PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis during ageing.

Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson's disease gene) is a pacemaker regulating PDH activity in CD4+ regulatory T cells (Treg cells). DJ-1 binds to PDHE1-ß (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Park7 (Dj-1) deletion impairs Treg survival starting in young mice and reduces Treg homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis (EAE). Dj-1 deletion also compromises differentiation of inducible Treg cells especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As Treg homeostasis is dysregulated in many complex diseases, the DJ-1-PDHB axis represents a potential target to maintain or re-establish Treg homeostasis.

A roadmap towards personalized immunology.

Big data generation and computational processing will enable medicine to evolve from a "one-size-fits-all" approach to precise patient stratification and treatment. Significant achievements using "Omics" data have been made especially in personalized oncology. However, immune cells relative to tumor cells show a much higher degree of complexity in heterogeneity, dynamics, memory-capability, plasticity and "social" interactions. There is still a long way ahead on translating our capability to identify potentially targetable personalized biomarkers into effective personalized therapy in immune-centralized diseases. Here, we discuss the recent advances and successful applications in "Omics" data utilization and network analysis on patients' samples of clinical trials and studies, as well as the major challenges and strategies towards personalized stratification and treatment for infectious or non-communicable inflammatory diseases such as autoimmune diseases or allergies. We provide a roadmap and highlight experimental, clinical, computational analysis, data management, ethical and regulatory issues to accelerate the implementation of personalized immunology.

Isolation of an HIV-1 neutralizing peptide mimicking the CXCR4 and CCR5 surface from the heavy-chain complementary determining region 3 repertoire of a viremic controller.

OBJECTIVES: The recent identification of neutralizing antibodies able to prevent viral rebound reemphasized the interest in humoral immune responses to control HIV-1 infection. In this study, we characterized HIV-1-inhibiting sequences from heavy-chain complementary determining region 3 (HCDR3) repertoires of a viremic controller. DESIGN AND METHODS: IgM and IgG-derived HCDR3 repertoires of a viremic controller presenting plasma-neutralizing activity and characterized by over 20 years of infection with a stable CD4 T-cell count were displayed on filamentous phage to identify HCDR3 repertoire-derived peptides inhibiting HIV-1 entry. RESULTS: Screening of phage libraries against recombinant gp120 led to the identification of an HCDR3-derived peptide sequence (LRTV-1) displaying antiviral properties against both X4 and R5 viruses. The interaction of LRTV-1 with gp120 was enhanced upon CD4 binding and sequence comparison revealed homology between LRTV-1 and the second extracellular loop of C-X-C chemokine receptor type 4 (CXCR4) (11/23) and the N-terminus of C-C chemokine receptor type 5 (CCR5) (7/23). Alanine scanning experiments identified different clusters of residues critical for interaction with the viral envelope protein. CONCLUSIONS: LRTV-1 peptide is to date the smallest human HCDR3 repertoire-derived peptide identified by phage display inhibiting HIV entry of R5 and X4 viruses. This peptide recognizes a CD4-dependent gp120 epitope critical for coreceptor binding and mimics the surface of CXCR4 and CCR5. Our data emphasize the potential of human HCDR3 immune repertoires as sources of small biologically active peptides for HIV cure.

Phages and HIV-1: from display to interplay.

The complex hide-and-seek game between HIV-1 and the host immune system has impaired the development of an efficient vaccine. In addition, the high variability of the virus impedes the long-term control of viral replication by small antiviral drugs. For more than 20 years, phage display technology has been intensively used in the field of HIV-1 to explore the epitope landscape recognized by monoclonal and polyclonal HIV-1-specific antibodies, thereby providing precious data about immunodominant and neutralizing epitopes. In parallel, biopanning experiments with various combinatorial or antibody fragment libraries were conducted on viral targets as well as host receptors to identify HIV-1 inhibitors. Besides these applications, phage display technology has been applied to characterize the enzymatic specificity of the HIV-1 protease. Phage particles also represent valuable alternative carriers displaying various HIV-1 antigens to the immune system and eliciting antiviral responses. This review presents and summarizes the different studies conducted with regard to the nature of phage libraries, target display mode and biopanning procedures.

Chemopreventive and therapeutic effects of curcumin.

