2--7--bis(carboxyethyl)-5(6)-carboxyfluorescein and Leukemia--Myeloid--Acute

The NADPH oxidase is one of the main microbicidal systems of granulocytes. Stimulation of the oxidase during infection leads to a burst of metabolic acid generation. Potentially deleterious cytosolic acidification is prevented by the simultaneous activation of homeostatic H+ extrusion mechanisms, including a recently described H+ conductance. Studies in granulocytes from chronic granulomatous disease patients have suggested a relationship between the oxidase and the H+ conductive pathway. In this report we compared the expression of the H+ conductance and the NADPH oxidase during granulocytic differentiation of dimethyl sulfoxide-induced HL-60 cells. Patch-clamp determinations demonstrated that the H(+)-selective current detectable in differentiated HL-60 cells is virtually absent in uninduced cells. The H+ conductance was also estimated fluorimetrically, measuring changes in the cytosolic pH of suspended cells. Imposition of an inward protonmotive force failed to induce significant cytosolic acidification. In contrast, a sizable conductive H+ extrusion was detected in acid-loaded differentiated cells, consistent with the rectifying properties of the current measured electrophysiologically. By the spectroscopic method, the H+ conductance was not detectable in uninduced cells, developing gradually during granulocytic differentiation. Development of the conductive pathway was found to parallel the biochemical and functional appearance of the NADPH oxidase. These findings suggest that the H+ extrusion mechanisms required for the maintenance of the intracellular pH during granulocyte activation develop pari passu with the acid generating systems and suggest a functional and possibly structural association between the H+ conductance and the NADPH oxidase.

Topics: Amino Acid Sequence; Antibodies; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Electric Conductivity; Fluoresceins; Fluorescent Dyes; Gene Expression; Humans; Hydrogen-Ion Concentration; Immunoblotting; Leukemia, Myeloid, Acute; Molecular Sequence Data; Molecular Weight; NADH, NADPH Oxidoreductases; NADPH Oxidases; Peptides; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.

Topics: Cell Cycle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Dimethyl Sulfoxide; DNA; Drug Interactions; Flow Cytometry; Fluoresceins; G1 Phase; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Immunoblotting; Lamin Type B; Lamins; Leukemia, Myeloid, Acute; Nuclear Proteins; Phosphoproteins; Phosphorylation; S Phase

The changes in activation of Na+-H+ exchange on granulocytic differentiation of human leukemic promyelocytic HL60 cells have been studied by measuring changes in intracellular pH with the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). It was found that the Na+-H+ exchanger is activated by stimulation of protein kinase C in the dimethyl sulfoxide (DMSO)-differentiated (neutrophil-like) HL60 cell, but not in the undifferentiated (promyelocyte-like) cell. In contrast, osmotic shrinkage of the cells triggers the exchanger in the undifferentiated cells, but not in the DMSO-differentiated cells. The data suggest that activation of Na+-H+ exchange by osmotic shock does not occur exclusively through stimulation of kinase C.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Carrier Proteins; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Fluoresceins; Granulocytes; Humans; Hydrogen-Ion Concentration; Leukemia, Myeloid, Acute; NADH, NADPH Oxidoreductases; NADPH Oxidases; Protein Kinase C; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate