Cyclopamine induces eosinophilic differentiation and upregulates CD44 expression in myeloid leukemia cells

Leukemia Research 35 (2011) 638–645

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Cyclopamine induces eosinophilic differentiation and upregulates CD44 expression in myeloid leukemia cells Tsutomu Takahashi a , Koshi Kawakami a , Seiji Mishima b , Miho Akimoto a , Keizo Takenaga a , Junji Suzumiya c , Yoshio Honma a,∗ a b c

Department of Life Science, Shimane University School of Medicine, 89-1 Enya, Izumo, Shimane 693-8501, Japan Central Clinical Laboratory, Shimane University School of Medicine, Izumo, Shimane, Japan Cancer Center, Shimane University School of Medicine, Izumo, Shimane, Japan

a r t i c l e

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Article history: Received 23 June 2010 Received in revised form 27 August 2010 Accepted 27 September 2010 Available online 23 October 2010 Key words: Myeloid leukemia cell Differentiation Cyclopamine CD44 Eosinophil Luxol-fast-blue staining

a b s t r a c t Cyclopamine, a plant-derived steroidal alkaloid, inhibits the hedgehog (Hh) signaling pathway by antagonizing Smoothened. This drug can induce the differentiation of myeloid leukemia cell lines and acute myeloid leukemia (AML) cells in primary culture. The treated cells were stained with Luxol-fast-blue, which is specific for eosinophilic granules. Ligation of CD44 with some specific monoclonal antibodies can reverse the differentiation of AML cells. Combined treatment with cyclopamine and a monoclonal antibody to ligate CD44 more than additively induced the differentiation of HL-60 cells. These results may provide useful information for the development of a CD44-targeted therapy in AML. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Hedgehog (Hh) signaling plays an important role in the regulation of stem/progenitor cell expansion [1,2]. The Hh pathway is activated in some leukemia cell lines and primary leukemia cells [3–5]. Loss of Smoothened (Smo), an essential component of the Hh pathway, impairs hematopoietic stem cell renewal and decreases the induction of leukemia by Bcr-Abl oncogene [3,4]. These results indicate that Hh pathway activity is required for the maintenance of normal and neoplastic stem cells. The fact that the Hh pathway is constitutively activated in a wide range of tumors suggests that the Hh pathway may be important as a novel therapeutic target. Cyclopamine, a plant-derived teratogenic steroidal alkaloid, inhibits the Hh pathway by antagonizing Smo [6]. The first evidence regarding the antiproliferative effect of cyclopamine was reported by Dahmane et al. [7]. Recent reports have indicated that cyclopamine inhibits proliferation and induces apoptosis in several malignancies in vitro and in vivo [3–5]. Glucocorticoids have been reported to induce the monocytic differentiation of mouse myeloid leukemia cells [8] and apoptosis of human myeloid leukemia cells [9]. In contrast, glucocorticoids effi-

ciently inhibit erythroid differentiation of erythroleukemia cells [10]. Bufalin, a cardiotonic steroid enhanced the retinoid-induced differentiation of acute promyelocytic leukemia cells in primary culture [11]. Cyclopamine blocked the erythroid differentiation of normal progenitor cells and leukemia cells [12]. These results suggest that some steroids may switch on or off the cellular regulatory mechanism of differentiation. Adhesion molecule CD44 is a cell-surface transmembrane glycoprotein. As a receptor for hyaluronic acid, CD44 is involved in lymphocyte activation, recirculation and homing, adhesion of extracellular matrix, and migration [13]. CD44 has been shown to play an important role in normal myelopoiesis, and ligation of CD44 with some specific monoclonal antibodies can reverse the differentiation of AML cells, which raises the possibility of CD44-targeted differentiation therapy in AML [14–16]. The expression of CD44 may affect the ability of some AML cells to be induced to undergo differentiation.

2. Materials and methods 2.1. Materials

∗ Corresponding author. Tel.: +81 853 20 2351; fax: +81 853 20 2340. E-mail address: [email protected] (Y. Honma). 0145-2126/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2010.09.022

Cyclopamine and other Hh signaling inhibitors were obtained from Calbiochem. Purified anti-CD44 monoclonal antibody (clone A3D8) was purchased from Sigma Chemical.

