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.

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.

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

No description available.

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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.

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.

Diss. (sammanfattning) Umeå : Umeå universitet, 2006. / Härtill 3 uppsatser.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 140-162). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2003. / Vita. Bibliography: 142-166.