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

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

Ceramides are bioactive lipids that can promote splicing of apoptosis-related genes, including caspase 9 and BCL-x. A recent study demonstrated that expression of neutral sphingomyelinase (NSMase), an enzyme that hydrolyzes sphingomyelins to generate ceramide, is regulated by Group VIA phospholipase A2 (iPLA2β)-dependent mechanism during β-cell apoptosis. This prompted us to hypothesize that iPLA2 is upstream of ceramide generation in the process regulating splicing of apoptotic genes. To test this, Jurkat T cells were treated with the selective inhibitor of iPLA2β, bromoenol lactone (BEL), RNA was isolated and converted to cDNA, and caspase 9 and BCL-x mRNA viii species were amplified using RT-PCR. Inhibition of iPLA2β activity with BEL caused a significant shift in splicing favoring variants encoding the anti-apoptotic forms of caspase 9 (caspase 9b) and BCL-x (BCL-x(L)). This shift was consistent with previously reported effects of ceramide and suggested that iPLA2β regulates splicing of these pre-mRNAs. We next determined whether iPLA2β regulates splicing events during a biological response. Caspase-9 and BCL-x splice variants were compared in human and mouse islets, mouse islet cell lines, and in rat insulinoma (INS1) cells. INS-1 insulinoma cells were treated with thapsigargin to induce ER stress, which can eventually lead to apoptosis. Thapsigarin-treated INS-1 cells exhibited an increase in the ratio of BCL-x(s) (pro-apoptotic) to BCL-x(L) (anti-apoptotic) but BEL prevented this shift in splicing. Splicing data obtained from genetically modified rodent mice (iPLA2β knockouts and transgenics) also demonstrated the involvement of iPLA2β in alternative splicing. Together, these observations indicate that iPLA2β plays an important role in the regulation of pre-mRNA splicing of key apoptotic factors. Our findings therefore suggest a novel role for iPLA2β in determining whether cells survive or undergo apoptosis.

The pro-apoptotic, caspase 9a, and the anti-apoptotic, caspase 9b, are derived from the caspase 9 gene by alternative splicing. This study demonstrates that the alternative splicing of caspase 9 is dysregulated in a large percentage of non-small cell lung cancer (NSCLC) tumors of the adenocarcinoma type. Furthermore, modulation of the levels of splice variants of caspase 9 had dramatic effects on the anchorage-independent growth and tumorigenic capacity of NSCLC cells. Due to these findings, the molecular mechanisms regulating the post-transcriptional processing of caspase 9 were therefore examined and an exonic splicing silencer (ESS) regulating the pre-mRNA processing of caspase 9 was identified. To study the possible RNA trans-factors interacting with this RNA sequence, we utilized an electromobility shift assay (EMSA) coupled with competitor studies and demonstrated three specific protein:RNA complexes for this ESS. Affinity purification and mass spectrometry analysis identified hnRNP L as part of these protein:RNA complexes. Downregulation of hnRNP L induced a significant increase in caspase 9a/caspase 9b mRNA ratio, which translated to the protein level. Expression of hnRNP L verified the siRNA specificity lowering the caspase 9a/9b ratio, but expression of hnRNP L produced the contrasting effect in non-transformed cells suggesting a post-translational modification specific for NSCLC cells. Indeed, the phospho-status of hnRNP L was significantly increased in NSCLC cells, and mutagenesis studies identified Ser52 as a critical residue regulating the ability of hnRNP L to repress the inclusion of the exon 3,4,5,6 cassette into the mature caspase 9 mRNA. The biological relevance of this mechanism was demonstrated by stable downregulation of hnRNP L in NSCLC cells, which induced a complete loss of both anchorage-independent growth and tumorigenic capacity. This effect of hnRNP L downregulation was due to distal modulation of the alternative splicing of caspase 9 as the loss of both phenotypes was “rescued” by ectopic expression of caspase 9b. Therefore, this study identifies cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase 9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interferes with hnRNP U for binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Overall, a mechanistic model has been revealed where hnRNP U competes with hnRNP L for C9/E3 binding to enhance the inclusion of the four-exon cassette, and this splice-enhancing effect is blocked by the AKT pathway via phosphorylation of hnRNP L. As to the latter aim, it is unknown about the mechanistic roles of caspase-9b besides the inhibitory effect on caspase-9a processing. In this study, caspase-9b has been demonstrated to have a dual function in regulating the survival/oncogenic nuclear factor κB (NF-κB) pathway, which is independent from modulating caspase-9a activation. In particular, caspase-9b has been shown to activate the canonical arm and inhibit the non-canonical arm of the NF-κB pathway by destabilizing NF-κB inhibitor alpha (IκB-α) and NF-κB-inducing kinase (NIK). Importantly, this new role for caspase-9b contributes to the enhanced survival and anchorage-independent growth of NSCLC cells conferred by caspase-9b expression. Further mechanistic studies have demonstrated a direct association of caspase-9b with the cellular inhibitor of apoptosis 1 (cIAP1), a regulatory factor in both arms of the NF-κB network, via its IAP-binding motif. Through this interaction, caspase-9b induces the E3 ligase activity of cIAP1, which regulates NF-κB activation, and promotes the survival, anchorage-independent growth and tumorigenicity of NSCLC cells. Overall, a novel tumorigenic mechanism has been identified, by which alternative mRNA processing regulates the NF-κB signaling independent of external agonist.

