Browsing by Subject "Apoptosis"
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Item Aging and apoptosis(1994-07-07) Yarbrough, W. C.Item Apoptosis Determinants in Drosophila Melanogaster(2007-12-17) Chew, Su Kit; Abrams, John M.Apoptosis is a form of programmed cell death (PCD) that is governed by a core set of genes conserved across diverse metazoan phyla. Cells dying by apoptosis exhibit a characteristic series of morphological and biochemical changes that is also conserved. This form of PCD plays pivotal roles in homeostatic regulation of cell numbers, developmental sculpting of organs, damage and infection responses; conversely, its disregulation has profound implications in diseases such as cancers, immune disorders infertility and dystrophies. Common parallels in the regulation of the core apoptosis machinery have been elucidated in human and experimental model organisms, though many fundamental questions in our understanding of its regulation remain. A conserved node in the apoptosis pathway is the apoptosome, comprising the apical caspase and its adaptor protein. To understand the functions of this node, I generated a null allele of the apical caspase Dronc in the experimental model organism Drosophila melanogaster. Dronc is required for developmentally regulated apoptosis in multiple tissues during embryogenesis and larval development. Failure of apoptosis correlated with tissue hyperplasia. Notably, the removal of Dronc eliminated the cellular apopototic response to stresses in cells. In some of the stress contexts tested, Dronc depletion partially rescued cell viability to the same levels as pan-caspase inhibition by small peptide inhibitors, suggesting that Dronc functions map specifically to caspase activation and apoptosis. These and similar observations in its adaptor protein Dark point to the apoptosome as a key node for apoptosis in Drosophila. From these observations, I sought to use the induced apoptosis cellular response as a means to identify novel components and regulators in the apoptosis pathway. I optimized a cell culture system for high-throughput cell-based screening using RNA interference (RNAi) mediated gene silencing and a synthetic antagonist of inhibitors of apoptosis proteins (IAPs). From a genome-wide Drosophila RNAi library, I identified 42 potential genes required for apoptosis, of which I characterized 13 highly validated targets for their requirements in multiple stress contexts. One of these hits, Tango7, regulates pro-Dronc protein and represents an unprecedented point of apoptosis regulation. Collectively, my studies bolster the model for the crucial requirement of the apoptosome in apoptosis and identify new regulation entry-points into the apoptosis pathway.Item Bcl-2 Function in Drosophila(2007-12-17) Galindo, Kathleen A.; Abrams, John M.Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced an authentic null allele at the Debcl locus. Although gross development and lifespans were unaffected, we found that debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf-1counterpart, dark, but was not required for killing by RPR proteins. Surprisingly, in a model of caspaseindependent cell death, we found that heterologous killing by Murine Bax required debcl to exert its pro-apoptotic activity. DebclKO mutants were also significantly affected for mitochondrial density. Taken together, these findings suggest that evolutionary functions impacting mitochondrial properties represent ancient activities which preceded the evolution of these proteins as central regulators of PCD.Item Biochemical Pathways in Apoptosis(2005-05-03) Nijhawan, Deepak; Wang, XiaodongCaspases are a family of proteases that once activated execute apoptosis, a cellular suicide pathway. Activated caspases have a unique property to cleave and activate themselves. Once the first caspase is activated, it generates a chain reaction resulting in robust caspase activity and rapid death. The central question in apoptosis is to understand how the first caspase is activated. To address this question, we present an assay that recapitulates de novo caspase activation in vitro. We describe how this assay was used to purify three proteins that were sufficient to reconstitute caspase activation in vitro: Apaf-1, cytochrome c, and caspase-9. The mechanism of caspase activation in vivo, however, is complicated by these proteins subcelluar localization. In the living cell, Apaf-1 and caspase-9 are cytoplasmic whereas cytochrome c is mitochondria, however, during apoptosis, cytochrome is released to the cytoplasm. In vitro, cytochrome c induces the formation of a stable Apaf-1/caspase-9 complex and caspase-9 autoactivation suggesting that cytochrome c release from the mitochondria to the cytosol is the rate-limiting event that leads to caspase activation. This conclusion