Apoptosis serves as a natural barrier to cancer development, and the resistance to apoptosis represents one of the key capabilities acquired during tumor development or progression. Impairment of the intrinsic apoptotic pathway exemplifies one of the established mechanisms of constitutive or acquired drug resistance. As most of the currently used cytotoxic drugs initiate tumor cell death by direct or indirect triggering of the intrinsic apoptotic pathway, impairment of the intrinsic pathway is associated with therapy failure. Targeting of the death receptors, however, enables induction of apoptosis even in the chemotherapy resistant cancer cells. TRAIL is a death ligand belonging to the TNFα superfamily that specifically kills tumor cells while sparing healthy tissues. Much enthusiasm has been generated for TRAIL as a highly promising targeted anti-cancer agent. However, many primary tumors have been shown to be TRAIL resistant. In attempt to overcome such an intrinsic TRAIL resistance a wide array of agents have been shown to sensitize tumor cells to TRAIL. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. In this study we analyzed the sensitivity of diverse hematologic malignancies to TRAIL-induced apoptosis and measured the...

Transcription and DNA replication of herpes simplex virus type 1 (HSV-1) occur in nuclear domains adjacent to structures named ND10. The HSV-1 single-stranded DNA binding protein ICP8 localizes to these nuclear domains to direct the assembly of the pre- and replication compartments. Inhibition of cyclin dependent kinases with roscovitine inhibits HSV-1 DNA replication, even in the presence of all required HSV-1 proteins, at an unidentified step. Here I show that roscovitine inhibits the localization of pre-expressed ICP8 to new replication sites. Therefore, the inhibition of HSV-1 DNA replication occurs at a step prior to initiation. I next evaluated the mechanisms of inhibition of proper ICP8 localization. ICP8 was extracted at lower salt concentrations from roscovitine-treated than untreated cells, but the affinity of ICP8 for ssDNA in vitro was not affected. I propose that roscovitine inhibits HSV-1 DNA replication by inhibiting DNA accessibility. I also discuss alternative mechanisms.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.

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No description available.

Herpes Simplex Virus Type 1 (HSV-1) establishes latent infections in neurons in vivo and lytic infections in epithelial cells and fibroblasts. During latent infections, HSV-1 transcription is restricted and the genomes are not replicated. Latent HSV-1 genomes are chromatinized, such that digestion with micrococcal nuclease (MCN) releases DNA fragments with sizes characteristic of nucleosomal DNA. During lytic infections, in contrast, all HSV-1 genes are expressed, the genomes are replicated, and their digestion produces primarily heterogeneously sized fragments. However, as evaluated by ChIP assays, HSV-1 DNA interacts with histones during lytic infections, although in most cases only a small percentage of HSV-1 DNA co-immunoprecipitates with histones (or is cleaved to nucleosome sizes following MCN digestion). Therefore, although current models propose that chromatin regulates HSV-1 transcription, it remains unclear how the association of histones with only a small percentage of HSV-1 DNA can globally regulate viral transcription. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. My objective was therefore to evaluate the biophysical properties of the HSV-1 DNA-containing complexes during lytic infection. Differing from pervious studies, however, I used classical chromatin purification techniques. I show that most HSV-1 DNA is in unstable nucleoprotein complexes and, consequently, more accessible to MCN than DNA in cellular chromatin. This HSV-1 DNA is protected from MCN redigestion only after crosslinking, similar to unstable cellular nucleosomes. HSV-1 DNA is in such complexes throughout lytic infection. Using unrelated small-molecule inhibitors, I further show that inhibition of HSV-1 transcription is associated with a decrease in MCN accessibility of HSV-1 DNA. Roscovitine, a cyclin-dependent kinase inhibitor, prevents activation but not elongation of IE, E, and L HSV-1 transcription. Consistent with a functional association between accessibility and transcription, roscovitine only decreases the accessibility of DNA templates of which it also inhibits transcription, independent of specific promoter sequences. In summary, I show that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infection and that accessibility to HSV-1 DNA likely plays a key role in regulating HSV-1 transcription.

