Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina

Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death.

RASAGILINE INTERFERES WITH NEURODEGENERATION IN THE PRPH2/RDS MOUSE

Rasagiline (N-propargyl-1(R)-aminoindan) is a second-generation propargylamine with neuroprotective effects. We used the Prph2/rds mouse to assess the effect of rasagiline on photoreceptor cell death and to examine the possible modulation of different pathways of programmed cell death.

Ultrabithorax Confers Spatial Identity in a Context-Specific Manner in the Drosophila Postembryonic Ventral Nervous System

In holometabolous insects such as Drosophila melanogaster, neuroblasts produce an initial population of diverse neurons during embryogenesis and a much larger set of adult-specific neurons during larval life. In the ventral CNS, many of these secondary neuronal lineages differ significantly from one body segment to another, suggesting a role for anteroposterior patterning genes. Here we systematically characterize the expression pattern and function of the Hox gene Ultrabithorax (Ubx) in all 25 postembryonic lineages. We find that Ubx is expressed in a segment-, lineage-, and hemilineage-specific manner in the thoracic and anterior abdominal segments. When Ubx is removed from neuroblasts via mitotic recombination, neurons in these segments exhibit the morphologies and survival patterns of their anterior thoracic counterparts. Conversely, when Ubx is ectopically expressed in anterior thoracic segments, neurons exhibit complementary posterior transformation phenotypes. Our findings demonstrate that Ubx plays a critical role in conferring segment-appropriate morphology and survival on individual neurons in the adult-specific ventral CNS. Moreover, while always conferring spatial identity in some sense, Ubx has been co-opted during evolution for distinct and even opposite functions in different neuronal hemilineages.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

TRAIL mediated signaling in human mast cells: the influence of IgE-dependent activation

BACKGROUND: Mast cells activation through FcepsilonRI cross-linking has a pivotal role in the initiation of allergic reactions. The influence of this activation on programmed cell death of human mast cells has not yet been clarified. This study evaluates the influence of IgE-dependent activation alone and in synergy with TRAIL on the expression of molecules involved in the apoptotic signal transduction. METHODS: Human cord blood derived mast cells (CBMC) were cultured with myeloma IgE followed by activation with anti-human IgE. The expression of proteins involved in apoptotic signal transduction was assessed by immunoblot analysis. To test the effect of activation on a pro-apoptotic stimulus, activated, IgE-treated and resting CBMC were incubated with TRAIL, or in a medium with suboptimal concentrations of stem cell factor (SCF). RESULTS: In accordance with a previous study of ours, it was found that IgE-dependent activation increased TRAIL-induced caspase-8 and caspase-3 cleavage. However, it did not have a significant influence on CBMC death induced by SCF withdrawal. IgE-dependent activation increased the expression of FLIP and myeloid cell leukemia 1 (MCL-1) anti-apoptotic molecules as well as the pro-apoptotic one, BIM. In addition, a decrease in BID expression was observed. TRAIL could reverse the increase in FLIP but did not influence the upregulation of MCL-1 and of BIM. CONCLUSIONS: These findings suggest that IgE-dependent activation of human mast cells induces an increase in both pro-survival and pro-apoptotic molecules. We therefore hypothesized that IgE-dependent activation may regulate human mast cell apoptosis by fine-tuning anti-apoptotic and pro-apoptotic factors.

Characterisation of the metacaspase gene family in Arabidopsis thaliana

Caspases are known to be involved in animal programmed cell death (PCD). The objective of this thesis was to use gene expression analysis and reverse genetics to determine if Arabidopsis metacaspase (AtMC) genes play a role in plant PCD. The majority of AtMC genes were found to be expressed nearly constitutively in various tissues, developmental stages, and under various inductive treatments. Transgenic Arabidopsis plants generated with AtMCpromoter::AtMCgene::GUS fusions showed expression of the reporter gene in leaves, vasculature, trichomes, siliques, anthers, and during embryo development. Preliminary phenotypic characterization of single and double Arabidopsis AtMC loss-of-function mutants suggested that the expression of the AtMC genes are highly functionally redundant. Nevertheless, our results suggest that AtMC1, 2, 4, 6 and 9 may be directly involved in rosette and/or stem development. Although this study does not provide a definitive role of MCs in plant PCD, it lays the foundation for their further in-depth analysis.

