TPA-treated HL-60 Cells Research Articles

12-o-Tetradecanoylphorbol 13-acetate prevents baicalein-induced apoptosis via activation of protein kinase C and JNKs in human leukemia cells

In the present study, we found that baicalein (BE), but not its glycoside baicalin (BI), induced apoptosis in human leukemia HL-60 and Jurkat cells, but not in primary murine peritoneal macrophages (PMs) or human polymorphonuclear (PMN) cells, by the MTT assay, LDH release assay, and flow cytometric analysis. Activation of the caspase 3, but not caspase 1, enzyme via inducing protein processing was detected in BE-induced apoptosis. The ROS-scavenging activity of BE was identified by the anti-DPPH radical, DCHF-DA, and in vitro plasmid digestion assay, and none of chemical antioxidants including allpurinol (ALL), N-acetyl-cystein (NAC), and diphenylene iodonium (DPI) affected BE-induced apoptosis in HL-60 cells. This suggests that apoptosis induced by BE is independent of the production of ROS in HL-60 cells. Interestingly, the apoptotic events such as DNA ladders formation and activation of the caspase 3 cascade were significantly blocked by TPA addition in the presence of membrane translocation of PKCalpha, and TPA-induced protection was reduced by adding the PKC inhibitors, GF-109203X and staurosporin. TPA addition induces the phosphorylation of JNKs and ERKs, but not p38, protein in HL-60 cells, and incubation of HL-60 cells with JNKs inhibitor SP600125, but not ERKs inhibitor, PD98059 or the p38 inhibitor SB203580, suppressed the protective effect of TPA against BE-induced apoptotic events including DNA ladders, apoptotic bodies, caspase 3 and D4-GDI protein cleavage in according with blocking JNKs protein phosphorylation. In addition, PKC inhibitor GF-109203X treatment blocks TPA-induced ERKs and JNKs protein phosphorylation, which indicates that activation of PKC locates at upstream of MAPKs activation in TPA-treated HL-60 cells. Additionally, a loss in mitochondrial membrane potential with a reduction in Bcl-2 protein expression, the induction of Bad protein phosphorylation, and translocation of cytochrome c from mitochondria to the cytosol were observed in BE-treated HL-60 cells, and these events were prevented by the addition of TPA. GF-109203X and SP600125 suppression of TPA against cytochrome c release induced by BE was identified. This suggests that activation of PKC and JNKs participate in TPA's prevention of BE-induced apoptosis via suppressing mitochondrial dysfunction in HL-60 cells.

Transcriptional repression of vimentin gene expression by pyrroline dithiocarbamate during 12-O-tetradecanoylphorbol-13-acetate-dependent differentiation of HL-60 cells

Vimentin is a member of the intermediate filament family, and the NF-kappaB binding site is located in the human vimentin promoter. To gain insight into the role of NF-kappaB in the regulation of the vimentin gene during 12-O-tetradecanoylphorbol-13-acetate (TPA)-dependent differentiation of HL-60 cells, the effect of pyrrolidine dithiocarbamete (PDTC) has been investigated using Northern blot hybridization and DNA mobility shift assay. PDTC inhibited macrophage-like morphologic change of HL-60 cells by TPA. TPA-dependent increase of vimentin mRNA level was decreased in a time- and dose-dependent manner by pretreatment with PDTC. One DNA-protein complex was formed by DNA mobility shift assay when the NF-kappaB or AP-1 binding sites were incubated with nuclear extract prepared from TPA-treated HL-60 cells, but no protein bound in control HL-60 cells without TPA. After PDTC pretreatment, NF-kappaB binding activity vanished but AP-1 binding activity was unchanged. Taken together, these results suggest that NF-kappaB may be an essential transacting factor for transcriptional repression of the vimentin gene by PDTC during TPA-dependent differentiation of HL-60 cells.

