N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis - PubMed (original) (raw)

N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis

K Boon et al. EMBO J. 2001.

Abstract

The myc oncogenes are frequently activated in human tumors, but there is no comprehensive insight into the target genes and downstream cellular pathways of these transcription factors. We applied serial analysis of gene expression (SAGE) to identify targets of N-myc in neuroblastomas. Analysis of 42,000 mRNA transcript tags in SAGE libraries of N-myc- transfected and control neuroblastoma cells revealed 114 up-regulated genes. The majority of these genes have a role in ribosome assembly and activity. Northern blot analysis confirmed up-regulation of all tested transcripts. Induction was complete within 4 h after N-myc expression. The large majority of the ribosomal proteins were induced, as well as genes controlling rRNA maturation. Cellular rRNA content was 45% induced. SAGE libraries and northern blot analysis confirmed up-regulation of many of these genes in N-myc-amplified neuroblastomas. As N-myc can functionally replace c-myc, we analyzed whether N-myc targets were induced by c-myc as well. Approximately 40% of these N-myc targets were up-regulated in a c-myc-transfected melanoma cell line. These data suggest that myc genes function as major regulators of the protein synthesis machinery.

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Figures

Fig. 1. Northern blot analysis of N-myc downstream target genes. Equal amounts of total RNA from exponentially growing SHEP-2 and SHEP-21N cells were loaded. Northern blots were hybridized with probes for the 19 indicated N-myc targets. RNA quantification was checked by ethidium bromide staining; the 28S band is shown.

Fig. 2. Level of induction of the 56 ribosomal protein genes identified as N-myc targets (P <0.01) in SHEP-21N cells. (A) Fold induction by N-myc in SHEP-21N cells as a function of the basic expression levels in SHEP-2. _x_-axis, basic expression level in SHEP-2 cells normalized per 10 000 tags; _y_-axis, fold induction in SHEP-21N cells. (B) Increase in the same 56 ribosomal protein genes in the N-_myc_-amplified neuroblastoma N159 as a function of the basic expression level in N-myc single-copy neuroblastoma N52. _x_-axis, expression level in N52 normalized per 10 000 tags; _y_-axis, fold increase in N159 relative to N52.

Fig. 3. Time course analysis of N-myc and downstream target gene induction in SHEP-21N cells. SHEP-21N cells were treated for 24 h with 10 ng/ml tetracycline, washed and grown for an additional 36 h without tetracycline. Cells were harvested at 0, 8 and 24 h of tetracycline treatment. Subsequent samples were taken at 1, 2, 4, 8, 10, 12, 24 and 36 h after removal of the antibiotic. (A) Northern blot analysis of total RNA at the indicated time points. (B) Western blot analysis of N-myc protein at the indicated time points. A 10 µg aliquot of total protein samples of the time course experiment were fractionated through a 10% SDS–polyacrylamide gel, blotted on an Immobilon membrane and probed with a monoclonal anti-N-myc antibody.

Fig. 4. Expression of N-myc, nucleolin, nucleophosmin, translation elongation factors EEF1γ and EEF1β and total RNA content of SHEP-2 and SHEP-21N cells. (A) Western blot analysis. Total cell extracts (10 µg) were fractionated through an acrylamide gel, blotted and probed with monoclonal antibodies against N-myc and nucleo phosmin, and with polyclonal antibodies against nucleolin, EEF1γ and EEF1β. SHEP-21N cells were treated for 0–8 days with tetracycline (lanes 2–8) and subsequently cultured for 1 or 7 days without tetracycline (lanes 9 and 10). A Coomassie Blue staining is shown as control for loading. (B) Total RNA content of SHEP-2 and SHEP-21N cells. RNA was isolated from 10 samples of 106 cells of each cell line and analyzed spectrophotomerically. Error bars give the SD.

Fig. 5. Northern blot analysis of total RNA from neuroblastoma cell lines and tumors. Filters were hybridized with the indicated probes. RNA quantification was checked by ethidium bromide staining; the 28S band is shown. (A) Panel of 21 neuroblastoma cell lines. (B) Panel of 16 fresh tumors. Tumors in lanes 1–9 are N-myc amplified.

Fig. 6. Northern blot analysis of induction of N-myc target genes in a c-myc-transfected melanoma cell line. Clone 3 is a c-myc-transfected clone of the IGR39D melanoma cell line. Equal amounts of total RNA of IGR39D and clone 3 were loaded. Filters were hybridized with the indicated probes.

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N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis - PubMed (original) (raw)