The cadherins are a family of calcium-dependent cell adhesion molecules. The regulators of cadherin expression have not been identified. We have examined the ability of hormones to regulate the expression of cadherin subtypes present in the mouse testis, ovary and uterus. 17-$ beta$ estradiol caused a rapid and significant increase in N-cadherin mRNA levels in the Sertoli cells of the testis and granulosa cells of the ovary. This hormone was also capable of stimulating E-cadherin mRNA levels in the epithelial cells of the ovary and uterus. Progesterone was capable of increasing E-cadherin mRNA levels in the uterus but not in the ovary. These studies are the first demonstration that steroids are capable of regulating cadherin mRNA levels in vivo. Furthermore, this is the first demonstration of a physiological role for 17-$ beta$ estradiol in the testis. / Follicle stimulating hormone was capable of increasing N-cadherin mRNA levels in mouse testes and in cultured mouse Sertoli cells. The stimulatory effects of FSH on Sertoli cells were mimicked by cholera toxin and forskolin, suggesting the involvement of the cAMP signal transduction pathway. Furthermore, there was a synergistic effect between 17-$ beta$ estradiol and elevated levels of cAMP on N-cad mRNA levels in cultured Sertoli cells. Our observations indicate that steroids and follicle-stimulating hormone are key regulators of cadherin expression.

The distribution of glucocorticoid receptors (GR) in the rat testis was examined in vivo using (3H)-dexamethasone 21-mesylate (DM) which binds covalently to GR. After injection with $ sp3$H-DM, the testes of adrenalectomized, adult rats were processed for light microscope (LM) radioautographic (RAG) analysis. Control rats received simultaneously a 25-or 50- fold excess of cold dexamethasone. Quantitation of the label confirmed the presence of specific DM binding sites in both Leydig cells (86% of the label) and to a lesser degree, in the cellular layers of the seminiferous epithelium (14% of the label). These binding data were confirmed by LM immunocytochemistry. Interstitial macrophages were non-specifically labeled. In the Leydig cell, an electron microscope (EM) quantitative RAG analysis of 3H-DM binding sites showed that smooth endoplasmic reticulum (sER) and mitochondria were heavily labeled, with 53% and 31% of the total label, respectively. Cytosol (exclusive of all structures) and nucleus showed comparatively weak labeling, with 9% and 7% of the label, respectively. All other structures showed little or no labeling.

An immunocytochemical procedure for the human glucocorticoid receptor (GR) was developed herein in order to assess its level and subcellular distribution in a well-studied system of childhood acute lymphoblastic leukemia cells (CEM), in which sensitive and resistant subclones have been established, and in different tissue preparations (lymph node biopsies, bone marrow aspirates and peripheral blood) from patients with various forms of leukemia or lymphoma. In the absence of preincubation with steroid, the GR was localized predominantly in the cell cytoplasm and to a weaker extent in the nucleus of CEM cells. Following incubation with glucocorticoid, an increase in nuclear GR was observed in the steroid-sensitive CEM-C7 and C7E$ sb2$A cell lines but not in the resistant C7 subclones (4R4, 3R43 and ICR27). Incubation of human peripheral mononuclear cells with 10$ sp{-6}$M dexamethasone for 30 or 60 minutes in vitro resulted in a marked increase in nuclear GR immunoreactivity. Since nuclear translocation of GR is a critical step in glucocorticoid hormone action on target cells, assessment of the subcellular distribution of GR may provide functional tests for steroid responsiveness. Furthermore, the effect of glucocorticoids on the regulation of GR and its mRNA was studied by immunocytochemistry, RIA and in situ hybridization.

No description available.