Chemoprevention is a promising anti-cancer approach with reduced secondary effects in comparison to classical chemotherapy. Curcumin, one of the most studied chemopreventive agents, is a natural compound extracted from Curcuma longa L. that allows suppression, retardation or inversion of carcinogenesis. Curcumin is also described as an anti-tumoral, anti-oxidant and anti-inflammatory agent capable of inducing apoptosis in numerous cellular systems. In this review, we describe both properties and mode of action of curcumin on carcinogenesis, gene expression mechanisms and drug metabolism.

Expression of glutathione S-transferase P1-1 in leukemic cells is regulated by inducible AP-1 binding.

Glutathione S-transferases (GST) are involved in cellular protection against xenobiotics, oxidative stress as well as in resistance against chemotherapeutic compounds such as doxorubicin. Levels of human placental type GSTP1-1 are known to be increased in many tumors and hematopoietic diseases. In this work, we compare transcriptional mechanisms in cells that express or not GSTP1-1. Transient transfection assays are used to show that different GST-promoter reporter constructs generate cell-type specific levels of luciferase activity. In expressing cells, transcriptional activity is strongly dependent on AP-1 binding elements within the -65 to -75 bp region of the GSTP1 gene as shown by site-directed mutagenesis. Electrophoretic mobility shift assays show that DNA binding activity is exclusively observed in GSTP1-1-expressing cells and is increased after stimulation with hydrogen peroxide, TPA, tert-butylhydroquinone and doxorubicin. Non-expressing cells present neither constitutive nor inducible AP-1 binding. Taken together, our results provide evidence for the induction of the GSTP1 gene via AP-1 binding activity in leukemia cells and contribute to a better understanding of the molecular events regulating genes involved in drug resistance mechanisms.

Effect of chemopreventive agents on glutathione S-transferase P1-1 gene expression mechanisms via activating protein 1 and nuclear factor kappaB inhibition.

Glutathione S-transferase P1-1 (GSTP1-1) is a phase II drug metabolism enzyme implicated in carcinogenesis and development of resistance to anti-cancer drugs. It was previously shown that both activating protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) are involved in its regulation. In the present study we examined the inhibitory effect of several chemopreventive agents on the tumor necrosis factor (TNF) alpha- or 12-O-tetradecanoylphorbol 13 acetate (TPA)-induced promoter activity of GSTP1-1, as demonstrated by transient transfection experiments in K562 and U937 leukemia cells. Our results provide evidence for a differential effect of chemopreventive agents such as beta-lapachone, emodin, sanguinarine and capsaicin, which significantly inhibit reporter gene expression as well as TNFalpha- and TPA-induced binding of AP-1 and NF-kappaB, whereas trans-anethole and silymarin do not produce any inhibitory effect. Our results demonstrate the ability of selected chemopreventive agents to decrease GSTP1-1 gene expression mechanisms and could thus contribute to reduce the incidence of glutathione related drug resistance in human leukemia.

Increased glutathione S-transferase P1-1 expression by mRNA stabilization in hemin-induced differentiation of K562 cells.

GSTP1-1 gene expression mechanisms were investigated in hemin-induced erythroid differentiation of K562 cells. Hemoglobin production during differentiation was followed by a significant increase in GSTP1-1 mRNA (1.7-fold, P < 0.01) and protein (1.2-fold, P < 0.01) after 4 days of induction. This increase in mRNA production was not due to transcriptional up-regulation by GATA-1 previously shown to regulate GSTP1-1 during erythroid and megakaryocytic differentiation. Moreover, a drastic decrease in differentiation-specific GATA-1 mRNA expression was correlated to a reduction in GATA-1 promoter binding activity. Neither AP-1 nor NF-kappaB transcription factor binding activities could provide an explanation to the GSTP1-1 mRNA overexpression in hemin-treated cells. GSTP1-1 mRNA stability analysis using actinomycin D as an inhibitor of mRNA neosynthesis showed that mRNA half-life was doubled in hemin-induced erythroid differentiation of K562 cells. These results allow us to add stabilization of GSTP1-1 mRNA as a novel regulatory mechanism during hemin-mediated differentiation of K562 cells.

Regulation of glutathione S-transferase P1-1 gene expression by NF-kappaB in tumor necrosis factor alpha-treated K562 leukemia cells.

