The activation of PI 3-K and PKC zeta in PMA-induced differentiation of HL-60 cells

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol3P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 microM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC zeta is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC zeta during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC zeta activation. The activity of PI 3-K is essential for PKC zeta activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC zeta during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC zeta in the process of PMA-induced monocytic differentiation.     

Inhibition by bryostatin 1 of the phorbol ester-induced blockage of differentiation in hexamethylene bisacetamide-treated Friend erythroleukemia cells

Phorbol esters inhibit chemically induced differentiation in Friend erythroleukemia cells. This study examines the effect of the macrocyclic lactone bryostatin 1 on phorbol ester responses in a Friend erythroleukemia cell clone, PS 7. In several biological systems, bryostatin 1 was reported to mimic phorbol ester action, including activation of protein kinase C, but in HL-60 cells it blocked phorbol ester-induced differentiation. We report here that bryostatin 1 blocks phorbol ester action in Friend cells (clone PS 7), a second differentiating system. In this system, in contrast to HL-60 cells, the phorbol esters inhibit rather than induce differentiation. Bryostatin 1 restores the differentiation response [50% effective dose, 15 +/- 3.5 nM (SEM)] as well as blocks a second phorbol ester effect, induction of cellular adherence. The inhibition of erythroid differentiation by dexamethasone, a nonphorbol compound whose action presumably is not protein kinase C mediated, is unaffected by bryostatin 1. Although bryostatin 1 inhibits [3H]phorbol 12,13-dibutyrate binding in intact Friend erythroleukemia cell clone PS 7, the mechanism for the antagonism of phorbol ester action by bryostatin 1 in Friend cells cannot be explained by simple competition at the binding site.     

The level of c-myc transcript in differentiation-defective mouse erythroleukemia cells

We isolated several mouse erythroleukemia (MEL) cell lines, deficient in in vitro erythroid differentiation, and examined the change in level of c-myc mRNA after exposing the cells to dimethyl sulfoxide (DMSO). The results showed all the mutant MEL cells to exhibit a similar change in c-myc mRNA level to that in parental (DS19) cells with regard to both an initial decline and a subsequent increase to the pretreatment level. The c-myc mRNA level did not, however, decline again thereafter, as observed in the parental cells, but remained at the pretreatment level, even on prolonged culture in the presence of DMSO. Essentially the same results were obtained for the parental cells in the presence of DMSO and either 12-O-tetradecanoylphorbol 13-acetate (TPA) or dexamethasone, specific inhibitors of MEL cell differentiation. These results suggest that the crucial stage in MEL cell differentiation, regarding c-myc mRNA level, is the late stage, when the MEL cells become committed to differentiation.     

C-Jun modulates apoptosis but not terminal cell differentiation in murine erythroleukemia cells.

The proto-oncogene c-Jun has been implicated in the control of cell proliferation and differentiation and more recently in the regulation of apoptosis. We have previously reported the involvement of c-Jun in the erythroid differentiation block in murine erythroleukemia (MEL) cells. As reported here, we investigated the role of c-Jun in the regulation of terminal differentiation and apoptosis of MEL cells by studying different stable transfectant clones containing c-jun constructs in sense or antisense orientation. c-Jun did not prevent cell growth arrest in G0/G1 and p21 induction that are normally associated with terminal differentiation induced by DMSO treatment, suggesting that c-Jun may uncouple phenotypic differentiation and terminal cell division in the MEL cell system. Spontaneous apoptosis was accelerated in c-jun expressing MEL cells before and after DMSO treatment. Moreover, c-Jun sensitized apoptosis induced by various drugs. Drug-induced apoptosis was associated with c-Jun N-terminal kinase (JNK) activation and c-Jun N-terminal phosphorylation (JNP). In contrast, overexpression of c-jun delayed apoptosis in serum-starved cells, indicating that c-Jun may reduce or accelerate apoptosis in MEL cells depending on the nature of the apoptotic stimulus. These results suggest that the proto-oncogene c-Jun may modulate differentiation and apoptosis of leukemic cells.     