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Table 1 Clinical profiles of AML patients and CD44 expression. Case

Diagnosis (FAB)

Age/gender

1 2 3 4 5 6 7 8 9 10 11 12 13 14

M2 M5 M6 M2 M2 M1 Biphenotypic M2 M5 M2 M5b M2 M2 M2

74/F 63/F 77/M 51/F 27/M 60/M 59/M 57/M 57/M 67/M 70/M 45/F 52/F 71/M

WBC (/␮l) 17,930 4800 1840 8290 15,500 42,800 1300 9800 8290 4960 67,100 2560 61,100 5470

Therapy outcome CR, relapse CR, BMT NR CR CR, PBT CR, BMT NR, BMT CR CR, relapse NR NR CR, relapse CR, BMT NR

Clinical characteristics

Supportive care t(8;21) CD10,19,13,33,34(+) t(8;21) Therapy-related, 47XY,+11 54XY CD7,13,33,34(+) t(8;21), CD56(+)

CD44 expression −/+ cyclopamine +++/+++ ++/+++ ++/++a ++/++a ++/++ ++/++b ++/++ ++/+++ ++/+++ +++/+++ +++/+++ ++/+ +++/+++ +++/+++

Evaluation of CD44 expression; +++, >80% positivity; ++, 50–80% positivity; +, 20–50% positivity. CR, complete remission; NR, no response; BMT, allogenic bone marrow transplantation; PBT, allogenic peripheral blood transplantation. Bold significantly increased (P < 0.05). a Slightly decreased. b Slightly increased.

2.2. Leukemia patients AML was diagnosed and classified according to the FAB criteria. Leukemic bone marrow specimens were collected at diagnosis, after all of the patients gave their written informed consent for sample collection in accordance with institutional policy. This study was approved by the Institutional Review Board at Shimane University. The clinical profiles are shown in Table 1. 2.3. Cells and cell culture Human myeloid leukemia cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 80 ␮g/ml gentamicin. Heparinized bone marrow aspirations were diluted with RPMI-1640 medium supplemented with 10% fetal bovine serum, overlaid on 15 ml of Ficoll-Paque Plus and centrifuged at 500 × g for 30 min. Only specimens that contained at least 70% leukemia cells were studied. 2.4. Assay of cell growth and properties of differentiated cells Suspensions of cells were incubated with or without the test compounds in multidishes. Cells were treated with anti-CD44 monoclonal antibody after dialysis against phosphate-buffered saline (PBS) to remove preservative. Superoxidegenerating oxidase was determined by the ability of the cells to reduce NBT [17]. Metachromatic cells in smears were determined by toluidine blue staining [18]. The cells were stained with Luxol-fast-blue for the evaluation of eosinophils [19]. Surface expression of myelomonocytic antigens was determined by monoclonal antibody labeling and flow cytometry, as described previously [17].

induced HL-60 cells to differentiate into neutrophils with fine granules (Fig. 2A–C). VD3 induced the monocytic differentiation of HL-60 cells (Fig. 2D). Undifferentiated HL-60 cells were weakly positive for metachromasia by toluidine blue staining (a marker of basophils), but metachromatic cells decreased and completely disappeared upon treatment with cyclopamine (Fig. 2E and F). On the other hand, untreated HL-60 cells showed weak and diffuse staining by the Luxol-fast-blue method (a marker of eosinophils) and differentiated cells contained granules that were strongly positive for Luxol-fast-blue staining (Fig. 2G and H). Cyclopamine significantly enhanced the expression of the surface antigen CD11b in a concentration-dependent manner when the cells were treated for 2 days (Fig. 1C). The drug also enhanced the expression of CD44 antigen, and reduced the expression of CD33. The expression of other CD markers was not significantly affected by cyclopamine. However, the expression of CD44 was not affected by other differentiation inducers such as ATRA and VD3, although these inducers efficiently upregulated CD11b expression in HL-60 cells (Fig. 1D). Cyclopamine also enhanced the expression of CD44 antigen in another leukemia cell line (THP-1) (Fig. 1E). These results indicate that cyclopamine preferentially enhances the expression of CD44 antigen during the induction of differentiation.

