(VK2) synergistically enhances cell dif - Nature

Leukemia (2002) 16, 1519–1527  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu

Combination of 22-oxa-1,25-dihydroxyvitamin D3, a vitamin D3 derivative, with vitamin K2 (VK2) synergistically enhances cell differentiation but suppresses VK2inducing apoptosis in HL-60 cells K Funato, K Miyazawa, M Yaguchi, A Gotoh and K Ohyashiki First Department of Internal Medicine (Hematology/Oncology), Tokyo Medical University, Tokyo, Japan

We originally reported that vitamin K2 (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D3 derivative, 22-oxa-1,25-dihydroxyvitamin D3 (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1␣,25(OH)2D3. In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias. Leukemia (2002) 16, 1519–1527. doi:10.1038/sj.leu.2402614 Keywords: vitamin K2; vitamin D3; apoptosis; leukemia cell; differentiation

Introduction Differentiation therapy in patients with acute promyelocytic leukemia (APL) using all-trans retinoic acid (ATRA) has now been well established as an effective strategy for the treatment of leukemia.1,2 Although there are other potent differentiationinducing reagents including 1␣, 25(OH)2-vitamin D3 (VD3) for leukemia cells in vitro, few of the inducers known to date seem to have therapeutic value for treatment of leukemia because of their severe adverse effects or the unusually high concentrations required.3 We originally reported that vitamin K2 (menaquinone: VK2) has a potent apoptosis-inducing effect in leukemia cell lines and primary cultured leukemia cells.4,5 Furthermore, several case studies have shown the clinical benefits of using VK2 for

Correspondence: K Miyazawa, First Department of Internal Medicine (Hematology/Oncology), Tokyo Medical University, 6-7-1, Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan; Fax: 81-5381-6651 KF and KM contributed equally to this report and should be regarded as co-first authors Received 19 April 2001; accepted 17 April 2002

the treatment of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS).6–8 Since VK2 has much lower toxicity as compared with other anti-cancer reagents, and can also be administered long term, VK2 is expected to open up novel strategies including chemoprevention for the management of patients with hematopoietic malignancies. Others have reported that VK2 also induces differentiation of acute myeloid leukemia cell lines such as HL-60 and U937.9 Recently, we observed that VK2 treatment induced monocytic differentiation in HL-60-bcl-2 cells, which enforced overexpression of BCL-2 by gene transfection, whereas VK2 induced apoptosis in HL-60-neo cells.10 It is noteworthy that although HL-60-bcl-2 became almost completely resistant to apoptosis induction by VK2, monocytic differentiation via G0/G1 arrest was still observed.10 This suggests that VK2 promotes not only apoptosis induction but also a differentiation-inducing effect against the leukemia cells, which are resistant to VK2-inducing apoptosis. It has been well documented that VD3 is capable of inhibiting proliferation and inducing differentiation of leukemic myeloid cells into the monocyte/macrophage lineage.3,11 Furthermore, clinical trials of VD3 in patients presenting with MDS produced a transient increase in peripheral blood cell numbers.12,13 However, about 50% of the patients treated developed hypercalcemia.12,13 22-oxa-1,25-dihydroxyvitamin D3 (OCT: oxa carcitriol) is a unique vitamin D analogue with less calcemic activity than calcitriol, and it effectively suppresses parathyroid hormone (PHT) secretion in uremic patients receiving dialysis therapy.14–16 In the present study, we focused on the combined effects of VK2 and OCT on leukemia cells. Our data clearly demonstrates that a combination of OCT and VK2 synergistically enhances induction of monocytic differentiation of HL-60 cells, suggesting the clinical benefit of the combined use of these vitamins.

Materials and methods

Reagents and antibodies Menaquinon-4 was donated by Eisai Co. (Tokyo, Japan). 1␣,25(OH)2-vitamin D3 and 22-oxa-1␣,25(OH)2-vitamin D3 (OCT: oxacalcitriol) were supplied by Chugai Pharmaceutical Co. (Tokyo, Japan).15 Fluorescein isothiocyanate (FITC)-conjugated human CD33 monoclonal antibody (mAb) was purchased from DAKO (Glostrup, Denmark), and CD11b, CD13 and CD15 mAbs were from Immunotech (Marseille, France). Phycoerythrin (PE)-conjugated human CD11c mAb, phycoerythrin cyamine 5 (PC5)-conjugated anti-human CD14, CD45, and APO2.7 mAbs were also obtained from Immunotech.

