pp75: A novel tyrosine-phosphorylated protein that heralds differentiation of HL-60 cells

The human promyeloid cell line HL-60 differentiates toward monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or dibutyryl cAMP, respectively. When nondifferentiated cells were incubated for 20 min with 2 mM H2O2 and 0.1 mM sodium orthovanadate to inhibit their protein-tyrosine-phosphatase activity (Heffetz, D., Bushkin, I., Dror, R., and Zick, Y. (1990) J. Biol. Chem. 265, 2896-2902), we found marked tyrosine phosphorylation of a single major protein of 53 kDa. Induction of differentiation of HL-60 cells was accompanied by the appearance of an additional major cytosolic tyrosine-phosphorylated protein of 75 kDa (pp75). In dibutyryl cAMP-treated cells, tyrosine phosphorylation of pp75 peaked after 24 h and then declined rapidly. In 1,25(OH)2D3-treated cells, increased tyrosine phosphorylation was detected as early as 2 h and peaked after 3 days, whereas the presence of differentiated phenotypes, assessed by the capacity of the cells to reduce nitro blue tetrazolium, was detected no earlier than 24 h. Doses of 1,25(OH)2D3 as low as 1 nM induced the appearance of pp75 at a stage where almost no differentiation measured by nitro blue tetrazolium reduction was detected. Phosphorylation of pp75 was not stimulated by adriamycin, which induced growth arrest without initiation of differentiation. pp75 could also be detected in U-937, a monocytic cell line that is more advanced in its differentiation state, and also in terminally differentiated circulating human monocytes treated with H2O2/vanadate. pp75 underwent in vitro tyrosine phosphorylation in cytosolic extracts derived from 1,25(OH)2D3-induced HL-60 cells, but not in extracts derived from uninduced cells. Our results raise the possibility that tyrosine phosphorylation of pp75 may be a common early event that heralds the differentiation of HL-60 cells into both the monocytic and granulocytic pathways.     

Identification of phosphoproteins as possible differentiation markers in all-trans-retinoic acid-treated neuroblastoma cells

Background

Neuroblastic tumors account for 9–10% of pediatric tumors and neuroblastoma (NB) is the first cause of death in pre-school age children. NB is classified in four stages, depending on the extent of spreading. A fifth type of NB, so-called stage 4S (S for special), includes patients with metastatic tumors but with an overall survival that approximates 75% at five years. In most of these cases, the tumor regresses spontaneously and regression is probably associated with delayed neuroblast cell differentiation.

Methodology/Principal Findings

In order to identify new early markers to follow and predict this process for diagnostic and therapeutics intents, we mimicked the differentiation process treating NB cell line SJ-NK-P with all-trans-retinoic acid (ATRA) at different times; therefore the cell proteomic pattern by mass spectrometry and the phosphoproteomic pattern by a 2-DE approach coupled with anti-phosphoserine and anti-phosphotyrosine western blotting were studied.

Conclusions/Significance

Proteomic analysis identified only two proteins whose expression was significantly different in treated cells versus control cells: nucleoside diphosphate kinase A (NDKA) and reticulocalbin-1 (RCN1), which were both downregulated after 9 days of ATRA treatment. However, phosphoproteomic analysis identified 8 proteins that were differentially serine-phosphorylated and 3 that were differentially tyrosine-phosphorylated after ATRA treatment. All proteins were significantly regulated (at least 0.5-fold down-regulated). Our results suggest that differentially phosphorylated proteins could be considered as more promising markers of differentiation for NB than differentially expressed proteins.     

A nuclear cAMP binding protein in retinoic acid-treated HL-60 cells

A cAMP binding protein was detected in HL-60 cells using photoaffinity labeling with 8-azido [32P]cAMP. The binding protein was found in a 0.35 M NaCl nuclear protein extract from untreated HL-60 cells and from the HL-60 cells induced to mature with retinoic acid. While the quantity of the cAMP binding protein did not change following the induced differentiation, a second form of the subunit, altered in charge, was present at 3 and 5 days after retinoic acid treatment. The findings indicate that the regulatory subunit of the type II cAMP-dependent protein kinase could be involved in nuclear functions associated with human myeloid cell differentiation.     