2.5. CD44 mRNA expression analysis by RT-PCR

3.2. Increase in CD44 mRNA expression by cyclopamine

Total RNA (1 ␮g) from leukemia cells was converted to first-strand cDNA primed with random hexamer in a reaction volume of 20 ␮l using an RNA PCR kit (TaKaRa Bio), and 4 ␮l of this reaction was used as a template in the polymerase chain reaction [17]. The quantitative RT-PCR reaction was performed using an ABI PRISM 7000 Real-Time PCR System (Applied Biosystems).

We examined whether this increase in CD44 expression is transcriptionally regulated by cyclopamine. Fig. 3 shows that cyclopamine significantly enhanced the expression of CD44 mRNA in a concentration-dependent manner. The time-course of the expression of CD44 mRNA revealed that cyclopamine significantly induced the expression of the gene within 24 h (Fig. 3C), suggesting that the increase in CD44 cell-surface antigen was transcriptionally regulated. Ligation of CD44 with some monoclonal antibodies against CD44 can reverse the differentiation-blockage of leukemia cells [14–16]. A3D8 is a CD44-ligating monoclonal antibody that activates the signaling pathway. We analyzed the effect of cyclopamine on the expression of CD44 antigen as recognized by A3D8 (Fig. 3D and E). After 7 ␮g/ml cyclopamine was added, the expression (ligation) of CD44 was significantly increased at day 2 (34.2 ± 2.9% CD44-positive cells with mean fluorescence intensity 14.1 ± 3.4% versus 23.2 ± 2.3%, 7.3 ± 1.8 in control), while the expression of CD44 as detected by a pan-CD44 antibody was slightly increased. These results indicate that cyclopamine significantly enhanced the expression of “functional” CD44 in HL-60 cells, as well as the upregulation of mRNA synthesis.

2.6. Statistical evaluation Statistical analyses were performed using an unpaired two-tailed Student’s ttest. Pearson’s correlation coefficient was used to evaluate the correlations between paired values.

3. Results 3.1. Effect of cyclopamine on the growth and differentiation of HL-60 cells Cyclopamine concentration-dependently inhibited the proliferation of human myeloid leukemia HL-60 cells, and induced NBT reduction, which is a typical marker of myelomonocytic differentiation, when the cells were treated for 6 days (Fig. 1A and B). Cyclopamine also induced morphological changes in HL-60 cells toward granulocytes with large reddish granules, while ATRA

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Fig. 1. Effects of cyclopamine on growth and differentiation of human myeloid leukemia cells. (A) Cell numbers in the presence of 0 (), 2 (), 4 (), and 6 () ␮g/ml cyclopamine. Cells were cultured with various concentrations of cyclopamine. The values are the means of three separate experiments. (B) Induction of NBT reduction by treatment with cyclopamine for 6 days. The values are the means (±SD) of four separate experiments. (C) Effects of cyclopamine on the expression of cell-surface antigens in HL-60 cells. Cells were cultured with cyclopamine for 2 days. The values are the means of three separate experiments. (D) Expression of cell-surface antigens on HL-60 cells treated with 6 ␮g/ml cyclopamine (CY), 60 nM ATRA or 90 nM VD3 for 2 days. The values are the means (±SD) of three separate experiments. (E) Expression of cell-surface antigens on THP-1 cells treated with 6 ␮g/ml cyclopamine for 5 days.