Effectsof OCT plus VK2 on leukemia cells K Funato et al

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Cell line and primary cultured leukemia HL-60 cells obtained from the American Type Culture Collection (Rockville, MD, USA) were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (100 ␮g/ml). Exponentially growing HL-60 cells were used for further experiments. In some experiments, leukemia cells freshly isolated from six patients, consisting of three de novo AML and three secondary AML were used. All patients gave informed consent prior to the study. Mononuclear cells were obtained from the heparininzed bone marrow aspirate by Ficoll–Hypaque fractionation. Isolated mononuclear cells consisting of more than 80% of leukemic blasts were cultured as well as HL-60 cells.

tion by 2% agarose gel electrophoresis in a limiting dilution system using apoptotic cells and non-apoptotic cells.5

Assays for caspase-3 activity Caspase-3 activity was examined by flow cytometry using a substrate reagent kit containing PhiPhiLux-G6D2, a rhodamine-containing specific substrate with amino acid sequence GDEVDGI (Oncolmmunin, College Park, MD, USA). After treatment with/without VK2 and OCT, cells were washed with PBS and incubated with a substrate reagent at 37°C for 60 min. Fluorescence of the profluorescent substrate cleaved by activated caspase-3 was analyzed by flow cytometry at FL2 channel with excitation at 488 nm.

Flow cytometric analysis Cellular antigens and viable cell numbers were analyzed by flow cytometry using EPICS XL II (Beckman-Coulter Japan, Tokyo, Japan). Before staining with antibodies, cells were preincubated with a human ␥-globulin, Venoglobulin-IH (Welfide Co., Osaka, Japan), for 30 min at room temperature for blocking antibody binding to Fc receptors. Thereafter, cells were incubated with FITC-, PE- or PC5-conjugated mAbs for 30 min at 4°C. After washing three times with PBS, fluorescent intensities were analyzed. For the assessment of the viable cell count, HL-60 cells treated with/without either VK2 and/or OCT were stained with a solution containing 1% (v/v) propiodium iodide (PI) (Sigma Chemical Co., St Louis, MO, USA) for 30 min at 4°C. First, the gating area of a cytogram for detecting viable HL-60 cells was established according to the PI staining-negative area (indicating viable cells) and the forward- and side-scatter intensities. Then the cell cultures were pipetted gently to obtain uniform cell suspension, and were introduced to a flow cytometer. The number of cells in the gating area for viable HL-60 cells was assessed for 60 s. The number relative to the cells treated with a control proved revealed to be well-correlated with the results obtained from a Cell Counting Kit (Dojin East, Tokyo, Japan), with absorption measurements at 450 nm.17

Cell cycle analysis Cells were fixed and stained with a solution containing 1% PI, 100 ␮g/ml digitonin, 0.01% NaN3, 200 ␮g/ml RNAase (Sigma), and 2.5% FCS for 10 min at room temperature. Cells were analyzed by flow cytometry with a cell cycle analysis program, MultiCycle AV (Phoenix Flow Systems, San Diego, CA, USA).

Assessment of apoptosis The cells undergoing apoptosis were assessed by flow cytometry using PC5-conjugated APO2.7 mAb (clone 2.7; Immunotech), which was raised against the 38 kDa mitochondrial membrane protein (7A6 antigen) specifically expressed by cells undergoing apoptosis.18 We have reported that detection of the percentage of APO2.7-positive cells was consistent with the results of the TUNEL method and DNA ladder formaLeukemia

Assessment of cell differentiation Differentiation of HL-60 cells was assessed by morphology and cell surface antigen expressions using flow cytometry as described above. For morphologic observations, the cell suspension was introduced in Shandon Cytospin 2 (Shandon, Pittsburgh, PA, USA) and the preparations were stained with May-Giemsa. For assessment of monocytic differentiation, non-specific esterase staining was further performed using an Esterase Staining kit (Muto Chemical Co., Tokyo, Japan).

