The Bcl-2 family of proteins strictly regulates the intrinsic pathway of apoptosis. Direct physical interactions between Bcl-2 proteins regulate mitochondrial outerpermeabilisation (MOMP), which occurs in response to various cell stresses andapoptotic stimuli. How changes in Bcl-2 protein activity regulate apoptosiscommitment is still unclear, especially with regard to how they interact with eachother within the context of the mitochondrial membrane. Recent studies haveshown that Bcl-2 proteins exist in a dynamic equilibrium between the mitochondriaand the cytosol. In this thesis, by using FRAP, I have measured changes in Bcl-XLand Mcl-1 dynamics in single cells. Surprisingly, individual cells within a populationshow widely differing Bcl-XL and Mcl-1 dynamics. There is a corelation betweenBcl-XL and Mcl-1 dynamics with BH3-only protein expression. Anti-apoptotic andpro-apoptotic Bcl-2 proteins stabilise each other on the OMM. Together, theseresults indicate that cells constantly fine tune mitochondrial priming and thatanalysing anti-apoptotic Bcl-2 proteins by FRAP allows this to be measured at asingle cell level in real time before MOMP.

Neste trabalho foram analisados 30 casos de linfomas de células B da região oral, fixados em solução de formaldeído e incluídos em parafina, através da técnica de imuno-histoquímica para as proteínas c-Myc, Bcl-2, Bcl-6 e ciclina D1. Dos casos analisados 40% foram positivos para a marcação para c-Myc, 33,3% para a marcação para ciclina D1, 83,3% para a marcação para Bcl-2 e 53,3% para a marcação para Bcl-6. Todos os casos foram diagnosticados como linfomas difusos de grandes células B, o subtipo de linfoma com a maior casuística. A análise destas proteínas é de fundamental importância para o diagnóstico e direcionamento do tratamento de doenças hematopoiéticas, pois estão envolvidas em vários processos de controle da transcrição gênica, do ciclo celular e dos processos apoptóticos e o aumento do conhecimento sobre sua ação em diferentes subtipos de linfomas pode corroborar outros estudos. / In this study, 30 cases of formalin-fixed and paraffin-embedded B-cell lymphomas of the oral region were submitted to immunohistochemistry for the detection of proteins c-Myc, Bcl-2, Bcl-6 and cyclin D1. Fourty percent (40%) of the studied cases were positive for c-Myc, 10% for cyclin D1, 83.3% for Bcl-2 and 53.3% for Bcl-6. The analysis of these proteins has fundamental importance for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription and cell cycle. All cases were diffuse large B-cell lymphomas, the subtype with the highest incidence. The analysis of these proteins is very important for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription, cell cycle and apoptotic processes and an increase in the knowledge of their action in different subtypes of lymphomas can corroborate to other studies.

Head and neck cancer accounts for approximately 3 percent of all cancers in the United States, and over 90% of them are head and neck squamous cell carcinoma (HNSCC). Chemotherapeutic drugs that treat HNSCC can activate BCL-2 family dependent apoptosis. Pro-apoptotic NOXA induced by adenovirus (Ad-NOXA) or fenretinide inactivates anti-apoptotic MCL-1, while ABT-263 can inactivate other anti-apoptotic BCL-2 family members such as BCL-2 and BCL-XL. We used p53 inactive HN8 and HN12, p53 wild-type UMSCC1, and HPV-positive UMSCC47 human HNSCC cell lines and five mouse HNSCC cell lines. Cells were treated with Ad-NOXA, ABT-263, and fenretinide alone or in combinations. Combinational treatment of ABT-263 with Ad-NOXA or fenretinide enhanced cell death among all cell lines we tested regardless of p53 status. These findings support the hypothesis that combinational treatment of Ad-NOXA or fenretinide with ABT-263 increases cell death by simultaneously inhibiting all anti-apoptotic BCL-2 family proteins in HNSCC cells.