Les agents permettant de créer des dommages sur l'ADN sont principalement utilisés dans les traitements contre le cancer. L'activation de points de contrôle du cycle cellulaire après lésion de l'ADN entraîne un arrêt du cycle des cellules. De la connaissance des mécanismes moléculaires de l'arrêt du cycle cellulaire par ces points de contrôle dépend l'efficacité du traitement. Dans les cellules humaines, ces points de contrôle sont primordiaux puisque leur inactivation entraîne la carcinogenèse (génération de cancers). Après traitement par des agents chimiothérapiques et des rayons X, les cellules s'arrêtent en phase G-1 et G-2/Mitose (M) du cycle cellulaire. Si de nombreuses études ont permis de clarifier les mécanismes de l'arrêt en phase G-1 pour des cellules dont l'ADN est endommagé, peu de données sont disponibles concernant l'arrêt en phase G-2/M. Parmi ces points de contrôle, le point de contrôle G-2/M est particulièrement important car il prévient l'entrée en mitose (phase M) des cellules dont l'ADN est endommagé. <br /> Nous avons analysé le rôle du complex appelé APC (Anaphase-Promoting Complex) dans les points de contrôle G-2/M après lésion de l'ADN. Les lésions de l'ADN sont induites dans les cellules synchronisées en phase S. Suite à ces dommages, les cellules montrent un retard et s'arrêtent en phase G-2 avec 4N chromosomes. Afin d'identifier les bases biochimiques de l'arrêt en G-2/M après traitement avec des agents endommageant l'ADN, nous allons concentrer notre recherche sur un complexe composé de multiples protéines possédant une activité de ligation de l'ubiquitine de type E3 (ubiquitin-ligase E3). Ce complexe APC est necessaire pour la dégradation des inhibiteurs d'entrée en anaphase, cyclins mitotiques, et plusieurs kinases mitotiques pour la complétion de la sortie de la mitose. Nous avons analysé et déterminé que l'absence d'activité du complexe APC inhibe l'activation du point de contrôle G-2/M lors de dommages de l'ADN.

La ICSI porcina es una herramienta con gran potencial aplicativo en diversos campos, entre los que destacan la producción de animales transgénicos, y la recuperación de razas en peligro de extinción. Aunque en la actualidad existen referencias de obtención de descendencia viva, el rendimiento es inferior al de otras especies, posiblemente debido al desconocimiento de las condiciones idóneas, y la dificultad de los cigotos para alcanzar el estadío de blastocisto in vitro. El presente trabajo se llevó a cabo para determinar diferentes factores que podrían afectar al rendimiento de la técnica, estudiando el efecto de 1) la secuencia de cultivo de los zigotos recién inyectados; 2) modificaciones en el sistema de MIV tradicional, y por último 3) la activación exógena del ovocito mediante la inyección de inositol trifosfato con el espermatozoide. El objetivo global de este estudio fue el de incrementar el rendimiento final de la ICSI en la especie porcina. / ICSI in pigs is a tool with an important applicable potential in diverse fields. One of this is the production of transgenic animals, and the conservation of endangered species. Even though there are some cases of living offspring, its output is still quite low comparing to other species, possibly due to unknown factors referring to ideal conditions for the development, and to the difficulty of the zygotes to reach the blastocyst stage in vitro. The goal of this study was to evaluate different factors affecting the ICSI performance. This was done by studying 1) the sequence of culture of the injected oocytes; 2) In vitro maturation (IVM) modifications, through meiotic inhibitors, such as roscovitine, and changes in IVM duration time, and finally 3) the exogenous oocyte activation through inositol triphosphate (InsP3) injection together with the sperm. The main objective of this study was to increase the final performance of ICSI in pigs.