A novel method to establish glucocorticoid resistant acute lymphoblastic leukemia cell lines

CEM-C7–14(GC sensitive) and CEM-C1–15 (GC resistant) cell lines were subcloned from the CCRF-CEM cell line [11], which was established from a patient with T-ALL [12]. The two cell lines were kindly provided by Dr. Thompson (The University of Texas Medical Branch). The GC-sensitive B-ALL cell line, NALM-6 was purchased from Shanghai Institute Cell Resources Bank; HXEX-ALL1 was established from a B-ALL patient by our laboratory. All cell lines were maintained in RPMI 1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone) at 37 °C under a humidified atmosphere with 5% carbon dioxide (CO₂) and 21% oxygen (O₂; normoxic condition).

Logarithmically growing cells were harvested and seeded in 6-well sterile plastic culture plates (Corning Inc., Corning, NY, USA) at a density of 1~3 × 10⁴/ml in RPMI-1640 medium supplemented with 10% FBS at 37 °C, and cultured in a tri-gas CO₂ incubator (Thermo Fisher, Carlsbad, CA, USA) with a 5% CO₂ and 1% O₂ atmosphere (hypoxic condition). When the cell density reached 1~3 × 10⁵/ml, various concentrations of Dex (0.10 μM, 0.25 μM and 0.50 μM) were added to the culture plates. After 10~14 days in the lag-phase, the cells began to grow. The medium was replaced every 3~4 days to maintain the cells at a density of 5~10 × 10⁵/ml in the next 2~3 weeks. After 5~6 weeks of culture under the hypoxic condition, the cells were transferred to the normoxic condition and cultured in drug-free medium continuously for 1 year.

Logarithmically growing cells were harvested and seeded in 6-well sterile plastic culture plates at a density of 5 × 10²/ml in methylcellulose RPMI-1640 medium containing 0.9% methylcellulose (MethoCult GFH4434; Sigma, St. Louis, MO, USA) and 10% FBS at 37 °C under a humidified atmosphere with 5% CO₂ and 21% O₂. Random aspiration of individual colonies growing in methylcellulose was undertaken on day 8 of the culture. Next, each colony was cultured in RPMI-1640 complete medium.

Cells were cultured in a 6-well sterile plastic culture plates at 1 × 10⁵/ml in RPMI-1640 medium with 10% FBS and grown for 7 days. Viable cells were counted using trypan blue staining every day. Doubling time (Td) was calculated for cells in exponential growth with the following equation: Td (h) = t × lg2/lg(N_t/N₀), where t is the time of continuous culture, N_t is the final number of cells, and N₀ is the initial number of cells. Cell viability was evaluated by MTT assay. Briefly, cells were seeded in 96-well plates. Next, 0.5 mM MTT (final concentration) was added to each well for 4 h at 37 °C. Then, solubilization buffer (10% SDS in 0.01 M HCl) was added to each well, and the plates were further incubated for 24 h at 37 °C. The spectrophotometric absorbance was measured at 570 nm (reference 690 nm) using a multiplate reader (Multiskan Spectrum, Thermo Electron Co., Waltham, MA, USA). Values were obtained by comparing the experimental cells with their respective controls. Mean values were calculated from triplicate cultures.

The chemotherapeutic drugs, Dex, daunorubicin, vincristine, arabinoside, methotrexate and asparaginase, were purchased from Sigma. All cells were treated with increasing concentrations of different drugs for 48 h, followed by assessment of cell viability by MTT assay. The IC₅₀ was calculated by linear interpolation.

For each analysis, 10⁶ cells were harvested 48 h after treatment and fixed overnight in 70% ethanol at 4 °C. The cells were then washed and stained with 5 μg/ml PI in the presence of DNAse-free RNAse (Sigma). After 30 min at room temperature, the cells were analyzed via flow cytometry (Cytomics FC 500 and CXP & Multicycle software, Beckman Coulter Inc., Miami, FL, USA), acquiring 30,000 events.

For the detection of the immunophenotype of the CEM-C7/HDR and CEM-C7–14 cells, we used antibodies against the following targets: CD33, CD34, HLA-DR, cyTdT, cCD3, CD3, CD4, CD5, CD7, CD8, CD117, CD1α, CD2, CD10, CD19, CD20, CD13 and CD45 (Becton Dickinson Inc., Franklin Lakes, NJ, USA). Positivity for the antigens was determined using a FACSCalibur flow cytometer (Becton Dickinson Inc.).

The identity of the CEM-C1–15, CEM-C7–14, CEM-C7/HDR and CEM-C7/H cell lines was checked using DNA fingerprinting. DNA was prepared from these cells using the Qiagen DNeasy Blood Kit (Qiagen), according to the instructions provided by the manufacturer. The following 22 highly polymorphic short tandem repeat (STR) loci were tested by a multiplex PCR: Amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, vWA, Penta E, Penta D, D2S441, D2S1338, D3S1358, D6S1043, D8S1179, D10S1248, D12S391, D18S51, D19S433, D21S11 and FGA.

