Neuroblastoma is the most common extracranial solid tumor in children. Despite dose intensive chemotherapy, the disease is frequently incurable. Clinical and biological risk-stratification implicates amplification of the MYCN oncogene (observed in 25-35% of cases) as a robust marker for poor prognosis. Although the binding of this transcription factor to promoter consensus sequences has been well characterized, the direct downstream targets of MYCN, that might couple its amplification and over-expression to the malignant phenotype, remain largely undefined. In order to identify these targets and advance our knowledge of the pathogenesis of this aggressive tumor we have undertaken a molecular analysis of gene expression in an in vitro model of MYCN-amplified and non-amplified neuroblastoma. We used the TET21 neuroblastoma cell line stably transduced with MYCN under the control of a tetracycline regulated promoter (a gift from Dr. Manfred Schwab). This cell line undergoes well characterized changes in proliferation, morphology and growth requirements upon MYCN induction which correlate with known properties of MYCN-amplified tumors. Representational difference analysis (RDA) was used to isolate a series of differentially expressed genes either trans-activated or trans-repressed as a consequence of MYCN expression using mRNA from the MYCN induced [tet(-)] and non-induced [tet(+)]forms of the cell line. Thirty seven distinct genes were identified from 115 sequenced clones. These represent known genes, homologues to known genes and several previously non-annotated molecules with EST, genomic BAC or Unigene cluster matches. Differential expression was confirmed by Northern, cDNA Southern and amplicon blots. As a validating internal control, MYCN was found in the tet(-), MYCN-induced cells. MYCN induction stimulated high expression of FUS/TLS, an oncogene product which binds nuclear RNA and is implicated in the pathogenesis of liposarcomas and acute myelogenous leukemia. Northern blots demonstrated FUS expression increased at least three fold upon MYCN induction and this gene was markedly upregulated in MYCN amplified cell line IMR-32 with low levels found in SKN-SH and SK-N-AS non-amplified cell lines. Several other differentially expressed genes potentially involved in control of cellular proliferation, adhesion and drug resistance were regulated by MYCN expression including CD151 (endothelial tetraspanan antigen), leu 13 (17kd interferon-induced transmembrane antigen), and TRIP-6 (zinc-finger protein associated with tumoregenesis). Further characterization of the expression pattern and biologic function of these molecules in vitro as well as in pédiatrie tumors is ongoing and should enable the development of novel therapies for these high risk tumors.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - Jan 1 2000|
ASJC Scopus subject areas
- Cell Biology