Effects of dimethylsulfoxide on friend erythroleukemic cell proliferation and on the activity of enzymes involved in this process

Variations in catalase, glutathione peroxidase (GP) and adenylate cyclase (AC) activity in murine erythroleukemic (MEL) cells were studied during multiplication and dimethylsulfoxide (DMSO)-induced differentiation. The results demonstrated that, although DMSO favors the incorporation of ³H-thymidine into DNA of treated cells, it slows down cell multiplication. Increased incorporation was also observed in superoxide dismutase (SOD)-treated cells. DMSO also determined an early and significant drop in AC activity and a late fall in catalase activity, whereas there was no significant variation in GP activity in parallel with the decreased cell multiplication that accompanied cell differentiation. We hypothesize that DMSO and SOD favor ³H-thymidine incorporation by neutralizing the reactive forms of oxygen and that the reduction in catalase and AC activity is closely related to the mitotic activity of MEL cells.     

Amiloride potentiation of differentiation of human promyelocytic cell line HL-60

Cells of the human acute promyelocytic cell line HL-60 undergo differentiation when exposed to dimethyl sulfoxide (DMSO); in this report, amiloride, an inhibitor of passive intracellular Na+ flux, potentiated the DMSO-induced differentiation of HL-60 cells. This effect was seen at several concentrations of DMSO. Amiloride alone did not affect HL-60 differentiation. Various analogues of amiloride were tested for their ability to potentiate differentiation of HL-60 cells. The synergistic induction of myeloid differentiation by a membrane solvent (DMSO) and an Na+ transport inhibitor (amiloride) suggested membrane cation flux as being important in initiating differentiation.     

Activation of protein kinase C enhances TNF-alpha-induced differentiation by preventing apoptosis via rapid up-regulation of c-Myc protein expression in HL-60 cells

Tumor necrosis factor-alpha (TNF-alpha) induces both rapid onset of apoptosis and monocytic differentiation in HL-60 human myeloid leukemia cells. In this study, we examined the effect of activation of protein kinase C (PKC) in c-Myc protein expression in association with the induction of apoptosis and differentiation in TNF-alpha-treated HL-60 cells. Pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, prevented TNF-alpha-induced rapid onset of apoptosis, which occurs at 3 h culture with TNF-alpha, concomitantly with the up-regulation of c-Myc protein expression. In addition, PMA enhanced TNF-a-induced differentiation at 24h treatment. This was documented by the expression of integrin Mac-1 molecule (CD11b) on the cell surface and the cellular adhesion to the plastic bottom of the flask, indicating the differentiation along with the monocyte/macrophage lineage. These results indicate that activation of PKC not only counteracts apoptosis but also enhances differentiation in TNF-alpha-treated HL-60 cells. Up-regulation of c-Myc protein evoked by pretreatment with PMA for a short time could disturb the signaling pathway of the ceramide and sphingosine, which are known to function as the endogenous modulators mediating the apoptotic signal of TNF-alpha. Our results strongly suggest the role of c-Myc protein as a mediator of cytoprotective effect of PKC pathway, and PKC pathway opposes apoptosis and consequently undergo differentiation via rapid up-regulated c-Myc protein expression during TNF-alpha signaling of HL-60 cells. Our findings provide a new insight for a role of PKC and c-Myc protein with special reference to the regulatory mechanisms in the decision of cellular fate, differentiation or apoptosis.     

Induction of differentiation in murine erythroleukemia cells by 1α,25-dihydroxy vitamin D3

The Friend murine erythroleukemia (MEL) cells can be stimulated to differentiate in response to a variety of chemical inducing agents. In the present study, the effect of 1α,25-dihydroxyvitamin D₃ on differentiation of MEL cells was investigated. Vitamin D₃ induced differentiation of MEL cells in culture as determined by elevated hemoglobin content, a rise in the number of benzidine-positive cells and increase in acetylcholine esterase activity. The optimum concentration of the vitamin required to induce differentiation of MEL cells was found to be 750 nM. The pattern of induction of differentiation was similar to that observed with DMSO and the induction of differentiation by vitamin D₃ was inhibited by dexamethasone.     

Decursin and PDBu: two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Phorbol 12,13-dibutyrate (PDBu) induces the megakaryocytic differentiation of K562 human erythroleukemia cells through PKC activation. Decursin, a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. We report here the differences between two PKC activators, tumor-suppressing decursin and tumor-promoting PDBu, in their actions on the megakaryocytic differentiation of K562 cells. First of all, decursin inhibited PDBu-induced bleb formation in K562 cells. Decursin also inhibited the PDBu-induced megakaryocytic differentiation of K562 cells that is characterized by an increase in substrate adhesion, the secretion of granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6), and the surface expression of integrin β3. The binding of PDBu to PKC was competitively inhibited by decursin. Decursin induced the more rapid down-regulation of PKC and βII isozymes than that induced by PDBu in K562 cells. Unlike PDBu, decursin promoted the translocation of PKC and βII to the nuclear membrane. Decursin-induced faster down-regulation and nuclear translocation of PKC and βII were not affected by the presence of PDBu. All these results indicate that decursin and phorbol ester are PKC activators distinctively acting in megakaryocytic differentiation and PKC modulation in K562 leukemia cells.     

