Two separate differentiation inducing proteins for human myeloid leukemia cells and their isolation from normal lymphocytes

Conditioned medium from mitogen stimulated normal peripheral blood lymphocytes (PBL) has been demonstrated to contain a maturation inducer activity mediating the differentiation of human myeloid leukemia cells to monocytes and macrophages. The maturation inducer activity was isolated by salt precipitation, Sepharose CL-6B ion exchange and affinity chromatographies and electrophoresis. Two separate activities with M.W. ranges of 52-56 and 32-35 kDa capable of mediating the terminal differentiation of leukemic HL-60 promyelocytes to monocytes and macrophages were detected. The higher molecular weight species was determined to be a 54 kDa single polypeptide and was found to be distinct from IL-3 and IL-6 by ELISA and differentiation blocking assay. The inducing activity of the 32-35 kDa material was largely neutralized after treatment with anti-IL-3, but not with other antibodies. Employing the immunofluorescent antibody technique, the 54 kDa protein was detected on the surface membranes of PBL. The proportions and number of maturation inducer bearing lymphocytes in patients with acute myelogenous leukemia (0.4% and 35/mm3, respectively) were significantly lower than that of healthy donors (7.9% and 178/mm3) The role of these physiological factors in leukemia cell differentiation is discussed.     

Presence of a functional vitamin D receptor does not correlate with vitamin D3 phenotypic effects in myeloid differentiation

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exhibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function, we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.     

A system for monocytic differentiation of leukemic cells HL 60 by a short exposure to 1,25-dihydroxycholecalciferol

The human promyelocytic cell line HL 60 can be induced to differentiate toward more mature myeloid or monocytic forms by a variety of agents. This process is thought to require several days of exposure to the inducer, thus making it difficult to identify the early cellular changes which are fundamental to the differentiation program, and to relate the induction to phases of the cell cycle. In order to study the kinetics of leukemic cell differentiation we have developed a system for the induction of rapid monocytic maturation in a subpopulation of HL 60 cells. The cells are exposed to 10(-7) M 1,25-dihydroxycholecalciferol for 4 hr in serum-free medium. Subsequent incubation in a complete medium results in cellular differentiation recognizable by several criteria (phagocytosis, nonspecific esterase reaction, adherence to substratum, cell morphology) beginning at 10 hr from the exposure to the inducer. Approximately 20 hr later 30-40% of the cells in culture show the differentiated phenotype and are capable of phagocytosis. The proportion of differentiated cells in culture decreases thereafter. This system has been utilized to study the expression of c-myc oncogene in relation to the kinetics of maturation, and it was found that the inhibition of the expression of this gene precedes the onset of phenotypic differentiation by approximately 8 hr, is transient, and is accompanied by a brief retardation of cell proliferation, which resumes the normal rate within 24 hr of the exposure to the inducer.     

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro: 4: Preculture proto-oncogene expression and the behaviour of myeloid leukemia cells in vitro

Studies were conducted to determine the relationship between the pretherapy characteristics of leukemia cells and their behaviour during culture in vitro. Leukemia cells which proliferated well in vitro also proliferated well in vivo. Cells which manifested myeloid or monocytic differentiation in vivo tended to manifest differentiation along these lines in vitro. Cells which manifested high levels of expression of c-fms, c-fes, or triose phosphate isomerase prior to culture were likely to differentiate in vitro, with high levels of c-fes expression being related to myeloid maturation. These observations suggest that differentiation at the molecular level prior to culture is a requisite for leukemia cell differentiation in vitro. The same may be true for differentiation in vivo under the influence of exogenously administered agents such as cytotoxic chemotherapy or recombinant growth factors.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Securinine, a myeloid differentiation agent with therapeutic potential for AML

As the defining feature of Acute Myeloid Leukemia (AML) is a maturation arrest, a highly desirable therapeutic strategy is to induce leukemic cell maturation. This therapeutic strategy has the potential of avoiding the significant side effects that occur with the traditional AML therapeutics. We identified a natural compound securinine, as a leukemia differentiation-inducing agent. Securinine is a plant-derived alkaloid that has previously been used clinically as a therapeutic for primarily neurological related diseases. Securinine induces monocytic differentiation of a wide range of myeloid leukemia cell lines as well as primary leukemic patient samples. Securinine's clinical potential for AML can be seen from its ability to induce significant growth arrest in cell lines and patient samples as well as its activity in significantly impairing the growth of AML tumors in nude mice. In addition, securinine can synergize with currently employed agents such as ATRA and decitabine to induce differentiation. This study has revealed securinine induces differentiation through the activation of DNA damage signaling. Securinine is a promising new monocytic differentiation inducing agent for AML that has seen previous clinical use for non-related disorders.     

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.     

