Cell surface marker and cell cycle analysis, Hedgehog signaling, and flow cytometry.

Detailed cytological analysis of cells undergoing differentiation often reveals clues to the regulation of multiple cell features. The Hedgehog (Hh) signaling cascade is a master regulator of cell fate during differentiation and is implicated in the development of some neoplasias. Hh signaling affects the expression of cell surface markers of differentiation.

Hedgehog signaling and cell cycle control in differentiating erythroid progenitors.

Hedgehog (Hh) signaling regulates differentiation in numerous systems, but its functions in the control of hematopoietic differentiation have not been extensively explored. Initial studies have indicated that hedgehog signaling affects the proliferation and differentiation of erythroid progenitors (Detmer, K., et al.

Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic-like stem cells.

Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self-renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin.

Adult reserve stem cells and their potential for tissue engineering.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher orders. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve precursor cells.

Isoproterenol inhibits transcription of cardiac cytokine genes induced by reactive oxygen intermediates.

Background: Cytokines such as tumor necrosis factor alpha (TNF-alpha) are produced by the myocardium in heart disease and might be stimulated by reactive oxygen. In some cell types, cyclic adenosine monophosphate (AMP) inhibits TNF-alpha production. The authors tested the hypothesis that stimulation of cardiac beta-adrenergic receptors would inhibit cytokine gene transcription induced by reactive oxygen.

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors.

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed.

An IkappaB-alpha mutant inhibits cytokine gene expression and proliferation in human vascular smooth muscle cells.

Background: Inflammatory reaction and intimal proliferation of smooth muscle cells are characteristics of vascular stenotic lesions. Nuclear factor kappaB (NF-kappaB) is involved in regulation of inflammation and cell survival in a variety of cell types. We tested a hypothesis that selective inhibition of NF-kappaB by expression of a mutated, nondegradable inhibitor of NF-kappaB, IkappaB-alphaM, would inhibit proinflammatory cytokine expression and proliferation in human vascular smooth muscle cell.

Endotoxin stimulated cytokine production in rat vascular smooth muscle cells.

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) mRNA increased in response to LPS. Activation of nuclear factor-kappaB (NF-kappaB) presumably results in NF-kappaB binding to regulatory regions of target genes and activating transcription.

Erythroid differentiation in vitro is blocked by cyclopamine, an inhibitor of hedgehog signaling.

Adult hematopoietic differentiation is a developmental process that employs many of the same molecular mechanisms as embryogenesis. To explore the possibility that hedgehog signaling is involved in the control of hematopoietic differentiation, we screened a panel of human leukemia cell lines for the expression of Patched1 and Smoothened, the receptor and coreceptor for hedgehog ligands. Expression was found in multiple cell lines, and Patched1 expression was detected in normal marrow.

Lineage-restricted expression of bone morphogenetic protein genes in human hematopoietic cell lines.

To explore the possibility that bone morphogenetic proteins (BMPs) are autocrine/paracrine regulators of hematopoietic differentiation and function, we screened a panel of human cell lines encompassing the hematopoietic lineages for expression of members of this family of genes. Expression of BMP-2, BMP-4, BMP-6, BMP-7, Growth and Differentiation Factor-1 (GDF-1), Placental Bone Morphogenetic Protein (PLAB), and Transforming Growth Factor-beta3 (TGF-beta3) was detected in one or more cell lines. BMP-2, BMP-4, BMP-7, and TGF-beta3 expression was also found in normal hematopoietic tissue.

Acute hypoxia increases alveolar macrophage tumor necrosis factor activity and alters NF-kappaB expression.

Alterations in alveolar macrophage (AM) function during sepsis-induced hypoxia may influence tumor necrosis factor (TNF) secretion and the progression of acute lung injury. Nuclear factor (NF)-kappaB is thought to regulate the expression of endotoxin [lipopolysaccharide (LPS)]-induced inflammatory cytokines such as TNF, and NF-kappaB may also be influenced by changes in O2 tension. It is thus proposed that acute changes in O2 tension surrounding AMs alter NF-kappaB activation and TNF secretion in these lung cells.

Human pluripotent and progenitor cells display cell surface cluster differentiation markers CD10, CD13, CD56, and MHC class-I.

Each year millions of people suffer tissue loss or end-stage organ failure. While allogeneic therapies have saved and improved countless lives, they remain imperfect solutions. These therapies are limited by critical donor shortages, long-term morbidity, and mortality.

Expression of class I homeobox genes in fetal and adult murine skin.

We examined the expression patterns of several class I homeobox genes in mouse fetal and adult skin. All the genes of the Hox-B locus, except Hoxb-1, are expressed in skin from murine fetuses of 17 and 18 d gestation, at which time the epidermis is undergoing stratification and differentiation. The amount of individual Hox gene message varies considerably, but expression of all genes is detectable by RNase protection except Hoxb-1, which could not be detected even by the reverse transcription-polymerase chain reaction (RT-PCR) assay.

Expression of HOX C homeobox genes in lymphoid cells.

The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines.

Expression of the Hox 2.2 homeobox gene in murine embryonic epidermis.

The expression of the Hox 2.2 gene was studied in mouse fetal skin by in situ hybridization with an antisense RNA probe derived from the homeobox region of this gene. In contrast to the expression of Hox 2.

Modulation of HOX2 gene expression following differentiation of neuronal cell lines.

The expression of the genes in the human HOX2 locus has been studied during differentiation of two human neuroblastoma (SH-SY5Y and Kelly), a human glioblastoma (251-MG), and the murine F9 embryonal carcinoma cell lines. Cells were differentiated with retinoic acid (RA), or with RA together with dibutyral cyclic AMP (db-cAMP) and nerve growth factor (NGF) in order to assess the changes in the expression patterns of these homeobox genes during neuronal differentiation. We show that the genes of the HOX2 locus are expressed in a complex transcription pattern that varies with cell type.

Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines.

We have previously reported that certain genes of the HOX2 cluster of homeobox genes on human chromosome 17 are specifically expressed in human leukemic cell lines with erythroid potential, suggesting that these genes are involved in hematopoietic differentiation. We now show that the expression of the HOX 2.2 gene decreases during erythropoietin-induced differentiation of the erythroid cell line MB02.

Erythroid-restricted expression of homeobox genes of the human HOX 2 locus.

We have previously reported that certain members of the HOX 1 and HOX 2 clusters of class 1 homeobox-containing genes showed lineage-restricted patterns of expression in a small series of human hematopoietic cell lines. We now report on the expression patterns of the entire HOX 2 cluster, consisting of nine homeobox genes, in a broad survey of leukemic cell lines of different phenotypes. The most striking observation is that all but one of the HOX 2 genes are consistently expressed in cells with erythroid character and/or potential, but, with rare exception, not in cells with myelomonocytic or T- or B-lymphoid phenotype.

A human Hox 1 homeobox gene exhibits myeloid-specific expression of alternative transcripts in human hematopoietic cells.

As part of a survey of the expression of homeobox-containing genes in human hematopoietic cells, we identified a novel gene (PL1) expressed only in cells of the myelomonocytic lineage (Shen et al., Proc. Natl.

Alternative splicing of the HOX 2.2 homeobox gene in human hematopoietic cells and murine embryonic and adult tissues.

The HOX 2.2 homeobox gene is expressed in human hematopoietic cell lines with erythroid features (W.-F.

Lineage-restricted expression of homeobox-containing genes in human hematopoietic cell lines.

We investigated the role of homeobox-containing genes in human hematopoiesis because homeobox genes (i) control cell fate in the Drosophila embryo, (ii) are expressed in specific patterns in human embryos, and (iii) appear to function as transcription factors that control cell phenotype in other mammalian organs. Using four homeobox probes from the HOX2 locus and a previously undescribed homeobox cDNA (PL1), we screened mRNAs from 18 human leukemic cell lines representing erythroid, myeloid, and T- and B-cell lineages. Complex patterns of lineage-restricted expression are observed: some are restricted to a single lineage, while others are expressed in multiple lineages.

Expression of retinoic acid receptor alpha mRNA in human leukemia cells.

The expression of the newly described human retinoic acid receptor alpha (RAR alpha) in six nonlymphoid and six lymphoid leukemia cell lines and nine freshly obtained samples of leukemia cells from patients with acute nonlymphoid leukemia was assessed by Northern blot analysis, using a full length cDNA clone of RAR alpha as probe. RAR alpha was expressed in all 12 cell lines and in all fresh leukemia samples as two major transcripts of 2.6 and 3.

Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography.

An enzymatic activity that catalyzes ATP-dependent homologous pairing and strand exchange of duplex linear DNA and single-stranded circular DNA has been purified several thousand-fold from a human leukemic T-lymphoblast cell line. The activity was identified after chromatography of nuclear proteins on a Z-DNA column matrix. The reaction was shown to transfer the complementary single strand from a donor duplex linear substrate to a viral circular single-stranded acceptor beginning at the 5' end and proceeding in the 3' direction (5'----3').

Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase.

The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1.

Effect of monovalent anions on the mechanism of phenol hydroxylase.

The mechanism of phenol hydroxylase (EC 1.14.13.