Cryopreservation of oocytes is a challenge. Studies were conducted to vitrify mouse zygotes and cumulus-intact bovine oocytes from follicles of different diameters, small (≤ 4 mm) and medium (4 to 10 mm), using nylon mesh. The specific goals were to assess changes in apoptotic gene expression (Fas-FasL, Bax, Bcl-2, and survivin) in conjunction with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase assays. Mouse zygotes were exposed to increasing concentrations of ethylene glycol (EG), Ficoll-70 and sucrose in phosphate buffered saline (PBS) for vitrification on nylon mesh and plunged into liquid nitrogen. Warming resulted in 81.7% morphological survival. The rate of blastocyst formation was 59.9% for vitrified zygotes but, this was significantly lower than that of non vitrified embryos (66.2%). There was no difference in the hatching rates between groups. Both Fas and FasL mRNA were detected at the 4-cell and morula stages, suggesting Fas signaling was operational in early embryos. The level of expression of Bax mRNA tended to increase, while expression of survivin mRNA was not different for 2- and 4-cell embryos. Fragmented embryos showed an increase in Bax mRNA levels, while survivin mRNA level was reduced. In the second experiment, vitrification of bovine oocytes was carried out. Pre-cooled cryovials resulted in 98.9% morphological survival. The oocytes from small and medium size follicles had a significant impact on cleavage (53.7 ± 1.6% vs. 43.8 ± 1.6%, respectively) and blastocyst rates were 16.9 ± 1.0% and 11 ± 1.2%, respectively. Follicle size for oocytes had no impact on the expression of apoptotic genes. The Fas-FasL and Bax-Bcl-2 mRNA showed increased expression after vitrification, but tended to decrease after 9 h of maturation. Yet, results from TUNEL and caspase assays did not support the evidence of the downstream apoptotic signaling pathway in embryos. The semen utilized for in vitro fertilization in both vitrified and control oocytes responded differently in the 4 tested bulls than their field fertility record. The altered transcriptional activities of apoptotic genes, Fas-FasL and Bax-Bcl-2, and survivin were indicative of possible apoptotic activity in vitrified embryos and oocytes subjected to in vitro fertilization. / Ph. D.

Par‐4 is a pro‐apoptotic tumor suppressor that is mutated, suppressed or inactivated in cancer. Par‐4 exploits components of the extrinsic pathway to cause apoptosis selectively of cancer cells. This study identified Par‐4 as an essential component of the apoptotic pathway induced by TRAIL, which selectively targets cancer cells. RNA interference‐mediated knockdown of Par‐4 rendered cancer cells unresponsive to TRAIL‐induced apoptosis. Cells with knocked‐down levels of Par‐4 were deficient in the activation of the apoptosis‐initiator caspase‐8 and the apoptosis‐effector caspase‐3 in response to TRAIL. Par‐4 was identified as a critical mediator of membrane translocation of caspase‐8 and the adapter protein FADD. Surprisingly, Par‐4 was also found to interact with caspase 8 in untreated cells, and was cleaved at the N‐terminus at aspartic acid residue 123 in response to TRAIL. This, along with another cleavage by caspase‐9 effectively generated a fragment containing the functional module of Par‐4, the SAC domain, which is sufficient for apoptosis of cancer cells. Moreover, TRAIL activated caspase‐8 was also found to be involved in nuclear translocation of Par‐4, a crucial step during apoptosis induction by Par‐4. Together, our findings suggest that Par‐ 4 is an essential downstream target of caspase‐8 that is activated by TRAIL signaling and that, in turn, activates caspase‐8 and the downstream apoptotic pathway in response to TRAIL.

During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound in order to retain significant catalytic activity. Nevertheless, in apoptotic cells, the vast majority of processed caspase-9 is paradoxically observed outside of the complex. We demonstrate herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Due to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based “molecular timer”, wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer “ticks” over. To understand the physiological relevance of molecular timer in vivo, we are currently generating caspase-9 knock-in mouse models. These mouse models will enhance our understanding of the importance of caspase-9 processing within the apoptosome. / text

No description available.

Prostate cancer is one of the most malignant tumors in the world. It has been demonstrated that prostate cancer could metastasis to bones, bladder, rectum, and lymph nodes. It is the most common type of cancer found in adult males, and the mortality is elevated. From screening our chemical library of 398 small molecule compounds, we have found that TCH derivatives showed anti-proliferative activities using prostate cancer cell line PC-3. The results of MTT assay allow us to identify TCH-1038w as potential candidat for developing anticancer drug. Morphological investigation on PC-3 cells after TCH-1038w treatment showed that PC-3 cells rounded up and combined with cell shrinkage, abridge, membrane blebbing. Our results of flowcytometric analysis showed that TCH-1038w can cause the percentage increase of sub-G1 that indicated the DNA fragmention in TCH treated PC-3 cells. By DAPI staining , we observed that TCH-1038w can induce the DNA fragmentation in PC-3 cells. Moreover by immunoblotting analysis, we have demonstrated that procaspase 3 and PARP were cleavaged and activated after TCH treatment. These results indicsted TCH drugs can induce caspase activation, DNA fragmentation, and consequently cause apoptotic cell death. Together, TCH-1038w may serve as a potential chemotherapy candidate for treating prosate cancer in the future.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 116-142.

