeed, 1) its abrogation by CAPE or protein synthesis inhibition sensitizes U937 to BSO; 2) in a panel of four tumor lines, BSO-resistant (U937, HepG2, and HGB1) but not BSO-sensitive (BL41) cells can up-regulate Bcl-2 following GSH depletion; remarkably, only the latter are chemosensitized by BSO.