cell line (HL-60) by retinoic acid* (retinoids/myeloid differentiation/hematopoiesis)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 77, No. 5, pp , May 198 Medical Sciences Induction of differentiation of the human promyelocytic leukemia cell line (HL-6) by retinoic acid* (retinoids/myeloid differentiation/hematopoiesis) T. R. BREITMAN, STUART E. SELONICKt, AND STEVEN J. COLLINSf Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 225 Communicated by Marshall Warren Nirenberg, January 31, 198 ABSTRACT The HL-6 cell line, derived from a patient with acute promyelocytic leukemia, proliferates continuously in suspension culture and consists predominantly (>9%) 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-6 at concentrations as low as nm. Maximal differentiation (approximately 9%) occurs at 1 MuM, a concentration 1/5th to 1/16,th the concentrations of butyrate (.5 mm) and dimethyl sulfoxide (16 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-6. Retinol (vitamin A), retinal, and retinyl acetate are approximately 1/1th 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. Retinoids (vitamin A and its natural and synthetic analogs) influence the normal differentiation of epithelium. Retinoid deficiency in rats leads to premalignant lesions in a wide variety of epithelial tissue that are reversed by the administration of retinoids (1). Retinoids inhibit carcinogenesis in vio (2, 3) and suppress malignant transformation in vitro (4, 5). Certain of these compounds inhibit the growth of transformed cells (6, 7) and induce differentiation of mouse embryonal carcinoma cells in vitro (8-1). The antineoplastic effects of retinoids suggest that these compounds could be used therapeutically for the chemoprevention of cancer (11). The HL-6 cell line, derived from a patient with acute promyelocytic leukemia (12), proliferates continuously in suspension culture and consists predominantly of promyelocytes. These cells manifest a transformed phenotype, as shown by karyotypic abnormalities, continuous proliferation for over 3 years in culture, tumorigenicity in nude mice, and morphological, histochemical, and karyotypic similarity to the patient's leukemic cells (13). However, these cells can be induced to terminally differentiate to morphologically mature granulocytes by incubation with a wide variety of compounds, including butyrate, hypoxanthine, actinomycin D, and polar planar compounds such as dimethyl sulfoxide (Me2SO) and hexamethylene bisacetamide (HMBA) (14, 15). Moreover, these The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact induced HL-6 cells have many of the functional characteristics of normal peripheral blood granulocytes, including phagocytosis, complement receptors, chemotaxis, and the ability to reduce nitroblue tetrazolium (NBT) (14, 16, 17). Thus, this cell line provides a unique system for studying human myeloid differentiation in vitro. In the present report we describe the induction of terminal differentiation of HL-6 cells by retinoic acid. This compound induces differentiation of HL-6 cells at concentrations 1/5th to 1/16,th the concentrations of other inducers. This study suggests a new approach to the therapy of some human leukemias and indicates that retinoids may be involved in the differentiation of certain hematopoietic tissues. MATERIALS AND METHODS Cells. The HL-6 cell line has been maintained in continous suspension culture for almost 3 years (12); passages 15 through 5 were used for these experiments. Cells (2 X 15 cells per ml) were grown in Corning polystyrene tissue culture flasks containing growth medium consisting of equal volumes of Dulbecco's modified Eagle's minimal essential medium and Ham's F12 medium and supplemented with 1.2 g of NaHCO3 per liter, 15 mm Hepes, and 1% (vol/vol) heat inactivated fetal calf serum (Flow Laboratories, McLean, VA). Substitution of RPMI 164 (GIBCO) supplemented with 1% inactivated fetal calf serum for the above medium gave identical results. Cell counts were determined with a Coulter Counter (Coulter Electronic, Hialeah, FL), and viability was assessed by trypan blue dye exclusion. Morphologic assessment of the cells was made on Cytospin slide preparations stained with Wright- Giemsa. Differential counts were then performed under light microscopy on a minimum of 2 cells. Chemicals. Retinoic acid, retinol, retinal, and retinyl acetate were obtained from Sigma and stored at -2 C; 13-cis-retinoic acid was a gift of Hoffman-La Roche. 