Glutathione S-transferases (GSTs) play an important role in the protection of cells against xenobiotics and lipid hydroperoxides generated by oxidative stress. In human, the GSTP1-1 expression is commonly increased in many tumors and involved in the development of antineoplastic drug resistance. Reactive oxygen species are released at inflammation sites and oxidative stress conditions enhance the expression of genes encoding antioxidant enzymes such as GSTs. Here we investigated the regulation of the GSTP1-1 gene expression in the K562 cell line by nuclear factor kappaB (NF-kappaB) and the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). By studying GSTP1-1 mRNA expression and NF-kappaB/GSTP1-1 promoter interactions, we showed the implication of NF-kappaB in the GSTP1-1 gene expression and we described a new specific TNFalpha-inducible NF-kappaB binding site upstream of the minimal promoter. Moreover, TNFalpha treatment as well as cotransfection of NF-kappaB signaling pathway intermediates induced an activation of the GSTP1-1 gene promoter in K562 cells. Site-directed mutagenesis of the NF-kappaB site strongly inhibited TNFalpha- and NF-kappaBp65-induced promoter activation. Altogether, we showed that a sequence located at -323/-314 within the GSTP1-1 promoter bound NF-kappaB p50/65 and p65/p65 dimers and that this kappaB site was involved in the regulation of the gene by TNFalpha.

A beginner's guide to NF-kappaB signaling pathways.

Nuclear factor kappaB (NF-kappaB) belongs to a family of heterodimeric transcription factors that play a key role in inflammatory and stress responses as well as in tumor cell resistance to apoptosis. These effects are due to the NF-kappaB-dependent transcription of many proinflammatory and antiapoptotic genes, whose products ensure various cell responses to environmental conditions. The signal transduction pathways leading to NF-kappaB activation are well characterized, and the different steps implicated in these pathways involve proteins that could constitute targets for NF-kappaB inhibition. Several inhibitors aiming to prevent NF-kappaB activity and thus the transcription of target genes are studied, and a few compounds seem particularly promising. We try here to summarize the advantages that can issue from various studies on NF-kappaB.

Curcumin stability and its effect on glutathione S-transferase P1-1 mRNA expression in K562 cells.

To investigate the stability of curcumin in physiological media, the absorption variation of a curcumin solution was measured in 0.1% and 10% FCS. Under daylight conditions, curcumin degraded very rapidly in 0.1% FCS and was found to be more stable in higher serum concentrations. Under dark conditions, almost no decomposition could be observed after 2 h, whether the measurements were performed in 0.1% or 10% FCS. Furthermore, depending on the medium concentration, differential glutathione S-transferase P1-1 mRNA expression could be observed in K562 cells after incubation with curcumin. Indeed, incubation in 0.1% FCS led to a decrease of mRNA expression, whereas incubation in 10% FCS induced an increase of mRNA production.

Expression of glutathione S-transferase P1-1 in differentiating K562: role of GATA-1.

Glutathione S-transferase P1-1 (GSTP1-1) conjugates glutathione to electrophilic compounds and its expression is correlated to chemotherapeutic drug resistance. Results show that GSTP1-1 mRNA as well as protein expressions are increased during Aclarubicin (Acla)- and Doxorubicin (Dox)-induced erythroid differentiation of human K562 cells. In contrast, during megakaryocytic differentiation by 12-O-tetradecanoyl phorbol 13-acetate (TPA), GSTP1-1 expression decreased at both mRNA and protein levels. In order to clarify the molecular mechanisms leading to these variations, we identified a GATA sequence located at -1208 relative to the transcriptional start site of the GSTP1-1 promoter. By gel shift, competition, and supershift analyses we show here the specificity of the GATA-1 binding regulated by both anthracyclines and TPA. Altogether, these results demonstrate for the first time the implication of GATA-1 in differentiation-specific variations of GSTP1-1 expression.

Induction of apoptosis by curcumin: mediation by glutathione S-transferase P1-1 inhibition.

