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- Walz, Thomas M., 1960-
(author)
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Transforming growth factor a in human hematopoietic cells
- 1994
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Doctoral thesis (other academic/artistic)abstract
- TGF-a, transforming growth factor a, is a potent growth factor belonging to the family of EGF (epidermal growth factor) -related proteins. Binding to the same receptor as EGF it has a ubiquitous repertoire of target cells, including, mesenchymal, epithelial and neuronal cells. The occurrence and role of TGF-a in hematopoietic cells has not been elucidated. Therefore the present work was initiated to (I) examine the expression of TGF-a and the EGF receptor in nmmal circulating human blood cells; (2) study the effects of specific cytokines on TGFa mRNA levels and protein release in mature blood cells; (3) examine the occurrence of TGF-a and the EGF receptor in normal human bone marrow cells; (4) determine TGF-a gene expression and protein production during granulocyte and monocyte/macrophage differentiation in vitro.TGF-a mRNA was consistently found in white blood cells from normalhealthy donors as shown by Northern blot analysis. In situ hybridizationexperiments assigned the TGF-a gene expression to the eosinophils; no other cell types were recognized by the complementary TGF-a RNA probe. Incubation of white blood cells led to liberation of TGF-a to the culture medium, as determined by ELISA.White blood cells exposed in vitro to the cytokines GM-CSF (granulocytemacrophage colony stimulating factor) or interleukin (IL)-3 showed increased levels of TGF-a mRNA. The effect of GM-CSF was different from that of IL-3, since only GM-CSF augmented the release of TGF-a protein from the cells.Immunohistochemical examination of human normal bone marrow cells, using a monoclonal TGF-a antibody, revealed TGF-a-reactive material on erythroid cells, at all stages of differentiation. The nature of the stain, in conjunction with the fact that the cells could be pulled out by immunomagnetic cell sorting would seem to indicate a membrane-bound, extracellular configuration of TGF-a, rather than an intracellular one. Using a different antibody a different staining pattern was obtained, indicating the presence of TGF-a in eosinophilic precursor cells and in promyelocytes and neutrophilic myelocytes.Attempts were made to identify target cells for TGF-a, i.e. EGF receptorcarrying cells. Intron-differential reverse transcriptase PCR was used to detect the EGF receptor signal. Immunohistochemistry revealed the EGF receptor protein in a small but distinct population of immature, blast-like cells of myelomonocytic appearance.The expression of TGF-a was monitored, at the mRNA and protein level, in human leukemic cells induced to differentiate in culture. Differentiation of the promyelocytic cell line, HL-60, along the granulocytic pathway was accompanied by increased TGF-a mRNA levels and TGF-a protein release.When tbe HL-60 cells were brought towards monocytes/macrophages tbe effect on TGF-a expression depended on the inducing agent used, irrespective of a number of differentiation criteria.Differentiation of tbe monocytoid cell line, U-937, witb different inducers had different effects on TGF-a mRNA levels as well as TGF-a release. This supports tbe idea of phenotypic heterogeneity in tbe differentiated cells.
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- Wolfraim, Lawrence A, et al.
(author)
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Loss of Smad3 in acute T-cell lymphoblastic leukemia
- 2004
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In: New England Journal of Medicine. - 0028-4793 .- 1533-4406. ; 351:6, s. 552-559
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Journal article (peer-reviewed)abstract
- BACKGROUND: The receptors for transforming growth factor β (TGF-β) and their signaling intermediates make up an important tumor-suppressor pathway. The role of one of these intermediates - Smad3 - in the pathogenesis of lymphoid neoplasia is unknown. METHODS: We measured Smad3 messenger RNA (mRNA) and protein in leukemia cells obtained at diagnosis from 19 children with acute leukemia, including 10 with T-cell acute lymphoblastic leukemia (ALL), 7 with pre-B-cell ALL, and 2 with acute nonlymphoblastic leukemia (ANLL). All nine exons of the SMAD3 gene (MADH3) were sequenced. Mice in which one or both alleles of Smad3 were inactivated were used to evaluate the role of Smad3 in the response of normal T cells to TGF-β and in the susceptibility to spontaneous leukemogenesis in mice in which both alleles of the tumor suppressor p27Kip1 were deleted. RESULTS: Smad3 protein was absent in T-cell ALL but present in pre-B-cell ALL and ANLL. No mutations were found in the MADH3 gene in T-cell ALL, and Smad3 mRNA was present in T-cell ALL and normal T cells at similar levels. In mice, the loss of one allele for Smad3 impairs the inhibitory effect of TGF-β on the proliferation of normal T cells and works in tandem with the homozygous inactivation of p27Kip1 to promote T-cell leukemogenesis. CONCLUSIONS: Loss of Smad3 protein is a specific feature of pediatric T-cell ALL. A reduction in Smad3 expression and the loss of p27Kip1 work synergistically to promote T-cell leukemogenesis in mice.
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