Die Bcl-2-Familienmitglieder A1 und sein humanes Homolog Bfl-1 gewährleisten das Überleben der Zelle. Gleichzeitig trägt eine Dysregulation der Expression von A1/ Bfl-1 zur Krebsentstehung bei. Die Stabilität von A1/ Bfl-1 wird durch deren Ubiquitinylierung sowie die anschließende proteosomale Degradation gesteuert. Mit Hilfe eines Yeast-Two-Hybrid-Screens wurde die E3-Ubiquitinligase HectD1 als potentieller Interaktionspartner von A1/ Bfl-1 identifiziert. Die Interaktion von A1 und HectD1 des Yeast-Two-Hybrid-Screens konnte in Säugerzellen bestätigt werden. Desweiteren konnte gezeigt werden, dass lediglich 87 Aminosäuren für eine Interaktion von HectD1 und A1 nötig sind. Da membrangebundenes HectD1 zu einer Translokation von zytosolischem A1 an die Zellmembran führt, kann man davon ausgehen, dass beide Proteine auch in vivo miteinander interagieren. Eine dominant negative HectD1-Mutante schließlich beeinflusst die Ubiqutinylierung von A1 und führt somit zu dessen Stabilisierung. Diese Daten legen nahe, dass HectD1 ein wichtiger negativer Regulator von A1/ Bfl-1 ist und dass HectD1 für die Regulierung der A1/ Bfl-1-Proteinmenge in (Krebs)zellen sehr wichtig ist. / The Bcl-2 family members A1 and its human orthologue Bfl-1 support survival of cells. Dysregulation of their expression contributes to cancer. Stability of A1/ Bfl-1 is controlled by ubiquitination followed by degradation via the proteasome. Using a yeast two-hybrid screen we identified the E3 ubiquitin-ligase HectD1 as potential A1/ Bfl-1-interacting partner. We confirmed interaction of these two proteins in mammalian cells. Only 87 amino acids of HectD1 are necessary for the interaction of the protein with A1. Membrane-bound HectD1 recruits A1 to the membranes further supporting the notion that the two proteins interact in vivo. Importantly, dominant negative versions of HectD1 interfered with ubiquitination of A1 stabilizing the protein. These findings indicate that HectD1 maybe an important negative regulator of the A1/ Bfl-1 anti-apoptotic protein, providing an important target for interfering with dysregulation of A1/ Bfl-1 in cancer.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Η απόπτωση ή προγραμματισμένος κυτταρικός θάνατος είναι ένα φαινόμενο στρατηγικής σημασίας για την ανάπτυξη, διαφοροποίηση και διατήρηση της ακεραιότητος ενός ιστού. Είναι πλέον γνωστό όμως ότι τόσο η υπέρμετρη όσο και η ανεπαρκής ενεργοποίηση των αποπτωτικών μηχανισμών μπορούν να οδηγήσουν στην εκδήλωση διαφόρων παθήσεων. Η απόπτωση χαρακτηρίζεται από συγκεκριμένες μορφολογικές και βιοχημικές μεταβολές ενώ η ρύθμισή της ελέγχεται γενετικά μέσω της εκφράσεως ή καταστολής διαφόρων ογκογονιδίων. Ιδιαίτερη σημασία για την ρύθμιση της αποπτώσεως έχουν τα γονίδια της οικογένειας του bcl-2. Συγκεκριμένα το bcl-2 και το bcl-xL καταστέλλουν την απόπτωση ενώ το bax την προάγει. Η ψωρίαση είναι μία χρόνια υποτροπιάζουσα φλεγμονώδης δερματοπάθεια η οποία προσβάλλει ποσοστό 1%-3% του γενικού πληθυσμού. Η ψωρίαση κατά πλάκας αποτελεί την συχνότερη μορφή της νόσου, η οποία χαρακτηρίζεται από την εμφάνιση σε γενετικώς προδιατεθειμένα άτομα, ερυθηματολεπιδωδών πλακών με σαφή αφορισμό από το πέριξ υγιές δέρμα. Χαρακτηρίζεται από έντονη υπερπλασία της επιδερμίδος, μειωμένη ωρίμανση των κερατινοκυττάρων, φλεγμονώδη διήθηση στην επιδερμίδα (CD8+) και το χόριο (CD4+) και νεοαγγειογένεση. Πρωταρχικό ρόλο στην έναρξη και διατήρηση των ψωριασικών αλλοιώσεων παίζουν τα Τ- λεμφοκύτταρα, τα οποία εκκρίνουν πληθώρα κυτταροκινών Τh1 τύπου. Παρά τα σημαντικά βήματα προόδου που έχουν γίνει τα τελευταία χρόνια στην έρευνα της ψωριάσεως, η παθογένεια της νόσου αυτής παραμένει ακόμα αδιευκρίνιστη. Τα κύτταρα της ψωριασικής επιδερμίδας αποτελούν έναν από τους ταχύτερα αναπτυσσόμενους και πολλαπλασιαζόμενους κυτταρικούς πληθυσμούς του ανθρώπινου οργανισμού. Θεωρητικά τουλάχιστον, η ακάνθωση της ψωριασικής επιδερμίδος θα μπορούσε να προκύπτει όχι μόνο από την αυξημένη μιτωτική δραστηριότητα των κερατινοκυττάρων αλλά επίσης