Programmed cell death-4 tumor suppressor protein contributes to retinoic acid-induced terminal granulocytic differentiation of human myeloid leukemia cells

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.     

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.     

Inhibition of mitochondrial function in HL60 cells is associated with an increased apoptosis and expression of CD14.

The myelomonocytic cell line HL60 can be induced by a variety of chemical agents to differentiation to either neutrophils or monocytes. Examination of gene expression, by differential display, in cells induced to monocytes with 1alpha,25-dihydroxyvitamin D(3) or neutrophils with all-trans retinoic acid (ATRA) identified a number of clones with altered patterns of expression over the period of differentiation. One of these clones was the mitochondrial gene NADH dehydrogenase subunit 4 (ND4) which showed a differential pattern of expression between the neutrophil and monocyte lineages. The potential of mitochondrial inhibitors to induce differentiation was investigated by treating the HL60 cells with either the NADH dehydrogenase inhibitor, Rotenone, the complex III inhibitor, Antimycin A, or the highly specific mitochondrial ATP-synthase inhibitor, Oligomycin. Although functional assays of differentiation did not produce any positive results, all the inhibitors resulted in a dramatic increase in CD14 expression at day 1, with CD38 markers not observed until day 3. The increased expression of CD14 was accompanied by a decrease in viability and all CD14 positive cells were also positive for Annexin V, a marker of apoptosis. These results suggest that inhibition of the components of the mitochondrial pathways may lead to the marking of some cells, via CD14, for cell death, whilst allowing commitment to differentiation to occur in the surviving population.     

Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type I cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type I cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

An RXR-Selective Analog Attenuates the RARα-Selective Analog-Induced Differentiation and Non-G1-Restricted Growth Arrest of NB4 Cells

NB4, a human acute promyelocytic leukemia cell line expressing the promyelocyte–retinoic acid receptor α (PML–RARα) hybrid protein was treated with RAR- and retinoid X receptor (RXR)-selective analogs to determine their effects on cell proliferation, retinoblastoma (RB) tumor-suppressor protein phosphorylation, and differentiation. An RAR- or just RARα-selective analog alone induced similar cell population growth arrest, cell cycle arrest without restriction to G₁, hypophosphorylation of RB, and myelomonocytic cell surface differentiation marker expression (CD11b). In addition, an RARα antagonist could inhibit the effects of the RARα agonist completely. The RARα-selective analog-elicited response was attenuated by simultaneous addition of various RXR-selective analogs. In contrast, each of the RXR-selective analogs was unable to induce any of the cellular responses analyzed. The growth arrest of NB4 cells is not G₁-restricted and occurs at all points in the cell cycle. Cells growth arrested by treatment with an RARα-selective analog show primarily hypophosphorylated RB. When these cells are sorted into G₁or S + G₂/M subpopulations by flow cytometry, hypophosphorylated RB protein was in G₁as well as S + G₂/M cells. This suggests that the hypophosphorylated RB protein may be mediating the growth arrest of NB4 cells at all points in the cell cycle. These results are consistent with an involvement of PML–RARα and/or RARα in the transduction of the retinoid signal in NB4 cells.     

Vav1 modulates protein expression during ATRA-induced maturation of APL-derived promyelocytes: a proteomic-based analysis

Overexpression of Vav1 promotes the overcoming of the differentiation blockade that characterizes acute promyelocytic leukemia cells. At variance, down-modulation of Vav1 prevents ATRA-induced maturation, and in particular, the inhibition of its tyrosine phosphorylation prevents the neutrophil differentiation-related changes of cell morphology. These findings allowed to identify Vav1 as a crucial protein in the ATRA-dependent differentiation of tumoral promyelocytes. By means of a proteomic approach, here we have investigated a possible role for Vav1 in modulating protein expression during ATRA treatment of tumoral promyelocytes. We have performed high-resolution 2-DE coupled with mass spectra analysis of HL-60 and NB4 promyelocytic cell lines induced to differentiate with ATRA when the amounts or the tyrosine phosphorylation of Vav1 were forcedly reduced. We have found that the down-regulation of Vav1 affects the expression level of a number of proteins, including cell cycle/apoptosis- and cytoskeleton-related proteins. In particular, the expression of 14-3-3epsilon, alpha-enolase, alpha-tubulin and splice isoform 2 of alpha3 proteasome subunit changed as a consequence of the down-modulation of Vav1 during the differentiation of both HL-60 and NB4 cell lines, suggesting that these proteins may constitute a common part of the ATRA-induced pathway during maturation of APL-derived promyelocytes. These results indicate an unprecedented role for Vav1 in the maturation of myeloid cells as a regulator of protein expression.     

