Synergistic effects of clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate in combination with all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and sodium butyrate on differentiation in HL-60 cells.

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1alpha,25-dihydroxyvitamin D3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 microM) increased the number of nitroblue tetrazolium (NBT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 microM), VD3 (1 nM), or NaB (100 microM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P < or = 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.     

Up-regulation of the BTG2 gene in TPA- or RA-treated HL-60 cell lines

The key pathogenesis of leukemia is the defection of the differentiation processes of hematopoietic stem cells. There are five APRO (anti-proliferative) genes, BTG1, BTG2, BTG3, TOB and TOB2, and it was reported that certain APRO genes are associated with cell differentiation. However, it is still unknown whether APRO genes are related with the differentiation process of blood cells. In this study, we investigated the expression of APRO genes in 12-O-tetra-decanoylphorbol-13-acetate (TPA) or retinoic acid (RA)-treated HL-60 cell lines. Our data show that the expression of the BTG2 gene was increased in 32 nM TPA-treated and 1 microM RA-treated HL-60 cells, but the expression of the BTG1 and BTG3 genes was not increased. The expression of BTG2 in TPA- or RA-treated HL-60 cells was also increased even in the presence of cyclohexamide, which is an inhibitor of translation, implying that the increased expression of BTG2 mRNA did not require the new synthesis of another protein. Notably, the up-regulation of BTG2 in TPA- or RA-treated HL-60 cells was observed prior to the increased expression of p21. Our data show that PKC pathways are uninvolved with the up-regulation of BTG2 in TPA- or RA-treated HL-60 cells. Thus, the up-regulation of BTG2 genes may be involved in the differentiation process of HL-60 cells after TPA or RA treatment. Furthermore, this event occurred prior to p21 expression, implying that the BTG2 expression plays a role at a very early point during the differentiation processes of hematopoietic cells.     

The activation of PI 3-K and PKC zeta in PMA-induced differentiation of HL-60 cells

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol3P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 microM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC zeta is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC zeta during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC zeta activation. The activity of PI 3-K is essential for PKC zeta activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC zeta during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC zeta in the process of PMA-induced monocytic differentiation.     

Inhibition by palmitoylcarnitine of adhesion and morphological changes in HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

Effects of DL-palmitoylcarnitine (PC), an inhibitor of calciumactivated, phospholipid-dependent protein kinase (protein kinase C), on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell differentiation were investigated in human promyelocytic leukemia cells (HL-60). TPA caused HL-60 cell adhesion concomitant with morphological changes, and an increase in acid phosphatase activity. The median effective concentration was 1 nM, which corresponded well to the dissociation constant of [3H]TPA binding to the cell extract. [3H]TPA binding to the cell extract was saturable and reversible. The maximal number of [3H]TPA-binding sites was 1.5 pmol/mg protein and a Hill coefficient was unity, indicating noncooperative interactions. PC, but neither palmitic acid nor DL-carnitine, inhibited the TPA-induced cell adhesion and morphological changes with the median inhibitory concentration of 1 microM, whereas a TPA-induced increase in acid phosphatase activity was not affected by 3 microM PC. Addition of PC 1 or 2 days after the addition of TPA was also effective in inhibiting the cell adhesion. Among various acylcarnitines, PC had the largest effect. [3H]TPA binding to the cell extract was not inhibited by PC at the concentration which was effective in inhibiting the TPA-induced cell adhesion. These results indicate that protein kinase C possibly mediates HL-60 cell differentiation induced by TPA.     

