Alteration in insulin receptor expression accompanying differentiation of HL-60 leukemia cells.

Differentiation of leukemic cells in vitro is characterized by the sequential appearance of morphological, functional, and biochemical markers of maturation. The interaction of insulin with its receptor may be a regulator of growth and differentiation of leukemic cells. Human promyelocytic leukemia cells (HL-60) demonstrate specific reversible insulin binding consistent with properties of human insulin receptor. HL-60 cells treated with 500 microM N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate, 1 microM 1 alpha, 25-dihydroxyvitamin D3, or 41 nM phorbol-12-myristate-13-acetate expressed monocytic markers of differentiation and an increase in insulin receptor expression. The change in insulin receptor expression with 1 microM 1 alpha, 25-dihydroxyvitamin D3 and N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate induction was further characterized by Scatchard analysis. High affinity binding (Kd) constant was not altered, and the change in binding was attributed to receptor number. Commitment to increased insulin receptor expression was demonstrated after 1-h exposure to 1 microM 1 alpha, 25-dihydroxyvitamin D3. Agents which induced granulocytic differentiation, such as 160 mM dimethyl sulfoxide and 100 nM retinoic acid, significantly decreased insulin receptor expression compared to monocytic inducing agents. This difference in insulin receptor expression correlated with binding characteristics in normal human peripheral granulocyte and monocytes. The HL-60 cell line offers a model for the study of the molecular events which lead to the contrasting insulin receptor expression during myeloid and monocytoid hematopoiesis.

Effect of retinoic acid on phorbol ester-stimulated differentiation and protein kinase C-dependent phosphorylation in the U937 human monoblastoid cell.

Phorbol esters stimulate differentiation of certain human leukemic cell lines. Although activation of protein kinase C may mediate certain effects of phorbol esters, controversy exists as to the role of protein kinase C activation in phorbol ester-induced differentiation. Retinoic acid modulates responses to phorbol esters in several cell types. Retinoic acid has also been found to alter protein kinase C-dependent phosphorylation in leukemic cells. We correlated the effects of retinoic acid on protein kinase C-dependent phosphorylation and differentiation stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester, in the human monoblastoid U937 cell line. At concentrations less than 1 nM, which were 100-fold less than those directly stimulating differentiation, retinoic acid potentiated TPA-induced differentiation of the U937 cell as assessed by enhanced adherence to plastic and acquisition of nonspecific esterase activity. TPA-stimulated decreases in cellular proliferation were not affected by retinoic acid treatment. Without altering the sensitivity to TPA, retinoic acid increased the maximal response to this agent. Retinoic acid enhanced TPA-stimulated phosphorylation of a Mr 48,000 substrate in intact 32P-labeled U937 cells and also increased the protein kinase C-dependent phosphorylation of a similar Mr 48,000 substrate and a Mr 80,000 substrate in cellular extracts. In cellular extracts the retinoic acid-induced enhancement of protein kinase C-dependent phosphorylation was predominantly localized to the cytosolic fraction. Increases in protein kinase C-dependent phosphorylation were evident within a 12-h exposure to 1 nM retinoic acid and were observed at retinoic concentrations of 0.01 to 1 nM. A retinoic acid-induced increase in the protein kinase C-dependent phosphorylation of an exogenous substrate, histone, was observed following diethylaminoethyl extraction of cytosol, but not a solubilized particulate fraction. The conditions of retinoic acid treatment increasing protein kinase C activity and enhancing protein kinase C-dependent phosphorylation of endogenous substrates were similar to those conditions potentiating phorbol ester-induced differentiation. Thus, the retinoic acid-induced amplification of phorbol ester signal transduction at the level of protein kinase C activation could mediate the effects of this vitamin on phorbol ester-induced differentiation.

1,25-Dihydroxyvitamin D3 enhances phorbol ester-stimulated differentiation and protein kinase C-dependent substrate phosphorylation activity in the U937 human monoblastoid cell.

In the U937 human monoblastoid cell line, 1,25-dihydroxyvitamin D3[1,25(OH)2D3] through a specific interaction with the 1,25(OH)2D3 receptor promotes differentiation toward a more mature phenotype. In addition to this direct effect, 1,25(OH)2D3 also potentiates differentiation in response to lymphokines and (Bu)2cAMP. We examined the effect of 1,25(OH)2D3 on phorbol ester-stimulated differentiation. Either preincubation with or simultaneous exposure to 1,25(OH)2D3 enhanced phorbol ester-stimulated differentiation. Over a 72-h period, the increase in phorbol ester responsiveness was dependent on the duration of 1,25(OH)2D3 exposure. Enhancement of phorbol ester-induced differentiation was observed with 1,25(OH)2D3 concentrations ranging from 0.1-10 nM. The 1,25(OH)2D3 vitamin D metabolite was more potent than the 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 metabolites in potentiating phorbol ester-induced differentiation. Phorbol esters can exert at least a portion of their effects on cellular function by activating protein kinase C. Thus, one mechanism by which 1,25(OH)2D3 could amplify signal transduction leading to potentiation of phorbol ester-stimulated differentiation would be by enhancing phorbol ester-stimulated phosphorylation. To examine this possibility, we measured protein kinase C-dependent substrate phosphorylation in extracts derived from cells pretreated with 1,25(OH)2D3. In extracts derived from cells treated with 1,25(OH)2D3, the protein kinase C-dependent phosphorylation of endogenous U937 substrates stimulated by calcium, phosphatidyl serine, and diolein was increased compared to that observed in vehicle-treated cells. The conditions required for 1,25(OH)2D3 to increase protein kinase C-dependent phosphorylation of endogenous substrates (concentration, duration of exposure, and metabolite specificity) were similar to those required to enhance phorbol ester-stimulated differentiation. Possibly mediating this enhanced phosphorylation was an increase in protein kinase C activity observed in extracts derived from 1,25(OH)2D3-treated cells.

