Cooperative effects of gamma interferon and 1-alpha,25-dihydroxyvitamin D3 in inducing differentiation of human promyelocytic leukemia (HL-60) cells

Various agents induce differentiation of human leukemia cells in vitro. Most of these agents cause myeloid differentiation, but phorbol diesters, 1-alpha,25-dihydroxyvitamin D3 (1,25[OH2]D3), and certain lymphokines cause differentiation to monocyte-like cells. The purpose of this study was to determine the cooperative effects of 1,25(OH2)D3 and the lymphokine gamma interferon (IFN-gamma) on HL-60 cell differentiation. The recombinant human IFN-gamma or 1,25(OH2)D3 caused a slight reduction in the proliferation of the HL-60 cells (30%-40% reduction at doses of 100-200 U/ml [0.25-0.50 nM] IFN-gamma, or 5-25 nM 1,25[OH2]D3). HL-60 cells treated with 100 U/ml IFN-G had an eightfold increase in expression of nonspecific esterase (NSE) and a twofold increase in H2O2 production in response to phorbol myristate acetate (PMA). 1,25(OH2)D3 enhanced NSE expression eight- to 30-fold and H2O2 secretion twofold in response to PMA. There was also enhanced expression of HLA-DR and the receptor for C3bi. The 1,25(OH2)D3- and IFN-gamma-differentiating effects appeared to be additive or synergistic. Populations of IFN-gamma-treated HL-60 cells (but not the 1,25[OH2]D3-treated cells) had multinucleated giant cells. The polykaryons had NSE activity and had some properties of macrophage polykaryons or osteoclasts. 1,25(OH2)D3 did not augment the IFN-gamma-induced polykaryon formation.     

Detection of luxol-fast-blue positive cells in human promyelocytic leukemia cell line HL-60

Differentiation of HL-60 cells toward the eosinophilic series has not been reported previously. Eosinophil granule specific staining with Luxol-fast-blue was used to determine if HL-60 cells could differentiate into the eosinophilic lineage. The specificity of the Luxol-fast-blue stain for cells of the eosinophilic series was substantiated by comparison of the staining of cells from a patient with an eosinophilic syndrome by Wright-Giemsa and Luxol-fast-blue. Luxol-fast-blue positivity was most notable in cells found in colonies formed from HL-60 clonogenic cells in semisolid agar medium. Colony and cluster formation was spontaneous but in the presence of medium conditioned by either human placental cells or the human monocyte-like cell line, GCT, Luxol-fast-blue positive colonies and clusters were detected at a higher frequency.     

Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells

Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry.     

Recombinant human interferon alpha enhancement of retinoic-acid-induced differentiation of HL-60 cells

We have previously demonstrated that a combination of interferon beta and a differentiation agent, dimethyl sulfoxide (DMSO), is cytotoxic for HL-60 cells, a human promyelocytic leukemic cell line. We now report that a combination of recombinant interferon alpha (Intron; Schering) and retinoic acid is synergistically cytostatic for HL-60 cells. Retinoic acid (RA) induced the differentiation of HL-60 cells into granulocytes. Interferon (IFN) alone at 1-1000 IU/ml had no effect on either differentiation or proliferation of HL-60 cells. The addition of 1000 IU/ml of IFN and 10(-7) M RA at the initiation of culture reduced the number of viable cells to 28% of that observed for cells treated with RA alone. The decreased number of cells was a result of decreased cellular proliferation, rather than of a cytotoxic effect of the combination. IFN-RA-treated cells differentiated more rapidly than cells treated with RA alone. In addition, the final percentage of mature cells was increased at day 7 in IFN-RA-treated cultures, as compared with RA-treated cells. Simultaneous treatment of the cells with IFN and RA decreased the concentration of RA needed to induce differentiation or to exert a cytostatic effect. Significant changes in the nuclear structure of RA-treated HL-60 cells after 24 h have been reported. Cells were pulsed with RA for 24 h, washed, and IFN added. At day 7, cell growth was inhibited to the same extent as that of cells continuously exposed to IFN-RA. However, while 70% of the continuously exposed cell differentiated, cells pulsed with RA and subsequently treated with IFN did not differentiate. The results of this investigation further support our findings that combinations of IFN and inducers of differentiation may be of importance in the treatment of leukemia.     

