Human Myelogenous Leukemia Cell Line HL-60 Cells Resistant to Differentiation Induction by Retinoic Acid DECREASED CONTENT OF GLYCOSPHINGOLIPIDS AND GRANULOCYTIC DIFFERENTIATION BY NEOLACTO SERIES GANGLIOSIDES*

We have recently reported that neolacto series gan- gliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulo- cytes when the cella are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In calntrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, eslpecially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingo- lipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid- resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, as described (14). The release of 0; was calculated from the linear portion of cytochrome c reduction for PMA and from cytochrome c reduced for 5 min after the addition of fMLP. Surface membrane antigens were assessed by cytofluorometry in an Ortho Spectrum 111 (Ortho Diagnostic Systems, Inc., Westwood, MA) using the author- ized monoclonal antibodies including anti-OKM1 (CDll), anti-OKB2 (CD24), anti-OKM5 (Ortho Diagnostic Systems), and anti-Mo2 (CDW14) (Coutler Immunology, Hialeah, FL).

Human Myelogenous Leukemia Cell Line HL-60 Cells Resistant to Differentiation Induction by Retinoic Acid DECREASED CONTENT OF GLYCOSPHINGOLIPIDS AND GRANULOCYTIC DIFFERENTIATION BY NEOLACTO SERIES GANGLIOSIDES* (Received for publication, October 5, 1988, and in revised form, May 30, 1989) Seiichi KiltagawaS, Hisao NojiriS, Mitsuru NakamuraS, Robert E. Gallaghers, and Masaki SaitoST We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cella are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263,[7443][7444][7445][7446]. In calntrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, eslpecially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acidresistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The (differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2 3 sialylparagloboside and 2 + 3 sialylEhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLc molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and ( b ) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.
We have recently reported that the particular ganglioside molecules play an important role in the regulation of the differentiation of humlan myelogenous leukemia cell line HL-60 cells (1)(2)(3)(4)(5). The ganglio series ganglioside I13NeuAc-LacCer (GM3)l is increased during monocytic differentiation of HL-60 cells induced by phorbol 12-myristate 13-acetate (PMA).' When HL-60 cells are cultivated in the presence of G M~, the cells are differentiated into monocytic cells (1)(2)(3)(4). On the other hand, neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of HL-60 cells induced by retinoic acid or dimethyl sulfoxide. When HL-60 cells are cultivated in the presence of NeuAc-nLc, the cells are differentiated into granulocytic cells (1,2,5). These findings suggest that the appearance or accumulation of particular ganglioside molecules on the cell surface membrane plays an important role in the triggering of differentiation and the determination of differentiation direction in HL-60 cells.
In order to clarify further the role of gangliosides in differentiation induction, we analyzed in this paper the glycosphingolipids in HL-60 cells resistant to differentiation induction by retinoic acid. We found that (a) the content of gangliosides (sialic acid-containing glycosphingolipids), especially NeuAc-nLc, in retinoic acid-resistant HL-60 cells was markedly decreased as compared with that in parental wild-type HL-60 cells; ( b ) the content of gangliosides in retinoic acid-resistant HL-60 cells was not increased by cultivation with rctinoic acid; (c) retinoic acid-resistant HL-60 cells were differentiated into mature granulocytes when the cells were cultivated in the presence of NeuAc-nLc; and ( d ) wild-type HL-60 cells differentiated by NeuAc-nLc showed changes in ganglioside composition that were similar to those in wild-type HL-60 cells differentiated by retinoic acid.

MATERIALS AND METHODS
Cells and Cell Culture-Human myelogenous leukemia cell line HL-60 cells were grown in a serum-free synthetic medium (DME/F-12) as described (4,5). The retinoic acid-resistant HL-60 subline was selected by cultivating wild-type HL-60 cells in the presence of retinoic acid, the concentration of which was sequentially increased up to 1 pM as described previously (6, 7). Retinoic acid (1 p~) was always added to the culture medium for retinoic acid-resistant HL-60 cells unless otherwise indicated. For induction of the differentiation of HL-60 cells, cells were seeded at 2 X lo6 cells/ml and grown in the presence or absence of retinoic acid (Sigma) or NeuAc-nLc. The preparation of NeuAc-nLc used in the present experiments was a mixture of gangliosides isolated from peripheral blood granulocytes that were obtained from the patients with chronic myelogenous leukemia. The ganglioside preparation contained 96.5% NeuAc-nLc The nomenclature for glycosphingolipids follows the recommendations of the Nomenclature Committee of the International Union of Pure and Applied Chemistry (43).
