Inhibition of phorbol-ester-induced adhesion of differentiating human myeloid leukemic cells by pentamidine-isethionate

Abstract. Human myeloid leukemia cells can be induced to differentiate into macrophage-like cells by various phorbol esters, particularly 12-O-tetradecanoyl-phorbol-14-acetate (TPA). In this study, the effect of several known protease inhibitors on TPA-induced differentiation of human acute promyelocytic leukemia cells (line HL-60) was tested. Among the tested compounds, only pentamidine-isethionate (PI), an inhibitor of trypsin-like enzymes, prevented one early marker of differentiation, e.g. cell adherence to plastic and glass surfaces. However, PI failed to affect other markers of differentiation and did not inhibit readherence of scraped and resuspended TPA-treated cells. Exposure to TPA resulted in a decrease in the cellular alkaline proteolytic activity and an increase in the acid proteolytic activity. PI further inhibited the residual activity of the alkaline protease in the 36,000 g pellet fraction of the TPA-treated cells, but did not reduce this activity in control cells. The present results indicate, on the basis of the differential effects of PI, that the emergence of differentiation markers in HL-60 cells following exposure to TPA is independent of the induction of adherence.     

Sialic acid lyase in human promyelocytic leukemic cells (HL-60) during phorbol-ester-induced differentiation

There is a marked increase in the activity of sialic acid lyase (N-acetylneuraminate lyase; EC 4.1.3.3; also known as sialic acid aldolase) in HL-60 cells induced to differentiate into macrophages by the phorbol ester, tetradecanoylphorbol 12-myristate 13 acetate (TPA). Exposure of HL-60 cells to retinoic acid, butyric acid or dimethyl sulfoxide has little or no effect. The level of the enzyme remains unaltered in HL-60 cells grown in the presence of an inactive analog of TPA, nor does it change in variants of HL-60 cells resistant to TPA.     

Induction of differentiation in human myeloid leukemic cells by proteolytic enzymes

Exogenous serine proteases were found to induce differentiation in human myeloid leukemic cells from either in vitro established long-term cell lines or in primary cultures of cells derived directly from patients with acute myeloid leukemia. Exposure of the human promyelocytic cell line HL-60 to trypsin, chymotrypsin, or elastase induced the appearance, within 3-6 days, of neutrophilic granulocytes defined by their morphology, their ability to reduce nitroblue tetrazolium, and their efficient phagocytosis of latex particles. Upon further incubation monocyte-like cells appeared. While these cells developed into fully mature macrophages other types of cells disappeared and on day 12 the culture consisted of a pure macrophage population. The inducing effect could be observed when the enzyme was presented alone, whereas a synergistic effect was noted when the protease was added in the presence of subthreshold concentrations of chemicals known to induce differentiation in this cell line such as dimethylsulfoxide, retinoic acid, butyric acid, or hexamethylene bisacetamide. Optimal induction of differentiation by trypsin required a 48 hr continuous exposure to the enzyme. When the protease was removed earlier no appreciable differentiation was noticed. The protease-induced differentiation involved a direct interaction with the cells and was not due to a proteolytic cleavage of a serum component because it could be obtained in serum-free cultures. The enzymatic activity of the protease was needed for its effect on cell maturation: Addition of protease inhibitors such as soybean-trypsin inhibitor or trasylol completely blocked differentiation induced by the proteases but had no effect on differentiation induced by the other inducers. It is still to be determined whether a proteolytic process is a general molecular event in cell differentiation or induction by chemicals involves a mechanism different from that initiated by exogenous proteases.     

GALECTIN-3 expression in differentiating human myeloid cells

Galectin-3 is an endogenous mammalian carbohydrate-binding protein with affinity for ABH group carbohydrate epitopes and polylactosamine glycans present on cell surface and extracellular matrix glycoproteins. It has been shown to play a role in cell differentiation, morphogenesis, adhesion and cell proliferation regulation. Progenitor cell proliferation in bone marrow depends on stem cell factors including those modulating their adhesion to the bone marrow stroma. The present study shows that the 32 kD galectin-3 is developmentally expressed in human myeloid cells and is strongly upregulated on the cell surface of late mature myeloid cells. Despite the fact that the expression of the galectin-3 is very low in CD34+ early myeloid cell, a 70 kD protein is found by Western blotting using antibodies specific to galectin-3, exclusively in those cells. Finally, exogenous human recombinant galectin-3 binds strongly to CD34+ early myeloid cells and emphasizes granulocyte-colony stimulating factor (G-CSF) driven proliferation in vitro.     

