RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

DAOY (wild-type), DAOY V11 (empty vector-transfected), and DAOY M2 (c-MYC-transfected) human MB cells have been described previously [24]. D341 and D425 human MB cells were the kind gift of Dr Henry Friedman, Duke University, Durham, NC, USA. DAOY, D341, and D425 MB cell lines are derived from three different MB obtained at craniotomy of patients aged 3–5 years [21, 29, 30]. All MB cells were cultured in Richter's zinc option medium (Invitrogen; Basel, Switzerland) supplemented with 10% fetal bovine serum. Non-essential amino acids were added to the medium of D341 and D425 cells to a final concentration of 1%, and G418 was added to the medium of DAOY V11 and DAOY M2 to a final concentration of 500 μg/ml. All cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂.

The Silencer c-MYC validated siRNA targeting the 3'-untranslated region of the human c-MYC mRNA sequence [31, 32], referred to here as c-MYC siRNA, and Silencer scrambled c-MYC negative control siRNA, referred to here as control siRNA, having no significant homology to any known gene sequences from mouse, rat, or human, were purchased from Ambion, and used according to the manufacturer's instructions [33]. In brief, small interfering RNAs (siRNAs) for c-MYC and scrambled control siRNA were transfected at a final concentration of 30–60 nM with siPORT amine (Ambion) in the case of DAOY MB cells and with siPORT NeoFX (Ambion) in the case of D341 and D425 MB cells as described before [34, 35]. Comparable with other reports we found reproducible inhibition of RNA and protein expression at 48–72 h post transfection [34, 35].

Total RNA isolation, reverse transcription reactions, and RT-PCR were performed as described previously [36]. Kinetic real-time PCR quantification of target genes was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems; Rotkreuz, Switzerland), as described previously [36]. Primers and probes for c-MYC (assay ID: Hs00153408_m1) and the endogenous control 18S rRNA (assay ID: Hs99999901_s1) were purchased from Applied Biosystems (Rotkreuz, Switzerland). Primers and probes for hTERT were purchased from Microsynth (Balgach, Switzerland): forward primer, 5'-TGACACCTCACCTCACCCAC-3', reverse primer, 5'-CACTGTCTTCCGCAAGTTCAC-3', probe, 5'-ACCCTGGTCCGAGGTGTGTCCCTGAG-3'. Experiments were performed in triplicate for each data point. Relative mRNA expression of target genes was calculated by using the comparative C_T method [37].

The expression of c-MYC, cyclin-dependent kinase (CDK) inhibitor p21 ^(waf1/Cip1), caspase-9 and β-actin protein was assessed by Western blot analysis. In brief, human MB cells transfected with siRNAs for 72 h or treated with chemotherapy for 72 h after 48 h of transfection with siRNAs, were lysed with lysis buffer (1 ml/10⁷ cells, 50 mM Tris-HCl buffer [pH 8.0], 150 mM NaCl, 1% Nonidet P40, 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (Complete, Roche; Basel, Switzerland) and incubated on ice for 30 min. After measuring the protein concentration by the BCA method (Pierce; Rockford, USA), 12 μg total protein lysates were separated by 10% SDS-polyacrylamide gels and the gels were subjected to immunoblotting. Nonspecific binding sites were blocked by 3 h incubation in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05% Tween-20) supplemented with 5% non-fat dry milk. Membranes were incubated overnight at 4°C with a 1:2000 dilution of rabbit polyclonal anti-c-MYC antibody (Santa Cruz Biotechnology; Heidelberg, Germany), with a 1:1000 dilution of mouse monoclonal anti-p21 ^(waf1/Cip1) antibody (Upstate), and with a 1:1000 dilution of rabbit polyclonal antibody specific for cleaved caspase-9 (Cell signaling).

Membranes were then washed three times at room temperature in TBST for 30 min each time, and bound Ig was detected using anti-isotype monoclonal secondary antibody coupled to horseradish peroxidase (Santa Cruz Biotechnology; Heidelberg, Germany). The signal was visualized by enhanced chemiluminescence ECL (Amersham Biosciences; Dübendorf, Switzerland) and autoradiography. Next, immunoblotting with a 1:5000 dilution of a mouse monoclonal primary β-actin antibody (Sigma; Basel, Switzerland) was performed to verify the protein loading. For densitometry, the zymographic profiles of the gels were scanned. Relative band intensities were determined using Quantity One analysis software (Bio-Rad).

The binding activation of c-MYC was measured using the Mercury TransFactor assay (BD Clontech, Basel, Switzerland), an enzyme-linked immunosorbent assay (ELISA)-based assay, according to the manufacturer's instructions, as previously described [36].

