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BIOPHA 3403 1–8 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
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Original article
Differentiation-stimulating potency of differentiated HL60 cells after drug treatment Q1 Cong
Wang, Qun Zhang, Bao-Di Gou, Tian-Lan Zhang * , Kui Wang
Department of Chemical Biology, Peking University School of Pharmaceutical Sciences, 38, Xueyuan Road, Beijing 100191, P.R. China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 April 2014 Accepted 28 May 2014
Differentiation therapy in the treatment of leukemia is often hampered by limitations on using certain pharmaceutical regents or on the required doses due to various reasons, such as drug-resistance and retinoic acid syndrome. To circumvent these problems, a strategy might be developed on the basis of the ability of drug-differentiated cells to stimulate differentiation in leukemia cells. Using the promyelocytic leukemia cell line HL60 as a cell model, we assessed the differentiation-stimulating potency of differentiated granulocytes and monocytes/macrophages after treatments with all-transretinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. ATRA- and TPA-differentiated cells were able to stimulate differentiation in fresh HL60 cells, accompanied by inhibition on cell growth to various extents. The differentiated cells of the second generation, especially those originated from TPA treatment, were as potent as the drugs themselves in stimulating differentiation in fresh HL60 cells. On the basis of ‘‘differentiation induced by differentiated cells’’, we explored the feasibility of ex vivo therapy. ß 2014 Published by Elsevier Masson SAS.
Keywords: Leukemia Drug-differentiated cell Cell-induced differentiation Tetradecanoylphorbol acetate Phorbol ester Retinoic acid
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1. Introduction
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Now that leukemia is characterized by the blockade of differentiation in cellular maturation, differentiation therapy [1,2] would be a reasonable choice in clinical practice. Indeed, since 1980s, differentiation therapy has achieved great success in the treatment of acute promyelocytic leukemia (APL). This specific type of leukemia accounts for 10% to 15% of new cases of adults with acute myeloid leukemia (AML) per year in the United States [3]. In the treatment of APL, all-transretinoic acid (ATRA) is most widely used as a differentiation inducer, providing ‘‘the best proof of principle for differentiation therapy’’ [4]. Through binding to retinoic acid receptor, ATRA induces a profound change in the phenotype of APL blasts, making them quickly shift from immature promyelocytes to short-lived, terminally differentiated, granulocytes, either ex vivo or in vivo [5,6]. In combination with chemotherapy, the ATRA-based differentiation therapy has made APL the most curable subtype of all the AML cases [3,4]. However, ATRA by itself only rarely yields prolonged remissions in clinical practice [7]. Besides, the development of retinoic acid syndrome (RAS; or more generally, differentiation syndrome, DS [8]) ranges
* Corresponding author. Tel.: +8610 82801539; fax: +8610 62015584. E-mail addresses: [email protected], [email protected] (T.-L. Zhang).
from 2% to 27% in clinical trials and case reports, which has been recognized as a distinct complication and a potential lifethreatening adverse reaction [9]. Since the emerging extramedullary relapse was not observed frequently prior to the ATRA treatment era, this unique phenomenon may be related to the development of the RAS [10]. Another efficient differentiation inducer is 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA has a broad range of cellular and pharmacological effects. By mimicking the second messenger diacylglycerol to activate protein kinase C (PKC) and to modulate cell-signaling pathways, TPA affects a variety of cellular responses including cell death, cell survival, cell-cycle progression, and differentiation [11]. It has been shown that the TPA-evoked PKC activation leads to the differentiation of several myelocytic leukemia cell lines along the monocyte/macrophage pathway [12], including the human promyelocytic leukemia cell line HL60. TPA can lead HL60 cells to differentiate into macrophage-like phenotype [13,14], a distinct pathway from the ATRA-led granulocytic phenotype. Besides, TPA exhibits cytotoxicity by modulating the ERK signaling pathway in primary AML cells [15]. Importantly, clinical studies have shown that TPA is likely to have pharmacological activity at doses that are well tolerated without irreversible adverse effects [16–18]. The reduction in leukemic blasts and the improvement in blood counts have been seen in a pilot trial of TPA for patients with myeloid malignancies [19].
http://dx.doi.org/10.1016/j.biopha.2014.05.001 0753-3322/ß 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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doses of ATRA (0.1, 1 mM) and TPA (10, 100 nM), respectively, for indicated period of time. In the treatment with ATRA and TPA, the amount of DMSO in cell culture medium never exceeded a final concentration of 0.1%.
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2.4. Assessment of the differentiation-stimulating potency of drugtreated cells
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Cocultures were prepared using Millicell inserts (Millipore Corporation, Billerica, MA, USA) and six-well culture plates. As shown in Fig. 1, fresh HL60 cells were seeded in the lower part of a chamber at the density of 4 105 cells in 2 mL culture (middle of Fig. 1), and the drug-differentiated cells (left of Fig. 1), after removal of the excessive drug by washing twice with PBS, were added to the upper part (4 105 cells in 2 mL). The coculture was thereafter incubated for indicated period of time.
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2.5. Assessment of the inheritability of the differentiation-stimulating potency of drug-treated cells
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After being cocultured with drug-differentiated cells (middle of Q2 Fig. 1), the HL60 cells in the lower part of the chamber, which had undergone differentiation, were cocultured with equal number of fresh HL60 cells in the upper part for indicated period of time (right of Fig. 1).
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2.6. Analysis of cell proliferation
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To examine the effect of a drug or differentiated cells on cell proliferation, cells were collected and counted using a hemocytometer after treatment or coculture.
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2. Materials and methods
2.7. Determination of cell differentiation
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2.1. Chemicals and reagents
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RPMI-1640 medium, certified fetal bovine serum, antibiotics (penicillin, streptomycin), trypsin-EDTA solution, and phosphate buffer solution (PBS) were products of Corning Co. (Corning, NY, USA). ATRA and TPA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). PE mouse anti-human CD11b/Mac1 and PE mouse IgG1 isotype control were from BD Biosciences (San Jose, CA, USA). PE mouse anti-human CD38, PE mouse anti-human CD14 and PE mouse IgG1 isotype control were purchased from Biolegend (San Diego, CA, USA). Other reagents used were commercially available products of the highest purity. ATRA and TPA were dissolved in dimethyl sulfoxide (DMSO) at concentration of 100 mM and 200 mM, respectively, and stored at – 20 8C in the dark. The working solutions were made by dilution with RPMI-1640 medium.
Differentiation was assessed by measuring the cell surface antigen CD11b, CD38, and CD14 with flow cytometric analysis. CD11b is a cell surface marker for differentiation into either monocytes or granulocytes [26], while CD38 and CD14 are expressed in the differentiated granulocytes [27] and monocytes [28] respectively. After treatment with a drug or differentiated cells, samples of 106 cells were washed twice with PBS, and suspended in 100 mL of RPMI-1640 medium containing 2% fetal bovine serum. Then 10 mL of PE mouse IgG1 isotype control was added to the cell suspension, for setting threshold parameters. Cells of no less than 104 were examined on FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with Cell Quest Pro.