3.3. Induction of differentiation of HL-60 cells by combined treatment with anti-CD44 monoclonal antibody and cyclopamine Previous reports have indicated that ligation of CD44 with some specific monoclonal antibodies can reverse the differentiation of AML cells [14]. However, HL-60 cells were not efficiently induced to undergo differentiation by anti-CD44 monoclonal antibody (clone A3D8) under our experimental conditions (Fig. 3F), although HL-60 cells significantly expressed cell-surface CD44 antigen. When the cells were treated with anti-CD44 and cyclopamine, NBT reduction was induced in a concentration-dependent manner and cell proliferation was greatly inhibited. Morphological changes were also induced by the combined treatment. These results indicate that anti-CD44 can induce the differentiation of HL-60 cells with the aid of cyclopamine, which enhances the surface expression of CD44 antigen. 3.4. Enhancing effect of cyclopamine on the differentiation of leukemia cells treated with suboptimal concentrations of differentiation inducers ATRA and VD3 effectively induce the myelomonocytic differentiation of several myeloid leukemia cell lines. We examined the effect of cyclopamine on the differentiation of HL-60 cells induced by VD3. VD3 at 1 nM did not inhibit the proliferation of HL-60 cells, but, when combined with the same concentration of VD3, cyclopamine at concentrations of up to 6 ␮g/ml effectively inhibited the proliferation of HL-60 cells. VD3 induced NBT reduction,

and cyclopamine effectively enhanced NBT reduction induced by suboptimal concentrations of VD3 (Fig. 4B). ATRA-induced NBT reduction was also enhanced by cyclopamine when the cells were simultaneously treated with ATRA and cyclopamine for 5 days (Fig. 4C). To determine whether simultaneous treatment yields the best results, we examined the effects of pre- and posttreatment with cyclopamine on ATRA-induced NBT reduction by HL-60 cells (Fig. 4C). For pretreatment, cells were treated with various concentrations of cyclopamine for 2 days, washed in fresh medium, and incubated with ATRA for 5 days. For posttreatment, cells were treated with ATRA for 5 days, and then with cyclopamine for 2 days. Pretreatment with cyclopamine is more effective than posttreatment, which suggests that cyclopamine has a priming effect on the ATRA-induced differentiation of HL-60 cells. The enhancing effects of cyclopamine were also observed in other leukemia cells (Fig. 4D and E). Next, we examined the effects of other Hh inhibitors on the ATRA-induced differentiation of HL-60 cells. While SANT-1, AY9944 and U1866A alone hardly induced NBT reduction of HL-60 cells when the cells were treated for 4 days, these drugs significantly enhanced ATRA-induced NBT reduction (Fig. 5A). However, jervine, a potent Hh inhibitor, did not essentially affect ATRAinduced NBT reduction (Fig. 5B). On the other hand, tomatidine, which is similar to cyclopamine but does not inhibit the Hh pathway, efficiently enhanced ATRA-induced NBT reduction (Fig. 5C). These results suggest that the differentiation-enhancing effects are not associated with the inhibitory activities toward Hh signaling.

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Fig. 2. Morphological differentiation of HL-60 cells induced by cyclopamine, ATRA or VD3. Cells were treated without (A) or with 5 ␮g/ml cyclopamine (B), 60 nM ATRA (C), or 90 nM VD3 (D) for 7 days. Cytospin smear preparations were stained with May-Grünwald-Giemsa. (E and F) Toluidine blue staining. (G and H) Luxol-fast blue staining. (E and G) untreated HL-60 cells. (F and H) HL-60 cells treated with 5 ␮g/ml cyclopamine for 7 days. Magnification 1000×.

3.5. Effect of cyclopamine on the expression of CD44 antigen and differentiation-associated CD antigens of leukemia cells in primary culture Leukemia cells from patients with myeloid malignancies (Table 1) were freshly isolated and incubated with various concentrations of cyclopamine for 5–7 days. The CD44 cell-surface antigen was expressed on all of the leukemia cells we tested, and cyclopamine did not enhance the expression of CD44 in strongly positive cases (cases 1, 10, 11, 13 and 14, Table 1). Among the 10 cases with moderate expression, cyclopamine significantly enhanced CD44 expression in 3 cases (cases 2, 8 and 9). Both the percentage of positive cells and the expression intensity were concentration-dependently increased by treatment with cyclopamine (Fig. 6A–D). A slight increase in CD44 expression was observed in one case (AML-M1), a significant decrease was seen in one case, and a slight decrease was observed in two cases (Table 1). Next, we examined the expression of myelomonocytic differentiation-associated surface markers on leukemia cells treated with cyclopamine in primary culture. In all cases in which cyclopamine upregulated CD44 expression, the upregulation of CD11b, CD14 or CD15 and downregulation of CD33 or CD34 were also observed (Fig. 6F–H). Moreover, changes in these differentiation-associated markers were also observed in