Following transfection with siRNAs, a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (Promega; Wallisellen, Switzerland) was used to quantitate cell viability of human MB cells, as previously described [36]. Each experiment was performed in triplicate. The absorbance values of each well were measured with a microplate spectrophotometer (Molecular Devices; Sunnyvale, CA) at 490 nm. All proliferation assays were repeated as independent experiments at least twice.

A clonogenic survival assay was performed using cells transfected with siRNAs for 48 h, as described previously [38]. The number of single cells seeded was adjusted to obtain around 100 colonies per cell culture dish [39]. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and allowed to grow for 9 (DAOY wt, V11, M2), 14 (D341), or 12 (D425) days respectively, before fixation in methanol/acetic acid (75%:25%) and staining with Giemsa. Only colonies with more than 50 cells were counted. All clonogenic assays were repeated as independent experiments at least twice.

Cells transfected with siRNAs were harvested after 48 h by trypsinization, washed with PBS 1×, and fixed with 1 ml of 70% ethanol [38]. After washing twice in PBS, the cells were stained with a solution containing 50 μg/ml propidium iodide (Becton-Dickinson; Allschwil, Switzerland) and 100 U/ml RNase A (Qiagen; Hombrechtikon, Switzerland) in PBS for 30 min at room temperature. The percentage of cells in the different phases of the cell cycle was determined by evaluating DNA content according to methods previously described [36]. A total of 30'000 events per sample were acquired. Flow cytometric analysis was performed on a FACSCalibur flow cytometer (BD Biosciences; Allschwil, Switzerland) with CELLQuest software (BD Biosciences). The percentages of cell cycle distribution were calculated on linear PI histograms using the mathematical software ModFit LT 2.0 (Verity Software House; Topsham, ME)

Telomerase activity in MB cell lines was measured using the Telomerase PCR ELISA kit (Roche Diagnostics; Rotkreuz, Switzerland) as previously described [40]. For this procedure, 10⁶ cells were lysed and homogenized in 200 μl CHAPS buffer. After 30 min incubation on ice, the lysates were centrifuged at 16,000 g for 30 min at 4°C. Protein concentration was measured using the BCA method (Pierce; Rockford, USA). For the elongation step, 2 μl of cell extract (2 μg protein/μl) were incubated in 50 μl reaction mixture at 25°C for 30 min to allow the telomerase to add telomeric repeats (TTAGGG) to the end of the biotin-labeled synthetic P1-TS primer. The products extended by telomerase were then amplified by PCR in the presence of the biotin-labeled P1-TS primer and another primer, P2. Amplification products from the PCR were next immobilized in the well of a StreptAvidin-coated microplate and hybridized to a digoxigenin (DIG)-labeled probe specific for the telomeric repeat sequence. Finally, the DIG-labeled hybrids were visualized with a peroxidase-conjugated anti-DIG antibody and a colorimetric peroxidase substrate and quantified photometrically. In order to visualize the typical 6-nucleotide-ladder resulting from the TRAP assay, the amplification products from the PCR were separated by 12.5% polyacrylamide gel electrophoresis (PAGE), blotted onto a positively charged membrane, and incubated with a StreptAvidin alkaline phosphatase conjugate. The blotted products were visualized by enhanced chemiluminescence (Biotin Luminescence Detection Kit Roche Applied Science; Rotkreuz, Switzerland) and autoradiography.

Cell lysates were obtained from exponentially growing MB cells transfected with siRNAs for 72 h and from MB cells treated with etoposide and cisplatin for 72 h after 48 h of transfection with siRNAs. A photometric enzyme immunoassay (Cell Death Detection ELISA; Roche Diagnostics, Basel, Switzerland) was used for the quantitative determination of cytoplasmic histone-associated DNA fragments, as described previously [36]. In brief, after the different treatment schedules, cells were counted, and cell lysates of equal number of cells (5 × 10⁴ cells/ml) were placed in a StreptAvidin-coated microtiter plate. A mixture of biotin-labeled monoclonal histone antibody and peroxidase-conjugated monoclonal DNA antibody was then added, followed by incubation for 2 h. After washing to remove unbound antibodies, the amount of mono- and oligonucleosomes was measured at 405 nm (reference wavelength 490 nm).

After 48 h of transfection with c-MYC siRNA or control siRNA, appropriate numbers of exponentially growing MB cells were seeded in 96-well plates. Cells were treated for 72 h with various concentrations of etoposide and cisplatin or were irradiated with 4 Gy and 10 Gy, using a Pantak Therapax 300 kV X-ray unit at 0.7 Gy/min. Dosimetry was controlled with a Vigilant dosimeter. Cell viability of human MB cells was quantified using the MTS assay 72 h after adding the chemotherapeutic substances and irradiation, respectively.