Phosphorylation of octamer-binding transcriptional factor may be correlated with H2B histone gene repression during 12-O-tetradecanoylphorbol 13-acetate-dependent differentiation of HL-60 cells

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA), the binding pattern of nuclear proteins to various elements in the human H2B histone gene upstream region have been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA rapidly reduced at 24 h in TPA-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of TPA. In DNase I footprinting analysis, one nuclear factor (octamer-binding transcription factor, OTF) bound at -42 bp (octamer motif), before and after TPA-induced differentiation of HL-60 cells. One DNA-protein complex (OTF) was formed by DNA mobility shift assay when octamer element was incubated with nuclear extract of undifferentiated HL-60 cells. In DNA mobilith shift assay, OTF vanished, and phosphorylated OTF (p-OTF) newly appeared during TPA-induced differentiation. p-OTF was not detected after pretreatment of the protein kinase C inhibitor, staurosporin, but was not changed after CHX treatment. TPA-induced repression of H2B histone mRNA was also restored after pretreatment of staurosporin. These results suggest that OTF is phosphorylated by protein kinase C during TPA-induced differentiation of HL-60 and the transcriptional repression of the H2B histone gene may be mediated by protein kinase C-dependent phosphorylation of OTF.

Ubiquitin-proteasome system is involved in induction of LFA-1/ICAM-1-dependent adhesion of HL-60 cells.

Membrane-permeable proteasome inhibitors, lactacystin (LC) and N-acetyl-Leu-Leu-norleucinal (ALLN), but not calpain inhibitor Z-Leu-leucinal (ZLL), prevented LFA-1/ICAM-1-dependent cellular adhesion of TPA-stimulated HL-60 cells. These proteasome inhibitors affected neither the induction of monocytic differentiation nor the accompanying protein-tyrosine phosphorylation. They suppressed the increase in the avidity of LFA-1 to ICAM-1 without changing the expression of these molecules. Immunoblotting using monoclonal antibody FK-1, which reacts specifically with polyubiquitinated proteins, demonstrated that the proteasome inhibitors caused the drastic accumulation of the polyubiquitinated proteins in the membrane fraction of TPA-treated HL-60 cells. This indicates that accompanying activation of LFA-1, TPA induces the polyubiquitination of the membrane proteins, which are rapidly degraded by proteasomes. These data taken together show that proteolysis mediated by the ubiquitin-proteasome system is a prerequisite for the induction of LFA-1-dependent adhesion of HL-60 cells.

Reduced level of ATF is correlated with transcriptional repression of DNA topoisomerase II alpha gene during TPA-induced differentiation of HL-60 cells.

DNA topoisomerase II is a marker for the proliferation state of mammalian cells in culture, and the protein levels are markedly higher in exponentially growing cells than quiescent cells and can be downregulated by growth of the cells at high density and serum starvation. Correlation between ATF and TPA-repressed DNA topoisomerase II alpha (Topo II alpha) mRNA has been investigated during TPA-induced differentiation of HL-60 cells. Topo II alpha mRNA and unknotting activity were reduced at 24 hours in TPA-treated HL-60 cells. The level of Topo II alpha mRNA and the activity were gradually decreased in proportion to the concentration of TPA. Two DNA-protein complexes were formed by DNA mobility shift assay when ATF-binding site was incubated with nuclear extract prepared from TPA-free HL-60 cells, and the amount of ATF was vanished after TPA treatment. TPA-repressed Topo II alpha mRNA and ATF levels were partially restored after pretreatment of staurosporin. These results suggest that the reduced level of ATF may be important to the transcriptional repression of Topo II alpha gene during TPA-induced differentiation in HL-60 cells and related to protein kinase C signal pathway.

Regulation of 92-kD Gelatinase Release in HL-60 Leukemia Cells: Tumor Necrosis Factor-α as an Autocrine Stimulus for Basal- and Phorbol Ester-Induced Secretion

Matrix metalloproteinase 9 (MMP-9), also known as 92-kD type IV collagenase/gelatinase, is believed to play a critical role in tumor invasion and metastasis. Here, we report that MMP-9 was constitutively released from the human promyelocytic cell line HL-60 as determined by zymographic analysis. Tumor necrosis factor-alpha (TNF-alpha) enhanced the enzyme release threefold to fourfold and the protein kinase C (PKC) activator and differentiation inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) eightfold to ninefold. Gelatinase induction by TNF-alpha and TPA was inhibited by actinomycin D or cycloheximide, indicating that de novo protein synthesis was required. Neutralizing monoclonal antibodies to TNF-alpha (anti-TNF-alpha) decreased the basal MMP-9 release of these cells. In addition, these antibodies also significantly interfered with the TPA-induced enzyme release. Agents that inhibit TNF-alpha expression in HL-60 cells, such as pentoxifylline and dexamethasone, completely abrogated both the constitutive and TPA-evoked MMP-9 release. Diethyldithiocarbamate, which is known to stimulate TNF-alpha production in HL-60 cells, exerted a positive effect on MMP-9 release in untreated cells but was inhibitory in TPA-treated HL-60 cells. The PKC inhibitor staurosporine at low concentrations (100 ng/mL) caused a significant augmentation of MMP-9 release in untreated cultures that was blocked by the addition of anti-TNF-alpha. High concentrations (2 mumol/L) of staurosporine completely abolished the extracellular enzyme activity both in untreated and TPA-stimulated cells. These results suggest, that TNF-alpha is required for basal and PKC-mediated MMP-9 release in HL-60 leukemia cells. Thus, MMP-9 secretion may be regulated by TNF-alpha not only in a paracrine but also in an autocrine fashion. This may potentiate the matrix degradative capacity of immature leukemic cells in the processes of bone marrow egress and the evasion of these cells into peripheral tissue.