Evidence for multiple sites of regulation of heme synthesis in murine erythroleukemia cells.

Friend murine erythroleukemia (MEL) cells can be induced to differentiate along the erythroid pathway by such dissimilar agents as dimethyl sulfoxide (DMSO), butyrate, inhibitors of DNA synthesis, and certain highly polar agents. This study showed differential biochemical effects of the potent inducers DMSO and butyrate on the heme biosynthetic pathway in three clones of Friend MEL cells. When the cells were incubated with combinations of DMSO and butyrate, hemoglobin production was less than that amount produced when each inducer was incubated singly with the cells. Procaine, a local anesthetic that competes with Ca2+ and thus affects membrane permeability, slightly inhibited DMSO-mediated hemoglobin production but almost tripled the level stimulated by butyrate alone. Similarly, EDTA, which also can bind Ca2+ and which can modify the activity of heme biosynthetic enzymes, also inhibited hemoglobin production mediated by DMSO, whereas it stimulated hemoglobin production in cells exposed to butyrate. Total porphyrin accumulation was greater in DMSO-treated cells than in butyrate-treated cells, which suggests that butyrate induces the enzymes of the heme pathway more efficiently than does DMSO. DMSO and butyrate may affect the heme biosynthetic pathway by multiple mechanisms or, alternatively, they may affect the multistep pathway at various points, producing partial blocks or incomplete enzyme induction.     

Molecular and cellular mechanisms of leukemic hemopoietic cell differentiation: an analysis of the Friend system

Murine erythroleukemia (MEL or Friend) cells grown in culture and induced to differentiate into cells resembling orthochromatic normoblasts provide a suitable system for uncovering molecular and cellular mechanisms of hemopoiesis and for understanding globin gene regulation. Inducer-treated cells undergo an irreversible commitment to maturation and accumulate large amounts of hemoglobin. Clonal analysis of commitment of individual cells combined with biochemical measurements has revealed that MEL cell differentiation is a highly coordinated set of events (program) leading to the differentiated erythroid state. The developmental program of MEL cells consists of early and late processes. The early events appear to be membrane-mediated processes which operate independently of each other and lead to commitment to terminal maturation and hemoglobin synthesis. Inducer-treated cells express an ability to remember ("memory response") previous exposure to inducer and to continue their differentiation after discontinuous exposure to inducer; expression of "memory response" occurs early in differentiation and affects both the initiation of commitment and accumulation of globin mRNA in a similar manner in inducer-treated cells. Commitment to maturation appears to be the central process responsible for determining the pattern of gene expression, limitation of proliferative activity and nuclear condensation. Commitment, however, can occur independently of hemoglobin synthesis. Although initiation of commitment is associated with early membrane-mediated events (e.g., ion-transport), maintenance and completion of maturation erythroid state is a result of a number of cellular processes. These processes are discussed in relation to the molecular and cellular mechanisms of initiation and completion of MEL cell differentiation. The role of the MEL system as a model for studying mouse and human globin gene regulation is presented.     

Differentiation-inducing and growth-inhibitory activities of erythroid differentiation factor (EDF/activin A) toward mouse erythroleukemic cells in vivo

The in vivo differentiation-inducing activity of a purified human erythroid differentiation factor (EDF) toward mouse erythroleukemic cells (MEL cells) was examined. BDF1 mice with diffusion chambers implanted in the peritoneal cavity were treated with continuous i.p. administration of EDF. MEL cells within a diffusion chamber differentiated into hemoglobin-positive cells when treated with EDF, the percentage of the positive cells being 32.3 +/- 28.3 on day 5 as compared to 0.2 +/- 0.3 in the controls. The anti-tumor activity of EDF was also examined in a nude mouse MEL solid tumor model. Daily intra-tumor treatment with EDF for 10 days resulted in 73% suppression of tumor growth on day 25. A histological study revealed that EDF-treated solid tumor cells became hemoglobin-positive, indicating the anti-tumor activity of EDF through induction of differentiation in vivo. EDF could induce in vitro the differentiation of human erythroleukemic cell lines K562 and HEL, as well as the murine cell line. These results indicate the possibility of differentiation therapy for erythroleukemia using EDF.     

