Recent progress in molecular medicine has provided important tools to identify antigen-specific T cells. In most cases, the approach is based on oligomeric combinations of recombinant major histocompatibility complex-peptide complexes fixed to various rigid supports available for binding by the T-cell receptor. These tools have greatly increased our insight into mechanisms of immune responses mediated by CD8+ T cells. Examples of the diverse fields of application for this technology include immunization, viral infections and oral tolerance induction.
HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.
Adenine deoxynucleosides, such as 2-chlorodeoxyadenosine (2CdA) and fludarabine, induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. We previously demonstrated that that the 5-triphosphate metabolite of 2CdA (2CdATP), similar to dATP, can cooperate with cytochrome c and apoptosis protein-activating factor-1 (APAF-1) to trigger a caspase pathway in a HeLa cell-free system. We used a fluorometry-based assay of caspase activation to extend the analysis to several other clinically relevant adenine deoxynucleotides in B-chronic lymphocytic leukemia extracts. The nucleotide-induced caspase activation displayed typical Michaelis-Menten kinetics. As estimated by the V max /K m ratios, the relative efficiencies of different nucleotides were Ara-ATP > 9-fluoro-9--D-arabinofuranosyladenine 5-triphosphate > dATP > 2CdATP > 9--D-arabinofuranosylguanine 5-triphosphate > dADP > ATP. In contrast to dADP, both ADP and its nonhydrolyzable ␣,-methylphosphonate analog were strong inhibitors of APAF-1-dependent caspase activation. The hierarchy of nucleotide activation was confirmed in a fully reconstituted system using recombinant APAF-1 and recombinant procaspase-9. These results suggest that the potency of adenine deoxynucleotides as co-factors for APAF-1-dependent caspase activation is due both to stimulation by the 5-triphosphates and lack of inhibition by the 5-diphosphates. The capacity of adenine deoxynucleoside metabolites to activate the apoptosome pathway may be an additional biochemical mechanism that plays a role in the chemotherapy of indolent lymphoproliferative diseases.
In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using highaffinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.S ignal transducer and activator of transcription 3 (STAT3) is a key element in multiple signaling pathways and is aberrantly activated in many human cancers (1, 2). STAT3 promotes cell proliferation, survival, angiogenesis, and immune-evasion (1-3). Phosphorylation at Tyr705 (pTyr705), catalyzed by Janus kinases (JAK) and other tyrosine kinases, induces STAT3 dimerization through the interaction of the SH2 domain (SH2D), nuclear accumulation, and target gene transcription (1, 3, 4). Emerging evidence indicates that STAT3 also localizes to mitochondria and controls mitochondrial functions (2, 5-7). Mitochondrial localized STAT3 (mSTAT3) is critical for survival of RAStransformed mouse embryo fibroblasts (MEF) under glucosestarvation, reflecting a specific dependency of cancer cells on mitochondria in certain conditions (6). Interestingly, mSTAT3 is prevalently phosphorylated at Ser727 (pSer727), which enhances its mitochondrial functions (5, 6). Furthermore, constitutive pSer727 is found in many human cancers and is apparently sufficient to drive tumorigenesis in various model systems (8-10).STAT3 is an attractive cancer therapeutic target because of its central role in multiple oncogenic processes and great effort has been devoted in recent years to discover STAT3 inhibitors (STAT3i) (11,12). To date, small-molecule STAT3i have shown relevant activity in preclinical models and few of them are currently investigated in clinical trials (11,(13)(14)(15)(16)(17). However, an important gap persists in our knowledge of the biological mechanisms of antitumor activity, the critical cellular processes affected, and the factors determining sensitivity of cancer cells to STAT3i, hindering further clinical development of these highly promising anticancer drugs. Indeed, great atten...
Adenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to the pro-apoptotic factor Apaf-1 and the activation of the caspase-9 and -3 pathways. In this study it is shown, by means of several assays on whole cells and isolated mitochondria, that 2-chloro-2′-deoxyadenosine (2CdA) and 2-choloro-2′-ara-fluorodeoxyadenosine (CaFdA) disrupt the integrity of mitochondria from primary chronic lymphocytic leukemia (B-CLL) cells. The nucleoside-induced damage leads to the release of the pro-apoptotic mitochondrial proteins cytochrome c and apoptosis-inducing factor. The other adenine deoxynucleosides tested displayed comparable DNA-damaging potency but did not affect mitochondrial function. Interference with mitochondrial integrity, thus, may be a factor in the potent cytotoxic effects of 2CdA and CaFdA toward nondividing lymphocytes.
