TPA-induced Differentiation Of HL-60 Cells Research Articles

Ubiquitin-specific peptidase 3 induces TPA-mediated leukemia cell differentiation via regulating H2AK119ub

ABSTRACTAcute myeloid leukemia (AML) is the most common type of leukemia in adults. Owing to the chemotherapy associated side effects and toxicity, it is necessary to find a new mechanism, which can identify new potential therapeutic targets at the molecular level. Here, we identified new target genes that are induced during the TPA-induced HL-60 cell differentiation by ChIP-seq and microarray data analysis. Using q-PCR and ChIP assay, we confirmed that the target genes including USP3, USP35, TCF4, and SGK1 are upregulated during TPA-mediated HL-60 cell differentiation. Levels of USP3, one of the deubiquitinating enzymes (DUBs), increased by TPA treatment, resulting in the reduction of H2AK119ub levels. In addition, we revealed that depletion of USP3 inhibits TPA-mediated leukemia cell differentiation q-PCR and FACS analysis. Taken together, our data indicate that USP3 promotes TPA-mediated leukemia cell differentiation via regulating H2AK119ub levels.

Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation.

The human myeloid leukemia cell line HL-60 differentiate into monocytes following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the mechanism underlying the differentiation of these cells in response to TPA has not been fully elucidated. In this study, we performed ChIP-seq profiling of RNA Pol II, HDAC2, Acetyl H3 (AcH3), and H3K27me3 and analyzed differential chromatin state changes during TPA-induced differentiation of HL-60 cells. We focused on atypically active genes, which showed enhanced H3 acetylation despite increased HDAC2 recruitment. We found that HDAC2 positively regulates the expression of these genes in a histone deacetylase activity-independent manner. HDAC2 interacted with and recruited paired box 5 (PAX5) to the promoters of the target genes and regulated HL-60 cell differentiation by PAX5-mediated gene activation. Taken together, these data elucidated the specific-chromatin status during HL-60 cell differentiation following TPA exposure and suggested that HDAC2 can activate transcription of certain genes through interactions with PAX5 in a deacetylase activity-independent pathway.

Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.

Characterization of various promoter regions of the human DNA helicase-encoding genes and identification of duplicated ets (GGAA) motifs as an essential transcription regulatory element

DNA helicases are important in the regulation of DNA transaction and thereby various cellular functions. In this study, we developed a cost-effective multiple DNA transfection assay with DEAE-dextran reagent and analyzed the promoter activities of the human DNA helicases. The 5'-flanking regions of the human DNA helicase-encoding genes were isolated and subcloned into luciferase (Luc) expression plasmids. They were coated onto 96-well plate and used for co-transfection with a renilla-Luc expression vector into various cells, and dual-Luc assays were performed. The profiles of promoter activities were dependent on cell lines used. Among these human DNA helicase genes, XPB, RecQL5, and RTEL promoters were activated during TPA-induced HL-60 cell differentiation. Interestingly, duplicated ets (GGAA) elements are commonly located around the transcription start sites of these genes. The duplicated GGAA motifs are also found in the promoters of DNA replication/repair synthesis factor genes including PARG, ATR, TERC, and Rb1. Mutation analyses suggested that the duplicated GGAA-motifs are necessary for the basal promoter activity in various cells and some of them positively respond to TPA in HL-60 cells. TPA-induced response of 44-bp in the RTEL promoter was attenuated by co-transfection of the PU.1 expression vector. These findings suggest that the duplicated ets motifs regulate DNA-repair associated gene expressions during macrophage-like differentiation of HL-60 cells.

Characterization of the promoter region of the human PARG gene and its response to PU.1 during differentiation of HL‐60 cells

The metabolism of poly(ADP-ribose) plays important roles in the nuclear function of mammalian cells. Previously, we analyzed expression of the poly(ADP-ribose) glycohydrolase (PARG) gene during HL-60 cell differentiation and found that expression was greatly reduced by 4 h after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and returned to the initial level within 20 h. In the present study, a 2.1-kb fragment of the 5'-flanking (promoter) region of the human PARG gene was isolated from the HL-60 genome by polymerase chain reaction and ligated into a luciferase-expression vector, pGL3, to generate the pPARG-Luc#2 reporter plasmid. Deletion analysis revealed that a 75-nt sequence is required for basal promoter activity and TPA responsiveness. Mutations in this 75-nt sequence reduced promoter activity and the TPA response of HL-60 cells. TFSEARCH analysis revealed that Ets family binding motifs are located in the 75-nt sequence. Chromatin immunoprecipitation assay, electrophoretic mobility shift assay and competition analysis indicated that PU.1 (Spi-1) binds to the 75-nt sequence. Moreover, co-transfection of HL-60 cells with a PU.1 expression plasmid and pPARG-Luc indicated that PU.1 down-regulate the PARG promoter. These results suggest that PARG gene expression is modulated by PU.1 during TPA-induced 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.