Statistics Data are given as the mean ± s.d. Comparisons between the two groups were assessed with a Student’s t-test.

Results

OCT induces differentiation of HL-60 cells more potently as compared with 1␣,25(OH)2D3 It was previously reported that OCT showed more differentiation-inducing effects on leukemia cells in vitro as compared to the effects of 1␣,25(OH)2D3.19 To confirm this evidence, we first examined the differentiation-inducing activities of OCT and 1␣,25(OH)2D3 in HL-60 cells. As shown in Figure 1, 72 h exposure to various concentrations of either OCT or 1␣,25(OH)2D3 revealed that a significant enhancement of the expression of CD14 was induced by 5 × 10−11 M of OCT, whereas the equivalent effect was induced over 5 × 10⫺10 M of 1␣,25(OH)2D3 in vitro. This result was consistent with the expressions of CD11c and C13 and also monocytic morphologic changes in HL-60 cells as evaluated by May-Giemsa staining after 72 h of treatment with either OCT or 1␣,25(OH)2D3 (data not shown). However, both over 5苲10 nM of OCT and 1␣,25(OH)2D3 exerted the maximal effect of differentiation induction and reached a plateau. This more potent ability of OCT19 shown previously in vivo for promoting differentiation with a lesser extent of inducting hypercalcemia might be an important clinical benefit for treating patients with AML. Therefore, we used OCT in stead of 1␣,25(OH)2D3 for further experiments.


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Figure 1 Induction of CD14 expression after treatment with OCT and 1␣,25(OH)2D3 in HL-60 cells. HL-60 cells (2 × 105 cells/ml) were treated with either OCT or 1␣,25(OH)2D3 at various concentrations (0.01苲50 nM) for 72 h. Thereafter, the cells were processed by flow cytometry to evaluate CD14 expression as described in Materials and methods. This is a reproducible result from one of three separate experiments.

Combination of VK2 plus OCT suppressed VK2induced apoptosis but enhanced induction of G0/G1 arrest in HL-60 cells VK2 was reported to potently induce apoptosis of primary cultured leukemia cells and leukemia cell lines including HL-60 cells.4,10 Therefore, we first examined growth inhibition and apoptosis induction of HL-60 cells after treatment with VK2 and OCT. Flow cytometry revealed that treatment with VK2 activates caspase-3 activity and induces apoptosis in HL-60 cells as previously reported (Figure 2).10 In the presence of 10 nM of OCT alone, no caspase-3 activity and apoptosis induction was observed, and rather suppressed spontaneous caspase-3 activation of HL-60 cells cultured in control medium containing 10% FCS. Notably, by the combined treatment with VK2 plus OCT, VK2-induced caspase-3 activation and apoptosis induction was significantly suppressed. As shown in Figure 3, viable cell number after 96 h exposure to VK2 was also significantly suppressed in the presence of OCT. Treating the cells with OCT alone resulted in some increase in viable cell number as compared with the cells cultured in control medium (P = 0.048). Cell cycle analysis demonstrated that the accumulation of the cells in the G0/G1 phase was increased after 48 h exposure to VK2 alone, as compared to the cells cultured in control medium (Figure 4). Furthermore, cell cycle arrest in the G0/G1 phase was further enhanced in the presence of OCT plusVK2.

Combined treatment of OCT plus VK2 dramatically promoted monocytic differentiation of HL-60 cells Many lines of evidence suggest that G1 arrest is a critical checkpoint for the cells to differentiate in various cell culture systems.20–24 Based on the result of enhanced G0/G1 arrest

Figure 2 Induction of apoptosis in the presence of VK2 and OCT in HL-60 cells. After treatment of HL-60 cells with either VK2 (10 ␮M) and/or OCT (10 nM) for 72 h, caspase-3 activity and apoptosis induction were assessed by flow cytometry as described in Materials and methods. The number in each panel indicates the percentage of the fluorescence substrate cleaved by activated caspase-3 and the HL-60 cells positive for APO 2.7, respectively. This is one of the representative results from five separate experiments.

