<p>The E-protein transcription factors E2A and HEB regulate various cell processes during the development of B and T lymphocytes, including cell differentiation, lineage commitment, recombination of immune receptor genes, proliferation, and survival. B cell development is dependent on E2A from the earliest stages whereas T cell development relies on the cooperative efforts of both E2A and HEB. Established work demonstrates that the timing and dosage of E-protein expression is critical for mediating these diverse functions. The goal of this dissertation is to develop and utilize new genetic tools to manipulate the timing and dosage of E2A and HEB expression in order to enhance our understanding of E-protein function. Here we develop two new mouse models to identify novel lineage and stage specific roles of E-proteins during B lineage commitment and thymocyte selection.</p><p>First, we have generated an E2A inducible mouse model to allow reversible regulation of E2A function and precise timing of induction at the protein level. This system was created by inserting a tamoxifen responsive region of the estrogen receptor ligand binding domain (ER) at the carboxyl end of the <italic>tcfe2a</italic> gene, encoding E2A, to generate E2AER fusion proteins. To our knowledge, the ER fusion system has not yet been tested from an endogenous locus in live animals. Using the E2AER system, we have demonstrated rapidly induced E2AER activity upon tamoxifen treatment that is capable of supporting B cell development in an <italic>ex vivo</italic> culture system. In addition to characterizing the kinetics and reversibility of this inducible system, we have utilized tamoxifen treatment of E2AER B cell progenitors to identify potential novel E2A target genes driving B lineage commitment.</p><p>Second, we have analyzed E-protein function during the double positive (DP) stage of alpha beta T cell development by using a Cre-loxp conditional deletion system. Here, E-protein dosage was manipulated by removal of both E2A and HEB, and the timing of deletion was controlled by using a CD4Cre transgene. During development, survival through the DP stage and initiation of differentiation to the subsequent single positive (SP) stage for generation mature alpha beta T cells is dependent on the production of a functional alpha beta T cell receptor (TCR). The mechanism that maintains cells at the DP stage prior to expression of a mature TCR remains unclear. In this study, we have shown that E2A and HEB together are required to maintain DP fate and regulate the transition to the SP stage. Loss of E2A and HEB in DP thymocytes was sufficient to trigger DP to SP differentiation, even in the absence of a TCR. Deletion of E2A and HEB allowed cells to bypass the requirement for a TCR-mediated positive selection signal. These findings identify E2A and HEB as key regulators enforcing thymocyte positive selection to ensure maturing T cells express a functional receptor.</p> / Dissertation

Non-steroidal anti-inflammatory drugs (NSAIDs) and their NO derivatives (NO-NSAIDs), and synthetic analogs are highly effective as anticancer agents that exhibit relatively low toxicity compared to most clinically used drugs. However, the mechanisms of action for NSAIDs and NO-NSAIDs are not well defined and this has restricted their clinical applications and applications for combined therapies. Earlier studies from our laboratory reported that specificity protein (Sp) transcription factors (Sp1, Sp3 and Sp4) are overexpressed in several types of human cancers including colon cancer and many Sp-regulated genes are pro-oncogenic and individual targets for cancer chemotherapy. Based on published results showing that NSAIDs downregulate several putative Sp-regulated genes, we hypothesized that the anticancer properties of NSAIDs may be due, in part, to downregulation of Sp transcription factors. NSAIDs including aspirin and tolfenamic acid (TA) and nitro derivatives of NSAIDs such as GT-094 have been investigated in colon cancer cells and in vivo xenograft models. Aspirin and TA induced apoptosis and decreased colon cancer cell growth and tumor growth in vivo and downregulated genes associated with cell growth, survival, and angiogenesis. Previous RNA interference studies in this laboratory have shown that many of these genes are regulated, in part, by Sp transcription factors Sp1, Sp3 and Sp4 that are overexpressed in colon and other cancer cell lines. Not surprisingly, these NSAIDs also decreased Sp1, Sp3 and Sp4 proteins and Sp-regulated gene products in colon cancer cells and this was due to caspase-dependent proteolysis of Sp1, Sp3 and Sp4 proteins. Aspirin-induced activation of caspases and degradation of Sp1, Sp3 and Sp4 was due to sequestration of zinc and could be reversed by addition of zinc sulphate, whereas TA mediated induction of caspases was independent of zinc ions and is currently being investigated. GT-094 is a novel NO chimera-containing NSAID, which also inhibited colon cancer cell proliferation and induced apoptosis; these effects were accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated Sp and Sp-dependent gene products and was due to decreased expression of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp transcriptional repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to ROS-dependent disruption of miR-27a:ZBTB10. The importance of ROS induction in targeting Sp transcription factors was also confirmed using pro-oxidants such as ascorbic acid, hydrogen peroxide and t-butyl hydroperoxide and similar results have been observed in collaborative studies with other ROS inducers in colon cancer cells. Many cancer cell lines and tumors exhibit addiction to non-oncogenes such as Sp1, Sp3 and Sp4 for maintaining the oncogenic phenotype and future research will focus on the mechanisms of ROS-mediated targeting of Sp transcription factors which represents a novel approach for cancer chemotherapy.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 108-125.