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Surface Functionalization and Analysis Thereof for an Ovarian Cancer Diagnostic BiosensorAhmad, Asad Ali 01 January 2011 (has links)
Ovarian cancer is the fifth leading cause of cancer death among women in United States and has an alarming 1.4% (1 in 71) lifetime risk. The lack of overt symptoms and the absence of a reliable screening test to detect ovarian cancer result in over 70% of women being diagnosed after the disease has spread beyond the ovary resulting in a poor prognosis. A key characteristic of ovarian cancer is the ability of tumor cells to evade apoptosis, or programmed cell death contributing to the limitless replicative potential, which is a hallmark of all carcinogenesis. There is conclusive evidence that levels of bcl-2 are elevated in ovarian cancer patients' indication that this protein is an ovarian cancer biomarker. The overall goal of this thesis is to functionalize a substrate for specific, sensitive and cost-effective bcl-2 capture. This surface will ultimately be incorporated into an acoustic wave-based diagnostic device for worldwide point-of-care (POC) ovarian cancer detection.
This research looks to assess the capture of this analyte protein on a series of bioconjugated surfaces. For the research to be diagnostically applicable, certain factors reveal themselves as more important than others. Since the surface-bound capture antibody must recognize the bcl-2 protein, it is vital to ensure upright orientation of this specific antibody with high affinity for the analyte. Furthermore once integrated with a nanosensor, the surface will sense a change in the mass on the surface, which requires that the surface is highly resistant to non-specific binding. Bioconjugation techniques were employed to initiate self-assembled monolayers (SAM) of silanes, immobilize antibodies (via amine-crosslinking or direct adsorption of protein A/G) and disperse polyethylene glycol (PEG) reagents to reduce non-specific binding on the glass substrates. 3-aminopropyltrimethoxysilane (3-APTMS) and chlorodimethyloctylsilane (ODMS) were deposited on the surface to create initial hydrophilic and hydrophobic properties on which molecular self-assembly could occur. Testing a variety of assemblies with and without the presence of silanes, amine-crosslinking and PEGylation reagents, the substrate displaying the highest efficacy of bcl-2 capture was revealed. These various surfaces were assessed through contact angle and a novel sandwich enzyme linked immunosorbent assay (ELISA) for sensitivity and specificity of bcl-2 standard capture.
The consistently low background and facile assembly of the ODMS based substrate with direct adsorption of protein A/G and the PEGylation reagent, Pluronic, was deemed the best functionalized surface for non-specific recruitment of the bcl-2 protein. The substrate also consistently displayed low signal-to-noise ratio which was of extreme importance in this research to guarantee the prevention of false-positive results when detecting nascent carcinogenic behavior. Elucidation of this substrate assembly is the first step towards the long term objective of this thesis, which is to construct a cost-effective early ovarian cancer detection device which can be implemented at the point-of-care to those who need it the most. This is ultimately expected to dramatically improve health outcomes for females worldwide.
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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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Heat shock-induced apoptosisMahajan, Indra Maria 21 January 2014 (has links)
Apoptosis is a conserved program of cell death that promotes organism homeostasis in all stages of life. Two main pathways activate caspases, which are cysteinyl-aspartate proteases that execute apoptosis. The extrinsic pathway is initiated by cell surface death receptors, while the intrinsic pathway is initiated by intracellular signals that cause permeabilization of the outer mitochondrial membrane (MOMP). The Bcl-2 protein family regulates MOMP, which causes the release of several pro-apoptotic proteins (such as cytochrome c, Smac) into the cytosol. Bcl-2 proteins share homology in up to four "BH" domains and are subdivided into three subgroups. Pro-apoptotic Bax and Bak catalyze pore formation in the mitochondria, while anti-apoptotic members (Bcl-2, Mcl-1) inhibit MOMP. The third subgroup, termed BH3-only, promotes MOMP by either antagonizing Bcl-2 proteins or by directly activating Bax/Bak, and initiate apoptosis in response to various stressors, including heat shock (HS). Hyperthermia or acute HS reportedly induces apoptosis through caspase-2-mediated cleavage of BID, engaging the intrinsic pathway. However, additional evidence suggests that this pathway could represent an amplification loop. Thus we hypothesized that during HS, another BH3-only protein such as BIM, that does not require cleavage, could engage MOMP. Herein, we report that BIM mediates an alternative HS-induced apoptosis pathway. Cells lacking BIM are resistant to HS and exhibit better short and long-term survival than either Bid[superscript -/-] or Bax[superscript -/-]Bak[superscript -/-]. Moreover, caspase-2 induces apoptosis in Bim[superscript -/-] but not Bid[superscript -/-] cells, implying that caspase-2 kills exclusively through BID. Interestingly, Bim[superscript -/-] and Bax[superscript -/-]Bak[superscript -/-] cells are entirely resistant to MOMP, but the Bax[superscript -/-]Bak[superscript -/-] cells still undergo caspase-3 activation and remain partially sensitive to HS, indicating that BIM triggers caspase-3 activation upstream of mitochondria. Thus, BIM plays an important role in HS-induced apoptosis. Hyperthermia has clinical applications for the treatment of solid tumors. Unfortunately, a practical limitation is the development of thermotolerance, which confers resistance not only to subsequent HS but also to radiotherapy and chemotherapy. Therefore, a better understanding of the molecular mechanisms involved both in heat-induced apoptosis and thermotolerance could lead to new therapeutic interventions. Here we also show evidence for a putative role for the stress kinase JNK signaling pathway in the regulation of thermotolerance. / text
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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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Mitochondrial Priming Determines Chemotherapeutic Response in Acute Myeloid LeukemiaVo, Thanh-Trang January 2012 (has links)
