Regulation of MYCNexpression in human neuroblastoma cells

Tumour samples were aliquoted in two parts to isolate both DNA and RNA. Total DNA was isolated with the QIAamp isolation-kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. All samples were RNAse treated. Total RNA was isolated with TriZol reagent (Invitrogen, Carlsbad, CA, USA) and treated with Deoxyribonuclease I (Dnase I; Invitrogen). DNase-treated RNA was reverse-transcribed using oligo(dT) primers with the SuperScript First-Strand Synthesis System (Invitrogen).

MYCN and splice variants were amplified from cDNA by using the GC-RICH PCR System (Roche Applied Science, Almere, The Netherlands). Primers were developed by the primer3 program (http://​frodo.​wi.​mit.​edu/​cgi-bin/​primer3/​primer3_​www.​cgi[19]). Primer sequences are shown in table 1 and the position of the primers in MYCN are depicted in figure 1. PCR products were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing V2.0 Ready Reaction Kit and analysed with the ABI PRISM 3730 DNA analyser (Applied Biosystems, Foster City, CA, USA).

QPCR and gQPCR was performed by SYBR Green-based quantification (Bio-Rad, Veenendaal, The Netherlands). PCRs were performed on an iCycler (MyiQ single color Real-Time detection System, Bio-Rad, Veenendaal, The Netherlands). Sequences of the primers used to quantify cDNA transcript levels and genomic DNA are shown in table 1 and the position of the primers is depicted in figure 1. PCR products were between 80- and 100-bp. Validation of the primer pairs and (g)QPCR experiments were performed as described previously [20, 21]. Differences in expression of a gene of interest or in genomic DNA copy number between two samples were calculated by the comparative Ct or 2^ΔΔCt method [22, 23]. Hoebeeck et al. described and validated a similar assay for the determination of MYCN copy numbers in tumor samples [24].

NB-samples were homogenized in RIPA-buffer (50 mM Tris-HCl pH 7.4, 0.2% sodium dodecyl sulfate (SDS), 0.2% sodium deoxycholate, 1% triton X-100, 1 mM EDTA) supplemented with 1 mM DTT, 1 mM PMSF, aprotinin 2 μg/ml and leupeptin 2 μg/ml. Total protein concentration was determined according to the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).

For immunoprecipitation, 5 μg C-19 mAb (Santa Cruz Biotechnology, Heidelberg, Germany) was coupled to Prot A sepharose CL-4B beads (Pharmacia Biotechnologies, Uppsala, Sweden) for 1 hour at 4°C to). NB-lysates were precleared O/N with 50 μl packed Prot A sepharose CL-4B beads. To the precleared lysates, 20 μl C-19-coupled beads was added and incubated for 24 h at 4°C. Subsequently, the beads were washed with PBS and resuspended in SDS sample buffer and stored at -80°C until SDS-polyacrylamide gel electrophoresis (PAGE).

Samples (30 μg homogenized NB-sample or 20 μl precipitation beads) were separated a 10% polyacrylamide gels and transferred to nitrocellulose (Hi-bond, Amersham Biosciences, Little Chalfont, UK). Ponceau S staining was used to confirm that equal amounts of protein were loaded in each lane (additional file 1). The nitrocellulose blot was blocked with 1% BSA in 20 mM Tris-HCl pH 7.4 and 0.1% Tween (Tris-buffered saline/Tween 20; TBS-T) for 1 h. The blot was washed for 5 min with TBS-T followed by incubation with the C-19 anti-MYCN antibody 1:200 diluted in TBS-T for 1 h at RT. After washing 3× with TBS-T, the blot was incubated with HRP-conjugated swine-anti-rabbit antibody (1:5000 diluted in TBS-T) for 1 h at RT. Subsequently, the blot was washed and incubated for 1 min with ECL substrate (Amersham Biosciences, Little Chalfont, UK) and exposed to film (Kodak, Rochester, NY, USA).

Primers for the amplification of MYCNOS were developed by using the primer3 program (Table 1). MYCNOS was amplified from DNA isolated from a healthy control using the GC RICH PCR System (Roche, Woerden, The Netherlands). Subsequently, MYCNOS was cloned into the Gateway donor vector pDONR-201 (Invitrogen). Using the Gateway cloning system, MYCNOS was subsequently subcloned into the pcDNA3 expression vector and integrity of the construct was validated by sequence analysis. IMR-32 NB cells at 50% confluence in a 25 cm² flask were co-transfected with 8 μg pcDNA3-MYCNOS and C1-GFP using 120 μl lipofectamine (Invitrogen) in 3 ml Opti-MEM-I for 20 min at RT.

Statistically significant differences in expression of MYCN-transcripts between NBs with or without MYCN-amplification were calculated with Students' T-test. Correlation of MYCN-transcipt expression with disease stage was calculated using the Spearman rank correlation and correlation of MYCN-transcripts with the MYCN-amplification numbers was calculated with the Pearson correlation test. All statistical tests were two-sided, significance was determined as p < 0.05.

We analysed fresh-frozen NBs from 16 pediatric patients (age range: 0–6 years old). The NBs are classified according to the Children's Cancer Group Neuroblastoma Staging System [25] and treated according to Pediatric Oncology Group-protocols (Table 2). Six out of 16 NBs carried a MYCN-amplification initially detected by Southern blot and/or FISH.