IRF-2 Is Involved in Up-regulation of Nonmuscle Myosin Heavy Chain II-A Gene Expression during Phorbol Ester-induced Promyelocytic HL-60 Differentiation

Transcription of the nonmuscle myosin heavy chain II-A (NMHC-A) gene is regulated by various factors, including cell type, proliferation and differentiation stage, and extracellular stimuli. We have identified an intronic region (designated 32kb-150), which is located 32 kb downstream of the transcription start sites in the human NMHC-A gene, as a transcriptional regulatory region. 32kb-150 contains an interferon-stimulated response element (ISRE). By using HeLa and NIH3T3 cells, in which NMHC-A is constitutively expressed, interferon regulatory factor (IRF)-2 was found to be the only major protein, among the IRF family proteins, that bound to the ISRE in 32kb-150 both in vitro and in intact cells. IRF-2, which is known to either repress or activate target gene expression, acts as a transcriptional activator in the context of the 32kb-150 reporter gene. The carboxyl-terminal basic region of IRF-2 serves as an activation domain in this context. This is in contrast to its acting as a repressor domain in the context of the synthetic core ISRE. Furthermore, after treatment of promyelocytic HL-60 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), which triggers differentiation into macrophages, both NMHC-A expression and IRF-2 expression were found to be up-regulated with a similar time course. TPA treatment leads to recruitment of IRF-2 to 32kb-150 of the endogenous NMHC-A gene and acetylation of the core histones surrounding this region. In addition, the ISRE in the 32kb-150 reporter gene recruits IRF-2 and mediates TPA-induced activation of a reporter gene in HL-60 cells. Together, these results indicate that IRF-2 contributes to transcriptional activation of the NMHC-A gene via 32kb-150 during TPA-induced differentiation of HL-60 cells.

Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.

Up-regulation of CD13/aminopeptidase N induced by phorbol ester is involved in redox regulation and tumor necrosis factor alpha production in HL-60 cells.

Here, we demonstrate the possible involvement of oxidative stress in altered CD13/aminopeptidase N (APN) expression during myeloid cell differentiation induced by TPA. In flow cytometrical analysis, CD13/APN protein was constitutively expressed in HL-60 cells. When the cells were treated with TPA, CD13/APN expression was up-regulated with increased intracellular peroxides and a morphological change into macrophage-like cells. This increase in CD13/APN expression was suppressed by treatment with N-acetylcysteine. Transfection of Mn-superoxide dismutase (MnSOD) gene to the cells also suppressed the up-regulated CD13/APN expression. Furthermore, a neutralizing antibody to TNFalpha partially blocked this up-regulation. These results indicate that the change in intracellular redox state could be involved in the up-regulation of CD13/APN expression during TPA-induced differentiation of HL-60 cells, suggesting that TNFalpha may serve as, at least, one of the signals stimulated by TPA.

Effects of bacterial glyceroglycolipid M874B on growth and TPA-induced differentiation of HL60 cells.

Bacterial monogalactosyldiacylglycerol M874B (MGDAG), which protects against oxygen radicals, was found to increase the growth of the human promyelocytic leukemia cell HL60 when added to the cell culture, but suppresses the 12-O-tetradecanoyl phorbol-13-acetate-induced differentiation. Analogous MGDAG, S365B had weak, but similar effects. These activities were not observed with analogous plant glyceroglycolipids and diacylglycerol.

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.

Modulation of phorbol ester-induced HL-60 differentiation by prostaglandin E 2

When treated with phorbol tumor promoters, HL-60 cells undergo terminal differentiation evidenced by a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity. Internalization of fluorescent particles by cells exhibiting the phagocytic positive phenotype (phag +) provides a sensitive indication of promoter-induced differentiation, and the resulting fluorescent cells can be quantitatively analyzed by flow cytometry. The current study was initiated to further test the predicitive power of a flow cytometry based HL-60 differentiation assay in the detection of agents associated with tumor promotion. Specifically, experiments were designed to assess the sensitivity of the test system to co-promoters which enhance promoter activity in vivo. Prostaglandin E 2 (PGE 2) was chosen as a model co-promoter since it has been shown to potentiate phorbol ester (i.e. 12- O-tetradecanoyl phorbol-13-acetate; TPA) induced biological effects in vivo. Results detailed in the current report indicate that PGE 2 enhances TPA-induced differentiation of HL-60 cells in a dose-dependent manner. As with in vivo co-promotion experiments, PGE 2 exhibited a maximum potentiating effect when administered prior to TPA. These data indicate that HL-60 cells are not only sensitive to phorbol promoters, but also to the co-promoter PGE 2. These experiments support the hypothesis that a flow cytometry based HL-60 assay may prove useful for studying chemical agents or intrinsic cellular factors that are involved in the tumor promotion phase of carcinogenesis.

Changes in the kinetics of inositol transport during TPA-induced differentiation of HL60 cells towards monocytes

When exposed to the phorbol ester TPA, HL60 cells undergo growth arrest and differentiate towards monocytes. During TPA-induced differentiation there was a 2.6-fold increase in the rate of inositol transport (Vmax), a 2.1-fold increase in intracellular inositol and a 1.5-fold increase in inositol lipid. An increase in the Vmax of inositol transport did not occur when the variant cell line HL60Ast3 was exposed to TPA, which has been shown in this cell line to induce growth arrest but not differentiation. This observation suggests that the change in inositol transport during HL60 monocyte differentiation is specifically associated with the process of cell differentiation as opposed to growth arrest.

