D. Thoraval

Co-amplification of a novel gene, NAG, with the N- myc gene in neuroblastoma

Substantial evidence implicates amplification of the N-myc gene with aggressive tumor growth and poor outcome in neuroblastoma. However some evidence suggests that this gene alone is not the sole determinant of outcome in N-myc amplified tumors. We have searched for genes that co-amplify with N-myc in neuroblastoma by means of two-dimensional analysis of genomic restriction digests. Using this approach, we have identified and cloned a novel genomic fragment which is co-amplified with N-myc in neuroblastomas. This fragment was mapped in close vicinity to N-myc on chromosome arm 2p24. It was amplified in 5/8 N-myc amplified neuroblastoma cell lines and in 9/13 N-myc amplified tumors. Using a PCR-based approach we isolated a 4.5 kb c-DNA sequence that is partly contained in the genomic fragment. The open reading frame of the cDNA encodes a predicted protein of 1353 amino acids (aa). The homology of the predicted protein, which we designated NAG (neuroblastoma amplified gene), to a C. elegans protein of as yet unknown function, and its ubiquious expression suggest that NAG may serve an essential function. By Northern blot analysis we showed that amplification of the cloned gene correlates with over-expression in neuroblastoma cell lines. Amplification and consequent over-expression of NAG may, therefore, contribute to the phenotype of a subset of neuroblastomas.

Demethylation of repetitive DNA sequences in neuroblastoma

Altered genomic methylcytosine content has been described for a number of tumor types, including neuroblastoma. However, it remains to be determined for different tumor types whether specific loci or chromosomal regions are affected by a methylation change or whether the change is random. We have implemented a computer‐based approach for the analysis of two‐dimensional separations of human genomic restriction fragments. Through the use of methylation‐sensitive restriction enzymes, methylation differences in genomic DNA between tumor and normal tissues can be detected. We report the cloning and sequencing of two fragments detectable in two‐dimensional separations of genomic DNA of neuroblastomas. These fragments were found to be a part of repetitive units that exhibited demethylation in neuroblastoma relative to other tumor types. Our finding of a distinct pattern of methylation of repetitive units in neuroblastoma suggests that altered methylation at certain loci may contribute to the biology of this tumor. Genes Chromosom Cancer 17:234–244 (1996).

Two-Dimensional DNA Electrophoresis Identifies Novel CpG Islands Frequently Coamplified With MYCN in Neuroblastoma

BACKGROUND Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours. PROCEDURE We have implemented an approach for the two-dimensional separation of human genomic restriction fragments to detect and isolate as yet unknown amplified sequences in the MYCN amplicon in neuroblastoma. Using this approach we have recently cloned a novel gene referred to as NAG that is frequently coamplified with MYCN in neuroblastoma. RESULTS AND CONCLUSIONS We report here the identification and cloning of two additional CpG islands that are amplified in neuroblastoma. One contains a sequence that is identical to the first intron of DDX1. The other represents a novel CpG island that is associated with an as yet unidentified gene. We show that the novel CpG island is located in close proximity to the MYCN locus on chromosome 2 and is as frequently coamplified with MYCN in neuroblastoma as NAG and DDX1.

DNA Methylation Changes in Lung Cancer

Recent evidence has suggested a role for DNA methylation changes and for DNA (cytosine 5-) methyltransferase in some aspect of tumor development. Limited data are currently available related to changes in the methylation process in lung cancer. Using a novel two-dimensional genome scanning approach to detect gene dosage alterations in tumors, we have identified loci that exhibited hypermethylation and others that exhibited hypomethylation in lung adenocarcinoma. The data suggest that some of the methylation changes observed are unlikely to be due to methylation errors. Full implementation of the genome scanning approach will probably uncover a large number of loci affected by methylation change in lung carcinogenesis.