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Drug Resistance Mechanisms to Gamma-secretase Inhibitors in Human Colon Cancer CellsTimme, Cindy R. 01 January 2013 (has links)
Colorectal cancer is the third leading cause of cancer-related mortality. Much progress has been achieved in combating this disease with surgical resection and chemotherapy in combination with targeted drugs. However, most metastatic patients develop drug resistance so new modalities of treatment are needed.
Notch signaling plays a vital role in intestinal homeostasis, self-renewal, and cell fate decisions during post-development and is activated in colorectal adenocarcinomas. Under debate is its role in carcinomas and metastatic disease. In theory, blocking Notch activation using gamma-secretase inhibitors (GSIs) may show efficacy alone or in combination with chemotherapy in the treatment of colon cancer.
In Chapter Three, we tested the capacity for GSIs to synergize with oxaliplatin in colon cancer cell lines and evaluated the underlying molecular mechanisms. GSI alone had no effect on colon cancer cell lines. Surprisingly, we show that GSIs blocked oxaliplatin-induced apoptosis through increased protein levels of the anti-apoptotic Bcl-2 proteins Mcl-1 and/or Bcl-xL. Restoration of apoptosis was achieved by blocking Mcl-1 and/or Bcl-xL with obatoclax (an anti-apoptotic Bcl-2 agonist) or siRNA. An unexpected result was the induction of cell death with the combination of GSI and obatoclax.
In Chapter Four, we examined the mechanism of GSI + obatoclax-mediated cell death. We found that apoptosis played a minimal role. Rather, we identified blockage of cytoprotective autophagy played a causative role. Interestingly, we also saw autophagy induction in GSI-treated cells, which could explain the insensitivity of colon cancer cells to GSI. When autophagy was blocked in GSI-treated cells, cells became sensitive to GSI and cell death was elicited. The mechanism by which induction of autophagy occurs in GSI- treated cells is an area for further research.
Overall, our work questions the validity of the use of GSIs in the treatment of colorectal cancers. We show that GSIs may block apoptosis and induce cytoprotective autophagy simultaneously, resulting in increased drug resistance and cellular survival. Whether these two cellular survival processes occurs in patients needs to be examined before GSIs can be utilized in a clinical setting. If so, these two hurdles must be overcome.
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Synthesis of small molecule inhibitors targeting signal transduction pathwaysRamamoorthy, Divya 01 June 2009 (has links)
The main aim of the study described in this thesis is the development of small molecules as inhibitors targeting signal transduction pathways, thereby treating cancer. We attempted to synthesize compounds based on the hits obtained from high throughput screening of the Chemdiv diversity set compounds. Chapter One is a general introduction to cancer, history of chemotherapeutic drugs and an introduction to signal transduction pathways. The following two chapters briefly introduce the biological targets in the authors study. Chapter Two describes the role of B-cell lymphoma type xL (Bcl-xL), in apoptosis and the development of drugs targeting Bcl-xL. Examples of Bcl-xL drugs relevant to this study have been provided.
Chapter Three introduces Src homology 2 (SH2) domain containing tyrosine phosphatase Shp2, a protein tyrosine phosphatase, as an oncogene, its role in signal transduction pathways and the recent developments in drug development towards the inhibition of this oncogene. Chapter Four gives a general introduction to microwave-assisted organic synthesis and its advantages. This chapter also describes the use of flow reactors in organic synthesis and its advantages. The following two chapters describe the author's own findings. Chapter Five focuses on the design, synthesis and biological evaluation of small molecules as inhibitors of Bcl-xL. Isoquinolinols, NSC-131734 and HL2-100 emerged as lead compounds from high throughput screening for Bcl-xL. Our strategy focused on identifying an isoquinolinol lead with increased potency.
Based on isatin hits obtained earlier through HTS screen and SAR studies in our lab, more isatin derivatives were synthesized focusing on developing inhibitors with increased cell permeability and improved potency.
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BAD-interacting proteins in breast cancer cellsCraik, Alison C Unknown Date
No description available.
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ARK5 Regulates Subcellular Localization of hnRNP A1 During Hypertonic StressRichard, Travis January 2017 (has links)
During cellular stress, the regulation of protein synthesis is a key adaptive mechanism used by cells to survive. In response to various stresses, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), an RNA binding protein principally found within the nucleus, is phosphorylated and consequently accumulates in the cytoplasm. Among other roles, cytoplasmic hnRNP A1 functions as an auxiliary translation factor for internal ribosome entry site (IRES)-mediated translation of specific mRNA, including the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL). To identify which kinases control the cytoplasmic accumulation of hnRNP A1, an RNAi-based kinome-wide screen was performed in hypertonically stressed U2OS cells, from which AMPK-related kinase 5 (ARK5) was identified as a potential regulator of hnRNP A1’s localization. Here we show that ARK5 directly phosphorylates hnRNP A1 and that the inhibition of ARK5 expression blocks the stress induced cytoplasmic accumulation of hnRNP A1, modulates expression of Bcl-xL protein and increases cell viability. Our data points to a novel role for ARK5 and provides further insight into the mechanisms regulating cellular stress response.
