Yves Labelle

The EWS/TEC fusion protein encoded by the t(9;22) chromosomal translocation in human chondrosarcomas is a highly potent transcriptional activator

The EWS/TEC gene fusion generated by the t(9;22) chromosomal translocation found in extraskeletal myxoid chondrosarcomas encodes a fusion protein containing the amino-terminal domain of the EWS protein fused to the whole coding sequence of the orphan nuclear receptor TEC. We have compared the DNA-binding and transcriptional activation properties of various TEC isoforms and the corresponding EWS/TEC fusion proteins. Band-shift experiments show that the full-length TEC receptor can efficiently bind the NGFI-B Response Element (NBRE), whereas an isoform lacking the entire carboxyl-terminal domain of the receptor binds much less efficiently the NBRE. Addition of the amino-terminal domain of EWS to either isoforms does not alter significantly their DNA-binding properties to the NBRE. Co-transfection experiments of COS cells and human chondrocytes indicate that whereas TEC moderately activates transcription from a NBRE-containing promoter, the corresponding EWS/TEC fusion protein is a highly potent transcriptional activator of the same promoter, being approximately 270-fold more active than the native receptor. EWS/TEC may thus exert its oncogenic potential in chrondrosarcomas by activating the transcription of target genes involved in cell proliferation.

p21WAF1/CIP1 Upregulation through the Stress Granule-Associated Protein CUGBP1 Confers Resistance to Bortezomib-Mediated Apoptosis

Background p21WAF1/CIP1 is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown. Methodology/Principal Findings We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis. Conclusions/Significance We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1.

Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase.

Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

Triphasic expression of interleukin-1 receptor type I in human endometrium throughout the menstrual cycle of fertile women and women with unexplained infertility.

OBJECTIVE To evaluate expression of interleukin-1 receptor type I in the endometrium of fertile women throughout the menstrual cycle and to investigate whether unexplained infertility may be associated with abnormal expression of IL-1 receptor type I. DESIGN Retrospective study using immunohistochemical technique, Western blot assay, and reverse transcription polymerase chain reaction. SETTING Gynecology clinic and human reproduction research laboratory. PATIENT(S) 39 fertile women and 25 women with unexplained infertility. INTERVENTION(S) Endometrial biopsy of samples obtained at laparoscopy. MAIN OUTCOME MEASURE(S) Immunostaining intensity, molecular weight, and messenger RNA levels of IL-1 receptor type I. RESULT(S) Immunostaining showed that two threshold days (13 and 22) separate the menstrual cycle into three distinct periods of IL-1 receptor type I expression, both in epithelial and stromal cells. Results of Western blot assay and reverse transcription polymerase chain reaction confirmed the immunohistochemical data. Statistical analyses showed that the pattern of IL-1 receptor type I expression was similar in women with unexplained infertility and fertile women. CONCLUSION(S) IL-1 receptor type I exhibits three distinct levels of expression throughout the menstrual cycle in the endometrium of fertile women, suggesting different physiologic roles of the receptor within the cycle. However, IL-1 receptor type I does not seem to be involved in unexplained infertility.

The EWSR1/NR4A3 fusion protein of extraskeletal myxoid chondrosarcoma activates the PPARG nuclear receptor gene

The NR4A3 nuclear receptor is implicated in the development of extraskeletal myxoid chondrosarcoma (EMC), primitive sarcoma unrelated to conventional chondrosarcomas, through a specific fusion with EWSR1 resulting in an aberrant fusion protein that is thought to disrupt the transcriptional regulation of specific target genes. We performed an expression microarray analysis of EMC tumours expressing the EWSR1/NR4A3 fusion protein, comparing their expression profiles to those of other sarcoma types. We thereby identified a set of genes significantly overexpressed in EMC relative to other sarcomas, including PPARG and NDRG2. Western blot or immunohistochemical analyses confirm that PPARG and NDRG2 are expressed in tumours positive for EWSR1/NR4A3. Bioinformatic analysis identified a DNA response element for EWSR1/NR4A3 in the PPARG promoter, and band‐shift experiments and transient transfections indicate that EWSR1/NR4A3 can activate transcription through this element. Western blots further show that an isoform of the native NR4A3 receptor lacking the C‐terminal domain is very highly expressed in tumours positive for EWSR1/NR4A3, and co‐transfections of this isoform along with EWSR1/NR4A3 indicate that it may negatively regulate the activity of the fusion protein on the PPARG promoter. These results suggest that the overall expression of PPARG in EMC may be regulated in part by the balance between EWSR1/NR4A3 and NR4A3, and that PPARG may play a crucial role in the development of these tumours. The specific up‐regulation of PPARG by EWSR1/NR4A3 may also have potential therapeutic implications. Copyright

