Acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation by overexpression of Lyn tyrosine kinase.

Stimulation with granulocyte colony-stimulating factor (G-CSF) induces myeloid precursor cells to differentiate into neutrophils, and tyrosine phosphorylation of certain cellular proteins is crucial to this process. However, the signaling pathways for neutrophil differentiation are still obscure. As the Src-like tyrosine kinase, Lyn, has been reported to play a role in G-CSF-induced proliferation in avian lymphoid cells, we examined its involvement in G-CSF-induced signal transduction in mammalian cells. Expression plasmids for wild-type Lyn (Lyn) and kinase-negative Lyn (LynKN) were introduced into a murine granulocyte precursor cell line, GM-I62M, that can respond to G-CSF with neutrophil differentiation, and cell lines that overexpressed these molecules (GM-Lyn, GM-LynKN) were established. Upon G-CSF stimulation, both the GM-Lyn and GM-LynKN cells began to differentiate into neutrophils, showing early morphological changes within a few days, much more rapidly than did the parental cells, which started to exhibit nuclear lobulation about 10 days after the cells were transferred to G-CSF-containing medium. However, the time course of expression of the myeloperoxidase gene, another neutrophil differentiation marker, was not affected by the overexpression of Lyn or LynKN. Therefore, in normal cells, protein interactions with Lyn, but not its kinase activity, are important for the induction of G-CSF-induced neutrophilic nuclear lobulation in mammalian granulopoiesis.     

Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells

Triphenyltin (TPT) is an environmental endocrine disruptor and toxic substance, but little information is available on its immunological effects. To assess the effect of TPT on leukocyte differentiation, we investigated its effect on the neutrophilic differentiation of HL-60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At a low concentration, 10(-7)M, TPT increased superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 45% and increased expression of CD18, a component of the OZ-receptor, by about 90%. Real-time PCR analysis revealed that TPT augmented the expression not only of CD18 but also of components of superoxide-generating NADPH-oxidase, p47phox, 2.7-fold, and p67phox, 2.0-fold, and of granulocyte colony-stimulating factor receptor (G-CSFR), 3.0-fold, whereas various other endocrine disruptors, including parathion, vinclozolin, and bisphenol A, had no such enhancing effects. The results of a DNA macroarray analysis showed that TPT enhanced the expression of G-CSFR and certain other neutrophil functional proteins, including CD14 and myeloid leukemia cell differentiation protein (MCL-1), and that TPT induced a decrease in expression of LC-PTP, leukocyte protein-tyrosine phosphatase, to about half the control level. The TPT-dependent suppression of LC-PTP was confirmed by real-time PCR analysis, and the results of immunoblotting indicated that TPT enhances the expression of myeloid specific tyrosine kinase hck by about 30% at the protein level, and this together with the reduction of LC-PTP may enhance tyrosine phosphorylation, in turn resulting in enhancement of superoxide production. These findings suggest that TPT may have an enhancing effect on the neutrophilic maturation of leukocytes.     

Src homology 2 domain of overexpressed Lyn kinase is responsible for the acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation

We have previously shown that the overexpression of a Src family kinase, Lyn, and its kinase-negative form, LynKN, in a granulocyte progenitor cell line, GM-I62M, accelerates neutrophilic nuclear lobulation when the cells are cultured in the presence of granulocyte colony-stimulating factor. In this study, we investigated the role of the Src homology 2 (SH2) and SH3 domains of Lyn in the accelerated induction of nuclear lobulation. In contrast to wild-type Lyn, the overexpression of its SH2 domain mutant did not induce the accelerated nuclear morphological changes, but the overexpressed SH3 domain mutant had the same effects as wild-type Lyn. Therefore, the SH2 domain of Lyn is responsible for the accelerated induction of neutrophilic nuclear lobulation upon G-CSF stimulation.     

Requirement of tyrosine-phosphorylated Vav for morphological differentiation of all-trans-retinoic acid-treated HL-60 cells.