All data are expressed as mean ± SD. Student's t-test was used to test statistical significance. P < 0.05 was considered to be significant. GraphPad Prism 4 (GraphPad Software, San Diego, California) software was used to calculate IC₅₀ values and their 95% confidence intervals and to statistically compare the fitted midpoints (log IC₅₀) of the two curves.

To establish an efficient method of specifically down-regulating c-MYC expression in MB, we used a siRNA approach. Transfection of human MB cells with siRNA directed against c-MYC resulted in a pronounced down-regulation of c-MYC mRNA expression to 21% in DAOY wt (p < 0.001), 22% in DAOY V11 (p < 0.001), 6% in DAOY M2 (p < 0.001), 33% in D341 (p < 0.001), and 65% in D425 (p = 0.017) compared with negative control siRNA-transfected cells, as determined by quantitative RT-PCR at 48 h following transfection (Figure 1A). At the protein level, transfection of human MB cells with c-MYC siRNA resulted in a significant down-regulation of c-MYC protein expression compared with negative control siRNA-transfected cells as determined by Western blotting 72 h post transfection in all MB cell lines tested (Figure 1B). Transfection of human MB cells with c-MYC siRNA resulted in a significant reduction of c-MYC binding activation to 10% in DAOY wt (p = 0.015), 28% in DAOY V11 (p = 0.009), 21% in DAOY M2 (p = 0.008), 74% in D341 (p = 0.05), and 63% in D425 (p = 0.03), compared with MB cells transfected with negative control siRNA at 72 h following transfection (Figure 1C).

To test whether siRNA-mediated reduction of c-MYC alters cellular proliferation of human MB cells, we assessed cell viability at 0, 2, 4, and 6 days following c-MYC siRNA or control siRNA transfection. When compared with control siRNA-transfected cells, growth was lower in c-MYC siRNA-transfected MB cell lines (Figure 2A). This effect was independent of basal c-MYC expression. We then determined clonogenic survival using MB cells transfected with control or c-MYC siRNA. After 48 h of exposure to either c-MYC siRNA or to control siRNA, the siRNA was removed, and clonogenic survival was determined. When compared with control siRNA-transfected cells, the clonogenic survival of c-MYC siRNA-transfected cells was significantly reduced, as observed by decreased number of large colonies (colonies with more than 50 cells), to 67% in DAOY wt (p = 0.011), 78% in DAOY V11 (p = 0.026), 26% in DAOY M2 (p = 0.038), 52% in D341 (p = 0.003), and 59% in D425 (p = 0.001) human MB cells (Figure 2B). We then examined whether alterations in the cell cycle underlie the c-MYC siRNA-mediated effect on proliferation of MB cells. Flow cytometric analysis of cells transfected with c-MYC siRNA for 48 h showed consistent changes in the cell cycle distribution compared with control siRNA-transfected cells. In all cell lines tested, there was an increase in the number of cells in the G0/G1 phase, which was significant in DAOY wt (p = 0.028), DAOY V11 (p = 0.001), DAOY M2 (p = 0.033), and D341 (p = 0.006), but slightly below the significance level in D425 (p = 0.062) (Figure 2C). In all MB cells under study there was a decrease in the number of cells in the S-phase, which was significant in DAOY V11 (p = 0.005) and DAOY M2 (p = 0.05), compared with control siRNA-transfected cells. The observation that c-MYC siRNA transfection resulted in an up-regulation of CDK inhibitor p21 ^(waf1/Cip1) expression compared with control siRNA-transfected cells to 153% in DAOY wt, 128% in DAOY V11, 239% in DAOY M2, 142% in D341, and 198% in D425, as determined by Western blotting and photometrical quantification (Figure 2D), was in accordance with the G1 arrest induced by c-MYC down-regulation. Taken together, these results demonstrate that down-regulation of c-MYC inhibits tumor cell growth at least partly by induction of cell cycle arrest

To investigate whether the decrease in cellular growth induced by c-MYC down-regulation was also caused by increased levels of apoptosis, we determined apoptotic cell death in siRNA-transfected MB cells (72 h). This analysis revealed that down-regulation of c-MYC resulted in a reduction of apoptosis in all human MB cell lines tested (Figure 3; DAOY wt: 79% (p = 0.12); DAOY V11: 76% (p = 0.10); DAOY M2: 48% (p = 0.001); D341: 49% (p = 0.002), D425: 54% (p = 0.001)). This inhibitory effect on apoptosis was more pronounced in human MB cell lines expressing high levels of c-MYC. Cell cycle analysis of control and c-MYC siRNA-transfected cells revealed a decrease in the proportion of cells in sub-G1 when exposed to c-MYC siRNA to 74% in DAOY, to 50% in DAOY V11, to 47% in DAOY M2, to 48% in D341, and to 53% in D425 MB cells.