Regulation of 92-kD gelatinase release in HL-60 leukemia cells: tumor necrosis factor-alpha as an autocrine stimulus for basal- and phorbol ester-induced secretion

Matrix metalloproteinase 9 (MMP-9), also known as 92-kD type IV collagenase/gelatinase, is believed to play a critical role in tumor invasion and metastasis. Here, we report that MMP-9 was constitutively released from the human promyelocytic cell line HL-60 as determined by zymographic analysis. Tumor necrosis factor-alpha (TNF-alpha) enhanced the enzyme release threefold to fourfold and the protein kinase C (PKC) activator and differentiation inducer 12-O-tetradecanoylphorbol-13- acetate (TPA) eightfold to ninefold. Gelatinase induction by TNF-alpha and TPA was inhibited by actinomycin D or cycloheximide, indicating that de novo protein synthesis was required. Neutralizing monoclonal antibodies to TNF-alpha (anti-TNF-alpha) decreased the basal MMP-9 release of these cells. In addition, these antibodies also significantly interfered with the TPA-induced enzyme release. Agents that inhibit TNF-alpha expression in HL-60 cells, such as pentoxifylline and dexamethasone, completely abrogated both the constitutive and TPA-evoked MMP-9 release. Diethyldithiocarbamate, which is known to stimulate TNF-alpha production in HL-60 cells, exerted a positive effect on MMP-9 release in untreated cells but was inhibitory in TPA-treated HL-60 cells. The PKC inhibitor staurosporine at low concentrations (100 ng/mL) caused a significant augmentation of MMP-9 release in untreated cultures that was blocked by the addition of anti-TNF-alpha. High concentrations (2 mumol/L) of staurosporine completely abolished the extracellular enzyme activity both in untreated and TPA-stimulated cells. These results suggest, that TNF- alpha is required for basal and PKC-mediated MMP-9 release in HL-60 leukemia cells. Thus, MMP-9 secretion may be regulated by TNF-alpha not only in a paracrine but also in an autocrine fashion. This may potentiate the matrix degradative capacity of immature leukemic cells in the processes of bone marrow egress and the evasion of these cells into peripheral tissue.

Effects of 1 alpha,25-dihydroxyvitamin D3 on macrophage colony- stimulating factor production and proliferation of human monocytic cells

1 alpha-25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates the proliferation of human monocytes in vitro. In the present study, we investigated a possible role of macrophage colony-stimulating factor (M-CSF) in 1 alpha,25(OH)2D3-induced proliferation of human circulating monocytes and the effects of 1 alpha,25(OH)2D3 on M-CSF production by human monocytic cells. Both 1 alpha,25(OH)2D3 and recombinant human M-CSF increased 2.5-fold the nucleus number of human circulating monocytes on day 6 of the culture. These effects were inhibited by antihuman M-CSF antibody as well as by anti-c-fms antibody, although these antibodies themselves did not affect the nucleus number when added to control culture. These results indicated that M-CSF is required for 1 alpha,25(OH)2D3-stimulated monocyte proliferation. In addition, 1 alpha,25(OH)2D3 stimulated M-CSF secretion from human circulating monocytes. Secretion and mRNA expression of M-CSF by 12-0-tetradecanoylphorbol-13-acetate (TPA)-treated THP-1 cells (human monocytic leukemia cell line) and TPA-treated HL-60 cells (human promyelocytic leukemia cell line) were also increased by 1 alpha,25(OH)2D3. M-CSF secretion from TPA-treated THP-1 cells was increased by 1 alpha,25(OH)2D3 in a dose-dependent and metabolite-specific manner. The present study demonstrates that 1 alpha,25(OH)2D3 is a potent stimulator for M-CSF production by human monocytic cells and that the proliferative effect of 1 alpha,25(OH)2D3 on human monocytes may be attributed, at least in part, to the stimulated secretion of M-CSF.

