Xavier Dolcet

Receptors of the Glial Cell Line-Derived Neurotrophic Factor Family of Neurotrophic Factors Signal Cell Survival through the Phosphatidylinositol 3-Kinase Pathway in Spinal Cord Motoneurons

The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promote in vivo and in vitro survival of different neuronal populations, including spinal cord motoneurons. These factors signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of the GDNF family receptor α (GRFα) family of glycosylphosphatidylinositol-linked coreceptors. Activation of the receptor induces Ret phosphorylation that leads the survival-promoting effects. Ret phosphorylation causes the activation of several intracellular pathways, but the biological effects caused by the activation of each of these pathways are still unknown. In the present work, we describe the ability of the GDNF family members to promote chicken motoneuron survival in culture. We show the presence of Ret and GFRα-1, GFRα-2, and GFRα-4 in chicken motoneurons using in situ hybridization and reverse transcription-PCR techniques. By Western blot analysis and kinase assays, we demonstrate the ability of these factors to induce the phosphatidylinositol 3 kinase (PI 3-kinase) and the extracellular regulated kinase (ERK)–mitogen-activated protein (MAP) kinase pathways activation. To characterize the involvement of these pathways in the survival effect, we used the PI 3-kinase inhibitor LY 294002 and the MAP kinase and ERK kinase (MEK) inhibitor PD 98059. We demonstrate that LY 294002, but not PD 98059, prevents GDNF-, neurturin-, and persephin-induced motoneuron survival, suggesting that PI 3-kinase intracellular pathway is responsible in mediating the neurotrophic effect.

NF-κB signalling regulates the growth of neural processes in the developing PNS and CNS

The proper growth and elaboration of neural processes is essential for the establishment of a functional nervous system during development and is an integral feature of neural plasticity throughout life. Nuclear factor-kappa B (NF-κB) is classically known for its ubiquitous roles in inflammation, immune and stress-related responses and regulation of cell survival in all tissues, including the nervous system. NF-κB participation in other cellular processes remains poorly understood. Here we report a mechanism for controlling the growth of neural processes in developing peripheral and central neurons involving the transcription factor NF-κB. Inhibiting NF-κB activation with super-repressor IκB-α, BAY 11 7082 (IκB-α phosphorylation inhibitor) or N-acetyl-Leu-Leu-norleucinal (proteosomal degradation inhibitor), or inhibiting NF-κB transcriptional activity with κB decoy DNA substantially reduced the size and complexity of the neurite arbors of sensory neurons cultured with brain-derived neurotrophic factor while having no effect on their survival. NF-κB exerted this effect during a restricted period of development following the phase of naturally occurring neuronal death when the processes and connections of the remaining neurons are extensively modified and refined. Inhibiting NF-κB activation or NF-κB transcriptional activity in layer 2 pyramidal neurons in postnatal somatosensory cortical slices reduced dendritic arbor size and complexity. This function of NF-κB has important implications for neural development and may provide an explanation for reported involvement of NF-κB in learning and memory.

Activation of Phosphatidylinositol 3‐Kinase, but Not Extracellular‐Regulated Kinases, Is Necessary to Mediate Brain‐Derived Neurotrophic Factor‐Induced Motoneuron Survival

Abstract : Chick embryo spinal cord motoneurons develop a trophic response to some neurotrophins when they are maintained in culture in the presence of muscle extract. Thus, after 2 days in culture, brain‐derived neurotrophic factor (BDNF) promotes motoneuron survival. In the present study we have analyzed the intracellular pathways that may be involved in the BDNF‐induced motoneuron survival. We have observed that BDNF activated the extracellular‐regulated kinase (ERK) mitogen‐activated protein (MAP) kinase and the phosphatidylinositol (Pl) 3‐kinase pathways. To examine the contribution of these pathways to the survival effect triggered by BDNF, we used PD 98059, a specific inhibitor of MAP kinase kinase, and LY 294002, a selective inhibitor of Pl 3‐kinase. PD 98059, at doses that significantly reduced the phosphorylation of ERKs, did not show any prominent effect on neuronal survival. However, LY 294002 at doses that inhibited the phosphorylation of Akt, a down‐stream element of the Pl 3‐kinase, completely abolished the motoneuron survival effects of BDNF. Moreover, cell death triggered by LY 294002 treatment exhibited features similar to those observed after muscle extract deprivation. Our results suggest that the Pl 3‐kinase pathway plays an important role in the survival effect triggered by BDNF on motoneurons, whereas activation of the ERK MAP kinase pathway is not relevant.

Molecular pathology of endometrial carcinoma: practical aspects from the diagnostic and therapeutic viewpoints

This article reviews the main molecular alterations involved in endometrial carcinoma. Five molecular features (microsatellite instability, and mutations in the PTEN, k-RAS, PIK3CA and β-catenin genes) are characteristic of endometrioid carcinomas, whereas non-endometrioid carcinomas show alterations of p53, loss of heterozygosity (LOH) on several chromosomes, as well as other molecular alterations (STK15, p16, E-cadherin and C-erb B2). The review also covers the phenomenon of apoptosis resistance, as well as the results obtained from cDNA array studies, and the perspectives for targeted therapies. A group of practical applications of molecular pathology techniques are also mentioned: diagnosis of hereditary non-polyposis colon cancer syndrome in patients with endometrial carcinoma; evaluation of precursor lesions; prognosis; diagnosis, particularly for synchronous endometrioid carcinomas of the uterus and the ovaries; and targeted therapies.

