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

Background With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. Methods To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). Results siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. Conclusion In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.


Background
Medulloblastomas (MB) are the most common malignant pediatric neoplasms of the central nervous system. MB constitute 20% of all pediatric brain tumors [1] and are characterized by their aggressive clinical behavior and a high risk of leptomeningeal dissemination. With current treatment strategies, nearly half of all patients eventually die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal.
Developing therapeutic approaches to inhibit c-MYC would have an enormous impact on the treatment of a wide range of human cancers [25][26][27]. Many strategies are currently under development to target c-MYC in tumor cells, including inhibitors that block c-MYC expression, such as antisense oligonucleotides and small interfering RNA (siRNA) [28]. One aspect to be considered by evaluating the potential of c-MYC as a novel therapeutic target in MB, is its impact on cellular sensitivity to radio-and chemotherapy. In the present study, siRNA-mediated c-MYC inhibition was used to study the biological role of c-MYC in a representative panel of human MB cells expressing different levels of c-MYC.

Methods
Human MB cell lines 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% CO 2 .

Down-regulation of c-MYC
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].
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).

c-MYC transcription factor binding activation assay
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].

Clonogenic survival assay
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% CO 2 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.

Cell cycle analysis
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
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, 10 6 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.

Apoptosis assay
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 × 10 4 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).

Chemo-and radio-sensitivity assays
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.

Statistical analysis
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 IC 50 values and their 95% confidence intervals and to statistically compare the fitted midpoints (log IC 50 ) of the two curves.

Targeting c-MYC by siRNA in MB cells
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 downregulation 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 pro-tein 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).

siRNA-mediated c-MYC down-regulation reduces cellular proliferation, inhibits clonogenic growth, induces G1 cell cycle arrest and CDK inhibitor p21 (waf1/Cip1) expression in MB cells
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 siRNAmediated 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

siRNA-mediated c-MYC down-regulation reduces hTERT mRNA expression and telomerase activity in 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 siRNAtransfected cells, as determined by real-time RT-PCR (Figure 4A). We then determined the effect of c-MYC downregulation 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 siRNAtransfected 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). siRNA-transfected cells were more resistant to killing by IR ( Figure 5).

siRNA-mediated c-MYC down-regulation attenuates the cellular apoptotic response of DAOY MB cells to chemotherapy that is associated with reduced caspase-9 activity
To investigate whether the reduction of chemo-sensitivity induced by c-MYC down-regulation, as measured by cell viability (Figure 6 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, trans-fected 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.

Discussion
Whereas the impact of c-MYC gene amplification and c-MYC mRNA over-expression on MB histology and prognosis has been investigated intensively [19][20][21][22][23][24], the biological functions of c-MYC in MB cells remain largely elusive. To our knowledge, the current study represents the first comprehensive analysis of c-MYC targeting in a representative panel of human MB cells expressing different levels of c-MYC.
c-MYC siRNA-mediates reduced susceptibility to ionizing radiation  siRNA-mediated c-MYC down-regulation resulted in an anti-proliferative effect in all human MB cell lines tested. Moreover, the ability of c-MYC siRNA-treated MB cells to form large colonies was significantly reduced. Down-regulation of c-MYC expression resulted in an activation of the CDK inhibitor p21 and in a cell cycle arrest at the G0/ G1 phase. These results are supported by findings from others, demonstrating that c-MYC represses the p21 promoter [44], and showing that c-MYC over-expression in primary human fibroblasts results in inhibition of p21 transcription [45]. Moreover, the G1-S progression of eukaryotic cells is in part controlled by c-MYC [2, 4,7]. Ectopic expression of c-MYC in quiescent cells promotes the entry into the S-phase [46,47]. On the other hand, down-regulation of c-MYC expression by antisense meth-ods resulted in anti-proliferative effects by preventing Sphase entry [48,49]. Our investigation shows that c-MYC inhibition reduces tumor cell growth suggesting that c-MYC might be an attractive target in MB to inhibit tumor growth.
siRNA-mediated c-MYC down-regulation resulted in a down-regulation of hTERT mRNA expression and in a reduction of telomerase activity in all but D425 human MB cells. D425 cells are characterized by relatively low hTERT mRNA expression and TRAP-negativity [40]. However, the absence of significant correlation between c-MYC and hTERT expression and the difference seen between hTERT mRNA and activity inhibition could be attributed to the presence of several binding sites for other transcrip- A tion factors -than c-MYC -on hTERT gene promoter and to the fact that expression of hTERT mRNA is not the only regulator of telomerase activity [50][51][52]. Alternative splicing [53], posttranscriptional modification [54,55], subcellular hTERT localization [56] and the presence of enzyme inhibitors may alter telomerase activity. Moreover, it has been suggested that a threshold level of hTERT protein is required in order for telomerase to become active, therefore low levels of hTERT mRNA may not indicate active telomerase [57]. In this respect, hTERT expression has not been found to correlate with telomerase activity in colorectal tumors [58]. Furthermore, hTERT mRNA expression has been detected in lymphocytes regardless of telomerase activity [59] and in some normal telomerase negative tissues such as human brain, prostate liver and ovary [60]. Telomerase impairment can lead to the induction of apoptosis through a mechanism that relies on the inhibition of telomerase catalytic activity and on the consequent telomere shortening [61,62]. However, depending on the telomere length, this mechanism is rather slow. Several generations and a long lag time may be required before telomeres shorten below a critical length [62][63][64]. Holt et al. have showed that cells with elongated telomeres are more resistant to induction of apoptosis than their parental counterparts with short telomeres [65]. Our experiments were executed in a short time window (72 h) due to cell distress upon treatment with c-MYC siRNA.
DAOY M2 cells engineered to stably over-express c-MYC show higher basal apoptotic activity compared with DAOY wt and DAOY V11 cells, and are characterized by enhanced apoptosis rate in vivo [24]. D425 and D341 MB cells (both with c-MYC gene amplification and high c-MYC expression) also show a higher basal apoptotic rate when compared with DAOY wt cells. Moreover, c-MYC inhibition resulted in a significant decrease in the apoptotic rate which was most pronounced in MB cells with high c-MYC expression. It has been shown that c-MYC can drive cells to undergo apoptosis [7][8][9]. However, this might not be the case for all cell lines. In UW-228 human MB cells, down-regulation of c-MYC by an antisense approach resulted not only in an inhibition of cell growth but also in an increase of apoptosis as determined by FACS analysis [66].
We then analyzed whether c-MYC down-regulation alters sensitivity to IR and chemotherapeutic agents and found a decrease in the sensitivity to IR, cisplatin, and etoposide. This effect was most prominent in cells expressing high c-MYC levels. The differences of cytotoxic effect of cisplatin and etoposide on siRNA-transfected MB cells may be due to the different mode of action that the two drugs exert. The cytotoxicity of cisplatin is mediated by its interaction with DNA to form DNA adducts, which activate several signal transduction pathways and terminate in the activation of apoptosis [67], whereas etoposide belongs to the class of topoisomerase II poison with the ability to stabilize complex between DNA topoisomerase II and DNA that results in a high level of DNA damage [43]. By addressing the question of whether c-MYC alters the response of cells to radio-and chemotherapy, results have been conflicting [68][69][70][71][72][73][74]. Our results are in agreement with studies demonstrating that c-MYC sensitize a variety of cells to different cytotoxic treatments [68][69][70], and that absence of c-MYC expression might confer resistance to anti-cancer agents [71]. However, this effect of c-MYC also seems to be cell type-specific. In human melanoma cells, a decrease in c-MYC expression has been reported to enhance the effect of IR [72] and cisplatin treatment [73,74].
Cancer chemotherapies induce apoptosis [42]. Changes that decrease the ability to activate the apoptotic machinery might affect the sensitivity of cells to a range of chemotherapeutic agents. In order to evaluate whether c-MYC down-regulation affects the susceptibility to apoptosis, we investigated the apoptotic response of siRNAs-transfected DAOY M2 cells following cisplatin and etoposide treatment. This cell line was used, because c-MYC inhibition caused significant reduction of chemo-sensitivity to cisplatin and etoposide. This analysis revealed that down-regulation of c-MYC attenuated drugs-induced apoptosis that was associated with a reduced caspase-9 processing. These results are consistent with findings from other groups showing that c-MYC inhibition mediates resistance to chemotherapy-induced apoptosis by preventing activation of caspase-mediated pathways [68,69,71].

Conclusion
Taken together, our data show for the first time that targeting c-MYC in human MB cells decreases cell growth, induces cell cycle arrest and reduces telomerase activity. However, c-MYC down-regulation also causes decreased apoptosis and resistance towards IR, cisplatin, and etoposide. Following in vivo validation, targeting c-MYC might represent a promising strategy to reduce tumor growth in pretreated MB patients whose tumors have ceased to respond to chemotherapy. Our data also indicate that targeting c-MYC might not be used concomitantly with chemotherapy or radiotherapy in MB because of the risk of reducing the effectiveness of these therapies.
Emerging evidence indicates that the different precursor cell populations that form the cerebellum and the cell signaling pathways that regulate its development likely represent distinct compartments from which the variable subtypes of MB arise [75,76]. It remains to be tested whether down-regulation of c-MYC has MB subtype specific effects or is rather MB subtype independent.