Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

BACKGROUND: Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. METHODS: In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. RESULTS: Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. CONCLUSION: From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells

Clear Cell Sarcoma of the Kidney and Congenital Mesoblastic Nephroma: Cellular and Molecular Characterization of Primary Tumors, Tumor Cell Lines, and Nude Mouse Xenografts Delineate Wilms\u27 Tumor-Distinct Histopathogenetic Properties

Clear cell sarcoma of the kidney (CCSK) and Congenital mesoblastic nephroma (CMN) are both pediatric renal tumors that differ from Wilms\u27 tumor based on their distinctive histopathologic characteristics and clinical behavior. CCSK is a very malignant tumor characterized by a propensity for bone metastases, contrasting the relatively benign course characteristic of the majority of Wilms\u27 tumors. CMN, in contrast, is a tumor of the newborn usually diagnosed within the first three months of life that is generally cured with surgery alone. To investigate further the cellular and molecular relationships of CCSK, CMN, and Wilms\u27 tumor, a nude mouse xenograft and cell culture system was established for both CCSK and CMN; the immunohistochemical, gene expression, and functional analysis of the p53 tumor suppressor in cell cycle control signal transduction was performed; and Northern blot analysis of genes relevant to normal nephrogenesis was examined. The histologic, ultrastructural, and molecular features of CCSK and CMN nude mouse xenografts, some of which have been passaged over several years, retained the characteristics of the primary tumors. Similarly, cell cultures derived from either primary tumors or mouse xenografts maintained cellular and molecular profiles consistent with findings for primary tumors. p53 mutation is a marker for poor prognosis in Wilms\u27 tumors. p53 immunoreactivity was absent from a large panel of CCSK and CMN primary tumor specimens examined. However, immunohistochemical analysis of CCSK and CMN tumor cell lines surprisingly revealed p53 immunoreactivity in both CMN cell lines but in none of the CCSK cells examined suggesting that p53 was mutated in the benign CMN tumors. Functional analysis of p53 in the same CCSK and CMN cell culture system using a DNA damaging agent cis­ diamminedicholroplatinum (100 µM) demonstrated the presence of functional p53 as determined by the upregulation of p2l Waf-1/Cip-1 mRNA and G1 cell cycle arrest over an eight hour period. The histogeneses of CCSK and CMN is not known. CCSK and CMN primary tumor tissue was examined for mRNA expression of the genes WT-1, PAX-2, Pax-8, and tenascin- C, all genes relevant to normal kidney development. CCSK and CMN were generally negative for WT-1, PAX-2, and Pax-8 expression. CMN, but not CCSK tumor specimens, were positive for tenascin-C mRNA. To evaluate further the histogenesis of these tumors, cultured CCSK and CMN cells were treated with the differentiation agent all-trans retinoic acid (1µM) over three days. Induction of several genes were noted during this period such as PAX-2 and tenascin. The expression of IGF-2, IGF-BP2, and p21waf-1/cip1 were also modulated by this treatment. There were no light microscopically evident changes in cellular morphology in either CCSK or CMN cells. However, transmission and scanning electron microscopic analysis of the cells demonstrated closer cellular juxtaposition and flattening. These studies indicate that the in vivo and in vitro model system for CCSK and CMN will be useful in further investigating the molecular pathological relationships and tumorigenic mechanisms in pediatric renal tumors. The apparent absence of p53 functional impairment in CCSK distinguishes this aggressive tumor type from anaplastic Wilms\u27 tumors which often bear mutation of p53 and suggests an alternative genetic explanation for the poor prognosis of CCSK. Further, a deeper understanding of the gene expression profile of pediatric renal tumors and renal development will be useful in the diagnosis and treatment of CCSK, CMN, and Wilms\u27 tumor. Both the CCSK and CMN xenografts and cell lines also have potential utility in understanding the pathobiology of the developing fetal kidney as well as other pathologies affecting both children and adults

Coordinate Interstitial Deletions Of Retinoblastoma (Rb1) And Neurobeachin (Nbea) Genes On Chromosome 13 In Mgus And Multiple Myeloma

ABSTRACT OF THE DISSERTATION Coordinate interstitial deletions of Retinoblastoma: RB1) and Neurobeachin: NBEA) genes on chromosome 13 in monoclonal gammopathy of undetermined significance: MGUS) and multiple myeloma By Julie O\u27Neal Doctor of Philosophy in Biology and Biomedical Sciences: Molecular and Cellular Biology) Washington University in Saint Louis, 2009 Assistant Professor Michael H. Tomasson, Chair Numeric or structural chromosomal abnormalities are detected in nearly all patients with plasma cell dyscrasias, including primary amyloidosis, monoclonal gammopathy of undetermined significance: MGUS) and multiple myeloma: MM). Chromosome 13 deletions, most frequently monosomy 13, are detected in 10- 20% of MM cases by routine cytogenetics or metaphase fluorescent in situ hybridization: FISH) and are a significant predictor of shortened survival. Previous efforts to map somatically acquired DNA copy number losses on chromosome 13 have been limited by their low resolution. To identify DNA copy number losses on chromosome 13 at high resolution, we used genomic DNA isolated from CD138 enriched bone marrow cells: tumor) from twenty patients xiv with MM, monoclonal gammopathy of undetermined significance: MGUS) or amyloidosis. We used matched skin biopsy: normal) genomic DNA to control for copy number polymorphisms and a novel aCGH array dedicated to chromosome 13 to map somatic DNA gains and losses at unprecedented resolution: \u3e385,000 probes; median probe spacing 60bp). We identified RB1 and NBEA as being coordinately affected by copy number loss in MGUS and MM. To characterize these genes in the context of myeloma biology, we performed sequence and expression analysis on RB1 and found exonic mutations affecting RB1 were extremely rare, RB1 levels were decreased in patient samples harboring monosomy 13, and RB1 protein phosphorylation was not common. Expression analysis of NBEA revealed most patient samples harboring monosomy 13 had reduced NBEA, but to our surprise, a subset harbored high levels. Analysis of Nbea in hematopoietic tissues revealed although it was detected in thymus and spleen, using a fetal liver transplantation assay, Nbea was dispensable for hematopoietic development. Future studies investigating cooperation of RB1 and NBEA in plasma cell dyscrasias are warranted

Involvement of the WT1 and p16 genes in Wilms' tumour and human malignant mesothelioma

Normal cellular homeostasis is established and maintained by the positive and negative regulatory activities of many genes. When these genes are mutated to forms that can contribute to tumorigenesis, either by dominant, gain of function mutations in positive regulators or recessive, loss of function mutations in negative regulators, they are termed oncogenes or tumour suppressor genes, respectively. Identified human tumour suppressor genes, though still relatively few in number, are found to be active in many different cellular processes. I have studied two such genes, the WT1 and p16 genes, which in their wild type forms are known to contribute to development of the urogenital system and the mesothelium (WTI) and to control of the cell cycle (pi6).WT1 was identified as a gene repeatedly disrupted in Wilms' tumour, a common paediatric renal malignancy. This study is one of many which sought to confirm and understand some of the roles of WTI in both normal development and tumorigenesis. Mutation analysis of WTI in samples from patients with sporadic, bilateral and syndrome-associated Wilms' tumour has produced a pattern of results consistent with the findings of other groups. No mutations were detected in the sporadic and bilateral tumours, however exonic point mutations were detected in 7 out of 9 syndrome-associated Wilms' tumour samples that were analysed in detail. Studies examining parental WTI status, genomic imprinting and allele loss distal to the WTI locus are detailed for the syndrome-associated Wilms' tumour samples. Additionally, analysis of WTI in samples from patients with testicular tumours or malignant mesothelioma did not identify any mutations believed to be significant in tumorigenesis.The contribution, resulting effects and complementary nature of WTI and p16 mutations in Wilms' tumour and malignant mesothelioma is discussed

Claudin-1 is the direct target of RUNX3 in gastric epithelial cells

Ph.DDOCTOR OF PHILOSOPH

The Identification of the novel gene SENEX and its role in Endothelial Cell Senescence and Survival

The Identification of the novel gene SENEX and its role in Endothelial Cell Senescence and Survival Cellular senescence was originally described as a mechanism to inhibit the growth of mammalians cells after oncogenic activation, in response to damage or following stress. Senescence is a permanent halt in the cell cycle and causes major alterations in the genetic profile of the cell. Recent research has demonstrated a significant role for senescence in human pathologies, such as tumour development and cardiovascular disease. We describe here the identification of a novel gene, which we have called SENEX, that regulates stress induced premature senescence pathways in endothelial cells (EC) involving p16INK4a and Rb activation. Endogenous levels of SENEX remain unchanged during replicative senescence but are upregulated by H2O2 mediated stress. In contrast to that previously described for senescence in other cell types, the SENEX induced senescent EC are profoundly anti-inflammatory. The cells are resistant to TNFα induced adhesion of neutrophils and mononuclear cells and the surface expression of E-selectin and VCAM1 is decreased in SENEX induced senescent cells. SENEX is also essential for EC survival since depletion by siRNA causes apoptosis. Together, these findings expand our understanding of the role of senescence in the vasculature and identifies SENEX as a fulcrum for driving the resultant phenotype of the endothelium

