Transfection Of Mutant Research Articles

Nuclear Transcription Factor Kappa B Downregulation Reduces Chemoresistance in Bone Marrow-derived Cells Through P-glycoprotein Modulation

Nuclear transcription factor kappa B (NF-κB) is associated with many types of refractory cancer. However, despite multiple strategies to treat cancer and novel target drugs, multidrug resistance still causes relapses. The best-characterized mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein (P-gp). Because the direct inhibition of this protein is very toxic, other methods of multidrug resistance (MDR) regulation have been proposed. The MDR-1 promoter sequence contains a κB site, which is recognized by NF-κB. The aim of this work was to characterize whether NF-κB modulation changes the response of bone marrow-derived cells (BMDCs) to chemotherapy. We exposed BMDCs to etoposide and doxorubicin, two of the most used antineoplastic drugs. BMDCs presented high tolerance to these drugs, which correlated with high intrinsic P-gp activity and strong protein expression of NF-κB. To determine the mechanism behind the poor sensitivity of BMDCs to chemotherapy, we blocked the activity of the heterodimer protein NF-κB using the pharmacological inhibitor Bay 11-7085 and through the transfection of an adenovirus negative mutant of I kappa B alpha. The multidrug resistance phenotype of BMDCs was reversed by inhibiting the NF-κB pathway, and this change was accompanied by a decrease in P-gp activity. NF-κB is a possible target for improving the antineoplastic response.

Melanoma antigen-D2: A nucleolar protein undergoing delocalization during cell cycle and after cellular stress

Melanoma antigen D2 (MAGE-D2) is recognized as a cancer diagnostic marker; however, it has poorly characterized functions. Here, we established its intracellular localization and shuttling during cell cycle progression and in response to cellular stress. In normal conditions, MAGE-D2 is present in the cytoplasm, nucleoplasm, and nucleoli. Within the latter, MAGE-D2 is mostly found in the granular and the dense fibrillar components, and it interacts with nucleolin. Transfection of MAGE-D2 deletion mutants demonstrated that Δ203–254 leads to confinement of MAGE-D2 to the cytoplasm, while Δ248–254 prevents its accumulation in nucleoli but still allows its presence in the nucleoplasm. Consequently, this short sequence belongs to a nucleolar localization signal. MAGE-D2 deletion does not alter the nucleolar organization or rRNA levels. However, its intracellular localization varies with the cell cycle in a different kinetic than nucleolin. After genotoxic and nucleolar stresses, MAGE-D2 is excluded from nucleoli and concentrates in the nucleoplasm. We demonstrated that its camptothecin-related delocalization results from two distinct events: a rapid nucleolar release and a slower phospho-ERK-dependent cytoplasm to nucleoplasm translocation, which results from an increased flux from the cytoplasm to nucleoplasm. In conclusion, MAGE-D2 is a dynamic protein whose shuttling properties could suggest a role in cell cycle regulation.

Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats.

Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine.

Analysis of 54 Follicular Lymphomas By Whole Exome Sequencing Identifies Multiple Novel Recurrently Mutated Pathways