5 of 10 cases in which the upregulation of CD44 was hardly observed, although these changes were less than those in the CD44 modulation-positive cases. In some cases, cyclopamine reduced the expression of CD44 and differentiation-associated surface antigens. We then examined the correlation between CD44 expression and differentiation-associated surface antigens. Fig. 6E shows that CD44 expression is closely associated with CD11b expression in leukemia cells treated with cyclopamine (correlation coefficient r = 0.76). 3.6. Combined effects of cyclopamine and differentiation inducers on the expression of differentiation-associated surface markers of leukemia cells in primary culture Leukemia cells from AML-M5, which were sensitive to the cyclopamine-enhanced expression of CD44, were incubated with cyclopamine and various inducers for 5 days. The expression of CD44 was increased by treatment with cyclopamine (Fig. 6A–D), but ATRA and VD3 did not further enhance the cyclopaminestimulated expression of CD44. However, CD11b was greatly enhanced by combined treatment with cyclopamine and VD3, but not ATRA (Fig. 6I). Untreated leukemia cells from an AML-M2 patient did not express CD15, a typical marker of granulocytes, and neither cyclopamine nor VD3 affected the expression. The expression of CD15 was slightly increased by ATRA or cotylenin A,

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Fig. 3. Effect of cyclopamine on the expression of CD44 mRNA (A–C) and cell-surface CD44 antigen (D and E). HL-60 cells were treated with various concentrations of cyclopamine for 2 days. (A and B) The expression of CD44 mRNA was examined by RT-PCR analysis. (C) Time-course of CD44 mRNA expression in HL-60 cells treated with 6 ␮g/ml cyclopamine. The levels of GAPDH expression are shown to demonstrate that equal amounts of RNA were used for RT-PCR. (D and E) () Cell-surface CD44 detected by pan-CD44 antibody; () cell-surface CD44 detected by A3D8 antibody. The values are the means (±SD) of four separate experiments. (F) Combined effects of cyclopamine and A3D8 antibody on NBT reduction of HL-60 cells. Cells were treated with A3D8 antibody in the absence (䊉) or presence of 3 () or 6 () ␮g/ml cyclopamine for 4 days. The values are the means (±SD) of four separate experiments.

Fig. 4. Combined effects of cyclopamine and VD3 on growth inhibition (A) and NBT reduction (B) of HL-60 cells. Cells were treated with various concentrations of cyclopamine in the presence of 0 (), 3 (), or 30 () nM VD3 for 4 days. The values are the means (±SD) of four separate experiments. (C) Enhancement by cyclopamine of ATRA-induced differentiation of HL-60 cells. (i) Cells were cultured with ATRA in the presence of 0 (), 2 (), or 4 () ␮g/ml cyclopamine for 5 days. (ii) Cells were cultured with 0 (), 2 (), or 4 () ␮g/ml cyclopamine for 2 days. The cells were then washed with fresh medium and reincubated with ATRA for 5 days. (iii) Cells were cultured with ATRA for 2 days and then recultured with 0 (), 2 (), or 4 () ␮g/ml cyclopamine for 5 days. The values are the means of four separate experiments. (D and E) Combined effects of ATRA and cyclopamine on NBT reduction of THP-1 (D) and U937 (E) cells. Cells were cultured with 6 ␮g/ml cyclopamine and/or 30 nM ATRA for 5 days. The values are the means (±SD) of four separate experiments.

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Fig. 5. Effects of various Hh inhibitors on NBT reduction of HL-60 cells in the presence of ATRA. (A) Cells were cultured with 0 (♦), 0.5 (), 1 (), or 1.5 () ␮g/ml of SANT-1 in the presence or absence of ATRA for 4 days. (B) Cells were cultured with 0 (♦), 3 (), 6 (), or 9 () ␮g/ml jervine in the presence or absence of ATRA for 4 days. (C) Cells were cultured with 0 (♦), 3 (), 6 (), or 9 () ␮g/ml tomatidine in the presence or absence of ATRA for 4 days. The values are the means of four separate experiments.