Effects of uridine on the growth and differentiation of HL-60 leukemia cells

HL-60 leukemia cells, induced to differentiate, activate a Na(+)-dependent nucleoside transport system, concomitant with a reduction in the nitrobenzylthioinosine (NBMPR)-sensitive facilitated transport of nucleosides. The consequence of these changes lead to the formation of intracellular pools of uridine. To examine the possible role of accumulated uridine in the commitment of HL-60 leukemia cells to undergo maturation, the effects of uridine on the growth and differentiation of HL-60 cells were monitored. Uridine at millimolar levels caused a concentration-dependent inhibition of cellular growth, resulting in the accumulation of cells in the G2/M phases of the cell cycle, phenomena that preceded the formation of differentiated cells. These effects of uridine were reduced by 10 microM NBMPR, an inhibitor of the facilitated transport of nucleosides. The effects of 24 mM uridine on growth and differentiation of HL-60 cells were also prevented by 5 mM inosine, and partially prevented by either 2 mM hypoxanthine or 20 microM adenosine. Pretreatment of HL-60 cells with 24 mM uridine for 6 days, followed by a 2 h exposure to TPA, resulted in the rapid attachment of cells to the tissue culture dish, and the extension of long processes. Although the concentrations of uridine required for the above effects are greater than those achieved during differentiation, these observations suggest that uridine may play a role in regulating the maturation process.     

JAK-STAT signaling involved in phorbol 12-myristate 13-acetate- and dimethyl sulfoxide-induced 2'-5' oligoadenylate synthetase expression in human HL-60 leukemia cells

The JAK-STAT signal transduction cascade participates in various cellular processes, including immune response, cell replication, differentiation and oncogenesis. Here, we report that this cascade is induced in two human myeloid HL-60 leukemia cell variants by the granulocyte differentiation inducer dimethyl sulfoxide (DMSO) and macrophage differentiation inducer phorbol 12-myristate 13-acetate (PMA). DMSO and PMA also induced the expression and catalytic activity of 2'-5' oligoadenylate synthetase (2-5A synthetase), a known interferon (IFN) inducible enzyme. The HL-60 cell variants included HL-205, which is susceptible to DMSO- and PMA-induced differentiation, and HL-525, which is susceptible to DMSO- but not to PMA-induced differentiation. Treatment of HL-205 and HL-525 cells with DMSO and HL-205 cells with PMA-induced JAK1 phosphorylation, JAK1/STAT1 association, formation of STAT1-STAT2 heterodimers, and the binding of the active IFN stimulating growth factor 3 (ISGF3) to the IFN-stimulated response element (ISRE) fragment isolated from the 2-5A synthetase promoter. These events were either reduced or absent in the resistant HL-525 cells treated with PMA. Taken together, our data implicate the above signaling cascade in DMSO- and PMA-induced 2-5A synthetase expression and catalytic activity in the HL-60 cell system.     

Activation of protein kinase C induces differentiation in the human T-lymphoblastic cell line MOLT-3

We attempted to determine whether or not activation of calcium phospholipid-dependent protein kinase C (PKC) is associated with the induction of differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the human T-lymphoblastic cell line MOLT-3. PKC activities were assayed in MOLT-3 and its five subclones resistant to TPA-induced cell differentiation. The cytosolic PKC activities of TPA-resistant subclones were 36-53% of that of the parental MOLT-3 cells. TPA treatment led to a rapid decrease in PKC activities in the cytosol, together with a concomitant increase in PKC activities in the particulate fraction, in both MOLT-3 and a TPA-resistant subclone. Thus, translocation of PKC from the cytosol to the membrane occurred following treatment with TPA, in both cell lines. However, the amount of PKC translocated from the cytosol to particulate fraction for 60 min in a TPA-resistant subclone was about 20% of that of the parental MOLT-3 cells. These findings suggest that the quantity of cytosolic PKC activity and the extent of translocation may relate to responses to TPA-induced cell differentiation in this T-cell line.     

Glycosaminoglycan metabolism of HL-60 cells during differentiation induction by tetradecanoylphorbol-13-acetate

Many biochemical responses to phorbol ester differentiation inducers have been reported, including alterations in synthesis of specific gene products such as glycoproteins. Stage-specific glycosaminoglycan changes have previously been associated with the differentiation process, including a dramatic reduction in cellular chondroitin 4-sulfate during human myeloid leukemia cell maturation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). We have demonstrated that treatment of HL-60 human promyelocytic leukemia cells with 4-methyl-umbelliferyl-beta-D-xyloside increases precursor incorporation into glycosaminoglycans linked to beta-D-xyloside, rather than core protein, eliminating the need for core protein and xylosyltransferase. Therefore, these beta-D-xyloside-treated cells were used to study the decreased glycosaminoglycan production during TPA-induced HL-60 differentiation. Exposure of these pretreated HL-60 cells to TPA, which induces macrophage-like maturation, resulted in a 70% reduction of incorporation of [35S]sulfate into cell-associated glycosaminoglycans. Thus, even in HL-60 cells in which glycosaminoglycan production is maximally stimulated by beta-D-xyloside, TPA is a strong inhibitor of free glycosaminoglycan chain production, and this biochemical effect is associated with other features of leukocyte maturation.     

Combination of 22-oxa-1,25-dihydroxyvitamin D(3), a vitamin D(3) derivative, with vitamin K(2) (VK2) synergistically enhances cell differentiation but suppresses VK2-inducing apoptosis in HL-60 cells.

We originally reported that vitamin K(2) (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D(3)derivative, 22-oxa-1,25-dihydroxyvitamin D(3) (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1alpha,25(OH)(2)D(3). In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.     

Phorbol ester-induced macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells occurs independently of transferrin availability

Human promyelocytic leukemia (HL-60) cells can be induced to differentiate into macrophage-like cells by the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Addition of this agent to HL-60 cells causes a rapid internalization of surface transferrin receptor, followed by long-term receptor down-regulation at the level of gene expression. These effects precede the inhibition of proliferation and the acquisition of differentiation markers, and it has been suggested that transferrin receptor down-regulation may play a mediating role in these later events. Here we show that HL-60 cells will grow indefinitely in serum-free medium supplemented with either 5 micrograms ml-1 transferrin or 300 microM ferric citrate and that TPA inhibits cell proliferation (assayed by cell density and rate of thymidine incorporation) and induces macrophage-like differentiation (assayed by induction of cell adhesion and increased nonspecific esterase activity) with identical dose curves in both media. Furthermore, a neutralizing anti-transferrin antibody completely inhibits transferrin-dependent cell proliferation but has no effect on differentiation in the presence or absence of transferrin. We conclude that TPA-induced down-regulation of transferrin binding and internalization does not mediate the subsequent growth arrest and differentiation of HL-60 cells.     

Alteration in insulin receptor expression accompanying differentiation of HL-60 leukemia cells

Differentiation of leukemic cells in vitro is characterized by the sequential appearance of morphological, functional, and biochemical markers of maturation. The interaction of insulin with its receptor may be a regulator of growth and differentiation of leukemic cells. Human promyelocytic leukemia cells (HL-60) demonstrate specific reversible insulin binding consistent with properties of human insulin receptor. HL-60 cells treated with 500 microM N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate, 1 microM 1 alpha, 25-dihydroxyvitamin D3, or 41 nM phorbol-12-myristate-13-acetate expressed monocytic markers of differentiation and an increase in insulin receptor expression. The change in insulin receptor expression with 1 microM 1 alpha, 25-dihydroxyvitamin D3 and N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate induction was further characterized by Scatchard analysis. High affinity binding (Kd) constant was not altered, and the change in binding was attributed to receptor number. Commitment to increased insulin receptor expression was demonstrated after 1-h exposure to 1 microM 1 alpha, 25-dihydroxyvitamin D3. Agents which induced granulocytic differentiation, such as 160 mM dimethyl sulfoxide and 100 nM retinoic acid, significantly decreased insulin receptor expression compared to monocytic inducing agents. This difference in insulin receptor expression correlated with binding characteristics in normal human peripheral granulocyte and monocytes. The HL-60 cell line offers a model for the study of the molecular events which lead to the contrasting insulin receptor expression during myeloid and monocytoid hematopoiesis.     