In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies identified serine 199, serine 201, serine 227, and serine 234 as critical residues regulating the alternative splicing of caspase 9, as well as playing a role in the anchorage-independent growth of A549 cells. Since dysregulation of this splicing mechanism correlated with NSCLC tumors/cell lines and constitutively active Akt, oncogenic factors for NSCLC known to activate the PI3-Kinase/Akt pathway were examined in HBEC-3KT cells. In contrast to k-ras V12 expression, the overexpression/mutation of EGFR affected the alternative splicing of caspase 9 in a pro-oncogenic manner, dramatically lowering the caspase 9a/9b mRNA ratio. Stable downregulation of caspase 9b by shRNA blocked the ability of E746-A750 del EGFR expressing HBEC-3KTs to induce anchorage-independent growth, suggesting a role for caspase 9b as a cooperative oncogenic factor. These findings were further corroborated by the ability of caspase 9b expression to completely block the inhibition of clonogenic colony formation by erlotinib. Therefore, this study demonstrates that oncogenic factors activating the PI3-Kinase/Akt pathway regulate the alternative splicing of caspase 9, to produce caspase 9b, via a coordinated mechanism involving the phosphorylation of SRp30a. In additional studies, we demonstrate that the PI3-Kinase/PKCι pathway, a pathway important for cancer cell survival and transformation of lung epithelial cells, regulates the alternative splicing of Bcl-x pre-mRNA via modulation of SAP155 expression to produce an anti-apoptotic phenotype in NSCLC. Therefore, these studies link oncogenic mechanisms in NSCLC to the therapeutically relevant and distal target mechanisms of caspase 9 and Bcl-x pre-mRNA splicing.

La mort cellulaire programmée ou apoptose est un mécanisme conservé chez les eucaryotes multicellulaires qui contribue au développement embryonnaire et à l'homéostasie cellulaire des organismes. Dans les cellules vivantes, l'activité des protéases qui exécutent le programme de mort cellulaire, les caspases, est contrôlée par des signaux de survie provenant de l'environnement cellulaire. Les caspases initiatrices de l'apoptose régulée par l'environnement, la caspase 9 et la caspase 8 sont activées respectivement par l'apoptosome et par les récepteurs de mort. Les signaux environnementaux, parmi lesquels le contact avec la matrice extracellulaire ou la présence de facteurs de croissance, activent des voies de signalisation contrôlant la machinerie de mort cellulaire. La voie des MAPK1/3 est une voie de signalisation contrôlée par le proto-oncogènes Ras et comportant les kinases Raf, MEK1/2 et MAPK1/3 (ERK2/1 ou p42/p44). La voie des MAPK1/3, qui est impliquée dans la prolifération et la différentiation cellulaire, joue un rôle essentiel dans la survie cellulaire. L'objectif de cette thèse a été de caractériser les mécanismes moléculaires impliqués dans le contrôle de la mort cellulaire par la voie des MAPK1/3. Ce travail est basé sur l'utilisation d'une forme active et inductible de la kinase Raf-1 (DRaf-1:ER) dont l'activation forte et prolongée correspond à une induction pathologique de la voie des MAPK1/3. Nous avons montré que, selon le type cellulaire, l'activation de deltaRaf-1:ER favorise la survie ou la mort cellulaire. Dans les cellules fibroblastiques CCL39, l'activation de deltaRaf-1:ER protège de la mort cellulaire mitochondriale induite par la privation en sérum du milieu de culture. Dans ces conditions, nous avons montré que la stimulation de Raf-1 :ER bloque l'activation de la caspase-9 mais n'empêche pas la délocalisation du cytochrome c, la multimérisation d'APAF1 ni le recrutement de la procaspase 9 dans l'apoptosome. Ce mécanisme post mitochondrial de protection contre la mort cellulaire dépend de la néo-synthèse des protéines et nécessite une activité continue de la kinase MEK. A l'inverse, dans les cellules HEK 293 issues de rein embryonnaire et présentant des caractéristiques neuronales, nous avons montré que l'activation soutenue de la voie des MAPK1/3 par DRaf1-ER induit une mort cellulaire massive. Celle-ci est caractérisée par l'activation des caspases et la fragmentation de l'ADN. La mort cellulaire est détectée plus de 24 heures après l'activation de Raf1-ER, elle est maximale à 48h. L'induction de la mort cellulaire ne requière la synthèse protéique que durant la phase précoce d'activation mais nécessite l'activité continue du module MEK/MAPK. La mort cellulaire résulte de l'activation de la caspase 8 et n'implique pas la voie mitochondriale, elle est caractérisée par une vacuolisation importante du cytoplasme des cellules qui l'apparente à une forme particulière d'apoptose. L'inactivation des fonctions du récepteur fas et de son adaptateur FADD indique que le processus d'activation de la caspase 8 est indépendant de la voie des récepteurs de mort. L'ensemble de ces travaux apporte des connaissances nouvelles sur le contrôle de la mort cellulaire par la voie Raf/MAPK1/3. Nous avons montré que la voie de signalisation peut, selon le contexte cellulaire, favoriser la survie cellulaire ou induire la mort. Dans les deux cas, le contrôle de la mort cellulaire dépend à la fois de la synthèse protéique et de mécanismes post-traductionnels. Les mécanismes moléculaires affectés par l'activation prolongée des MAPK1/3 seraient impliqués aussi bien dans la résistance des cellules tumorales aux traitements proapoptotiques que dans le développement des maladies neurodégénératives.

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 148-168).

A novel marine active polypeptide (PCF), isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCF. Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining. The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42-5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.