Our previous data demonstrated that cellular and nuclear tyrosine-phosphorylated Vav associate with phosphoinositide 3-kinase during all-trans-retinoic acid-dependent granulocytic differentiation of HL-60 cells. In this study, aimed to analyze the mechanism by which Vav is recruited and activated, we report that the Src homology 2 domain of Vav interacts with tyrosine-phosphorylated proteins in a differentiation-dependent manner. Two adaptor proteins, Cbl and SLP-76, were identified, showing a discrete distribution inside the cells, with Cbl absent from the nuclei and SLP-76 particularly abundant in the nuclear compartment. Of note, Vav interacts with the tyrosine kinase Syk, which is also present in the nuclear compartment and may phosphorylate Vav in vitro when cells differentiate. Inhibition of Syk activity by piceatannol prevents both in vitro and in vivo Vav tyrosine phosphorylation, its association with the regulatory subunit of phosphoinositide 3-kinase, and the nuclear modifications typically observed during granulocytic differentiation of this cell line. These findings suggest that tyrosine-phosphorylated Vav and its association with phosphoinositide 3-kinase play a crucial role in all-trans-retinoic acid-induced reorganization of the nucleoskeleton, which is responsible for the changes in nuclear morphology observed during granulocytic differentiation of HL-60 cells.     

Granulocyte colony-stimulating factor promotes the translocation of protein kinase Ciota in neutrophilic differentiation cells

Previously, we suggested that the phosphatidylinositol 3-kinase (PI3K)-p70 S6 kinase (p70 S6K) pathway plays an important role in granulocyte colony-stimulating factor (G-CSF)-dependent enhancement of the neutrophilic differentiation and proliferation of HL-60 cells. While atypical protein kinase C (PKC) has been reported to be a regulator of p70 S6K, abundant expression of PKCiota was observed in myeloid and lymphoid cells. Therefore, we analyzed the participation of PKCiota in G-CSF-dependent proliferation. The maximum stimulation of PKCiota was observed from 15 to 30 min after the addition of G-CSF. From 5 to 15 min into this lag time, PKCiota was found to translocate from the nucleus to the membrane. At 30 min it re-translocated to the cytosol. This dynamic translocation of PKCiota was also observed in G-CSF-stimulated myeloperoxidase-positive cells differentiated from cord blood cells. Small interfering RNA for PKCiota inhibited G-CSF-induced proliferation and the promotion of neutrophilic differentiation of HL-60 cells. These data indicate that the G-CSF-induced dynamic translocation and activation processes of PKCiota are important to neutrophilic proliferation.     

Cultivation of HL-60 cells in a serum-free medium containing granulocyte/macrophage colony-stimulating factor.

In order to develop a defined cultivation medium for HL-60 cells, we cultivated these cells in a serum-free suspension medium and tested the effect of various growth factors. Of the factors tested, granulocyte/macrophage colony-stimulating factor was most active in growth stimulation. A much lower effect was obtained with granulocyte colony-stimulating factor and transferrin. No effect was found with interleukin-3 and insulin. Granulocyte colony-stimulating factor was the only growth factor tested that also induced differentiation as judged by the nitroblue tetrazolium test. Growth of HL-60 cells in medium containing granulocyte/macrophage colony-stimulating factor (125 U/ml) and transferrin (5 micrograms/ml) as the only protein factors was similar to growth in medium containing 10% serum. No increase in spontaneous differentiation of HL-60 cells in this defined medium was observed. Physiological concentrations of retinol bound to retinol-binding protein and retinyl ester in chylomicron remnants reduced proliferation as well as the level of c-myc oncoprotein and induced differentiation of HL-60 cells cultivated in defined medium. Hence, this defined medium may be useful when studying the function of retinoids in HL-60 cells.     

Commitment of neutrophilic differentiation and proliferation of HL-60 cells coincides with expression of transferrin receptor. Effect of granulocyte colony stimulating factor on differentiation and proliferation.