after combined treatment with VK2 plus OCT, we next evaluated the differentiation-inducing ability using these vitamins. Expression levels of CD14, which is a differentiation marker for monocytic cells,25 were quantitatively assessed by flow cytometry. Since VK2 induces apoptosis rather than differentiation as shown in previous reports,4,5,10 the gating area was established for detecting the viable HL-60 cells on the cytogram for the assessment of CD14 expressions. As shown in Figure 5a, the expression levels of CD14 on the viable cells gated in flow cytogram significantly increased after 72 h exposure to either 10 ␮M of VK2 or 1 nM of OCT. It was noteworthy that combined treatment with VK2 plus 1nM of OCT dramatically enhanced CD14 expression. This effect far exceeded the effect of treatment with 10 nM OCT, at an optimal condition for leading the maximal differentiation induction in HL-60 cells. These results were consistent with the morphologic changes of HL-60 cells shown in Figure 5b and c; either VK2 or OCT alone for 72 h treatment induced some monocytic differentiation such as condensation of nuclei, and enlargement of cytoplasm as compared with the cells treated with control medium. The cells treated with VK2 plus OCT further increased evidence of morphologic differentiation into mature monocytic cells. Enhanced staining for non-specific esterase also supports the maturation of HL-60 cells after exposure to VK2 plus OCT. Leukemia


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small population of HL-60 cells, which appeared be exempted from apoptosis induction of VK2 pre-exposure and were still viable at 96 h (Figure 7b). Pre-exposure to OCT followed by VK2 treatment showed some induction of apoptosis but no significant enhancement of differentiation as compared to the effects of OCT alone. These data suggest that the simultaneous exposure to both OCT and VK2 appears to be required to promote the maximal monocytic differentiation.

Enhancement of differentiation of primary cultured leukemia cells by combined treatment of OCT plus VK2

Figure 3 HL-60 cell growth after exposure to VK2 and OCT. HL60 cells (2 × 105 cells/ml) were cultured in the presence or absence of either OCT (10 nM), VK2 (10 ␮M), or OCT+VK2 for 96 h, and then the viable cell numbers were assessed as described in Materials and methods. This is one of the representative results from three separate experiments.

To examine whether this enhanced differentiation can reproduce in primary cultured leukemia cells, we next treated leukemia cells freshly isolated from six patients with AML. Their background and leukemic cell phenotypes are summarized in Table 1. After 48 to 72 h treatment of freshly isolated leukemia cells with OCT and/or VK2 in vitro, induction or enhancement of the lineage-specific antigen expressions including CD11c, CD13 and CD14 were examined. Pronounced differentiation after exposure to OCT plus VK2 was observed in four out of six cases of primary cultured leukemia cells (Table 2 and Figure 8). These data demonstrated that, as well as in HL-60 cells, enhancement of differentiation induction can be reproduced in some cases of primary cultured leukemia cells. Discussion

Figure 4 Cell cycle analysis after exposure to either VK2 and/or OCT in HL60 cells. HL-60 cells were treated with OCT (10 nM), VK2 (10 ␮M), and OCT plus VK2 for 72 h, then cell cycle analysis was performed as described in Materials and methods. This is one of the representative results from five separate experiments.

We next performed the kinetics analysis of differentiation induction in response to OCT plus VK2. HL-60 cells were treated with either VK2 at a fixed concentration of 10 ␮M plus 0.1 to 10 nM of OCT or with OCT fixed at 1 nM plus 0.1 to 10 ␮M of VK2 for various lengths of time, then CD14 expression levels were examined (Figure 6). Compared with the cells treated with either VK2 or OCT alone, a prominent enhancement of CD14 expression can be detected within 24 h by combining 10 ␮M of VK2 plus over 1 nM of OCT. Figure 7 demonstrates the effects of sequential treatments with these vitamins in apoptosis and monocytic induction. After pretreatment with either OCT or VK2 for 48 h, HL-60 cells were subsequently exposed to either VK2 and/or OCT for 48 h. When the cells were pretreated with VK2 and followed by OCT exposure, apoptosis induction was prominent, which resulted in reduction of viable cell number at 96 h. In this case, there was no significant difference in apoptosis induction and viable cell number as compared with the cells treated with VK2 alone. However, it was of interest that the dramatic enhancement of monocytic differentiation was observed in a Leukemia

