The mesenchyme homeobox genes, MEOX1 and MEOX2, encode homeodomain transcription factors. Studies of Meox1/Meox2 knockout mice established that these proteins are partially redundant during development, suggesting that they may regulate common target genes. In the adult vasculature, MEOX2 is expressed in vascular smooth muscle and endothelial cells. MEOX2 has been demonstrated to: i) inhibit proliferation, ii) activate apoptosis and iii) induce senescence. In contrast, the role of MEOX1 has not been studied in the vasculature. Currently, there are two known target genes of MEOX2: cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1/WAF1) and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a), which regulate transient (quiescent) and permanent (senescent) cell cycle arrest. Senescence is postulated to contribute to the development of atherosclerotic vascular disease by promoting endothelial dysfunction. We hypothesized that MEOX1 and MEOX2 would activate both p21CIP1/WAF1 and p16INK4a expression, as well as induce apoptosis, cell cycle arrest and senescence in endothelial cells. Furthermore, we postulated that the majority of newly identified MEOX target genes in endothelial cells would be regulated by both MEOX1 and MEOX2. MEOX proteins were expressed in human endothelial cells via adenoviral transduction. Levels of target gene expression were measured by luciferase reporter gene assays, western blot and quantitative real-time PCR. Electrophoretic mobility shift assays were used to demonstrate MEOX binding to DNA. Cellular proliferation, senescence, and apoptosis were evaluated. For the identification of novel target genes, microarrays were used to compare levels of gene expression in endothelial cells transduced with MEOX constructs or control virus. Both MEOX1 and MEOX2 activated p21CIP1/WAF1 and p16INK4a gene transcription, inhibited proliferation and induced apoptosis and senescence in endothelial cells. MEOX activation of p21CIP1/WAF1 transcription occurs via a DNA-binding independent mechanism that requires the SP1 transcription factor. In contrast, MEOX activation of p16INK4a transcription is dependent upon DNA-binding. Microarray analysis revealed that both MEOX1 and MEOX2 increased the expression of intercellular adhesion molecule 1 (ICAM-1) and decreased the expression of nitric oxide synthase 3 (NOS3/eNOS). Taken together, we conclude that MEOX1 and MEOX2 have similar target genes in endothelial cells including p21CIP1/WAF1, p16INK4a and eNOS. As increased endothelial senescence and decreased nitric oxide production are hallmarks of endothelial dysfunction, this study proposes a role for the MEOX proteins in the progression of atherosclerotic vascular disease.

The mesenchyme homeobox genes, MEOX1 and MEOX2, encode homeodomain transcription factors. Studies of Meox1/Meox2 knockout mice established that these proteins are partially redundant during development, suggesting that they may regulate common target genes. In the adult vasculature, MEOX2 is expressed in vascular smooth muscle and endothelial cells. MEOX2 has been demonstrated to: i) inhibit proliferation, ii) activate apoptosis and iii) induce senescence. In contrast, the role of MEOX1 has not been studied in the vasculature. Currently, there are two known target genes of MEOX2: cyclin-dependent kinase inhibitor 1A (CDKN1A/p21CIP1/WAF1) and cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a), which regulate transient (quiescent) and permanent (senescent) cell cycle arrest. Senescence is postulated to contribute to the development of atherosclerotic vascular disease by promoting endothelial dysfunction. We hypothesized that MEOX1 and MEOX2 would activate both p21CIP1/WAF1 and p16INK4a expression, as well as induce apoptosis, cell cycle arrest and senescence in endothelial cells. Furthermore, we postulated that the majority of newly identified MEOX target genes in endothelial cells would be regulated by both MEOX1 and MEOX2. MEOX proteins were expressed in human endothelial cells via adenoviral transduction. Levels of target gene expression were measured by luciferase reporter gene assays, western blot and quantitative real-time PCR. Electrophoretic mobility shift assays were used to demonstrate MEOX binding to DNA. Cellular proliferation, senescence, and apoptosis were evaluated. For the identification of novel target genes, microarrays were used to compare levels of gene expression in endothelial cells transduced with MEOX constructs or control virus. Both MEOX1 and MEOX2 activated p21CIP1/WAF1 and p16INK4a gene transcription, inhibited proliferation and induced apoptosis and senescence in endothelial cells. MEOX activation of p21CIP1/WAF1 transcription occurs via a DNA-binding independent mechanism that requires the SP1 transcription factor. In contrast, MEOX activation of p16INK4a transcription is dependent upon DNA-binding. Microarray analysis revealed that both MEOX1 and MEOX2 increased the expression of intercellular adhesion molecule 1 (ICAM-1) and decreased the expression of nitric oxide synthase 3 (NOS3/eNOS). Taken together, we conclude that MEOX1 and MEOX2 have similar target genes in endothelial cells including p21CIP1/WAF1, p16INK4a and eNOS. As increased endothelial senescence and decreased nitric oxide production are hallmarks of endothelial dysfunction, this study proposes a role for the MEOX proteins in the progression of atherosclerotic vascular disease.

by Kwok-keung Chan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 177-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

Thesis (Ph. D.)--University of Virginia, 2006. / Bracketed B in title is the Greek letter beta. Includes bibliographical references (leaves150-169). Also available online through Digital Dissertations.

No description available.