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2.8. Statistical analysis
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2.2. Cell line
Results were presented as mean SD of three independent experiments conducted in triplicate. Means were compared by the single factor analysis of variance (ANOVA) test. Statistical significant
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The promyelocytic leukemic cell line HL60 was acquired from Cell Resource Center of Peking Union Medical College and kept in low passage (< 2 months). HL60 cells were maintained in RPMI1640 growth medium supplemented with 10% heat-inactivated fetal bovine serum, containing 100 U/mL penicillin and 100 mg/ mL streptomycin at 37 8C, 5% CO2 and humidity. In bulk culture, cells were in continuous logarithmic growth (less than 106 cells/ mL).
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2.3. Treatment of HL60 cells with drugs
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HL60 cells were cultured in six-well plates (Costar, Cambridge, MA, USA) with complete growth medium and treated with various
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Nevertheless, since only a minority of leukemia samples underwent apoptosis after TPA treatment, experts have called for more extensive investigation before a broader use of TPA in clinical trials [20]. ATRA and TPA represent two typical drugs in the treatment of leukemia: one is currently used with some drawbacks (such as RAS and drug-resistance), and the other, not having been authorized for clinical use though, demonstrates pharmacological potency. It would be ideal to develop a strategy by which therapists could overcome or circumvent the negative effects while, at the same time, allowing these drugs to play their roles to the full extent. In this regard, the differentiation-stimulating potency of drugdifferentiated cells might be exploited. Reportedly, mononuclear blood cells are able to produce differentiation-inducing factor(s) upon stimulation with various mitogens [21]. The conditioned medium from normal blood mononuclear cells has been shown to make HL60 cells undergo macrophage differentiation [22–24]. The TPA-differentiated macrophages might likewise be capable of inducing differentiation in leukemic cells. As for ATRA-differentiated granulocytes, the production of inflammatory and hematopoietic cytokines [25] has been reported, of which some may facilitate cell differentiation. The aim of the present study was to assess the differentiation-stimulating potency, as well as its inheritability, of ATRA- and TPA-differentiated cells. And further, on the basis of ‘‘differentiation induced by differentiated cells’’, we intended to explore the feasibility of ex vivo therapy. Since HL60 cells undergo differentiation along the granulocytic and monocytic/macrophage pathways upon ATRA and TPA treatments, respectively, it may serve as a suitable cell model for the investigation.
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Fig. 1. Schematic of the coculturing experiments for the assessment of differentiation-stimulating potency of differentiated cells in fresh HL60 cells.
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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analysis was determined by Bonferroni’s method, and differences regarded as significant when P < 0.05.
3.2. ATRA-differentiated granulocytes inhibit proliferation of fresh HL60 cells
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3. Results
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3.1. ATRA-differentiated granulocytes stimulate differentiation in fresh HL60 cells
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Fig. 2A–C shows the percentages of CD11b positive cells of various samples. After treatment of cells with ATRA at either of the two concentrations, the CD11b expression was higher in the cells treated for 3 days than that treated for 2 days, but the concentration-caused difference was not significant (Fig. 2A). The ATRA-differentiated cells, when cocultured with fresh HL60 cells after the removal of residual ATRA, induced the latter batch of cells to differentiate time- and concentration-dependently (Fig. 2B), where the term ‘‘concentration’’ refers to the drug ATRA in obtaining the effector cells (Fig. 2A). Notably, the effector cells from the ATRA treatment for 2 days, when cocultured with fresh HL60 cells for 3 days (referred to as ‘‘2 d + 3 d’’, Fig. 2B), were more potent in stimulating differentiation than those treated by ATRA for 3 days (3 d + 3 d). Using the cells stimulated by the ATRA (1 mM)-differentiated cells, we further assessed the differentiation-inducing potency of the differentiated cells of the second generation. As shown in Fig. 2C, the cells that were originated from the ATRA treatment for 2 days (2 d + 3 d + 3 d) were much more potent than those from the ATRA treatment for 3 days (3 d + 3 d + 3 d). It is worth mentioning that the percentage of CD11b expression of the third generation dropped to 22%, significantly lower than those (around 50%) of the first two generations. Fig. 2D shows the CD38 expression in the differentiated cells of the three generations, confirming the granulocytic differentiation pathway, as well as the dropping potency of the effector cells. The shorter and the longer periods of ATRA treatment (2 d, 3 d) did not make any significant difference in CD38 expression.
Fig. 3A shows the inhibited proliferation of HL60 cells caused by ATRA treatment in 5 days. ATRA at the two concentrations (0.1 mM and 1 mM) did not cause any significant difference in the antiproliferative effect. Fig. 3B shows the effects of the ATRA-treated cells and the differentiated cells of the second generation (referred to as ‘‘G2 cells’’) on the proliferation of fresh HL60 cells, respectively. The ATRA-treated cells (2 d or 3 d) caused only slight inhibition of cell growth, being less efficient than the drug itself. As for the differentiated cells of the second generation (G2 cells, both ‘‘2 d + 3 d’’ and ‘‘3 d + 3 d’’), which had been stimulated by the ATRA-treated cells, no significant change was observed in the proliferation of the cocultured fresh HL60 cells, compared to the control group.
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3.3. TPA-differentiated cells stimulate differentiation in fresh HL60 cells
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Fig. 4A–C shows the percentages of CD11b positive cells of various samples. The TPA-induced differentiation appeared to be insensitive to either TPA concentration or the incubation time within the examined range, as indicated by the approximately same levels of upregulation of CD11b expression (Fig. 4A). The TPA-differentiated cells, when cocultured with fresh HL60 cells after the removal of residual TPA, induced the latter to differentiate. Notably, the differentiation induced by the TPAdifferentiated cells was both time- and concentration-dependent (Fig. 4B), different from that induced by TPA itself (Fig. 4A). As shown in Fig. 4C, the differentiated cells obtained from coculturing with the TPA-differentiated cells, i.e. the differentiated cells of the second generation, showed strong potency in stimulating differentiation in fresh HL60 cells, with the levels of CD11b comparable to that induced by TPA itself. Fig. 4D shows the CD14
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Fig. 2. Differentiation of HL60 cells induced by ATRA and differentiated cells. (A) CD11b expression stimulated by ATRA. (B) CD11b expression stimulated by ATRA-treated cells. After treatment by ATRA at the indicated concentrations for 2 or 3 days, the differentiated cells were cocultured with fresh HL60 cells for 3 days (referred to as ‘‘2 d + 3 d’’ and ‘‘3 d + 3 d’’, respectively). (C) CD11b expression stimulated by the differentiated cells of the second generation. The differentiated cells from (B) were cocultured with fresh HL60 cells for 3 days. (D) CD38 expression of differentiated cells stimulated by ATRA (2 d, 3 d), by ATRA-treated cells (2 d + 3 d), and by the differentiated cells of the second generation (2 d + 3 d + 3 d). *, #, $, &, P < 0.05 versus respective control.
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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expression in the differentiated cells of the three generations, confirming the monocytic/macrophage phenotype of the differentiation pathway.
