sq-23377 and Leukemia--Erythroblastic--Acute

This study was conducted on human K562 lymphocytes to investigate the mechanisms implicated in the regulation of the serotonin transport process. The uptake of serotonin in these cells was saturable (Km, 3.37 microM; Vmax, 2.03 nmol/10(6) cells) and Na+ dependent; isoosmotic replacement of Na+ with choline chloride in the assay medium resulted in the decreased uptake process. Augmentation of intracellular free calcium, [Ca2+]i, by thapsigargin decreased the uptake of serotonin in these cells. Similarly, addition of calcium ionophores (A23187) and ionomycin also inhibited serotonin transport. In Fura-2-loaded cells, these agents increased the [Ca2+]i contents. These results suggest that an increase in [Ca2+]i is implicated with a decrease in serotonin transport. Since an increase in [Ca2+]i is known to activate calmodulin (CaM), we employed CaM antagonists. Calmodulin antagonists W-7 (N-[6-aminohexyl]-5-chloro-1-naphthalene-sulfonamide) and mellitin inhibited serotonin uptake in K562 cells, suggesting that CaM is involved in serotonin transport regulation. Furthermore, acute exposure of K562 cells to known protein kinase C (PKC) activators, phorbol-12-myristate-13-acetate (PMA) and sn-1,2-dioctanoylglycerol (DiC8), curtailed serotonin uptake by these cells. However, staurosporine (a PKC inhibitor) failed to abolish the inhibitory effects of PMA on serotonin transport in these cells, indicating that the target of PMA is not PKC. Nonetheless, the PMA-induced inhibitory effects are specific as 4 alpha-phorbol-12,13,didecanoate (a phorbol ester known not to activate PKC) failed to mimic PMA-like actions on serotonin transport in K562 cells. DiC8 not only exerted higher inhibitory effects than PMA but also had additive effects in the presence of the latter on serotonin transport. These results suggest that in addition to PKC, there are other cellular targets (of PMA) implicated in serotonin transport regulation.

Topics: Biological Transport; Calcimycin; Calcium; Calmodulin; Enzyme Activation; Humans; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Lymphocytes; Protein Kinase C; Serotonin; Serotonin Antagonists; Thapsigargin; Tumor Cells, Cultured

Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Cells, Cultured; Collagen; Epinephrine; Humans; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Large B-Cell, Diffuse; Megakaryocytes; Rats; Receptors, Purinergic; Thrombin; Tumor Cells, Cultured

The effects of calcium ionophores A23187 and ionomycin on the c-myb and c-myc mRNA levels have been investigated in the Friend erythroleukemia cell line F4-6 using Northern blot analysis. Treatment of the cells with 0.5-4 microM A23187 or 1-4 microM ionomycin induced a concentration-dependent decrease in c-myb mRNA; this decrease was abolished by EGTA. c-myc mRNA levels were only moderately affected. After 12-24 h of calcium ionophore exposure, c-myb mRNA returned to pretreatment levels. No similar decrease in c-myb mRNA was seen with the sodium ionophore monensin (up to 16 microM). The dimethyl sulfoxide-induced suppression of c-myb and also of c-myc mRNA levels was not prevented in Ca(2+)-free medium and thus appeared Ca(2+)-independent. A23187 and ionomycin were capable of inducing beta-globin mRNA synthesis in F4-6 cells. Prolonged calcium ionophore exposure, however, strongly reduced cell viability and resulted only in a slight hemoglobin increase at lower concentrations. These results suggest that a rise in [Ca2+]i may be a signal leading to a transient decrease in c-myb mRNA and the initiation of erythroid differentiation in Friend cells. The transient suppression of c-myb mRNA levels represents a common feature of the action of dimethyl sulfoxide and calcium ionophores.