Expression of histamine H1 receptors on cultured histiocytic lymphoma cells.

Histamine H1 receptors were identified in U937 human histiocytic lymphoma cells using a radioligand binding technique with [3H]mepyramine. Reversible high-affinity binding with this ligand was obtained, and specificity of binding for selected H1 agonists and antagonists was demonstrated. Competition binding experiments with mepyramine and histamine yielded results consistent with single-site binding for mepyramine and two-site binding for histamine. Dissociation constants for the high- and low-affinity histamine binding states were 6.8 X 10(-6) and 2.4 X 10(-4) M respectively. The high-affinity state for histamine binding was abolished when the membranes were coincubated with 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S). Saturation binding studies yielded an average of 66 fmol/mg protein binding sites (6000 receptors per cell) with a [3H]mepyramine KD of 9.6 nM. When differentiation of these cells was induced by phorbol-myristate-acetate, receptor density increased by 73% to 114 fmol/mg protein. This increase in receptor density was inhibited by actinomycin D and cycloheximide. Exposure of native and differentiated U937 cells to 10(-5) and 10(-4) M histamine for 24 hr resulted in a dose-dependent down-regulation in receptor density. The data indicate that U937 cells may provide a model cell line for the study of histamine receptor gene expression.

Study of differentiation of fresh myeloid leukemic cells by physiologic agents that induce a human promyelocytic leukemic line (HL-60) to differentiate.

A promyelocytic cell line known as HL-60 can be induced to mature to granulocytes or monocytes after exposure to a variety of physiologic agents including 1-alpha 25 dihydroxy Vitamin D3 (Vit D), retinoic acid, cyclic AMP cell permeant compounds, and stimulators of adenylate cyclase. These compounds were used in primary culture of blast cells from 12 patients with acute nonlymphocytic leukemia. Maturation was assessed by morphology, superoxide production, development of esterase activity and chemotactic peptide receptor expression. Morphologic maturation and superoxide production correlated with chemotactic peptide receptor expression. The majority of blast cells treated with inducer showed no significant change in morphologic or functional markers compared to the blast cells cultured in fresh media alone. Chemotactic peptide receptor expression increased 3 to 30-fold in 13 of 14 cases studied. In 4 patients, the highest receptor expression was without inducer and in 4 patients the highest increase was with dibutyryl cyclic AMP treatment. Our study suggests that physiologic inducers of HL-60 differentiation do not consistently have the same effect on primary suspension culture of freshly isolated human leukemia cells.

Induction of insulin receptor expression of human leukemic cells by 1 alpha, 25 dihydroxyvitamin D3.

The HL-60 and U-937 leukemic cell line and explants of fresh human leukemia cells were differentiated in vitro by incubation with 1 alpha, 25 dihydroxyvitamin D3 (Vit D). Morphologic change to monocytes was demonstrated in the promyelocytic (HL-60) line but not in the U-937 line and four of five leukemic cells. One patient with chronic granulocytic leukemia in the myeloid blast phase had incomplete morphologic change with Vit D treatment. In all cells studied, an increase in specific insulin receptor binding was independent of morphologic maturation. An increase in insulin receptor expression by Vit D was an early monocytic membrane marker dissociated from morphologic and functional alterations.

Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line.

FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O-tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine-independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.

Influence of gravity on cardiac performance.

Results obtained by the investigators in ground-based experiments and in two parabolic flight series of tests aboard the NASA KC-135 aircraft with a hydraulic simulator of the human systemic circulation have confirmed that a simple lack of hydrostatic pressure within an artificial ventricle causes a decrease in stroke volume of 20%-50%. A corresponding drop in stroke volume (SV) and cardiac output (CO) was observed over a range of atrial pressures (AP), representing a rightward shift of the classic CO versus AP cardiac function curve. These results are in agreement with echocardiographic experiments performed on space shuttle flights, where an average decrease in SV of 15% was measured following a three-day period of adaptation to weightlessness. The similarity of behavior of the hydraulic model to the human system suggests that the simple physical effects of the lack of hydrostatic pressure may be an important mechanism for the observed changes in cardiac performance in astronauts during the weightlessness of space flight.