JAK-STAT signaling involved in phorbol 12-myristate 13-acetate- and dimethyl sulfoxide-induced 2'-5' oligoadenylate synthetase expression in human HL-60 leukemia cells

The JAK-STAT signal transduction cascade participates in various cellular processes, including immune response, cell replication, differentiation and oncogenesis. Here, we report that this cascade is induced in two human myeloid HL-60 leukemia cell variants by the granulocyte differentiation inducer dimethyl sulfoxide (DMSO) and macrophage differentiation inducer phorbol 12-myristate 13-acetate (PMA). DMSO and PMA also induced the expression and catalytic activity of 2'-5' oligoadenylate synthetase (2-5A synthetase), a known interferon (IFN) inducible enzyme. The HL-60 cell variants included HL-205, which is susceptible to DMSO- and PMA-induced differentiation, and HL-525, which is susceptible to DMSO- but not to PMA-induced differentiation. Treatment of HL-205 and HL-525 cells with DMSO and HL-205 cells with PMA-induced JAK1 phosphorylation, JAK1/STAT1 association, formation of STAT1-STAT2 heterodimers, and the binding of the active IFN stimulating growth factor 3 (ISGF3) to the IFN-stimulated response element (ISRE) fragment isolated from the 2-5A synthetase promoter. These events were either reduced or absent in the resistant HL-525 cells treated with PMA. Taken together, our data implicate the above signaling cascade in DMSO- and PMA-induced 2-5A synthetase expression and catalytic activity in the HL-60 cell system.     

Early decrease in hyaluronidase-sensitive cell surface charge during the differentiation of Friend erythroleukemic cells by dimethyl sulfoxide.

Early membrane events in erythroid differentiation were investigated by means of cell electrophoresis utilizing cultured Friend erythroleukemia cell clones of different inducibility. The cell electrophoretic mobility decreased by 18% within 30 min of treatment with 1.5% dimethyl sulfoxide (DMSO) in highly inducible clones but not in noninducible clones. The reduced mobility persisted for 5 days of incubation with DMSO until hemoglobin synthesis. DMSO treatment for less than 16 hr and subsequent incubation without the drug resulted in the complete recovery of the mobility and no hemoglobin synthesis. Longer exposure to DMSO resulted in the loss of recovery of mobility and an increasing fraction of benzidine-positive cells seen on Day 5. Measurement of the electrophoretic mobility after the removal of acidic sugars by their specific enzymes suggested that hyaluronidase-sensitive negative charges were lost from the cell surface only in highly inducible clones. The mobility reduction associated with hyaluronic acid was also caused by other potent inducers (sodium butyrate, N-methylacetamide, and N,N-dimethylacetamide). These results suggest that the decrease in cell surface glycocalyx might be an early step in the induction of differentiation of Friend erythroleukemia cells.     

Induction of morphological and functional differentiation of human promyelocytic leukemia cells (HL-60) by componuds which induce differentiation of murine leukemia cells

Various compounds active in promoting in vitro differentiation of certain murine leukemia cell lines (Friend erythroleukemia cells and mouse myeloid leukemia cells) were tested for their capacity to induce differentiation of HL-60 cells, a human promyelocytic leukemia cell line capable of terminally differentiating in vitro to functionally mature granulocytes. Polar planar compounds including hexamethylene bisacetamide (HMBA), certain purines (particularly hypoxanthine), and actinomycin-D induced morphological and functional (as assessed by the capacity to reduce NBT dye) differentiation of HL-60. In contrast, hemin, ouabain, prostaglandin E1, X-irradiation, dexamethasone and some other anti-leukemic chemotherapeutic agents induced little if any significant differentiation of HL-60 cells. These results, together with previous observations with murine leukemia cells, suggest that the human HL-60 cells share common cellular target sites for the inducing action of polar planar compounds, hypoxanthine and actinomycin-D with some murine leukemic cells. In contrast, hemin, ouabain and prostaglandin E1 may be specific for mouse erythroleukemia cells, while X-irradiation and chemotherapeutic agents induce differentiation of both types (erythroid and myeloid) of mouse leukemic cells, but have little effect on HL-60 cells.