Bryostatin, an activator of the calcium phospholipid-dependent protein kinase, blocks phorbol ester-induced differentiation of human promyelocytic leukemia cells HL-60.

Phorbol esters bind to and activate a calcium phospholipid-dependent protein kinase (C kinase). Some researchers believe that activation of C kinase is necessary for the induction of phorbol ester biologic effects. Our research indicates that bryostatin, a macrocyclic lactone that binds to the phorbol ester receptor in human polymorphonuclear leukocytes, also binds to this receptor in the human promyelocytic leukemia cell line, HL-60. Bryostatin activates partially purified C kinase from HL-60 cells in vitro, and when applied to HL-60 cells in vivo, it decreases measurable cytoplasmic C kinase activity. Unlike the phorbol esters, bryostatin is unable to induce a macrophage-like differentiation of HL-60 cells; however, bryostatin, in a dose-dependent fashion, blocks phorbol ester-induced differentiation of HL-60 cells and, if applied within 48 hr of phorbol esters, halts further differentiation. These results suggest that activation of the C kinase by some agents is not sufficient for induction of HL-60 cell differentiation and imply that some of the biologic effects of phorbol esters may occur through a more complex mechanism than previously thought.     

Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL- 60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation- induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O- tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.     

The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression

The HL-60 cell line, derived from a single patient with acute promyelocytic leukemia, provides a unique in vitro model system for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic cells of the granulocyte/monocyte/macrophage lineage.     

Synergistic enhancement by 12-O-tetradecanoylphorbol-13-acetate and dibutyryl cAMP of 1alpha,25-dihydroxyvitamin D3 action in human promyelocytic leukemic HL-60 cells.

We have reported that dibutyryl cAMP (dbcAMP), an activator of cAMP-dependent protein kinase (PKA), potentiated the effects of 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3)-induced 24-hydroxylation activity in HL-60 cells by increasing 1,25-(OH)2D3 receptor (VDR). The present study demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent phorbol ester, also potentiated the effect of 1,25-(OH)2D3 on HL-60 cells and that TPA and dbcAMP acted in a synergistic manner to enhance the effect of 1,25-(OH)2D3. It is interesting that TPA induced 24-hydroxylation activity far more efficiently than dbcAMP, in addition to their effects in increasing VDR. TPA increased basal levels of c-fos mRNA to the maximum by 1 h after the treatment, whereas dbcAMP failed to affect c-fos gene expression. Together with the previous data indicating the presence of AP-1-like sequence in the promoter of 24-hydroxylase gene, it was suggested that TPA potentiated the effect of 1,25-(OH)2D3 through an activation of c-fos gene expression. This notion was further supported by the data showing that TPA and dbcAMP also acted in a synergistic manner to activate c-fos gene expression. Neither TPA nor dbcAMP affected c-jun early response gene in the HL-60 clone used in the present study. The present study suggested that the activation of early c-fos response gene by TPA might be another mechanism to enhance the effect of 1,25-(OH)2D3, besides up-regulation of VDR.     