Proteasome Inhibitors Induce Death but Activate NF-κB on Endometrial Carcinoma Cell Lines and Primary Culture Explants

Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-κB, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-κB, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKKα/β, phosphorylation and degradation of IκBα, and phosphorylation of the p65 NF-κB subunit on serine 536. Proteasome inhibitor-induced NF-κB activity can be blocked by a non-degradable form of IκBα or dominant negative forms of either IKKα or IKKβ. Lentiviral delivery of shRNAs to either IKKα or IKKβ cause blockade of NF-κB transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IκBa degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-κB activation. Accordingly, siRNA knockdown of p65 inhibits proteasome inhibitor-induced NF-κB transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-κB instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NF-κB activation should be carefully examined in particular cell types.

Cytokines Promote Motoneuron Survival through the Janus Kinase-Dependent Activation of the Phosphatidylinositol 3-Kinase Pathway ☆

To determine which intracellular pathways mediate the survival effects of ciliary neurotrophic factor and cardiotrophin-1 cytokines on motoneurons, we studied the activation of the Jak/STAT, the PI 3-kinase/Akt, and the ERK pathways. At shorter time points, cytokines induced the activation of STAT3 and ERK, but not PI 3-kinase. Jak3 inhibitor suppressed cytokine- and muscle extract-induced survival. In contrast, PD 98059, a MEK inhibitor, was not able to prevent cytokine-induced survival, demonstrating that ERK is not involved. Surprisingly, the PI 3-kinase inhibitor LY 294002 prevented the survival-promoting effects of cytokines. When assays of PI 3-kinase activity were performed at later stages following cytokine treatment a significant increase was observed compared to control cultures. This delayed increase of activity could be completely prevented by treatment with protein synthesis or Jak3 inhibitors. Collectively, these results demonstrate that cytokines induce motoneuron survival through a PI 3-kinase activation requiring de novo protein synthesis dependent on Jak pathway.

Immunohistochemical analysis of PTEN in endometrial carcinoma: a tissue microarray study with a comparison of four commercial antibodies in correlation with molecular abnormalities

The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r=−0.249, P=0.037). The monoclonal antibody 10P03 exhibited an association with phospho-AKT that did not have statistical significance. Both 6.H2.1 and 10P03 antibodies stained PTEN-transfected cells, and were negative in the PTEN-deficient cell line blocks. The polyclonal antibody and the monoclonal antibody 28H6 produced positive staining in PTEN-deficient cell line blocks, suggesting nonspecific staining. The results indicate that monoclonal antibody 6.H2.1 may be a suitable alternative for tumors with inactivation of PTEN.

Endometrial carcinoma: molecular alterations involved in tumor development and progression

In the western world, endometrial carcinoma (EC) is the most common cancer of the female genital tract. The annual incidence has been estimated at 10–20 per 100 000 women. Two clinicopathological variants are recognized: the estrogen related (type I, endometrioid) and the non-estrogen related (type II, non-endometrioid).The clinicopathological differences are paralleled by specific genetic alterations, with type I showing microsatellite instability and mutations in phosphatase and tensin homologue deleted on chromosome 10, PIK3CA, K-RAS and CTNNB1 (β-catenin), and type II exhibiting TP53 mutations and chromosomal instability. Some non-endometrioid carcinomas probably arise from pre-existing endometrioid carcinomas as a result of tumor progression and, not surprisingly, some tumors exhibit combined or mixed features at the clinical, pathological and molecular levels. In EC, apoptosis resistance may have a role in tumor progression. Understanding pathogenesis at the molecular level is essential in identifying biomarkers for successful targeted therapies. In this review, the genetic changes of endometrial carcinogenesis are discussed in the light of the morphological features of the tumors and their precursors.

HGF regulates the development of cortical pyramidal dendrites

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.

Abnormalities in the NF-κB family and related proteins in endometrial carcinoma

The NF‐κB family of transcription factors regulates a wide variety of cellular processes including cell growth, differentiation, and apoptosis. A tissue microarray was constructed from paraffin wax‐embedded blocks from 95 endometrial carcinomas (EC), previously studied for microsatellite instability, as well as for alterations in PTEN, k‐RAS and beta‐catenin. Immunohistochemical evaluation included members of the NF‐κB (p50, p65, p52, c‐Rel, Rel‐B) and the IκB (IκBα, IκBβ, IκBε, Bcl‐3) families, as well as putative targets of NF‐κB such as Flip, Bcl‐xL, Cyclin D1, and oestrogen and progesterone receptors. Results were correlated with the clinical and pathological data. Nuclear immunostaining for members of the NF‐κB family was frequent in EC (p50, 20%; p65, 16.5–21.9%; p52, 9.3%; c‐Rel, 48.9%; Rel‐B, 15.7%); and it correlated with negativity for members of the IκB family in some cases. There was a statistically significant association between immunoreaction for p50 and p65 (p = 0.006), suggesting activation of the so‐called ‘classic form’ of NF‐κB, similar to that described in breast cancer. Bcl‐3 nuclear immunostaining was detected in 60.7% of cases. The vast majority of p52‐positive tumours showed Bcl‐3 nuclear immunoreaction (p = 0.038). Immunostaining for putative targets of NF‐κB was as follows: Bcl‐xL, 76.2% (p = 0.001); Flip 43.0%; Cyclin D1, 64.79%. p65 immunostaining correlated with increased immunoreaction for steroid hormone receptors. No correlation was found between NF‐κB nuclear pattern and the presence of microsatellite instability, or alterations in PTEN, k‐RAS, or beta‐catenin. These results suggest that the NF‐κB and IκB families of genes may be important in endometrial carcinogenesis, by controlling apoptosis and cell proliferation. Copyright