Evidence for a cytoskeleton attachment domain at the N-terminus of the NF2 protein.

Neurofibromatosis type 2 is a hereditary cancer syndrome characterized by the development of bilateral vestibular schwannomas. Underlying the disease are inactivating mutations of the NF2 tumor suppressor gene, located on chromosome 22, encoding a 595-amino-acid protein. The NF2 protein, also known as merlin or schwannomin, is reported to act as a membrane-cytoskeleton linking protein. This assumption is based on the homology of the NF2 protein to a group of band 4.1-related proteins, ezrin, radixin, and moesin. The cytoskeletal association of the NF2 protein has in part been confirmed by its ability to resist extraction from cells by nonionic detergents. We performed detergent extraction on COS cells transfected with NF2 cDNA constructs. The extracts were analyzed by Western blotting and immunofluorescent staining with monoclonal anti-NF2 antibodies. The results provide evidence for a high-affinity cytoskeleton attachment domain at amino acids 29-131 and a putative lower affinity domain between amino acids 321 and 470.

Clonal analysis of a case of multifocal oesophageal (Barrett's) adenocarcinoma by comparative genomic hybridization.

Oesophageal adenocarcinomas arising in Barrett's epithelium occasionally present as multiple lesions. This could be due to either a multifocal presentation of the same tumour, or different neoplasms arising simultaneously in a dysplastic Barrett's oesophagus ('field cancerization'). This is a report of the genetic analysis of multiple neoplastic sites in a Barrett's oesophagus with an extensive area of dysplasia. In addition, the dysplastic Barrett's epithelium was evaluated. For the genetic screening, comparative genomic hybridization (CGH) allowed evaluation of the whole genome of each specimen. Five cancerous regions were selected and subsequently dissected from paraffin-embedded tissue blocks. The use of archival materials enabled a targeted collection of representative tumour locations. Multiple genetic aberrations were detected by CGH in all cancer sites. Losses on 3p, 4, 7q, 18q, and Y, as well as gains on 8q, 9q, 12p, 13q, 17q, 20p and X, were found in each specimen. In four out of the five lesions, simultaneous losses on 9p, 15q, and 16q, with concomitant gains on 5p, 7q, and 10p, were disclosed by CGH. Adjacent high-grade dysplastic Barrett's mucosa shared the losses on 3p, 4, 7q, 9p, 18, and Y, as well as the gains on 5p, 7q, 13q, 17q, and X, thereby confirming its precursor status. Within this single and rare case of multifocal Barrett's adenocarcinoma, a monoclonal genotype was present. This must have been caused by an extensive outgrowth of a single tumour.

Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence.

BACKGROUND: The structure-specific ERCC1/XPF endonuclease complex that contains the ERCC1 and XPF subunits is implicated in the repair of two distinct types of lesions in DNA: nucleotide excision repair (NER) for ultraviolet-induced lesions and bulky chemical adducts; and recombination repair of the very genotoxic interstrand cross-links. RESULTS: Here, we present a detailed analysis of two types of mice with mutations in ERCC1, one in which the gene is 'knocked out', and one in which the encoded protein contains a seven amino-acid carboxy-terminal truncation. In addition to the previously reported symptoms of severe runting, abnormalities of liver nuclei and greatly reduced lifespan (which appeared less severe in the truncation mutant), both types of ERCC1-mutant mouse exhibited an absence of subcutaneous fat, early onset of ferritin deposition in the spleen, kidney malfunction, gross abnormalities of ploidy and cytoplasmic invaginations in nuclei of liver and kidney, and compromised NER and cross-link repair. We also found that heterozygosity for ERCC1 mutations did not appear to provide a selective advantage for chemically induced tumorigenesis. An important clue to the cause of the very severe ERCC1-mutant phenotypes is our finding that ERCC1-mutant cells undergo premature replicative senescence, unlike cells from mice with a defect only in NER. CONCLUSIONS: Our results strongly suggest that the accumulation in ERCC1-mutant mice of endogenously generated DNA interstrand cross-links, which are normally repaired by ERCC1-dependent recombination repair, underlies both the early onset of cell cycle arrest and polyploidy in the liver and kidney. Thus, our work provides an insight into the molecular basis of ageing and highlights the role of ERCC1 and interstrand DNA cross-links.