To examine the regulatory mechanisms of proliferation and maturation in neutrophilic lineage cells, we have tried to sort dimethyl sulfoxide (Me(2)SO)-treated HL-60 cells into transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells. Differentiated Trf-R(-) cells expressed more formyl-Met-Leu-Phe receptor (fMLP-receptor) and ability of O-(2) genaration, as markers of differentiation, than Trf-R(+) cells, and Trf-R(-) cell differentiation was markedly accelerated by the incubation with granulocyte colony stimulating factor (G-CSF). On the other hand, Trf-R(+) cells had a tendency to proliferate rather than differentiate, and proliferation was enhanced by G-CSF. These results indicate that Trf-R expression coincides with the commitment to proliferate or differentiate of HL-60 cells, and G-CSF accelerates these commitments. G-CSF-induced tyrosine phosphorylation of STAT 3 in Trf-R(-) cells much more than in Trf-R(+) cells. Protein 70 S6 kinase expression was higher in Trf-R(+) cells than in Trf-R(-) cells. Furthermore, p70 S6 kinase was hyperphosphorylated by G-CSF in Trf-R(+) cells, but not in Trf-R(-) cells. Rapamycin, an inhibitor of p70 S6 kinase activity, inhibited G-CSF-dependent proliferation of Trf-R(+) cells and increased fMLP-R expression on these cells. These results suggest that commitment to proliferation and differentiation in Me(2)SO-treated HL-60 cells is preprogrammed and correlated with Trf-R expression, and G-CSF potentiates the cellular commitment. STAT 3 may promote differentiation of Me(2)SO-treated HL-60 cells into neutrophils, while p70 S6 kinase may promote proliferation and negatively regulate neutrophilic differentiation.     

Monocytic differentiation of HL-60 cells is characterized by the nuclear translocation of phosphatidylinositol 3-kinase and of definite phosphatidylinositol-specific phospholipase C isoforms.

Immunochemical and immunocytochemical data indicate that nuclei of HL-60 cells contain different enzymes involved in the phosphoinositide cycle, such as PI 3-K and the phosphatidylinositol-specific PLC isoforms beta3, gamma1 and gamma2. These enzymes translocate differently to the nuclear fraction when HL-60 cells are treated with differentiating doses of vitamin D3: PI 3-K translocated progressively to the nucleus in parallel with full differentiation until 96 hours. PLC beta3 increased until 72 hours of treatment and then lowered its intranuclear amount and PLC gamma1 was unchanged at all the examined times. PLC gamma2 nuclear translocation increased progressively until 96 hours of vitamin D3 administration. A fourth PLC isozyme, beta2, present in the cytoplasm of untreated cells, translocates to the cytoplasm after vitamin D3 addition and reaches the highest concentration at the end of monocytic differentiation. Terminal monocytic differentiation was characterized at the nuclear level by high levels of PI 3-K and PLC gamma2 and by the novel expression of PLC beta2. We then observed that the xi isoform of PKC, constitutively present in nuclei of HL-60 cells, translocated to the nucleus when cells were induced to differentiate along the monocytic lineage, but the nuclear translocation of PKC xi was blocked as a consequence of PI 3-K inhibition by Wortmannin. These findings indicate that the main components of the noncanonical and canonical inositol lipid signal transduction pathways, including PI 3-K, PLC beta2 and beta3, PLC gamma2, undergo nuclear translocation and may therefore play a relevant role during monocytic differentiation at the nuclear level. Furthermore, PKC xi nuclear translocation appears to be related to PI 3-K activity.     

Intranuclear distribution of the human myeloid cell nuclear differentiation antigen in HL-60 cells

Based on solubility properties, the human myeloid cell nuclear differentiation antigen exists as at least two distinct populations. Most is easily extracted from isolated nuclei in 0.35 M NaCl, while 20 percent resists such treatment. Compared to undigested nuclei, both the amount of myeloid cell nuclear differentiation antigen (MNDA) released from nuclei after DNase I treatment and the amount resisting further extraction in 0.35 M NaCl increased after DNA was digested with DNase I. Under these conditions, there was a concomitant decrease in the amount of MNDA that was extractable with 0.35 M NaCl. Mixing nuclear protein extracts that contain MNDA with nuclei from cells that do not express this protein demonstrated that the MNDA redistributes from the freely soluble form to the nuclear residual fraction as a consequence of DNase I digestion. These data are consistent with a model in which the amount of MNDA that is tightly bound to salt-washed nuclei is held constant in the presence of an excess of unassociated MNDA in the nucleus, and that the level of MNDA binding to this nuclear fraction increases in proportion to the extent of DNA damage resulting from DNase I digestion.     