Effects of hexadecylphosphocholine on protein kinase C and TPA‐induced differentiation of HL60 cells

Several structural analogs of alkylphosphocholine (APC) were studied for their effects on protein kinase C (PKC) and 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited biochemical and cellular events in HL60 cells. Hexadecylphocholine (He-PC2), the APC prototype, inhibited PKC competitively with respect to phosphatidylserine an noncompetitively with respect to CaCl2, both with an apparent Ki of about 15 microM. Inhibition of PKC by He-PC2 was selective, since cyclic AMP dependent protein kinase and Ca2+/calmodulin dependent protein kinase II were relatively unaffected. He-PC2 inhibited TPA-induced depletion of PKC and TPA-stimulated phosphorylation of cellular proteins in HL60 cells. TPA-induced differentiation of HL60 cells was also inhibited by He-PC2, and this inhibition was synergistic or additive to the effects of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor. The present findings are consistent with the hypothesis that inhibition of PKC might be related, in part, to the antineoplastic effect of He-PC2 and ether lipid analogs such as ET-18-OCH3 (1-octadecyl-2-methyl-glycero-3-phosphocholine).

Expression of src family genes during monocytic differentiation of HL-60 cells.

It has been reported that src family protein-tyrosine kinases were expressed specifically in a certain lineage or differentiation stage of hematopoietic cells. To understand the molecular basis for differentiation and function of monocyte/macrophage, we investigated the expressions of src family genes by the HL-60 cells stimulated with differentiation-inducing agents. TPA and vitamin D3 (D3) were used as stimulants for monocytic development, since each agent has been known to induce phenotypically specific differentiation of HL-60 cells. The fyn, fgr, and lyn genes were characteristically expressed concomitantly with phenotypic changes and expressions of nuclear proto-oncogenes, whereas src, lck, hck, and yes genes were not. In TPA-induced differentiation of HL-60 cells, both fyn and lyn genes, but not fgr gene, were expressed. In contrast, both fgr and lyn genes, but not fyn gene, were expressed in D3-induced differentiation of the cells. The independent and characteristic expressions of these genes were observed in the further advanced differentiation of HL-60 cells induced by TPA plus D3 or D3 plus human transforming growth factor-beta 1. The granulocytic differentiation of the cells treated with retinoic acid was accompanied by intense expression of fgr, but weak or no expression of lyn and fyn gene. These data indicate that each protein-tyrosine kinase encoded by src family genes may play distinct roles in development and/or functions of monocyte/macrophage-lineage cells.

Phorbol esters activate the pathway for phosphatidylethanol synthesis in differentiating HL-60 cells

12- O-Tetradecanoylphorbol 13-acetate (TPA) has been shown to induce the formation of an unusual acidic phospholipid, phosphatidylethanol, in HL-60 cells. The synthesis of this lipid is dependent upon the presence of ethanol in the culture medium of TPA-treated cells; however, other exogenous alcohols can substitute as headgroup precursors with the formation of the corresponding phosphatidyl alcohol. The activation of the pathway for phosphatidyl alcohol synthesis appears to be mediated through protein kinase C. Studies of the time-course for the synthesis and accumulation of phosphatidylethanol suggest a possible involvement of the pathway for phosphatidyl alcohol synthesis in the TPA-induced differentiation of HL-60 cells.

1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7), a potent inhibitor of protein kinases, inhibits the differentiation of HL-60 cells induced by phorbol diester

Treatment of the human promyelocytic leukemia cell line HL-60, with 12-o-tetradecanoylphorbol acetate (TPA) results in the differentiation into macrophage-like cell. A potent inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7), suppressed the proliferation of HL-60 cells and also inhibited TPA-induced cell differentiation of these cells. N-(2-guanidinoethyl)- 5-isoquinolinesulfonamide(HA-1004), a weaker analog of H-7, failed to inhibit this TPA-induced cell differentiation. H-7 also inhibited TPA-induced protein phosphorylation in these cells. Thus, protein kinase C-mediated phosphorylation may be involved in the process of TPA-induced HL-60 cell differentiation.

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells.

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.

Plasminogen activator activity in differentiating leukemia cells.

Plasminogen activator (PA) activity of human promyelocytic leukemia cell line HL-60 was assayed by following the conversion of plasminogen to plasmin and the plasmin-mediated hydrolysis of 14C-labeled globin. When HL-60 cells were induced to differentiate into macrophages by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cell-associated PA activity and secretion of PA into the conditioned medium increased profoundly. PA activity increased earlier and as a result of lower concentrations of TPA than the ability of the cells to adhere. Exposure to 10(-6)M dexamethasone did not prevent TPA-induced adherence and produced a slight inhibition of cellular PA activity. These findings imply that TPA-induced differentiation of HL-60 cells to macrophage-like cells is associated with induction of PA activity.