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Biomedical subjects

K L Blanchard

Publications and source records attributed to K L Blanchard.

13 recordsLinked to original sources
PubMed2006

Gene amplification and associated loss of 5' regulatory sequences of CoAA in human cancers.

CoAA is an RRM-containing transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. We show that the CoAA gene is amplified at the chromosome 11q13 locus in a subset of primary human cancers including non-small cell lung carcinoma, squamous cell skin carcinoma and lymphoma. Analysis of 42 primary tumors suggests that CoAA amplifies independently from the CCND1 locus. Detailed mapping of three CoAA amplicons reveals that the amplified CoAA gene is consistently located at the 5' boundaries of the amplicons. The CoAA coding and basal promoter sequences are retained within the amplicons but upstream silencing sequences are lost. CoAA protein is overexpressed in tumors containing the amplified CoAA gene. RNA dot blot analysis of 100 cases of primary tumors suggests elevated CoAA mRNA expression. CoAA positively regulates its own basal promoter in transfection assays. Thus, gene amplification, loss of silencing sequence and positive feedback regulation may lead to drastic upregulation of CoAA protein. CoAA has transforming activities when tested in soft agar assays, and CoAA is homologous to oncoproteins EWS and TLS, which regulate alternative splicing. These data imply that CoAA may share a similar oncogenic mechanism with oncogene EWS and that CoAA deregulation may alter the alternative splicing of target genes.

Animals↗
PubMed2000

DNA methylation represses the expression of the human erythropoietin gene by two different mechanisms.

The human erythropoietin gene is expressed predominantly in the kidney and liver in response to hypoxia. Although the signaling cascade for hypoxia is present in many different cell types, the expression of erythropoietin is restricted to only a few tissues. The authors show that the promoter and 5'-untranslated region (5'-UTR) of the erythropoietin gene comprise a CpG island and that methylation of the CpG island correlates inversely with expression. Methylation represses the expression of the erythropoietin gene in 2 ways: high-density methylation of the 5'-UTR recruits a methyl-CpG binding protein to the promoter, and methylation of CpGs in the proximal promoter blocks the association of nuclear proteins. (Blood. 2000;95:111-119)

5' Untranslated Regions↗
PubMed1998

Acute mixed lineage leukemia with an inv(8)(p11q13) resulting in fusion of the genes for MOZ and TIF2.

Chromosomal abnormalities in acute leukemia have led to the discovery of many genes involved in normal hematopoiesis and in malignant transformation. We have identified the fusion partners in an inv(8)(p11q13) from a patient with acute mixed lineage leukemia. We show by fluorescence in situ hybridization (FISH) analysis, Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) that the genes for MOZ, monocytic leukemia zinc finger protein, and TIF2, transcriptional intermediary factor 2, are involved in the inv(8)(p11q13). We demonstrate that the inversion creates a fusion between the 5' end of MOZ mRNA and the 3' end of TIF2 mRNA maintaining the translational frame of the protein. The predicted fusion protein contains the zinc finger domains, the nuclear localization domains, the histone acetyltransferase (HAT) domain, and a portion of the acidic domain of MOZ, coupled to the CREB-binding protein (CBP) interaction domain and the activation domains of TIF2. The breakpoint is distinct from the breakpoint in the t(8;16)(p11;p13) translocation in acute monocytic leukemia with erythrophagocytosis that fuses MOZ with CBP. The reciprocal TIF2-MOZ fusion gene is not expressed, perhaps as a result of a deletion near the chromosome 8 centromere. The MOZ-TIF2 fusion is one of a new family of chromosomal rearrangements that associate HAT activity, transcriptional coactivation, and acute leukemia.

Acetyltransferases↗
PubMed1997

In vivo analysis of DNA-protein interactions on the human erythropoietin enhancer.

The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.

Base Sequence↗
PubMed1993

Regulation of the erythropoietin gene.

Erythropoietin (Epo), the hormone that stimulates red blood cell production, is induced by hypoxia. We have utilized the human hepatoma cell line, Hep3B, to investigate the regulation of the Epo gene. We present evidence that the oxygen sensor in Hep3B cells is a heme protein. Hypoxic and cobalt induction of Epo protein is paralleled by a 50- to 100-fold increase in Epo mRNA which we have accurately quantified by means of an assay based on competitive polymerase chain reaction. This increase in Epo mRNA is due primarily to increased transcription. Transfection experiments utilizing the sensitive luciferase reporter gene show that the minimal portions of the Epo gene required for hypoxic induction include a 53 bp promoter element and a 43 bp enhancer located downstream from the polyadenylation site. Gel shift experiments show that these two regions cross-compete for specific DNA binding proteins. The enhancer contains a hexanucleotide direct repeat with a two bp insert which footprints with nuclear extracts from Hep3B cells and, when mutated, results in loss of hypoxic induction. This sequence is likely to bind to a member of the steroid/thyroid hormone receptor family of DNA binding proteins. These enhancer and promoter elements appear to cooperate in enabling the Epo gene to respond to hypoxia in a physiologically appropriate manner.

Erythropoietin↗
PubMed1992

Development of Lhermitte's sign after bone marrow transplantation.