In the present study, we have demonstrated that combined treatment with OCT plus VK2 dramatically enhances monocytic differentiation of HL-60 cells. This enhanced effect far exceeds the maximal differentiation-inducing ability of OCT alone (Figure 5). In addition, in four out of six cases of patients with AML, freshly isolated myeloid leukemia cells as well as HL-60 cells showed pronounced differentiation in response to combined treatment with OCT plus VK2 in vitro as compared to the cells cultured with either OCT or VK2 alone (Table 2). The mechanism of this enhancement of differentiation induction is still not understood. However, the population of HL-60 cells in the G0/G1 phase was significantly increased after treatment with OCT plus VK2 (Figure 4). Many lines of evidence support the concept that cell cycle arrest and differentiation are tightly linked processes:20–24 retinoic acid-dependent growth arrest of the LAN-5 neuroblastoma cell line is associated with accumulation of p27KIP1 leading the cells to G1 arrest and induced terminal differentiation.20 Overexpression of p21CIP1 induces cell differentiation along with G1 arrest in a human glioma cell line.21 In U937 cells, treatment with 1,25(OH)2D3 induced up-regulation of the gene expression of cyclin-dependent kinase inhibitors p21, p25, p15 and p18 along with G1 arrest during myeloid differentiation.22 Growth arrest in the G1 phase also resulted in the production of melanin in murine melanoma B16 cells after treatment with mannosylerythriol lipid.23 Furthermore, in normal human keratinocytes, the introduction of eta and delta isoforms of protein C leads to G1 arrest and differentiation.24 Theses data suggest that G1 arrest is the critical process during which the cell undergoes differentiation, although some reports argue that G1 arrest is part of a broader molecular program during differentiation, but is not enough by itself.25–27 As shown in Figure 4, G0/G1 arrest was indeed enhanced along with pronounced differentiation after combined exposure to


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Figure 5 Induction of monocytic differentiation after treatment with either OCT and/or VK2 in HL-60 cells. (a) CD14 expressions: After 72 h exposure to OCT (1 nM, 10 nM), VK2 (10 ␮M), and OCT (1 nM) plus VK2, CD14 expression levels were assessed by flow cytometry. The upper number of each panel represents the mean channel number of fluorescent intensity for CD14 in HL-60 cells. The number in parentheses represents the percentage for CD14-positive HL-60 cells. (b) May-Giemsa staining: HL-60 cells were treated with OCT (10 nM) , VK2 (10 ␮M), and OCT (1 nM) plus VK2 (10 ␮M) for 72 h. (original magnification was ×1000). (c) Non-specific esterase staining: HL-60 cells were treated with OCT and/or VK2 as described above in (b). Not shown is that the esterase-positive cells showing brown granular staining which disappeared when the cytospin preparations were pre-treated with NaF solution (original magnification was ×1000). These are one of the representative results from three separate experiments.

OCT and VK2 in HL-60 cells. Not shown is that combined treatment with OCT plus VK2 further reduced the expression levels of phosphorylated Rb along with enhanced molecular interaction between p27KIP1 and cdk2 in HL-60 and U937 (Iguchi and Miyazawa, manuscript in preparation). These data may in part explain the synergistic enhancement for differentiation. However, comparing the differentiation-inducing effect in HL-60 cells between OCT and VK2 alone, VK2 induced cell cycle arrest more potently as compared to OCT (71.4% vs 61.8%), while OCT showed more prominent differentiation (Figures 4 and 5). Therefore, pronounced differentiation induction is not simply explained by enhanced cell

cycle arrest in the G0/G1 state. It has also been reported that non-genomic effects of VD3 such as an increase of intracellular free calcium,28 rapid and transient activation of phosphatidylinositol 3-kinase,29 and extracellular signal-regulated kinases (ERKs)30 are of major importance for the induction of the monocytic maturation process, and all these events appear to be independent of cell cycle regulation. Since the synergistic enhancement of monocytic differentiation can be detected even after 24 h exposure to OCT and VK2 (Figure 6), and also the simultaneous exposure to both vitamins appeared to be required for the maximal differentiation induction (Figures 6 and 7), some modulations of these initial signaling events may Leukemia