12-O-Tetradecanoylphorbol 13-acetate, obtained from the National Institutes of Health Carcinogen Repository, was stored at -2 C in acetone at 2,ug/ml. NBT was obtained from Aldrich. HMBA was a gift of Roberta Reuben. Induction of Differentiation. All retinoids were dissolved in 95% (vol/vol) ethanol at concentrations varying from 1-3 to 1-7 M, and the solutions were diluted 1-fold into the growth medium such that the final ethanol concentration was Abbreviations: Retinoic acid, all-trans-f3-retinoic acid; Me2SO, dimethyl sulfoxide; NBT, nitroblue tetrazolium; HMBA, hexamethylene bisacetamide. * A portion of this work was presented at the Annual Meeting of the American Federation for Clinical Research, May 1-12, 198, Washington, DC. t Present address: Department of Medicine, Johns Hopkins Hospital, 61 North Broadway, Baltimore, MD i Present address: Department of Medicine, V.A. Hospital, 4435 Beacon Avenue South, Seattle, WA 9818.

2 Medical Sciences: Breitman et al. Proc. Natl. Acad. Sci. USA 77 (198) _/_ 6~~~~~~~~~ ~~~~~~ ~~~~~~~~..a no higher than.1%. Varying the concentration of ethanol from.1% to.1% had no effect on the extent of retinoic acidinduced differentiation. Cells were incubated at 37 C in a humidified atmosphere of 5% CO2 in air. All manipulations were performed in subdued light. NBT Reduction. NBT reduction was assayed as described (16). Cells (2 X 16 per ml of medium) were incubated for 25 min at 37 C with an equal volume of.2% NBT dissolved in Dulbecco's phosphate-buffered saline containing 2 ng of freshly diluted tetradecanoylphorbol acetate per ml. The per- 4- X O// 1 7' 2- -*Control Time in culture, days FIG. 2. Differentiation of HL-6 cells induced by various concentrations of retinoic acid. Cells were seeded at 2 X 15 per ml. Numbers identifying each curve are the molar concentrations of retinoic acid. Differentiation was determined by NBT reduction. FIG. 1. Morphology of uninduced and retinoic acid-induced HL-6 cells. Cytospin slide preparations of suspension cell cultures stained with Wright-Giemsa. (X11.) (Upper) Cells cultivated without inducer consist of promyelocytes with characteristic cytoplasmic granules, large nuclei, and prominent nucleoli. (Lower) Cells cultured in 1,M retinoic acid for 5 days consist of metamyelocytes and banded and segmented neutrophils. centage of cells containing intracellular blue-black formazan deposits was then determined on Wright-Giemsa-stained Cytospin slide preparations. At least 2 cells were assessed. RESULTS Morphological Changes in HL-6 Cells Induced by Retinoic Acid. In the absence of an inducer, cultured HL-6 cells are predominantly promyelocytes, with 3-1% of the cells more mature granulocytes (myelocytes, metamyelocytes, and banded and segmented neutrophils). Retinoic acid induced striking changes in the morphology of HL-6 cells characteristic of terminal differentiation of myeloid cells (Fig. 1). After 6 days of incubation in 1,uM retinoic acid, over 9% of HL-6 cells resembled mature granulocytes. The extent of this differentiation as assessed by differential counts indicates that with retinoic acid there was a "shift to the right" when compared with other HL-6 inducers, including Me2SO and. sodium butyrate (Table 1). Thus, retinoic acid induces relatively extensive morphological differentiation of HL-6 at concentrations some 1/5th to 1/16,th the concentrations of other inducers. Table 1. Differential counts of HL-6 after incubation with various inducing compounds Myeloid cell type, % of total cells Banded Segmented Inducer Myeloblasts Promyelocytes Myelocytes Metamyelocytes neutrophils neutrophils None Me2SO (16 mm) HMBA (2. mm) Butyrate (.5 mm) Retinoic acid (1 IAM) HL-6 cells were incubated under standard conditions with the indicated concentration of inducer. After 6 days, differential counts were performed on Wright-Giemsa-stained Cytospin slide preparations of the cell suspensions.