To investigate the effects of c-MYC down-regulation on telomerase in MB cells, we determined hTERT mRNA expression by using quantitative RT-PCR at 48 h following transfection with c-MYC siRNA or control siRNA. We found a significant reduction of hTERT mRNA expression in c-MYC siRNA-transfected cells to 34% in DAOY wt, 54% in DAOY V11, 21% in DAOY M2, 15% in D341, and 35% in D425 compared with negative control siRNA-transfected cells, as determined by real-time RT-PCR (Figure 4A). We then determined the effect of c-MYC down-regulation on the enzymatic activity of telomerase and performed a modified TRAP assay on cell lysates 72 h post transfection. This analysis revealed that telomerase activity was inhibited in c-MYC siRNA-transfected DAOY wt, DAOY V11, DAOY M2, and D341 MB cells (Figure 4B). Telomerase activity was found to be reduced to 77% in DAOY wt, 79% in DAOY V11, 63% in DAOY M2, and 30% in D341, compared with negative control siRNA-transfected cells (Figure 4C). In D425 MB cells, which are characterized by low TRAP activity [40], c-MYC inhibition did not alter the telomerase activity (Figure 4B,C).

DAOY, D341, and D425 human MB cells transfected with c-MYC or control siRNA were irradiated with 0, 4, and 10 Gy and cell viability was quantified after 72 h. D341 and D425 were more sensitive to IR than DAOY cells harboring mutant p53 [41] (Figure 5). DAOY M2 cells engineered to express higher c-MYC levels were more susceptible to IR than DAOY wt or DAOY V11 (Figure 5). Compared with control siRNA-transfected cells, c-MYC siRNA-transfected cells were more resistant to killing by IR (Figure 5).

To investigate whether siRNA-mediated down-regulation of c-MYC alters chemo-sensitivity of MB cells, we measured the cytotoxic response to etoposide and cisplatin. In the case of cisplatin treatment, c-MYC siRNA transfection resulted in a significant decrease in chemo-sensitivity in DAOY M2 (p < 0.001), D425 (p = 0.003), and D341 (p = 0.013) (Figure 6A). However, no changes in chemo-sensitivity were observed in DAOY wt and DAOY V11 human MB cells (Figure 6A). In the case of etoposide treatment, c-MYC siRNA transfection resulted in a significant decrease in chemo-sensitivity in DAOY wt (p < 0.0001), DAOY V11 (p < 0.0001), DAOY M2 (p < 0.0001), and D341 (p < 0.001), and a minor decrease in D425 (p = 0.12) (Figure 6B).

To investigate whether the reduction of chemo-sensitivity induced by c-MYC down-regulation, as measured by cell viability (Figure 6), was caused by decreased levels of apoptosis, we measured the apoptotic cell death in siRNA-transfected (48 h) DAOY M2 cells – showing significant difference in viability following c-MYC down-regulation and treatment with cisplatin and etoposide – after treatment with either cisplatin or etoposide for 72 h. DAOY V11 was included as a control. Following c-MYC down-regulation, siRNA-transfected DAOY V11 cells were equally sensitive to cisplatin in terms of viability and induction of apoptosis (Figure 6A, 7A). In the case of etoposide treatment, c-MYC siRNA-treated DAOY V11 cells showed reduced cellular sensitivity (Figure 6B) and only a moderate induction of apoptosis compared to control siRNA-transfected cells (Figure 7B). Treatment of control siRNA-transfected DAOY M2 cells with cisplatin and etoposide resulted in a dose-dependent increase of apoptotic cell death when compared with c-MYC siRNA-transfected DAOY M2 cells. Most of the chemotherapeutics, including cisplatin and etoposide [42, 43], induce apoptosis mainly through the mitochondrial pathway. Mitochondria-mediated apoptosis involves the cleavage of pro-caspase-9 into the active caspase-9. Next, we examined processing of caspase-9 in DAOY M2 cells, transfected with siRNAs for 48 h and treated for 72 h with cisplatin and etoposide. More caspase-9 cleavage was detected in control siRNA-transfected cells compared to c-MYC siRNA-transfected DAOY M2 cells as determined by Western blotting. This analysis suggests that down-regulation of c-MYC inhibits the apoptotic response to chemotherapy that appears at least partly mediated by caspase-9.