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3.4. TPA-differentiated cells inhibit proliferation of fresh HL60 cells
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Fig. 5A shows the inhibited proliferation of HL60 cells caused by TPA treatment in 5 days. No significant difference was observed in the inhibitive effects of TPA at the two concentrations. However, the TPA-differentiated cells derived from TPA treatment at different concentrations did exhibit different effect on the proliferation of fresh HL60 cells. When cocultured with the 10 nM TPA-differentiated cells, the proliferation of fresh HL60 cells was only slightly inhibited (Fig. 5B); whereas the inhibitive effect of the 100 nM TPA-differentiated cells was as strong as that of TPA itself (Fig. 5C).
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3.5. The macrophage-like, adherent cells generated by TPA treatment are more potent than the floating ones in stimulating differentiation in fresh HL60 cells
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Fig. 6A shows the round, floating HL60 cells of the control group. Most of them became adherent to culture plate, as well as to each other, with pseudopodia formation after TPA treatment (Fig. 6B) or after cocultured with TPA-differentiated cells (Fig. 6C) for 2 days. The presence of adherent cells is indicative of macrophage-like differentiation. We examined the differentiation-stimulating potency of the adherent cells and the floating cells separately. The adherent cells caused an upregulation of CD11b expression in the cocultured fresh HL60 cells (Fig. 6D). This percentage (83.3%) of CD11b positive cells is not only much higher than that induced by the floating cells (34.1%), but also higher than that produced by TPA treatment (72.3%, Fig. 4A). Besides, the inhibitive effect of the
Fig. 3. Effect of ATRA and differentiated cells on the proliferation of fresh HL60 cells. Relative ratio = (cell number at indicated time)/(cell number at day 0). (A) Proliferation inhibition caused by prolonged ATRA treatment. (B) Comparison of the cell proliferation after 3 day-treatment by ATRA (1 mM), by ATRA-treated cells (2 d or 3 d), and by the differentiated cells of the second generation which are referred to as G2 cells (2 d + 3 d) or G2 cells (3 d + 3 d). *P < 0.05 versus respective control.
Fig. 4. Differentiation of HL60 cells induced by TPA and differentiated cells. (A) CD11b expression stimulated by TPA. (B) CD11b expression stimulated by TPA-treated cells. After treatment by TPA at the indicated concentrations for 2 days, the differentiated cells were cocultured with fresh HL60 cells for 2 days (referred to as ‘‘2 d + 2 d’’) or 3 days (2 d + 3 d). (C) CD11b expression stimulated by the differentiated cells of the second generation. The differentiated cells from (B) were cocultured with fresh HL60 cells for 2 or 3 days (2 d + 2 d + 2 d, 2 d + 2 d + 3 d, 2 d + 3 d + 3 d). (D) CD14 expression of differentiated cells stimulated by TPA (2 d), by TPA-treated cells (2 d + 3 d), and by the differentiated cells of the second generation (2 d + 3 d + 3 d). *, #, $, P < 0.05 versus respective control.
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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Fig. 5. Effect of TPA and differentiated cells on the proliferation of fresh HL60 cells. Relative ratio = (cell number at indicated time)/(cell number at day 0). (A) The inhibited proliferation after TPA treatment. (B) The reduced inhibitive effect of 10 nM TPA-treated cells. After 10 nM TPA treatment for 2 days, the cells were cocultured with fresh HL60 cells for the indicated times. (C) Maintenance of the inhibitive effect of 100 nM TPA-treated cells. *, #, P < 0.05 versus respective control.
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adherent cells on the proliferation of fresh HL60 cells is as intensive as that of TPA itself (data not shown). It is worth mentioning that some HL60 cells also became adherent to culture plates after cocultured with the floating cells generated by TPA treatment (data not shown).
3.6. Assessment of the effect of residual drugs in the coculture on the differentiation of fresh HL60 cells
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As demonstrated in Fig. 7, the drug-differentiated cells washed twice and those washed once showed almost the same level of
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Fig. 6. Morphological changes of HL60 cells (A to C, 250) and differentiation-stimulating potency of adherent and floating cells (D). (A) Control group. (B) Cells treated with 100 nM TPA for 2 days. (C) Cells cocultured with the TPA-differentiated cells for 2 days. (D) The CD11b expression of fresh HL60 cells induced by the floating and the adherent cells. After 100 nM TPA treatment for 2 days, the floating and adherent cells were separated. The two kinds of cells were cocultured with fresh HL60 cells for the indicated times, respectively. *, #, P < 0.05 versus respective control.
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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Fig. 7. Influence of washing drug-treated cells on the differentiation of cocultured fresh HL60 cells. After treatment with ATRA or TPA for 2 days, the differentiated cells were washed with phosphate buffer solution 1 or 2 times. They were then cocultured with fresh HL60 cells for 3 days, followed by the examination of the latter for CD11b expression.
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differentiation-stimulating effect in cocultured fresh HL60 cells. If the effect were mainly caused by the residual drug that is carried over into the cocultured cells, the twice-washed cells should remove larger amount of drug and thus be less effective than those washed only once. Therefore, the differentiation of the second generation, as well as the third generation, was caused mainly by the cytokines released from the differentiated cells, and the contribution from the residual drug must be minor.
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4. Discussion
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The differentiation induced by either of the two drugs (ATRA and TPA), as shown in the left part of Fig. 1, has extensively been studied before [29]. The main focus of the present study is the potency of the drug-differentiated cells, as well as the differentiated cells induced by them, in stimulating differentiation in fresh HL60 cells (Middle and Right of Fig. 1). Totally three generations of differentiation were examined, with the second and the third ones serving as the indicatives of the differentiationstimulating efficacy of the first and the second generations, respectively. The ATRA-differentiated granulocytic cells are able to stimulate differentiation in fresh HL60 cells, and the two generations of cellstimulated differentiation share some common features. As shown in Fig. 2A, the CD11b expression was higher in the cells after ATRA treatment for 3 days than for 2 days. However, the differentiated cells which had originally been generated from the ATRA treatment for 2 days (‘‘2 d + 3 d’’ in Fig. 2B and ‘‘2 d + 3 d + 3 d’’ in Fig. 2C) were more potent in stimulating differentiation than those treated for 3 days (‘‘3 d + 3 d’’ in Fig. 2B and ‘‘3 d + 3 d + 3 d’’ in Fig. 2C). Since the differentiated cells of the first and the second generations were washed twice each (Fig. 1), it is unlikely that the differentiation of the third generation was caused merely by the residual ATRA. Hence the phenomenon presented in Fig. 2A may reflect the intrinsic difference between the cells treated by ATRA for different periods of time. Cells at different stages of differentiation secrete different cytokines. Upon ATRA treatment, HL60 cells can express various cytokines, including interleukin 1a (IL-1a), IL-1b, IL-6, tumor necrosis factor-a (TNF-a), and stem cell factor (SCF) [25]. A number of cytokines have been shown to have differentiation-stimulating efficacy in various human leukemic cell lines, and two or more cytokines may act in synergy in stimulating a particular cell to differentiate [2]. For the differentiated cells that were treated with ATRA for 3 days, their reduced efficacy may be attributed to the lower level of secretion of the positive cytokines. Along with the functional maturation, the