Multidrug resistance (MDR) in cancer is a phenomenon that impairs efficient treatment that has now become a major hinderance to chemotherapy. ABC transporters such as the P-glycoprotein (P-gp or ABCB1) and the multidrug resistance-associated protein 1 (MRP1 or ABCC1) are known to actively efflux chemotherapeutic agents out of cells thereby inducing MDR. A promising approach in order to specifically target MDR cells is to take advantage of their increased sensitivity (collateral sensitivity) to certain compounds. The second chapter of this thesis aims to elucidate the mechanism of collateral sensitivity in MRP1 cells. Overall, the results show a hypersensitivity of these cells to glutathione (GSH) modulating agents. Such hypersensitivity in cells is caused by the presence of MRP1, which extrudes GSH out of the cell. Collateral sensitivity can be achieved by stimulating the extrusion with verapamil (Vrp), Apigenin (Api) or by inhibiting the GSH synthesis with butathione sulfoximine (BSO). Finally, GSH depletion leads to reactive oxygen species (ROS) accumulation, which likely triggers apoptosis. The third and fourth chapters aim toresolve the collateral sensitivity mechanism induced in P-gp expressing cells. The study of Vrp, progesterone (Pro) and deoxycorticosterone (DOC) induced collateral sensitivity lead to the conclusion that two requirements ar necessary for validating this phenomenon. The first requirement, common to the three compounds, is the presence of and active P-gp. This P-gp sees its ATPase activity stimulated and the electron transport chain higher demand generated leads to the formation of higher levels of ROS. It seems that these ROS alone are not sufficient and they need to potentiate something else. In the case of Vrp it seems that increase Ca2+ level would be the second requirement. For Pro and DOC, the presence of cholesterol at the membrane levels seems to be enough. This allows the / La résistance multiple à la chimiothérapie (MDR) dans le cancer est un phénomène qui nuit à l'efficacité du traitement et est devenue un important problème. Les transporteurs ABC comme la P-glycoprotéine (P-gp ou ABCB1) et la protéine associée à la résistance multiple 1 (MRP1 ou ABCC1) sont reconnue pour pomper de façon active les médicaments hors des cellules et de ce fait induisent la MDR. Une approche prometteuse visant à cibler spécifiquement ces cellules MDR est de prendre avantage de leur sensibilité accrue (sensibilité collatérale) face à certains composés.Le second chapitre de cette thèse visait à élucider le mécanisme de sensibilité collatérale chez les cellules exprimant MRP1. Ensemble les résultats montre une sensibilité accrue de ces cellules face aux agents modulant la GSH. Cette hypersensibilité est causée par la présence de MRP1 qui pompe la GSH hors de la cellule. La sensibilité collatérale a pue être obtenue en stimulant le pompage en utilisant la verapamil (Vrp) ou l'Apigenin (Api), ou en inhibant la synthèse de la GSH avec la buthionine sulfoximine (BSO). Finalement, la diminution de la GSH résulte en une accumulation d'espèce réactive d'oxygène (ROS) déclanchant l'apoptose.Les chapitres trois et quatre étaient dirigés vers la résolution du mécanisme de sensibilité collatérale induite chez les cellules exprimant la P-gp. L'étude de la sensibilité collatérale induite par la Vrp, la progestérone (Pro) et la désoxycorticostérone (DOC) a mené conclusion que deux pré-requis sont nécessaire pour l'obtention du phénomène. Le premier, commun pour les trois composés, est la présence d'une P-gp active. Cette P-gp voit sont activité ATPase être stimulé et l'élévation de la demande de la chaîne de transport d'électrons entraîne la formation de niveau élevés de ROS. Il semble que ces ROS seuls ne sont pas suffisant et ils on bes

The paucity of CAR in malignant glioma cell lines and grade IV primary astrocytomas (glioblastoma multiforme) suggest that CAR expression may be disadvantageous to malignant gliomas. The present study directly tested this hypothesis by determining the effect of forced CAR expression on the invasion and growth of the human glioma cell line U87-MG expressing no detectable levels of CAR. We also studied the requirement of the cytoplasmic domain of CAR in mediating for these effects. We have found that forced expression of the full-length forms of mCAR1 isoform in U87-MG cell lines (i) does not affect their growth in vitro in monolayer, but does significantly reduce the tumor volumes generated by implanted glioma cells in mouse brains in vivo; and (ii) reduced invasion by glioma cells in a three-dimensional spheroid model. In contrast, expression in U87 cells of the tailless-CAR having the entire cytoplasmic domain deleted except for the first two intracellular juxtamembrane cysteine-cysteine amino acids did not significantly reduce the invasion rate of the glioma spheroid in vitro, nor did it reduce the tumor volume generated in vivo in the brain. Together these results suggest that the paucity of CAR expression in malignant gliomas may confer the tumors selective advantage in growth and invasion.