16149 and 3.5% GM3 (5). The concentration of NeuAc-nLc was determined on the basis of lipid-bound sialic acid. Cell viability was determined by the erythrosin B dye exclusion test. Analysis of Gangliosides-Gangliosides from the cells were prepared by chloroform/methanol extraction and DEAE-Sephadex chromatography and analyzed quantitatively by densitometric scanning on a high performance thin-layer chromatography plate (Merck, Darmstadt, Federal Republic of Germany) as described (1,2,5,8). Lacto series gangliosides were further confirmed by the immunostaining method using monoclonal antibodies (9,10). The lipid-bound sialic acid of gangliosides was estimated by the resorcinol-HC1 method (11). Neutral glycosphingolipids were analyzed quantitatively by densitometric scanning on a high performance thin-layer chromatography plate as described (12). The hexose content of the neutral glycosphingolipid fraction was measured by the phenol-sulfuric acid method (13).
Determination of Cell Differentiation-The morphological assessment of the cells was performed under a light and an electron microscope as described (4,5). The activity of naphthol AS-D chloroacetate esterase or a-naphthyl butyrate esterase was determined by the esterase double staining method (5). The phagocytic activity toward polystyrene latex particles and the nitro blue tetrazolium reducing activity were measured as described (5). Superoxide (0;) release stimulated by PMA (100 ng/ml) or formylmethionylleucylphenylalanine (fMLP, 1 p~) was assayed spectrophotometrically by superoxide dismutase-inhibitable reduction of ferricytochrome c, and the continuous assay was performed in a Hitachi 557 spectrophotometer (a double wavelength spectrophotometer, Hitachi Ltd., Tokyo) as described (14). The release of 0; was calculated from the linear portion of cytochrome c reduction for PMA and from cytochrome c reduced for 5 min after the addition of fMLP. Surface membrane antigens were assessed by cytofluorometry in an Ortho Spectrum 111 (Ortho Diagnostic Systems, Inc., Westwood, MA) using the authorized monoclonal antibodies including anti-OKM1 (CDll), anti-OKB2 (CD24), anti-OKM5 (Ortho Diagnostic Systems), and anti-Mo2 (CDW14) (Coutler Immunology, Hialeah, FL).

RESULTS
Decreased Content of Gangliosides in Retirwic Acid-resistant HL-60 Cells-The thin-layer chromatograms of glycosphingolipids in wild-type and retinoic acid-resistant HL-60 cells are shown in Fig. 1, and the data are summarized in Table I. The total content of gangliosides of retinoic acid-resistant HL-60 cells was approximately one-sixth that of parental wild-type HL-60 cells. The content of G M~ was almost equivalent in both types of cells, whereas the content of NeuAc-nLc in retinoic acid-resistant HL-60 cells was markedly diminished as compared with that in wild-type HL-60 cells.   Other neutral glycosphingolipids with longer sugar chains.
When wild-type HL-60 cells were cultivated in the presence of retinoic acid (1 p~) for 4 days, the cells were differentiated into mature granulocytes with a concomitant increase in the content of NeuAc-nLc (Table I) (1,2,5). During retinoic acidinduced granulocytic differentiation of wild-type HL-60 cells, 2 + 3 sialylparagloboside and 2 4 3 sialylEhexaosylceramide were increased, whereas 2 6 sialylparagloboside was decreased. On the other hand, when retinoic acid-resistant HL-60 cells were cultivated in the presence of retinoic acid (1 p~) for 4 days, the cells were not differentiated and remained promyelocytes morphologically, and no increase in the content of NeuAc-nLc was observed (Table I).
