Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

Doxorubicin (Dox) has been employed in cancer chemotherapy for a few decades. However its clinical application became restricted because of dose-dependent cardiomyopathy. Recent studies suggest that Dox-induced cardiomyocyte apoptosis is a primary cause of cardiac damage. Vascular endothelial growth factor (VEGF) is a major factor for endothelial cell survival and angiogenesis. We have previously shown that VEGF16S significantly attenuates oxidative stress-induced cardiomyocytes apoptosis. We hypothesized that VEGF165 will protect the cardiomyocytes from Dox-induced apoptosis. To evaluate our hypothesis, we transfected cardiomyocytes H9c2 with adenovirus expressing VEGF16S 24 hours before the cells were challenged with Dox at a concentration of 2 uM. Cardiomyocyte apoptosis was evaluated by Annexin V-FITC staining and by Western blot detection of cleaved caspase-3. The hypothesis was confirmed, and the protective mechanisms involve the inhibition of death receptor-mediated apoptosis and up-regulation of the prosurvival Akt/NF-κB/BcI-2 signaling pathway.

Our previous studies indicate that 3, 4-(methylenedioxy)-1-(2′, 3′-epoxypropyl)-benzene (safrole oxide), a newly synthesized compound, induces apoptosis in vascular endothelial cells (VECs) and A549 lung cancer cells. To our knowledge, the inhibition of angiogenesis by safrole oxide has not been reported yet. We report here that cultured rat aorta treated with safrole oxide exhibited a significant microvessel reduction as determined by counting the number of microvessels in a phase contrast microscope. There were more microvessels formed in the presence of A549 lung cancer cells in rat aorta model, while a dramatic inhibition of angiogenesis was obtained by adding 220-450 μmol l- 1 of safrole oxide to the growth medium (P <. 01). The culture of rat aorta treated with safrole oxide produced only some abortive endothelial cells but not microvessels. Furthermore, safrole oxide induced antiangiogenic effect in the chorioallantoic membranes (CAM) as a dose dependent manner. Eggs treated with 2-11 μmol 100 μl- 1 per egg of the safrole oxide for 48 h exhibited a significant reduction in blood vessel area of the CAM, a process likely mediated by apoptosis as demonstrated by DNA fragmentation. Our results suggest that safrole oxide has antiangiogenic activity and this effect might occur by induction of cellular apoptosis.

We investigated the role of Fas ligand in the development of anti-tumor immunity. The LSA tumor specific cytotoxic T lymphocyte (CTL) clone, PE-9, expressed both Fas and Fas ligand. This CTL clone upregulated Fas and Fas ligand expression upon activation through the T-cell receptor and induced apoptosis in Fas+, LSA tumor cells using the FasL-based pathway. However, LSA and EL-4 tumor cells constitutively expressed Fas ligand and killed Fas+ PE-9 CTLs and Fas+, but not Fas-negative (Fas-) activated T cells and thymocytes. These data suggested that T cells and cancer cells can kill each other and that cancer cells may use Fas ligand to evade the action of the immune T cells. In addition to the expression of membrane-bound form, FasL+ LSA and EL-4 tumor cells produced a soluble form of Fas ligand when they grew in vivo and in vitro. Serum from EL-4 or LSA-bearing wild type mice contained significant levels of Fas ligand. The soluble FasL induced apoptosis in liver and thymus of C57BL/6 wild type (Fas+) mice, but not C57BL/6 lpr/lpr (Fas-) mice. The detection of apoptosis in the liver of C57BL/6 gld/gld (FasL-defective) mice suggested that the source of Fas ligand found in the sera of EL-4 or LSA-bearing mice was from the tumor cells rather than the host cells. CTL or NK cells used FasL-based apoptosis to kill the target cells when activated. To this end, we tested whether constitutive expression of Fas on tumor cells generate enhanced anti-tumor immunity. IL-2 or poly-I-C induced/ activated NK/LAK cells displayed higher cytotoxicity against L1210 Fas+, but not L1210 Fas- tumor cells. Furthermore, growth of L1210 Fas+, but not Fas- tumor, in vivo, generated Fas-specific cytotoxic T lymphocytes. Therefore, mice bearing L1210 Fas+ tumor cells survived for a longer time than mice bearing L1210 Fas- tumor cells. To determine the role of the Fas, FasL, and perforin in the initiation of tumor, C57BL/6 +/+ (FasL+, Fas+), C57BL/6 lpr/lpr (Fas-), C57BL/6 gld/gld (FasL-), and perforin knock-out (PKO) (FasL+, Fas+, but perforin-deficient) mice were injected with methylcholanthrane (MCA). Tumor development in lpr or gld mice was faster and uncontrollable, compared to C57BL/6 (wild-type) and PKO mice. However, wild-type and PKO mice showed delayed tumor appearence and were able to suppress tumor growth. In addition to the deficiency of Fas or FasL, high levels of TGF-b and IL-10 expression detected in lpr and gld mice were also responsible for the early tumor development. Together these data suggested that interactions between Fas and Fas ligand, expressed on immune cells and tumor cells, play an important role in the generation of anti-tumor immunity. Tumor cells use FasL to evade the action of the immune system, and upregulation of FasL makes T cells more cytolytic. Tumor growth may depend on the number of cancer cells vs. the number of cancer specific T cells. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18184号 / 医科博第49号 / 新制||医科||4(附属図書館) / 31042 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 松田 道行, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Final publication is available at http://www.sciencedirect.com/science/article/pii/S0006291X1530276X / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19563号 / 医博第4070号 / 新制||医||1013(附属図書館) / 32599 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 藤田 潤, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins. Cellular IAP 1 and 2 (cIAP1 and 2) are two members of the IAP protein family that regulate the activity of the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family. The role and mechanism of the IAPs in ubiquitination are not yet completely understood due to the complexity of this posttranslational modification process. Additionally, The IAPs are involved in a myriad of cellular processes, and many of the process-specific mechanisms by which the IAPs are involved is unknown. I aim to delve deeper into the signalling pathways that are controlled by cIAP1 and cIAP2 by discovering currently unknown protein-protein interactions. In doing so, I will determine which proteins interact with the cIAPs and what signalling pathways these proteins are involved in. Using a BioID approach, I sought to characterize the cIAP1 interactors involved in the canonical and non-canonical NF-κB pathways. I generated a stable cell line expressing TurboID-cIAP1 fusion protein in HEK 293T cells that expressTurboID-cIAP1 at levels comparable to endogenous cIAP1. I identified multiple potential cIAP1 interactors that have ties to the NF-κB pathway. These proteins regulate NF-κB signalling in multiple ways including influencing acetylation and nuclear retention of the NF-κB transcription factors, phosphorylation of NF-κB transcription factors, and RNA splicing of genes involved in the TNFR1 complex I. Further work needs to be done to confirm these interactions and to discover the mechanisms by which these interactions occur. NF-κB signalling is known to have widespread function within the cell and within diseases such as cancer; it will be beneficial to study these interactions to better understand how cancer develops and how to treat it best, especially in patients with a poor prognosis.