The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH(2)-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer.

Involvement of the ABC-transporter ABCC1 and the sphingosine 1-phosphate receptor subtype S1P(3) in the cytoprotection of human fibroblasts by the glucocorticoid dexamethasone

Glucocorticoids (GC) represent the most commonly used drugs for the treatment of acute and chronic inflammatory skin diseases. However, the topical long-term therapy of GC is limited by the occurrence of skin atrophy. Most interestingly, although GC inhibit proliferation of human fibroblasts, they exert a pronounced anti-apoptopic action. In the present study, we further elucidated the molecular mechanism of the GC dexamethasone (Dex) to protect human fibroblasts from programmed cell death. Dex not only significantly alters the expression of the cytosolic isoenzyme sphingosine kinase 1 but also initiated an enhanced intracellular formation of the sphingolipid sphingosine 1-phosphate (S1P). Investigations using S1P (3) ((-/-)) -fibroblasts revealed that this S1P-receptor subtype is essential for the Dex-induced cytoprotection. Moreover, we demonstrate that the ATP-binding cassette (ABC)-transporter ABCC1 is upregulated by Dex and may represent a crucial carrier to transport S1P from the cytosol to the S1P(3)-receptor subtype.

Mechanisms of Adenovirus-Mediated Autophagy

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered how viruses have evolved to manipulate autophagy for their benefit. Extensive studies of viral-induced autophagy provide a rationale to investigate other viruses, such as the adenovirus, which may be developed as part of a therapy against cancers resistant to apoptosis. Despite the present and relatively poor understanding of the mechanisms behind adenoviral-induced autophagy, adenovirus is a promising candidate, because of its ability to efficiently eradicate tumors. A better understanding of how the adenovirus induces autophagy will allow for the development of viruses with increased oncolytic potency. We hypothesized that adenovirus induces autophagy in order to aid in lysis. We found that replication, not infection, was required for adenovirus-mediated autophagy. Loss of function analysis of early genes revealed that, of the early genes tested, no single gene was sufficient to induce autophagy alone. Examination of cellular pathways for their role in autophagy during adenovirus infection revealed a function for the eIF2α pathway and more specifically the GCN2 kinase. Cells lacking GCN2 are more resistant to adenovirus-mediated autophagy in vitro; in vivo we also found these cells fail to undergo autophagy, but display more cell death. We believe that autophagy is a protective mechanism the cell employs during adenoviral infection, and in the in vivo environment, cells cannot recover from virus infection and are more susceptible to death. Congruently, infected cells deficient for autophagy through deletion of ATG5 are not able undergo productive cell lysis, providing evidence that the destruction of the cytoplasm and cell membrane through autophagy is crucial to the viral life cycle. This project is the first to describe a gene, other than a named autophagy gene, to be required for adenovirus- mediated autophagy. It is also the first to examine autophagic cell death as a means to aid in viral-induced cell lysis.