The decreased content of gangliosides in retinoic acidresistant HL-60 cells may come from the specific defect of terminal sialylation or a general decrease of the synthesis of glycosphingolipids. If the specific defect of terminal sialylation is responsible, it is expected that neutral glycosphingolipids, the precursors of gangliosides, would be accumulated in retinoic acid-resistant HL-60 cells. As shown in Fig. 1 and Table 11, neutral glycosphingolipids were not accumulated in retinoic acid-resistant HL-60 cells. The total content of neutral glycosphingolipids of retinoic acid-resistant HL-60 cells

Gangliosides and Differentiation
of HL-60 16151 was rather slightly less than that of wild-type HL-60 cells.
The major component of neutral glycosphingolipids was LacCer in both types of cells. When the cells were cultivated in the presence of retinoic acid (1 p~) for 4 days, the total content of neutral glycosphingolipids in wild-type HL-60 cells was consistently increased and that in retinoic acid-resistant HL-60 cells was rather slightly decreased (Table 11).
Granulocytic Differentiation of Retinoic Acid-resistant HL-60 Cells by NeuAc-nLc-When wild-type HL-60 cells are cultivated in the presence of retinoic acid or NeuAc-nLc, the cells are differentiated into mature granulocytes (5). If NeuAc-nLc mediate the differentiation induction by retinoic acid and the defective synthesis of NeuAc-nLc in response to retinoic acid is responsible for the lack of differentiation in retinoic acid-resistant HL-60 cells, it is expected that retinoic acidresistant HL-60 cells could be differentiated into mature granulocytes when NeuAc-nLc are supplemented exogenously. When retinoic acid-resistant HL-60 cells were cultivated with 1 p~ NeuAc-nLc prepared from. mature granulocytes in the serum-free medium, an apparent morphological change reflecting differentiation along the granulocytic lineage was observed on day 2. On day 4, approximately 90% of the cells showed mature granulocytic cells morphologically (Fig. 2). The electron microscopic picture confirmed that HL-60 cells differentiated by NeuAc-nLc were mature granulocytes (Fig. 2). The granulocytic differentiation induced by NeuAc-nLc was also confirmed by the cytochemical lineagespecific esterase staining. When retinoic acid-resistant HL-60 cells were differentiated by NeuAc-nLc, the number of cells strongly positive for the granulocytic lineage-specific naphthol AS-D chloroacetate esterase activity was increased, whereas the number of cells positive for the monocytic lineage-specific a-naphthyl butyrate esterase activity was not altered ( Table 111). The number of cells capable of ingesting latex particles and reducing nitro blue tetrazolium was markedly increased by cultivating the cells in the presence of NeuAc-nLc (Table 111)   normal granulocytes (14). The effect of NeuAc-nLc on the 0;-releasing capacity was dependent on the concentration of NeuAc-nLc used as the inducer of differentiation (Figs. 3 and   4). As shown in Table IV, retinoic acid-resistant HL-60 cells differentiated by NeuAc-nLc expressed the surface membrane antigens recognized by monoclonal antibodies anti-OKM1 and anti-OKB2, whereas these cells did not express the antigens recognized by anti-OKM5 or anti-Mo2. Anti-OKM1 reacts on granulocytes and monocytes (15); anti-OKB2 reacts on granulocytes but not on monocytes (16); and anti-OKM5 and anti-Mo2 react on monocytes but not on granulocytes (17,18). Thus, the findings with surface membrane antigens indicated that retinoic acid-resistant HL-60 cells differentiated by NeuAc-nLc were granulocytic cells. The differentiation of retinoic acid-resistant HL-60 cells by NeuAc-nLc was accompanied by inhibition of cell growth (Fig. 5). Inhibition of cell growth by NeuAc-nLc was observed without a significant loss of cell viability. The higher concentrations of NeuAc-nLc (>2 p M ) were toxic to the cells.