Regulation of integrin-mediated myeloid cell adhesion to fibronectin: influence of disulfide reducing agents, divalent cations and phorbol ester.

Three different agents, dithiothreitol (DTT), Mn2+, and phorbol ester (TPA), were found to induce HL-60 cell adhesion to fibronectin through distinct mechanisms. The binding of HL-60 cells to fibronectin and a 120-kDa fibronectin fragment is completely dependent on the alpha 5 beta 1 integrin, the adhesion activators, and appropriate divalent cations such as Mg2+. Mn2+ alone was able to induce maximal adhesion in the absence of these other activators. With any of the three activators, Ca2+ inhibited adhesion to fibronectin substrates by inhibiting alpha 5 beta 1-fibronectin binding. DTT and Mn2+ were both found to enhance the binding of fibronectin to purified alpha 5 beta 1, which suggests that both agents can directly stimulate the integrin-ligand binding reaction. TPA acts by inducing intracellular phosphorylation whereas neither DTT nor Mn2+ induced protein phosphorylation. TPA-treated HL-60 cells adhere and spread on fibronectin substrates, whereas DTT- and Mn(2+)-treated cells adhere but do not spread. The actin cytoskeletal inhibitor, cytochalasin B, markedly blocks TPA-induced adhesion, has an intermediate effect on DTT-induced adhesion, and has a minimal effect on Mn(2+)-induced adhesion. Collectively, the data suggest that TPA seems to act by inducing phosphorylation events that lead to cytoskeletal changes and alpha 5 beta 1 integrin activation. In contrast, DTT and Mn2+ seem to act primarily by directly influencing the alpha 5 beta 1-fibronectin binding reaction. These studies characterize in detail a regulatory system for studying leukocyte alpha 5 beta 1-fibronectin adhesion and identify DTT as a novel activator of alpha 5 beta 1-fibronectin binding.

Tyrosine-phosphorylation of tubulin during monocytic differentiation of HL-60 cells.

We have demonstrated that the 53- and 55-kDa proteins were markedly phosphorylated at tyrosine residues during tetradecanoyl phorbol acetate (TPA)-induced monocytic differentiation of HL-60 cells and that the inhibition of such phosphorylation resulted in suppression of the differentiation. Western blotting, using antiphosphotyrosine antibody PY-20, of antitubulin immunoprecipitate from the lysate of TPA-treated HL-60 cells revealed that tubulin was tyrosine-phosphorylated in TPA-treated HL-60 cells, but not in nontreated HL-60 cells. The tyrosine-phosphorylated 53- and 55-kDa proteins in TPA-treated HL-60 cells were absorbed with antitubulin antibody indicating that the predominantly tyrosine-phosphorylated proteins during the monocytic differentiation were tubulins. By combination of immunoprecipitation and in vitro kinase assay, the protein tyrosine kinases, Fyn and Lyn, which were abundantly induced during monocytic differentiation of HL-60 cells, were demonstrated to be associated with tubulin in the cells. The reorganization of microtubules was observed by immunostaining using rhodamine-conjugated anti-mouse Ig and mouse anti-alpha-tubulin mAb. These data suggest that tyrosine-phosphorylation of tubulin, in which Fyn and Lyn might involve, regulate monocytic differentiation through alteration of microtubule assembly.

Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO.

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.

Transcriptional and post-transcriptional regulation of c-jun expression during monocytic differentiation of human myeloid leukemic cells.

AP-1, the polypeptide product of c-jun, recognizes and binds to specific DNA sequences and stimulates transcription of genes responsive to certain growth factors and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). We studied the effects of TPA on the regulation of c-jun gene expression in HL-60 cells during monocytic differentiation. Low levels of c-jun transcripts were detectable in untreated HL-60 leukemic cells, increased significantly by 6 h, and reached near maximal levels by 24 h of exposure to 32 nM TPA. Similar kinetics of c-jun induction by TPA were observed in human U-937 and THP-1 monocytic leukemia cells. Similar findings were obtained with bryostatin 1 (10 nM), another activator of protein kinase C and inducer of monocytic differentiation. Furthermore, 1,25-dihydroxyvitamin D3 (0.5 microM), a structurally distinct agent which also induces HL-60 monocytic differentiation, increased c-jun expression. TPA treatment of HL-60 cells in the presence of cycloheximide was associated with superinduction of c-jun transcripts. Run-on analysis demonstrated detectable levels of c-jun gene transcription in untreated HL-60 cells, and that exposure to TPA increases this rate 3.3-fold. Treatment of HL-60 cells with both TPA and cycloheximide had no effect on the rates of c-jun transcription. The half-life of c-jun RNA as determined by treating HL-60 cells with TPA and actinomycin D was 30 min. In contrast, the half-life of c-jun RNA in TPA-treated HL-60 cells exposed to cycloheximide and actinomycin D was greater than 2 h. These findings suggested that the increase in c-jun RNA observed during TPA-induced monocytic differentiation is mediated by both transcriptional and post-transcriptional mechanisms.