The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells

The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.     

Phospholipase A2 has a role in proliferation but not in differentiation of HL-60 cells

The role of phospholipase A₂ (PLA₂) and its metabolite arachidonic acid (AA) in the proliferation and differentiation of HL-60 cells was investigated. Addition of either 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) or retinoic acid (RA) to HL-60 cells for 2 h inhibited PMA-stimulated PLA₂ activity measured by [³H]AA release. The inhibitor of PLA₂ activity, p-bromophenacyl bromide (BPB), significantly inhibited the proliferation of HL-60 cells and of fibroblast L929 and Swiss 3T3 cells in a dose-dependent manner. The effect of BPB on proliferation is probably through its inhibitory effect on PLA₂ activity, since the same doses of BPB which inhibited proliferation also inhibited PLA₂ activity determined by [³H]AA release. The importance of PLA₂ activity for cell growth was further supported by the effect of two other PLA₂ inhibitors, AACOCF₃ and scalaradial, which inhibited HL-60 proliferation in a dose-dependent manner. BPB, AACOCF₃ and scalaradial significantly increased the doubling time to 32.4 h, 34.0 h and 31.8 h, respectively, compared with 24.6 h in the control. The inhibitory effect of BPB on HL-60 proliferation was reversed by addition of exogenous free AA to HL-60 cells, indicating the importance of this metabolite for the proliferation process. This reversible effect is specific for AA since it was not achieved by other fatty acids like linolenic acid (LA) or oleic acid (OA). Addition of free AA to HL-60 cells did not induce differentiation, as expected. Although BPB, AACOCF₃, or scalaradial inhibited proliferation, they did not induce differentiation nor affect the differentiation induced by 1,25(OH)₂D₃ or RA. These results implicate that PLA₂ activity has no regulatory role in differentiation of HL-60 cells. The differential effect of PLA₂ inhibitors on proliferation and differentiation of HL-60 cells suggests that these two processes function under different regulatory mechanisms.     

The differentiation of HL-60 cells in the synthetic medium induced by GM3 ganglioside

GM3 ganglioside, added exogenously to a promyelocytic leukemia cell line (HL-60 cells) in serum-free synthetic medium, induced differentiation into macrophage-like cells. Macrophagic morphology and function of differentiation-induced cells were determined by cell growth behavior, May-GriJnwald-Giemsa staining, activities of nonspecific esterase, phagocytosis and nitroblue tetrazolium (NBT) reduction. GM3 ganglioside may play a role in triggering differentiation of HL-60 cells into macrophage-like cells.     

ATRA-regulated Asb-2 gene induced in differentiation of HL-60 leukemia cells

Suppressors of cytokine signaling (SOCS) proteins possess common structures, a SOCS box at the C-terminus and a SH2 domain at their center. These suppressors are inducible in response to cytokines and act as negative regulators of cytokine signaling. The ASB proteins also contain the SOCS box and the ankyrin repeat sequence at the N-terminus, but do not have the SH2 domain. Although Socs genes are directly induced by several cytokines, no Asb gene inducers have been identified. In this study, we screened the specific genes expressed in the course of differentiation of HL-60 cells, and demonstrated that ASB-2, one of the ASB proteins, was rapidly induced by all-trans retinoic acid (ATRA). Typical retinoid receptors (RARs) or retinoid X receptors (RXRs) binding element (RARE/RXRE) were presented in the promoter of the Asb-2 gene. We showed that RARalpha, one of the RARs, binds to the RARE/RXRE in the Asb-2 promoter. In addition, we demonstrated by luciferase reporter assay that this element was a functional RARE/RXRE. These findings indicate that ASB-2 is directly induced by ATRA and may act as a significant regulator, underlying such physiological processes as cell differentiation.     

An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation.