differentiated cells express both neutrophil-like functional activity and susceptibility to spontaneous cell death [30]. Apoptosis has been proposed to account for the cell death of terminally differentiated HL60 cells after ATRA treatment [31,32], which could be a cause for the marked inhibition of cell growth after prolonged ATRA treatment (Fig. 3A). However, in most cases in the present study, the cells were examined for differentiation after only 2 or 3 day-treatment (Fig. 2), periods which were too short to observe obvious apoptosis (data not shown). Consistently, the changes are insignificant in the proliferation of the cells that were stimulated by the differentiated cells (Fig. 3B), as compared with the control group. Thus the contribution of apoptotic cells to the cell-induced differentiation can be excluded. The TPA-differentiated cells, as well as the differentiated cells stimulated by them, are more potent than those induced by ATRA in stimulating differentiation in fresh HL60 cells, and the differentiation induced by differentiated cells exhibits distinct features from that induced directly by TPA itself. For example, the TPA-induced differentiation appeared insensitive to TPA concentration and incubation time within the examined ranges (Fig. 4A). As for the differentiation stimulated by the TPA-treated cells (Fig. 4B), however, the percentage of CD11b positive cells was markedly higher when the effector cells had been generated from TPA treatment at the higher concentration (100 nM vs. 10 nM). Besides, a time-dependency was observed in the differentiation induced by the differentiated cells of both the first generation (Fig. 4B) and the second generation (‘‘2 d + 2 d + 2 d’’ versus ‘‘2 d + 2 d + 3 d’’, Fig. 4C). In assessing the differentiation-stimulating potency of the differentiated cells of the second generation, we compared the effector cells that had been cocultured with TPAtreated cells (100 nM, 2 d) for 2 days (referred to as ‘‘2 d + 2 d + 3 d’’, Fig. 4C) and those treated for 3 days (‘‘2 d + 3 d + 3 d’’, Fig. 4C). As judged from the percentage of the differentiated cells of the third generation, the efficacy of the two kinds of effector cells (of the second generation) were at approximately the same level (‘‘2 d + 2 d + 3 d’’ versus ‘‘2 d + 3 d + 3 d’’, Fig. 4C). So it seems that a coculturing period of two days is enough for the fresh HL60 cells to acquire all the necessary stimuli for the development of differentiation-stimulating potency. Notably, the differentiated percentages of the second generation (100 nM, 2 d + 3 d, Fig. 4B) and the third generation (‘‘2 d + 2 d + 3 d’’ and ‘‘2 d + 3 d + 3 d’’, Fig. 4C), both belonging to the cell-induced differentiation, are all comparable with that induced directly by TPA itself. These facts greatly reduce the likelihood that the residual drug was carried over to the coculture system in any significant amount and played a major role in the differentiation-stimulating potency of differentiated cells. As demonstrated in Fig. 7, more extensive washing did not led to a lower differentiation-stimulating potency of the drug-treated cells, indicating very small amount of residual drug. Since the differentiated cells of the second generation had been washed twice before they were cocultured with another bench of fresh HL60 cells, the amount of the residual drug must be even less. Therefore, a marked decrease in the differentiationstimulating potency should be expected for the residual drug, a deduction that disagrees with the observed facts. Besides, the percentage of CD11b positive cells stimulated by TPA-differentiated adherent cells (Fig. 6D) is higher than that produced by TPA itself (Fig. 4A), also making it unlikely that the residual drug in the differentiated cells played a major role in the differentiationstimulating potency. Hence the differentiation of three generations observed in the present study is a clear indication of the inheritability of the differentiation-stimulating potency and, to the best of our knowledge; no such data are available in the literature concerning the inheritable differentiation-stimulating potency of drug-differentiated leukemic cells. The expression of CD14 confirms the monocytic/macrophage differentiation path
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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(Fig. 4D). A kind of macrophage differentiation-inducing factor has reportedly been purified from the culture of THP-differentiated macrophage-like cells [33]. It is noteworthy that normal monocytes have been shown to induce differentiation in other types of cells [22–24]. However, it remains unclear whether the monocytes/ macrophages from a healthy person or a promyelocytic leukemia patient possess the same potency and inheritability as the TPAdifferentiated macrophages do. TPA-induced macrophage differentiation was accompanied by a rapid inhibition of cell growth, more prominent than that observed in ATRA-induced differentiation (Fig. 5A). Notably, the TPA-differentiated cells, just as TPA, also showed an inhibitive effect on the proliferation of fresh HL60 cells (Fig. 5C). The presence of the adherent cells after TPA treatment (Fig. 6B), agreement with the observations by others [34–36], is characteristic of matured macrophages. The remarkable potency of the adherent cells may be an intrinsic property of differentiated macrophages distinct from that of differentiated granulocytes. The differentiation-stimulating potency of drug-differentiated cells might be of significance for the treatment of human leukemia. First, the chemically induced potency of differentiated cells may represent a ‘‘hidden mechanism’’, by which some clinically used drugs are currently playing their roles in patients. Second, this inheritable potency may reveal the feasibility of ‘‘ex vivo therapy’’. In the treatment of human leukemia, drug-resistance often develops in patients, especially in those with recurrent leukemia. Unfortunately, the required higher dose is frequently too high for a patient to endure. However, some powerful pharmaceutical regents, after costly development for years, cannot pass the clinical trial due to serious side effects. In both cases, such a pharmaceutical regent may be applied to patient cells at the required dose ex vivo, followed by transfusion the differentiated cells back to the patient. Thus, on the basis of ‘‘differentiation induced by differentiated cells (DID)’’, an ex vivo therapy might be developed. A recent report demonstrates that macrophages can self-renew by local proliferation of mature differentiated cells [37], supporting for the idea of ex vivo therapy. Actually, the autologous leukemia-derived dendritic cell-based immunotherapy in acute and chronic myeloid leukemia has been shown feasible in a subgroup of patients, which could serve as an alternative to allogeneic stem cell transplantation that is limited to those patients less than 60 years old due to graft-versus-host disease and infections [38]. The drug/HL60 system demonstrated in the present study merely serves as a prototype for DID investigation. For any specific subtype of leukemia, a corresponding cell line could be selected; for any individual with specific leukemia, primary leukemia cells could be separated from the patient for DID investigation. According to the conditions of a patient, selections can even be made of the specific differentiation pathway (granulocyte or monocyte/macrophage), the suitable drugs (for example, TPA or VD3 for monocyte/macrophage differentiation), and even the suitable generation of differentiated cells (i.e. the drugdifferentiated cells or the second generation of differentiated cells). With the optimized conditions derived from a pilot ex vivo experiment, more leukemia cells could be separated from the patient, treated ex vivo, and then transfused back to the patient. In this way an exact personalized therapy could be developed for a patient, at least for those patients with relapsed/refractory malignancies or those who cannot endure the side effects of conventional therapies. Since an ex vivo treatment imposes much fewer constraints on a (potential) drug than an in vivo one, there would be more regents for a therapist to choose.
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Disclosure of interest
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The authors declare that they have no conflicts of interest concerning this article.