3 2938 Medical Sciences: Breitman et al. Functional Changes in HL-6 Cells Induced by Retinoic Acid. NBT, a water-soluble dye, is reduced to insoluble intracellular blue-black formazan by phagocytizing or membrane-stimulated granulocytes (18). Morphologically mature HL-6 cells, but not the immature promyelocytes, reduce NBT when stimulated by tetradecanoylphorbol acetate (16, 17). The NBT reaction provides a sensitive and easily quantitated HL-6 differentiation marker, eliminating observer subjectivity associated with morphological assessment alone. After 5 days of incubation in various concentrations of retinoic acid, the percentage of HL-6 cells reducing NBT varied with retinoic acid concentration, reaching a maximum of 95% at 1,M (Fig. 2). HL-6 cells treated with retinoic acid also exhibited the capacity to phagocytose Candida albicans (data not shown). Decreased HL-6 Cell Growth in Presence of Retinoic Acid. With the addition of retinoic acid, alterations in the rate of cell growth were apparent by 3 days (Fig. 3). With 1 AM retinoic acid, growth ceased by day 4. These cells no longer proliferated even when resuspended in growth medium without added retinoic acid. Thus, as expected, morphologically and functionally mature HL-6 cells induced by retinoic acid have also lost the capacity for further proliferation. The high percentage of viable cells (Fig. 3) eliminated the possibility that all or a major part of the retinoic acid-induced differentiation was a result of selective enrichment for differentiated cells. Induction of HL-6 Differentiation by Other Retinoids. Retinoic acid and 13-cis-retinoic acid were equally effective and superior to other retinoids in inducing differentiation of HL-6 (Fig. 4). Retinol (vitamin A), retinal, and retinyl acetate were approximately 1/1th as potent as retinoic acid. 2 - ' ' / Control lo-9- (86%) 7E 7 -n {6% 6 (7%) ,, 1-8 o- % 1-6_ Time in culture, days FIG. 3. Growth of HL-6 cells in various concentrations of retinoic acid. Cell counts were determined with a Coulter Counter. Numbers identifying each curve are the molar concentrations of retinoic acid. Numbers in parentheses are percentage of viable cells on day 5 as determined by trypan blue exclusion a- to 2- Proc. Natl. Acad. Sci. USA 77 (198) 1-9 lo Retinoid concentration, M FIG. 4. Differentiation of HL-6 cells induced by various concentrations of retinoids. Cells were seeded at 2 X 15 per ml. Differentiation was determined by NBT reduction after 5 days of growth in the indicated concentration of retinoid., All-trans-retinoic acid;, 13-cis-retinoic acid;,, retinol; o, retinyl acetate; *, retinal. Exposure Time Necessary for Optimal HL-6 Differentiation. To determine how long an exposure to retinoic acid was required for HL-6 cells to differentiate, we incubated cells with 1,uM retinoic acid for various time intervals, washed them, and then resuspended them in fresh growth medium without retinoic acid. After a total of 5 days of incubation, the percentage of cells reducing NBT was assessed. Continuous exposure to retinoic acid was necessary for optimal differentiation, with the percentage of mature cells in the culture directly related to the time of retinoic acid exposure (Fig. 5). In other experiments, an increase in differentiation was noted after only 3 hr of exposure. DISCUSSION Retinoic acid induces morphological and functional terminal differentiation of thehuman promyelocytic leukemia cell line HL-6. Maximal differentiation (approximately 9%) occurs at a retinoic acid concentration of 1,uM, a concentration 1/ 5th to 1/16,th the concentrations of sodium butyrate (.5 mm), HMBA (2. mm), and Me2SO (16 mm) that promote a similar increase in differentiation (Table 1). Moreover, the extent of morphological differentiation of retinoic acid-treated HL-6 cells is as great as that found with any other inducer (Table 1). Although other retinoids increase HL-6 cell differentiation, retinoic acid is clearly superior to retinol (vitamin A), retinal, and retinyl acetate (Fig. 4). This compound has been shown to have the same high activity with respect to retinol analogs in other culture systems, including reversal of keratinization in retinoid-deficient tracheal epithelium (19), inhibition of growth of transformed cells (6), and enhanced production of plasminogen activator in mouse embryonal carcinoma cells (8, 1). HL-6 cells may provide a similar convenient in vitro system