1 alpha,25-dihydroxyvitamin D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation

Cyclosporin A and FK506, at concentrations that inhibited phosphatase activity of calcineurin in HL-60 cellular lysates, augmented the proliferation of leukemic HL-60 cells. These immunosuppressants did not affect 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced monocytic differentiation of HL-60 cells, but did abrogate the 1,25(OH)2D3- induced inhibition of HL-60 cell growth. Treatment with 20 nmol/L 1,25(OH)2D3 led to a progressive increase in calcineurin phosphatase activity in subcellular fractions from HL-60 cell extracts, the increase in this activity appeared to parallel the phenotypic and functional changes of HL-60 cells during monocytic differentiation induced by 1,25(OH)2D3. Immunoblot analysis indicated that increase in calcineurin activity was concordant with the increased expressions of calcineurin catalytic subunit isozymes, calcineurin A alpha (CNA alpha), and calcineurin A beta(CNA beta), and a regulatory calcineurin B subunit (CNB) proteins, which were preceded by a coordinate increase in the levels of CNA alpha, CNA beta and CNB mRNAs. The expression of calmodulin remained unaltered throughout 1,25(OH)2D3-induced monocytic differentiation. These results suggest that calcineurin activation has a net negative effect on HL-60 cell proliferation, and that the increased expression of calcineurin may be involved in 1,25(OH)2D3- induced inhibition of HL-60 cell proliferation.     

Synergistic growth inhibitory and differentiating effects of trimidox and tiazofurin in human promyelocytic leukemia HL-60 cells

Increased ribonucleotide reductase (RR) activity has been linked with malignant transformation and tumor cell growth. Therefore, this enzyme is considered to be an excellent target for cancer chemotherapy. We have examined the effects of a newly patented RR inhibitor, trimidox (3,4,5-trihydroxybenzohydroxamidoxime). Trimidox inhibited the growth of human promyelocytic leukemia HL-60 cells with an IC50 of 35 mumol/L. Incubation of HL-60 cells with 50 mumol/L trimidox for 24 hours decreased deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) pools to 24% and 39% of control values, respectively. Incubation of HL-60 cells with 20 to 80 mumol/L trimidox even up to a period of 4 days did not alter the distribution of cells in different phases of cell cycle. Sequential incubation of HL-60 cells with trimidox (25 mumol/L) for 24 hours and then with 10 mumol/L tiazofurin (an inhibitor of inosine monophosphate dehydrogenase) for 4 days produced synergistic growth inhibitory activity, and the cell number decreased to 16% of untreated controls. When differentiation- linked cell surface marker expressions were determined in cells treated with trimidox and tiazofurin, a significantly increased fluorescence intensity was observed for the CD 11b (2.9-fold). CD 33 (1.9-fold), and HLA-D cell surface antigens. Expression of the transferrin receptor (CD71) increased 7.3-fold in cells treated with both agents, compared with untreated controls. Our results suggest that trimidox in combination with tiazofurin might be useful in the treatment of leukemia.     

Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid.

The HL-60 cell line, derived from a patient with acute promyelocytic leukemia, proliferates continuously in suspension culture and consists predominantly (greater than 90%) of promyelocytes. These cells can be induced to differentiate to morphologically and functionally mature granulocytes by incubation with a wide variety of compounds, including butyrate and hypoxanthine and polar planar compounds such as dimethyl sulfoxide and hexamethylene bisacetamide. We have now found that retinoic acid (all-trans-retinoic acid) induces differentiation (as measured morphologically and by the ability to reduce nitroblue tetrazolium) of HL-60 at concentrations as low as 1 nM. Maximal differentiation (approximately 90%) occurs at 1 micro M, a concentration 1/500th to 1/160,000th the concentrations of butyrate (0.5 mM) and dimethyl sulfoxide (160 mM) that promote a similar increase in differentiation. Continuous exposure to retinoic acid is necessary for optimal differentiation, with the percentage of mature cells in the culture directly related to the length of time of exposure to retinoic acid. Retinoic acid and 13-cis-retinoic acid are equally effective in inducing differentiation of HL-60. Retinol (vitamin A), retinal, and retinyl acetate are approximately 1/1000th less potent. This study suggests that retinoids could provide a therapeutic tool in the treatment of acute myeloid leukemia, a disease that has been looked upon as primarily involving a block in myeloid differentiation, and indicates that retinoids, in addition to their well-characterized involvement in epithelial cell differentiation, may also be involved in the differentiation of certain hematopoietic cells.     