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. FL carries multiple recurrently mutated genes that are under active investigation. However, due to the relatively small number of published sequenced cases, knowledge regarding the coding genome in FL is still evolving.Methods: To further our understanding of the genetic basis of FL, we used solution exon capture of sheared and processed genomic DNA isolated from highly purified light chain restricted B-cells and paired CD3+ T-cells from 54 FL cases for paired-end massively parallel sequencing (WES). Data were subsequently analyzed using bioinformatics pipelines including the variant callers MuTect v.1.1.4, Strelka v.1.0.13, and VarScan2 v.2.3.7. Candidate somatically acquired gene mutations with variant allele frequencies (VAFs) >0.15 were confirmed using Sanger sequencing. Selected mutations were validated in an expansion cohort of 120 FL.Results: We identified heterozygous missense mutations in the mTOR regulator RRAGC in 10% of FL. The RRAGC mutations targeted multiple hotspot residues (amino acid 115, 118 and 119). RRAGC forms heterodimers with either RRAGA or RRAGB that under conditions of amino acid sufficiency facilitate recruitment of mTOR through the raptor subunit to lysosomal membranes. At the lysosomal surface, multiple protein complexes, each containing various proteins regulate mTOR activation through RHEB.To gain insights into the functional consequences of RRAGC mutations, we performed 3-dimensional modeling of FL-associated RRAGC mutations and located the mutations into relatively close proximity to the RRAGC GTP/GDP binding site. Energy calculations did not identify strong effects of mutated amino acid residues on the binding of GTP/GDP to RRAGC.We performed studies of the effects of RRAGC mutants on mTOR activity as measured through S6-kinase phosphorylation. In transient transfection systems (293T and HELA) achieving expression slightly above endogenous RRAGC levels, performed under conditions of leucine starvation or sufficiency, we did not identify differences in baseline mTOR activation. In stably transfected 293T cell lines (expressing RRAGB and RRAGC proteins above endogenous levels), that were starved for leucine for 1 hour, we detected modestly elevated p-S6K levels in RRAGC mutant versus wild type transfectants, suggesting a mild intrinsic activation phenotype of RRAGC mutations. Experiments in lentivirally-transfected lymphoma cell lines, including RRAGC binding studies to raptor and folliculin (a RRAGC regulator) are in progress and will be updated at the meeting. Curiously, we did not identify mutations in the other three small GTP binding proteins that are part of the same amino acid sensing pathway (RRAGA, RRAGB or RRAGD), potentially pointing to a unique advantage conferred by RRAGC mutants on FL B cells.We identified additional mutations (combined ~15%) in other mTOR components linked to lysosomal amino acid sensing, including recurrent mutations in the v-ATPase subunit ATP6V1B2 and the accessory subunit ATP6VAP1. The mutations in RRAGC and v-ATPase together highlight a previously unidentified role of the amino acid sensing pathway that regulates mTOR in FL pathogenesis.We have discovered a high frequency of mutations (40%) in the surrogate light chain gene IGLL5 in FL, a critical component of the pre-B-cell receptor. Mutations sharply cluster in the N-terminal 70 amino acid of IGLL5, a region known as the non-Ig domain of IGLL5. The non-Ig domain of IGLL5 has been implicated in influencing pre-B-cell receptor signaling and receptor surface expression as well as interaction with extracellular ligands. The mutational data suggest an unexpected role of IGLL5 in the pathogenesis of FL and work is in progress studying IGLL5 expression in primary FL samples.Conclusion: This large WES study of 54 FL identifies novel recurrently mutated genes and pathways in FL, including frequent mutations in genes involved in amino acid signaling to mTOR (RRAGC and v-ATPase) as well as pre-B-cell receptor signaling (the surrogate light chain gene IGLL5) and multiple other novel recurrently mutated genes that will be updated at the meeting. These data substantially broaden our understanding of the genetic basis of FL and provide clues to therapeutically targeting specific pathways in FL. DisclosuresMalek:Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership; Janssen Pharmaceuticals: Research Funding.

Sphingosine-1-phosphate receptor 1 transmits estrogens’ effects in endothelial cells

We have previously reported that the steroid hormone estrogens stimulate activation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) receptors in breast cancer cells. Both estrogens and S1P are potent biological modulators of endothelial function in vasculature able to activate multiple effectors, including endothelial nitric oxide synthase (eNOS). In this study we report that treatment of endothelial cells (ECs) with 17β-estradiol (E2) resulted in a rapid, transient, and dose-dependent increase in SphK activity and increased S1P production. The effect was not reproduced by the inactive E2 analogue 17α-E2. Expression of the dominant-negative mutant SphK1G82D or transfection of SphK1-targeted siRNA in ECs caused not only a defect in SphK activation by E2, but also a significant inhibition of E2-induced activation of Akt/eNOS. Furthermore, E2 treatment induced internalization of plasma membrane S1P1 receptor, accompanied with an increase in the amount of cytosolic S1P1. By down-regulating S1P1 receptor expression, the S1P1-specific antisense oligonucleotides significantly inhibited E2-induced activation of Akt/eNOS in ECs. E2-induced EC migration and tube formation were also inhibited by S1P1 down-regulation. Thus, the findings indicate an important role of the SphK1/S1P1 pathway in mediating estrogen signaling and its actions in vasculature.