PRECISE AND EFFICIENT THERAPEUTIC GENOME EDITING FOR THE CORRECTION OF GENETIC DISEASES IN ANIMALS

There are more than 6,052 identified genetic mutations linked to disease in humans and animals. Thanks to the advent of gene editing based on programmable nucleases and the advances in DNA sequencing and writing technologies, it is now possible to make precise changes in eukaryotic genomes with the potential to correct monogenic diseases, from affected cells, tissues, organisms and eventually whole populations. This is the concept behind therapeutic genome editing, which arises out of the idea that instead of pursuing palliative care, the ideal therapy for monogenic diseases would be to develop a method that can directly correct the disease-causing mutations. Many of these disease alleles have been have been unknowingly co-selected when performing phenotypic genetic selection on plants and animals. Although selected breeding has been successful in the establishment and improvement of many different strains of plants and breeds of animals, we have been propagating these disease alleles in the populations. One of these deleterious alleles is the Glycogen Branching Enzyme Deficiency (GBED), which is caused by a nonsense mutation (C > A) in the first exon of the GBE1 gene that severely disrupts glycogen metabolism. This mutation is lethal in homozygotes and an estimated 9% of Quarter Horse and Paint Horse lineages are heterozygote carriers. In this work, we corrected this mutation in a heterozygous cell line derived from a high genetic merit American Quarter Horse stallion, by using CRISPRCas9. The long-term goal is to use the corrected cell lines for somatic cell nuclear transfer (SCNT) thereby generating a cloned animal that maintains the genetic merit of its predecessor, but is free of the GBED mutation. Precise genome editing requires the introduction of a double stranded break (DSB) at an exact location in the genome and the correct DNA repair outcome. Although CRISPRCas9 has allowed for the introduction of precise DSBs in a very efficient manner, the lack of control over cell-autonomous repair mechanisms namely non-homologous end-joining (NHEJ) and homologous recombination (HR), is still the major bottle neck for seamless genome editing. The DNA-dependent protein kinase (DNA-PK), composed of the Ku 70 - Ku 80 heterodimer and the DNA-PK catalytic subunit (DNA-PKvcs), is best known as the NHEJ molecular sensor for DNA damage, but has been also identified as a pattern recognition receptor (PRR) that defends against the invasion of foreign nucleic acids. Here we devised a novel strategy that capitalizes on the natural ability of the Vaccinia virus (VACV) C16 protein that evolved as an elegant subversion mechanism to inhibit the detection of the VACV genome by the host cytoplasmic PRR defenses, specifically the Ku-mediated DNA sensing

The role of interleukin 10 in children with juvenile idiopathic arthritis

This thesis investigates the function of the IL-10 5' flanking region haplotypes and the association of these haplotypes in children with Juvenile Idiopathic Arthritis (JIA). We have shown that children with extended oligoarticular JIA are less likely to have a genotype containing the ATA haplotype than children with oligoarticular JIA (p<0.05, OR = 1.9,95% Cl: 1 to 3.5). The ATA haplotype is associated with lower reporter gene expression in transfection studies (Crawley, 1999c). The ATA/ATA genotype was shown to be associated with lower IL-10 production using whole blood culture in controls (p<0.02). This is consistent with the observation that mean IL-10 production was lower in the parents of those with extended oligoarticular disease when compared to the parents of children with oligoarticular disease (p=0.03, 95% Cl 88 to 2016). A similar effect was seen in asthma where those with severe asthma were less likely to have ATA containing genotypes than controls (p=0.04, OR1.57, Cl: 1.01 to 2). We did not show a difference in genotype distribution between patients with SLE and controls. The transmission disequilibrium test (TDT) demonstrated an increase in transmission of the ATA haplotype to patients with oligoarticular onset JIA (p=0.05). There was also increased transmission of both the ACC and the ATA haplotype to patients with uveitis (p=0.014 for each transmission). Strong linkage disequilibrium was demonstrated (p<0.0005) between these haplotypes and a microsatellite at -1000 in the IL-10 5' flanking region which has previously been shown to be associated with rheumatoid arthritis and systemic lupus erythematosus. Treatment of patients with methylprednisolone was associated with large changes in ex vivo stimulated IL-10 production which was strongly correlated with the change in ESR (correlation coefficient 0.922, p<0.01). To investigate whether the IL-10 5' flanking region haplotypes affected transcription, THP-1 cells were transfected with IL-10 luciferase DNA reporter constructs. Overall, reporter gene expression was higher with the GCC than the ATA construct when the THP-1 cells were transfected using Effectene Liposomes (median fold difference 1.71, range 1.12 to 5.49)