Involvement of nuclear factor-κB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin-1β in ectopic endometrial cells

OBJECTIVE To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN Prospective study. SETTING Human reproduction research laboratory. PATIENT(S) Nine women with endometriotic lesions. INTERVENTION(S) Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S) The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S) This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S) This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

PARP-1-induced cell death through inhibition of the MEK/ERK pathway in MNNG-treated HeLa cells

Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N′-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.

Localization of cells in the rat brain expressing fumarylacetoacetate hydrolase, the deficient enzyme in hereditary tyrosinemia type 1.

Fumarylacetoacetate hydrolase (FAH) is the terminal enzyme in the catabolic pathway of tyrosine. This enzyme which is mainly expressed in the liver and kidney is deficient in hereditary tyrosinemia type 1. As some affected individuals present neurologic abnormalities, we studied the expression of FAH in the rat and human brain. The FAH gene was shown to be expressed in the rat brain by immunoblot and Northern blot analysis. The FAH protein was also detected in human brain by the immunoblot assay. An immunohistochemical study was undertaken to localize the FAH-producing cells in the rat central nervous system. This analysis showed that the majority of FAH-producing cells are localized in the axonal nerve fibers of the white matter, although positive cells could also be found throughout the brain. The greatest number of FAH-positive cells were found in structures consisting essentially of white matter, such as the corpus callosum. This specific localization in the white matter indicates that some type of glial cells are responsible for the expression of the FAH protein in the rat central nervous system. The characteristic linear organization found in some of the FAH-positive cells in the corpus callosum suggests that these glial cells are oligodendrocytes. These findings are discussed with respect to the neurologic symptoms observed in some tyrosinemia patients.

Deficient expression of tumor necrosis factor receptor type 2 in the endometrium of women with endometriosis.

Problem: Tumor necrosis factor‐α (TNF‐α) is secreted mainly during the menstrual phase and has been suggested to play a role in induction of apoptosis in endometrial cells and menstrual shedding. TNF‐α receptor type 2 (TNF‐RII) is believed to play a central role in TNFα‐mediated cytotoxic, mitogenic, anti‐proliferative and apoptotic effects. The aim of this study was to assess whether TNF‐RII maybe expressed differentially in the endometrium of women with different degrees of endometriosis.

The AF2 domain of the orphan nuclear receptor TEC is essential for the transcriptional activity of the oncogenic fusion protein EWS/TEC

The EWS/TEC fusion protein encoded by the t(9:22) chromosomal translocation in human extraskeletal myxoid chondrosarcoma tumors is thought to participate in the tumoral process at least in part by deregulating the expression of specific target genes involved in the control of cell proliferation. In this work we show that the activation function-2 (AF2) domain of TEC is essential for the transcriptional activity of the EWS/TEC fusion protein. Significantly, deleting only the last 15 amino acids of the fusion protein, which contains 949 amino acids in its full form, results in a loss of over 70% of its transcriptional activity in transfected human chondrocyte cell lines. Point mutation analyses indicate that within the AF2 domain, amino acid residues I939, D940 and F943 all play a crucial role in the activity of EWS/TEC. Comparable results were obtained with the native TEC receptor. These results suggest that EWS/TEC interacts at least in part with the same transcriptional coactivators as the native TEC receptor, and that these coactivators may be involved in the tumoral process leading to human chondrosarcoma tumors.