Impaired nuclear localization of vitamin D receptor in leukemia cells resistant to calcitriol-induced differentiation

Calcitriol, the hormonal form of vitamin D₃, induces differentiation of monocytic leukemia cell lines in vitro, without inducing cytotoxicity of the cells. Besides this broad in vitro activity, a clinical implementation of calcitriol, or its analogs, as agents for differentiation therapy has been unsuccessful until now. A better understanding of cellular activities of calcitriol necessary for completion of cell differentiation program could help find better solutions for differentiation therapy of myeloid leukemias. In this paper we describe work carried on subline of acute monocytic leukemia, THP-1 resistant to calcitriol induced differentiation. This resistance correlates with impaired nuclear localization of vitamin D receptor, but not with its total expression in the cells. It also correlates with the resistance to calcitriol-induced growth inhibition, and to phorbol myristate acetate (PMA)-induced cell differentiation.     

Leukemic cell maturation: variability of the myeloid leukemic cell phenotype

Leukemia is characterized by a proliferation of cells that exhibit an arrest in the normal differentiation sequence. The HL-60 promyelocytic leukemia is a useful model of the phenomenon of maturation arrest, particularly as modulated by inducers that partially restore myeloid differentiation. In this investigation, we report the development of two sublines of HL-60 and the acquisition of two others that are clonal variants of the parent cell line. Each of the sublines demonstrates an altered pattern of differentiation and resistance to one or more of the drugs that serves as an inducer of the parent line. The two cell lines developed in this study, HL-60S and HL-60I, are resistant to arabinosylcytocine (ARA-c); HL-60I is also resistant to PMA. A study of the phenotype as expressed by the granule-associated cytoplasmic enzymes revealed that each subline had a slightly different pattern of maturation in response to dimethylsulfoxide (DMSO) or ARA-c as inducers. The proliferative rate of all sublines was similar. These data demonstrate that the maturation arrest observed in this model is in part reversible. The maturation arrest observed in myeloid leukemias is due to a reversible block secondary to altered proliferative activity.     

Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor

Human macrophage differentiation inducing factor (DIF) can induce differentiation of human myeloid leukemic cells into macrophage-like cells in vitro. A procedure is described for purification of DIF from serum-free human monocytic leukemia THP-1 cell-conditioned medium. The procedure included concentration of a conditioned medium by ultrafiltration, lentil lectin-Sepharose affinity chromatography, and fast protein liquid chromatography using Mono S and Mono Q. DIF-A of pI 9.0 and DIF-B of pI 8.8 were obtained after a final purification with a Mono Q column, and both DIF gave a single peak with a molecular weight of approximately 51,000 determined by gel chromatography. NH2-terminal amino acid analysis of DIF-A showed a noticeable homology with murine leukemia inhibitory factor. Human DIF-A was found to induce maturation of human and murine leukemic cells into both macrophage-like cells with nitro blue tetrazolium reducing activity and phagocytic cells, but was found to suppress proliferation of these leukemic cells.     

Modulation of c-myc expression in the HL-60 cell line

A decrease in the expression of the myc proto-oncogene of HL-60 cells has been reported as an accompaniment of myeloid differentiation induced by either dimethylsulfoxide or retinoic acid. We report herein that several inhibitors of poly(ADP-ribose)-polymerase induced myeloid differentiation in HL-60 cultures. Studies on the expression of the c-myc gene in total cell RNA populations indicate that expression of this gene is inversely correlated with the state of differentiation, either myeloid or monocytic, of the cultured cells independent of the inducer and the rate of cell proliferation.     

Inducers of leukemic cell differentiation cause down-regulation of RB gene expression.

Expression of the retinoblastoma (RB) tumor suppressor gene during cell differentiation induced by dimethyl sulfoxide or sodium butyrate was studied in HL-60 human promyelocytic leukemia cells. As cells progressed through the cell cycle, the amount of RB protein per cell increased with homeostasis maintained, so that the amount of RB protein relative to the total cell mass remained almost constant. Dimethyl sulfoxide was used to induce these promyelocytic leukemia cells to undergo terminal differentiation into mature myeloid cells. There was an early reduction in the RB protein expressed per cell. The reduction in expression was similar for cells in all cell cycle phases. There was also progressively reduced expression at later times as cells terminally differentiated. This was compared to the case in which sodium butyrate was used to induce the differentiation of HL-60 cells into mature monocytic cells. An early reduction in RB protein expression per cell also occurred. It occurred for cells in all cell cycle phases as well. Thus, the induced differentiation of HL-60 cells along either the myeloid or the monocytic differentiation lineage involves an early reduction in RB expression, which is common to both pathways. The reduction anteceded proliferative arrest or differentiation. In both cases, the final, resulting G0-differentiated cells had less RB protein per cell than the proliferating, immature, leukemic precursor cells.     

Ganglioside GM3 as a modulator of differentiation of mouse myeloid leukemia cells (M1-T22)

The effects of exogenously added glycosphingolipids on the differentiation of mouse myeloid leukemia cells (M1-T22) have been studied. Eight gangliosides and ten neutral glycosphingolipids were tested in terms of their induction of phagocytic activities on the leukemia cells. N-Acetyl-neuraminosyllactosylceramide (NAc-GM3) was the most effective glycolipid for inducing the activity. By the addition of 25 micrograms/ml of NAc-GM3, about 70 percent of the cells acquired phagocytic activity within 20 h incubation. GM1a showed about half the activity of the GM3. In the case of the neutral glycosphingolipids, lactosylceramide (CDH) and globotriaosylceramide (CTH) showed significant effects on the induction of phagocytic activity. Preincubation of the cells with the NAc-GM3 enhanced the effect of dexamethasone as a differentiation inducer on M1-T22 cells. When a human promyelocytic leukemia cell line, HL-60, was preincubated with the NAc-GM3 ganglioside, induction of the phagocytic activity, together with inhibition of the cell growth by phorbol ester (TPA), were markedly enhanced. From these observations, the NAc-GM3 ganglioside seems to act as a modulator of differentiation of mouse myeloid leukemia cells and also of HL-60 cells.     