Alterations in histone phosphorylation in HL-60 cells specific to monocytic differentiation

In order to examine the role of histone phosphorylation in regulation of the pathway of HL-60 cell differentiation, cells were labelled with [32P]phosphoric acid and histones fractionated by two-dimensional polyacrylamide gel electrophoresis. The monocytic inducer 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to specifically stimulate phosphorylation of histone H2B in a concentration-dependent manner. At a concentration of 100 mM, H2B phosphorylation was stimulated 2.3-fold after 4 h. A second monocytic inducer 1,25-dihydroxy-cholecalciferol (100 nM) also induced phosphorylation specifically in histone H2B. In contrast, the granulocytic inducers DMSO (1.5%) or retinoic acid (1 microM) did not increase phosphorylation in any histone species.     

Modulation of 6-thioguanine activity by guanine in human promyelocytic leukemia HL-60 cells

The effects of guanine coadministration on the metabolism and biological activity of 6-thioguanine (6-TG) were studied in human promyelocytic leukemia cells (HL-60). Cell growth, cytotoxicity (cloning assay), and cell differentiation were measured, along with nucleotide metabolism. Guanine was efficiently salvaged by HL-60 cells; at 200 microM, guanine suppressed the formation of 6-TG mononucleotides and abolished 6-TG incorporation into nucleic acids. Similarly, guanine antagonized 6-TG cytotoxicity in a dose dependent fashion. Furthermore, guanine (200 microM) fully suppressed the 6-TG (10 microM) induced HL-60 cell differentiation, which suggests that cell differentiation at pharmacological 6-TG concentrations is dependent on the anabolism of the drug to active nucleotides. 6-TG given alone reduced GTP levels and DNA synthesis rates in HL-60 cells, while a major intracellular 6-TG metabolite, 6-thioguanosine 5'-monophosphate, accumulated to high levels (approximately 100 microM). It is suggested that accumulation of 6-thioguanosine 5'-monophosphate and a resultant partial block of the de novo biosynthesis of guanine nucleotides is responsible for 6-TG induced cell differentiation in HL-60 cells.     

Alteration of intracellular actin levels induced by phorboldiester in human HL-60 leukemia cells susceptible or resistant to differentiation, and the effects of protein kinase inhibitors

During monocyte-macrophage differentiation of HL-60 cells by 12-O-tetradecanoyl phorbol 13-acetate, the intracellular globular(G)-actin and polymerized(F)-actin increased 3-fold and 1.7-fold, respectively. Time course studies showed that these changes of actin levels were nearly coincident with the development of macrophage characteristics, including adhesiveness, positive reactivity against OKM-1 antibody and elevated lysozyme activity. When exposed to 5 nM TPA, these different properties of differentiation were detectable as early as 12 h after TPA treatment and reached a maximum by 24 h. Phosphorylation of 17 K and 27 K proteins, induced by TPA, occurred early (within 30 min) during TPA-induced differentiation. On the other hand, HL-60R cells, which are resistant to TPA in terms of the development of adhesiveness and differentiation, showed no change in both G- and F-actin levels, after the TPA treatment. TPA did not induce phosphorylation of these proteins in the HL-60R cells. In the presence of the protein kinase inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 20 microM) and staurosporine (10 nM), the increase in actin levels induced by TPA was inhibited as well as other later evidence of differentiation. These results suggest that the phosphorylation of specific proteins is closely associated with the process of differentiation of HL-60 cells into macrophages.     

Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer

Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G(0)/G(1) cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.     

The effects of lithium on the growth and phorbol ester (TPA) induced differentiation of two HL-60 sublines

Two sublines of a human promyelocytic cell line, HL-60, were used to study the effect of lithium on TPA (12-o-tetradecanoylphorbol-13-acetate) induced macrophage-like differentiation. Although these sublines, HL-60 M and HL-60 JE, had different growth rates, both showed enhanced proliferation when treated with 5 mM lithium (128 +/- 2 and 141 +/- 1% in comparison to controls after 5 days of incubation, respectively). Treatment of the sublines with TPA for 72 h resulted in macrophage-like differentiation (assessed by cell adhesion) of about 90% at 10 nM TPA in HL-60 JE, whereas a maximum of 50% at 100 nM TPA was obtained in HL-60 M. Differentiation was also confirmed by non-specific esterase activity. However, incubation of both sublines with TPA and 5 mM lithium revealed that lithium has little or no effect on the macrophage-like differentiation of the HL-60 cell line.     