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Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells / ヒトiPS細胞から誘導した自己複製能をもつ巨核球細胞株は臨床応用における血小板の安定供給を可能にするNakamura, Sou 24 November 2015 (has links)
京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12971号 / 論医科博第2号 / 新制||医科||5(附属図書館) / 32409 / 新制||医科||5 / 横浜市立大学大学院国際総合科学研究科バイオ科学専攻 / (主査)教授 長船 健二, 教授 中畑 龍俊, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Comparative Characterization of the Major Human Glutaredoxin Isozymes and Identification of a Mechanism by which Grx1 Regulates Apoptosis in CardiomyocytesGallogly, Molly Megan 13 October 2009 (has links)
No description available.
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Survival is an essential component of self-renewal induced by a Class II leukemia oncogeneChou, Fu-Sheng, M.D. 20 September 2011 (has links)
No description available.
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Design, synthesis, and evaluation of thiazolidinedione derivatives inhibiting Bcl-2/Bcl-xL or ablating androgen receptor in prostate cancerYang, Jian 26 August 2009 (has links)
No description available.
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Characterization of the Activation Mechanism of BaxKale, Justin January 2017 (has links)
Mitochondrial outer membrane permeabilization (MOMP) is regulated by protein-protein and protein-membrane interactions between Bcl-2 family proteins. These interactions are governed by the concentrations and relative binding affinities of the proteins for each other. These affinities are altered by conformation changes of Bcl-2 family proteins resulting from interactions with each other and with membranes. How Bcl-2 proteins transition into and out of the conformations that controls their functions, and ultimately the fate of the cell, is not well understood. Here, kinetic analysis of the pore-forming Bcl-2 family member, Bax, revealed that Bax undergoes a conformational rearrangement through at least one structurally distinct intermediate that is a necessary precursor to pore formation. We discover that four cancer-associated Bax point mutants are trapped in the intermediate state, suggesting that transitions into and out of this intermediate can be modulated independently with consequences for the execution of apoptosis. Furthermore we report that the conformation changes Bax undergoes can be regulated by phosphorylation of Bax on residue S184 by the pro-survival kinase, Akt. Phosphorylation converts Bax into an anti-apoptotic protein that functions in a dominant-negative fashion. Bioinformatics revealed that in human cancers, higher levels of Bax are positively associated with high levels of PI3K/AKT pathway genes representing an added mechanism for cancer cells to evade apoptosis. Additionally we studied the interactions between Bax, the anti-apoptotic protein Bcl-XL, the sensitizer BH3 protein Bad and the BH3 activator protein Bid. We uncover a new mechanism of apoptosis regulation whereby Bad binds to one monomer of a Bcl-XL dimer eliciting an activating conformation change in a tBid bound to the other monomer of the Bcl-XL dimer. This allows Bad to function as a non-competitive inhibitor of Bcl-XL, and represents a novel mechanism that significantly enhances the potency of Bad to elicit apoptosis. / Thesis / Doctor of Philosophy (PhD) / Every day the human body creates billions of cells replacing damaged or unwanted cells. The death of these cells is tightly controlled and can result in disease when misregulated. Cancers arise when there is too little cell death and neurodegenerative diseases, such as Alzheimer’s, arise from too much cell death. Much research, including this thesis, is focused on understanding how cells die because once understood, cell death can be manipulated to treat disease. Cell death ironically occurs at the mitochondria, a cellular organ normally responsible for creating the energy required for the cell to live. When cell death is initiated, the mitochondria get holes poked into them, releasing pro-death factors that irreversibly commit the cell to dying. The work presented here uncovers new information about the regulation of the hole poking process, how it is blocked in breast cancer and how the process may be modulated to treat cancers.