EMF induces differentiation in HL-60 cells

This investigation provides evidence that a 60-Hz electromagnetic field (EMF) at 1 gauss (G) can drive differentiation of cultured hematopoietic progenitor cells. HL-60 cells are known to differentiate from a nonphagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity in the presence of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbal-13-acetate (TPA). The effect of 60-Hz EMF at 1 G on differentiation is approximately equivalent to treatment of the cells with 250-500 pg/ml TPA. Furthermore, the effect of both EMF and TPA treatment on differentiation is additive at low TPA concentrations. The results strongly suggest similarities between the effects of TPA treatment and EMF exposure and thus provide an approach for tracing the origins of the molecular effects of EMF exposure, as many transduction pathways in the differentiative process are defined.     

S-Adenosylhomocysteine hydrolase activity during differentiation of HL-60 cells

Early changes in S-adenosylhomocysteine (SAH) hydrolase activity during DMSO-induced granulocytic differentiation of HL-80 ceils were followed. Within 24 h a decrease of activity of SAH hydrolase could be detected in induced cultures but not in control cultures. This decrease could be shown to be associated with G1 phase of the cell cycle and was detected prior to phenotypic changes of the ceils.     

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.     

Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells-a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells

Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.     

Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of alpha-tubulin does not significantly change; but the centrosomal-microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome-MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.     

Low-dose ionizing radiation effects on differentiation of HL-60 cells

The biological effects of low-dose radiation have attracted attention, but data are currently insufficient to fully understand the beneficial role of the phenomenon. In the present study, we have investigated the effects of low doses of gamma-irradiation alone and in combination with all-trans-retinoic acid (RA) on proliferation, apoptosis and differentiation of the human promyelocytic leukemia HL-60 cells. Changes in cell behavior and protein expression were determined with the use of light and fluorescent microscopy, immunocytochemical and Western blot analysis. Low-dose irradiation with 1–100 cGy caused a dose-dependent inhibition of HL-60 cell proliferation, and induced apoptosis and differentiation to granulocytes with an increase in the number of CD15-positive cells. Pre-irradiation with 1–100 cGy for 24 h before treatment with RA promoted apoptosis but did not impair RA-induced differentiation. Both processes were associated with a decrease in the expression of the proliferating cell nuclear antigen (PCNA), BCL-2, c-MYC, and changes in both cytosolic and nuclear levels of protein tyrosine-phosphorylation as well as protein kinase C alpha or beta isoforms. These results demonstrate the beneficial role of low-dose irradiation in modulating leukemia cell proliferation, differentiation and apoptosis.     

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.     

Alterations in glycosyltransferases during myeloid and monocytoid differentiation of HL-60 cells.

HL-60, a human promyelocytic leukemia cell line, can be differentiated to myeloid lineage by all- trans retinoic acid (ATRA), dimethylsulfoxide (DMSO) and n -butyric acid (n -BA), or to monocytoid(monocytic/macrophagic) lineage by phorbol-12-myristate-13-acetate (PMA) and ganglioside GM(3). The activity alterations of N -acetylglucosaminyltransferase III and V (GnT-III, GnT-V) as well as alpha-1,6-fucosyl-tranferase (alpha1,6 Fuc T) were studied during the differentiation of HL-60 cells by the above-mentioned five inducers using the fluorescence (PA)-labeled glycan-HPLC method for GnT assays and biotin-labeled glycan-LCA affinity chromatography combined with the HRP-avidin colorimetric method for alpha1,6 Fuc T assay. It was observed that after 3 days, all three enzymes decreased in HL-60 cells induced by 1 micromol/l ATRA and 0.6 mmol/l n-BA, while GnT-III and alpha1,6 Fuc T increased, but GnT-V still decreased after induction by 1% DMSO. GnT-V and alpha1,6 Fuc T declined, while GnT-III was elevated after induction by 0.1 micromol/l PMA for 3 days. In contrast, GnT-III increased after the treatment with 50 micromol/l GM(3)for 3 or 6 days, but GnT-V was not appreciably changed and alpha1,6 FucT was elevated after 6 days of GM(3)treatment. It may be concluded that the decrease of GnT-V is the common change in myeloid differentiation and the increase of GnT-III is the general alteration in monocytoid differentiation. The changes in the activities of glycosyltransferases were consistent with the structural changes in surface N -glycans previously found in our laboratory, i.e. that the antennary number of N -glycans decreased during myeloid differentiation by ATRA, and the amount of bisecting GlcNAc in N -glycans increased during monocytoid differentiation by PMA.     