Combinations of recombinant human interferons and retinoic acid synergistically induce differentiation of the human promyelocytic leukemia cell line HL-60

The human acute promyelocytic leukemia cell line HL-60 is induced to differentiate into morphologically and functionally mature monocytelike cells by incubation with a combination of 10 nmol/L retinoic acid (RA) and various concentrations of recombinant immune interferon (rIFN- gamma). These induced cells show marked increases in antibody-dependent cellular cytotoxicity (ADCC), antibody-coated erythrocyte (EA) rosettes, nonspecific esterase, and 5'-nucleotidase activity. rIFN- gamma alone at concentrations of 10 to 1,000 U/mL has essentially no effect on morphological maturation, nitroblue tetrazolium reduction, and immunophagocytosis. However, rIFN-gamma at these concentrations increases EA rosetting in a concentration-dependent manner that is not affected by 10 nmol/L RA. At a concentration of 1,000 U/ml, rIFN-gamma induces moderate increases in nonspecific esterase, 5'-nucleotidase, and ADCC. These parameters are markedly increased by the addition of 10 nM RA, a concentration which alone has no effect on these markers. Based on units of antiviral activity, rIFN-gamma is tenfold more active than rIFN-alpha D in inducing EA rosettes and 40-fold more active in inducing nitroblue tetrazolium reduction and immunophagocytosis. These results, indicating that combinations of rIFN-gamma or rIFN-alpha and RA synergistically induce differentiation of HL-60, suggest that this combination may have clinical utility in the treatment of patients with certain leukemias.     

Glycosyltransferase alterations are cell type related when human promyelocytic leukemia (HL-60) cells are treated with various inducers of differentiation

We have assayed glycosyltransferase activities during the granulocytic and macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells. Functional granulocytic differentiation was assayed by the decarboxylation of 2-deoxyglucose in addition to nitroblue tetrazolium reduction. Dimethylsulfoxide (DMSO) treated HL-60 cells, induced to granulocytic differentiation, had higher 2-deoxy-glucose decarboxylation activity, and contained less sialyltransferase (ST), more fucosyltransferase (FT), and more N-acetylglucosaminyltransferase (NGT) activities than untreated cells. HL-60 cells treated with another granulocytic differentiator, retinoic acid, also had higher 2-deoxyglucose decarboxylation activity, and contained less ST, more FT, and more NGT activities than untreated cells. In contrast, cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reported to differentiate HL-60 to macrophage-like cells, but did not show an increased level of 2-deoxyglucose decarboxylation activity, but contained more galactosyltransferase (GT) and FT activities as compared to untreated cells. These findings suggest that the alterations of glycosyltransferase levels during the differentiation of precursor cells may not depend upon different inducers, but are characteristic of the phenotypic expression of the mature cell type.     

Establishment of eosinophilic sublines from human promyelocytic leukemia (HL-60) cells: demonstration of multipotentiality and single- lineage commitment of HL-60 stem cells

Recent observations indicating that the HL-60 human acute promyelocytic leukemia cell line contains a minor eosinophil population in addition to neutrophil and mononuclear phagocyte progenitors suggest the multipotentiality of HL-60 stem cells. In order to clarify multilineage differentiation and commitment to single-lineage progenitors we analyzed HL-60 colonies formed in methylcellulose. In an HL-60 parent line with a relatively high eosinophil content (5.5%), 36% of the spontaneous colonies consisted partly or wholly of eosinophilic cells. After two rounds of subcloning in methylcellulose, two eosinophilic sublines and two neutrophilic sublines were established. These lines have been in continuous liquid culture for more than four months, and they show stable single-lineage differentiation. Purified biosynthetic GM-CSF, which stimulates normal CFU-GM and CFU-EO, induced monocytic differentiation but no eosinophilic differentiation in the neutrophilic sublines and no neutrophilic or monocytic differentiation in the eosinophilic sublines. These observations indicate that HL-60 stem cells are multipotent and capable of spontaneous commitment to single- lineage progenitors. The eosinophilic HL-60 sublines should facilitate studies on the production and function of human eosinophils and the single-lineage sublines will allow further analysis of leukemic cell differentiation and stem cell commitment.     