και από έναν μειωμένο κυτταρικό θάνατο στις ζώσες στιβάδες της επιδερμίδος. Συμπερασματικά, στην παρούσα εργασία διαπιστώθηκε ότι υπό αγωγή και με τα δύο θεραπευτικά σχήματα επέρχεται ομαλοποίηση της εκφράσεως της bcl-2 στην ψωριασική διατυπωθεί η υπόθεση ότι τα ευρήματα της παρούσης εργασίας σε σχέση με αυτές τις δύο πρωτεΐνες συνδέονται με την υποστροφή των ψωριασικών αλλοιώσεων και όχι με τους μηχανισμούς θεραπευτικής δράσεως των χορηγηθέντων φαρμάκων. Τόσο υπό ασιτρετίνη όσο και υπό ανθραλίνη + καλσιποτριόλη παρατηρήθηκε μια σημαντική μείωση της εκφράσεως της πρωτεΐνης bcl-x στα κύτταρα της ακανθωτής στιβάδος της ψωριασικής επιδερμίδος. Το εύρημα αυτό σε συνδυασμό με την αντιαποπτωτική δράση της πρωτεΐνης bcl-x στην ψωριασική επιδερμίδα έχει ιδιαίτερη σημασία, αφού η μείωση της εκφράσεως της πρωτεΐνης αυτής υπό θεραπεία ίσως να συμμετέχει στους μηχανισμούς αντιψωριασικής δράσεως των χορηγηθέντων φαρμάκων αφού συνεπάγεται την είσοδο των ψωριασικών κερατινοκυττάρων στην απόπτωση. / Apoptosis or programmed cell death is a phenomenon of crucial importance for the growth, differentiation and maintenance of the integrity of tissues. However, it is well known that excessive or insufficient activation of apoptotic mechanisms can lead to the clinical manifestation of various diseases. Apoptosis is characterized by specific morphological and biochemical alterations, whereas its regulation is genetically determined by the induction or suppression of various oncogenes. Regulatory control of apoptosis is achieved through mechanisms in which proteins encoded by the bcl-2 gene family are involved. Thus, bcl-2 and bcl-xL inhibit the apoptotic process, whereas bax is a proapoptotic protein. Psoriasis is a chronic relapsing inflammatory genodermatosis affecting 1%-3% of general population. Plaque-type psoriasis, the most common form of the disease, is clinically characterized by the appearance of well-circumscribed erythematosquamous lesions in genetically predisposed individuals. On light microscopy, psoriatic lesions reveal marked acanthosis, incomplete maturation of keratinocytes, inflammatory infiltration of the dermis (CD4+) and the epidermis (CD8+) and enhanced angiogenesis. The role of T-cell-mediated immune mechanisms in the triggering of the disease is of particular importance. Despite the considerable progress in the research on various aspects of psoriasis, the pathogenesis of this disorder still remains unclear. Psoriatic keratinocytes represent one of the most rapidly developing and proliferating cellular populations of human body. Theoretically, acanthosis of psoriatic epidermis may have been caused from both, keratinocyte hyperproliferation and decreased cell death in the living epidermal layers. In the present study a normalization of bcl-2 protein expression was observed in psoriatic epidermis under both types of treatment. After 3 and 6 weeks of treatment, the reduction in the expression of bax in the spinous layer of psoriatic epidermis was higher under acitretin, as compared to that seen under topical application of anthralin + calcipotriol, as compared to the pretreatment status. Taking into account the limited differences in the expression of bcl-2 and bax proteins between the two groups (with the intensity of staining inside the normal limits), as well as the apparent absense of contributory role of bcl-2 and bax proteins in the regulation of the apoptosis of psoriatic keratinocytes, we can suppose that the findings of the present study are related to the regression of psoriatic lesions and not to the therapeutic action of the drugs used. Under both therapeutic regimens bcl-x immunoreactivity was reduced in the spinous layer of psoriatic epidermis. This finding, in combination with the antiapoptotic action of bcl-x protein in psoriatic epidermis, is of particular importance since the reduction in the expression of this protein under treatment is possibly implicated in the mechanisms of therapeutic action of these drugs.