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Figure 6 Kinetics of induction of CD14 expression after exposure to OCT plus VK2. (a) HL-60 cells were treated with either 10 ␮M of VK2 plus OCT (0.1 to 10 nM) or 1 nM of OCT plus VK2 (0.1 to 10 ␮M) for various lengths of time. Then CD14 expression levels were assessed by flow cytometry. A concentration of VK2 was fixed at 10 ␮M on the left panel, whereas OCT was fixed at 1nM on the right panel. This is one of the representative results from three separate experiments.

occur by combined treatment with OCT plus VK2. Another possible explanation for this evidence is molecular modulation of nuclear receptor bindings and co-transcription factors leading activation of specific gene transcriptions for OCT and VK2. 1␣,25(OH)2D3 has been well-known to mediate its biological activity through specific binding to the nuclear vitamin D receptor (VDR).3 The VDR, bound to 1␣, 25(OH)2D3, forms a heterodimer with a nuclear accessory factor, retinoid X receptor (RXR), and subsequently binds to specific nucleotide sequences or a vitamin D-responsive element (VDRE) to induce a series of gene transcription.31–33 In addition, various transcriptional co-activators such as SRC-1, TIF2 and AIB-1 have been known to participate in these processes.33 As well as 1␣,25(OH)2D3, OCT has been shown to bind VDR with an extremely low binding affinity but with potent transcription activities for genes having VDREs.34 In contrast, although it was proposed that the nuclear binding protein for vitamin K2 exists in nuclei similar to other vitamin receptors and that the molecular structure is very close to that Table 1

Case No. 1 2 3 4 5 6

of human GAPDH,35 the specific receptor for VK2 is still not identified as yet. It is of interest that VK deficiency in rats has been reported to markedly increase the VDR receptor binding to DNA and VK2-dependent ␥-carboxylation of VDR,36 suggesting some specific transcriptional modulation and interaction between OCT and VK2 may exist. The data shown here suggest that the HL-60 cell system appears to be a good model for further studies of the cross-talk between VD3 and VK2 signaling pathways. In contrast to pronounced differentiation, apoptosis induction of HL-60 cells by VK2 was significantly suppressed in the presence of OCT (Figure 2). Since OCT alone did not activate caspase-3 (Figure 2), and rather supports the survival of HL60 cells (Figure 3), OCT seemed to function as an inhibitor for VK2-inducing apoptosis in HL-60 cells. However, others have reported that, in U937 cells, subcellular localization of p21CIP1 has been changed from the nucleus to the cytosol along with monocytic differentiation in response to VD3 treatment, and this cytosolic p21CIP1 blocked apoptotic stimuli by binding to the stress-activated ASK1 and inhibiting its kinase activity.37 Therefore, becoming resistant to VK2-inducing apoptosis might be simply due to achieving monocytic maturation itself in response to OCT and VK2 treatment. In this aspect, monocytic differentiation and apoptosis induction appeared to be regulated inversely, and it appears not to become a disadvantage for the clinical application of a combined VK2 plus OCT therapy. In clinical trials of VD3 in patients with MDS, 1␣(OH)D3 treatment produced a transitory increase in the peripheral blood cell number. However, 50% of these patients treated developed hypercalcemia.13 Furthermore, oral administration of 1␣(OH)D3 to patients with MDS produced a sustained hematological response, in terms of increases in neutrophil and platelet counts, in half of these patients tested.38 In a randomized study of alfacalcidol in refractory myelodysplastic anemias, hypercalcemia was also reported to be the major toxic side-effect and to function as a dose-limiting factor for a series of clinical trials.39 In contrast, OCT has been shown to have less effect on hypercalcemia and has already been widely used for the treatment of hemodialyzed patients with secondary hyperparathyroidism.14,40 In addition, patients with osteoporosis have been treated with daily oral VK2 (menaquinone-4) administration.41 Therefore, the safety and non-toxicity of both OCT and VK2 for long-term administration have been established. Promoting the remarkable differentiation induction of leukemia cells shown here suggests that OCT and VK2 are strong candidates for differentiationbased therapy of AML and MDS.

