Christine Le Roy

Autocrine regulation of Leydig cell differentiated functions by insulin-like growth factor I and transforming growth factor beta.

The expression and the maintenance of specific differentiated function of Leydig cells are regulated not only by gonadotropin but by locally produced factors, which may act as autocrine regulators. Many factors, in particular growth factors, have been postulated to have such a type of effect on testicular cells, but very few fulfilled the three criteria required to establish a paracrine/autocrine role: (a) presence of receptors and biological action on local cells; (b) local secretion regulated by physiological signals; and (c) blockade of the factor or its receptors must modify the function of local cells. In the present work we demonstrate that two factors, insulin-like growth factor 1 (IGF-I) and transforming growth factor beta1 (TGFbeta1) fulfilled the three criteria: (a) IGF-I stimulates the transcription of the genes encoding Leydig cell differentiated function, leading to an enhanced steroidogenic responsiveness to LH/hCG; (b) Leydig cells (LC) express and secrete IGF-I and this secretion is enhanced by hCG; and (c) incubation of LC with IgG anti-IGF-I, but not with IgG-control, markedly reduced the steroidogenic responsiveness to LH/hCG. In contrast to IGF-I, TGFbeta is a potent inhibitor of LC differentiated function. Moreover, LC express TGFbeta1 mRNA and secrete this peptide. To prove that the locally produced TGFbeta has an autocrine role, LC were transfected with 10 microM of an antisense oligonucleotide (AON) complementary to the translation initiation region of TGFbeta1 mRNA. Transfection with AON but not with sense deoxynucleotide induces a complete disappearance of TGFbeta immunoreactivity in LC and an enhanced hCG-induced testosterone production by LC. This increased steroidogenic responsiveness was associated with a significant enhancement of both LH/hCG receptor mRNA and P450c17 mRNA. Taken together, the above results show that both factors play an autocrine role, although opposite, on Leydig cell function.

2-Chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide (CMV423), a new lead compound for the treatment of human cytomegalovirus infections.

Human cytomegalovirus (HCMV) remains one of the major pathogens in immunocompromised patients (AIDS and transplants) and the main cause for congenital infections leading from slight cognitive defects up to severe mental retardation. The drugs that are currently available for the treatment of HCMV infections, i.e. ganciclovir, foscarnet and cidofovir, are all acting at the level of the viral DNA polymerase. Here we describe an entirely new molecule, the 2-chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide (CMV423), that shows very potent in vitro activity against HCMV. CMV423 is highly active against HCMV reference strains and clinical isolates, but also against those strains, isolated from patients or emerging after in vitro selection, that are resistant to either ganciclovir, foscarnet or cidofovir. CMV423 also showed activity in two ex vivo models, that are both highly relevant for the pathophysiology of HCMV, the retinal pigment epithelial and the bone marrow stromal cell assays. Viral antigen expression analysis by flow cytometry, as well as time of addition experiments, confirmed that CMV423 acts on a step of the viral replicative cycle that precedes the DNA polymerase step and, most likely, coincides with the immediate early (IE) antigen synthesis. Finally, CMV423 combined with either ganciclovir, foscarnet or cidofovir in checkerboard experiments demonstrated a highly synergistic activity.

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia

B-cell antigen receptor (BCR)-mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium-calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

AMD3100 disrupts the cross-talk between chronic lymphocytic leukemia cells and a mesenchymal stromal or nurse-like cell-based microenvironment: pre-clinical evidence for its association with chronic lymphocytic leukemia treatments.

Background Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface. Design and Methods Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model. Results AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies. Conclusions Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.

Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism

Technological developments of multiparametric flow cytometry come along with the generation of new dyes. The APC‐tandem dyes, which combine the fluorophores APC and Cy7/H7, allow the detection of a specific signal in the APC‐Cy7/H7 channel along with an unexpected nonspecific signal in the APC channel. Depending on the magnitude of the latter, it may be a handicap for interpreting the data of multicolor labeling experiments. We investigated the alteration of the APC‐tandem dyes by labeling peripheral blood cells with antibodies directed toward leukocyte surface proteins and by analyzing cells by flow cytometry. Our results show that the APC‐Cy7/H7 tandem fluorochromes degraded over time. Nonspecific APC signal was observed with the various antibodies tested only upon cell attachment but not under bead linkage. Moreover, the percentage of degradation of the APC‐Cy7/H7 dyes was dependent on the cell type analyzed. Interestingly, nonspecific APC signal strongly decreased when the metabolic activity of immunolabeled cells was inhibited or when cells were incubated with vitamin C. This study demonstrates that the APC‐tandem dyes are the target of cell‐dependent degradation, which may be antagonized. These findings will allow cytometer users to optimize their multicolor panels.

