Ors8 is a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent CV-1 (monkey kidney cells) DNA from replication bubbles active at the onset of S phase. A panel of deletion mutants of ors8 have identified a fragment of 186 bp that is essential for the in vitro and in vivo replication of ors8 containing plasmid. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro (Todd et al., 1995). / The objective of the research in this thesis is the purification and characterization of a mammalian factor(s) that specifically interact(s) with the 186 bp fragment of ors8, and is involved in DNA replication. Fractionation of HeLa cell extracts by chromatographic techniques have identified such a protein fraction. The OBA (Ors Binding Activity) enriched fraction is able to support the in vitro replication of ors8, and is also enriched for polymerases alpha and delta, replication protein A (RP-A) and topoisomerase II (Ruiz et al., 1995). OBA sediments at approximately 150 kDa in a glycerol gradient. / Band-shift competition experiments demonstrate that a subfragment of 59 bp, from the 186 bp fragment of ors8, competes for OBA binding as efficiently as the 186 bp fragment itself. The 59 bp subfragment shares 85% homology with a 36 bp sequence (A3/4) generated by comparison of several mammalian replication origins, including the ors (origin rich sequences). The A3/4 sequence is able to compete efficiently for OBA binding. Furthermore, the addition of increasing amounts of the A3/4 oligonucleotide to the in vitro reaction inhibits the replication of 186 bp-containing plasmid. Band-shift elution of the A3/4-OBA complex, followed by Southwestern analysis showed that a band of approximately 92 kDa is involved in the interaction of OBA with the A3/4 sequence. Microsequencing analysis of this band revealed that it is identical to the 86 subunit of Ku antigen. Affinity purification of OBA by DNA sequence-specific chromatography demonstrated that both subunits of Ku antigen (p70 and p86) are present in this fraction. In addition, the affinity purified OBA (apOBA) fraction is also enriched in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In vitro replication experiments using anti-Ku70, anti-Ku86, and anti-DNA-PKcs antibodies, resulted in the inhibition of p186 replication in vitro, suggesting the involvement of Ku in mammalian DNA replication.

The proteins and sequence elements necessary to initiate mammalian DNA replication have been, to date, poorly characterized. Here, we have further defined the minimal sequence elements required to support autonomous DNA replication of a mammalian early replicating DNA sequence, ors8. In addition, we have identified a protein, CBP, which binds specifically to DNA cruciforms, a DNA secondary structure that can form at inverted repents which are commonly found within origins of DNA replication. / Cloned fragments from a known mammalian origin from the DHFR locus, oribeta, have been shown to replicate autonomously following transient transfection into mammalian cells and when used as templates in an in vitro DNA replication system. These in vivo and in vitro DNA replication assays have been used to define the sequence elements present within the minimal origin of ors8, a monkey o&barbelow;rigin enr&barbelow;iched s&barbelow;equence activated in early S phase. The minimal origin, consisting of an internal sequence of 186 bp, was shown to contain bent DNA, an imperfect Oct-1 binding site, and an inverted repeat which has been shown previously to extrude into a cruciform structure in vivo . Cruciform structures have been hypothesized to form at or near origins of DNA replication to serve as sites of recognition for initiator proteins. To further investigate this possibility, a previously described cruciform binding protein, CBP, was identified as a member of the 14-3-3 family of proteins. 14-3-3 proteins are involved in regulating a wide array of cellular processes by acting as adaptor or stabilizing molecules. We have shown that 14-3-3 binding to cruciform structures is required for the in vitro DNA replication of the 186-bp minimal origin of ors8, further suggesting a crucial role for cruciform structures in DNA replication.

