APO-1 is a 48-kDa cell-membrane protein identical to the Fas antigen now designated CD95. It is a member of the NGF/TNF receptor superfamily. Anti-APO-1 monoclonal antibody induces apoptosis in a variety of cell types expressing this antigen. We immunohistochemically investigated APO-1 expression in normal colon mucosa, 20 adenomas, 258 colon carcinomas and 10 liver metastases and carried out in vitro studies using a panel of colon-carcinoma cell lines. Immunohistochemically, APO-1 was regularly expressed at the basolateral membrane of normal colon epithelia. In a minor fraction of colon adenomas and in 39.1% of colon carcinomas APO-1 expression was diminished and in 48.1% of carcinomas, predominantly of the non-mucinous type, APO-1 expression was completely abrogated. The normal level of APO-1 in carcinomas was correlated with the mucinous type. Reduced/lost APO-1 expression was more frequent in rectal carcinomas. Complete loss of APO-1 was more frequent in tumors that had already metastasized. APO-1 expression in liver metastases essentially corresponded to that of the primary tumors. Comparative analysis with data from previous studies revealed that the mode of APO-1 expression is correlated with that of HLA-A,B,C./beta 2m, HLA-DR, HLA-D-associated invariant chain and of the secretory component. Surface expression of APO-1 was heterogeneous in colon-carcinoma cell lines; SW480 expressed considerable amounts of APO-1 on all cells, while HT-29 constitutively did less so and only in a minority of cells. Surface density of APO-1 and the fraction of positive cells in HT-29 was enhanced by interferon-gamma (IFN-gamma) and, additively, by tumor necrosis factor-alpha (TNF-alpha), whereas in SW480 APO-1 expression was not modulated by these cytokines. We conclude that neoplastic transformation of colon epithelium often leads to a loss of the physiologic, high level of surface APO-1 by giving rise either to a stable lack of APO-1 or to an IFN-gamma/TNF-alpha-sensitive phenotype of inducible APO-1 expression.

Cytogenetic information on chordomas is rudimentary and restricted to GTG-banding analysis of 26 cases worldwide. In this study, we present the chromosomal imbalances detected in a series of 16 chordomas (10 sacrococcyeal, five sphenooccipital, and one spinal) from 13 patients using comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). On average, 3.2 losses and 4.2 gains were detected per tumor. The most common DNA copy number alterations were losses on chromosomal arms 3p (50%) and 1p (44%). Losses of 3p were detected in five of seven primary chordomas. Therefore, the loss of 3p might be an early event in chordoma genesis. The most common gains involved 7q (69%), 20 (50%), 5q (38%), and 12q (38%). Additionally, we raised the first human chordoma cell line, U-CH1, from a recurrence of a sacral chordoma. U-CH1 and its parent tumor had almost the same CGH profile. According to GTG-banding and multicolor FISH, U-CH1 has the following clonal chromosomal abnormalities: der(1)t(1;22), del(4), +del(5), +del(6), +7, del(9), del(10), +der(20)t(10;20), +21. Thus, the novel permanent human chordoma cell line U-CH1 has chordoma-typical cytogenetic aberrations. Our data suggest that tumor suppressor genes or mismatch repair genes (located at 1p31 and 3p14) and oncogenes (located in 7q36) might be involved in chordoma genesis.

The peptide hormone leptin plays a major role in the regulation of energy intake and expenditure and is predominantly expressed in mature adipocytes but not in preadipocytes. Using bisulfite genomic sequencing, we found that 32 CpGs, distributed within a 317-bp sequence of the proximal leptin promoter, were highly methylated in human preadipocytes (73.4% ؎ 9.0%). During maturation toward terminally differentiated adipocytes, this promoter region was extremely demethylated (9.4% ؎ 4.4%). CpG methylation-dependent transcriptional activity of the promoter fragment was determined in transfection experiments using a set of 5-truncated mock-, HhaI-, and SssI-methylated promoter-reporter constructs. Whereas the methylated CpG within the CCAAT/enhancer-binding protein ␣ recognition site down-regulated reporter expression, methylated CpGs proximal to the TATA motif and/or in a further upstream region abrogated promoter activity completely. These distinct promoter CpG sequences were found unmethylated in leptin-expressing mature adipocytes. As evidenced by electrophoretic mobility shift assays, nuclear protein complexes were specifically formed on methylated oligonucleotide probes corresponding to the dedicated promoter sequences, indicating that methyl-CpG binding proteins participate in transcriptional repression and regulation of the human leptin gene.

A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non-neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U-CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence-like phenotype. The U-CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r]gammanull mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro.

Primary mediastinal B-cell lymphoma (PMBL) is a well-defined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene because expression profiling showed high Janus kinase-2 (JAK2) transcript levels and JAK2 was found to be constitutively phosphorylated in mediastinal B-cell lymphomas. We confirm that in the MedB-1 mediastinal B-cell line, harboring a trisomy 9, JAK2 transcription is elevated and the product is highly