Androgen-dependent apoptosis in male germ cells is regulated through the proto-oncoprotein Cbl

The proto-oncoprotein Cbl is known to control several signaling processes. It is highly expressed in the testis, and because spermatogenesis is androgen dependent, we investigated the androgen dependency expression of Cbl through its testicular sublocalization and its expression levels in rats that were exposed to the antiandrogen flutamide or were hypophysectomized. We report the androgen dependency of Cbl as it localizes in pachytene spermatocytes during androgen-dependent stages, is down-regulated upon flutamide exposure, and is up-regulated with testosterone in hypophysectomized rats. Coculture experiments showed the key control exerted by the Sertoli cell on Cbl activity. As flutamide induces germ cell apoptosis, we investigate members of the Bcl-2 family upon flutamide exposure. We show that the proapoptotic Bcl-2 family member Bim mirrored Cbl expression through a posttranscriptional process. We also show that in Cbl knockout mouse testes, the imbalance between the high expression of Bim and Smac/Diablo and antiapoptotic factors such as cellular inhibitor of apoptosis 2 favors a survival process, which makes these mice unresponsive to androgen withdrawal and could explain their hypofertility

Rôle du proto-oncogène c-cbl dans la régulation de l'apoptose des cellules germinales testiculaires et des cellules de la prostate ventrale du rat et de la souris

Le proto-oncogène c-cbl code pour une proteine cytoplasmique, la p120cbl ou C-Cbl. Cette proteine possède une fonction E3-ligase permettant la désignation puis l'ubiquitination de certains récepteurs à tyrosine kinase activés, entraînant leur régulation négative. Elle possède également une fonction poly-adaptatrice due à de nombreux sites de liaison à des molécules portant sur des motifs SH2 ou / et SH3. C-Cbl a été impliquée dans le transport du glucose, le régulation positive de la prolifération passant par la Phospho-Inositol-3 kinase, ou encore l'organisation de l'actine du cytosquelette. Nous nous sommes intéressés dans ce travail au rôle de cette protéine dans l'apoptose des cellules germinales testiculaires en raison d'une expression élevée de c-Cbl dans le testicule et d'indications de la littérature sur la possible implication de c-Cbl dans l'apoptose. Par aileurs, il est largement reporté que l'apoptose joue un rôle-clé dans le bon déroulement de la spermatogenèse. Dans un second temps, afin de comparere le rôle de c-Cbl sur le processus de l'apoptose des cellules germinales par rapport aux cellules somatiques dans un autre organe androgéno-dépendant, nos efforts ont porté en collaboration avec le groupe sur la fonction de c-cbl dans les cellules épithéliales de la prostate. Ce travail a été réalisé sur le rat et la souris. Nous avons montré que c-Cbl est essentiellement exprimé dans les spermatocytes pachytènes dans le tesicule et dans les cellules épithéliales de la prostate ventrale. Nous avons établi l'androgéno-dépendance du niveau d'expression de c-Cbl dans ces cellules, suggérant le rôle de régulation de cette protéine dans des organes androgéno-dépendants. Nous avons ensuite révélé la dérégulation de la voie apoptotiquue dans ces deux organes : sur-expression des facteurs pro-apoptotiques Bim, Smac / DIABLO et altération de l'expression des Inhibiteurs de l'Apoptose (IAP) dépendante du tissu étudié. Finalement, nous avons montré le rôle pro-apoptotique de c-Cbl fortement exprimée dans le testicule soutenant son effet pro-apoptotique dans cet organe . Ces perturbations sont à l'origine d'une hypofertilité que nous avons mise en évidence chez la souris KO pour c-cblThe proto-oncogene c-cbl codes for a cytoplasmic protein p120cbl or c-Cbl. It is a E3-ligase targeting and then allowing the ubiquitination and finally the down-regulation of several cellular activated tyrosine kinase receptors.c-Cbl is also considered as a molecular poly-adaptor, as numerous SH2- or SH3-bearing molecules specifically bind to it. c-Cbl has been reported involved in the glucose uptake, positive regulation of cell proliferation through PI3K activation, or cyto-skeleton regulation. As c-Cbl is highly expressed in testis and according to a few works could be involved in apoptosis, we explored this possibility in this tissue. Moreover, apoptosis is crucial in spermatogenesis. In collaboration with the team, we also investigated prostate epithelial cells, another androgen-dependent tissue, in order to compare c-Cbl apoptotic function in germ cells to the one in somatic cells. This work has been realized on rats and mice. We showed that c-Cbl is highly expressed in testis pachyten spermatocytes and in ventral prostate epithelial cells. We established the androgen-dependency of c-Cbl expression in those two androgen-dependent tissues, suggesting a c-Cbl regulation function. We then revealed the disruption of the apoptotic pathway in testis and prostate of c-Cbl KO mice : the pro-apoptotic factors Bim and Smac / DIABLO were over-expressed and the IAP expression was differently alterd depending on the considered tissue. We conclusively showed that c-Cbl is pro-apoptotic in prostate. This ambiguous result was understood when the team showed the over-expression of a c-Cbl pro-apoptotic isoform in testis. We finally demonstrated the hyperfertility of c-CBL KO mice, probably due to their apoptotic alterationsLYON1-BU.Sciences (692662101) / SudocSudocFranceF

The apoptotic and anti-proliferative activity of 'Origanum majorana' extracts on human leukemic cell line

Scientists are constantly searching for phytochemicals and compounds with anti-cancer and antioxidant activity. In this study, the anti-proliferative activity of plant extracts from 'Origanum majorana' (marjoram) was tested on human lymphoblastic leukemia cell line Jurkat. Cytotoxicity was examined using non-radioactive cytotoxicity assay and the IC(50) was calculated. At non-cytotoxic concentrations, the viability of cells decreased with increase of concentration of plant extract. The anti-proliferative effect was also found to be dose-dependent. Analysis via flow cytometry shows that marjoram extracts stimulated apoptosis. Induction of apoptosis was caused by an up-regulation of p53 protein levels and down-regulation of Bcl-2alpha. Marjoram exhibited a strong scavenging activity (SC(50)=0.03mg dry weight). The conclusions from this study suggest that marjoram extracts exhibit anti-proliferative effect and high antioxidant activity. For that it merits further investigation as a potential therapeutic agent