It has been postulated that intracellular binding sites for platelet activating factor (PAF) contribute to pro-inflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCEC) produced PAF-molecular species in response to H2O2 . Using fluorescent-activated cell sorter analysis, we demonstrated the expression of PAF receptors (PAFR) on cell and nuclear surfaces of PCEC. Confocal microscopy studies performed on PCEC, Chinese hamster ovary cells stably overexpressing PAFR and on isolated nuclei from PCEC also showed a robust nuclear distribution of PAFR. Presence of PAFR at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with PAF evoked a decrease in cAMP production and a pertussis toxin (PTX)-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a PTX-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei C-PAF evoked the expression of pro-inflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), and was associated with augmented ERK1/2 phosphorylation and NF-kappaB binding to DNA consensus sequence; COX-2 expression was prevented by MEK/ERK1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of pro-inflammatory gene COX-2.

In this thesis, the mechanism of endoplasmic reticulum localization of Saccharomyces cerevisiae alpha1,2-mannosidase is studied. alpha1,2-mannosidase is a type II membrane glycoprotein localized in the endoplasmic reticulum, yet it does not have any of the known endoplasmic reticulum localization signals. / Immunofluorescence studies show that the endoplasmic reticulum localization of alpha1,2-mannosidase depends on the Golgi protein Rer1p, since in rer1-deleted cells, alpha1,2-mannosidase migrates to the vacuoles. Furthermore, alpha1,2-mannosidase acquires Golgi-specific carbohydrate modification. These results show that the steady state endoplasmic reticulum localization of alpha1,2-mannosidase involves recycling from the Golgi apparatus. The transmembrane domain of alpha1,2-mannosidase is important for endoplasmic reticulum localization since fusing it to the Golgi protein Kre2p results in the endoplasmic reticulum localization of the chimera in an Rer1p-dependent manner. Mutation of the polar residues in the transmembrane domain do not affect endoplasmic reticulum localization of alpha1,2-mannosidase, nor Rer1p-dependent recycling, indicating that the polar residues are not important for these processes. alpha1,2-Mannosidase and Rer1p interact, determined using the split-ubiquitin system, a genetic method adapted to study membrane protein interactions in vivo. Therefore, the transmembrane domain of alpha1,2-mannosidase mediates recycling from the Golgi apparatus in a mechanism that involves interaction with the Golgi protein Rer1p. / When solubilized and subjected to gel filtration analysis, endogenous alpha1,2-mannosidase is eluted on Sephacryl S-200 as twice the molecular weight of the purified recombinant enzyme lacking its transmembrane domain. Immunoprecipitation studies show that alpha1,2-mannosidase can form a homodimer. Furthermore, mutation of the asparagine residue at position 3, or the tyrosine residues at positions 20 and 21, prevents dimerization.