The Met protooncogene encodes a receptor tyrosine kinase that is deregulated by point mutation, and overexpression/amplification in a number of human tumours. The Met receptor is also oncogenically activated following genomic rearrangement which generates a cytoplasmic, constitutively activated fusion protein, Tpr-Met. In addition to autophosphorylation sites within the catalytic domain, the carboxy terminus of Tpr-Met/Met contains a single major site of autophosphorylation, tyrosine 489. This tyrosine residue represents a unique multisubstrate binding site, capable of binding numerous intracellular proteins, and is critical for the biological activities of both the Met receptor and Tpr-Met oncoprotein. Addition of the c-src myristoylation sequence to the amino terminus of the normally cytoplasmic Tpr-Met, localizes Tpr-Met to plasma membranes and enhances cellular transformation, in vitro invasion, and tumourigenicity. Furthermore, a membrane targetted Tpr-Met is localized to a similar subcellular compartment as the Met receptor, and alters the complement of signalling proteins required for efficient transformation. In this respect, a membrane localized Tpr-Met resembles oncogenic forms of the transmembrane Met receptor, and provides a model with which to study transformation by Met receptor oncoproteins. Significantly, membrane localization of Tpr-Met induces a phosphoinositide 3' kinase (PI3' K) dependent autocrine loop, involving the production of hyaluronic acid (HA), and post-translational modification of the cell surface receptor for HA, CD44. PI3'K activity and the HA/CD44 autocrine loop, are dependent on the multisubstrate binding site, tyrosine 489, and tyrosine residue 498, a residue with no previously described biochemical function. Although the exact mechanisms by which PI3'K regulates HA production are unclear, the induction of a HA/CD44 autocrine loop may represent a novel mechanism by which deregulated receptor tyrosine kinases increase their onco

Ubiquitin dependent protein degradation is now emerging as an important mechanism of cellular regulation. Specificity for the multitude of different substrate proteins is provided by a series of E2, E3 and deubiquitinating enzymes. The structural basis by which ubiquitin (Ub) conjugating enzymes (E2s, UBCs) and deubiquitinating enzymes determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217k, and identified it as a homologue of S. cerevisiae UBC7. UBC7 differs from other class I E2 enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. By deletion of the loop in UBC7 and insertion of the loop in a typical class I E2 enzyme, E214K, we found that the loop interferes with the ability of E2 to bind to E1 charged with Ub as well as with E3s or substrates. We also identified two isoforms of a novel testis specific ubiquitin specific processing protease, UBP-t1 and UBP-t2, which contain identical core regions but different N-termini. Both isoforms were germ cell specific and developmentally regulated. UBP-t1 was induced in step 16--19 spermatids and primarily in the nucleus while UBP-t2 was expressed only in step 18--19 spermatids and located extra-nuclearly and in residual bodies. By expressing UBP-t1, UBP-t2 and UBP-core with myc tag, and fusions of the two distinct amino terminal ends with green fluorescent protein in COS-7 cells, we found that the N-terminus of UBP-t2 targets the protein to the centrosome and N-terminus of UBP-t1 targets the protein to nucleur. The N-termini also play a critical role in recognition of specific substrates. These findings indicate that divergent sequences, sometimes quite small, within conserved families of E2s or deubiquitinating enzymes can determine specificity of ubiquitination by influencing their subcellular location, and their abilities to recogni

As the limited utility of islet transplantation becomes established, interest turns to other means of restoring β-cell mass in diabetes. Specifically, reports suggest that the endogenous pancreas may be stimulated to regenerate β-cells, thereby restoring normoglycemia. However, the source of this regeneration is debated, with the islet proper proposed as a source, via replication or neogenesis. The mechanism is uncertain, given in vitro models of islet plasticity whereby islets can be induced to dedifferentiate, proliferate, and subsequently redifferentiate into functional endocrine tissue. We sought to examine the cells and signals involved in islet plasticity. Isolation stimulated the expression of TNFα in islets, whereas TNFα inhibition reduced apoptosis and improved β-cell function. Conversely, insulin secreted by islets improved islet survival. Thus, isolation induces the expression of both pro- and anti-apoptotic signals. Cultured islets produced EGF ligands that induced islet dedifferentiation. This effect was countered by TGFβ expression, which inhibited endocrine dedifferentiation. Thus it appears that within the context of this culture model, the major determinants of the differentiated state of the islet are signals provided by the islet itself. Cell tracking experiments were performed to identify the cells involved in islet dedifferentiation. Cultures of dedifferentiated islets were comprised primarily of two cell types: a cytokeratin+ epithelial population derived from α-, δ- and PP-cells, and a nestin+ fibroblast-like population derived from β-cells. While the origins of islet redifferentiation were not ascertained, the process appears to require the presence of β-cell-derived nestin+ cells. Islets from control and type 2 diabetic sources were compared, with islets from diabetic sources overexpressing nestin and vimentin, markers of β-cell dedifferentiation. Moreover, while cultures derived from diabetic donor tissue were equally capable / Alors que l’on découvre les limites du succès de la transplantation d’ilots, l’intérêt se porte sur d’autres moyens de restaurer la masse de cellules β chez le diabétique. Plus spécifiquement, certaines études suggèrent que le pancréas endogène peut être stimulé afin de régénérer des cellules β et rétablir une glycémie normale. Toutefois, la source de cette régénération est débattue, les ilots eux-mêmes étant proposés comme en étant la source, via réplication ou néogénese. Si le mécanisme de cette régénération reste incertain, des modèles in vitro montrent la capacité des ilots à se dédifférencier, proliférer et se redifférencier à nouveaux en ilots fonctionnels. Nous avons examiné les cellules et signaux impliqués dans la plasticité des ilots.L’isolation stimule l’expression de TNFα dans les ilots, alors qu’il a été montré que l’inhibition de TNFα réduit l’apoptose et améliore la fonction de la cellule β. Réciproquement, l’insuline secrétée par les ilots améliore la survie des ilots. Ainsi, l’isolation induit l’expression à la fois de signaux pro- et anti-apoptotiques.Les ilots en culture produisent des ligands EGF qui induisent leur dédifférenciation. Cet effet est contrecarré par l’expression de TGFβ qui lui inhibe la dédifferenciation. Il apparaît donc que dans les contraintes de ce modèle, les déterminants majeurs de l’état différencié des ilots sont des signaux produits par l’ilot lui-même.Nous avons conduit des expériences de traçage cellulaire pour identifier les cellules impliquées dans la dédifférenciation des ilots. Nous avons montré que les cultures d’ilots dédifférenciés comprenaient principalement deux types cellulaires : une population de cellules épithéliales cytokératine+ dérivée des cellules α, δ et PP, et une population de cellules fibroblastiques nestine+ dérivée des cellules β. Alors que l’origine cellulaire de la rediff