Gangliosides and Differentiation of HL-60
NeuAcnLc ( Changes in the Ganglioside Composition of HL-60 Cells Differentiated by NeuAc-nLc-To determine whether the content of NeuAc-nLc was actually increased in HL-60 cells differentiated by NeuAc-nLc, we analyzed the ganglioside composition in wild-type and retinoic acid-resistant HL-60 cells differentiated by NeuAc-nLc. As shown in Table I, the total content of gangliosides in wild-type HL-60 cells differentiated by NeuAc-nLc was not altered. However, the ganglioside composition was changed during NeuAc-nLc-induced granulocytic differentiation of wild-type HL-60 cells; 2 + 3 sialylparagloboside and 2 + 3 sialylEhexaosylceramide were increased, whereas 2 + 6 sialylparagloboside was decreased. The changes in ganglioside composition of wild-type HL-60 cells differentiated by NeuAc-nLc were similar to those of wild-type HL-60 cells differentiated by retinoic acid ( Table  I). As with wild-type HL-60 cells, the total content of gangliosides was not significantly altered during NeuAc-nLcinduced granulocytic differentiation of retinoic acid-resistant HL-60 cells ( 4 . 0 pg of lipid-bound sialic acid/lOS cells). Although minimal changes in ganglioside composition might occur in these cells, it was difficult to perform accurate analysis of ganglioside composition because of the limited amount of NeuAc-nLc available for differentiation induction and the low content of NeuAc-nLc in retinoic acid-resistant HL-60 cells.

DISCUSSION
By using wild-type HL-60 cells, we have recently demonstrated that retinoic acid induces granulocytic differentiation with a concomitant increase in the content of NeuAc-nLc and that NeuAc-nLc added exogenously to the medium induce granlocytic differentiation (1, 2, 5). These findings support the hypothesis that retinoic acid stimulates the synthesis of endogenous NeuAc-nLc, which, in turn, trigger or promote the differentiation of HL-60 cells into the granulocytic lineage. In this context, NeuAc-nLc added exogenously to the culture medium may be incorporated into the plasma membrane (19, ZO), the lipophilic ceramide moiety being inserted into the lipid layer, and work as endogenous NeuAc-nLc do. In fact, we have observed that L3H]GM3 is rapidly incorporated into the cells. 3 The results of the present experiments with retinoic acid-resistant HL-60 cells provide additional evidence to support this hypothesis. The results presented here show that (a) the content of NeuAc-nLc of retinoic acid-resistant HL-60 cells was markedly diminished as compared with that of wild-type HL-60 cells; ( 6 ) retinoic acid induced neither granulocytic differentiation nor any increase of the content of NeuAc-nLc in retinoic acid-resistant HL-60 cells; and (c) NeuAc-nLc added exogenously to the medium induced differentiation of retinoic acid-resistant HL-60 cells into granulocytic lineage. These findings taken together suggest (a) that the synthesis of endogenous NeuAc-nLc is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and ( b ) that the defective synthesis of endogenous NeuAc-nLc is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.
The analysis of the ganglioside composition of wild-type HL-60 cells differentiated by NeuAc-nLc revealed that although the total content of gangliosides was not altered, the ganglioside composition was changed during granulocytic differentiation. The changes in ganglioside composition of wildtype HL-60 cells differentiated by NeuAc-nLc were similar to those of wild-type HL-60 cells differentiated by retinoic acid, supporting the concept that granulocytic differentiation of HL-60 cells is closely associated with the changes in particular ganglioside molecules. The changes in the ganglioside composition of wild-type HL-60 cells differentiated by NeuAc-M. Nakamura and M. Saito, unpublished results.

Differentiation of
HL-60 16153 nLc do not necessarily implicate simple accumulation of exogenous gangliosides since we have recently observed that a large part of exogenous ['H]GM~ incorporated into wild-type HL-60 cells was rapidly degraded in the cells. 3 It is possible that the changes in ganglioside composition in these cells may reflect the increased g1:ycosphingolipid metabolism during differentiation. These findings and the negligible alteration in the total content of gangliosides during NeuAc-nLc-induced granulocytic differentiation of retinoic acid-resistant HL-60 cells taken together suggest that persistent accumulation of a large amount of NeuAc-nLc is not necessarily required for promotion of cell differentiation. It is likely that either accumulation of a small amount of a particular NeuAc-nLc molecules or a transient association of particular NeuAc-nLc molecules with the plasma membrane may be sufficient for promotion of cell differentiation.