Opposite regulation of vitamin D receptor by ATRA in AML cells susceptible and resistant to vitamin D-induced differentiation

Some leukemic cell lines can be driven to differentiate to monocyte-like cells by 1,25-dihydroxyvitamin D(3) (1,25D) and to granulocyte-like cells by all-trans retinoic acid (ATRA). Acute myloid leukemias (AMLs) are heterogeneous blood malignancies characterized by a block at various stages of hematopoietic differentiation and there are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML. Because of the multiplicity in the genetic lesions causing the disease, AMLs are particularly difficult to treat successfully. In particular, various AML cells to a variable degree respond to 1,25D-based differentiation and only one type of AML undergoes successfully ATRA-based differentiation therapy. In this paper we describe that AML cell line KG-1 is resistant to 1,25D-induced monocytic differentiation, while sensitive to ATRA-induced granulocytic differentiation. We show that KG-1 cells have very low level of VDR protein and that expression of VDR mRNA is upregulated by ATRA. We show for the first time that this regulation is cell context-specific, because in another AML cell line, HL60, VDR mRNA is downregulated by ATRA. ATRA-induced VDR protein in cytosol of KG-1 cells can be further activated by 1,25D to induce monocytic differentiation of these cells.     

ICAT as a potential enhancer of monocytic differentiation: implications from the comparative proteome analysis of the HL60 cell line stimulated by all‐trans retinoic acid and NSC67657

A novel sterol mesylate compound (NSC67657) was recently identified and reported by National Cancer Institute that could efficiently induce the differentiation of HL60 cells into monocytes in vitro and in vivo. The expression of many proteins would have been changed during the differentiation process, and some proteins may have played key roles in the differentiation of HL60 cell line induced by this drug. Therefore, we treated HL60 cells with NSC67657 and all‐trans retinoic acid (ATRA) to identify the differentially expressed proteins and determine their functions in cellular differentiation. Of the 45 differentially expressed protein spots investigated, 24 were either elevated or decreased in both the monocytic and granulocytic differentiating HL60 cells, 8 showed significant changes only when induced by NSC67657, and 13 showed significant changes only when induced by ATRA. After verification by RT‐PCR, Western blotting, and immunocytochemistry, only the protein ICAT was found to be elevated by NSC67657 treatment alone. Although the over‐expression of ICAT is not sufficient to induce the differentiation of HL60 cells into monocytes, it did increase the proportion of CD14+ cells in cells pretreated with NSC67657. Successful application of multiple techniques including two‐dimensional gel electrophoresis, matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry, Western blotting, and eukaryotic electroporation revealed that proteomic and molecular biological analyses provide valuable tools in drug development research. Copyright © 2009 John Wiley & Sons, Ltd.     

Retinoic acid-induced CD38 expression in HL-60 myeloblastic leukemia cells regulates cell differentiation or viability depending on expression levels

Retinoic acid-induced expression of the CD38 ectoenzyme receptor in HL-60 human myeloblastic leukemia cells is regulated by RARalpha and RXR, and enhanced or prevented cell differentiation depending on the level of expression per cell. RARalpha activation caused CD38 expression, as did RXR activation but not as effectively. Inhibition of MAPK signaling through MEK inhibition diminished the induced expression by both RARs and RXRs. Expression of CD38 enhanced retinoic acid-induced myeloid differentiation and G0 cell cycle arrest, but at higher expression levels, induced differentiation was blocked and retinoic acid induced a loss of cell viability instead. In the case of 1,25-dihydroxyvitamin D3, induced monocytic differentiation was also enhanced by CD38 and not enhanced by higher expression levels, but without induced loss of viability. Expression levels of CD38 thus regulated the cellular response to retinoic acid, either propelling cell differentiation or loss of viability. The cellular effects of CD38 thus depend on its expression level.     

Retinoic acid acylation (retinoylation) of a nuclear protein in the human acute myeloid leukemia cell line HL60

all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.     

Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system

Retinoic acid, which induces the differentiation of HL 60 cells to granulocytes, produces a cell alkalinization from pHi = 7.03 to pHi = 7.37. The half-maximum effect of retinoic acid is observed at 10 nM. The effect of retinoic acid on the pHi develops slowly, and it precedes the differentiation of the cells. A cell alkalinization is also observed after differentiation of the cells by dimethyl sulfoxide. It is not observed using etretinate, a synthetic retinoid that does not promote the differentiation of HL 60 cells. Two pHi regulating mechanisms coexist in HL 60 cells. The Na+/H+ exchange system is the major mechanism that allows HL 60 cells to recover from an intracellular acidosis. A second mechanism is a Na-HCO3 cotransport system. During differentiation of the cells by retinoic acid, a 2-fold increase in the activity of the Na+/H+ exchange system is observed, while the activity of the NaHCO3 cotransport remains constant. The properties of interaction of the Na+/H+ exchanger with internal H+, external Na+, and Li+ as well as with amiloride and its derivatives are defined. The Na+/H+ exchanger of HL 60 cells is characterized by unusually low affinities for alkali cations and a high affinity for amiloride and its derivatives. The pHi dependence of the exchanger is not modified after differentiation by retinoic acid. It is concluded that the mechanism of activation of the Na+/H+ exchanger by retinoic acid is distinct from the short-term effect produced by mitogens and phorbol esters which change the pHi dependence of the system.     

Proanthocyanidins from Barley Bran Potentiate Retinoic Acid-induced Granulocytic and Sodium Butyrate-induced Monocytic Differentiation of HL60 Cells

Polyphenol extract from barley bran (BPE) induced nitro blue tetrazolium (NBT) reducing activity and alpha-naphthyl butyrate esterase activity in HL60 human myeloid leukemia cells. Because BPE induced the biochemical markers of HL60 cell differentiation, we investigated the effects of proanthocyanidins isolated from BPE on the HL60 cell differentiation of HL60 cells. Prodelphinidin B-3, T1, T2, and T3 induced 26-40% NBT-positive cells and 22-32% alpha-naphthyl butyrate esterase-positive cells. Proanthocyanidins potentiated retinoic acid (all-trans-retinoic acid)-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells.     

Differentiation-linked expression of the plasminogen activator inhibitor type-2 gene in the human HL-60 promyelocytic cell line

HL-60 is a human promyelocytic cell line which was found to be capable of differentiating toward a macrophage-like or granulocyte-like phenotype. Histochemical analysis demonstrated that incubation of cells in the presence of phorbol myristate acetate (PMA) or 1,25-dihydroxyvitamin D3 induced varying degrees of monocytic differentiation, while incubation in the presence of retinoic acid (RA) or dimethyl sulfoxide (DMSO) induced granulocytic differentiation. The differentiation induced by PMA, RA, and to a lesser extent DMSO, was accompanied by the induction of plasminogen activator inhibitor expression. mRNA analysis of control and PMA-induced cultures revealed the induction of a 2-kb message in treated cells which hybridized with a PAI-2-specific oligonucleotide probe. This is consistent with the literature concerning the expression of PAI by macrophages and granulocytes. No hybridization was detected with a PAI-1 specific probe. Expression of PAI by cells of hematopoietic origin appears to be associated with differentiation or stimulation of committed cells. Furthermore, PAI-2 expression by HL-60 cells is not restricted to one specific hematopoietic lineage. Since other cells of hematopoietic origin such as platelets express PAI-1, future studies using pluripotential cell lines could provide information on the initial events of lineage commitment and gene expression.     

Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells

Activation of ERK1 and ERK2 protein kinases has been implicated in diverse cellular processes, including the control of cell proliferation and cell differentiation (Marshall [1995] Cell 80:179). In human myeloblastoid leukemia HL60 cells rapid (ca. 15 min) but transient activation of ERK1/2 has been reported following induction of macrophage/monocyte differentiation by phorbol esters, or by very high (10(-6) M) concentrations of 1,25-dihydroxyvitamin D(3) (1,25D3), while retinoic acid-induced granulocytic differentiation was accompanied by sustained activation of ERK1/2. We report here that monocytic differentiation of HL60 cells induced by moderate (10(-9) to 10(-7) M) concentrations of 1,25D3 could be divided into at least two stages. In the first phase, which lasts 24-48 h, the cells continued in the normal cell cycle while expressing markers of monocytic phenotype, such as CD14. In the next phase the onset of G1 cell cycle block became apparent and expression of CD11b was prominent, indicating a more mature myeloid phenotype. The first phase was characterized by high levels of ERKs activated by phosphorylation, and these decreased as the cells entered the second phase, while the levels of p27/Kip1 increased at that time. Serum-starved or PD98059-treated HL60 cells had reduced growth rate and slower differentiation, but the G1 block also coincided with decreased levels of activated ERK1/2. The data suggest that the MEK/ERK pathway maintains cell proliferation during 1,25D3-induced monocytic differentiation of HL60 cells, but that ERK1/2 activity becomes suppressed during the later stages of differentiation, and the consequent G1 block leads to "terminal" differentiation.