The function of prolactin in human breast cancer was studied using four different approaches. First, purification and characterization of the prolactin receptor from breast cancer cells indicated that the receptor has a molecular mass of 88 000 Da, 67 000 Da being protein, and the other 21 000 Da presumably carbohydrate. Secondly, prolactin was tested for mitogenic activity in breast cancer in vitro. No consistent mitogenic response to prolactin could be demonstrated in these experiments. Thirdly studies upon the regulation of the prolactin receptor in breast cancer cells indicated that the prolactin receptor is stimulated by lactogen, estrogen and progesterone at the protein level. Estrogen, progesterone, thyroid hormone, and forskolin (but not lactogen) increase prolactin receptor steady state RNA levels, and the phorbol ester PMA and retinoic acid inhibited receptor RNA levels. However, effects at the RNA level were of a much lesser magnitude than effects at the protein level. Mechanisms other than transcriptional regulation alone are likely involved in prolactin receptor regulation. Fourthly, prolactin receptor and prolactin inducible protein/gross cystic disease fluid protein (PIP/GCDFP-15) RNA levels were examined in breast cancer tumors. Highly significant correlations were observed between the prolactin receptor and the progesterone receptor; the prolactin receptor and PIP/GCDFP-15; and PIP/GCDFP-15 and progesterone receptor.