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.     

Control of HL-60 myeloid differentiation. Evidence of uncoupled growth and differentiation control, S-phase specificity, and two-step regulation

Myeloid differentiation of HL-60 human promyelocytic leukemia cells was studied during DMSO-induced differentiation. G 1/0-specific growth arrest could occur without the usual associated subsequent phenotypic differentiation into mature myeloid cells, suggesting that growth arrest and phenotypic differentiation are separately regulated. In the course of differentiating, the cells achieved a semi-stable intermediate state where they had a labile, pre-commitment memory of exposure to inducer, but were not yet committed to differentiation. This state was associated with a nuclear structural change previously found to be associated with the precommitment memory state. The process of differentiation could thus be resolved into two steps, early events up through development of pre-commitment memory and late events subsequents to pre-commitment memory. The kinetics of terminal cell differentiation indicated that the cellular regulatory event initiating a program of differentiation in response to inducer was S phase-specific. A comparison of the present results for DSMO to previous results for retinoic acid (RA)-induced HL-60 myeloid differentiation showed that the two inducers effect different cellular pathways for differentiation of HL-60 cells to mature myeloid cells, but with certain common features including the above S-phase specificity and pre-commitment memory.     

Myeloid cell differentiation in normal bone marrow and acute myeloid leukemia assessed by multi-dimensional flow cytometry.

A detailed analysis of normal myeloid differentiation was performed using mutlidimensional flow cytometry. Based on two light scattering and three color immunofluorescence signals, the normal maturation pathways of both the monocyte and neutrophil lineages could be elucidated. Gradual changes of light scattering properties and cell surface antigen expression defined the pathways of each of the lineages. The consistency of the location of these lineage specific pathways found in normal individuals provided the basis for the discrimination between normal and leukemic cells in acute myeloid leukemia (ANLL). The position of leukemic cells in patients with ANLL in a five-dimensional space was compared with the position of the maturation tracks in normal individuals. The expression of normal antigens on leukemic cells provided the tools to: (1) distinguish normal from clonal populations of leukemic cells in all 15 patients; (2) detect a lineage predominance, either monocytic or neutrophilic, in all 15 patients; (3) detect maturation heterogeneity in all 15 patients. Although maturation pathways of the monocytic and the neutrophilic lineages were analogous to the normal patterns they were distinct in several ways. The expression of normal antigens on leukemic cells may provide the tools to: (1) obtain a new frame-work for classification of leukemia based on the ability to quantify the aberrant antigen expression and to define a 'distance from normal' based on the characteristics studied (the maturation heterogeneity of the leukemic cells also can be correlated with the clinical outcome of the patients); (2) detect minimal residual disease using the difference in locations of the leukemic cells in the multidimensional space from the normal maturation pathways (3) monitor relapse and changes in phenotypes which may accompany chemotherapy, suggesting the appearance of variant or new clones.     

Induction of differentiation of human leukemia cells by combinations of COX inhibitors and 1, 25-dihydroxyvitamin D3 involves Raf1 but not Erk 1/2 signaling

Differentiation therapy of cancer is being explored as a potential modality for treatment of myeloid leukemia, and derivatives of vitamin D are gaining prominence as agents for this form of therapy. Cyclooxygenase (COX) inhibitors have been reported to enhance 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation of promyeloblastic HL60 cells, but the mechanisms of this effect are not fully elucidated, and whether this potentiation can occur in other types of myeloid leukemia is not known. We found that combination treatment with 1,25D and non-specific COX inhibitors acetyl salicylic acid (ASA) or indomethacin can robustly potentiate differentiation of other types of human leukemia cells, i.e. U937, THP-1, and that ASA +/- 1,25D is effective in primary AML cultures. Increased cell differentiation is paralleled by arrest of the cells in the G1 phase of the cell cycle, and by increased phosphorylation of Raf1 and p90RSK1 proteins. However, there is no evidence that this increase in phosphorylation of Raf1 is transmitted through the ERK module of the MAPK signaling cascade. Transfection of small interfering (si) RNA to Raf1 decreased differentiation of U937 cells induced by a combination of ASA or indomethacin with 1,25D. However, phosphorylation levels of ERK1/2, though not of p90RSK, were increased when P-Raf1 levels were decreased by the siRNA, suggesting that in this system the ERK module does not function in the conventional manner. Identification of the strong antiproliferative activity of ASA/1,25D combinations associated with monocytic differentiation has implications for cancer chemoprevention in individuals who have a predisposition to myeloid leukemia.