HL-60 and MV4-11 cell lines were purchased from American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. All cell lines were maintained in an incubator at 37°C and 5% CO₂. All experiments with cell lines were performed within 6 months after thawing or obtaining from ATCC.

All-trans-retinoic acid (ATRA) was purchased from Sigma (St Louis, MO). JQ1 was purchased from Selleck chemicals (Houston, TX). They were all dissolved by dimethyl sulfoxide (DMSO) as a stock solution at 50 mM and 10 mM respectively.

Cells were seeded at a density of 1×10⁵ cells to 2×10⁵ cells per ml and rested for 5 hours. Cells were then treated with ATRA and/or JQ1 for the indicated days. The number of viable cells was assessed using the Cell counting kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The coefficient of drug interaction (CDI) is calculated as follows: CDI=AB/(A×B). According to the absorbance of each group, AB is the ratio of the combination groups to control group; A or B is the ratio of the single agent group to control group. Thus, CDI value<1, =1 or >1 indicates that the drugs are synergistic, additive or antagonistic, respectively.

Myeloid differentiation was assessed by detection of the cell surface CD11b expression. Cells were pre-incubated with 0.5 μg Fc block (BD Biosciences, San Jose, CA) for 10 minutes at RT and stained with anti-CD11b-PE antibody (BD Biosciences, San Jose, CA) for 30 min at 4°C. The labeled cells were then washed and analyzed for CD11b expression. The apoptosis assay was performed using Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK). Briefly, cells were washed with staining buffer and resuspended in 300 μl of binding buffer. 100 μl of the samples were incubated with FITC-labeled Annexin V for 15 min in the dark at room temperature. The reaction was stopped by adding 300 μl of binding buffer and analyzed. Flow cytometry was performed on a FACSCalibur equipped with CellQuest Pro software (BD systems, San Jose, CA). FlowJo software (Version7.6.1, Treestar, Ashland, OR) was used for subsequent analysis.

Cells were washed with ice-cold PBS and lysed in RIPA buffer (Thermo Scientific, Waltham, MA) containing Complete protease inhibitor cocktail tablets (Roche, Indianapolis, IN) and PhosSTP phosphatase inhibitor cocktail (Roche, Indianapolis, IN). After incubation on ice for 30 minutes, cell lysates were centrifuged at 13,000 rpm for 15 minutes at 4°C. Supernatants were collected and protein concentrations were determined by Bradford’s protein assay (Bio-Rad, Hercules, CA) according to manufacturer’s protocol. 20 μg protein extracts were subjected to 8%~12% SDS-PAGE gels according to molecular weight of detected protein bands and then transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The blots were blocked for 1 h in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (TBS-T), and incubated with primary antibody at 4°C overnight. The blots were washed three times and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA) at room temperature for 1 h. After washing, blots were developed by enhanced chemiluminescence (ECL) according to manufacturer’s instructions (Cell Signaling Technology, Danvers, MA) and imaged with a LAS-4000 imaging system (Hugifilm Life Science). The following primary antibodies were used: anti-c-Myc (Abcam, Cambridge, UK). anti-C/EBPɛ (GeneTex. Irvine, CA). anti-β-actin (Cell Signaling Technology, Danvers, MA).

Total RNA was extracted from cells with the Trizol reagent (Invitrogen), and cDNA was synthesized using High Capacity RNA-to-cDNA kits (Applied Biosystems, Foster City, CA). Real-time PCR was performed in duplicate using SYBR green reagents (Roche, Indianapolis, IN). The PCR protocol consisted of thermal cycling as follows: pre-amplification at 95°C for 2 min followed by 45 cycles of 95°C for 10 sec; 60°C for 10 sec; and 72°C for 10 sec using LightCycler 96 System (Roche, Indianapolis, IN). The target mRNA expression was quantified using the Δ ΔC_T method and normalized to GAPDH expression.

We first explored the ability of ATRA to inhibit the proliferation of HL-60 and MV4-11 cells by CCK-8 assay. Both the HL-60 and MV4-11 cells were exposed to increasing concentrations of ATRA for the indicated days. ATRA concentrations of 20~100 nM induced similar growth inhibition in both cell lines (Fig. 1A). Therefore, we selected a dose of 100 nM ATRA which has been used in most in vitro studies (7–9). To determine the combined effects of ATRA and JQ1 on AML cell proliferation, we selected the dose of 50 nM and 100 nM JQ1 which were not enough for complete growth inhibition.