Two splice variants have been described for the proto-oncogene MYCN, the classical transcript that consists of three exons and a shortened ΔMYCN transcript that lacks exon 2. ΔMYCN is expressed in several fetal tissues, but its expression has not been reported in NBs. We used primers spanning exon 2 (Figure 1A and Table 1) to visualize by reverse transcriptase PCR whether or not both transcripts are present in MYCN-amplified IMR-32 neuroblastoma cells. Two fragments were identified that corresponded with the expected product lengths of MYCN and ΔMYCN of respectively 1007 and 100 bp (Figure 1D). Sequence analyses on the excised products confirmed that these fragments were MYCN and ΔMYCN. Absence of additional fragments in the IMR-32 NB cell line suggests that there are no other major MYCN splice variants. To determine whether the ΔMYCN protein is expressed in IMR-32 NB cells, MYCN proteins were visualized with the C-19 antibody that recognizes the c-terminal epitope of both MYCN and ΔMYCN proteins. In the lysate of IMR-32 cells, two protein bands were recognized at approximately 65 and 45 kD, which are the predicted molecular weights of MYCN and ΔMYCN respectively [18] (Figure 2A). For comparison, in a lysate of the melanoma cell line (BLM), which does not carry MYCN-amplification, no reactivity could be observed. We conclude from these experiments that the fetal MYCN isoform ΔMYCN is co-expressed with MYCN in IMR-32 cells.

mRNA expression levels of MYCN, ΔMYCN and MYCNOS were measured in 16 human neuroblastoma samples (Table 2) by QPCR relative to three reference genes: GUSB, TFRC and RNFIII [21]. Both MYCN and MYCNOS were found to be expressed in all 16 NBs. In addition, ΔMYCN expression was detected in all NB samples, except for patient 9, who did not carry an amplification of the MYCN region. For MYCN, it has been demonstrated that the relative expression-levels are significantly higher in NBs with MYCN-amplification as compared to non-amplified tumours [26]. Here, we show that besides MYCN, the relative mRNA expression levels of ΔMYCN and MYCNOS are also significantly increased in NBs with MYCN-amplification (p < 0.01; Figure 3A).

Correlation of NB stage with MYCN:ΔMYCN-ratio showed that the MYCN:ΔMYCN-ratio remains constant and does not change with either MYCN-amplification (Figure 3C; two-tailed p-value = 0.09, calculated with the Students' T-test) or NB stage (Figure 3F; two-tailed p-value = 0.12, calculated with the Spearman rank correlation). However, there is a significant correlation between the MYCN:MYCNOS-ratio and both MYCN-amplification (Figure 3C; two-tailed p-value = 0.025) and NB-stage (Figure 3E; two-tailed p-value = 0.007). The MYCNOS:ΔMYCN-ratio did not significantly change with either MYCN-amplification or NB-stage (two-tailed p-values = 0.58 and 0.24, respectively; data not shown). These data show that in more advanced NB tumours, mRNA expression of MYCNOS increases relative to MYCN.

To more exactly determine MYCN copy number in the tumours that were studied, we performed a genomic quantitative PCR (gQPCR) using genomic primers recognizing five different locations within the MYCN-gene (Figure 1A; Table 1). Amplification of these DNA fragments was calculated relative to three reference genes elsewhere on the genome, CFTR, TBX22, and SLC16A2. Among these three reference genes, there were no copy number differences noted in any of the NB samples. All 10 samples with a normal MYCN-copy number based on Southern blotting and/or FISH, carried two to four MYCN copies as determined by gQPCR. The presence of a MYCN duplication in NB cells that lack an overt amplification of MYCN is more often found, although the implications for the progression of the NB are still unclear [5, 27]. All 6 samples with multiple copies of MYCN, as determined by Southern blotting and/or FISH, had in between 20 and 139 MYCN gene amplifications (Table 2), which is within the normal range of gene copy numbers observed in NBs with MYCN-amplification [28].

We observed that MYCN mRNA-expression does not linearly correlate with MYCN-amplification, consistent with earlier reports [26]. In addition, also ΔMYCN and MYCNOS do not correlate linearly with the number of MYCN gene copies (Figure 3B). As shown in figure 3D, the relative mRNA expression ratio of MYCN:MYCNOS decreases with an increasing number of MYCN gene copies although this is not significant (non-interrupted line, slope = -0.4; two-tailed p-value = 0.18, calculated with the Pearson correlation test). The MYCN:ΔMYCN ratio does not change with higher MYCN copy numbers (Figure 3D; dotted line, slope = -0.1; two-tailed p-value = 0.88).

The pre-mRNA of MYCNOS, which represents the MYCN antisense transcript, shows overlap with the first exon of MYCN. Therefore MYCNOS may potentially modulate MYCN mRNA expression levels at the mRNA level via RNA-interference or RNA-editing, or direct MYCN splicing by RNA masking [11]. To test this premise, we transfected IMR-32 NB cells with C1-GFP and either the pcDNA3-vector containing MYCNOS or an empty vector. IMR-32 cells have relatively high endogenous expression levels of MYCN, MYCNOS and ΔMYCN, which enables quantification of all three mRNA levels. Flow cytometric analyses showed that there was a transfection efficiency of 74% after 72 hours (Figure 4A). Although there was a 50-fold increase of MYCNOS gene expression in the MYCNOS-transfected cell line relative to the empty vector control cell line (Figure 4B), expression of endogenous MYCN and ΔMYCN was not affected either at the mRNA level or at the protein level (Figure 4B, C). We conclude that although increased expression of MYCNOS relative to MYCN is correlated with an advanced disease state, RNA-interference or RNA-editing are not the mechanisms by which MYCNOS downregulates MYCN expression. In addition, the unchanged MYCN:ΔMYCN ratio in cells with MYCNOS overexpression shows that MYCNOS does not affect splicing by RNA-masking.