Synergistic action of resveratrol, an ingredient of wine, with Ara-C and tiazofurin in HL-60 human promyelocytic leukemia cells

OBJECTIVE: Resveratrol, a naturally occurring stilbene derivative, is a potent free-radical scavenger causing a number of biochemical and antineoplastic effects. It was shown to induce differentiation and apoptosis in leukemia cells. Resveratrol was also identified as an inhibitor of ribonucleotide reductase (RR), a key enzyme of DNA synthesis. We report about the biochemical effects of resveratrol on the concentration of deoxyribonucleoside triphosphates (dNTPs), the products of RR, and on the incorporation of 14C-labeled cytidine into the DNA of HL-60 human promyelocytic leukemia cells. MATERIALS AND METHODS: Incorporation of 14C-labeled cytidine into the DNA of resveratrol-treated HL-60 cells was measured. Concentration of dNTPs was determined by a HPLC method. Cytotoxic effects of resveratrol, Ara-C, and tiazofurin were analyzed using growth inhibition and clonogenic assays. Induction of apoptosis was studied using a Hoechst/propidium iodide staining method. RESULTS: We found that resveratrol effectively inhibited incorporation of 14C-labeled cytidine into DNA. Furthermore, incubation of HL-60 cells with resveratrol significantly decreased intracellular dCTP, dTTP, dATP, and dGTP concentrations. Based on these results, we investigated the combination effects of resveratrol with Ara-C or tiazofurin, both antimetabolites, which are known to exhibit synergistic effects in combination with other inhibitors of RR. In growth inhibition, apoptosis, and clonogenic assays, resveratrol acted synergistically with both Ara-C and tiazofurin in HL-60 cells. CONCLUSIONS: We conclude that resveratrol could become a viable candidate as one compound in the combination chemotherapy of leukemia and therefore deserves further testing.     

Sodium butyrate and a T lymphocyte cell line-derived differentiation factor induce basophilic differentiation of the human promyelocytic leukemia cell line HL-60

Sodium butyrate induces basophilic differentiation of HL-60 promyelocytic leukemia cells that have been previously passaged in alkaline medium. A factor present in Mo conditioned medium (Mo-CM) acts synergistically with sodium butyrate to promote basophilic maturation in a dose-dependent fashion. The induced HL-60 cells exhibit nuclei at various stages of maturity and cytoplasmic granules staining azurophilic with May-Grunwald-Giemsa and metachromatically with toluidine blue. The histamine content of induced HL-60 cells is 50 ng/10(6) cells with sodium butyrate alone or 190 ng/10(6) cells with butyrate in combination with Mo-CM. Induced cells release histamine in response to anti-IgE and have receptors for the Fc portion of human IgE. The basophilic cell-differentiating activity present in Mo-CM appears to be distinct from several other cytokines including recombinant human interleukin-1 alpha, interleukin-2, interferon-gamma, interferon-alpha, murine interleukin-3, erythroid-potentiating activity, and purified human granulocyte/macrophage colony-stimulating factor. This is the first demonstration of a cell line that is capable of differentiation along the basophil lineage and could provide a useful model for examining biochemical and molecular events associated with basophil differentiation.     

cGMP-induced differentiation of the promyelocytic cell line HL-60.