Topics: Actins; Animals; Blotting, Northern; Calcimycin; Cell Survival; Dimethyl Sulfoxide; Egtazic Acid; Friend murine leukemia virus; Gene Expression Regulation, Neoplastic; Genes, myc; Hemoglobins; Ionomycin; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; RNA, Messenger; Tumor Cells, Cultured

The relationship between calcium mobilization and phospholipase D (PLD) activation in response to E-series prostaglandins (PGEs) was investigated in human erythroleukemia cells. Intracellular free Ca2+ concentration ([Ca2+]i) was increased by PGE1 and PGE2 over the same concentration range at which PLD activation was seen. Pretreatment of cells with pertussis toxin greatly inhibited the PGE-stimulated increase in [Ca2+]i, implying that a G protein participates in the PGE receptor signaling process. The peak level and also the plateau level of Ca2+ mobilization stimulated by these prostaglandins were markedly decreased in Ca(2+)-depleted medium, indicating that both extracellular and intracellular Ca2+ stores contribute to the changes in [Ca2+]i. Likewise, activation of PLD by PGE1 and PGE2 was abolished by pertussis toxin pretreatment or incubation in Ca(2+)-depleted medium. U73122, a putative phospholipase C inhibitor, blocked both Ca2+ mobilization and PLD activation in PGE-stimulated cells. Furthermore, the intracellular loading of BAPTA, a Ca2+ chelator, inhibited both Ca2+ mobilization and PLD activation by PGE1 and PGE2 in a similar dose-dependent manner. Simultaneous measurement of [Ca2+]i and PLD activity in the same cell samples indicated that PLD activity increases as a function of [Ca2+]i in a similar fashion in cells stimulated either by PGEs or by the calcium ionophore ionomycin. Taken together, these findings suggest that a rise in [Ca2+]i is necessary for PGE-stimulated PLD activity in human erythroleukemia cells.

Topics: Alprostadil; Calcium; Chelating Agents; Cyclic AMP; Dinoprostone; Enzyme Activation; Estrenes; Humans; Ionomycin; Leukemia, Erythroblastic, Acute; Pertussis Toxin; Phospholipase D; Prostaglandins E; Pyrrolidinones; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid-specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Calcimycin; Calcium; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Erythroid Precursor Cells; Gene Expression; Globins; Ionomycin; Leukemia, Erythroblastic, Acute; Mice

Regulation of cytoplasmic pH (pHi) of the human monoblastic U-937 and erythroleukemic K-562 cell lines was investigated. The apparent resting pHi, as assessed by the fluorescent pH probe quenel, were 6.61 and 6.75 for the U-937 and K-562 cells, respectively. When extracellular Na+ was substituted by equimolar choline+, pHi decreased by about 0.2 units. The protein kinase C activating beta-form of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10(-10) and 10(-7) M) induced a dose-dependent alkalinization in both cell types of 0.03-0.12 units, whereas the alpha-form was inactive. The response was detectable after about 2 min and reached steady-state 10-15 min later. In the K-562 cells the alkalinization was mediated by Na+/H+ exchange as it was accompanied by stimulation of H+ extrusion and abolished by Na+ removal. The TPA response in the U-937 cells, however, was unaffected by Na+ removal, not accompanied by H+-efflux, and thus unrelated to Na+/H+ exchange. Since electron microscopy indicated development of multivesicular bodies with an acidic interior, the alkalinization can probably be accounted for by an intracellular mechanism. Ionomycin (10(-5) M) induced a rapid increase in the cytoplasmic Ca2+ concentration of both cell types and this response was accompanied by acidification followed by a Na+-dependent recovery. In the U-937, but not in the K-562, cells this recovery was followed by a net alkalinization. It is concluded that both cell types possess a Na+/H+ exchange of importance for pHi but that this mechanism is regulated differently in the U-937 and K-562 cells.

Topics: Calcium; Carrier Proteins; Cell Compartmentation; Cell Line; Cytoplasm; Ethers; Humans; Hydrogen-Ion Concentration; Ionomycin; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Microscopy, Electron; Monocytes; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate