Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.     

Sphingosine kinase 1 is involved in dibutyryl cyclic AMP-induced granulocytic differentiation through the upregulation of extracellular signal-regulated kinase, but not p38 MAP kinase, in HL60 cells

The role of sphingosine kinase (SPHK) in the dibutyryl cyclic AMP (dbcAMP)-induced granulocytic differentiation of HL60 cells was investigated. During differentiation, SPHK activity was increased, as were mRNA and protein levels of SPHK1, but not of SPHK2. Pretreatment of HL60 cells with N,N-dimethylsphingosine (DMS), a potent SPHK inhibitor, completely blocked dbcAMP-induced differentiation. The phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK was also increased during dbcAMP-induced differentiation. Pretreatment of HL60 cells with the MEK inhibitor, U0126, but not the p38 MAPK inhibitor, SB203580, completely suppressed dbcAMP-induced ERK1/2 activation and granulocytic differentiation, but did not affect the increase in SPHK activity. DMS inhibited dbcAMP-induced ERK1/2 activation, but had little effect on p38 MAPK activation. DMS had no effect on the dbcAMP-induced membrane translocation of protein kinase C (PKC) isozymes, and PKC inhibitors had no significant effect on ERK activation. The overexpression of wild-type SPHK1, but not dominant negative SPHK1, resulted in high basal levels of ERK1/2 phosphorylation and stimulated granulocytic differentiation in HL60 cells. These data show that SPHK1 participates in the dbcAMP-induced differentiation of HL60 cells by activating the MEK/ERK pathway.     

The rationale for deltanoids in therapy for myeloid leukemia: role of KSR-MAPK-C/EBP pathway

The evidence for the promising potential for derivatives of Vitamin D (deltanoids) in the treatment of myeloid leukemias is increasing, but currently is not matched by the understanding of the precise mechanisms by which these anti-neoplastic effects are achieved. Unlike solid tumors in which growth retardation by deltanoids appears to result from inhibition of cell proliferation and the promotion of cell death by apoptosis, control of myeloid leukemia proliferation by deltanoids results from the induction of differentiation of the immature myelo-monocytic cells towards functional monocytic cells. We present here the accumulating evidence that a pathway that is initiated by deltanoid activation of Vitamin D receptor (VDR) and leads to monocytic differentiation of human myeloblastic HL60 cells, includes the MEK-ERK and JNK mitogen-activated protein kinases (MAPKs), their positive and negative regulators and a downstream effector C/EBPbeta. As in other cells, the abundance of VDR protein increases shortly after an exposure of HL60 cells to 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2) D(3)). Other early events include a parallel upregulation of kinase suppressor of Ras (KSR-1) and the activation of the ERK MAPK pathway and data suggest that KSR-1 acts to amplify the signal provided by low concentrations of 1alpha,25(OH)(2) D(3). Maintenance of monocytic differentiation may be enhanced by JNK, but diminished by p38, MAPK signaling. Downstream, one of the targets of these pathways is C/EBPbeta, which can directly interact with the promoter for CD14, a gene characteristically expressed in monocytes. Importantly, in freshly obtained acute myeloid leukemia (AML)-M2 cells exposed to PRI-2191, a novel deltanoid with a modified side chain, upregulation of C/EBPbeta paralleled the induction of monocytic differentiation. These data provide a basis for the hypothesis that deltanoid-induced upregulation of C/EBPbeta bypasses the block to granulocytic differentiation in myeloid leukemia cells by redirecting the cells to monocytic differentiation.     

Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.

The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.     

Expression of p21WAF1 is dependent on the activation of ERK during vitamin E-succinate-induced monocytic differentiation

Vitamin E-succinate (VES) induced monocvtic differentiation of HL-60 human leukemia cells. Treatment with VES increased the nitroblue tetrazolium reduction activity, and the expression of monocyte specific cell surface antigen, CD14 and c-fms. During the monocytic differentiation of HL-60 cells that were induced by VES, the phosphorylation of extracellular signal-regulated protein kinase (ERK) was increased by 12 h and then gradually decreased to a level that was similar to that of the control. However, the phosphorylation levels of p38 and JNK, as well as the expression levels of ERK, p38, and JNK, were unchanged by the VES treatment. Treatment with VES also induced hypophosphorylation of the retinoblastoma protein and an increase of the p21WAF1 protein level. VES-induced ERK phosphorylation was abolished by the ERK inhibitor, PD98059, which resulted in a remarkable prevention of VES-induced monocytic differentiation. Inhibition of the ERK activity by PD98059 also diminished the VES-induced p21WAF1 protein expression, but did not change the phosphorylation state of the retinoblastoma protein. Collectively, these data suggest that the ERK signaling pathway mediates the up-regulation of the p21xWAF1 expression that is induced by VES, which is required for monocytic differentiation of HL 60 cells.     