Inhibition of the moloney murine leukemia virus cycle at a post reverse transcriptional step by the netropsin-intercalating hybrid molecule netropsin-oxazolopyridocarbazole

In a search for new antiretroviral agents acting at the nucleic acid level, two hybrid molecules composed of a bispyrrolecarboxamide chain related to netropsin, linked to the intercalating chromophore oxazolopyridocarbazole, were tested on the cycle of a defective Moloney murine leukemia virus (M.MuLV) derived from the SVX shuttle and expressing resistance to the G418 antibiotic. The drug netropsin-oxazolopyridocarbazole (Net-OPC), which displays a binding preference to duplex DNA containing A + T bases, inhibits the retroviral replicative cycle (IC50 = 4.8 microM). In contrast, the related molecule (bis)pyrollecarboxamide-oxazolopyridocarbazole (Bpc-OPC) devoid of sequence preference as well as the elemental components of Net-OPC, namely OPC, pentyl-OPC and netropsin, displays no significant action on the viral cycle. The estimation of cytosolic viral DNA in infected cells using quantitative polymerase chain reaction suggests that Net-OPC impairs a post retrotranscriptional step of the viral cycle.

Overexpression of a dominant-negative type II TGFbeta receptor tagged with green fluorescent protein inhibits the effects of TGFbeta on cell growth and gene expression of mouse adrenal tumor cell line Y-1 and enhances cell tumorigenicity.

Transforming growth factor beta (TGFbeta) has been reported to be a potent growth inhibitor of epithelial cells. The purpose of the present work was to study in vitro and in vivo the effects of overexpression of a dominant-negative type II TGFbeta receptor on the proliferation and differentiation of Y-1 cells. Stable transfections were performed with a mutant TbetaRII (TbetaRII-KR) fused with the Enhanced Fluorescent Green Protein (EGFP). The expression of this fusion protein and its overexpression were demonstrated by northern blot and immunoblot with EGFP and TbetaRII probes and antibodies respectively. The membrane localization of this fusion protein was confirmed by confocal microscopy. The functionality of this fusion protein was demonstrated by its blocking effects on TGFbeta action on DNA synthesis and on Y-1 expression of steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Moreover, in nude mice the tumorigenicity of cells stably transfected with the fusion protein was higher than that of cells stably transfected with EGFP alone. Taken together, the present results show that TbetaRII-KR/EGFP blocks the effects of TGFbeta1 on Y-1 cells and acts as a potent dominant-negative receptor preventing TGFbeta signaling.

Protein kinase D-dependent CXCR4 down-regulation upon BCR triggering is linked to lymphadenopathy in chronic lymphocytic leukaemia

In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser324/325. Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro. In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

Biological processes in solar lentigo: insights brought by experimental models

Common in ageing patient, the solar lentigo is a macular hyperpigmented skin lesion that results from chronic exposure to ultraviolet irradiations. Despite sharing numerous features with other pigmented spots, the diagnostic of this benign lesion is well characterized at the tissue level. Recent studies shed lights on several factors and their pathogenic mechanisms involved in the development of the solar lentigo. This review summarizes how diverse experimental approaches allowed the identification of several biomarkers, which contribute to a better understanding on the initiation and the maintenance of this pigmentary disorder.

Old DAT and new data: positive direct antiglobulin test identifies a subgroup with poor outcome among chronic lymphocytic leukemia stage A patients.

Only a minority of chronic lymphocytic leukemia (CLL) patients harboring a positive direct antiglobulin test (DAT) will develop autoimmune hemolytic anemia (AIHA). In a single institution cohort of 378 CLL patients, 56 patients (14.8%) had at least one positive DAT during the course of the disease, either at diagnosis or later. We found no relationship between the time of the first positive DAT and overall survival (OS). However, patients with a positive DAT who did not develop AIHA had the same adverse outcome as patients who developed AIHA. Of the patients who were in Binet stage A at diagnosis, those with a positive DAT had a significantly shorter OS, regardless of their IGHV mutational status, however, there was a strong association with VH1‐69. By multivariate analysis, a positive DAT was found to be an independent adverse prognostic factor for OS. Thus, DAT represents a strong adverse prognostic factor and its determination should be repeated during follow‐up. Am. J. Hematol. 90:E5–E8, 2015.