Hematopoietic transcription regulators and the origins of leukemia.
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Amino Acid Sequence↗
Biomedical subjects
R Hromas
Publications and source records attributed to R Hromas.
Retroviral-mediated gene transfer of bone marrow cells during autologous bone marrow transplantation for acute leukemia.
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Bone Marrow Transplantation↗
Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro.
Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.
Adult↗
Hem-1, a potential membrane protein, with expression restricted to blood cells.
Overlapping cDNAs 3.8 kb in length containing a long open reading frame were obtained that hybridized exclusively to transcripts from hematopoietic cells. Sequence analysis found eight potential membrane domains and two possible cAMP/cGMP phosphorylation sites. This sequence exhibited no homologies with the EMBL/Genbank nucleic acid, SwissProt or GenPept amino acid data bases. The gene is located at 12q13.1, a region of occasional translocations in hematopoietic neoplasia and a rare folic acid fragile site, Fra 12A.
Animals↗
A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells.
Zinc finger genes encode metal-binding proteins that can act as transcriptional regulators of other genes. In an effort to identify activators of the genetic cascade in hemopoietic differentiation, we used degenerate synthetic oligonucleotides to the conserved zinc finger histidine-cysteine link to probe a human myeloid lambda gt11 cDNA library. One of the cDNA clones obtained hybridized preferentially to mRNA from myeloid cells. This cDNA was used to isolate clones encompassing the coding region for this gene. Sequence analysis found 13 zinc finger regions and a glycine-proline-rich region between the fourth and fifth zinc finger domain. The gene was localized to chromosome 19q13.2-4, a chromosome that has a large cluster of zinc finger genes. The gene was preferentially expressed in myeloid leukemia cell lines with the highest mRNA levels noted in HL-60 cells induced to differentiate with retinoic acid. Thus, this new zinc finger gene (designated MZF-1) may be one regulator of transcriptional events during hemopoietic development.
Amino Acid Sequence↗
Cell-type specificity of interferon-gamma-mediated HLA class I gene transcription in human hematopoietic tumor cells.
Major histocompatibility complex class I gene expression plays a central role in cellular immunity and tumor surveillance. A substantial proportion of spontaneous tumors are class I-deficient and numerous experiments have suggested that alterations in class I expression may alter oncogenicity and, as a result, have potential therapeutic impact. Interferons (IFNs) are able to upregulate class I expression by mechanisms that remain to be elucidated, but which appear to be IFN- and cell-type specific. We have characterized in detail the in vivo class I transcriptional response to IFN-gamma in two human hematopoietic tumor cell lines, the class I-deficient K562 cell line and the class I-positive Ramos cell line. In each, IFN-gamma induces a rapid increase in class I transcription, which is sustained in Ramos cells, but transient in K562 cells. In each, stimulation by IFN-gamma is dependent on ongoing protein synthesis, suggesting the requirement for production of a "primary response" protein. These data suggest that more than one type of IFN-gamma-induced signal is operative in the transcriptional response to IFN-gamma. Cycloheximide alone is also capable of inducing a rapid increase in class I transcription in both cell types, suggesting that constitutive attenuation of class I transcription may be a common phenomenon, and that IFN-gamma may act, in part, by interfering with such attenuation.
Antibodies, Monoclonal↗
Identification of a second inducible DNA-protein interaction in the kappa immunoglobulin enhancer.
Control of kappa immunoglobulin light-chain gene expression requires the interaction of tissue specific and developmentally regulated DNA-binding proteins with the kappa gene enhancer. Deletion of enhancer sequences upstream from the NF-kB binding site has been shown to impair enhancer function, implying additional proteins may interact with these sequences. In surveying this region for sites of protein binding, a novel DNA-protein interaction, designated kBF-A, was detected. The binding activity of this factor appears to be specific to activated pre-B cells and correlates with high level induction of kappa transcription in these cells.
Animals↗
Transcriptional regulation of interleukin 3 gene expression in T lymphocytes.
Interleukin 3 (IL-3 or multi-colony-stimulating factor) plays an important role in the hematopoietic response to inflammatory stimuli through its action on both immature and mature blood cells. Like other lymphokines, IL-3 is produced in response to activation of the T-cell receptor and protein kinase C pathways. By using nuclear run-on assays of quiescent and stimulated T-cell lines, we demonstrate that IL-3 gene expression is controlled, at least in part, at the level of transcription. Functional reporter gene analysis was used to delineate two regions of the IL-3 5' flanking sequence responsible for transcriptional stimulation. DNA binding proteins that potentially mediate these responses were then recognized by mobility-shift and DNase footprinting assays. One region responsible for transcriptional enhancement was localized to the sequence GATGAATAAT, the cognate site of a transcription factor, here termed NF-IL3-A. A second region of functional activity and protein binding was localized to a single transcription factor AP-1 site. In addition three functionally inhibitory regions were identified. These results, along with the further characterization of NF-IL3-A, will contribute to the understanding of IL-3 gene regulation in stimulated T cells.
Base Sequence↗
Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions.
The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer.
Animals↗
Nuclear factors bind to regulatory regions of the mouse kappa immunoglobulin gene.
At least two regions of the mouse kappa immunoglobulin gene are necessary for appropriate transcription. One is located within the J-C intron; another is located 5' of the transcription start site in all variable region gene segments and contains a highly conserved octanucleotide sequence, ATTTGCAT. Trans-acting nuclear factors have been thought to interact with these regions on the basis of studies with transient transfection of modified immunoglobulin genes into lymphoid and fibroblast cell lines. Using a sensitive gel electrophoresis DNA binding assay, we have found nuclear factors that bind to these two regulatory regions of the kappa gene. Two patterns of specific binding of nuclear factors to DNA sequences containing the J-C intron enhancer were observed. One pattern was seen in cell lines which do not express immunoglobulin, while a different pattern was observed in cell lines which actively transcribe light chain genes. We also find factors which bind to the octanucleotide containing 5' sequence which are distinct from those which bind the J-C enhancer region.
Animals↗
Potentiation of DNA-reactive antineoplastic agents and protection against S-phase-specific agents by anguidine in Chinese hamster ovary cells.
Anguidine, a protein synthesis inhibitor, has been shown to induce a reversible cell cycle arrest in exponentially growing Chinese hamster ovary cells. The effect of pretreatment with anguidine on the cytotoxicity of subsequently administered various chemotherapeutic agents, hyperthermia, and radiation was investigated. We found that anguidine greatly potentiated the cytotoxic activity of cis-dichlorodiammineplatinum(II) and melphalan by abolishing the initial shoulder and steepening the subsequent exponential portion of the survival curves. Bleomycin-induced cell kill was also potentiated by anguidine pretreatment but to a lesser extent. However, anguidine pretreatment did not substantially alter radiation cytotoxicity. In contrast, anguidine markedly reduced the lethal effect of hydroxyurea, 5-fluorouracil, and hyperthermia, three modalities with S-phase activity. To investigate whether both anguidine-induced potentiation and protection of cells by different antitumor agents were due to its induction of complete suspension of cycle traverse, experiments were also conducted with plateau-phase cultures. Whereas anguidine potentiated cis-dichlorodiammineplatinum(II) cytotoxicity in an identical fashion as noted in exponentially growing cells, its protective effect against lethal damage from Adriamycin was absent. Thus, it appears that the two opposite effects of anguidine modification of cell kill by cytotoxic agents (protection and potentiation) come about by two different mechanisms, with cell cycle arrest underlying cytoprotection and the mechanism of synergistic toxicity remaining obscure.
Animals↗
Selective protection by anguidine of normal versus transformed cells against 1-beta-D-arabinofuranosylcytosine and Adriamycin.
Anguidine, a protein synthesis inhibitor, has been shown previously to induce a reversible arrest of cell progression through all phases of the mitotic cycle without inducing appreciable cell kill. This "frozen" cell cycle state provided protection of Chinese hamster ovary cells against the lethal effects of 1-beta-D-arabinofuranosylcytosine, Adriamycin, hydroxyurea, 5-fluorouracil, and hyperthermia. We now report on the preferential induction of cytostasis by anguidine in normal WI-38 fibroblasts, occurring at one-tenth of the dosage required to inhibit the cycle progression of WI-38 VA13 cells, the SV40 transformant. Pretreatment with anguidine at a concentration producing effective inhibition of normal cell cycle traverse while permitting sustained proliferation of transformed cells resulted in almost complete protection of WI-38 normal cells against the growth-inhibitory effects of 1-beta-D-arabinofuranosylcytosine and Adriamycin, without reducing the antiproliferative effects of these two agents against WI-38 VA13 transformed cells. Thus, this cytokinetic concept of preferential normal tissue protection should be explored in vivo to increase the therapeutic index of cancer chemotherapy.
Cell Cycle↗