Hoi-Yeung Li. / "May 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 151-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

HOX proteins are evolutionarily conserved homeodomain-containing transcription factors involved in hematopoiesis and patterning during embryogenesis. Their tasks as master regulators of embryonic development are achieved in large part through their ability to interact with co-factors of the PBX and MEIS/PREP families, which constitute the broader three amino-acid loop extension (TALE) class of homeodomain proteins. HOX, MEIS, and PBX have been implicated in leukemic hematopoiesis due to their association with hematological malignancies. The oncogenic function of MEIS1 in accelerating the onset of acute myeloid leukemia induced by HOX was mapped to its C-terminal transactivation domain, which is responsive to PKA signaling. This thesis extends our understanding regarding the mechanism by which MEIS1A executes its C-terminal transactivation function in vivo. We describe the involvement of CREB and its co-activators CBP and TORC in conferring the PKA-responsiveness of the ME1S1A C terminus. CREB mutants that fail to bind CBP or TORC also fail to promote PKA induction mediated by the C terminus of ME1S1A. TORC was further shown to be capable of bypassing the need for PKA to activate transcription by MEIS1, an ability endowed by its physical interaction with MEIS1. Chromatin immunoprecipitation (ChIP) demonstrated a concerted recruitment of endogenous MEIS1, TORC2, and CREB proteins on ME1S1 target genes. In addition, this thesis also characterizes the promoter of the murine Meis1 gene. Meis1 possesses multiple transcription start sites upstream of its translation initiation site. We identified a ME1S·PBX consensus recognition site within the Meis1 promoter and showed that PBX1 binds to this sequence in vitro. Our ChIP assay results further suggest an autoregulatory mode for the Meis1 gene as revealed by a co-occupancy of endogenous CREB, TORC2, PBX1, and MEIS1 itself on the Meis1 promoter. Collectively, this thesis proposes a mechanistic action conferred by CREB, CBP and TORC in the PKA-inducible transactivation of ME1S1A, and provides new information on the Meis1 promoter.

Prostate cancer is a frequently diagnosed malignancy which ranges from an indolent asymptomatic tumour to an aggressive, rapidly lethal systemic disease. Determination of chromosomal, genetic and epigenetic alterations associated with prostate carcinogenesis have led to the characterisation of functional consequences of these alterations, thereby elucidating pathways contributing to malignant growth that can be utilised clinically and therapeutically. FLJ22318 is a novel hypothetical protein that was identified by yeast two-hybrid analysis to interact with the prostatic homeodomain protein, NKX3-1. Expression of NKX3-1 is largely restricted to epithelial cells of the adult prostate where it is involved in maintaining the prostatic phenotype, while NKX3-1 expression is reduced or absent in prostate tumours. In contrast, FLJ22318 expression is documented in cDNA libraries derived from a variety of human adult and foetal tissues including the prostate, suggesting that it may be ubiquitously expressed and that it potentially interacts with a number of proteins in addition to NKX3-1. FLJ22318 expression is undocumented in human prostate tumours. Bioinformatic analyses have postulated multiple FLJ22318 mRNA transcripts however the proposed open reading frames encoded by these transcripts predict the FLJ22318 protein to contain three strong protein-protein interaction domains, a Lissencephaly type-1-like homology (LisH), a C-terminal to LisH (CTLH) and a CT11-RanBPM (CRA) domain. Of the 44 single nucleotide polymorphisms identified within the FLJ22318 gene, none are located within the protein coding region suggesting that FLJ22318 may be critical for cell survival and/or function. Comparison of the amino acid sequence between human FLJ22318 and its orthologues in a diverse range of mammalian species identified >97% sequence homology, providing further strong evidence of the critical cellular function of FLJ22318. To characterise the biological activity of FLJ22318 in prostate cancer cells, the FLJ22318 coding region was amplified by polymerase chain reaction (PCR) and ligated into mammalian and bacterial expression vectors to encode V5-, myc-, GFP-, HA-, and GST-FLJ22318 fusion proteins. Interaction between FLJ22318 and NKX3-1 was confirmed using (reverse) yeast two-hybrid, GST pull-down and co-immunoprecipitation assays. These data were supported by confocal microscopy which demonstrated the perinuclear and nuclear co-localisation of FLJ22318 and NKX3-1 in prostate cancer cells. Northern blotting identified expression of ~2Kb and ~4Kb FLJ22318 mRNA’s in prostate cancer cell lines, which was consistent with bioinformatic analyses of mRNA species. Transfection of prostate cancer cells to overexpress FLJ22318 did not alter endogenous NKX3-1 levels, however FLJ22318 exhibited transcriptional repressor function on an NKX3-1 responsive element and increased NKX3-1 transcriptional repressor activity on this element. To further investigate FLJ22318 function, additional yeast two-hybrid analyses were performed in prostate cancer cells to identify potential FLJ22318 binding proteins. These studies isolated cDNA’s encoding 33 different proteins involved in cell metabolism and apoptosis as well as transcriptional regulators associated with control of cellular proliferation. One of the candidate FLJ22318 interactors, protein kinase, interferon-inducible double stranded RNA dependent activator (PRKRA/PACT) was shown using confocal microscopy to extensively co-localise with FLJ22318 in the cytoplasm and perinuclear regions of prostate cancer cells. Preliminary co-immunoprecipitation and GST pull-down assays have provided additional evidence of the interaction of PRKRA and FLJ22318. Results of this thesis have generated important information characterising the structure of the FLJ22318 gene and protein, the interaction between FLJ22318 and NKX3-1 and the potential functions of FLJ22318 in prostate cancer cells. As the FLJ22318 gene is located on 5q35, a chromosomal region frequently disrupted in a variety of tumours, future studies of the biological activity of FLJ22318 will clarify its normal cellular functions and its contribution to tumorigenesis or malignant progression in the prostate and in other tissues.

Thesis (Ph. D.)--Cornell University, January, 2008. / Vita. Includes bibliographical references.