Dexamethasone inhibits chondrocyte differentiation by suppression of Wnt/β-catenin signaling in the chondrogenic cell line ATDC5.

Glucocorticoids (GCs) regulate proliferation and differentiation in cultured mesenchymal cells through the modulation of various molecules. However, the relationship between growth factor signaling and GCs in differentiating chondrocytes has not been elucidated. In this study, we examined the effects of Wnt/β-catenin signaling on chondrocyte differentiation and the effects of a GC analogue, dexamethasone (Dex), on Wnt/β-catenin signaling activity by using a chondrocyte progenitor cell line ATDC5. Western blot analysis and TCF/LEF-optimized promoter EGFP (TOPEGFP) assay showed that both β-catenin protein levels and TCF/LEF transcription were up-regulated during insulin-transferrin-sodium selenite (ITS)-induced chondrogenic differentiation. Morphological analysis showed that TCF/LEF transcription activity was most prominent in cartilage nodule-like structures. Furthermore, a β-catenin mutant with constitutive transcriptional activity (ΔN90) showed increased Alcian blue staining intensity and mRNA expression of Sox9, Col2a, aggrecan, Col10, and alkaline phosphatase, even in the absence of ITS stimulation. In contrast, Dex suppressed formation of ITS-induced cartilage nodule-like structures, TCF/LEF-mediated transcription, and β-catenin protein levels. Real-time PCR analysis showed that Dex increased the mRNA expression levels of secreted frizzled-related protein 1 (sFRP1) and Axin2. Furthermore, treatment with a sFRP1 inhibitor or the ΔN90 β-catenin mutant transfection attenuated Dex-induced suppression of cartilage matrix production by increasing Sox9 mRNA levels. These results suggest that Dex inhibits chondrocyte differentiation via down-regulation of Wnt/β-catenin signaling, which promotes chondrocyte differentiation in ATDC5 cells.

Noncanonical STAT3 activation regulates excess TGF-β1 and collagen I expression in muscle of stricturing Crohn's disease.

Increased TGF-β1 and TGF-β1-dependent Collagen I production in intestinal mesenchymal cells result in fibrosis in patients with Montreal B2 fibrostenotic Crohn's disease. Numerous cytokines, including IL-6, are produced by activated mesenchymal cells themselves and activate STAT3. The aim of the current study was to determine the mechanisms by which STAT-3 activation might result in intestinal fibrosis. Cytokine levels were measured by ELISA. STAT3 and suppressor of cytokine signaling 3 protein levels were measured by immunoblot, STAT3-TGFB1 DNA-binding activity by chromatin immunoprecipitation, and TGFB1 transcriptional activity by luciferase reporter assay. TGF-β1 (TGFB1), Collagen1α1, and connective tissue growth factor (CTGF) gene expression was measured by quantitative RT-PCR. The role of STAT3 activation was determined using STAT3 inhibitor, Stattic, and by transfection of STAT3 mutants. Autocrine production of cytokines was increased in muscle cells of B2 phenotype patients from strictures and normal intestine in the same patient and compared with other Crohn's phenotypes, ulcerative colitis, and non-Crohn's patients. A unique pattern of STAT3 phosphorylation emerged: high STAT3(S727) and low STAT3(Y705) in strictures and the opposite in unaffected intestine. TGFB1 transcriptional activity was regulated by phospho-STAT3(S727) and was decreased by Stattic or dominant-negative STAT3(S727A). TGF-β1, COL1A1, and CTGF expression was inhibited by Stattic or dominant-negative STAT3(S727A). Treatment of normal muscle cells with IL-6 or expression of constitutively active STAT3(S727E) phenocopied muscle cells from strictured intestine. Neutralization of autocrine IL-6 reversed STAT3 phosphorylation and normalized expression of TGF-β1 in strictured intestinal muscle. The ability of Stattic to improve development of fibrosis was confirmed in mice with 2,4,6-trinitrobenzenesulfonic acid-induced colitis. We observed a unique phospho-STAT3(S727) response in patients with Montreal B2 Crohn's disease, particularly in response to IL-6 leading to increased TGF-β1, collagen, and CTGF production in ileal strictures.