shifted the focus of our studies upstream from what initiates caspase activation to what triggers cytochrome c release. The second part of the dissertation uses a biochemical approach to identify how cytochrome c release is regulated after exposure to ultraviolet light. Ultraviolet light irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of such a response revealed that the initial step leading to cytochrome c release is the complete disappearance of the mRNA of Mcl-1, an anti-apoptotic member of the Bcl-2 family. This event leads to the elimination of Mcl-1 protein from cells due to the short half-life of its protein. The block or delay of Mcl-1 disappearance by either proteasome inhibitors or Mcl-1 over-expression prevents subsequent steps of this apoptotic pathway including the translocation of Bax and Bcl-xL from cytosol to mitochondria and dephosphorylation of BimEL on mitochondria. These sequential events lead to the oligomerization of Bax and Bak on the mitochondria, cytochrome c release and caspase activation.Item Characterization of a Small Molecule Smac Mimetic's Role in Inducing Apoptosis in Human Cancer Cells(2008-09-19) Yalcin-Chin, Asligul; Wang, XiaodongInhibitor of apoptosis proteins (IAPs) regulates apoptosis by inhibiting caspases. This inhibition mechanism is an escape from death used by some human cancers. Second mitochondria-derived activator of caspases (Smac), a mitochondria-released protein during apoptosis, binds to IAPs BIR domains with four amino acid residues (AVPI) and releases the inhibition caused on caspases by IAPs. With the idea of designing a Smac mimicking drug, that will induce apoptosis in cancer cells, we synthesized a small molecule Smac mimetic compound. I tested the ability of the Smac mimetic compound to induce apoptosis on several human cancer cells in combination with chemotherapeutic agents. Unexpectedly, in 25% of the cancer cells we tested, Smac mimetic treatment alone caused apoptosis. Of the cancer cells that were sensitive to Smac mimetic, MDA-MB231 human breast cancer cells and HCC44, HCC461, H2126 lung cancer cells had the highest sensitivity. In addition, a majority of the lung cancer cell lines I tested were sensitive to TNF and/or TRAIL in combination with Smac mimetic. We identified the target of Smac mimetic to be XIAP, cIAP1, and cIAP2 in both Smac mimetic induced and TNF/Smac mimetic induced apoptosis. Moreover, we were able to mimic the Smac mimetic effect by triple knockdown experiments of IAPs in TNF induced cell death. Furthermore, we identified the target of Smac mimetic to be XIAP in the TRAIL pathway. This work identifies the targets and mechanism of Smac mimetic induced cell death in cancer cells.Item Characterizaton of Mcl-1 in Regulating Different Forms of Cell Death(2007-12-17) Gao, Wenhua; Wang, XiaodongProgrammed Cell Deaths (apoptosis, autophagic cell death and necrosis) play essential roles in animal development and certain diseases. Autophagy is a cellular process that provides nutrients to starved cells by digesting cellular constituents. Defects in autophagy have been implicated in cancer and neurodegeneration, but how autophagy causes cell death is not understood. In mammals, there are two apoptotic pathways: the extrinsic one through death receptors in the cell membrane and the intrinsic one through mitochondria that release death proteins. The Bcl-2 family of proteins function upstream in the intrinsic pathway. They can be subdivided into anti-apoptotic and proapoptotic proteins. Mcl-1 has two different splice variants, Mcl-1L and Mcl-1S. Mcl-1L is an anti-apoptotic Bcl-2 family protein with a short half-life, which functions in the apical step of the intrinsic apoptotic pathway and can inhibit the release of death proteins from mitochondria. After genotoxic treatment, Mcl-1L is rapidly degraded, resulting in mitochondria damage and apoptosis. Prevention of Mcl-1L degradation with proteasome inhibitors blocks apoptosis. An Mcl-1L ubiquitin ligase was identified using biochemical purification method and named Mule, which has five recognizable domains including UBA, WWE, HECT and two ARM repeats like domains. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) that allows it to specifically interact with Mcl-1L. Depletion of Mule by RNA interference stabilizes Mcl-1L, resulting in an attenuation of apoptosis induced by DNA-damaging agents. To further understand Mcl-1 function, I generated inducible Mcl-1 knockdown stable U2OS cell lines. Depletion of Mcl-1L but not Mcl-1S causes the cells to commit autophagic cell death. The autophagic phenotype can be blocked by knocking down Beclin 1, which is a known upstream component of the autophagic pathway. Mcl-1L knockdown induced cell death is accompanied by and requires the upregulation of p53 and p21. Loss-of-function experiments confirmed the involvement of Mcl-1S in inducing autophagy. Taken together, my data showed that Mcl-1 genes function in the apical step of both apoptosis and autophagic cell death pathways.Item Communal Cell Death and P53 Mediated Transcriptional Control in Drosophila Melanogaster(2011-08-10) Link, Nichole Lynn; Abrams, John M.Apoptosis is essential for all metazoan development. The key component that functions in apoptosis, the apoptosome, is a molecular machine that initiates caspase activation and is conserved throughout the animal kingdom. Drosophila strains that are mutated for genes encoding the apoptosome show pronounced defects in programmed cell death (PCD). Using a characteristic phenotype associated with mosaic animals, we conducted a screen in Drosophila to discover new regulators or effectors of the apoptosome. Using this model, we also discovered a unique communal form of cell death where large regions of epithelial cells are eliminated within minutes. We also produced 'saturation tile' arrays by digital optical chemistry for an unbiased sampling of transcriptional activity in the Drosophila genome. We found that the scope of unannotated transcriptional activity is extensive and widespread. A dominant population of noncanonical transcripts was stress-responsive and required p53, a master regulator of conventional stress-responsive target genes in vertebrates and invertebrates. This prompted us to examine stimulus dependent activity surrounding a single p53 enhancer in our tiled region. Through genetic analyses, we showed that this enhancer coordinates stimulus dependent induction of multiple genes spanning over 300kb throughout the Reaper region. Surprisingly, this same enhancer regulated a gene positioned across the centromere at distances over 20Mb and also controlled at least one gene mapping to a different chromosome. Chromosome conformation capture analyses placed this enhancer in close proximity to these distant targets in vivo through specific DNA looping and these interactions were influenced by p53. Therefore, a single p53 enhancer is necessary and sufficient for long range, multigenic regulation in cis and in trans.Item Deconstructing Collective Cell Death in a Genetic Model(2015-07-27) Garcia Hughes, Gianella; Rothenfluh, Adrian; Abrams, John M.; Kraus, W. Lee; Krämer, HelmutElimination of cells and tissues by apoptosis is a highly conserved process. In Drosophila, the entire wing epithelium is completely removed shortly after eclosion. The cells that make up this epithelium are collectively eliminated through a highly synchronized form of apoptotic cell death, involving canonical apoptosome genes. Here I present evidence that transcription of the IAP antagonist, head involution defective (hid), is acutely induced in wing epithelial cells prior to this process. hid mRNAs accumulate to levels that exceed a component of the ribosome and likewise, Hid protein becomes highly abundant in these same cells. hid function is required for collective cell death, since loss of function mutants show persisting wing epithelial cells and, furthermore, silencing of the hormone bursicon in the CNS produced collective cell death defective phenotypes manifested in the wing epithelium. Taken together, these observations suggest that acute induction of Hid primes wing epithelial cells for collective cell death and that Bursicon is a strong candidate to trigger this process, possibly by activating the abundant pool of Hid already present. This model of collective cell death in the wing is predictable, easy to observe, and experimentally tractable. Previous studies have shown that mutants in the canonical apoptotic pathways share a late-onset blemishing and persisting cells phenotypes and here I present data of two other possible cell death gene candidates. First, I show that a compound deletion, 33B(del), which removes vps33B and part of fur1, generates late-onset blemishes and persisting cells in the wing blade. These phenotypes cannot be rescued by a vps33B genomic rescue, and the possibility exists that the phenotypes are due to fur1. Finally, I show that silencing of CTCF, a protein involved in chromosomal looping and 3D genomic organization, also generates late-onset blemishes and persisting cells in the wing blade. These results, together with the binding sites annotation in the Reaper region, suggest that CTCF might be coordinating the expression of the IAP antagonists, hid in particular, by chromosomal looping. Taken together, these studies contribute to the characterization of collective cell death in Drosophila.Item The Development of an Informational Video Using Three-Dimentional [sic] Animation to Teach the Fundamentals of the Cellular Process of Apoptosis(2003-06-01) Litton, Rebecca; Calver, Lewis E.The goal of this thesis was to create an animated video, with