The anthracycline quinone, doxorubicin (Adriamycin) is an antineoplastic agent that has substantial therapeutic activity against a broad variety of human cancers. Unfortunately, the use of this agent is limited by its cardiac toxicity, which is associated with free radical formation leading to apoptotic cell death. The goal of this work is to improve our understanding about doxorubicin induced cardiomyopathy and to identify compounds to limit doxorubicin induced cardiomyopathy. The knowledge gained here will have a generalized impact on all cardiac diseases involving oxidative stress and apoptosis. We show that doxorubicin induced apoptosis in primary neonatal rat cardiomyocytes can be attenuated by progesterone (PG). The anti-apoptotic action of PG was blocked by a progesterone receptor antagonist, Mifepristone (MF), indicating a progesterone receptor dependent pathyway. Affymetrix gene analyses found that PG treated cardiomyocytes increased the expression of 180 genes. Among the genes upregulated is NAD(P)H: Quinone Oxidoreductase-1 (NQO1) gene. NQO1 is a flavo-enzyme that can catalyze a two-electron reduction of Dox to a more stable hydroquinone, thereby acting as a defense mechanism against oxidative stress. The induction of NQO1 mRNA and NQO1 activity in cardiomyocytes was observed in a dose and time-dependent manner with PG treatment and was blocked by MF. Induction of NQO1 by b-naphoflavone, an inducer of NQO1, resulted in a decrease in caspase-3 activity. However, inhibition of NQO1 by dicoumarol did not attenuate the cytoprotective effect of PG. This data indicates that although induction of NQO1 can decrease Dox induced apoptosis, this is not the primary mechanism of cytoprotection induced by PG. Microarray analyses revealed that PG induced an increase of Bcl-XL mRNA. Inhibiting the expression of Bcl-XL using siRNA reduced the anti-apoptotic effect of PG, suggesting that Bcl-XL is a key player in PG induced cytoprotection. Western blot analyses indicated that PG induced the expression of Bcl-XL in a dose and time dependent manner consistent with the protective effect of PG. Induction of Bcl-XL by PG was blocked by cyclohexamide, but was not blocked by Actinomycin D indicating that a transcriptionally independent mechanism is responsible for the induction of Bcl-XL by PG. The activity of a bcl-x 3'UTR reporter was induced by PG and blocked by MF. These data suggest that PG may induce stabilization of the Bcl-X mRNA. We further explored the mechanism of PG induced Bcl-XL gene expression by comparing the effect of PG to two other steroids: corticosterone (CT) and retinoic acid (RA). Both CT and RA attenuate Dox induced apoptosis in cardiomyocytes. CT, but not RA or PG induced the activity of a GRE reporter plasmid. Analysis of the 5' region of the Bcl-XL promoter indicated that RA and CT, but not PG induced the activity of the 0.9kb region of the Bcl-XL promoter. The induction of the 0.9kb reporter plasmid by CT was glucocorticoid receptor dependent, since it was inhibited by MF. The Bcl-XL promoter does not contain any glucocorticoid or retinoid response elements, but does have AP-1 and NFkB response elements. CT, but not RA or PG induced the activity of an AP-1 reporter plasmid. RA, but not CT or PG induced the activity of an NFkB reporter plasmid. The induction of the 0.9kb Bcl-XL reporter plasmid by CT was blocked by expression of a dominant negative c-jun, TAM67 as well SB202190 indicating a nongenomic effect of CT in activating the Bcl-XL promoter through a p38 MAPK mediated AP-1 mechanism. Therefore although all three types of nuclear receptor ligands induce bcl-xL expression, the effect of CT is mediated by transcriptional activation by AP-1 signaling while NF-kB transcription factor appears to be involved in RA indced bcl-xL transcription.

Thèse (Ph. D.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 18 sept. 2007). Bibliogr.

Thèse (M.Sc.)--Université Laval, 2004. / Titre de l'écran-titre (visionné le 29 novembre 2004). Bibliogr.