Spry2 is a novel therapeutic target for periodontal tissue regeneration through fibroblast growth factor receptor signaling and epidermal growth factor signaling

Sprouty2 (Spry2) inhibits the activation of the extracellular signal-regulated kinase (ERK) pathway via receptor tyrosine kinase signaling. In a recent paper published in Journal of Cellular Biochemistry, we demonstrated that transfection of a dominant-negative mutant of Spry2 enhanced fibroblast growth factor (FGF)- and epidermal growth factor (EGF)-induced ERK activation in osteoblasts. In contrast, it decreased their activation in gingival epithelial cells. Consistent with these observations, the sequestration of Spry2 increased osteoblast proliferation by FGFR and EGFR stimulation, whereas it decreased gingival epithelial cell proliferation via the ubiquitination and degradation of EGF receptors (EGFR). In addition, reduction of Spry2 activity upregulated Runx2 expression and downregulated Twist, a negative regulator of Runx2 through FGFR and EGFR signaling, resulting in enhanced osteoblastogenesis in osteoblasts. Furthermore, we also found that suppression of Spry2 upregulated cell proliferation and migration in human periodontal ligament cell lines when they were stimulated by both FGF and EGF, and led to a shift in macrophage polarization, exerted immunosuppressive and tissue-repairing effects in macrophages. These results suggest that the application of a Spry2 inhibitor may effectively resolve inflammation by periodontitis and allow periodontal ligament and alveolar bone to grow and block the ingrowth of gingival epithelial cells in bony defects, biologically mimicking the barrier effect seen in conventional GTR. This approach has potential for developing a new regeneration strategy.

Metabolic synthesis of clickable glutathione for chemoselective detection of glutathionylation.

Glutathionylation involves reversible protein cysteine modification that regulates the function of numerous proteins in response to redox stimuli, thereby altering cellular processes. Herein we developed a selective and versatile approach to identifying glutathionylation by using a mutant of glutathione synthetase (GS). GS wild-type catalyzes coupling of γGlu-Cys to Gly to form glutathione. We generated a GS mutant that catalyzes azido-Ala in place of Gly with high catalytic efficiency and selectivity. Transfection of this GS mutant (F152A/S151G) and incubation of azido-Ala in cells efficiently afford the azide-containing glutathione derivative, γGlu-Cys-azido-Ala. Upon H2O2 treatment, clickable glutathione allowed for selective and sensitive detection of glutathionylated proteins by Western blotting or fluorescence after click reaction with biotin-alkyne or rhodamine-alkyne. This approach affords the efficient metabolic tagging of intracellular glutathione with small clickable functionality, providing a versatile handle for characterizing glutathionylation.

BAG1 is neuroprotective in in vivo and in vitro models of Parkinson's disease.

Bcl-2-associated athanogene-1 (BAG1) is a multifunctional protein comprising co-chaperone function, increasing Hsp70 foldase activity and chaperone-dependent protein degradation of misfolded substrates, with anti-apoptotic activity. It is neuroprotective in different models of neurological diseases, like cerebral ischemia and Huntington's disease. In the context of Parkinson's disease, it has recently been shown to restore DJ-1 function in an in vitro model of hereditary Parkinson's disease. Here, we demonstrate that BAG1 overexpression in SH-SY5Y cells reduces toxicity after transfection of disease-related α-synuclein mutants. Furthermore, it protects from rotenone-induced cell death in vitro and ameliorates neuronal demise in an in vivo 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) model for Parkinson's disease after adeno-associated virus (AAV)-mediated BAG1 gene transfer into the substantia nigra in mice but showed no protective effects in an in vitro 6-hydroxidopamine model. In conclusion, we present BAG1 as a potential therapeutic target in Parkinson's disease.

Cep126 is required for pericentriolar satellite localisation to the centrosome and for primary cilium formation.