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Acknowledgments
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T-.L. Zhang acknowledges the funding from the Project of Q3430 National Base for Talent Training in Basic Sciences (Grant No. 431 J0830836). We thank Prof. Nan-Yin Han, Prof. Xi-Hui He, and Ms. 432 Xin Chen for providing facilities for some experiments. Mr. Wei Liu 433 participated at the early stage of the study. 434 References
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Original article
Differentiation-stimulating potency of differentiated HL60 cells after drug treatment Q1 Cong
Wang, Qun Zhang, Bao-Di Gou, Tian-Lan Zhang * , Kui Wang
Department of Chemical Biology, Peking University School of Pharmaceutical Sciences, 38, Xueyuan Road, Beijing 100191, P.R. China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 April 2014 Accepted 28 May 2014
Differentiation therapy in the treatment of leukemia is often hampered by limitations on using certain pharmaceutical regents or on the required doses due to various reasons, such as drug-resistance and retinoic acid syndrome. To circumvent these problems, a strategy might be developed on the basis of the ability of drug-differentiated cells to stimulate differentiation in leukemia cells. Using the promyelocytic leukemia cell line HL60 as a cell model, we assessed the differentiation-stimulating potency of differentiated granulocytes and monocytes/macrophages after treatments with all-transretinoic acid (ATRA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively. ATRA- and TPA-differentiated cells were able to stimulate differentiation in fresh HL60 cells, accompanied by inhibition on cell growth to various extents. The differentiated cells of the second generation, especially those originated from TPA treatment, were as potent as the drugs themselves in stimulating differentiation in fresh HL60 cells. On the basis of ‘‘differentiation induced by differentiated cells’’, we explored the feasibility of ex vivo therapy. ß 2014 Published by Elsevier Masson SAS.
Keywords: Leukemia Drug-differentiated cell Cell-induced differentiation Tetradecanoylphorbol acetate Phorbol ester Retinoic acid
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1. Introduction
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Now that leukemia is characterized by the blockade of differentiation in cellular maturation, differentiation therapy [1,2] would be a reasonable choice in clinical practice. Indeed, since 1980s, differentiation therapy has achieved great success in the treatment of acute promyelocytic leukemia (APL). This specific type of leukemia accounts for 10% to 15% of new cases of adults with acute myeloid leukemia (AML) per year in the United States [3]. In the treatment of APL, all-transretinoic acid (ATRA) is most widely used as a differentiation inducer, providing ‘‘the best proof of principle for differentiation therapy’’ [4]. Through binding to retinoic acid receptor, ATRA induces a profound change in the phenotype of APL blasts, making them quickly shift from immature promyelocytes to short-lived, terminally differentiated, granulocytes, either ex vivo or in vivo [5,6]. In combination with chemotherapy, the ATRA-based differentiation therapy has made APL the most curable subtype of all the AML cases [3,4]. However, ATRA by itself only rarely yields prolonged remissions in clinical practice [7]. Besides, the development of retinoic acid syndrome (RAS; or more generally, differentiation syndrome, DS [8]) ranges
* Corresponding author. Tel.: +8610 82801539; fax: +8610 62015584. E-mail addresses: [email protected], [email protected] (T.-L. Zhang).
from 2% to 27% in clinical trials and case reports, which has been recognized as a distinct complication and a potential lifethreatening adverse reaction [9]. Since the emerging extramedullary relapse was not observed frequently prior to the ATRA treatment era, this unique phenomenon may be related to the development of the RAS [10]. Another efficient differentiation inducer is 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA has a broad range of cellular and pharmacological effects. By mimicking the second messenger diacylglycerol to activate protein kinase C (PKC) and to modulate cell-signaling pathways, TPA affects a variety of cellular responses including cell death, cell survival, cell-cycle progression, and differentiation [11]. It has been shown that the TPA-evoked PKC activation leads to the differentiation of several myelocytic leukemia cell lines along the monocyte/macrophage pathway [12], including the human promyelocytic leukemia cell line HL60. TPA can lead HL60 cells to differentiate into macrophage-like phenotype [13,14], a distinct pathway from the ATRA-led granulocytic phenotype. Besides, TPA exhibits cytotoxicity by modulating the ERK signaling pathway in primary AML cells [15]. Importantly, clinical studies have shown that TPA is likely to have pharmacological activity at doses that are well tolerated without irreversible adverse effects [16–18]. The reduction in leukemic blasts and the improvement in blood counts have been seen in a pilot trial of TPA for patients with myeloid malignancies [19].
http://dx.doi.org/10.1016/j.biopha.2014.05.001 0753-3322/ß 2014 Published by Elsevier Masson SAS.
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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doses of ATRA (0.1, 1 mM) and TPA (10, 100 nM), respectively, for indicated period of time. In the treatment with ATRA and TPA, the amount of DMSO in cell culture medium never exceeded a final concentration of 0.1%.
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2.4. Assessment of the differentiation-stimulating potency of drugtreated cells
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Cocultures were prepared using Millicell inserts (Millipore Corporation, Billerica, MA, USA) and six-well culture plates. As shown in Fig. 1, fresh HL60 cells were seeded in the lower part of a chamber at the density of 4 105 cells in 2 mL culture (middle of Fig. 1), and the drug-differentiated cells (left of Fig. 1), after removal of the excessive drug by washing twice with PBS, were added to the upper part (4 105 cells in 2 mL). The coculture was thereafter incubated for indicated period of time.
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2.5. Assessment of the inheritability of the differentiation-stimulating potency of drug-treated cells
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After being cocultured with drug-differentiated cells (middle of Q2 Fig. 1), the HL60 cells in the lower part of the chamber, which had undergone differentiation, were cocultured with equal number of fresh HL60 cells in the upper part for indicated period of time (right of Fig. 1).
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2.6. Analysis of cell proliferation
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To examine the effect of a drug or differentiated cells on cell proliferation, cells were collected and counted using a hemocytometer after treatment or coculture.
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2. Materials and methods
2.7. Determination of cell differentiation
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2.1. Chemicals and reagents
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RPMI-1640 medium, certified fetal bovine serum, antibiotics (penicillin, streptomycin), trypsin-EDTA solution, and phosphate buffer solution (PBS) were products of Corning Co. (Corning, NY, USA). ATRA and TPA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). PE mouse anti-human CD11b/Mac1 and PE mouse IgG1 isotype control were from BD Biosciences (San Jose, CA, USA). PE mouse anti-human CD38, PE mouse anti-human CD14 and PE mouse IgG1 isotype control were purchased from Biolegend (San Diego, CA, USA). Other reagents used were commercially available products of the highest purity. ATRA and TPA were dissolved in dimethyl sulfoxide (DMSO) at concentration of 100 mM and 200 mM, respectively, and stored at – 20 8C in the dark. The working solutions were made by dilution with RPMI-1640 medium.
Differentiation was assessed by measuring the cell surface antigen CD11b, CD38, and CD14 with flow cytometric analysis. CD11b is a cell surface marker for differentiation into either monocytes or granulocytes [26], while CD38 and CD14 are expressed in the differentiated granulocytes [27] and monocytes [28] respectively. After treatment with a drug or differentiated cells, samples of 106 cells were washed twice with PBS, and suspended in 100 mL of RPMI-1640 medium containing 2% fetal bovine serum. Then 10 mL of PE mouse IgG1 isotype control was added to the cell suspension, for setting threshold parameters. Cells of no less than 104 were examined on FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with Cell Quest Pro.