cGMP is a second messenger that mediates numerous metabolic events; in the present work a role in myeloid cell differentiation was demonstrated. Nitroprusside and NaNO2, which activate cytosolic guanylate cyclase and increase the intracellular cGMP concentration, induced granulocytic differentiation of the human promyelocytic cell line HL-60; differentiation was measured by acquisition of the OKM1 antigen, morphological changes, and nitroblue tetrazolium reduction. When theophylline, a phosphodiesterase inhibitor, which by itself induced modest differentiation, was added to nitroprusside or NaNO2, differentiation increased in an additive fashion. The degree of differentiation correlated with the increase in the intracellular cGMP concentration. 8-Bromoguanosine 3',5'-cyclic monophosphate, a membrane-permeable cGMP analogue, also induced differentiation of HL-60 cells but was much more effective in the presence of theophylline, with the two agents interacting synergistically. The effect of theophylline in these studies could not be attributed to increasing the intracellular cAMP concentration. Dimethyl sulfoxide, and established inducer of differentiation of HL-60 cells, markedly enhanced the differentiation induced by nitroprusside and NaNO2.     

Prothymosin alpha gene expression correlates with proliferation, not differentiation, of HL-60 cells

Accumulating evidence suggests that prothymosin alpha has an as yet undefined intracellular, perhaps intranuclear, function related to cell proliferation. Prothymosin alpha mRNA and/or peptide levels increase when cells are stimulated to proliferate. Because proliferation and differentiation events are often inversely correlated, we examined prothymosin alpha gene expression during proliferation and differentiation of HL-60 myeloid leukemia cells. Steady-state levels of prothymosin alpha mRNA, which are high in exponentially growing HL-60, decrease within hours after induction of HL-60 to differentiate along the neutrophil pathway with dimethylsulfoxide (DMSO) or along the macrophage lineage with either tetradecanoylphorbol acetate (TPA) or bryostatin 1. The decline in prothymosin alpha mRNA in response to these differentiation signals parallels that of c-myc mRNA under the same conditions. We then determined whether the downregulation of prothymosin alpha and c-myc mRNA were due to differentiation or cessation or proliferation. Recombinant human gamma-interferon induces monocytic differentiation of HL-60, but permits continued proliferation, and, under these conditions, expression of prothymosin alpha, as well as of c-myc, mRNA remains elevated. We conclude that prothymosin alpha and c-myc expression are coregulated in differentiating HL-60 and that their expression correlates with the proliferative state of HL-60 cells, rather than with the differentiated state.     

Induction of differentiation of human promyelocytic leukemia cell line HL-60 by retinoyl glucuronide, a biologically active metabolite of vitamin A.

We examined the differentiation activity of retinoyl beta-D-glucuronide, a biologically active physiological metabolite of vitamin A, using the human promyelocytic leukemic cell line HL-60, which can be induced to differentiate with retinoic acid. Retinoyl beta-D-glucuronide (1 microM) inhibited HL-60 cell proliferation by 55-75%, inhibited tritiated thymidine incorporation into DNA by 63-80%, and induced 38-50% of the cells to differentiate into mature granulocytes. The potency of growth inhibition and induction of differentiation by retinoyl beta-D-glucuronide was similar to that of all-trans-retinoic acid. The continuous presence of either retinoyl beta-D-glucuronide or all-trans-retinoic acid was not required to obtain maximum growth arrest and differentiation: a 1-hr exposure of HL-60 cells to the retinoids gave the same response (measured after a total incubation time of 48 hr) as a 24-hr or 48-hr continuous treatment. Retinoyl beta-D-glucuronide (0.1-0.2 mM) was 50% less cytotoxic to HL-60 cells than all-trans-retinoic acid at an equimolar concentration. Retinoyl beta-D-glucuronide was not significantly metabolized to other retinoids; retinoic acid was not formed during incubation. We conclude that retinoyl beta-D-glucuronide can arrest HL-60 cell proliferation and induce their differentiation into mature granulocytes; it may act by itself or by being hydrolyzed to retinoic acid, which could be immediately utilized and metabolized. The therapeutic use of this retinoid as an antineoplastic agent is suggested.