As shown in Fig. 1B, both HL-60 and MV4-11 cells were treated with indicated concentrations of JQ1 alone, ATRA (100 nM) alone, or the combination. To test the combination effect, the Coefficient and Drug Interaction (CDI) was calculated as described in materials and methods. As expected from the CCK-8 assay results (Fig. 1B), CDI values less than 1 indicated the combination of JQ1 and ATRA yielded synergistic interactions at both concentrations (50 nM and 100 nM) of JQ1 (Fig. 1C).

As the combination of JQ1 and ATRA synergistically inhibited proliferation of HL-60 and MV4-11 cells, we sought to examine whether the synergy resulted from differentiation. We assessed myeloid differentiation by increased CD11b expression using flow cytometry. Treatment of HL-60 cells with 100 nM of ATRA alone for 3 days resulted in 41.6±13.3 MFI of CD11b expression. When cells were treated with 50 nM or 100 nM JQ1 alone, MFI of CD11b expression was close to control or minimal respectively. However, combined treatment of ATRA with JQ1 for 3 days significantly induced CD11b expression dose dependently (96.8±16 MFI for 50 nM JQ1 and 142.3±9.7 MFI for 100 nM JQ1) (Fig. 2A). In MV4-11 cells, neither agent alone nor the combination induced CD11b expression (data not shown).

The enhanced differentiation was also confirmed by the expression changes in C/EBPɛ mRNA and protein in HL-60 cells. C/EBPɛ is a downstream target gene of RAR and a terminal differentiation marker of granulocytes (25). As shown in Fig. 2B, ATRA alone elevated C/EBPɛ mRNA level, but the combined treatment of ATRA with JQ1 synergistically increased the levels of C/EBPɛ mRNA. Similarly, Western blot analysis also revealed a strong upregulation of C/EBPɛ protein after the combined treatment of JQ1 and ATRA, compared to either treatment alone. Taken together, these results suggest that the combination of JQ1 and ATRA has the great potential to induce myeloid differentiation in HL-60 cells.

We next examined whether the synergistic effect of JQ1 and ATRA on suppression of AML cell growth resulted from apoptosis. Annexin V staining revealed that co-treatment with JQ1 and ATRA for 48 h did not have any synergistic or additive effect on apoptosis in HL-60 cells (Fig. 3A). In the case of MV4-11 cells, the combined treatment of JQ1 and ATRA enhanced apoptosis (Fig. 3B). Although the combined effect on apoptosis was not so significant, it may explain, at least in part, its effect on the synergistic growth inhibition.

To find a mechanism underlying the synergy between JQ1 and ATRA, we investigated expression levels of c-Myc in AML cells, because c-Myc is involved in AML cell proliferation, differentiation, and apoptosis (11, 26). Consistent with previous reports (13, 14), treatment with JQ1 for 24 hours dose-dependently decreased the levels of c-Myc mRNA. (Fig. 4A). ATRA alone also induced downregulation of c-Myc mRNA, and the combined treatment of ATRA with the indicated concentrations (50 nM and 100 nM) of JQ1 caused strong downregulation of c-Myc mRNA, compared to either treatment alone. (Fig. 4A). Corresponding to the decrease in c-Myc mRNA, the combination of JQ1 and ATRA also enhanced the down-regulation of c-Myc protein (Fig. 4B). Thus, the expression levels of c-Myc were correlated with the results of proliferation, differentiation and apoptosis analyses, and may contribute to the synergistic effect of ATRA and JQ1.

We next investigated the combined effect of ATRA and JQ1 on mRNA expression of hTERT, a direct target of c-Myc (27). Consistent with previous reports, treatment with 100 nM ATRA for 24 hours decreased the levels of hTERT mRNA in HL-60 cells (Fig. 4C, left). Treatment with indicated concentrations (50 nM and 100 nM) of JQ1 also downregulated hTERT mRNA, but the combined treatment of ATRA with JQ1 caused strong downregulation of hTERT mRNA, compared to either treatment alone in HL-60 cells (Fig. 4C, left). These effects were also observed in MV4-11 cell, although to a lesser extent (Fig. 4C, right). However, hTERT mRNA expression was much lower when cells were treated in the same way for longer time period (48 h, data not shown). These results suggest that the levels of hTERT mRNA are correlated with anti-proliferative synergism between JQ1 and ATRA.