No description available.

Les caspases forment une famille de protéases à cystéine impliquées dans divers processus comme Fapoptose et l’inflammation. Durant l'apoptose, une forme de mort cellulaire programmée, les caspases initiatrices (caspases 8, 9 et 10) activent par protéolyse les caspases exécutrices 3 et 7. Ces dernières cliveront par la suite divers substrats cellulaires pour ainsi mener au démantèlement contrôlé de la cellule qui est propre à l'apoptose. Durant mon doctorat, je me suis intéressé à l’implication de la caspase 7 durant l'apoptose, autant la façon dont elle est activée que la manière dont elle reconnait ses substrats. D’abord, j'ai voulu étudier l'implication des sites de clivage du connecteur interdomaine (CID) de la caspase 7 dans son activation par les caspases initiatrices et les déterminants primaires sous-jacents impliqués dans cette activation. Cette étude a révélé l'importance de la localisation du site de clivage dans le CID de la caspase 7 pour une activation adéquate par les caspases initiatrices. La mutagénèse de ces sites nous a permis de constater que la séquence des sites chez la caspase 7 était presque optimale pour son activation par les caspases 8 et 9 et qu’il contient des déterminants importants pour l'activité enzymatique de la caspase 7. Mes travaux soulignent également l’importance de la longueur du CID pour l’activité pré-clivage de la caspase 8, mais pas de la caspase 7. En somme, ces travaux ont permis d’éclaircir l’importance des sites de clivage de caspase 7 pour sa reconnaissance par les caspases initiatrices et pour son activité catalytique. Ces travaux ont fait l'objet du premier article de cette thèse. Puis, je me suis intéressé à la reconnaissance de certains substrats de la caspase 7 par des sites de reconnaissance situés à l'extérieur de la pochette de liaison du substrat, les exosites. J'ai identifié un exosite situé dans le domaine N-terminal de la caspase 7 qui est principalement contenu dans une séquence polybasique de quatre résidus lysine (K38-41). Cet exosite améliore le clivage des protéines poly(ADP ribose) polymérase 1 (PARP-1) et p23 sans toutefois changer l’activité catalytique basale de la caspase 7. Il est transférable sur la caspase 3 et est capable de lier PARP-1 seul. Cet exosite est également utilisé lors de l'apoptose pour favoriser le clivage de certains substrats par la caspase 7. L’existence de ce site explique les différentes activités catalytiques des caspases 7 et 3 sur des substrats apoptotiques précis. En somme, ces travaux constituent la première démonstration de l'existence d'exosites chez les caspases. Mes travaux ont donc permis de mieux comprendre la caspase 7, autant les mécanismes de son activation que ceux qu’elle utilise pour choisir ces substrats apoptotiques.

No description available.

Apoptosis and cell survival are two seemingly opposing fate-determining processes that are regulated by distinct and complex signaling pathways. Caspase-8, an apical caspase, plays a pivotal regulatory role in initiating apoptosis. c-Src, a prototypical member of the Src family kinases (SFKs), regulates a myriad of cellular processes including cell mitogenesis, proliferation, growth and migration. Although the regulation of caspase-8 by c-Src has been suggested, the reciprocal regulation of these two seemingly opposing signaling molecules, caspase-8 and c-Src, has never been explored. To study this reciprocal regulation, we asked three questions. (1) Can active caspase-8 negatively regulate c-Src activity to allow the propagation of apoptosis? (2) Can c-Src negatively regulate caspase-8 activity to prevent the propagation of apoptosis? (3) Can caspase-8, when its enzymatic activity is inhibited, further promote c-Src activity to allow the propagation of cell survival? To address these questions, we first investigated the effect of active caspase-8 on the activation and activity of c-Src. We discovered that active caspase-8 inhibited c-Src activation and some of its downstream effectors. Next, we investigated whether c-Src could tyrosine phosphorylate caspase-8. We discovered that c-Src could phosphorylate caspase-8 at multiple tyrosine sites. We then examined whether tyrosine phosphorylated caspase-8 prevents apoptosis. We found that phosphorylation of caspase-8 at Y465 prevented its cleavage, and activity towards activating caspase-3 and towards causing cell morphological changes associated with apoptosis. Finally, we studied whether tyrosine phosphorylation of caspase-8 could further promote the activation of c-Src. We showed that phosphorylation of caspase-8 at both Y465 and Y397 resulted in the activation of c-Src and extracellular signal-regulated kinase 1/2 (Erk1/2). In conclusion, this work demonstrated the reciprocal regulation of two opposing signaling molecules, caspase-8 and c-Src. These results also suggest an elegant mechanism for a cell to commit efficiently and rapidly to a fate-determining process, either apoptosis or survival, by further suppression of the opposing signaling pathway.