A series of isoquinolin-1-ones and quinazolin-4-ones and related derivatives were prepared and evaluated for their ability to inhibit tumor necrosis factor alpha (TNFalpha) production in human peripheral blood monocytes stimulated with bacterial lipopolysaccharide (LPS). In an effort to optimize the TNFalpha inhibitory activity, a homologous series of N-alkanoic acid esters was prepared. Several electrophilic and nucleophilic substitutions were also carried out. Alkanoic acid esters of four carbons were found to be optimum for activity in both the isoquinoline and quinazoline series. Ring substituents such as fluoro, bromo, nitro, acetyl, and aminomethyl on the isoquinoline ring resulted in a significant loss of activity. Likewise, similar groups on the quinazoline ring also reduced inhibitory activity. However, the 6- and 7-aminoquinazoline derivatives, 75 and 76, were potent inhibitors, with IC(50) values in the TNFalpha in vitro assay of approximately 5 microM for each. An in vivo mouse model of pulmonary inflammation was then used to evaluate promising candidate compounds identified in the primary in vitro assay. Compound 75 was selected for further study in this inhalation model, and was found to reduce the level of TNFalpha in brochoalveolar lavage fluid of LPS-treated mice by about 50% that of control mice. Thus, compounds such as 75, which can effectively inhibit proinflammatory cytokines such as TNFalpha in clinically relevant animal models of inflammation and fibrosis, may have potential as new antiinflammatory agents. Finally, a quinazoline derivative suitable to serve as a photoaffinity radiolabeled compound was prepared to help identify the putative cellular target(s) for these TNFalpha inhibitors.
Adenine deoxynucleosides, such as 2-chloro-2-deoxyadenosine (2CdA) induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. However, it has remained puzzling why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis. The present experiments demonstrate that the 5-triphosphate metabolite of 2CdA (2CdA-5-triphosphate), similar to dATP, can cooperate with cytochrome c and Apaf-1 to activate caspase-3 in a cell free system. Chronic lymphocytic leukemia cells and normal peripheral blood lymphocytes expressed both caspase-3 and apoptotic protease activating factor 1. Incubation of the lymphocytes with 2CdA induced caspase-3 activation prior to DNA degradation and cell death. Stimulation of the caspase proteolytic cascade by 2CdA-5-triphosphate, in the context of DNA strand break formation, may provide an explanation for the potent cytotoxic effects of 2CdA toward nondividing lymphocytes.The effectiveness of cancer chemotherapy often depends upon the induction of apoptosis in malignant cells. Among antimetabolites, the 2Ј-deoxyadenosine congeners 2-chloro-2Ј-deoxyadenosine (2CdA, cladribine) and 9--D-arabinofuranosyl-2-fluoroadenine (fludarabine) have the ability to induce apoptosis in nondividing lymphocytes, at concentrations that spare other cell types (1). For this reason, the deoxyadenosine analogs have achieved an important place in the treatment of indolent lymphoid malignancies, including hairy cell leukemia, chronic lymphocytic leukemia (CLL), and low grade lymphoma (2, 3).The cytotoxicity of 2CdA depends mainly upon the selective and progressive accumulation of its 5Ј-triphosphate metabolite (2CdATP) in lymphocytes that have a high ratio of deoxycytidine kinase (EC 2.7.1.74) to cytosolic 5Ј-nucleotidase (EC 3.1.3.5), compared with other cell types (1, 4). However, why 2CdATP triggers apoptosis in non-dividing cells is unclear.Various stimuli of apoptosis lead to the activation in the cytoplasm of cysteine proteases with specificity for aspartic acid residues, referred to as caspases. The activated caspases can cleave structural proteins and enzymes necessary for the survival of both proliferating and resting cells (reviewed in refs. 5-7). In addition, caspases have been shown to activate the endonuclease responsible for the internucleosomal cleavage of genomic DNA, a hallmark of apoptosis (8,9).One important component of the caspase cascade is caspase-3, which is activated by two sequential proteolytic events that cleave the 32-kDa precursor at aspartic acid residues to generate an active heterodimer of 20-and 12-kDa subunits (10). The activation can either be autocatalytic, or occur via a caspase cascade, similar to the serine protease cascade in the blood clotting process (7). In susceptible cells, caspase activation might amplify preexisting but sublethal apoptotic signals, leading to rapid and irreversible proteolysis.Recently, Wang and coworkers established a cell free system in ...
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