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2.8. Statistical analysis
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2.2. Cell line
Results were presented as mean SD of three independent experiments conducted in triplicate. Means were compared by the single factor analysis of variance (ANOVA) test. Statistical significant
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The promyelocytic leukemic cell line HL60 was acquired from Cell Resource Center of Peking Union Medical College and kept in low passage (< 2 months). HL60 cells were maintained in RPMI1640 growth medium supplemented with 10% heat-inactivated fetal bovine serum, containing 100 U/mL penicillin and 100 mg/ mL streptomycin at 37 8C, 5% CO2 and humidity. In bulk culture, cells were in continuous logarithmic growth (less than 106 cells/ mL).
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2.3. Treatment of HL60 cells with drugs
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HL60 cells were cultured in six-well plates (Costar, Cambridge, MA, USA) with complete growth medium and treated with various
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Nevertheless, since only a minority of leukemia samples underwent apoptosis after TPA treatment, experts have called for more extensive investigation before a broader use of TPA in clinical trials [20]. ATRA and TPA represent two typical drugs in the treatment of leukemia: one is currently used with some drawbacks (such as RAS and drug-resistance), and the other, not having been authorized for clinical use though, demonstrates pharmacological potency. It would be ideal to develop a strategy by which therapists could overcome or circumvent the negative effects while, at the same time, allowing these drugs to play their roles to the full extent. In this regard, the differentiation-stimulating potency of drugdifferentiated cells might be exploited. Reportedly, mononuclear blood cells are able to produce differentiation-inducing factor(s) upon stimulation with various mitogens [21]. The conditioned medium from normal blood mononuclear cells has been shown to make HL60 cells undergo macrophage differentiation [22–24]. The TPA-differentiated macrophages might likewise be capable of inducing differentiation in leukemic cells. As for ATRA-differentiated granulocytes, the production of inflammatory and hematopoietic cytokines [25] has been reported, of which some may facilitate cell differentiation. The aim of the present study was to assess the differentiation-stimulating potency, as well as its inheritability, of ATRA- and TPA-differentiated cells. And further, on the basis of ‘‘differentiation induced by differentiated cells’’, we intended to explore the feasibility of ex vivo therapy. Since HL60 cells undergo differentiation along the granulocytic and monocytic/macrophage pathways upon ATRA and TPA treatments, respectively, it may serve as a suitable cell model for the investigation.
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Fig. 1. Schematic of the coculturing experiments for the assessment of differentiation-stimulating potency of differentiated cells in fresh HL60 cells.
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analysis was determined by Bonferroni’s method, and differences regarded as significant when P < 0.05.
3.2. ATRA-differentiated granulocytes inhibit proliferation of fresh HL60 cells
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3. Results
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3.1. ATRA-differentiated granulocytes stimulate differentiation in fresh HL60 cells
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Fig. 2A–C shows the percentages of CD11b positive cells of various samples. After treatment of cells with ATRA at either of the two concentrations, the CD11b expression was higher in the cells treated for 3 days than that treated for 2 days, but the concentration-caused difference was not significant (Fig. 2A). The ATRA-differentiated cells, when cocultured with fresh HL60 cells after the removal of residual ATRA, induced the latter batch of cells to differentiate time- and concentration-dependently (Fig. 2B), where the term ‘‘concentration’’ refers to the drug ATRA in obtaining the effector cells (Fig. 2A). Notably, the effector cells from the ATRA treatment for 2 days, when cocultured with fresh HL60 cells for 3 days (referred to as ‘‘2 d + 3 d’’, Fig. 2B), were more potent in stimulating differentiation than those treated by ATRA for 3 days (3 d + 3 d). Using the cells stimulated by the ATRA (1 mM)-differentiated cells, we further assessed the differentiation-inducing potency of the differentiated cells of the second generation. As shown in Fig. 2C, the cells that were originated from the ATRA treatment for 2 days (2 d + 3 d + 3 d) were much more potent than those from the ATRA treatment for 3 days (3 d + 3 d + 3 d). It is worth mentioning that the percentage of CD11b expression of the third generation dropped to 22%, significantly lower than those (around 50%) of the first two generations. Fig. 2D shows the CD38 expression in the differentiated cells of the three generations, confirming the granulocytic differentiation pathway, as well as the dropping potency of the effector cells. The shorter and the longer periods of ATRA treatment (2 d, 3 d) did not make any significant difference in CD38 expression.
Fig. 3A shows the inhibited proliferation of HL60 cells caused by ATRA treatment in 5 days. ATRA at the two concentrations (0.1 mM and 1 mM) did not cause any significant difference in the antiproliferative effect. Fig. 3B shows the effects of the ATRA-treated cells and the differentiated cells of the second generation (referred to as ‘‘G2 cells’’) on the proliferation of fresh HL60 cells, respectively. The ATRA-treated cells (2 d or 3 d) caused only slight inhibition of cell growth, being less efficient than the drug itself. As for the differentiated cells of the second generation (G2 cells, both ‘‘2 d + 3 d’’ and ‘‘3 d + 3 d’’), which had been stimulated by the ATRA-treated cells, no significant change was observed in the proliferation of the cocultured fresh HL60 cells, compared to the control group.
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3.3. TPA-differentiated cells stimulate differentiation in fresh HL60 cells
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Fig. 4A–C shows the percentages of CD11b positive cells of various samples. The TPA-induced differentiation appeared to be insensitive to either TPA concentration or the incubation time within the examined range, as indicated by the approximately same levels of upregulation of CD11b expression (Fig. 4A). The TPA-differentiated cells, when cocultured with fresh HL60 cells after the removal of residual TPA, induced the latter to differentiate. Notably, the differentiation induced by the TPAdifferentiated cells was both time- and concentration-dependent (Fig. 4B), different from that induced by TPA itself (Fig. 4A). As shown in Fig. 4C, the differentiated cells obtained from coculturing with the TPA-differentiated cells, i.e. the differentiated cells of the second generation, showed strong potency in stimulating differentiation in fresh HL60 cells, with the levels of CD11b comparable to that induced by TPA itself. Fig. 4D shows the CD14
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Fig. 2. Differentiation of HL60 cells induced by ATRA and differentiated cells. (A) CD11b expression stimulated by ATRA. (B) CD11b expression stimulated by ATRA-treated cells. After treatment by ATRA at the indicated concentrations for 2 or 3 days, the differentiated cells were cocultured with fresh HL60 cells for 3 days (referred to as ‘‘2 d + 3 d’’ and ‘‘3 d + 3 d’’, respectively). (C) CD11b expression stimulated by the differentiated cells of the second generation. The differentiated cells from (B) were cocultured with fresh HL60 cells for 3 days. (D) CD38 expression of differentiated cells stimulated by ATRA (2 d, 3 d), by ATRA-treated cells (2 d + 3 d), and by the differentiated cells of the second generation (2 d + 3 d + 3 d). *, #, $, &, P < 0.05 versus respective control.
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expression in the differentiated cells of the three generations, confirming the monocytic/macrophage phenotype of the differentiation pathway.
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3.4. TPA-differentiated cells inhibit proliferation of fresh HL60 cells
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Fig. 5A shows the inhibited proliferation of HL60 cells caused by TPA treatment in 5 days. No significant difference was observed in the inhibitive effects of TPA at the two concentrations. However, the TPA-differentiated cells derived from TPA treatment at different concentrations did exhibit different effect on the proliferation of fresh HL60 cells. When cocultured with the 10 nM TPA-differentiated cells, the proliferation of fresh HL60 cells was only slightly inhibited (Fig. 5B); whereas the inhibitive effect of the 100 nM TPA-differentiated cells was as strong as that of TPA itself (Fig. 5C).