Background of patients and cell surface markers of leukemia cells

Initials

Age

Gender

MH NM NK KT YY IT

53 62 74 63 58 62

f f m m f m

Diagnosis

Surface antigens of leukemic clone

AML-M1 AML-M2 AML-M2 post-MDS AML post-CMPD AML post-CNL AML

CD13, CD33, CD13, CD16, CD13, CD13,

CD15, CD38, CD34, CD33, CD15, CD15,

CD33, CD41, CD38, CD34 CD33 CD34,

CD34, CD64, CD7 CD61 CD41, CD64, CD7 CD64

The immunophenotype of leukemic clones was assessed by flow cytometry. To detect leukemic clones, the cells showing CD45 dull positive with low side scatter intensity by flow cytometry were focused as ‘a blast gate’ and their immunophenotype were analyzed in all cases. CMPD, chronic myeloproliferative disease; CNL, chronic neutrophilic leukemia. Leukemia


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Figure 7 Effects of sequential exposures to OCT and VK2 on apoptosis and differentiation induction of HL-60 cells. HL-60 cells (1 × 105 cells/ml) were pretreated with either VK2 (10 ␮M) or OCT (1 nM) for 48 h. After washing twice with PBS, the cells were re-suspended with the medium containing either OCT (1 nM), VK2 (10 ␮M), or OCT (1 nM) plus VK2 (10 ␮M), and subsequently cultured for another 48 h. Then cell surface APO2.7 expression and viable cell numbers (a), and cell surface antigen expressions (b) were assessed by flow cytometry as described in Materials and methods. As a control, HL-60 cells (1 ⫻ 105 cells/ml were cultured with control medium for 48 h, washed twice with PBS, and the cell pellets were re-suspended with fresh control medium, cultured for another 48 h. This is one of the representative results from the two separate experiments.

Table 2

Case No. 1 2 3 4 5 6

Differentiation induction of primary cultured leukemia cells after 72 h treatment with either OCT and/or VK2

VK2 differentiationa ( ( ( ( ( (

− + + − + +

) ) ) ) ) )

OCT differentiationa ( ( ( ( ( (

− + + − − +

) ) ) ) ) )

VK2+OCT enhanced differentiationb ( ( ( ( ( (

− + + − + +

) ) ) ) ) )

a

Induction of differentiation (+) in response to either VK2 or OCT alone was defined when a greater than 15% increase in the mean channel number of fluorescent intensity for more than one lineage-specific antigens including CD11c, CD13 and CD14 was detected in the cells of ‘the blast gated area’ as compared with that in the cells cultured in the control medium. b Enhancement in differentiation by combination of VK2 plus OCT represents a greater than 15% further increase in the mean channel number of fluorescent intensity for more than one of the lineage-specific antigens in the cells of ‘the blast gated area’ as compared with the cells treated with either VK2 or OCT alone.

Leukemia


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Grant from Research Committee for Idiopathic Hemopoietic Disorders of the Ministry of Health and Welfare in Japan to KO. References

Figure 8 Flow cytograms of primary cultured leukemia cells in case No. 2 after treatment with OCT and VK2. Representative data demonstrating a pronounced induction of differentiation in primary cultured leukemia cells by the combined treatment with OCT plus VK2. Mononuclear bone marrow cells isolated from a patient with AML (case No. 2) were cultured in the presence of OCT (1 nM) and/or VK2 (10 ␮M) for 72 h. Thereafter, flow cytometry was performed to detect viable cell numbers and cell surface antigen expressions as described in Materials and methods. The upper number of each panel indicates the percentage for the CD11c-positive leukemic clone, and the number in parentheses indicates the mean channel number of fluorescent intensity for CD11c in leukemia cells.

Acknowledgements We thank Ayako Hirota for her excellent technical assistance. We also thank Eisai Co. (Tokyo, Japan) for providing VK2 (menaquinone-4), and Chugai Pharmaceutical Co. (Tokyo, Japan) for providing OCT and 1␣,25(OH)2D3. This study was supported by a Grant-in-Aid for Science Research (C) from The Ministry of Education, Science, Sports and Culture of Japan to KM (No. 11671017) and a Health Science Research Leukemia

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