The authors observed Lhermitte's sign in four patients after bone marrow transplantation (BMT) for hematologic malignancies. Three patients had acute myelogenous leukemia (AML), and one had chronic myelogenous leukemia. Before BMT, the patients with AML received daunorubicin, cytosine arabinoside and etoposide, whereas the patient with chronic myelogenous leukemia received hydroxyurea. One patient with AML received MY-9 antibody-depleted autologous BMT. The other patients received human lymphocyte antigen-identical, allogeneic BMT. Preparative therapy for BMT was cytosine arabinoside, cyclophosphamide, and total body exposure to radiation for two patients, and busulfan, cyclophosphamide, and no exposure to radiation in two other patients. Lhermitte's sign appeared 4 to 8 months after BMT and resolved spontaneously after 2 to 5 months. Neurologic sequelae had developed in none of the patients 16 to 34 months after BMT. No unifying etiologic factor could be identified in these patients. The development of Lhermitte's sign after BMT appears to be a benign, self-limited phenomenon that requires no specific treatment.

Adult↗
PubMed1992

Clonality in myeloproliferative disorders.

The myeloproliferative disorders are a group of hematologic diseases that are believed to arise from somatic mutations in an early hematopoietic stem cell. This statement is based on the demonstration of monoclonal involvement of terminally differentiated myeloid and lymphoid elements. The techniques for establishing clonal derivation of cells are discussed and the application of these techniques to myeloproliferative diseases is reviewed. The evidence for limited myeloid involvement, lineage heterogeneity, in some patients with myeloproliferative disorders is summarized.

Blood Cell Count↗
PubMed1992

Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.

Transcription of the human erythropoietin (Epo) gene is stimulated by exposure to hypoxia and/or cobalt in whole animals and in Hep3B cells. We have systematically investigated the promoter and 3' enhancer elements necessary for this induction by transient transfection of Hep3B cells. We define a promoter region of 53 bp and an enhancer region of 43 bp that confer hypoxia and cobalt inducibility. Each element gives rise to a 6- to 10-fold induction alone. In combination they produce a 50-fold induction after stimulation, similar to the 50- to 100-fold induction of the endogenous Epo gene. Two areas of DNA sequence homology are present in these regions. We demonstrate specific DNA-protein interactions in the enhancer and the ability of the promoter element to compete with these interactions in electrophoretic mobility shift assays. DNase I footprinting and methylation interference data further refine the cis-acting element in the 43-bp enhancer to a short region containing a direct repeat of a steroid/thyroid hormone receptor response element half-site separated by a 2-bp gap. Two half-site consensus sequences are also present in the 53-bp promoter. Site-specific mutation of the half-site sequences in the enhancer destroys the functional activity of the enhancer.

Base Sequence↗
PubMed1991

Clonality in myeloproliferative disorders: analysis by means of the polymerase chain reaction.

The myeloproliferative syndromes are acquired disorders of hematopoiesis that provide insights into the transition from somatic cell mutation to neoplasia. The clonal origin of specific blood cells can be assessed in patients with X chromosome-linked polymorphisms, taking advantage of random inactivation of the X chromosome. We have adapted the PCR for determination of clonality on as few as 100 cells, including individual colonies grown in culture. Amplifying a polymorphic portion of the X chromosome-linked phosphoglycerate kinase (PGK) gene after selective digestion of the active X chromosome with a methylation-sensitive restriction enzyme gave results fully concordant with standard Southern blotting of DNA samples from normal (polyclonal) polymorphonuclear cells (PMN) as well as clonal PMN from patients with myelodysplastic syndrome and polycythemia vera (PCV). We have used this technique to demonstrate heterogeneity of lineage involvement in patients with PCV. The same clinical phenotype may arise from clonal proliferation of different hematopoietic progenitors.

Base Sequence↗
PubMed1991

Clonality in acquired hematologic disorders.

Clonal populations of cells can be identified by a number of different independent approaches, including analyses of karyotype, gene rearrangements, deletions or point mutations, X-linked polymorphisms, and integration of virus into the genome. Assessment of clonality has yielded valuable and surprising clues to the pathogenesis of acquired hematologic disorders. In the myeloproliferative states, clonal expansion of a mutated pluripotent stem cell can induce production of blood cells having a normal phenotype. The transition to acute leukemia is often associated with additional mutations. A similar progression has also been noted in lymphoproliferative disorders, again supporting a multistep pathogenesis of malignancy. Thus, a mutation inducing clonal growth may be a necessary but not sufficient step in induction of malignancy.

Genetic Markers↗
PubMed1983

Two ribonucleic acid-dependent nucleoside triphosphate phosphohydrolases from rat liver nuclei.

Two nucleic acid-dependent ATPases have been isolated from hypotonic extracts of rat liver nuclei and partially characterized. Both enzymes are active with RNA and DNA but are most active with RNA. The most abundant enzyme, ATPase I, is a microheterogeneous, monomeric protein that consists of at least three forms with apparent molecular weights from 53,000 to 60,000. It specifically catalyzes the hydrolysis of ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ ions and nucleic acid cofactor and has a Km for ATP of 0.15 mM. The other enzyme, ATPase II, consists of a single protein with an apparent molecular weight of 37,000. It catalyzes the hydrolysis of all 4 ribonucleoside triphosphates and dATP to the corresponding nucleoside diphosphate and Pi. For both enzymes, nuclear ribonucleoprotein RNA and cytoplasmic poly(A+) RNA are particularly effective cofactors, while total polysomal RNA (primarily rRNA) is a poor cofactor. The properties of these enzymes make them appear similar to other eukaryotic nucleic acid-dependent ATPases that have been isolated but distinct from the prokaryotic enzyme, RNA synthesis termination factor rho. The biological role of these enzymes is unknown.

Adenosine Triphosphatases↗