Briefly, cells (10⁶) were washed twice in cold PBS and then lysed by Laemmli sample buffer (Bio-Rad). Samples were boiled for 5 min at 100 °C. Proteins were separated by 8%~ 15% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (0.22 μm or 0.45 μm, Millipore). Non-specific binding sites were blocked with 5% non-fat dry milk dissolved in TBS (10 mM Tris-HCl, pH 7.6, 137 mM NaCl) with 0.1% Tween 20 (TTBS) for 2 h at room temperature, followed by incubation with primary antibody for 2 h at room temperature or at 4 °C overnight. The membranes were then washed 3 times in TTBS and incubated for 2 h at room temperature with secondary HRP–conjugated donkey anti-rabbit antibody or HRP-conjugated sheep anti-mouse antibody diluted 1:5000 in TTBS with 5% non-fat milk. Proteins were visualized by incubation with ECL plus (Millipore). All experiments were conducted independently at least 3 times. The level of the β-Actin protein was used as a control for the amount of protein loaded into each lane.

Cultured 5 × 10⁶ cells were subcutaneously injected into the right flanks of 6-week-old female BALB/c (nu/nu) nude mice, and 0.1 ml of PBS was injected into the left flanks as the control. Once palpable tumors were established, animals were randomized into 2 groups, each containing 4 mice. Mice were injected intraperitoneally daily with 15 mg/kg/d Dex (Dex group), or PBS (Control group) for 14 days. Tumor size was measured by calipers every 2 days. The approximate tumor volume was calculated using the eq. V = (length×width×depth)/2. All animals were ear-tagged and monitored individually throughout the experiment. All animal care was in compliance with the guidelines established by the internal Institutional Animal Care and Use Committee and Ethics Committee of Sichuan University. After the mice were euthanized, the tumor mass was excised, fixed in 10% formalin, and routinely processed for paraffin embedding. Five-millimeter-thick sections were obtained and prepared for standard histopathological examination.

Logarithmically growing CEM-C7–14 cells were cultured under hypoxia condition for 5~6 weeks with or without dexamethasone (Dex) treatment. Then, the cells were transferred to the normoxic condition for 1 week. At that time, an MTT assay revealed that the IC₅₀ values of the Dex groups, with a single treatment of 0.10 μM, 0.25 μM or 0.50 μM Dex respectively, were 50~150 μM at 48 h. We chose the 0.25 μM Dex group to be cultured continuously and constructed a stable resistant cell line, which was designated CEM-C7/HDR. The IC₅₀ of Dex in the CEM-C7/HDR cells at a 10 cell population doubling level (PDL) was 128.1 ± 4.73 μM. Next, the IC₅₀ and the RI of those cells decreased slowly, resulting in the PDL increasing from 10 to 100, where it stabilized after 100 PDL (Fig. 1a). After 1 year of Dex-free continuously culturing, the IC₅₀ and RI were 60~70 μM and 1500~1800, respectively (Fig. 1a). Unexpectedly, the control group, which was cultured under the hypoxic condition with no Dex treatment, also gained Dex resistance. We named the control cell line CEM-C7/H, and its RI was 10~20 after 100 PDL (Fig. 1b). All CEM-C7/HDR subclones were resistant to Dex with RIs ranging from 1200 to 2000 (Fig. 1c). However, we obtained both resistant and sensitive subclones of CEM-C7/H, with RIs ranging from 0.3 to 33.4 (Fig. 1d). Interestingly, CEM-C7/H cells obtained cross-resistance to daunorubicin and vincristine, with RIs of 2.02 ± 0.23 and 1.71 ± 0.19, respectively. However, CEM-C7/HDR cells did not obtain cross-resistance to daunorubicin, vincristine, arabinoside, methotrexate or asparaginase. Figure 1e showed the procedure for constructing above cell lines. To clarify the mechanisms of resistance development, we selected two GC-sensitive CEM-C7–14 mono-clonal cells, named CEM-C7-SC2 and CEM-C7-SC14, with IC₅₀ values of 0.04 μM and 0.06 μM, respectively. This method resulted in the development of two resistant cell lines named CEM-C7-SC2/HDR and CEM-C7-SC14/HDR, with IC₅₀ values of 100~150 μM at 10PDL. The control groups, CEM-C7-SC2/H and CEM-C7-SC14/H, which were cultured under hypoxic condition with no Dex treatment, did not express the resistant phenotype. Additional file 1: Figure S1 showed the origin and name of the aforementioned cell lines.