narration, that explains the fundamentals of the process of apoptosis. The objectives were to produce a narrated 3D animation of apoptosis presented in an accurate efficient way, and format it for distribution on CD or DVD. Topics discussed in the video include: the difference between necrosis and apoptosis, the physical changes occurring in the cell during apoptosis, triggers of apoptosis and the effect apoptosis has on disease processes. The creation process began by determining subject, scope and audience. After these initial decisions were made a script was written and storyboards were produced. Narration was then recorded and combined with stills of the storyboards and preliminary animation to create an animatic. All animation was created in 3D Studio Max. Editing was accomplished using Adobe Premiere. The final product was then copied to CD and to DVD. This document discusses the process of creating this video from formation of the idea to DVD creation. Results of an informal test of the video are also iscussed as well as ideas for further research.Item Genetic Analysis of Hox Transcription Factors and Cofactors in the Regulation of Programmed Cell Death in C Elegans(2009-01-14) Potts, Malia Beth; Cameron, ScottIt has been well established that blocking apoptosis can promote cancer. Throughout the animal kingdom, apoptosis is exquisitely regulated in cell-specific and context-specific ways to ensure proper development and tissue homeostasis. In many cases, transcriptional pathways carry out this regulation by mechanisms that are not completely understood. Studies of programmed cell death in the nematode Caenorhabditis elegans provided an essential foundation for understanding the more complex pathways of apoptosis in mammals. More recent work, including my thesis research, has focused on the mechanisms that decide whether individual cells of C. elegans survive or undergo programmed cell death, and has revealed a striking concordance of transcription factors that regulate cell death and that cause cancer in humans when altered by mutation. These findings suggest that mutations affecting these transcriptional pathways can provide a survival advantage to cancer cells, and thus may represent promising novel therapeutic targets.Item Genetics of Stress-Induced Responses in Drosophila(2006-08-11) Akdemir, Fatih; Abrams, John M.Apoptosis is a highly conserved process responsible for elimination of cells during normal development and after cellular damage. Apical caspases that initiate caspase cascades are stimulated upon interaction with adaptor molecules. The Drosophila adaptor protein Dark, a homolog of nematode Ced-4 and mammalian Apaf-1, regulates the apical caspase Dronc, through interactions involving respective caspase recruitment domains (CARD). Dronc is the only caspase in the fly genome with a caspase recruitment domain. Here I pursue functional characterization of dronc and dark animals especially to find out whether they are required for all programmed cell death and whether they have distinct functions. dronc mutants have extensive hyperplasia of hematopoietic tissues and adult structures lacking dronc are disrupted for fine patterning. In diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. I also show the generation and functional characterization of dark null mutant animals. Using in vivo and ex vivo assays, I demonstrate a global apoptogenic requirement for dark and show that a required focus of dark- organismal lethality maps to the central nervous system. Finally I show functional similarities of dronc and dark null mutants by a diverse set of experiments. Together these findings illustrate broad requirements for Dark and Dronc in adaptive responses during stress-induced apoptosis and in normal cell death. Treatment of cells with DNA-damaging agents upregulates the transcription of many genes, many of which are not functionally characterized. In a series of independent studies I characterize an ionizing radiation (IR)-induced gene, CG17836, designated as xrp1. Xrp1 is robustly responsive to IR, and it is a nuclear protein with DNA-binding activity as inferred from domain structure. I have characterized two different loss-of-function mutants of xrp1. In a loss-of-heterozygosity (LOH) assay xrp1 mutant animals display higher genomic instability than wild types after IR challenge. Even though xrp1 is not required for apoptosis or cell cycle arrest after IR treatment in animals, surprisingly, its overexpression in cell culture prevents cell proliferation. Thus, Xrp1 might maintain genomic stability by modulating cell cycle checkpoints upon IR exposure.Item Mechanistic and Therapeutic Insights for Epigenetic Regulation in Cancer Development(2014-10-03) Patel, Amish Jayantibhai; Galindo, Rene; Martinez, Elisabeth; Brekken, Rolf A.; Le, Lu Q.Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are highly aggressive sarcomas that develop sporadically or in patients with Neurofibromatosis type 1 (NF1). Effective treatment options are lacking, and MPNSTs are typically fatal. To gain insights into MPNST pathogenesis, we utilized a novel MPNST mouse model that allowed us to study the evolution of these tumors at the transcriptome level. Strikingly, we found that progression to MPNST and loss of MPNST relevant tumor suppressors is associated with increased levels of chromatin regulator/BET bromodomain protein BRD4, and paradoxically, sensitivity and resistance to BET bromodomain inhibition with small molecule inhibitor JQ1. Indeed, genetic and pharmacological inhibition of BRD4 profoundly suppresses both growth and tumorigenesis of MPNSTs. Mechanistically, we uncovered that BET bromodomain inhibition leads to engagement of the ER stress/UPR pathway, and apoptosis through induction of pro-apoptotic effector molecule BIM and suppression of anti-apoptotic BCL-2 in MPNSTs. Moreover, we find that suppressed transcription of Cyclin D1 oncogene upon BRD4 inhibition correlates with reduced proliferation of MPNSTs. All together, this dual restraint on proliferation (via Cyclin D1 downregulation) and survival (via BIM induction) may indicate how BRD4 inhibition is exquisitely effective against MPNSTs and may represent a paradigm shift in therapy for MPNST patients. Moreover, these findings indicate an epigenetic mechanism underlying the balance of anti-/pro-apoptotic molecules, which suggests that BET bromodomain inhibition can shift this balance in favor of cancer cell death. Collectively, these studies provide new insights for developing strategies to overcome resistance to BET bromodomain inhibitor therapy for subverting cancer cell survival.Item Novel Small Molecule Induces Apoptosis in Malignant Peripheral Nerve Sheath Tumors of Neurofibromatosis Type I(2013-07-24) Chau, Vincent; White, Michael A.; Johnson, Jane E.; Parada, Luis F.; Le, Lu Q.Neurofibromatosis Type 1 (NF1) is an autosomal disease that affects neural crest-derived tissues, leading to a wide spectrum of clinical presentations. Patients commonly present with plexiform neurofibromas, benign but debilitating growths that can transform into malignant peripheral nerve sheath tumors (MPNSTs), a main cause of mortality. Currently, surgery is the primary course of treatment for MPNST, but with the limitation that these tumors are highly invasive. Radiotherapy is another treatment option, but is undesirable because it can induce additional mutations. MPNST patients may also receive doxorubicin as therapy, but this DNA-intercalating agent has relatively low tumor specificity and limited efficacy. In this study, we exploited a robust genetically-engineered mouse model of MPNST that recapitulates human NF1 associated MPNST to identify a novel small chemical compound that inhibits tumor cell growth. Compound 21 (Cpd21) inhibits growth of all available in vitro models of MPNST and human MPNST cell lines, while remaining non-toxic to normally-dividing Schwann cells or mouse embryonic fibroblasts. We show that this compound delays the cell cycle and leads to cellular apoptosis. Moreover, Cpd21 can reduce MPNST burden in a mouse allograft model, underscoring the compound's potential as a novel chemotherapeutic agent.Item p53 in a Genetic Model: Illuminating Adaptive Radiation Responses(2005-05-03) Sogame, Naoko; Abrams, John M.When cells are challenged by genotoxic stress, the tumor suppressor protein p53 promotes adaptive responses, including cell cycle arrest, DNA repair, or apoptosis. How these distinct fates are specified through an action of a single protein is not known. To study its functions in vivo we produced a targeted mutation at the Drosophila p53 (Dmp53) locus. I show that Dmp53 is required for damage-induced apoptosis but not for cell cycle arrest. Dmp53 function is also required for damage-induced transcription of two tightly linked cell death activators, reaper and sickle. When challenged by ionizing radiation, Dmp53 mutants exhibit radiosensitivity and genomic instability, indicating in our model, apoptosis is important for maintenance of genomic stability in response to ionizing radiation. I also examined a global transcriptional change in response to ionizing radiation in the absence of Dmp53. Only 35 genes were constantly radiation responsive in wild type animals and Dmp53 was required for induction of a vast majority of the genes. The Radiation Induced p53 Dependent (RIPD) genes include genes implicated in apoptosis and DNA repair as well as genes with unknown functions. The functional significance of RIPD genes for the activation of apoptosis was tested using RNAi. Thus far, I uncovered ribonucleotide reductase large subunit (RnrL) as a novel Dmp53 target