Fig. 6. Enhancement by cyclopamine of the expression of cell-surface CD44 antigen in primary culture of AML cells. (A and B) Case 9; (C and D) case 2. (A and C) % of CD44positive cells; (B and D) mean intensity of CD44. Cells were treated with various concentrations of cyclopamine for 7 days. The values are the means (±SD) of three separate experiments. (F–H) Expression of differentiation-associated antigens on AML cells in primary culture with cyclopamine. Cells were treated with various concentrations of cyclopamine for 7 days. (F) Case 9; (G) case 2; (H) case 8. The values are the means of three separate experiments. (E) Relationship between the levels of CD11b and CD44 on AML cells in primary culture with cyclopamine. Cells were treated with or without 6 ␮g/ml cyclopamine for 7 days. The ratio of surface expression in treated cells to that in untreated cells is plotted. (I and J) Combined effects of cyclopamine and other differentiation inducers on CD11b (l) and CD15 (J) expression for AML cells in primary culture. (I) Cells from case 2 were cultured with various concentrations of cyclopamine in the absence () or presence of 300 nM ATRA () or 10 nM VD3 () for 7 days. The values are the means (±SD) of three separate experiments. (J) Cells from case 8 were cultured with various concentrations of cyclopamine in the absence () or presence of 300 nM ATRA (), 10 nM VD3 (), or 5 mg/ml cotylenine A (䊉) for 5 days. The values are the means (±SD) of three separate experiments.

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another potent inducer of differentiation [20]. Combined treatment with cyclopamine and cotylenin A greatly induced CD15-positive cells (Fig. 6J). These results indicate that combined treatment with cyclopamine and other differentiation-inducers more than additively enhanced myelomonocytic differentiation in leukemia cells that were isolated from some AML patients.

4. Discussion Cyclopamine is a potent inhibitor of Hh signaling [1,2] and can induce the differentiation of myeloid leukemia cells. However, the present results indicate that the differentiation-inducing effects of cyclopamine and related compounds are not associated with their inhibitory activities toward Hh signaling. Some steroids can affect the differentiation of myeloid and erythroid leukemia cells [8–12]. These results suggest that cyclopamine acts as a ligand for a nuclear receptor(s) like steroid hormones. A recent study demonstrated that growth inhibition induced by cyclopamine in breast cancer cells is independent of Smo expression, suggesting that cyclopamine has unique secondary molecular targets [21]. CD44 expression is a function of eosinophil maturation of hematopoietic progenitor cells expressing CD34 antigen [22] and an activation marker of human eosinophils [23]. The ability of CD44 to bind to hyaluronic acid correlates with eosinophil peroxidase activity and major basic protein accumulation in the granules of maturing eosinophils [24]. Cyclopamine induced HL-60 cells to differentiate into granulocytes with eosinophilic granules and the upregulation of CD44 expression. HL-60 cells can differentiate to eosinophils when cultured under mildly alkaline conditions [25] or when treated with histone deacetylase inhibitors [26]. However, the upregulation of CD44 during eosinophilic differentiation has not been reported. CD44 is highly expressed in myeloid leukemia cells and downregulated during ATRA-induced differentiation [27,28]. Inducers of differentiation other than cyclopamine also suppressed the expression of CD44. The downregulation of CD44 mRNA expression was also observed in the ATRA-induced differentiation of HL-60 cells [28]. On the other hand, cyclopamine concentrationdependently upregulated the cell-surface CD44 antigen and its mRNA. The upregulation of CD44 by cyclopamine was also observed in other leukemia cell lines and in primary leukemia. These results suggest that the upregulation of CD44 during differentiation is a unique action of cyclopamine. However, the development of neutrophils from the human bone marrow has been associated with an initial decrease in CD44 expression followed by an increase during later stages of maturation [29]. The upregulation of CD44 may be more common in the maturation of other leukocytes. Hyaluronic acid stimulates the proliferation of CD34-positive cells into specifically differentiated mature eosinophils, and this augmented proliferation is completely attenuated by anti-CD44 antibody [22]. The present results in fresh leukemia cells suggest that the upregulation of CD44 expression is closely associated with cyclopamine-induced differentiation (upregulation of CD11b). However, the blockade of CD44 expression by treatment with antisense oligomer did not effectively suppress cyclopamine-induced differentiation (CD11b expression and morphological changes), which suggests that the expression of CD44 is independently regulated during cyclopamine-induced differentiation (data not shown). Cyclopamine did not further enhance CD44 expression in some fresh leukemia cells that strongly expressed CD44, which suggests that not all CD44 molecules are involved in cyclopamine-induced differentiation. CD44 proteins comprise a ubiquitously expressed family of cell-surface adhesion molecules that are involved in cell–cell and cell–matrix interactions. Multiple protein isoforms are encoded by a single gene by alternative splicing and are