Bezafibrate as differentiating factor of human myeloid leukemia cells.

Bezafibrate belongs to the class of fibric acid derivatives usually used as antihyperlipidemia agents. From the biochemical point of view, these drugs show intriguing properties which leads one to think they may promote a differentiation process in tumour cells. This new pharmacological activity of fibrates could partially depend on the induction of an oxidative stress. To test this hypothesis, the effect of bezafibrate, as well as of clofibric acid and gemfibrozil, on growth, functional and cytochemical characteristics of human leukaemia-derived cell lines HL-60, U-937 and K-562 has been studied in some details. The results show that bezafibrate, gemfibrozil and clofibric acid, do induce differentiation in human myeloid leukaemia cell lines as indicated by several differentiation markers. Moreover fibrates, in dose dependent manner, significantly alter the cell cycle distributions, mainly leading to G0/G1 phase increment and G2/M phase reduction. The differentiating activity of fibrates could have significant implications both for the pharmacotoxicological profile of this class of compounds and for the pathophysiology of neoplastic disease.     

Targeting of CD44 eradicates human acute myeloid leukemic stem cells

The long-term survival of patients with acute myeloid leukemia (AML) is dismally poor. A permanent cure of AML requires elimination of leukemic stem cells (LSCs), the only cell type capable of initiating and maintaining the leukemic clonal hierarchy. We report a therapeutic approach using an activating monoclonal antibody directed to the adhesion molecule CD44. In vivo administration of this antibody to nonobese diabetic-severe combined immune-deficient mice transplanted with human AML markedly reduced leukemic repopulation. Absence of leukemia in serially transplanted mice demonstrated that AML LSCs are directly targeted. Mechanisms underlying this eradication included interference with transport to stem cell-supportive microenvironmental niches and alteration of AML-LSC fate, identifying CD44 as a key regulator of AML LSCs. The finding that AML LSCs require interaction with a niche to maintain their stem cell properties provides a therapeutic strategy to eliminate quiescent AML LSCs and may be applicable to other types of cancer stem cells.     

Elevation of a potassium current in differentiating human leukemic (HL-60) cells

Human promyelocytic leukemia (HL-60) cells display a novel voltage-dependent outward current under voltage clamp. This current is present at low levels in the proliferative state and in granulocytes derived from HL-60 cells which were induced to differentiate with retinoic acid. It is elevated in macrophages derived from HL-60 cells after exposure to phorbol-12-myristate-13-acetate (PMA). The current is carried primarily by K+, is blocked by Cs+ and by increased intracellular concentrations of Cl-. From a holding potential of -80 mV, significant activation required depolarization to +20 mV membrane potential. Activation was not influenced by intracellular Ca2+ (1-2 X 10(-6) M). These properties appear to differ significantly from the Ca2+-activated K+ channel and the delayed rectifier. The increase of this voltage-activated current in differentiation toward the macrophage, but not the granulocyte, suggests that this current is correlated specifically with macrophage differentiation.     

Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection.

An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.     

Inhibition of DNA polymerase alpha from leukemic and normal human cells by partially thiolated human deoxyribonucleic acids