GBM is an advanced-stage form of brain cancer with an extremely low survival rate. Tumour reoccurrence is quite common, resulting from micropockets of tumour cells outside the primary mass, which makes surgical resection extremely difficult. Previous research in our laboratory suggested a link between GBM invasiveness and CRMP1 (Collapsin Response Mediator Protein 1), a member of a family of phosphoproteins involved in cytoskeletal dynamics and already considered a tumour invasion suppressor gene in Non-Small Cell Lung Cancer (NSCLC)1. The aim of this study was to examine the role of CRMP1 in GBM cell migration. Several CRMP members and longer alternatively spliced variants (long form - L, normal form - S) were also examined to determine common/distinct properties. CRMP cDNAs were transfected into glioma cell lines (U87, U251N) to generate stable pooled populations which were characterized in vitro and in vivo in terms of cell viability, cell migration and tumour growth. Although there was no significant difference in cell proliferation as compared to control neo-transfected glioma cell lines, significantly fewer cells migrated in a Boyden chemotactic assay in lines overexpressing CRMP1-S, CRMP2-S, or CRMP4-S. In athymic mice, intracerebral implantation of cells overexpressing CRMP1-S yielded tumours that were significantly smaller in volume as compared to those produced by control glioma cells. In addition, the analysis of gene expression profiles in GBM patient samples collected by the National Cancer Institute (REMBRANDT) established a link between patient survival and CRMP1 and CRMP4 levels. In conclusion, our results suggest that CRMPs 1, 2 and 4 may regulate glioma cell migration and CRMP1 and CRMP4, may be involved in GBM patient survival. / Le glioblastome multiforme (GBM) est un stade avancé de cancer du cerveau avec un très faible taux de survie. Sa récurrence est assez fréquente, causée par la présence de cellules tumorales en dehors de la masse primaire, ce qui rend difficile la résection chirurgicale. Des recherches antérieures dans notre laboratoire ont suggéré un lien entre le pouvoir envahissant du GBM et CRMP1 (Collapsin Response Mediator Protein 1), un membre d'une famille de phosphoprotéines impliquées dans la dynamique du cytosquelette et déjà considéré comme un gène suppresseur d’invasion dans le cancer du poumon. Le but de cette étude était d'examiner le rôle du CRMP1 dans la migration cellulaire des GBM. Plusieurs autres membres de la famille CRMP ainsi que leurs isoformes épissées differentiellement (forme longue - L, la forme normale - S) ont également été examinés afin de déterminer les propriétés en commun. L’ADN complémentaire des CRMP a été transfecté dans les lignées de cellules de gliome (U87, U251N) pour générer des populations stables qui ont été caractérisées in vitro et in vivo sur le plan de la viabilité cellulaire, la migration cellulaire et la croissance de la tumeur. Bien qu'il n'y ait pas eu de différence significative dans la prolifération cellulaire par rapport a la lignée contrôle, beaucoup moins de cellules migrèrent dans un essai de type Boyden dans les lignées surexprimant soit CRMP1-S, CRMP2-S, ou CRMP4-S. Dans les souris thimoprivées, l'implantation intracérébrale de cellules surexprimant CRMP1-S a produit des tumeurs qui ont été nettement plus faible en volume par rapport à celles produites par des cellules contrôles. En outre, l'analyse des profils d'expression génique dans les échantillons des patients GBM recueillis par l'Institut national du cancer (Rembrandt) a démontré un lien entre la survie des patients et les niveaux du CRMP1 et CRMP4. En conclusion, nos résultats suggèrent que CRMPs 1,

Hox-4.2 is a member of the vertebrate Hox genes. These homeobox genes encode sequence-specific DNA-binding proteins that play a role in vertebrate pattern formation. Their complex spatiotemporal expression patterns during embryogenesis are established in response to positional cues. To characterize regulatory regions involved in Hox-4.2 expression, transient expression assays in P19 murine embryonal carcinoma cells with luciferase reporter gene constructs, under the control of 5$ sp prime$ flanking sequences of Hox-4.2, were performed. Cotransfections of such luciferase reporter constructs with an expression vector for the Hox-4.2 product, resulted in the identification of a 217 bp fragment that functioned as an autoregulatory enhancer. Two binding sites for the Hox-4.2 product within this fragment were characterized by electrophoretic mobility shift assays and their role for autoregulation was demonstrated by site-directed mutagenesis. These findings suggest an autoregulatory mechanism in the control of Hox-4.2 expression and established the Hox-4.2 product as a transcriptional activator in a mammalian system. The use of luciferase reporter gene constructs in P19 embryonal carcinoma cells also led to the identification of a retinoic acid response element (RARE) in Hox-4.2 5$ sp prime$ flanking sequences which was critical for retinoic acid-mediated activation of reporter gene expression in cultured cells. The involvement of retinoic acid receptors in this response was demonstrated by mutation of the Hox-4.2 RARE and cotransfection of an expression vector for a dominant negative form of retinoic acid receptor $ alpha$, both of which inhibited the response to retinoic acid. Furthermore, retinoic acid receptors bound to the RARE as demonstrated in electrophoretic mobility shift assays. The characterization of the Hox-4.2 RARE represented one of the first direct indications for a retinoic acid receptor-mediated regulation of Hox genes. In addition, lacZ reporter gen