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.     

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).     

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.     

Effects of fluorescent derivatives of TPA on HL60 cells: dissociation between the differentiation-induced and protein kinase C activity

The four fluorescent derivatives of TPA--dansylaza-TPA, NBDaza-TPA, and (N)- and (P)-dansylamino-TPA--were synthesized and examined for their ability to induce differentiation in human promyelocytic leukemic HL60 cells. At a concentration of 20 nM, all the derivatives inhibited proliferation and induced 60-80% of the cells to differentiate into macrophage-like cells. Removal of dansylaza-TPA from the medium after 5 h did not arrest adherence or the expression of nonspecific esterase activity. However, upon removal of any of the other three compounds after 5 h, HL60 cells became nonadherent and expressed low nonspecific esterase activity after additional culture. To investigate the relationship between protein kinase C (PKC) activation and cell maturation, PKC activity and translocation were measured after 0.5, 5, 24, and 48 h of treatment with each compound. Cells induced to differentiate by dansylaza-TPA or (N)- or (P)-dansylamino-TPA exhibited enhanced PKC activity, 50-80% of which was located in the particulate fraction. In cells that differentiated with NBDaza-TPA, 65-70% of PKC activity remained in the cytosol. After removal of the TPA derivatives, all cells exhibited PKC activity in the cytosol. These results indicate that the fluorescent derivatives are as potent as TPA in inducing HL60 cell differentiation. However, in the case of NBDaza-TPA and (N)- or (P)-dansylamino-TPA, their continuous presence in the culture medium was required for the recruitment of cells to differentiate. Consequently, it is suggested that activation and translocation of PKC are among the early biochemical events that trigger HL60 cell differentiation. Nevertheless, these two events alone are not sufficient to induce differentiation.     

Cooperative interactions of protein kinase C and cAMP-dependent protein kinase systems in human promyelocytic leukemia HL60 cells

Interactions of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) systems were investigated in HL60 cells. It was found that the differentiating effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) were potentiated by dibutyryl cAMP (dbcAMP) or prostaglandin E2 (PGE2). In addition, dbcAMP or PGE2 inhibited TPA-induced binding of PKC to plasma membrane, leading to decreased protein phosphorylation, and promoted subsequent redistribution of enzyme to the nuclear membrane region. The findings are consistent with the hypothesis that PKC and PKA systems regulate cooperatively the phenotypical differentiation of leukemic cells.     

Protein Kinase C-δ Associates with Vimentin Intermediate Filaments in Differentiated HL60 Cells

The subcellular localization of protein kinase C (PKC)-δ was determined in HL60 cells differentiated toward monocytes/macrophages by treatment with TPA. PKC-δ was detected in the nucleus and cytoplasm of differentiated HL60 cells and, more specifically, associated with structures resembling intermediate filaments. Indirect immunostaining revealed that PKC-δ colocalized with vimentin in the cytosol and perinuclear region of these cells. Immunoprecipitation studies showed that PKC-δ was in an active (autophosphorylated) state in differentiated HL60 cells and that vimentin immunoprecipitated from these cells was also phosphorylated. Treatment of HL60 cells with the PKC-specific inhibitor chelerythrine decreased the phosphorylation of vimentin. These data suggest that vimentin is a substrate for PKC-δ and that this PKC isoenzyme may play a specific role in the regulation of shape change and cell adhesion during HL60 differentiation.     

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.     

Expression of the fgr protooncogene product as a function of myelomonocytic cell maturation

The fgr protooncogene is a member of the src family of protein tyrosine kinases. Recent studies have shown that normal myelomonocytic cells and tissue macrophages are the major sites of fgr mRNA expression. In the present study, we have identified the fgr protooncogene protein product in HL60 cells and have examined its expression as a function of HL60 cell maturation. Whether induced toward monocytic or granulocytic lineages, p55c-fgr accumulated in HL60 cells during maturation. In differentiated cells, the protein was active as a protein tyrosine kinase and was localized to peripheral cell membranes. Demonstration that a myristyl group was covalently bound to the protein probably accounted for its subcellular distribution. These findings establish developmental regulation of p55c-fgr in a lineage that represents its natural site of expression.