No description available.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

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.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22688号 / 医博第4632号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Apoptosis is the process by which organisms eliminate excess, damaged or hazardous cells in a controlled manner. This process is controlled by the Bcl-2 family of proteins. Bcl-2 and Bcl-XL are anti-apoptotic paralogues that can replace CED-9, the sole homologue in C. elegans. It has therefore been assumed that Bcl-2 and Bcl-XL are replaceable and functionally identical. However, evidence in some mammalian cells indicates that this may not be the case. The purpose of this project was to exhaustively compare the anti-apoptotic activities of Bcl-2 and Bcl-XL in one cell type. As Bcl-2 and Bcl-XL have been found to localize to the ER and the outer mitochondrial membrane, we also determined whether subcellular location affects the function of these proteins differently. MCF-7 breast cancer cells were stably transfected with Bcl-2 and Bcl-XL alternatively targeted to the ER or mitochondria, and exposed to various doses of doxorubicin; PARP cleavage was measured using quantitative Western blotting as an indication of apoptosis to obtain EC₅₀ values in the different cell lines. The levels of both Bcl-2 and Bcl-XL affected anti-apoptotic activity; specific degradation of both proteins was noted at higher doses of doxorubicin. Nevertheless, the results indicated clearly that there was a difference between Bcl-2 and Bcl-XL. Using EC₅₀ values Bcl-XL mutants were at least 8 times more protective than Bcl-2 mutants. Furthermore, most of the cleavage products of PARP in Bcl-XL expressing clones were due to non-caspase-7 proteases, a pattern not seen with Bcl-2. Bcl-2 and Bcl-XL localized to mitochondria were most effective, while cytosolic and ER localized Bcl-XL were less effective, and Bcl-2 at these sites did not inhibit apoptosis. / Thesis / Master of Science (MSc)

La protéine proapoptotique Bax joue un rôle fondamental au cours de la voie intrinsèque de l’apoptose. Elle participe au déclenchement de la mort en permettant la libération de facteurs apoptogéniques mitochondriaux vers le cytosol. Un des points-clé de la fonction de Bax est son activation, caractérisée par la transition entre une forme cytosolique, globulaire et inactive de la protéine et une conformation mitochondriale, membranaire et active. Les différentes étapes de l’activation de Bax sont relativement bien connues, toutefois un grand nombre de questions reste en suspens quant-à leur régulation.Ce travail s’est focalisé sur la régulation de l’activation de Bax par les kinases Akt et GSK-3β ainsi que par la protéine antiapoptotique Bcl-xL . Ces régulations ont été caractérisées en exprimant la protéine Bax humaine chez la levure Saccharomyces cerevisiae, un paradigme d’étude simplifié qui permet d’accéder aux composantes individuelles des mécanismes d’activation de Bax.Les données obtenues suggèrent qu’il existe deux étapes régulées indépendamment au cours de l’activation de Bax. Nous avons montré que la protéine kinase GSK-3β favorise l’adressage de Bax vers la mitochondrie mais qu’elle n’entraîne pas un changement de conformation suffisant à son activation complète et à la perméabilisation de la membrane mitochondriale externe. Des changements de conformations complémentaires de Bax sont requis pour conduire à une forme capable d’entraîner la libération des facteurs apoptogéniques mitochondriaux. La protéine kinase Akt est impliquée dans le contrôle de Bax via la phosphorylation de la sérine 184 et participe à l’inhibition de l’apoptose. Nous avons mis en évidence qu’une mutation phosphomimétique de la sérine 184 ou l’expression d’Akt, en l’absence de partenaires antiapoptotiques, stimulent un changement de conformation de Bax vers une forme active. Akt semble donc plus jouer un rôle sur la conformation de Bax qu’entraîner une inhibition directe. La présence de protéines antiapoptotiques serait ainsi requise pour l’inhibition de Bax en présence d’Akt.D’autre part, nous nous sommmes intéressés aux mécanismes d’action de la protéine antiapoptotique Bcl-xL . Nous avons déterminé que Bcl-xL pouvait favoriser l’adressage de Bax vers la membrane mitochondriale tout en exerçant un rôle antiapoptotique. Ceci suggère que Bcl-xL intervienne dans le contrôle des étapes tardives de l’activation de Bax. Ce contrôle est dépendant d’une interaction stable entre les deux protéines. Inversement, un variant de Bcl-xL n’interagissant que de façon transitoire avec Bax (Bcl-xL ∆C) entraîne l’activation de Bax. Cette observation est en faveur d’un modèle d’activation indirecte de Bax consécutive à la rupture de l’interaction avec Bcl-xL et dans lequel les protéines à BH3-seulement telles que Bad joueraient un rôle crucial. / Proapoptotic protein Bax plays a major role during apoptosis intrinsic pathway. Bax promotes cell death by inducing the release of apoptogenic factors from mitochondria to cytosol. Bax activation is a key step of its function which involves a change from a globular, cytosolic and inactive conformation to an active mitochondrial, membrane inserted conformation. Bax activation substeps are rather well known, however their regulation remains to be characterized.This work focuses on the study of the regulation of Bax activation by kinases Akt and GSK-3β and by antiapoptotic protein Bcl-xL . Human Bax regulations have been studied by expressing the protein in yeast Saccharomyces cerevisiae which represents a simplified paradigm for the understanding of the individual components of Bax activation mecha- nisms.Our data suggest that there are two independently regulated steps during Bax activation. We showed that GSK-3β expression led to Bax addressing to mitochondria but was not sufficent to promote a complete activation and mitochondrial outer membrane premeabilization. Further conformational changes are required to promote Bax full activation and the release of mitochondrial apoptotic factors. Protein kinase Akt is involved in Bax activation control through the phosphorylation of serine 184 and contributes to apoptosis inhibition. We observed that either a phosphomimetic mutation of serine 184 or coexpression of Akt, in the absence of antiapoptotic partners, were responsible of Bax conformational change into an active form. By itself Akt did not inhibit Bax but appeared more likely to control its conformational change. Thus, implication of antiapoptotic proteins seems to be critical in a model of Bax inhibition by Akt.Furthermore, we tried to understand the molecular mechanisms of antiapoptotic protein Bcl-xL inhibition on Bax. We determined that Bcl-xL could increase Bax mitochondrial localization while leading to its inhibition suggesting that Bcl-xL controled Bax late activation steps. Bax inhibition was dependent on a stable interaction with Bcl-xL . Conversely, a variant of Bcl-xL having a transitory interaction with Bax (Bcl-xL ∆C) was able to promote Bax activation. This supports a model of Bax indirect activation following the rupture of interaction with Bcl-xL in which BH3-only proteins like Bad would play an important role.