Biomedical subjects

Raymond Frade

Publications and source records attributed to Raymond Frade.

8 recordsLinked to original sources

PubMed2005

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human B lymphoma cell surface triggers Cbl tyrosine phosphorylation, its association with p85 subunit, Crk-L and Syk and its dissociation with Vav.

It is well established that CD21 activation on human B cell surface triggers B cell proliferation. We previously demonstrated that CD21 activation also triggers tyrosine phosphorylation of two components, p95 and p120, both interacting with SH2 domains of the p85 subunit of PI 3-kinase. We successively identified p95 as the nucleolin and the first signal transduction pathway specifically triggered by CD21 activation, i.e.: pp60Src activation, tyrosine phosphorylation of p95 nucleolin, its interaction with SH2 domains of p85 subunit and PI 3-kinase activation, followed by AKT-GSK-3 activations. We herein identified the p120 component as the protooncoprotein Cbl and the first steps associated to its activation. First, CD21 activation triggered Cbl tyrosine phosphorylation, which required c-Src kinase but not PI 3-kinase or Syk kinase activities. Involvement of Src kinase in this step was supported by inhibition of Cbl phosphorylation and its interactions with other components when cells were either preincubated with specific Src inhibitor or transfected with dominant-negative c-Src form. Second, once tyrosine phosphorylated, Cbl interacts with SH2 domains of p85 subunit, SH2 domains of Crk-L and with tyrosine phosphorylated Syk kinase. The third and unexpected feature was to found that, at the contrary of BCR or of CD19 (herein also analyzed for the first time), CD21 activation triggers dissociation of Cbl-Vav complex. Thus, these results provide the first molecular basis of a new signal transduction pathway specifically triggered by CD21 activation.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Infection of human B lymphoma cells by Mycoplasma fermentans induces interaction of its elongation factor with the intracytoplasmic domain of Epstein-Barr virus receptor (gp140, EBV/C3dR, CR2, CD21).

Human cell lines are often infected by mycoplama strains. We have demonstrated that when infected by Mycoplasma fermentans, human B lymphoma cell proliferation increased strongly. These infected B cells expressed a p45 kDa protein which interacted with the intracellular domain of CD21, the EBV/C3d receptor. p45 analysis demonstrated that this is a new gene which encodes an elongation factor originating from Mycoplasma fermentans. p45 interaction with CD21 was specific, there being no interaction with CD19. This is the first demonstration that Mycoplasma fermentans, in infecting human B cells, generates a p45 Mycoplasma component that interacts with CD21, which is involved in B cell proliferation.

Amino Acid Sequence↗

PubMed2005

RB18A enhances expression of mutant p53 protein in human cells.

RB18A (TRAP220/DRIP205) is a cofactor of transcription. We herein demonstrated that RB18A downregulated p53 and upregulated MDM2 promoters. These RB18A regulations, not modified by p53wt expression, were inhibited by mutant p53 (p53mut) expression, which directly interacts with RB18A D5 domain. In addition, RB18A via its D4 domain, also interacts directly and specifically with MDM2 protein inhibiting p53mut degradation. Altogether, these mechanisms contribute to maintain a high level of p53mut expression in tumor proliferating cells. Therefore, RB18A plays a central role to control p53wt and p53mut protein content and functions in cells through a loop of regulation, which involves MDM2.

Cell Line↗

PubMed2004

Inhibition of tumorigenicity and metastasis of human melanoma cells by anti-cathepsin L single chain variable fragment.

We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anti-cathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.

Animals↗

PubMed2003

Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI 3-kinase, respectively.

We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domains of p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-protein interaction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR.

Antigens, CD19↗

PubMed2002

Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells.

We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His. This 3D8 mAb interacted with an epitope localized on the 156-197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.

Amino Acid Sequence↗

PubMed2002

RB18A regulates p53-dependent apoptosis.

We previously demonstrated that RB18A, a member of TRAP220/DRIP205/PBP family, in vivo acted as a cofactor of transcription by differently regulating p53wt transactivating activity on physiological promoters. Using p53-negative cells transfected with different constructs, we herein demonstrated that RB18A down-regulated p53wt-dependent apoptosis. This biological regulation was due to a specific diminution of p53wt protein level, as level of p53mut and GAPDH proteins was not modified. This p53wt diminution was dependent on proteasome activity, as inhibited by MG-132 inhibitor. This specific p53wt degradation was correlated with an increase in expression of MDM2, which promoted p53wt degradation into proteasome. RB18A up-regulated MDM2 expression by activating MDM2 promoter, even in absence of p53wt. Altogether, these data emphasized that RB18A could regulate p53wt function not only by direct interaction between both proteins, but also by up-regulating promoter activity of MDM2, a p53-regulating partner.

Apoptosis↗

PubMed2002

Characterization of human cathepsin L promoter and identification of binding sites for NF-Y, Sp1 and Sp3 that are essential for its activity.

Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5'-flanking region of the gene encoding human cathepsin L was determined up to 3263 bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5' untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between -1489 and -1646 bp. In this region, two GC boxes (-1590/-1595 and -1545/-1550) and a CCAAT motif (-1571/-1575) were involved in specific DNA-protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.

Base Sequence↗