Mechanisms of Adenovirus-Mediated Autophagy

A patient diagnosed with a glioma, generally, has an average of 14 months year to live after implementation of conventional therapies such as surgery, chemotherapy, and radiation. Glioblastomas are highly lethal because of their aggressive nature and resistance to conventional therapies and apoptosis. Thus other avenues of cell death urgently need to be explored. Autophagy, which is also known as programmed cell death type II, has recently been identified as an alternative mechanism to kill apoptosis- resistant cancer cells. Traditionally, researchers have studied how cells undergo autophagy during viral infection as an immune response mechanism, but recently researchers have discovered how viruses have evolved to manipulate autophagy for their benefit. Extensive studies of viral-induced autophagy provide a rationale to investigate other viruses, such as the adenovirus, which may be developed as part of a therapy against cancers resistant to apoptosis. Despite the present and relatively poor understanding of the mechanisms behind adenoviral-induced autophagy, adenovirus is a promising candidate, because of its ability to efficiently eradicate tumors. A better understanding of how the adenovirus induces autophagy will allow for the development of viruses with increased oncolytic potency. We hypothesized that adenovirus induces autophagy in order to aid in lysis. We found that replication, not infection, was required for adenovirus-mediated autophagy. Loss of function analysis of early genes revealed that, of the early genes tested, no single gene was sufficient to induce autophagy alone. Examination of cellular pathways for their role in autophagy during adenovirus infection revealed a function for the eIF2α pathway and more specifically the GCN2 kinase. Cells lacking GCN2 are more resistant to adenovirus-mediated autophagy in vitro; in vivo we also found these cells fail to undergo autophagy, but display more cell death. We believe that autophagy is a protective mechanism the cell employs during adenoviral infection, and in the in vivo environment, cells cannot recover from virus infection and are more susceptible to death. Congruently, infected cells deficient for autophagy through deletion of ATG5 are not able undergo productive cell lysis, providing evidence that the destruction of the cytoplasm and cell membrane through autophagy is crucial to the viral life cycle. This project is the first to describe a gene, other than a named autophagy gene, to be required for adenovirus- mediated autophagy. It is also the first to examine autophagic cell death as a means to aid in viral-induced cell lysis.

Annexin A5-Conjugated Polymeric Micelles For Dual SPECT and Optical Detection of Apoptosis

Apoptosis, a form of programmed cell death, is critical to homoeostasis, normal development, and physiology. Dysregulation of apoptosis can lead to the accumulation of unwanted cells, such as occurs in cancer, and the removal of needed cells or disorders of normal tissues, such as heart, neurodegenerative, and autoimmune diseases. Noninvasive detection of apoptosis may play an important role in the evaluation of disease states and response to therapeutic intervention for a variety of diseases. It is desirable to have an imaging method to accurately detect and monitor this process in patients. In this study, we developed annexin A5-conjugated polymeric micellar nanoparticles dual-labeled with a near-infrared fluorescence fluorophores (Cy7) and a radioisotope (111In), named as 111In-labeled annexin A5-CCPM. In vitro studies demonstrated that annexin A5-CCPM could strongly and specifically bind to apoptotic cells. In vivo studies showed that apoptotic tissues could be clearly visualized by both single photon emission computed tomography (SPECT) and fluorescence molecular tomography (FMT) after intravenous injection of 111In-labeled Annexin A5-CCPM in 6 different apoptosis models. In contrast, there was little signal in respective healthy tissues. All the biodistribution data confirmed imaging results. Moreover, histological analysis revealed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of apoptosis. In sum, 111In-labeled annexin A5-CCPM allowed visualization of apoptosis by both nuclear and optical imaging techniques. The complementary information acquired with multiple imaging techniques should be advantageous in improving diagnostics and management of patients.