The role of granulocyte-macrophage colony-stimulating factor in induction of monocytic differentiation of HL-60 cells: synergistic interaction with 1 alpha, 25-dihydroxyvitamin D3 and interferon-gamma in inducing interleukin-1 beta.

1 alpha, 25-Dihydroxyvitamin D3 (D3) (100 nM) and interferon-gamma (IFN-gamma) (100 U/ml) cooperatively inhibited the proliferation of HL-60 cells, and synergistically induced their monocytic differentiation. The growth-promoting effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/ml) was inhibited appreciably by D3 and slightly by IFN-gamma. Despite the clear difference in their effects on growth of HL-60 cells, both IFN-gamma and GM-CSF in combination with D3 induced cell cycle changes, decreasing the number of cells in the S phase and increasing their percentage in the G1/0 phase. GM-CSF alone had no effect on differentiation, but enhanced differentiation induced by D3 distinctly though to a limited extent, and also enhanced monocytic differentiation, including morphological changes of HL-60 cells in the presence of D3 and IFN-gamma. GM-CSF as well as D3 and IFN-gamma induced interleukin-1 beta (IL-1 beta) production by the HL-60 cells, clearly indicating their importance in differentiation of these cells. IFN-gamma and GM-CSF had mutually potentiating effects and induced maximum IL-1 beta production in response to lipopolysaccharide (LPS) in the presence of D3. Thus despite its growth-promoting effect, GM-CSF is a potential inducer of monocytic differentiation of human myeloid leukemia cells, because in cooperation with IFN-gamma it induced monocyte-macrophage differentiation of HL-60 cells in the presence of D3.     

Dolastatin 10 and dolastatin 15: effects of two natural peptides on growth and differentiation of leukemia cells.

The effects of two natural peptides, dolastatin 10 and dolastatin 15, on growth and differentiation of hematopoietic cells were studied using freshly explanted leukemia cells and continuous leukemia cell lines. The proliferation of several myeloid cell lines and of growth-factor-stimulated peripheral blood cells from patients with acute myeloid leukemia (AML) was efficiently inhibited by the two agents at concentrations between 1 and 0.01 nM. Growth inhibition was dose-dependent and reversible. Neither of the dolastatins exhibited significant cytotoxicity on dividing cells, nor did they interfere with the viability of resting cells. The 12-O-tetradecanoylphorbol 13-acetate or bryostatin I induced differentiation of AML cells was not affected by the dolastatins. Short-term exposure to the phorbol ester conferred reduced sensitivity of the cell line HL-60 to the antiproliferative effect of the drugs. Our data suggest that the dolastatins alone or in combination with other drugs could exert a role in the treatment of human myeloid leukemia.     

Induction of morphological and functional differentiation of human promyelocytic leukemia cells (HL-60) by componuds which induce differentiation of murine leukemia cells

Various compounds active in promoting in vitro differentiation of certain murine leukemia cell lines (Friend erythroleukemia cells and mouse myeloid leukemia cells) were tested for their capacity to induce differentiation of HL-60 cells, a human promyelocytic leukemia cell line capable of terminally differentiating in vitro to functionally mature granulocytes. Polar planar compounds including hexamethylene bisacetamide (HMBA), certain purines (particularly hypoxanthine), and actinomycin-D induced morphological and functional (as assessed by the capacity to reduce NBT dye) differentiation of HL-60. In contrast, hemin, ouabain, prostaglandin E1, X-irradiation, dexamethasone and some other anti-leukemic chemotherapeutic agents induced little if any significant differentiation of HL-60 cells. These results, together with previous observations with murine leukemia cells, suggest that the human HL-60 cells share common cellular target sites for the inducing action of polar planar compounds, hypoxanthine and actinomycin-D with some murine leukemic cells. In contrast, hemin, ouabain and prostaglandin E1 may be specific for mouse erythroleukemia cells, while X-irradiation and chemotherapeutic agents induce differentiation of both types (erythroid and myeloid) of mouse leukemic cells, but have little effect on HL-60 cells.