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蛋白激酶 CK2 在大鼠腦部之抗細胞凋亡機制的探討 / The anti-apoptotic mechanisms of protein kinase CK2 in the brain of rat張家銘 Unknown Date (has links)
蛋白激酶 CK2 是一種具有多種功能的絲胺酸/蘇胺酸蛋白質激酶,CK2 作用的受質眾多且廣泛表現在哺乳類動物細胞中,對於細胞週期的發展、轉錄作用以及抗細胞凋亡等機制扮演非常重要的角色。在神經系統中,CK2 已知可以保護神經細胞以抵抗外來的傷害,但是其分子層面的機制目前尚未釐清。本篇論文的研究重點在於探討 CK2 保護作用可能參與的細胞分子機制。血清反應因子 SRF 是一種哺乳類動物細胞的轉錄因子,調控基因的轉錄作用來促進細胞的存活。Mcl-1 是抗細胞凋亡家族 Bcl-xL 家族蛋白成員之一,可以促進細胞的存活能力。先前研究指出,SRF 會受到 CK2 的磷酸化作用而增加本身的 DNA 結合能力。在其他研究也指出,Mcl-1 會受到 SRF 的調控。在本篇論文的第一部份,著重於 Mcl-1 的表現是否會受到 CK2 調控 SRF 的路徑所影響,實驗結果顯示,轉染野生型 CK2α 質體 DNA 可以增加海馬迴 CA1 腦區的 SRF 磷酸化,而轉染不活化的突變型 CK2αΑ156 質體 DNA 則會減少 SRF 的磷酸化。更進一步,轉染野生型 CK2α 會增加 Mcl-1 的 mRNA 及蛋白質表現,轉染突變型 CK2αΑ156 則減少 Mcl-1 的表現。此外,轉染突變型 SRF99A 也會減少 Mcl-1 的 mRNA 及蛋白質表現;而且在共同轉染實驗中,SRF99A 會拮抗野生型 CK2α 對促進的 Mcl-1 蛋白質表現的作用。
另一方面,DARPP-32 是一個在新紋狀體神經細胞中具有調控多巴胺訊息效力的訊息傳遞分子。先前研究指出,DARPP-32 具有抗細胞凋亡的功能,且發現在 DARPP-32 Ser102 氨基酸會受 CK2 的磷酸化作用。因此,本篇論文的第二部份主要是探討 CK2 的抗細胞凋亡能力是否是透過磷酸化 DARPP-32 來調控。實驗結果顯示,轉染野生型 CK2α 可以增加紋狀體 DARPP-32 的磷酸化,而轉染不活化的突變型 CK2αΑ156 則會減少 DARPP-32 的磷酸化。此外,轉染 CK2α 的小干擾 RNA (siRNA) 可以抑制內生性的 CK2 表現,同時也會減少 DARPP-32 的磷酸化以及抗細胞凋亡蛋白, Bcl-xL 的表現。綜合這些實驗結果,CK2α可以分別透過 SRF 或 DARPP-32 調控的訊息傳遞來促進 Mcl-1 或 Bcl-xL 的表現進而調控神經系統的抗細胞凋亡機制。 / Protein kinase CK2 is a multifunctional serine/threonine protein kinase with many protein substrares and is ubiquitously expressed in mammalian cells to play an important role in cell cycle progression, transcription, and anti-apoptosis. In the nervous system, CK2 is shown to protect neurons against injury, but the cellular mechanisms are not well studies. In the present studies, we investigate which cellular mechanism might involve in the CK2 protection effects. The serum response factor (SRF) is a mammalian transcription factor which mediates some gene transcriptions relevent to promote the cell survival. The Myeloid cell leukemin 1 (Mcl-1) is one of the anti-apoptotic Bcl-2 family members and is involved in promoting cell viability. Previous studied have revealed that the SRF phosphorylation by CK2 can enhance its DNA-binding activity. The regulation of Mcl-1 by SRF has also been reported in other studies. In the first part of the present studies, we investigate whether the Mcl-1 expression is regulated by CK2 through SRF mediated pathway. The results from wildtype CK2α plasmid DNA transfection revealed that the phosphorylated SRF were increased in hippocampus CA1 region, whereas transfection of the catalytically inactive CK2αA156 mutant plasmid DNA decreased phosphorylated SRF. Further, wildtype CK2α increased, whereas CK2αA156 mutant decreased the mRNA and protein levels of Mcl-1. Moreover, transfection of the mutant SRF99A also decreased the mRNA and protein levels of Mcl-1. Furthermore, the mutant SRF99A antagonized the upregulatory effects of wildtype CK2α on Mcl-1 protein level in the co-transfection experiments.
In the other side, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Previous studies have revealed that DARPP-32 might involve in the anti-apoptosis and its Ser102 residue is phosphorylated by CK2. Therefore, in the second part of this study, we investigate whether one of the anti-apoptotic effects of CK2 is through DARPP-32 phosphorylation by CK2 in the present study. The results revealed that the phosphorylated DARPP-32 is increased in stratum by wildtype CK2α transfection and decreased by catalytically inactive CK2αA156 mutant transfection. Further, transfection of CK2α siRNA can inhibit endogenous CK2 expression and also decrease phosphorylation of DARPP-32 as well as the anti-apoptotic protein, Bcl-xL. These results together suggest that CK2α-mediated anti-apoptotic effects are partially through SRF mediated or DARPP-32 mediated signaling to regulate Mcl-1 or Bcl-xL expression, respectively.
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