Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 164-183). Also available on the Internet.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

TAX1 Binding Protein 2 (TAX1BP2) has been found to be a centrosome duplication regulating protein. Previous findings have demonstrated that over-expression of TAX1BP2 suppresses centrosome over-duplication. Recently, our lab has revealed that TAX1BP2 is a novel tumor suppressor in hepatocellular carcinoma (HCC) regulated by cyclin-dependent protein kinase 2 (CDK2), nevertheless, the molecular mechanism of how TAX1BP2 regulates centrosome duplication and the link between its centrosome duplication regulatory ability and the tumor suppressing property remain elusive. With the aim to understand the roles of TAX1BP2 in HCC, the present study intended to investigate the link between centrosome duplication regulating ability and tumor suppressing property. Polo-like kinase 4 (PLK4) is a special member of the Polo-like kinase family as its structure is diverged from other family members. Instead of having two Polo-boxes, it carries one Polo-box and one cryptic Polo-box. It has been shown that PLK4 is involved in the formation of centrioles, an important component of centrosome, and is a key regulator of centrosome duplication. Based on the functional similarity, it was hypothesized that PLK4 may function as a regulator of TAX1BP2. To define if PLK4 regulate TAX1BP2, the interaction between PLK4 and TAX1BP2, both in vivo and in vitro, was first confirmed using affinity pulldown and co-immunoprecipitation assays. To understand the significance of the physical interaction, in vitro and in vivo kinase assay were used to study the phosphorylation activity between PLK4 and TAX1BP2. It was demonstrated that TAX1BP2 is a potential substrate of PLK4. Centrosome duplication assay was also performed to investigate if over-expression of PLK4 abolished the centrosome over-duplication suppressing ability of TAX1BP2. In order to delineate the signaling pathway of TAX1BP2, the interaction between TAX1BP2 and its cellular interacting partners was investigated in this study. Ten proteins were isolated as potential interacting partners of TAX1BP2 using Tandem affinity purification (TAP) coupled with Mass Spectrometry protein fingerprinting. Two of the ten proteins, the Ezrin and Mortalin, were confirmed to be binding partners of TAX1BP2 using affinity pull-down assay and TAP, respectively. The identification of the interacting partners suggested that TAX1BP2 may modulate centrosome duplication via alteration of the subcellular localization of Mortalin. These findings helped to delineate the signaling pathway of TAX1BP2 and enabled the better understanding of the roles of TAX1BP2 in tumor suppressor function of HCC. In summary, we demonstrated that TAX1BP2 contains a centrosome duplication regulatory domain (CDRD) and its centrosome duplication regulating ability is critical for its tumor suppressing property. Moreover, three novel interacting partners of TAX1BP2, including Ezrin, PLK4 and Mortalin, are identified. Our findings provide a new insight into the roles of TAX1BP2 in centrosome duplication, hepatocarcinogenesis and metastasis. / published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

The tumour suppressor p53 has evolved a MDM2-dependent feedback loop that has a dual role as either a stimulator of p53 protein translation through mRNA binding or a stimulator of p53 protein degradation through the ubiquitin-proteasome system. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic drug-binding pocket and we have evaluated whether the lid of MDM2 is a physiological regulator of this dual function of MDM2. Deletion of this flexible pseudosubstrate motif inhibits MDM2 indicating that this peptide stretch can function as a positive regulatory motif. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2S17D) stabilizes the binding of MDM2 towards p53. Molecular modeling orientates the pseudo-substrate motif over a charged surface patch on the MDM2 surface at Arg97/Lys98 and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phosphomimetic mutation. Transient or inducible low level expression of MDM2WT can promote an increase in p53 protein steady-state levels whilst the expression of MDM2S17D in cells results in p53 protein de-stabilization. Phospho-specific antibodies to the MDM2 lid demonstrate two physiological conditions that alter lid phosphorylation: (i) lid hypo-phosphorylation occurs after DNA damage where p53 protein is stabilized and (ii) lid hyper-phosphorylation occurs at high cell density under conditions where p53 protein is de-stabilized. Expression of MDM2S17D in cells also de-stabilizes hyperubiquitinated mutant p53 under conditions where MDM2WT has no effect on mutant p53 protein degradation. The lid functions as a flexible regulatory motif whose phosphorylation switches MDM2 from a synthesis mode to a degradation mode with implications for defining the physiological signals that control the MDM2-p53 feedback regulatory loop.

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 143-162).

EGR1, one of the immediate-early response genes, plays an important role as a mediator for transmitting extracellular stimuli. EGR1 is down regulated in many cancers. Many studies show that it functions as a tumor suppressor gene in a variety of cancers. EGR1 also acts as an oncogene in number of cancers. We found that in rhabdomyosarcoma (RMS), which is a muscle derived pediatric cancer, EGR1 was expressed in both RMS subtypes, embryonal and alveolar, but with a much higher expression profile in embryonal RMS. This suggests different mechanisms of down regulation of EGR1 in these two subtypes. Molecular and cellular approaches were used to characterize the functional role of EGR1 in RMS. We found that over expression of EGR1 in alveolar RMS significantly decreased cell proliferation, mobility, and anchorage-independent growth. We showed that exogenous EGR1 up regulated the cell cycle regulator, p21, which is normally repressed in RMS. EGR1 also promoted differentiation in RMS cells by up regulating several genes involved in muscle differentiation including myosin heavy chain (MyHC), MyoD and myogenin. We found that EGR1 interacts with the oncogene TBX2 in RMS cells and that TBX2 inhibits EGR1 function. To understand how TBX2 inhibits EGR1, we depleted TBX2 in RMS and we found an up regulation of the EGR1 targeted tumor suppressor gene, PTEN, and the cysteine protease inhibitor gene, CST6. Also, we performed luciferase assays and found that TBX2 decreased the expression of luciferase constructs fused with the PTEN promoter when TBX2 was co-transfected with EGR1. Our novel findings on the EGR1 function in RMS highlights the significant role of EGR1 in muscle development and tumor growth. Significantly, our work also suggests the EGR1 could promote tumor regression in RMS through inducing programmed cell death, or apoptosis. We found that EGR1 induced apoptosis through triggering the intrinsic apoptosis pathway and activating caspase cascades involving caspase 3 and caspase 9, which are essential mitochondrial apoptotic factors. Also, we observed the activation of two pro-apoptotic factors, BAX and dephosphorylated BAD, which are both located upstream of the caspase cascades in the intrinsic pathway. Also, we found in our study that EGR1 is repressed by the catalytic subunit of PRC2 complex, EZH2, which mediates gene silencing through methylation of lysine 27 on histone 3 (H3K27me3). EGR1 also sensitized RMS cells to chemotherapeutic agents, which could be a future direction for improved therapeutic targeting. Therefore, this work provides a novel and powerful molecular therapeutic target for RMS cancer.

No description available.