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3.5. The macrophage-like, adherent cells generated by TPA treatment are more potent than the floating ones in stimulating differentiation in fresh HL60 cells
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Fig. 6A shows the round, floating HL60 cells of the control group. Most of them became adherent to culture plate, as well as to each other, with pseudopodia formation after TPA treatment (Fig. 6B) or after cocultured with TPA-differentiated cells (Fig. 6C) for 2 days. The presence of adherent cells is indicative of macrophage-like differentiation. We examined the differentiation-stimulating potency of the adherent cells and the floating cells separately. The adherent cells caused an upregulation of CD11b expression in the cocultured fresh HL60 cells (Fig. 6D). This percentage (83.3%) of CD11b positive cells is not only much higher than that induced by the floating cells (34.1%), but also higher than that produced by TPA treatment (72.3%, Fig. 4A). Besides, the inhibitive effect of the
Fig. 3. Effect of ATRA and differentiated cells on the proliferation of fresh HL60 cells. Relative ratio = (cell number at indicated time)/(cell number at day 0). (A) Proliferation inhibition caused by prolonged ATRA treatment. (B) Comparison of the cell proliferation after 3 day-treatment by ATRA (1 mM), by ATRA-treated cells (2 d or 3 d), and by the differentiated cells of the second generation which are referred to as G2 cells (2 d + 3 d) or G2 cells (3 d + 3 d). *P < 0.05 versus respective control.
Fig. 4. Differentiation of HL60 cells induced by TPA and differentiated cells. (A) CD11b expression stimulated by TPA. (B) CD11b expression stimulated by TPA-treated cells. After treatment by TPA at the indicated concentrations for 2 days, the differentiated cells were cocultured with fresh HL60 cells for 2 days (referred to as ‘‘2 d + 2 d’’) or 3 days (2 d + 3 d). (C) CD11b expression stimulated by the differentiated cells of the second generation. The differentiated cells from (B) were cocultured with fresh HL60 cells for 2 or 3 days (2 d + 2 d + 2 d, 2 d + 2 d + 3 d, 2 d + 3 d + 3 d). (D) CD14 expression of differentiated cells stimulated by TPA (2 d), by TPA-treated cells (2 d + 3 d), and by the differentiated cells of the second generation (2 d + 3 d + 3 d). *, #, $, P < 0.05 versus respective control.
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Fig. 5. Effect of TPA and differentiated cells on the proliferation of fresh HL60 cells. Relative ratio = (cell number at indicated time)/(cell number at day 0). (A) The inhibited proliferation after TPA treatment. (B) The reduced inhibitive effect of 10 nM TPA-treated cells. After 10 nM TPA treatment for 2 days, the cells were cocultured with fresh HL60 cells for the indicated times. (C) Maintenance of the inhibitive effect of 100 nM TPA-treated cells. *, #, P < 0.05 versus respective control.
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adherent cells on the proliferation of fresh HL60 cells is as intensive as that of TPA itself (data not shown). It is worth mentioning that some HL60 cells also became adherent to culture plates after cocultured with the floating cells generated by TPA treatment (data not shown).
3.6. Assessment of the effect of residual drugs in the coculture on the differentiation of fresh HL60 cells
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As demonstrated in Fig. 7, the drug-differentiated cells washed twice and those washed once showed almost the same level of
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Fig. 6. Morphological changes of HL60 cells (A to C, 250) and differentiation-stimulating potency of adherent and floating cells (D). (A) Control group. (B) Cells treated with 100 nM TPA for 2 days. (C) Cells cocultured with the TPA-differentiated cells for 2 days. (D) The CD11b expression of fresh HL60 cells induced by the floating and the adherent cells. After 100 nM TPA treatment for 2 days, the floating and adherent cells were separated. The two kinds of cells were cocultured with fresh HL60 cells for the indicated times, respectively. *, #, P < 0.05 versus respective control.
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Fig. 7. Influence of washing drug-treated cells on the differentiation of cocultured fresh HL60 cells. After treatment with ATRA or TPA for 2 days, the differentiated cells were washed with phosphate buffer solution 1 or 2 times. They were then cocultured with fresh HL60 cells for 3 days, followed by the examination of the latter for CD11b expression.
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differentiation-stimulating effect in cocultured fresh HL60 cells. If the effect were mainly caused by the residual drug that is carried over into the cocultured cells, the twice-washed cells should remove larger amount of drug and thus be less effective than those washed only once. Therefore, the differentiation of the second generation, as well as the third generation, was caused mainly by the cytokines released from the differentiated cells, and the contribution from the residual drug must be minor.
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4. Discussion
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The differentiation induced by either of the two drugs (ATRA and TPA), as shown in the left part of Fig. 1, has extensively been studied before [29]. The main focus of the present study is the potency of the drug-differentiated cells, as well as the differentiated cells induced by them, in stimulating differentiation in fresh HL60 cells (Middle and Right of Fig. 1). Totally three generations of differentiation were examined, with the second and the third ones serving as the indicatives of the differentiationstimulating efficacy of the first and the second generations, respectively. The ATRA-differentiated granulocytic cells are able to stimulate differentiation in fresh HL60 cells, and the two generations of cellstimulated differentiation share some common features. As shown in Fig. 2A, the CD11b expression was higher in the cells after ATRA treatment for 3 days than for 2 days. However, the differentiated cells which had originally been generated from the ATRA treatment for 2 days (‘‘2 d + 3 d’’ in Fig. 2B and ‘‘2 d + 3 d + 3 d’’ in Fig. 2C) were more potent in stimulating differentiation than those treated for 3 days (‘‘3 d + 3 d’’ in Fig. 2B and ‘‘3 d + 3 d + 3 d’’ in Fig. 2C). Since the differentiated cells of the first and the second generations were washed twice each (Fig. 1), it is unlikely that the differentiation of the third generation was caused merely by the residual ATRA. Hence the phenomenon presented in Fig. 2A may reflect the intrinsic difference between the cells treated by ATRA for different periods of time. Cells at different stages of differentiation secrete different cytokines. Upon ATRA treatment, HL60 cells can express various cytokines, including interleukin 1a (IL-1a), IL-1b, IL-6, tumor necrosis factor-a (TNF-a), and stem cell factor (SCF) [25]. A number of cytokines have been shown to have differentiation-stimulating efficacy in various human leukemic cell lines, and two or more cytokines may act in synergy in stimulating a particular cell to differentiate [2]. For the differentiated cells that were treated with ATRA for 3 days, their reduced efficacy may be attributed to the lower level of secretion of the positive cytokines. Along with the functional maturation, the
differentiated cells express both neutrophil-like functional activity and susceptibility to spontaneous cell death [30]. Apoptosis has been proposed to account for the cell death of terminally differentiated HL60 cells after ATRA treatment [31,32], which could be a cause for the marked inhibition of cell growth after prolonged ATRA treatment (Fig. 3A). However, in most cases in the present study, the cells were examined for differentiation after only 2 or 3 day-treatment (Fig. 2), periods which were too short to observe obvious apoptosis (data not shown). Consistently, the changes are insignificant in the proliferation of the cells that were stimulated by the differentiated cells (Fig. 3B), as compared with the control group. Thus the contribution of apoptotic cells to the cell-induced differentiation can be excluded. The TPA-differentiated cells, as well as the differentiated cells stimulated by them, are more potent than those induced by ATRA in stimulating differentiation in fresh HL60 cells, and the differentiation induced by differentiated cells exhibits distinct features from that induced directly by TPA itself. For example, the TPA-induced differentiation appeared insensitive to TPA concentration and incubation time within the examined ranges (Fig. 4A). As for the differentiation stimulated by the TPA-treated cells (Fig. 4B), however, the percentage of CD11b positive cells was markedly higher when the effector cells had been generated from TPA treatment at the higher concentration (100 nM vs. 10 nM). Besides, a time-dependency was observed in the differentiation induced by the differentiated cells of both the first generation (Fig. 4B) and the second generation (‘‘2 d + 2 d + 2 d’’ versus ‘‘2 d + 2 d + 3 d’’, Fig. 4C). In assessing the differentiation-stimulating potency of the differentiated cells of the second generation, we compared the effector cells that had been cocultured with TPAtreated cells (100 nM, 2 d) for 2 days (referred to as ‘‘2 d + 2 d + 3 d’’, Fig. 4C) and those treated for 3 days (‘‘2 d + 3 d + 3 d’’, Fig. 4C). As judged from the percentage of the differentiated cells of the third generation, the efficacy of the two kinds of effector cells (of the second generation) were at approximately the same level (‘‘2 d + 2 d + 3 d’’ versus ‘‘2 d + 3 d + 3 d’’, Fig. 4C). So it seems that a coculturing period of two days is enough for the fresh HL60 cells to acquire all the necessary stimuli for the development of differentiation-stimulating potency. Notably, the differentiated percentages of the second generation (100 nM, 2 d + 3 d, Fig. 4B) and the third generation (‘‘2 d + 2 d + 3 d’’ and ‘‘2 d + 3 d + 3 d’’, Fig. 4C), both belonging to the cell-induced differentiation, are all comparable with that induced directly by TPA itself. These facts greatly reduce the likelihood that the residual drug was carried over to the coculture system in any significant amount and played a major role in the differentiation-stimulating potency of differentiated cells. As demonstrated in Fig. 7, more extensive washing did not led to a lower differentiation-stimulating potency of the drug-treated cells, indicating very small amount of residual drug. Since the differentiated cells of the second generation had been washed twice before they were cocultured with another bench of fresh HL60 cells, the amount of the residual drug must be even less. Therefore, a marked decrease in the differentiationstimulating potency should be expected for the residual drug, a deduction that disagrees with the observed facts. Besides, the percentage of CD11b positive cells stimulated by TPA-differentiated adherent cells (Fig. 6D) is higher than that produced by TPA itself (Fig. 4A), also making it unlikely that the residual drug in the differentiated cells played a major role in the differentiationstimulating potency. Hence the differentiation of three generations observed in the present study is a clear indication of the inheritability of the differentiation-stimulating potency and, to the best of our knowledge; no such data are available in the literature concerning the inheritable differentiation-stimulating potency of drug-differentiated leukemic cells. The expression of CD14 confirms the monocytic/macrophage differentiation path
Please cite this article in press as: Wang C, et al. Differentiation-stimulating potency of differentiated HL60 cells after drug treatment. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.05.001
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(Fig. 4D). A kind of macrophage differentiation-inducing factor has reportedly been purified from the culture of THP-differentiated macrophage-like cells [33]. It is noteworthy that normal monocytes have been shown to induce differentiation in other types of cells [22–24]. However, it remains unclear whether the monocytes/ macrophages from a healthy person or a promyelocytic leukemia patient possess the same potency and inheritability as the TPAdifferentiated macrophages do. TPA-induced macrophage differentiation was accompanied by a rapid inhibition of cell growth, more prominent than that observed in ATRA-induced differentiation (Fig. 5A). Notably, the TPA-differentiated cells, just as TPA, also showed an inhibitive effect on the proliferation of fresh HL60 cells (Fig. 5C). The presence of the adherent cells after TPA treatment (Fig. 6B), agreement with the observations by others [34–36], is characteristic of matured macrophages. The remarkable potency of the adherent cells may be an intrinsic property of differentiated macrophages distinct from that of differentiated granulocytes. The differentiation-stimulating potency of drug-differentiated cells might be of significance for the treatment of human leukemia. First, the chemically induced potency of differentiated cells may represent a ‘‘hidden mechanism’’, by which some clinically used drugs are currently playing their roles in patients. Second, this inheritable potency may reveal the feasibility of ‘‘ex vivo therapy’’. In the treatment of human leukemia, drug-resistance often develops in patients, especially in those with recurrent leukemia. Unfortunately, the required higher dose is frequently too high for a patient to endure. However, some powerful pharmaceutical regents, after costly development for years, cannot pass the clinical trial due to serious side effects. In both cases, such a pharmaceutical regent may be applied to patient cells at the required dose ex vivo, followed by transfusion the differentiated cells back to the patient. Thus, on the basis of ‘‘differentiation induced by differentiated cells (DID)’’, an ex vivo therapy might be developed. A recent report demonstrates that macrophages can self-renew by local proliferation of mature differentiated cells [37], supporting for the idea of ex vivo therapy. Actually, the autologous leukemia-derived dendritic cell-based immunotherapy in acute and chronic myeloid leukemia has been shown feasible in a subgroup of patients, which could serve as an alternative to allogeneic stem cell transplantation that is limited to those patients less than 60 years old due to graft-versus-host disease and infections [38]. The drug/HL60 system demonstrated in the present study merely serves as a prototype for DID investigation. For any specific subtype of leukemia, a corresponding cell line could be selected; for any individual with specific leukemia, primary leukemia cells could be separated from the patient for DID investigation. According to the conditions of a patient, selections can even be made of the specific differentiation pathway (granulocyte or monocyte/macrophage), the suitable drugs (for example, TPA or VD3 for monocyte/macrophage differentiation), and even the suitable generation of differentiated cells (i.e. the drugdifferentiated cells or the second generation of differentiated cells). With the optimized conditions derived from a pilot ex vivo experiment, more leukemia cells could be separated from the patient, treated ex vivo, and then transfused back to the patient. In this way an exact personalized therapy could be developed for a patient, at least for those patients with relapsed/refractory malignancies or those who cannot endure the side effects of conventional therapies. Since an ex vivo treatment imposes much fewer constraints on a (potential) drug than an in vivo one, there would be more regents for a therapist to choose.
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Disclosure of interest
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The authors declare that they have no conflicts of interest concerning this article.
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Acknowledgments
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T-.L. Zhang acknowledges the funding from the Project of Q3430 National Base for Talent Training in Basic Sciences (Grant No. 431 J0830836). We thank Prof. Nan-Yin Han, Prof. Xi-Hui He, and Ms. 432 Xin Chen for providing facilities for some experiments. Mr. Wei Liu 433 participated at the early stage of the study. 434 References
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