The promoter region of interferon regulatory factor 5 (IRF5) contains the rs2004640 T or G single nucleotide polymorphism (SNP) and a CGGGG indel. Both of these polymorphisms have been implicated as genetic risk factors for several autoimmune diseases, including systemic lupus erythematosus, whose pathology involves altered apoptosis and cytokine signaling. The polymorphisms' overall effect is to increase IRF5 levels. IRF5 is a transcription factor of several cytokines, including interferon, and is pro-apoptotic. Thus an alteration of cytokine levels and apoptosis signaling due to high IRF5 levels is the proposed source of autoimmune risk. Each of IRF5's four first exons (1A, 1B, 1C, 1D) has its own promoter and responds to specific stimuli. rs2004640 is a T or G polymorphism; T is the risk allele. The SNP creates a sequence-specific recognition site for the spliceosome, making exon 1B spliceable. Analysis of the 1B promoter showed putative p53 binding site. IRF5 and p53 are pro-apoptotic transcription factors, and the p53 site may provide a positive feedback loop. Apoptosis levels were altered in cells with the rs2004640 risk T/T allele when treated with DNA damaging agents (extrinsic apoptosis), but not when activating death receptors (intrinsic apoptosis). The 1B promoter was the only one to activate expression after inducing DNA damage in a luciferase reporter assay, and this activation was abolished after mutating the p53 site. The exon 1A promoter contains either three or four copies (4X) of CGGGG; the 4X variant is the risk allele. The 1A promoter is constitutively active and is responsive to the Toll-like receptor 7 agonist imiquimod. RNA folding analysis revealed a hairpin encompassing exon 1B. Mutational analysis showed that the hairpin shape decreased translation five-fold in a luciferase reporter assay. Cells with the CGGGG or rs2004640 risk allele exhibited higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR in cell lines with either risk polymorphism demonstrated decreased usage of exons 1C or 1D, although no other correlated splicing events were observed. Also, several mRNA splice variants of IRF5 were sequenced. The risk polymorphisms altered cytokine signaling as well. Expression of interferon, Toll-like receptor, and B cell receptor pathways were affected by a risk haplotype which includes the rs2004640 SNP. The CGGGG polymorphism decreased the levels of CC-chemokine receptor 7. Specific transcription factor binding sites define promoter activity and thus first exon usage and transcription levels. Translation levels are affected by mRNA folding. Overall, the rs2004640 SNP and the CGGGG indel cause high levels of IRF5. High IRF5 expression causes altered cytokine and apoptosis signaling, and may bias the immune system toward autoimmunity.