Background InformationThe centrosome is the primary microtubule-organising centre of animal cells and it has crucial roles in several fundamental cellular functions, including cell division, cell polarity, and intracellular transport. The mechanisms responsible for this are not completely understood.ResultsThe poorly characterised protein CEP126 localises to the centrosome, pericentriolar satellites and the base of the primary cilium. Suppression of CEP126 expression results in dispersion of the pericentriolar satellites and disruption of the radial organisation of the microtubules, and induces disorganisation of the mitotic spindle. Moreover, CEP126 depletion or the transfection of a CEP126 truncation mutant in hTERT-RPE-1 and IMCD3 cells impairs the formation of the primary cilium.ConclusionsWe propose that CEP126 is a regulator of microtubule organisation at the centrosome that acts through modulation of the transport of pericentriolar satellites, and consequently, of the organisation of cell structure.

Binding of the WASP/N-WASP-interacting protein WIP to actin regulates focal adhesion assembly and adhesion.

The actin cytoskeleton is essential for cell adhesion and migration, functions important for tumor invasion. In addition to binding N-WASP/WASP, WIP binds and stabilizes F-actin. WIP(-/-) fibroblasts were used to test the role of WIP in F-actin function. WIP(-/-) cells had defective focal adhesion (FA), stress fiber assembly, and adherence to substrates, functions that were restored by transduction of wild-type WIP. Protein and mRNA levels of several FA constituents regulated by the myocardin-related transcription factor (MRTF)–serum response factor (SRF) transcription factor complex were reduced in WIP(-/-) fibroblasts. The level of G-actin, which sequesters MRTF in the cytoplasm, was increased, and nuclear localization of MRTF-A and SRF was reduced, in WIP(-/-) fibroblasts. Transfection of an MRTF-A mutant that constitutively translocates to the nucleus or transfection of constitutively active SRF restored FA and stress fiber assembly. Fibroblasts from knock-in mice expressing a WIP mutant that fails to bind actin phenocopied WIP(-/-) fibroblasts. Thus, WIP is a novel regulator of FA assembly and cell adhesion.

Maintenance of chaperone-mediated autophagy activity in cultured cells expressing mutant huntingtin

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by mutant huntingtin (Htt) with an expanded polyglutamine tract. It has been reported that Htt regulates autophagy. However, it remains unclear whether mutant Htt affects chaperone-mediated autophagy (CMA). Our study aimed to investigate the effect of mutant Htt on CMA activity in cultured HEK293T cells. A HEK293T cell model of HD was produced by transient transfection of wild-type (20Q) or mutant (120Q) Htt plasmids. The effect of mutant Htt on two CMA components, lysosomal-associated membrane protein 2a (Lamp2a) and heat-shock cognate protein 70 (Hsc70), was determined by western blotting and immunofluorescent staining. We observed that mutant Htt did not significantly alter the expression of Lamp2a and Hsc70 when compared to normal Htt. These findings suggest that mutant Htt does not reduce CMA activity and that enhancing CMA activity to clear mutant Htt may be a novel strategy for the management of HD.

Modulation of angiotensin II-induced inflammatory cytokines by the Epac1-Rap1A-NHE3 pathway: implications in renal tubular pathobiology

