High-resolution analysis of chromosome rearrangements on 8p in breast, colon and pancreatic cancer reveals a complex pattern of loss, gain and translocation.

The short arm of chromosome 8, 8p, is often rearranged in carcinomas, typically showing distal loss by unbalanced translocation. We analysed 8p rearrangements in 48 breast, pancreatic and colon cancer cell lines by fluorescence in situ hybridization (FISH) and array comparative genomic hybridization, with a tiling path of 0.2 Mb resolution over 8p12 and 1 Mb resolution over chromosome 8. Selected breast lines (MDA-MB-134, MDA-MB-175, MDA-MB-361, T-47D and ZR-75-1) were analysed further. Most cell lines showed loss of 8p distal to a break that was between 31 Mb (5' to NRG1) and the centromere, but the translocations were accompanied by variable amplifications, deletions and inversions proximal to this break. The 8p12 translocation in T-47D was flanked by an inversion of 4 Mb, with a 100 kb deletion at the proximal end. The dicentric t(8;11) in ZR-75-1 carries multiple rearrangements including interstitial deletions, a triplicated translocation junction between NRG1 and a fragment of 11q (unconnected to CCND1), and two separate amplifications, of FGFR1 and CCND1 . We conclude that if there is a tumour suppressor gene on 8p it may be near 31 Mb, for example WRN; but the complexity of 8p rearrangements suggests that they target various genes proximal to 31 Mb including NRG1 and the amplicon centred around ZNF703/FLJ14299.

Cellular environment of metabolites and a metabonomic study of tamoxifen in endometrial cells using gradient high resolution magic angle spinning 1H NMR spectroscopy.

High resolution magic angle spinning (HRMAS) 1H NMR spectroscopy was used to metabolically characterise Ishikawa cells, a human cell line derived from endometrial adenocarcinoma. The spectra obtained had well-resolved resonances from the nucleotide derivatives of uridine and adenosine. Using a combination of diffusion- and relaxation-weighted spectroscopy, the cellular environment of key metabolites previously identified as related to cell growth was also investigated. As Ishikawa cells are hormone-responsive, the metabolic action of tamoxifen, a selective estrogen receptor modulator (SERM), was also investigated. Cells were exposed to 5, 1 and 0.1 microM tamoxifen. Using the statistical regression technique of prediction to latent structures by partial least squares, a predictive model was built modelling the metabolic profile of the cells against exposure to tamoxifen. These spectral changes were characterised by increased resonance intensities from ethanolamine (3.26 ppm), glucose (3.34-3.94 ppm), glutamate (2.14, 2.32 ppm), tyrosine (7.24 ppm), uridine (7.85 ppm) and adenosine (8.20 ppm), and a relative decrease in contributions from myo-inositol resonances (3.30, 3.62, 3.55 ppm). The nucleotide changes suggest that tamoxifen affects RNA transcription, while the changes in ethanolamine and myo-inositol concentrations are indicative of cell membrane turnover.

The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene.

Neuregulin-1 (NRG1) is both a candidate oncogene and a candidate tumour suppressor gene. It not only encodes the heregulins and other mitogenic ligands for the ERBB family, but also causes apoptosis in NRG1-expressing cells. We found that most breast cancer cell lines had reduced or undetectable expression of NRG1. This included cell lines that had translocation breaks in the gene. Similarly, expression in cancers was generally comparable to or less than that in various normal breast samples. Many non-expressing cell lines had extensive methylation of the CpG island at the principal transcription start site at exon 2 of NRG1. Expression was reactivated by demethylation. Many tumours also showed methylation, whereas normal mammary epithelial fragments had none. Lower NRG1 expression correlated with higher methylation. Small interfering RNA (siRNA)-mediated depletion of NRG1 increased net proliferation in a normal breast cell line and a breast cancer cell line that expressed NRG1. The short arm of chromosome 8 is frequently lost in epithelial cancers, and NRG1 is the most centromeric gene that is always affected. NRG1 may therefore be the major tumour suppressor gene postulated to be on 8p: it is in the correct location, is antiproliferative and is silenced in many breast cancers.