We previously identified the USP19 deubiquitinating enzyme. To further characterize the functions of USP19, we used siRNA to deplete this enzyme in cultured cells. Depletion of USP19 in FR3T3 fibroblasts resulted in an impairment of cell growth, delay in entry into S phase and accumulation of p27Kip1, an inhibitor of G1 to S phase progression. This increased p27Kip1 upon USP19 depletion was due to a decrease in the rate of degradation of p27Kip1 in G0 and G1 phases, but not at the G1/S transition. The increased p27Kip1 in USP19 depleted FR3T3 cells was associated with reduced levels of KPC1, the catalytic subunit of a ubiquitin protein ligase for p27Kip1, but no changes in levels of Pirh2 or Skp2, other ligases for p27Kip1. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Cell growth impairment upon USP19 depletion was partially reversed in cell lines stably expressing KPC1 or in p27Kip1−/− MEF cells. These findings indicated that in normal fibroblasts, USP19 exerts its effects on cell proliferation through KPC1/ p27Kip1 dependent mechanisms, by deubiquitinating and stabilizing KPC1 which in turn leads to increased ubiquitination of p27Kip1, and also by a KPC1/ p27Kip1 independent pathway(s). / To determine the importance of these pathways in other cells, in particular cancer cells, USP19 was depleted in a panel of cell lines, including prostate cancer DU145, PC-3, LNCaP and 22RV1, breast epithelial MCF10A, breast carcinoma MCF7 and MDA-MB-231 as well as transformed Hela and 293T cells. The ability of USP19 to regulate cell growth generally correlated with its ability to modulate p27Kip1 except in the Hela and 293T cells in which the regulation of p27Kip1 was maintained, but the regulation of cell growth was lost. These effects seemed to be independent of KPC1, Pirh2 or Skp2, as their levels were not changed upon USP19 depletion. These findings suggested that transformation can lead to loss of regulatory effects of USP19. Indeed, depletion of USP19 in Ras-transformed FR3T3 cells had no effects on cell growth, p27Kip1 or KPC1. / Thus, this work is conceptually significant in identifying USP19 as a new regulator of p27Kip1 and of G1 progression in normal fibroblasts and some prostate cancer cell lines. This work suggests USP19 as a potential novel target for treatment of prostate cancer. / Nous avons précédemment décrit la protéine USP19 comme étant une enzyme de déubiquitination. Maintenant, pour mieux comprendre le rôle de USP19, nous avons réduit les niveaux de l'enzyme en utilisant des ARNs interférents dans des cellules en culture. En réduisant la protéine USP19 dans des fibroblastes FR3T3, nous avons obtenu une altération de la croissance cellulaire, ainsi qu'un délai d'entrée en phase S du cycle cellulaire et une accumulation de p27Kip1, un inhibiteur de la progression cellulaire de la phase G1 à S. Cette augmentation de p27Kip1 suite à la réduction de USP19 est due à une diminution du taux de dégradation de p27Kip1 durant les phases G0 et G1 et non durant la transition G1/S. L'augmentation de p27Kip1 dans les cellules FR3T3 traitées avec l'ARN interférent contre USP19 est associée à une réduction des niveaux de KPC1, la sous-unité catalytique de KPC qui est une enzyme de ligation à l'ubiquitine pour p27Kip1. Par contre, aucun changement n'a été observé dans les niveaux de Pirh2 ou Skp2, deux autres enzymes de ligation à l'ubiquitine associée à p27Kip1. KPC1 est conjugué à l'ubiquitine in vivo et est stabilisé par des inhibiteurs du protéasome ou par la surexpression de USP19. KPC1 peut aussi être coimmunoprécipité avec USP19. L'altération de la croissance cellulaire suite à la réduction de USP19 peut être partiellement inversée dans des lignées cellulaires qui exprime KPC1 de façon stable ou dans des cellules MEF p27Kip1−/−. Ces résultats nous indiquent que dans des fibroblastes normaux, USP19 exerce son effet sur la prolifération cellulaire via des mécanismes dépendants de KPC1/ p27Kip1 et ce en déubiquitinant et en stabilisant KPC1 qui à son tour mènera à une augmentation de l'ubiquitination de p27Kip1. Toutefois, USP19 peut aussi exercer son effet par des voies indépendantes de KPC1/ p27Kip1. / Pour déterminer l'importance des ces mécanismes dans d'autres types de cellules, en particulier dans des cellules cancéreuses, nous avons réduit les niveaux de USP19 dans une panoplie de lignées cellulaires, incluant des lignées de cancer de prostate comme DU145, PC-3, LNCaP et 22RV1, des lignées de cancer du sein comme MCF7 et MDA-MB-231, d'épithélium mammaire comme MCF10A ainsi que dans des lignées transformées comme les cellules Hela et 293T. La capacité de USP19 à réguler la croissance cellulaire corrèle généralement avec sa capacité à moduler p27Kip1, à l'exception des cellules Hela et 293T dans lesquelles la régulation de p27Kip1 est maintenue mais où la régulation de la croissance cellulaire est perdue. Ces effets semblent être indépendants de KPC1, Pirh2 ou Skp2, étant donné que leurs niveaux protéiques restent inchangés suite à la réduction de USP19. Ces résultats suggèrent que la transformation cellulaire peut mener à la perte de l'effet de régulation de USP19. En effet, lorsque USP19 est réduit dans des cellules FR3T3 qui ont été transformées par l'oncogène Ras, aucun effet n'est observé sur la croissance cellulaire ou sur les niveaux de p27Kip1 ou KPC1. / Par conséquent, cette étude est conceptuellement significative car elle identifie USP19 comme étant un nouveau régulateur de p27Kip1 et de la progression G1 dans des fibroblastes normaux ainsi que dans certaines lignées cellulaires de cancer de prostate. Cette étude suggère que USP19 pourrait devenir une nouvelle cible pour le traitement du cancer de la prostate.