Besides the glomerulus, the tubulointerstitium is often concomitantly affected in certain diseases, e.g., diabetic nephropathy, and activation of the renin-angiotensin system, to a certain extent, worsens its outcome because of perturbations in hemodynamics and possibly tubuloglomerular feedback. Certain studies suggest that pathobiology of the tubulointerstitium is influenced by small GTPases, e.g., Rap1. We investigated the effect of ANG II on inflammatory cytokines, while at the same time focusing on upstream effector of Rap1, i.e., Epac1, and some of the downstream tubular transport molecules, i.e., Na/H exchanger 3 (NHE3). ANG II treatment of LLC-PK1 cells decreased Rap1a GTPase activity in a time- and dose-dependent manner. ANG II treatment led to an increased membrane translocation of NHE3, which was reduced with Epac1 and PKA activators. ANG II-induced NHE3 translocation was notably reduced with the transfection of Rap1a dominant positive mutants, i.e., Rap1a-G12V or Rap1a-T35A. Transfection of cells with dominant negative Rap1a mutants, i.e., Rap1a-S17A, or Epac1 mutant, i.e., EPAC-ΔcAMP, normalized ANG II-induced translocation of NHE3. In addition, ANG II treatment led to an increased expression of inflammatory cytokines, i.e., IL-1β, IL-6, IL-8, and TNF-α, which was reduced with Rap1a-G12V or Rap1a-T35A transfection, while it reverted to previous comparable levels following transfection of Rap1a-S17A or EPAC-ΔcAMP. ANG II-induced expression of cytokines was reduced with the treatment with NHE3 inhibitor S3226 or with Epac1 and PKA activators. These data suggest that this novel Epac1-Rap1a-NHE3 pathway conceivably modulates ANG II-induced expression of inflammatory cytokines, and this information may yield the impetus for developing strategies to reduce tubulointertstitial inflammation in various renal diseases.

TRAP1 role in endoplasmic reticulum stress protection favors resistance to anthracyclins in breast carcinoma cells

Adaptation to endoplasmic reticulum (ER) stress through the upregulation of the ER chaperone BiP/Grp78 favors resistance of cancer cells to anthracyclins. We recently demonstrated that the mitochondrial HSP90 chaperone TNF receptor-associated protein 1 (TRAP1) is also localized in the ER, where it is responsible for protection from ER stress and quality control on specific mitochondrial proteins contributing to its anti-apoptotic function and the regulation of the mitochondrial apoptotic pathway. Based on the evidence that Bip/Grp78 and TRAP1 are co-upregulated in about 50% of human breast carcinomas (BCs), and considering that the expression of TRAP1 is critical in favoring resistant phenotypes to different antitumor agents, we hypothesized that ER-associated TRAP1 is also favoring resistance to anthracyclins. Indeed, anthracyclins induce ER stress in BC cells and cross-resistance between ER stress agents and anthracyclins was observed in bortezomib- and anthracyclin-resistant cells. Several lines of evidence suggest a mechanistic link between the ER-stress protecting function of TRAP1 and resistance to anthracyclins: i) ER stress- and anthracyclin-resistant cell lines are characterized by the upregulation of TRAP1; ii) TRAP1 silencing in both drug-resistant cell models restored the sensitivity to bortezomib and anthracyclins; iii) the transfection of a TRAP1 deletion mutant, whose localization is restricted to the ER, in TRAP1 KD cells protected from apoptosis induced by anthracyclins; iv) the disruption of the ER-associated TRAP1/TBP7 pathway by a TBP7 dominant negative deletion mutant re-established drug sensitivity in drug-resistant cells. This process is likely mediated by the ability of TRAP1 to modulate the PERK pathway as TRAP1 KD cells failed to induce the phosphorylation of PERK in response to anthracyclins. Moreover, the downregulation of TRAP1 in combination with ER stress agents produced high cytotoxic effects in BC cells. These results suggest that ER-associated TRAP1 plays a role in protecting tumor cells against DNA damaging agents by modulating the PERK pathway.

Characteristics of TCR/CD3 complex CD3ɛ chains of regulatory CD4+ T (Treg) lymphocytes: role in Treg differentiation in vitro and impact on Treg in vivo