further modified by a range of post-translational modifications with glycosylation. The degree of glycosylation can affect the ligand-binding characteristics of the protein and therefore alter its function [30,31]. Cyclopamine more efficiently enhanced the cell-surface CD44 antigen as recognized with A3D8 monoclonal antibody (Fig. 3), which suggests that the cyclopamine-treated cells were more sensitive to differentiation induced by A3D8. A3D8 alone did not effectively induce NBT reduction of HL-60 cells, which suggests that the monoclonal antibody was not a potent inducer. However, the combination of A3D8 and cyclopamine significantly induced NBT reduction of HL-60 cells. The upregulation of “functional” CD44 molecules which are effectively bound by ligands and/or activating antibodies might be more important than the upregulation of total mRNA and proteins for CD44 in cyclopamine-induced differentiation. Due to the diversity of their extracellular domains, CD44 proteins may interact with numerous different partners (ligands). Many ligands have been described, e.g. hyaluronate, fibronectin, collagen types I and IV, serglycin and osteopontin [31]. Cyclopamine may also induce ligand(s) of CD44 during the differentiation of HL-60 cells. Further experiments will be needed for us to better understand the role of CD44 signaling in the cyclopamine-induced differentiation of leukemia cells. A cure for leukemia requires the elimination of leukemic stem cells, which are capable of initiating and maintaining the leukemic clonal hierarchy [32]. Targeting of CD44 by an activating monoclonal antibody eradicates human AML stem cells [33]. Cyclopamine stimulated the expression of cell-surface CD44 antigen in some primary AML cells. Moreover, cyclopamine significantly increased the surface antigen which can interact with an activating monoclonal antibody directed to the adhesion molecule CD44. These results suggest that combined treatment with cyclopamine and the monoclonal antibody provides a more effective strategy for eliminating quiescent AML stem cells. Cyclopamine has been shown to retard the growth of human cancer xenografts [34–36]. Cyclopamine significantly prolonged median survival without apparent adverse effects in a pancreatic cancer mouse model [36]. However, cyclopamine has the potential for causing serious adverse effects in normal tissues because Hh signaling plays an important role in the regulation of normal stem cell expansion [1,2] and cyclopamine has teratogenic activity [6]. Differentiation-inducing activity was also observed for tomatidine, which is an analog of cyclopamine that does not inhibit the Hh pathway. These results suggest that we may be able to obtain novel therapeutic agents that target CD44 signaling without the inhibition of Hh signaling. Screening of these agents may provide valuable information for the development of a therapeutic strategy through the alteration of CD44 regulation. Ligation of CD44 by the activation of monoclonal antibodies can reverse the differentiation block of AML cells [14–16] and eradicate AML stem cells [33]. However, these interesting effects were modest in any of the AML cases. Combined treatment with these agents and the activation of monoclonal antibodies directed to CD44 may be useful in therapy for AML. Conflict of interest The authors declare no competing financial interests. Acknowledgements This study was supported in part by a research grant from the Shimane University Medical Education and Research Foundation. Contributions. T.T, K.K. and S.M. performed experiments and analyzed the data; M.A., K.T., and J.S. commented on the paper and Y.H. wrote the paper and designed the research.

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