Mineralization of the extracellular matrix of bone is a cell-mediated process, which is tightly regulated by a delicate balance between stimulatory and inhibitory molecules. A disruption in the metabolism or levels of these mediating factors results in pathologically hypomineralized or hypermineralized bone. The experimental results presented in this thesis describe the effects of key proteins, peptides and small-molecule ions on an osteoblast cell-culture model of bone mineralization. / This study presents evidence that pyrophosphate inhibits mineralization by at least three different mechanisms that include direct binding to hydroxyapatite crystals, induction of osteopontin expression, and inhibition of alkaline phosphatase activity. The data presented also demonstrate that inhibition of mineralization by mineral-binding osteopontin and MEPE ASARM peptides is phosphorylation-dependent and that this inhibition can be rescued by enzymatic degradation of the peptides by PHEX. This result identifies a novel mechanism by which loss of PHEX activity can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia of X-linked hypophosphatasia. Finally, the thesis describes the novel effects of a potentially physiologic modulator of mineralization, inositol hexakisphosphate, on osteoblast activity – namely, that inositol hexakisphosphate inhibits osteoblast culture mineralization, adsorbs to mineral and induces expression of osteopontin. / In summary, the findings herein suggest a model whereby regulation of crystal propagation/growth within the extracellular matrix is maintained by the enzymatic removal (e.g. by alkaline phosphatase and PHEX) of mineralization inhibitors (e.g. pyrophosphate and ASARM peptides) and that these inibitors regulate mineralization by acting on both the mineral phase and on the cellular expression of mineral-regulating genes. / Minéralisation de la matrice extracellulaire de l'os est un processus de médiation cellulaire, qui est rigoureusement réglementé par un délicat équilibre entre les molécules stimulatoires et inhibitrices. Un dysfonctionnement dans le métabolisme de ces facteurs donnera soit une déficience ou un excès des minéraux dans les os. Les résultats expérimentaux présentés dans cette thèse, décrivent les effets des protéines et des ions principaux sur un modèle de cellule-culture d'osteoblaste de minéralisation d'os. / Cette étude présente les éléments de preuve que le pyrophosphate inhibe la minéralisation par au moins trois différents mécanismes qui incluent la liaison directe aux cristaux d'hydroxyapatite, à l'induction de expression d'osteopontin et à l'inhibition de l'activité de la phosphatase alcaline. Les données présentées démontrent également que l'inhibition de la minéralisation par la liaison de minéraux osteopontin/MEPE ASARM peptides est phosphorylation dépendante et que cette inhibition peut être sauvée par dégradation enzymatique avec PHEX. Ce résultat identifie un nouveau mécanisme par lequelle une perte d'activité de PHEX peut conduire à l'accumulation d'ASARM dans la matrice ce qui se traduit par la ostéomalacie de X-linked hypophosphatasia. / Enfin, la thèse décrit les effets d'un nouveau modulateur de minéralisation, l'inositol hexakisphosphate, de l'activité d'ostéoblaste. Nous avons trouver que l'inositol hexakisphosphate inhibe la minéralisation de culture d'ostéoblastes, s'attache aux minéraux et augmente l'expression d'osteopontin.