In continuing search for exploitable biochemical differences between cancer and normal cells at the level of DNA replication, leukemic and "normal" hematopoietic cells from four different, established human cell lines were grown in culture flasks, and both the DNA and the DNA polymerase alpha were isolated in each case from the harvested (5-10 g wet weight) cell pellets. The four selected cell lines included a "normal" lymphoblastoid B-cell line (RPMI-1788), a pre-B cell (NALM-6) and a T-cell (MOLT-4) acute lymphoblastic leukemias, and a promyelocytic leukemia (HL-60). The DNA polymerase alpha enzyme of the two B-cell lines (both the leukemic and the "normal") showed the usual sensitivity toward inhibition by aphidicolin, while those from the two other leukemic cell lines were remarkably resistant to the antibiotic. Partially thiolated polycytidylic acid (MPC) strongly inhibited only the DNA polymerase alpha of the "normal" cell line, whereas the corresponding enzymes of all three leukemic cell lines were relatively insensitive to MPC. In contrast, the partially thiolated DNAs derived from the leukemic cell lines more strongly inhibited the DNA polymerase alphas of the leukemic cell lines than that of the "normal" cell line. These results indicate the existence of some structural differences between the DNA polymerase alpha enzymes (as well as between the DNAs) of human cells of different lineage and, particularly, of leukemic vs. "normal" character; such differences could be exploited in the design of selective antitemplates for chemotherapy.     

Induction by recombinant human granulocyte colony-stimulating factor of differentiation of mouse myeloid leukemic M1 cells

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on induction of differentiation of mouse myeloid leukemic M1 cells was examined. Purified G-CSF caused dose-dependent induction of phagocytic activity and lysozyme activity in M1 cells. Its half-maximally effective concentration was 10 ng/ml. On treatment of M1 cells with G-CSF (100 ng/ml) for 4 days, 30-50% of the cells differentiated morphologically into macrophage cells; 30-40% of the cells were blast cells and 20-30% of the cells were forms intermediate between blastic cells and mature macrophages.     

Differential effect of recombinant human leukocyte interferon on human leukemic and normal myeloid progenitor cells

Two separate clones of recombinant leukocyte interferon (IFLrA and IFLrD) inhibited the cloning efficiency in soft agar of the human leukemia cell lines HL-60 and KG-1. Inhibition of the growth in agar of normal human bone marrow myeloid progenitors was also observed, but this required considerably higher concentrations. IFLrA and IFLrD also inhibited the growth of HL-60 and KG-1 cells in suspension culture. This antiproliferative effect did not appear to be due to induction of maturation of these cells. Our results suggest that homogeneous preparations of interferon may be capable of exerting selective antiproliferative effects on malignant human myeloid progenitor cells in comparison to their normal counterparts.     

Protein synthesis in differentiating normal and leukemic erythroid cells

Erythroleukemic cells transformed by the AEV or S13 strains of avian erythroblastosis virus differentiate in vitro either spontaneously (S13) or following a temperature induction (temperature-sensitive mutants of AEV). To study differentiation in these cells at the molecular level, homogeneous fractions of maturing cells at discrete stages of differentiation were prepared by Percoll density-gradient centrifugation. This method was also used for the fractionation of differentiating normal erythroid cells separated from total bone marrow by an immunological "panning" technique. Total protein synthesis in these cells was then analyzed by two-dimensional gel electrophoresis. The expression of several proteins was altered in differentiating leukemic cells but not in their normal counterparts. However, in general, the normal and leukemic cells from comparable stages of maturity showed closely related protein synthetic patterns. Similar early and late changes in the synthesis of a number of polypeptides were detected during maturation from early erythroid precursors to terminally differentiated erythrocytes. Further, the leukemic as well as the normal cells appeared to undergo a major switch in total protein synthetic pattern during late differentiation. These results demonstrate that normal and erythroleukemic cells differentiate along similar pathways.     

Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

Recombinant human leukocyte interferon (IFN-alpha A) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-alpha A is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-alpha A and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represents a novel means of reducing resistant leukemic cell populations.     

Induction of differentiation of cultured mouse myeloid leukemic cells by arginase.

Mouse myeloid leukemic cells (Ml) could be induced by a factor in ascitic fluid to phagocytize, migrate in agar, and change into forms that were morphologically similar to macrophages and granulocytes. Arginase also induced these differentiation-associated properties of the cells. The Ml cells did not differentiate in culture medium containing arginine, but they differentiated into macrophages and granulocytes during culture in arginine-deficient culture medium. Therefore, the effect of arginase may be attribute to arginase-mediated arginine depletion.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.