4 Cu ca co 4- c a._ 6F 4F 2h Medical Sciences: Breitman et al. 8k I Time of retinoic acid exposure, days FIG. 5. Differentiation of HL-6 cells after various exposure times to retinoic acid. Cells (2 X 15 per ml) were incubated in 1.M retinoic acid for the indicated period of time, washed, and resuspended in growth medium without added retinoic acid. After 5 days of total incubation, differentiation was determined by NBT reduction. and, Data from two independent experiments. for assessing the activity of various natural and synthetic retinoids and for studying the biological effects of retinoid antagonists. The mechanism by which retinoic acid enhances differentiation and exerts its antineoplastic effect on HL-6 and other cells is unknown. An appealing possibility is that in HL-6 retinoic acid suppresses the activity of an endogenously produced polypeptide growth factor in a manner analogous to the blockage by retinoids of the phenotypic cell transformation produced by sarcoma growth factor in normal rat kidney fibroblasts (2). Other mechanisms of action, such as exerting an effect in the nucleus after binding to a highly specific cytoplasmic receptor (cellular retinoic acid-binding protein) (21) and promoting glycosylation of membrane glycoproteins (22), cannot be ruled out. It is also unknown whether the mechanism of induction by retinoic acid is the same as that of polar compounds such as Me2SO and HMBA; in initial experiments the effects of combinations of retinoic acid and Me2SO were additive and not synergistic, suggesting that these two inducers may act by similar pathways. The ability of retinoic acid to convert proliferating HL-6 leukemic promyelocytes to terminally differentiated, functionally mature granulocytes suggests that this compound could provide a new therapeutic tool in the treatment of acute myeloid leukemia, a disease that has been looked upon as primarily involving a block in myeloid differentiation (23). Indeed, 13- cis-retinoic acid has been used for treatment of certain skin disorders (24, 25). If the in vitro studies described here reflect potential behavior of this drug in vivo, then serum levels as high as 1 ttm might be necessary over a prolonged period of time to induce maximal differentiation of the leukemic cells. Although this level is pharmacologically obtainable (26, 27), it is unknown i Proc. Natl. Acad. Sci. USA 77 (198) 2939 what toxicity would result from prolonged in vivo exposure. In comparison to other retinoids, retinoic acid is more toxic to tracheal cartilage in organ cultures (19). However, 13-cis-retinoic acid is less toxic than retinoic acid (28, 29) and in clinical studies 13-cis-retinoic acid has shown relatively little toxicity (24, 25). Leukemic cells other than HL-6 may not be susceptible to induction of differentiation by retinoic acid. In preliminary experiments we have not been able to induce the production of hemoglobin in Friend erythroleukemic cells or in K-562 (3) (a cell line derived from a patient with chronic myelogenous leukemia in blast crisis) with the addition of retinoic acid. Both of these cell lines produced hemoglobin when incubated with the appropriate inducer, Me2SO for Friend leukemic cells (31) and butyrate for K-562 (32). Retinoic acid also did not induce granulocytic differentiation of KG-1, a cell line derived from a patient with acute myelogenous leukemia (33). Thus, HL-6 is unusual among leukemic cells in exhibiting such a pronounced differentiative response to retinoic acid. 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