In retinoic acid-resistant HL-60 cells, the content of NeuAc-nLc was markedly diminished without any accumulation of neutral glycosphingolipids, the precursors of gangliosides, suggesting that the decreased content of gangliosides could not be explained by the defect of terminal sialylation alone. It is likely that in addition to the defect of terminal sialylation, a general decrease of the synthesis of glycosphingolipids may be responsible for the decreased content of gangliosides in retinoic: acid-resistant HL-60 cells.
Retinoic acid is believed to exert its biological effect in various types of cells by interacting with a specific cytoplasmic retinoic acid-binding protein (21)(22)(23)(24). However, it appears that modulation of tunnor cell growth rate by retinoic acid is not necessarily mediated by cytoplasmic retinoic acid-binding protein since the sensitivity of the cells to growth inhibition by retinoic acid does not correlate with the level of cytoplasmic retinoic acid-binding protein in the cell (25). In addition, leukemic cells, including HL-60 and KG-1 cells, are reported to lack such a. binding protein (26). Furthermore, it has been reported that retinoic acid-induced differentiation is initiated at the cell surface since granulocytic differentiation occurs when HL-60 cells are cultured with retinoic acid immobilized on a solid substrate (27). It is unknown how retinoic acid interacts with the cell surface membrane and what kinds of intracellular signals are produced by retinoic acid. The present and our previous experiments (5) suggest that the synthesis of endogenous NeuAc-nLc is an important step for granulocytic differentiation induced by retinoic acid. Thus it is possible that the defective enzyme system required for the synthesis of NeuAc-nLc may be responsible not only for the decreased content of NeuAc-nLc but also for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid. Another possibility is that the interaction between retinoic acid and the cell surface membrane may fail to activate the enzymes required for the synthesis of NeuAc-nLc in retinoic acid-resistant HL-60 cells.
It has been reported that exogenously added gangliosides alter the growth of various types of cells (28)(29)(30)(31)(32)(33)(34). Exogenous gangliosides inhibit the action of several growth factors as well as the tyrosine kinase activity associated with the growth factor receptors (31, 32); inhibit the proliferation of lymphocytes stimulated by leckins, antigens, or interleukin 2 (33); sensitize tumor cells to growth inhibitors (29); and stimulate the proliferation of astroglial (34) and neuroblastoma cells (30). Furthermore, the I3 subunit of cholera toxin, which binds specifically to ganglioside GMl on the cell surface membrane, has been shown to stimulate the proliferation of thymocytes (35) and quiescent nontransformed mouse 3T3 cells and to inhibit the growth of nzs-transformed 3T3 cells and rapidly dividing normal 3T3 cells (36). These observations and the present experiments strongly suggest that gangliosides play an important role in the regulation of cell differentiation as well as cell growth. The molecular basis for gangliosideinduced stimulatory or inhibitory effects remains to be determined. One possible mechanism is that the increased content of particular ganglioside molecules on the cell surface membrane may affect the cell metabolism by altering the functions or the enzyme activities of certain glycoproteins on the cell surface. It has been reported recently that protein kinases regulated by gangliosides are present in guinea pig brain (37, 38). Another possibility is that the interaction between the particular ganglioside molecules and the cell surface membrane may produce second intracellular messengers that are not yet identified. Ganglioside-specific binding protein has been demonstrated recently on rat brain membranes (39).
There are several reports of patients with promyelocytic leukemia, who have apparently benefited from the systemic therapy with retinoic acid (40-42). The present experiments indicate that NeuAc-nLc could be beneficial to the treatment of certain types of myelogenous leukemia in which leukemic cells become resistant to differentiation induction by retinoic acid. Further investigations into the mechanisms of retinoic acid-and NeuAc-nLc-induced differentiation of HL-60 cells may help to shed light on the pathophysiology in differentiation and proliferation of leukemic cells.