Tregs are anergic CD4(+)CD25(+)Foxp3(+) T lymphocytes exerting active suppression to control immune and autoimmune responses. However, the factors in TCR recognition underlying Treg differentiation are unclear. Based on our previous data, we hypothesized that Treg TCR/CD3 antigen receptor complexes might differ from those of CD4(+)CD25(-) Tconv. Expression levels of TCR/CD3, CD3ε,ζ chains, or other molecules involved in antigen signaling and the characteristics of CD3ε chains were analyzed in thymus or spleen Treg cells from normal mice. Tregs had quantitative and qualitatively distinct TCR/CD3 complexes and CD3ε chains. They expressed significantly lower levels of the TCR/CD3 antigen receptor, CD3ε chains, TCR-ζ chain, or the CD4 coreceptor than Tconv. Levels of kinases, adaptor molecules involved in TCR signaling, and early downstream activation pathways were also lower in Tregs than in Tconv. Furthermore, TCR/CD3 complexes in Tregs were enriched in CD3ε chains conserving their N-terminal, negatively charged amino acid residues; this trait is linked to a higher activation threshold. Transfection of mutant CD3ε chains lacking these residues inhibited the differentiation of mature CD4(+)Foxp3(-) T lymphocytes into CD4(+)Foxp3(+) Tregs, and differences in CD3ε chain recognition by antibodies could be used to enrich for Tregs in vivo. Our results show quantitative and qualitative differences in the TCR/CD3 complex, supporting the hyporesponsive phenotype of Tregs concerning TCR/CD3 signals. These differences might reconcile avidity and flexible threshold models of Treg differentiation and be used to implement therapeutic approaches involving Treg manipulation.

Cytoplasmic localization and redox cysteine residue of APE1/Ref-1 are associated with its anti-inflammatory activity in cultured endothelial cells.

Apurinic/apyrimidinic endonuclease1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in base excision DNA repair and transcriptional regulation of gene expression. APE1/Ref-1 is mainly localized in the nucleus, but cytoplasmic localization has also been reported. However, the functional role of cytoplasmic APE1/Ref-1 and its redox cysteine residue are still unknown. We investigated the role of cytoplasmic APE1/Ref-1 on tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) expressions in endothelial cells. Endogenous APE1/Ref-1 was mainly observed in the nucleus, however, cytoplasmic APE1/Ref-1 was increased by TNF-α. Cytoplasmic APE1/Ref-1 expression was not blunted by cycloheximide, a protein synthesis inhibitor, suggesting cytoplasmic translocation of APE1/Ref-1. Transfection of an N-terminus deletion mutant APE1/Ref-1(29-318) inhibited TNF-α-induced VCAM-1 expression, indicating an anti-inflammatory role for APE1/Ref-1 in the cytoplasm. In contrast, redox mutant of APE1/Ref-1 (C65A/C93A) transfection led to increased TNF-α-induced VCAM-1 expression. Our findings suggest cytoplasmic APE1/Ref-1 localization and redox cysteine residues of APE1/Ref-1 are associated with its anti-inflammatory activity in endothelial cells.

Cheliensisin A Inhibits EGF-Induced Cell Transformation with Stabilization of p53 Protein Via a Hydrogen Peroxide/Chk1-Dependent Axis

Cheliensisin A (Chel A), a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been shown to induce apoptosis in human promyelocytic leukemia HL-60 cells with Bcl-2 downregulation. Yet, the potential chemopreventive effect of Chel A has not been explored. Here, we showed that Chel A treatment with various concentrations (0.5, 1.0, 2.0, and 4.0 μmol/L) for 3 weeks could dramatically inhibit EGF-induced cell transformation in Cl41 cells (IC50 ∼2.0 μmol/L). Also, coincubation of Cl41 cells with Chel A (2.0 and 4.0 μmol/L) for 48 hours could induce cell apoptosis in a caspase-3-dependent manner. Mechanically, Chel A treatment could result in increased p53 phosphorylation at Ser15 and elevated p53 total protein expression. Moreover, we found that p53 induction by Chel A was regulated at the protein degradation level, but not at either the transcription or the mRNA level. Further studies showed that p53 stabilization by Chel A was mediated via induction of phosphorylation and activation of Chk1 protein at Ser345. This notion was substantiated by the results that transfection of dominant negative mutant of Chk1 (GFP-Chk1 D130A) significantly attenuated the p53 protein expression, cell apoptosis, and inhibition of cell transformation by Chel A. Finally, increased hydrogen peroxide was found to mediate Chk1 phosphorylation at Ser345, p53 protein induction, cell apoptotic induction, and transformation inhibition following Chel A treatment. Taken together, our studies identify Chel A as a chemopreventive agent with the understanding of the molecular mechanisms involved.