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.     

Glutamine promotes colony formation in bone marrow and HL-60 cells; accelerates myeloid differentiation in induced HL-60 cells

Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis.     

Glutamine promotes colony formation in bone marrow and HL-60 cells; Accelerates myeloid differentiation in induced HL-60 cells

Summary Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis. This work has been supported by USPHS Grants AM 31624 and CA 00859 and a Faculty Research Grant from Texas College of Osteopathic Medicine.     

Analysis of cell-surface protein changes accompanying differentiation of HL-60 cells

The cell-surface proteins of HL-60 human promyelocytic leukemia cells have been compared to those of normal human neutrophils. Proteins of HL-60 cells surface labeled with 125I differed markedly from those of normal neutrophils, as shown by immunoprecipitation and polyacrylamide electrophoresis. Differentiation of HL-60 cells by treatment with dimethylformamide, trans-retinoic acid, or 12-O-tetradecanoylphorbol acetate did not modify the predominant surface-labeled proteins of HL-60 cells to produce a pattern similar to that of normal, mature neutrophils. However, the agents did induce greater quantities of minor cell-surface proteins immunoprecipitated by hyperimmune anti-human neutrophil serum. These immunoprecipitated proteins resembled several of the surface-labeled polypeptides of normal human neutrophils.     

Suppression of ethanolamine-containing glycerophospholipid synthesis in HL-60 cells during retinoic acid-induced differentiation.

Synthesis and degradation of glycerophospholipids in HL-60 cells and retinoic acid (RA)-treated HL-60 cells were examined. The synthesis of each subclass of ethanolamine-containing glycerophospholipids was extremely suppressed in RA-treated HL-60 cells, while that of other glycerophospholipids was not seriously affected. A pulse-chase experiment revealed that about 88% of 1,2-diacyl and 28% of 1-alkenyl-2-acyl glycerophosphoethanolamine were degraded during 4 days in RA-treated HL-60 cells. These characteristics of metabolism observed in RA-treated HL-60 cells might be responsible for the change of subclass composition of ethanolamine-containing glycerophospholipids in HL-60 cells during differentiation to granulocytes.     

Calcium stores in electropermeabilized HL-60 cells before and after differentiation

Non-induced HL-60 cells (N-IND) and HL-60 cells induced to differentiate with 2 microM retinoic acid (IND) were electropermeabilized with electrical discharges, and the intracellular Ca2+ stores were measured in each type of cell. Both N-IND and IND cells accumulate Ca2+ in the presence of ATP after electropermeabilization. The Ca2+ is stored in at least two different compartments; accumulation in one of the compartments is inhibited by oligomycin and CCCP, and it is not releasable by Ins(1,4,5)P3. The maximal accumulation of Ca2+ by the Ins(1,4,5)P3 sensitive pool is about 0.3 nmol/10(6) cells and 0.9 nmol/10(6) cells for the N-IND and for the IND cells, respectively, and the half-maximal value occurs at a free Ca2+ concentration of 0.23 microM and 0.63 microM, respectively. The oligomycin + CCCP sensitive pool hardly accumulates any Ca2+ at this level of free Ca2+, but at higher free [Ca2+] (greater than microM) its maximal capacity is 80-100-fold higher than the Ins(1,4,5)P3-sensitive pool (about 17-18 nmol/10(6) cells). It is concluded that at physiological free Ca2+ concentrations, the non-mitochondrial Ca2+ pool is regulating the intracellular free Ca2+ in N-IND and IND HL-60 cells, and that this Ca2+ pool can be mobilized by Ins(1,4,5)P3. Furthermore, the capacity of this pool increases about 3-fold when the cells are induced to differentiate with retinoic acid.