Expression of glutathione S-transferase P1-1 (GSTP1-1) is correlated to carcinogenesis and resistance of cancer cells against chemotherapeutic agents. Curcumin, a natural compound extracted from Curcuma longa, has shown strong antioxidant and anticancer properties and also the ability to regulate a wide variety of genes that require activating protein 1 and nuclear factor kappaB (NF-kappaB) activation. In the present study, we examined the inhibitory effect of curcumin on the expression of GSTP1-1 mRNA as well as protein, and we correlated this inhibition with the apoptotic effect of curcumin on K562 leukemia cells. Curcumin efficiently inhibited the tumour necrosis factor alpha- and phorbol ester-induced binding of AP-1 and NF-kappaB transcription factors to sites located on the GSTP1-1 gene promoter. TNFalpha-induced GSTP1-1 promoter activity was also inhibited by curcumin as shown by reporter gene assay. In parallel, curcumin induced pro-caspases 8 and 9 as well as poly ADP ribose polymerase cleavage and thus leading to apoptosis in K562 cells. Our results overall add a novel role for curcumin as this chemoprotective compound could contribute to induce apoptosis by its ability to inhibit the GSTP1-1 expression at the level of transcription.

An introduction to the molecular mechanisms of apoptosis.

Apoptosis is a type of cell death that has been observed and studied for more than a century. The process of apoptosis was described as "programmed cell death" in 1964, and the term apoptosis, from a Greek word meaning "to fall away from" and describing the fall of dead leaves from trees in autumn, was only coined in 1972. During the last 30 years, this type of cell death has been extensively investigated and the molecular mechanisms underlying this cell suicide well characterized. Apoptosis is a physiological phenomenon necessary to tissue and body genesis and homeostasis, but defects in its regulation may cause numerous diseases, including cancer. Investigating the mechanisms of apoptosis is thus important to discover new cellular regulators that could be potential targets for new death-inducing drugs.

Identification of cytokine-induced nuclear factor-kappaB target genes in ovarian and breast cancer cells.

NF-kappaB is a pleiotropic transcription factor controlling the expression of many genes and viruses. NF-kappaB plays a role in immune response, cellular adhesion or acute phase response. It also inhibits apoptosis and favors cancer cell survival. We studied the expression of genes controlled by NF-kappaB in ovarian and breast adenocarcinoma cancer cells. We stably transfected OVCAR-3 and MCF7 A/Z cells with an expression vector coding for the mutated inhibitor IkappaBalpha, which sequesters NF-kappaB in the cytoplasm. We stimulated control and IkappaBalpha expressing cells with IL-1beta or TNF-alpha and extracted the RNA, which was reverse-transcribed and hybridized to DNA microarrays. Several of the genes identified were not known as NF-kappaB target genes. Among them, we confirmed the differential expression of ephrin-A1 and caveolin-1 by quantitative real-time polymerase chain reaction. Our results showed an NF-kappaB-dependent induction of ephrin-A1 and caveolin-1 mRNAs after stimulation with TNF-alpha and IL-1beta, confirming that NF-kappaB controls target genes implied in tumor angiogenesis and cell transformation.

NF-kappaB-dependent MnSOD expression protects adenocarcinoma cells from TNF-alpha-induced apoptosis.

NF-kappaB is known to exert a cytoprotective action against TNF-alpha-induced apoptosis. To study the role of NF-kappaB in various TNF-alpha-treated epithelial cell lines, we generated stable transfectants overexpressing a mutated unresponsive form of the IkappaBalpha inhibitor (MT cells). As NF-kappaB prevented TNF-alpha-induced apoptosis in various epithelial cancer cell lines, we searched for NF-kappaB target gene products responsible for this difference of sensitivity. We observed an increased Bcl-X(L) expression level in OVCAR-3 cells compared with OVCAR-3 cells expressing a mutated IkappaBalpha inhibitor (MT cells). Induction of the antioxidant enzyme MnSOD was detected only in TNF-alpha-treated OVCAR, MCF7A/Z and HCT116 cells but not in MT cells. Moreover, reactive oxygen species were involved in TNF-alpha-induced apoptosis, as various antioxidants partially protected these cells from apoptosis. At last, transfection of the MnSOD cDNA in MT cells, which do not express this protein after TNF-alpha stimulation, partially restored resistance to TNF-alpha-induced cell death, as observed by clonogenic assays. However, transfection of the Bcl-X(L) cDNA did not induce any protective effect. Therefore, MnSOD expression is induced by NF-kappaB in epithelial cancer cells in response to TNF-alpha, and is at least partially responsible for their resistance to TNF-alpha-induced apoptosis, presumably through the clearance of death-inducing ROS.