that is necessary for induction of caspase activation. Taken together, my study supports the notion that core ancestral functions of the p53 gene family are intimately coupled to cell death and possibly DNA repair as an adaptive response.Item Regulation of apoptosis: Implications for cancer biology and therapy(2002-01-24) Chaudhary, Preet M.Item Regulation of Cytochrome C Release in UV-Induced Apoptosis(2006-05-16) Traer, Elie; Mumby, Marc C.Apoptosis, or programmed cell death, is vitally important for maintaining cellular homeostasis. When damaged cells do not appropriately enact the cell death program they have the potential to accumulate genetic mutations and become cancerous. Therefore, a mechanistic understanding of how cells decide to die would provide the foundation for drug discovery to specifically target cancer cells. Current genotoxic chemotherapeutics, and other sources such as ultraviolet (UV) radiation, damage DNA, leading to induction of apoptosis through the mitochondrial pathway. Cytochrome c is released from the mitochondria, binds dATP and Apaf-1, and together they form a structure called the apoptosome. The apoptosome then binds and activates caspase-9, which cleaves caspase-3 and other caspases resulting the morphological changes characteristic to apoptosis. To understand how UV causes apoptosis, an assay was developed to reproduce cytochrome c release in vitro. This assay revealed that UV radiation causes a rapid decrease of the anti-apoptotic protein Mcl-1 in HeLa cells. Reduction of Mcl-1 on the mitochondria causes in vitro release of cytochrome c from mitochondria three hours before in vivo release of cytochrome c is observed, i.e. the mitochondria are primed to release cytochrome c. Removal of Mcl-1 is required for UV-induced apoptosis, but it is not sufficient to induce apoptosis. This means that, in addition to mitochondrial priming, there is another required event, a second hit. It was reasonable to think that this required second hit might be bound specifically by Mcl-1. To that end, Mcl-1 was found to bind BimEL with far greater affinity than any other pro-apoptotic Bcl-2 family member. In addition, BimEL is dephosphorylated after UV in an ERK1/2-dependent manner. Despite perfectly fitting the profile of the second hit, BimEL and ERK1/2 phosphorylation do not have any affect on induction of caspases after UV. These results have helped gain insight into the regulation of cytochrome c release, which remains the most perplexing and important question in apoptosis.Item Smac Mimetic Induced Cell Death: Mode of Action and Overcoming Resistance(2009-09-04) Petersen, Sean L.; Wang, XiaodongCancers are characterized by uncontrolled growth and proliferation. One of the key regulators that act to prevent tumor development is programmed cell death, or apoptosis. Defects in the ability of tumors to undergo apoptosis are as important in cancer progression as the loss of signaling controls that limit growth and proliferation. Hence, a fundamental approach, that to date has not been fully exploited, is to attempt to reestablish proper apoptotic signaling allowing cells that have lost normal regulatory controls to essentially commit suicide. Among key regulators of cell death is the inhibitor of apoptosis (IAP) family of proteins that act to suppress activation of enzymes, the caspases, responsible for carrying out the cell death program. An endogenous protein called second mitochondrial activator of caspases (smac) is released from the mitochondria upon genotoxic stress, such as DNA damage, and binds to IAPs, un-inhibiting caspases, allowing apoptosis to occur. In many cancers this process is defective, with the observation that IAPs are often over expressed and that cancer cells become resistant to genotoxic stress and do not release smac from the mitochondria. The nature of the interaction between smac and IAPs presents itself as an ideal target. A four amino acid motif of smac binds to and displaces caspases from the IAPs. As a means to bypass the need to induce genotoxic stress, a small molecule mimetic of the four amino acid motif was synthesized and shown to be able to synergize with proapoptotic stimuli to induce apoptosis, and was also shown to have single agent efficacy. My research has aimed to identify the mechanism of why some cell lines are sensitive to single agent smac mimetic. I identified autocrine TNF production, both basal and smac mimetic induced, as a key feature of cells able to respond to single agent treatment. Additionally, I was able to identify key components responsible for cell death to occur by conducting a limited, targeted siRNA knockdown screen of TNF signaling components to identify receptor interacting protein kinase I (RIPK1) as a key component involved in caspase-8 activation. Furthermore, I was also interested in