α-Lipoic acid prevents p53 degradation in colon cancer cells by blocking NF-κB induction of RPS6KA4

α-Lipoic acid (α-LA) is a biogenic antioxidant that has been used successfully in the treatment of diabetic polyneuropathy and its application to many oxidative stress-associated chronic diseases has increased. In this study, we investigated the effect of α-LA on colorectal cancer cell growth and its underlying mechanism. α-LA treatment resulted in a marked reduction in the growth of HCT116 colon cancer cells in a dose-dependent manner through the G1 arrest of the cell cycle and apoptosis induction. α-LA treatment significantly increased tumor cell response to various apoptotic stresses, such as etoposide, 5-fluorouracil, UVC, γ-irradiation, hypoxia, and tumor necrosis factor α (TNFα). Interestingly, α-LA increased p53 protein stability and its apoptosis-enhancing effect was more evident in wild-type p53-carrying cells compared with p53-deficient cells, suggesting that the proapoptotic role of α-LA is associated with its p53-stabilizing function. On the basis of our microarray data showing α-LA downregulation of the ribosomal protein p90S6K (RPS6KA4), which has been reported to inhibit p53 function, we tested whether α-LA regulation of RPS6KA4 is associated with its proapoptotic function. α-LA treatment led to a marked reduction in the RPS6KA4 mRNA level in multiple colorectal cancer cells and restoration of RPS6KA4 expression markedly attenuated α-LA induction of apoptosis in a p53-dependent manner. In addition, we observed that RPS6KA4 expression is activated by TNFα whereas both basal and TNFα induction of RPS6KA4 are inhibited by the nuclear factor-κB (NF-κB) inhibitor BAY11-7082 or transfection of a dominant-negative mutant of NF-κB, indicating that NF-κB plays a crucial role in RPS6KA4 gene expression. Finally, we found that α-LA exerts an inhibitory effect on the nuclear translocation of NF-κB triggered by TNFα. Collectively, our study shows that α-LA suppresses colorectal tumor cell growth at least partially by preventing RPS6KA4-mediated p53 inhibition through blockade of NF-κB signaling.

Resistance to paclitxel in breast carcinoma cells requires a quality control of mitochondrial antiapoptotic proteins by TRAP1

TRAP1 is a mitochondrial antiapoptotic protein up-regulated in several human malignancies. However, recent evidences suggest that TRAP1 is also localized in the endoplasmic reticulum (ER) where it is involved in ER stress protection and protein quality control of tumor cells. Based on the mechanistic link between ER stress, protection from apoptosis and drug resistance, we questioned whether these novel roles of TRAP1 are relevant for its antiapoptotic function. Here, we show for the first time that: i) TRAP1 expression is increased in about 50% of human breast carcinomas (BC), and ii) the ER stress protecting activity of TRAP1 is conserved in human tumors since TRAP1 is co-upregulated with the ER stress marker, BiP/Grp78. Notably, ER-associated TRAP1 modulates mitochondrial apoptosis by exerting a quality control on 18 kDa Sorcin, a TRAP1 mitochondrial client protein involved in TRAP1 cytoprotective pathway. Furthermore, this TRAP1 function is relevant in favoring resistance to paclitaxel, a microtubule stabilizing/ER stress inducer agent widely used in BC therapy. Indeed, the transfection of a TRAP1 deletion mutant, whose localization is restricted to the ER, in shTRAP1 cells enhances the expression of mitochondrial Sorcin and protects from apoptosis induced by ER stress agents and paclitaxel. Furthermore, BC cells adapted to paclitaxel or ER stress inducers share common resistance mechanisms: both cell models exhibit cross-resistance to single agents and the inhibition of TRAP1 by siRNAs or gamitrinib, a mitochondria-directed HSP90 family inhibitor, in paclitaxel-resistant cells rescues the sensitivity to paclitaxel. These results support the hypothesis that ER-associated TRAP1 is responsible for an extramitochondrial control of apoptosis and, therefore, an interference of ER stress adaptation through TRAP1 inhibition outside of mitochondria may be considered a further compartment-specific molecular approach to rescue drug-resistance.