To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer. This oligomer was stable in culture and entered cells, forming an intracellular duplex. Incubation of cells with the anti-myc oligomer decreased the steady-state levels of c-myc protein by 50 to 80%, whereas a control oligomer did not significantly affect the c-myc protein concentration. Direct inhibition of c-myc expression with the anti-myc oligomer was associated with a decreased cell growth rate and an induction of myeloid differentiation. Related antisense oligomers with 2- to 12-base-pair mismatches with c-myc mRNA did not influence HL-60 cells. Thus, the effects of the antisense oligomer exhibited sequence specificity, and furthermore, these effects could be reversed by hybridization competition with another complementary oligomer. Antisense inhibition of a nuclear proto-oncogene apparently bypasses cell surface events in affecting cell proliferation and differentiation.     

Changes in mechanical properties with DMSO-induced differentiation of HL-60 cells.

We measured changes in the deformability of human promyelocytic leukemic (HL-60) cells induced to differentiate for 5-6 days along the granulocyte pathway by 1.25% dimethylsulfoxide (DMSO). Differentiation resulted in an approximately 90% reduction in the transit times of the cells through capillary-sized pores over a range of aspiration pressures. Cell volume, as measured by two methods, decreased by an average of 35%. To account for the contribution of the volume decrease to the decrease in transit time, the liquid drop model, developed to describe neutrophil deformability, was used to calculate an apparent viscosity of the cells during this deformation. The apparent viscosity of both uninduced and induced HL-60 cells was a function of aspiration pressure, and an approximately 80% reduction in viscosity occurred with induction, as determined by regression analysis. The deformation rate-dependent viscosities of the induced cells were between 65 and 240 Pa-sec, values similar to those measured for circulating neutrophils. To assess the role of polymerized actin in these viscosity changes, intracellular F-actin content was measured, and the effect of dihydrocytochalasin B (DHB), an agent that disrupts actin polymerization, was determined. Despite the significant decrease in cellular viscosity, F-actin content per cell volume did not change significantly after induced differentiation. Treatment with 3 and 30 microM DHB lowered cellular F-actin content in a dose-dependent manner in both uninduced and induced cells. Cellular viscosity of both uninduced and induced cells decreased sharply with 3 microM DHB treatment (85% and 76% respectively). 30 microM DHB treatment caused a further significant reduction in the viscosity of uninduced cells, but for induced cells the additional decrease in viscosity was not significant. These data indicate that reductions in both cell volume and intrinsic viscosity contribute to the increased deformability of HL-60 cells with DMSO-induced differentiation. However, changes in the concentration of F-actin cannot account for the decrease in cellular viscosity that occurs.     

Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation

Sphingolipid metabolism was examined in human promyelocytic leukemia HL-60 cells. Differentiation of HL-60 cells with 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3; 100 nM) was accompanied by sphingomyelin turnover. Maximum turnover of [3H]choline-labeled sphingomyelin occurred 2 h following vitamin D3 treatment, with sphingomyelin levels decreasing to 77 +/- 6% of control and returning to base-line levels by 4 h. Ceramide and phosphorylcholine were concomitantly generated. Ceramide mass levels increased by 55% at 2 h following vitamin D3 treatment and returned to base-line levels by 4 h. The amount of phosphorylcholine produced equaled the amount of sphingomyelin hydrolyzed, suggesting the involvement of a sphingomyelinase. Vitamin D3 treatment resulted in a 90% increase in the activity of a neutral sphingomyelinase from HL-60 cells. The inferred role of sphingomyelin hydrolysis in the induction of cell differentiation was investigated using an exogenous sphingomyelinase. When a bacterial sphingomyelinase was added at concentrations that caused a similar degree of sphingomyelin hydrolysis as 100 nM vitamin D3, it enhanced the ability of subthreshold levels of vitamin D3 to induce HL-60 cell differentiation. This study demonstrates the existence of a "sphingomyelin cycle" in human cells. Such sphingolipid cycles (Hannun, Y., and Bell, R. (1989) Science 243, 500-507) may function in a signal transduction pathway and in cellular differentiation.