To evaluate the repeatability of the method, we constructed stable Dex-resistant cell lines, NALM-6/HDR and HXEX-ALL1/HDR, from the NALM-6 and HXEX-ALL1 cell lines, using the same method. NALM-6/HDR and HXEX-ALL1/HDR were 28,000- and 4- fold more resistant to Dex than their parental cells (Additional file 2: Figure S2).

The CEM-C7/HDR and CEM-C7/HDR-C2 (its monoclonal cell line) cells were grown in suspension as single cells, and Wright-Giemsa staining showed no obvious differences in morphology between the two resistant cell lines and the parental cell line, CEM-C7–14 (Fig. 2a and b). All three cells lines showed similar percentages of G₀/G₁ and S phase cells, with no statistically significant differences observed (P > 0.05) (Fig. 2c). They also exhibited similar growth curves (Fig. 2d). The three cell lines all stably proliferated in RPMI-1640 medium containing 10% FBS with population doubling times of 16~22 h. CEM-C7/HDR and CEM-C7–14 displayed identical immunophenotype with cCD3, cyTDT, CD4, CD5 and CD7 positive (Fig. 3). STR analysis confirmed that CEM-C7/HDR, CEM-C7/H, CEM-C7–14 and CEM-C1–15 were all derived from CCRF-CEM (Table 1).

Low expression of GR may be a major cause of GC resistance in leukemia cells [13, 14]. As shown in Fig. 4a, the expression of GR and p-GR (Ser211) were significantly lower in the CEM-C7/HDR cells than in the CEM-C7–14 and CEM-C7/H cells. In GC resistant cells, GR expression was lower in CEM-C7/HDR than in CEM-C1–15 (p < 0.01), the p-GR (Ser211) expression showed no significant difference between the two cell lines (p > 0.05). We obtained CEM-C7/HDR and CEM-C7/H cell lines from culturing under hypoxia condition. Therefore, we detected the expression of the energy sensor proteins, AMPK and p-AMPK. However, the expression of AMPK and p-AMPK in CEM-C7/HDR were both lower than in CEM-C7–14 and CEM-C1–15 (p < 0.01) (Fig. 4a). In CEM-C7/H, the expression of p-AMPK was lower than in CEM-C7–14, although the AMPK expression showed no significant difference (p > 0.05) (Fig. 4a). It is generally known that culturing under hypoxic conditions may induce a cellular switch in energy production, which is characterized by an increase in glycolysis phenotype in cancer cells [15]. In our study, although the CEM-C7/HDR cell line was cultured in hypoxic condition for 5 weeks, the expression of glycolysis-associated proteins, Glut-1, HKII, LDH and p-LDH, were all lower than that in CEM-C7–14 (p < 0.01) (Fig. 4b). Dysregulated activation of PI3K-Akt-mTOR pathway may relate to the chemotherapeutic resistance, including GC resistance, in hematological malignances [16, 17]. However, in CEM-C7/HDR, the expression of the two main downstream effectors of mTOR, 4E-BP1 and p70S6K, and their phosphorylated status p-4E-BP1 (Thr37/46) and p-p70S6K (Thr389), were all obviously lower than CEM-C7–14, CEM-C7/H and CEM-C1–15 cells (p < 0.01) (Fig. 4c). Thus, the molecular mechanisms of GC resistance in the CEM-C7/HDR cell line should be far different from that in the CEM-C1–15 cells.

In an in vivo transplantation test, subcutaneous injection of CEM-C7–14 and CEM-C7/HDR resulted in the development of tumors in all 12 mice (100%). After 20 days, the mean volume of the tumors generated by subcutaneous injection with CEM-C7–14 and CEM-C7/HDR were 717.8 ± 309.6 mm³ (n = 6) and 803.0 ± 294.9 mm³ (n = 6), respectively. Hematoxylin and eosin (HE) staining indicated that the tumor masses were composed of leukemia cells (Fig. 5a). Immunohistochemistry (IHC) staining showed that the expression locations of GR, Glut-1, HKII, and 4E-BP1 proteins were consistent in the xenografts of CEM-C7–14 and CEM-C7/HDR (Fig. 5b-e). The results from STR analysis indicated that subcutaneous tumors derived from the corresponding CCRF-CEM cells (Table 1).

Having shown the GC-resistant phenotype of CEM-C7/HDR in vitro, we examined the in vivo efficacy of Dex given intraperitoneally in CEM-C7/HDR and CEM-C7–14 xenografts in nude mice. As anticipated, 15 mg/kg/d Dex used alone showed almost no antitumor effect (p > 0.05) in CEM-C7/HDR xenografts mice, whereas inhibited tumor growth obviously in CEM-C7–14 xenografts mice (p < 0.05, Fig. 5f).