Wild‐ sequence of MYCN in neuroblastoma cell lines

Neuroblastoma, a tumor of the post-ganglionic sympathetic nervous system, is the most common extra-cranial solid neoplasm of childhood. Amplification of theMYCN protooncogene (from 5to 300-fold per haploid genome) occurs in about 25% of primary tumors and 90% of established cell lines (Brodeur et al., 1984). Amplification is associated with increasedMYCN expression at both the RNA and protein levels in virtually all cases (Nisenet al.,1988; Seegeret al.,1988). MYCN amplification is more frequent in advanced stages of disease, but it is correlated with rapid tumor progression and poor outcome independent of other clinical or biological prognostic markers (Seeger et al., 1985). Thus, amplification of MYCN remains the most important genetically based independent prognostic factor for neuroblastoma, and it is an intrinsic biological property of a subset of aggressive neuroblastomas. Amplification of MYCN does not, however, occur in all neuroblastomas of advanced stage or aggressive phenotype. There is no consistent evidence for altered expression, mutation or amplification of any other known oncogenes or tumorsuppressor genes in neuroblastoma (Brodeur t al., 1997; Kawamataet al., 1996), and there are as yet no alternative mechanisms of MYCN activation established in these tumors. Although studies have shown a correlation between MYCN expression and gene copy number, MYCN mRNA expression in single-copy tumors has not been shown to correlate with outcome (Grady-Leopardi et al.,1986; Nisenet al.,1988). However, MYCN protein over-expression, in the absence of gene amplification, appears to correlate significantly with outcome, though this has not been confirmed (Chan et al., 1997). Finally, about 90% of established neuroblastoma cell lines contain amplified MYCN, suggesting a strong selectional bias for cells with activation of this proto-oncogenein vitro. Therefore, we hypothesized that mutations within theMYCN coding region might substitute for gene amplification in neuroblastoma cell lines with single-copy MYCN, leading to activation of this oncogene. We have sequenced the MYCN gene in 5 cell lines with single-copyMYCN (NBL-S, SK-N-SH, NB-69, SK-N-AS and LA-N-6) and one cell line withMYCN amplification (LA-N-1). Of these, NBL-S and LA-N-1 have elevated steady-state MYCN protein levels; the remaining cell lines have low-level expression characteristic of tumors without MYCN amplification. Genomic DNA was amplified by PCR to generate overlapping fragments covering the entire coding region of MYCN. Direct sequencing of PCR-amplified products was accomplished using internal primers. Sequencing reactions were performed in duplicate and bi-directionally from independent template preparations to ensure that mutations were not the result of PCR artifact. All 6 cell lines showed only wild-type MYCN, and all confirmed a previously noted error in the first submitted human MYCN germ-line sequence (GenBank accession number Y00664), with an ATC (rather than GTC) at codon 363. This results in isoleucine rather than valine at this position. MYCN is a member of the myc family of proteins, which have significant sequence homology within highly conserved functional domains (DePinhoet al.,1987). Aberrant expression of myc family members following gene amplification or translocation has been implicated in the pathogenesis of numerous human cancers. In Burkitt’s lymphoma (BL), distinct nonrandom chromosomal translocations juxtapose MYC to one of the heavyor light-chain immunoglobulin gene loci, resulting in deregulation of the gene (Ledert al., 1983). Although the resultant deregulatedMYC expression has long been considered the major contributor to lymphomagenesis, it has subsequently been shown that mutations are present in a large proportion of these translocated MYC alleles (Bhatiaet al., 1993; Yanoet al.,1993). These mis-sense mutations may be of biological significance as they tend to cluster within highly conserved domains and affect regions critical to the transactivation function of the protein. In contrast toMYC in BL, we have shown that mutation within the coding region of MYCN is not a common mechanism of gene activation in neuroblastoma cell lines. These data extend the observations of Feng et al.(1997), who demonstated no mutations within theMYCN transactivation domain in 13 primary neuroblastomas, a region which accounts for 12% of the protein. ThatMYCN may be activated in the absence of gene amplification has been suggested previously. Cohn et al. (1990) demonstrated an increased MYCN protein half-life in the absence of gene amplification in the neuroblastoma cell line NBL-S. We now show that NBL-S has only wild-type MYCN coding sequence, suggesting that this prolonged half-life is not the result of sequence mutation resulting directly in altered protein stability or degradation. It is possible that NBL-S has a prolonged MYCN protein half-life due to post-translational modifications, alterations in a specific MYCN-degradation pathway or alterations in partner proteins affecting MYCN protein stability. It remains plausible that alternative mechanisms to amplification exist for the activation of MYCN and are relevant in neuroblastoma tumorigenesis, but mutations within the gene do not appear to be commonly involved. Yours sincerely, Michael D. HOGARTY* and Garrett M. BRODEUR Division of Oncology and Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA