Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein.     

Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils

Galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, was investigated with respect to its ability to activate the human neutrophil NADPH-oxidase. The galectin-3-induced activity was determined with in vivo exudated cells (obtained from a skin chamber) and compared with that of peripheral blood neutrophils. Galectin-3 was found to be a potent activator of the NADPH-oxidase only in exudated neutrophils and the binding of galectin-3 to the surface of these cells was increased compared with peripheral blood cells. Different in vitro priming protocols resulting in degranulation were used to mimic the exudation process in terms of increasing the receptor exposure on the cell surface. Galectin-3 could induce an oxidative response similar to that in exudated cells only after a significant amount of the intracellular organelles had been mobilized. This increase in oxidative response was paralleled by an increased binding of galectin-3 to the surface of the cells. The major conclusion of the study is that galectin-3 is a potent stimulus of the neutrophil respiratory burst, provided that the cells have first experienced an extravasation process. The results also imply that the neutrophil response to galectin-3 could be mediated through receptors mobilized from intracellular granules, and we report the presence of galectin-3-binding proteins in such organelles.     

Activation of c-myc gene expression by tumor-derived p53 mutants requires a discrete C-terminal domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3' end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

Epithelial galectin-3 during human nephrogenesis and childhood cystic diseases

Galectin-3 is a beta-galactoside-binding protein with putative roles in development, oncogenesis, and inflammation. Its expression in human nephrogenesis has not been previously reported. This study examines galectin-3 expression in early human embryos by Western blot and immunohistochemistry. This 33-kD protein was detected in the apical domain of distal tubules of the mesonephros and also in the mesonephric duct. In the metanephros, the adult kidney precursor, galectin-3 was detected in the apical domains of ureteric bud branches, and there was intense expression in fetal medullary and papillary collecting ducts in both the cytoplasm and plasma membranes. Low levels of galectin-3 were detected in the cytoplasm of a subset of cells in adult collecting ducts; these were alpha-intercalated cells because they expressed basal band 3 protein. In human multicystic dysplastic kidneys, all diseased epithelia had an embryonic apical expression pattern of galectin-3 and, in addition, all cystic epithelia in autosomal recessive polycystic kidneys expressed this molecule. It is concluded that galectin-3 is expressed by cells of the mesonephric duct/ureteric bud lineage, and it is speculated that the different subcellular locations may be implicated in both the regulation of normal growth and differentiation of this lineage, as well as in the pathogenesis of cystic epithelia.