Gain- and loss-of-function studies of the BCL-2 family of proteins have shown that they can impact chemotherapeutic sensitivity. However, cells contain myriad anti-apoptotic and pro-apoptotic BCL-2 family members making it difficult to predict cell fate decisions based on the initial conditions of these proteins. BH3 profiling is a tool that measures mitochondrial priming, the readiness of a cell to die through the intrinsic (or mitochondrial) apoptotic pathway. Priming is due to the cumulative effect of the BCL-2 family of proteins that act as the gate keepers of the mitochondrial apoptotic pathway. Priming is measured by determining the sensitivity of mitochondria to perturbation by peptides derived from the BH3 domains of pro-apoptotic proteins. Using BH3 profiling, we now have a functional readout that can quantify priming and assess its contribution to drug sensitivity. Here we show that priming affects the sensitivity of acute myeloid leukemia (AML) cell lines to various standard chemotherapeutics, especially topoisomerase II inhibitors. Priming predicts clinical response to conventional induction chemotherapy as well as the long term maintenance of remission in AML patients. Interestingly, the priming of normal hematopoietic stem cells (HSCs) sits at the boundary line between the priming of cured and refractory patient AML. This HSC priming likely defines the therapeutic index since AML that are lower primed than HSCs are often refractory and cannot be cured without transplantation. Additionally, our BH3 profiles revealed that AML cells are more sensitive to BCL-2 antagonism than normal HSCs, which are primarily dependent on MCL-1. Indeed, we were able to kill primary refractory AML cells in vitro with the BCL-2 antagonist ABT-737 at doses that left HSCs unharmed. Cumulatively, these findings show that priming is a major mechanistic determinant of AML response in vitro and in the clinic to standard induction chemotherapy. With the ability to predict outcome, BH3 profiling may offer physicians and patients a promising tool for treatment decision-making.
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SPLIT-PROTEIN REASSEMBLY METHODS FOR THE DETECTION AND INTERROGATION OF BIOMOLECULAR INTERACTIONS AND MODULATORS THEREOFPorter, Jason Robert January 2009 (has links)
The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.
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The role of Mcl-1 in the response of human colorectal cancer cells to treatment with dichloroacetateDelaney, Leanne 26 August 2013 (has links)
Dichloroacetate (DCA) it a metabolic reprogramming agent that is used to target the unique metabolism of cancer cells, but is not always effective in colorectal cancer cells. In HCT116 cells, DCA was unable to induce apoptosis, but did decrease proliferation when compared to untreated cells. A decrease in full length Mcl-1 protein expression 7 hours following DCA treatment did not correspond with changes in mRNA production or changes in expression of inhibitory binding partners, but may be due to altered proteasomal degradation. Similar reduction in levels of a lower molecular weight Mcl-1 band occurred, which did not result from alternative splicing or from caspase-mediated cleavage. Mcl-1 showed primarily nuclear localization within the cell, and expression changes in full-length Mcl-1 were seen in nuclear lysate but not cytoplasmic lysate after 7 hours of DCA treatment. Changes in nuclear Mcl-1 expression did not correspond with cell cycle arrest or progression. These results suggest that proteasomal degradation of Mcl-1 may be altered following treatment with DCA, and this change may be associated with decreased proliferation, independent of cell cycle arrest. This may indicate a novel role of nuclear Mcl-1 in response of colorectal cancer to DCA exposure. / Final thesis for Leanne Delaney in partial fulfillment of requirements for the degree of Master of Science in Biomedical Sciences / NSERC
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Implication des gènes de la famille Bcl-2 dans l'apoptose des hépatocytes chez le rat Long-Evans CinnamonCloutier, Alexis-Simon January 2007 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal
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Identification and characterization of TMEM 85, a novel suppressor of bax-mediated cell death in yeastRing, Giselle Natasha. January 2007 (has links)
The ability to evade apoptosis is an acquired characteristic associated with many normal and pathophysiological processes. TMEM 85 represents a novel transmembrane domain containing human protein isolated in our previous screen for Bax suppressors, but whose function is currently unknown. Using viability and growth assays, we confirmed that TMEM 85 is anti-apoptotic. Four unique human cDNA sequences containing regions distinct from and of perfect identity to our cDNA were present in the database. Analysis of TMEM 85 suggests that it consists of five exons, alternatively spliced to produce at least four different mRNA's and proteins (TMEM 85v1-v4). RT-PCR analysis using RNA isolated from mice and humane tissues show that all transcripts are expressed. Yeast contain an orthologue of the human TMEM 85v1 protein, YGL213C. Surprisingly, the viability assay indicated that mutants lacking YGL231c do not show a hyper-responsive apoptotic phenotype, however its overexpression shows that it is nevertheless anti-apoptotic. Using a yeast strain expressing chromosomally TAP-tagged YGL231c, we found no up-regulation of the endogenous gene due to stress. The deletion mutant is also known to expresses a synthetically lethal phenotype in the presence of alpha-synuclein. While expression of alpha-synuclein caused significant death in both the wild type and deletion mutants, TMEM 85v2 was unable to exhibit a protective role. These findings demonstrate the complexity of the TMEM 85 gene and its anti-apoptotic function in both yeast and human.
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Preferential Estrogen Receptor β Ligands Inhibit Proliferation and Reduce Bcl-2 Expression in Fulvestrant-resistant Breast Cancer CellsRuddy, Samantha 18 January 2013 (has links)
Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2.
Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland.
Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.
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