Low-density lipoprotein receptor-related protein 1B (LRP1B) is frequently deleted in tumors of various types, but its status and expression in esophageal squamous cell carcinomas (ESCs) have never been reported. In the course of a program to screen ESC cell lines for copy-number aberrations using array-based comparative genomic hybridization, we identified a homozygous deletion of LRP1B. Genomic PCR experiments revealed homozygous deletions of LRP1B in additional ESC cell lines (total, 6 of 43; 14.0%) and in primary esophageal tumors (30 of 70; 42.9%). Moreover, expression of LRP1B mRNA was frequently silenced in ESC lines without homozygous deletions (14 of 37; 37.8%). Using bisulfite-PCR analysis and sequencing, we found that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a CpG island of LRP1B, a sequence possessing promoter activity. Treatment with 5-aza-2-deoxycytidine restored expression of LRP1B in those ESC lines. Histone acetylation status correlated directly with expression of LRP1B and inversely with the methylation status of the CpG island. Methylation of LRP1B was also detected in primary esophageal tumors. Restoration of LRP1B expression in ESC cells reduced colony formation. These results suggest that loss of LRP1B function in esophageal carcinogenesis most often occurs either by homozygous deletion or by transcriptional silencing through hypermethylation of its CpG island.

We performed genome-wide screening for deoxyribonucleic acid copy-number aberrations in 31 gastric cancer (GC) cell lines by using custom-made comparative genomic hybridization (CGH)-array. Copy-number gains were frequently detected at 1q, 3q, 5p, 7p, 7q, 8q, 11q, 17q, 20p, 20q, Xp and Xq, and losses at 3p, 4p, 4q, 8p, 9p, 18p and 18q. With respect to histological subtypes, copy-number gains at 1p, 16p, 20p, 20q and 22q, and losses at 8p, 10p, 10q and 18q were significantly frequent in cell lines derived from tumors of the well-differentiated type, whereas copy-number gains at 1q, 7p, 7q, Xp and Xq were frequent in the undifferentiated type. Homozygous deletions were seen at five loci, whereas high-level amplifications were detected in 15 of the 31 GC cell lines; these had occurred at 24 loci, including the segment containing CDK6 (7q21.2). Amplification of that gene had never been reported in GC before. Immunohistochemical studies showed increased levels of CDK6 protein in 54 of the 292 primary GC samples we examined (18.5%). Cytoplasmic localization of CDK6, as well as CDK6 over-expression, was more frequent in well-differentiated GC than in undifferentiated tumors. Nuclear expression of CDK6 was more frequent in early stage GC than in advanced tumors, suggesting that nuclear localization of CDK6 is likely to be a prognostic factor for GC. Taken together, our data indicate that CDK6 might be involved in the pathogenesis of GC and, more generally, that CGH-arrays have a powerful potential for identifying novel cancer-related genetic changes in a variety of tumors. (Cancer Sci 2005; 96: 100-110)

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.