understanding why a majority of cells do not respond to smac mimetic, either as a single agent or in combination with TNF. I determined at least two mechanisms whereby this was achieved. Firstly, one of the key mechanisms of smac mimetic action is to induce the degradation of cellular IAP1 (cIAP1) and cIAP2. This degradation is key for the proper formation of an active RIPK1-caspases 8 complex. Some cells are highly sensitive to TNF induced up-regulation of cIAP2, which becomes refractive to degradation following the initial smac mimetic treatment, owing to loss of cIAP1. The return of cIAP2 blocks formation of the RIPK1-caspase 8 complex and the presence of cIAP2 accounts for resistance in some cell lines. Secondly, there is another class of cells that do not express cIAP2, basally or in response to TNF, that are nonetheless resistant. These cells are defective in responding to TNF and are thus unable to properly recruit RIPK1 to the TNF receptor. These cells are also defecting in nuclear factor-kappaB (NF-κB) signaling and possess, in relative terms, far less RIPK1 than do sensitive cells and simply do not have enough RIPK1 to incorporate into the death inducing complex. As a last goal, I wanted to determine if it was possible to force cells that are resistant to become sensitive. Given the key role that TNF plays in smac mimetic sensitivity, it seemed like a good bet that interfering with signaling downstream of the receptor might allow events at the receptor to still occur, but block downstream prosurvival events from happening. Utilizing both siRNAs and chemical inhibition of NF-κB I was able to sensitize previously resistant cells to smac mimetic and TNF treatment. Additionally, I was able to demonstrate that targeting parallel pathways that regulate cIAP2 also sensitized cells. Specifically, targeting protein kinase B (AKT) and targeting the epidermal growth factor receptor with erlotinib (Tarceva) were both highly effective. These results will hopefully expand the therapeutic use of smac mimetic as well as other established chemotherapeutic. Such combinatorial therapy offers the hope of limiting the toxicity of current therapies and expanding the pool of patients that might be responsive.Item [Southwestern News](2004-09-02) Siegfried, AmandaItem Transcriptional Regulation of Cardio-Pulmonary Development(2004-01-14) Aiyer, Aparna R.; Srivastava, DeepakOrganogenesis is a complex process, disruption of which results in developmental anomalies. In recent years, genetic dissection of the pathways involved in cardiogenesis, have shown a striking similarity in molecular mechanisms across species. One conserved protein is dHAND, a basic helix-loop-helix (bHLH) transcription factor that is required for normal development of the right ventricle, the pharyngeal arches and limb buds. Loss of dHAND leads to apoptosis in the aforementioned tissues and to embryonic lethality at E10.0. A differential display analysis was performed to identify genes dysregulated in dHAND-/- hearts. Characterization of such genes could potentially shed light on the molecular mechanisms involved in the defects seen in dHAND mutants, while also identifying genes required for normal embryonic development. This thesis represents work on two molecules that were identified in this screen. Bnip3, a hypoxia inducible, pro-apoptotic molecule that can induce mitochondrial damage, was upregulated in the dHAND-/- pharyngeal arches and heart, suggesting a role for mitochondrial damage in the observed apoptosis. I have shown that while Apaf-1, a downstream mediator of mitochondrial-induced apoptosis, is required for the apoptosis observed in dHAND-null pharyngeal and aortic arch mesenchyme, cardiomyocyte apoptosis in dHAND mutants is Apaf-1 independent. Rescue of pharyngeal arches revealed that premature closure of the pharyngeal arch arteries likely contributes to the early lethality observed in dHAND-/- embryos. The mouse ortholog of Bcl-2 associated transcription factor (Btf), which was similar to thyroid hormone receptor associated protein 150 (TRAP150), was down regulated in dHAND mutants. TRAPs are a family of transcriptional co-activators that are required for normal cardiac and embryonic development. Mice lacking Btf showed normal cardiac development, however, the animals had hypercellular lungs and died within 24 hours after birth. Analysis of lung ultrastructure and cell specific markers showed presence of immature secretory cells in the proximal airways of the lung and aberrant proximal-distal patterning. The ectopic presence of stem cell-like proximal epithelial cells (Clara cells) in the distal epithelium may explain the hypercellularity observed in btf-null lungs. These results show that Btf is required for normal maturation and patterning of the pulmonary epithelium and survival of the animal.