Two isozymes of PKC found in HL-60 cells show a difference in activation by the phorbol ester TPA

Cytosol from untreated cells and a detergent extract of the particulate fraction from TPA-treated HL-60 cells were analyzed for protein kinase C activity by consecutive column chromatography on Mono Q and hydroxyapatite. From both preparations two separate peaks of enzyme activity were obtained. The first peak, eluting at lower salt concentrations, is activated at lower TPA concentrations (3 × 10 −9 M) than the other (10 −7 M), which was eluted at higher salt concentrations.

Replication of herpes simplex virus type 1 in differentiated human promyelocytic HL-60 cells

The capability of herpes simplex virus type 1 (HSV-1), strain Angelotti (ANG), to replicate in human promyelocytic HL-60 cells treated with 1,2-tetradecanoyl-phorbol-13-acetate (TPA) and dimethyl sulfoxide (DMSO) was examined. Virus titrations and infectious center assays revealed that HSV-1 ANG replicated in nontreated HL-60 cells and in HL-60 cells treated with TPA. An abortive infection was observed in DMSO-stimulated HL-60 cells. Viral DNA synthesis was detected in nontreated and TPA-treated cells, but not in DMSO-treated cells. Analysis of HSV-1 transcripts revealed that albeit the differences in pretreatment, HL-60 cells synthesized viral immediate-early (ICP4) and early (tk and poll RNAs, whereas a late viral transcript (gC) was almost exclusively detected in nontreated and TPA-treated HL-60 cells. In line with these observations were the results obtained from studies on viral protein synthesis. The immediate-early protein ICP4 was found in all three cell types. Early (pol), delayed-early (gB), as well as late proteins (VP 5, gC) were identified in nontreated and TPA-treated cells, but only in reduced amounts in DMSO-treated cells. These data suggest a translational block of HSV replication in DMSO-treated HL-60 cells at the level of early gene expression.

Changes in glycosaminoglycan synthesis during the differentiation of HL-60 cells induced by 12-O-tetra-decanoylphorbol-13-acetate.

The changes in glycosaminoglycan (GAG) synthesis during the differentiation of HL-60 cells to macrophage-like cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) were studied by using 35S-sulfate or 3H-glucosamine as a tracer. The incorporation of 35S-sulfate into GAGs of HL-60 cells treated with TPA (TPA-treated HL-60 cells) decreased by 30% compared with untreated HL-60 cells (HL-60 cells). The profiles of the Sepharose CL-6B chromatography showed that the molecular weight size of proteoglycans (PGs) in the medium fraction of TPA-treated HL-60 cells was larger than that of HL-60 cells although the PGs in the cell fractions of both HL-60 and TPA-treated HL-60 cells were of the same molecular weight size. On the other hand, the molecular weight sizes of GAGs in both the medium and the cell fractions of TPA-treated HL-60 cells were larger than those of HL-60 cells. In order to examine the synthesis of GAG chains, HL-60 and TPA-treated HL-60 cells were incubated with 35S-sulfate in the presence of 4-methylumbelliferyl-beta-D-xyloside, an exogenous initiator. The incorporation of 35S-sulfate into total GAGs of the TPA-treated HL-60 cells exposed to the beta-D-xyloside increased 3-fold over that of HL-60 cells. HL-60 cells synthesized core protein-initiated GAGs and beta-D-xyloside-initiated GAGs but TPA-treated HL-60 cells synthesized beta-D-xyloside-initiated GAGs only. beta-D-Xyloside-initiated GAGs synthesized by both cells were obtained in the same fraction by Sepharose CL-6B chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Serological analysis of cell surface antigens of HL-60 cells before and after treatment with a phorbol ester tumor promoter