Atrial Natriuretic Factor (ANF) is a cardiac peptide hormone that has been implicated in the regulation of blood pressure. The physiological role of ANF was investigated by generating transgenic mice (designated MT/ANF) that overexpress ANF in the circulation as well as at several ectopic sites, most notably in the pituitary and adrenal glands. Systolic blood pressure was significantly decreased in MT/ANF mice, but was not accompanied by any changes in heart rate, hematocrit, body weight, water or food intake, urine output, or sodium or potassium excretion. The decreased blood pressure of transgenic mice therefore appears to be independent of the effects of ANF to elicit natriuresis and diuresis. Rather, the hypotensive phenotype was associated with a suppression of adrenocortical function, as evidenced by decreased circulating aldosterone and corticosterone levels, reduced adrenal steroidogenic enzyme expression, and atrophy of the adrenal cortex. Adrenal insufficiency was also reflected in an elevated urinary sodium to potassium excretion ratio. Decreased adrenocortical activity did not result from pituitary effects of ANF, as plasma adrenocorticotropin and pituitary pro-opiomelanocortin mRNA levels were increased in transgenic animals. The circulating renin-angiotensin system and hypothalamic vasopressin expression remained unchanged in transgenic mice. These findings support the conclusion that decreased adrenocortical activity in MT/ANF mice is the result of a direct effect of ANF on the adrenal gland. Therefore, these transgenic animals constitute a model for primary adrenal insufficiency associated with hypotension, and demonstrate a novel manner in which ANF can participate in the chronic regulation of blood pressure.

A coordinated response to DNA damage is vital to maintain cellular viability and prevent the onset of disease. In mammalian cells, the intra-S phase checkpoint regulator, ATR (Ataxia-telangiectasia mutated and RAD3-related) kinase coordinates the response to DNA damage to ensure the genome is accurately and completely replicated before the cell enters mitosis. Y box Binding Protein 1 (YB-1), a transcription and translation factor, has previously been implicated in cell proliferation and the development of chemotherapeutic resistance. YB-1 has been linked to a wide variety of cellular stresses, but has not been studied in the context of DNA replication. In this study, we determined that YB-1 associates to both the β-globin replication origin (origin-containing) and the control (origin-lacking) DNA regions. This observation suggested that YB-1 may be involved in DNA replication elongation instead of initiation. By immunoprecipiating YB-1, we identified that PCNA and MCM7 preferentially interact with YB-1 during S phase. The examination of the spatial and temporal dynamics of these interactions by immunofluorescence microscopy, however, did not reveal nuclear colocalization of these proteins. By treating cells with hydroxyurea to stall the replication fork, we re-examined the protein-protein interaction between YB-1 and MCM7 and found that following 8 hours of hydroxyurea treatment, YB-1 and MCM7 exhibited diffuse colocalization in the cell nucleus. This finding may implicate YB-1 in exerting a late-onset response to prolonged replication fork arrest either directly at stalled replication forks or at "dormant" origins bound by MCM complexes. A number of roles for YB-1 can be postulated, such as the requirement of YB-1 in facilitating the resumption of DNA replication, or the activation of additional origins to duplicate the genome in the presence of a replication stress. This finding may in turn account fo / Une réponse coordonnée lors de dommages à l'ADN est vitale pour le maintien de la viabilité cellulaire et pour éviter l'installation de maladies. Dans les cellules de mammifères, le point de contrôle de la phase S, la protéine kinase ATR (Ataxia-telangiectasia mutated and RAD3-related), coordonne la réponse aux dommages de l'ADN afin d'assurer une réplication complète et fidèle du génome avant l'entrée en mitose. La protéine YB-1 (Y box Binding Protein 1), un facteur de transcription et de traduction, est impliqué dans la prolifération cellulaire et la résistance aux chimiothérapies. YB-1 est également lié à une large variété de stress cellulaires but aucune donnée n'est disponible quant à son rôle dans la réplication de l'ADN. Lors de mon travail de Master, j'ai pu montrer qu'YB-1 s'associe à la fois à l'origine de réplication de la β-globine et dans les régions contrôles de l'ADN. Ce résultat suggère qu'YB-1 pourrait être impliqué dans la phase d'élongation de la réplication de l'ADN plutôt que dans celui de l'initiation. Par immunoprécipitation, j'ai identifié PCNA et MCM7 comme interacteurs préférentiels d'YB-1 en phase S. Par contre, en immunofluorescence, je n'observe pas de colocalisation nucléaire entre ces protéines. Lors du blocage de la fourche de réplication par un traitement à l'hydroxyurée, l'interaction entre YB-1 et MCM7 a été réexaminée et j'ai mis en évidence que 8h après le traitement, ces deux protéines présentent une co-localisation diffuse dans le noyau. Ces données indiquent qu'YB-1 pourrait être impliqué dans une réponse tardive suite à un arrêt prolongé de la fourche de réplication soit directement au point d'arrêt soit au niveau d'origines « dormantes » liées aux complexes MCM. YB-1 peut donc avoir plusieurs rôles tels que l'aide à la reprise de la réplication de l'ADN ou l'activation d'origines de réplicatio