ECHOGENIC LIPOSOMES FOR NITRIC OXIDE DELIVERY AND BREAST CANCER TREATMENT

Liposomes, also known as nontoxic, biodegradable, and non-immunogenic therapeutic delivery vehicles, have been proposed as a carrier for drugs and antitumor agents in cancer chemotherapy. Echogenic liposomes (ELIP) have the potential to entrap air or bioactive gas to enhance acoustic reflectivity in ultrasound and are used as a contrast agent. The innovative part of this study is based on a novel concept to encapsulate nitric oxide (NO) gas into ELIP, deliver it to breast cancer cells, and control its release via direct ultrasound exposure. Studies on the effect of NO in tumor biology have shown that a high levels of NO (> 300 nM) leads to cytostasis or apoptosis by decreasing the translation of several cell cycle proteins and stimulating cancer cell death by activating the p53 pathway. The central hypothesis is that NO gas can be packaged and delivered through a delivery methodology to breast cancer cells to facilitate tumor regression with minimal systemic toxicity. The primary goal of this thesis is to develop an echogenic liposomal solution that has the ability to encapsulate NO, to release NO locally upon ultrasound exposure, and to induce breast cancer cell death. NO-containing echogenic liposomes (NO-ELIP) were prepared by the freezing-under-pressure method previously developed in our laboratory. It was necessary to evaluate stability of NO-ELIP and release of NO from NO-ELIP by measuring echogenicity using intravascular ultrasound images. Breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were selected to investigate the cytotoxic effects of NO liberated from NO-ELIP and their response to NO concentration. Ultrasound-triggered NO release from NO-ELIP using ultrasound activation was studied. It was demonstrated that NO-ELIP remained stable for 5 hours in bovine serum albumin. Delivery of NO using NO-ELIP induced cytotoxicity and programmed cell death of MDA-MB-231 and MDA-MB-468 after 5 hours of incubation. Enhancement of the NO-ELIP effect for therapeutic application was observed with ultrasound activation. This work demonstrates that NO-ELIP can incorporate and deliver NO to breast cancer cells providing increased NO stability and ultrasound-controlled NO release. Improved therapeutic effect with the use of NO-ELIP is expected to be found for breast cancer treatment.

Characterization of the human tissue transglutaminase gene promoter

Transglutaminases are a family of calcium-dependent enzymes, that catalyze the covalent cross-linking of proteins by forming $\varepsilon(\gamma$-glutamyl)lysine isopeptide bonds. In order to investigate the molecular mechanisms regulating the expression of the tissue transglutaminase gene and to determine its biological functions, the goal of this research has been to clone and characterize the human tissue transglutaminase promoter. Thirteen clones of the tissue transglutaminase gene were obtained from the screening of a human placental genomic DNA library. A 1.74 Kb fragment derived from DNA located immediately upstream of the translation start site was subcloned and sequenced. Sequence analysis of this DNA fragment revealed that it contains a TATA box (TATAA), a CAAT box (GGACAAT), and a series of potential transcription factor binding sites and hormone response elements. Four regions of significant homology, a GC-rich region, a TG-rich region, an AG-rich region, and HR1, were identified by aligning 1.8 Kb of DNA flanking the human, mouse, and guinea pig tissue transglutaminase genes.^ To measure promoter activity, we subcloned the 1.74 Kb fragment of the tissue transglutaminase gene into a luciferase reporter vector to generate transglutaminase promoter/luciferase reporter constructs. Transfection experiments showed that this DNA segment includes a functional promoter with high constitutive activity. Deletion analysis revealed that the SP1 sites or corresponding sequences contribute to this activity. We investigated the role of DNA methylation in regulating the activity of the promoter and found that in vitro methylation of tissue transglutaminase promoter/luciferase reporter constructs suppressed their basal activity. Methylation of the promoter is inversely correlated with the expression of the tissue transglutaminase gene in vivo. These results suggest that DNA methylation may be one of the mechanisms regulating the expression of the gene. The tumor suppressor gene product p53 was also shown to inhibit the activity of the promoter, suggesting that induction of the tissue transglutaminase gene is not involved in the p53-dependent programmed cell death pathway. Although retinoids regulate the expression of the tissue transglutaminase gene in vivo, retinoid-inducible activity can not be identified in 3.7 Kb of DNA 5$\sp\prime$ to the tissue transglutaminase gene.^ The structure of the 5$\sp\prime$ end of the tissue transglutaminase gene was mapped. Alignment analysis of the human tissue transglutaminase gene with other human transglutaminases showed that tissue transglutaminase is the simplest member of transglutaminase superfamily. Transglutaminase genes show a conserved core of exons and introns but diverse N-terminuses and promoters. These observations suggest that key regulatory sequences and promoter elements have been appended upstream of the core transglutaminase gene to generate the diversity of regulated expression and regulated activity characteristic of the transglutaminase gene family. ^

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^