The human HL-60 cell line derived from acute promyelocytic leukemia, consisting of promyelocytic type of cells, was able to differentiate into adherent cells with monocyte-macrophage features by the treatment with 12- O-tetradecanoyl phorbol-13-acetate (TPA). Cell surface antigens of HL-60 cells before and after TPA treatment were studied with monoclonal antibodies and four hybridoma clones producing IgM antibodies were established. Two antibodies (HL-21 and HL-47) reacted only with the immunizing TPA-treated HL-60 cells, and HL-1 antibody produced against untreated cells was reactive with both TPA-treated and untreated cells, but HL-5 antibody reacted predominantly with the immunizing untreated cells. Serological reactivity against various types of normal hematopoietic cells and acute leukemias (diagnosed by the French-American-British classification) was studied by immune adherence assay and immuno-electron microscopy. HL-21 antibody was reactive with monocytes and most cases of M4 and M5 types of acute non-lymphocytic leukemia cells. HL-47 antibody did not react with the cells of myelocyte-monocyte lineage or mature lymphocytes, but it did react with one-third of acute lymphocytic leukemia (L1 and L2) cases. Since all HL-47 + cases were included in the group of common ALL antigen positive cases, it was estimated that HL-47 is a differentiation antigen present on lymphocyte precursors, from which null-cell type acute lymphocytic leukemia cells generally originate. HL-1 antibody reacted with the cells of myelocyte-monocyte lineage as well as those of most acute non-lymphocytic leukemias. HL-5 antibody reacted with granulocytes and M2 type of acute myelocytic leukemia cases, and also with M5 type of acute monocytic leukemia cases. Serological studies of these antibodies revealed that TPA can induce to differentiate HL-60 cells not only into HL-21 + macrophage-like cells, but also into HL-47 + lymphoid stem cells. In addition, these antibodies were demonstrated to be very valuable for differential diagnosis of acute leukemias.

Release of Platelet-Activating Factor From HL-60 Human Leukemic Cells Following Macrophage-Like Differentiation

Platelet-activating factors (PAF), a phospholipid mediator of anaphylaxis, is also known to be released in vitro from both phagocytic polymorphonuclear neutrophils (PMN) and monocytes in response to a variety of stimuli. The fact that human myeloid cells of the HL-60 line can be made to differentiate in vitro into macrophage-like cells by 12-O-tetradodecanoylphorbol-13-acetate (TPA) prompted us to investigate the generation and release of PAF during this transformation. Both passive release of PAF at pH 9.5, and active release, following phagocytosis of C3b- and C3d-opsonized yeast spores, and stimulation with C5a anaphylatoxin from untreated and TPA-treated HL-60 cells, PMN, and plastic-adherent normal human monocytes were studied. It was found that after 3 days of TPA treatment, HL-60 cells released PAF following phagocytosis of C3b- and C3d-opsonized yeast spores. Inhibition of PAF release by a selective inhibitor of phospholipase A2 and labeling of PAF with sodium 14C-acetate indicated that PAF generation is a two-step process: (1) release of PAF precursor from cell membranes and (2) its acetylation. A model for the in vivo study of mechanisms and metabolic events involved in PAF generation and release could perhaps be built on these findings.U

Release of platelet-activating factor from HL-60 human leukemic cells following macrophage-like differentiation

Platelet-activating factors (PAF), a phospholipid mediator of anaphylaxis, is also known to be released in vitro from both phagocytic polymorphonuclear neutrophils (PMN) and monocytes in response to a variety of stimuli. The fact that human myeloid cells of the HL-60 line can be made to differentiate in vitro into macrophage-like cells by 12- O-tetradodecanoylphorbol-13-acetate (TPA) prompted us to investigate the generation and release of PAF during this transformation. Both passive release of PAF at pH 9.5, and active release, following phagocytosis of C3b- and C3d-opsonized yeast spores, and stimulation with C5a anaphylatoxin from untreated and TPA-treated HL-60 cells, PMN, and plastic-adherent normal human monocytes were studied. It was found that after 3 days of TPA treatment, HL-60 cells released PAF following phagocytosis of C3b- and C3d-opsonized yeast spores. Inhibition of PAF release by a selective inhibitor of phospholipase A2 and labeling of PAF with sodium 14C-acetate indicated that PAF generation is a two-step process: (1) release of PAF precursor from cell membranes and (2) its acetylation. A model for the in vivo study of mechanisms and metabolic events involved in PAF generation and release could perhaps be built on these findings.