A new monoclonal antibody for specific immunocytochemical staining of nucleoli.

We have isolated a hybridoma cell line (clone 1E10) producing a monoclonal antibody which specifically recognizes nucleoli. The antibody (IgM, k isotype) was found to react in a nucleolar pattern with a variety of cell types. Specific staining was only obtained on cryostat sections of unfixed tissues. Paraffin embedding destroyed the epitope. Tissue specificity or species specificity was not observed. Nucleoli in neoplastic cells were highly reactive, presumably due to the larger size of nucleoli in these cells. Immunoelectron-microscopy (using a pre-embedding as well as a post-embedding technique) confirmed the specific nucleolar localization of the immunoreactivity. Immunoreactivity was confined to the granular component of the nucleolus. The intensity of the immunoreactivity increased after cell or tissue pretreatment with DNase, pronase or trypsin, indicating that the target epitope is not DNA or a protein. On Western blots of immunoreactive cells no specific signal was obtained, which supports the non-protein nature of the epitope. Acid hydrolysis and RNase digestion abolished the immunoreactivity. Parallel staining experiments with methylgreen pyronin and acridin orange confirmed the RNA nature of the epitope. In spot blots, immunoreactivity was not found with tRNA or mRNA. These observations indicate that 1E10 recognizes a conformational RNA epitope which occurs only in the nucleolus.

Cytogenetic heterogeneity and histologic tumor growth patterns in prostatic cancer.

Twenty-five prostatic adenocarcinomas were studied for the presence of intratumoral cytogenetic heterogeneity by interphase in situ hybridization (ISH) to routinely processed tissue sections. ISH with a chromosome Y-specific repetitive DNA probe provided a model to investigate patterns of chromosomal heterogeneity within and between different pathological grades. The Gleason grading system was used, since it is based on a detailed classification of growth patterns. Heterogeneity with respect to ploidy of the tumor was examined by ISH with a repetitive DNA probe specific for chromosome 1. The ploidy status of these cancers was confirmed by DNA flow cytometry (P < 0.001). Cytogenetic heterogeneity at the (Y) chromosomal level was observed between Gleason areas, within one area, and even within single tumor glands. The different patterns of chromosomal heterogeneity were seen in all tumor grades and stages. Differences in ploidy status were also found following the aforementioned histological patterns, again, in all grades and stages. Intraglandular heterogeneity was most frequently seen. No correlation was found between cytogenetic heterogeneity and proliferative activity (Ki-67 immunostaining). In contrast to current views on clonality, suggesting regional separation of subclones with different DNA content, this study demonstrates that these subclones can be interspersed.

Identification of numerical chromosome aberrations in archival tumours by in situ hybridization to routine paraffin sections: evaluation of 23 phaeochromocytomas.

We have applied non-isotopic in situ hybridization (ISH) to interphase cell nuclei of 23 phaeochromocytomas (18 primary and 5 metastatic tumours) within routine paraffin-embedded tissue sections. Each tumour was screened for numerical aberrations with a defined alphoid repetitive DNA probe set containing DNA probes specific for chromosomes 1, 7, 15, and Y. Normal adrenal medullas and other normal human cell types served as cytogenetic controls. Preservation of tissue morphology enabled targeted analysis of tumour cells. The presence of numerical chromosome changes in the tumour cells could easily be evaluated by comparing the ISH results of the DNA probes. Numerical abnormalities not previously reported in this neoplasm included overrepresentation of chromosomes 1 and 7, loss of chromosome 15, and both gain and loss of chromosome Y (P values < 0.01). The percentage of aneuploid cell nuclei in a tumour correlated well with the percentage of cells in the 4C peak of flow cytometric DNA histograms from these neoplasms. We conclude that interphase ISH can be used for the identification of new and reported cytogenetic changes in tumour cell nuclei within archival tissue sections. This novel procedure also allows for retrospective analysis of previously not karyotyped material.

Prognostic relevance of DNA flow cytometry in oligodendroglioma.

In a retrospective study of 85 cases, the prognostic value of DNA flow cytometry in oligodendrogliomas was evaluated. Paraffin-embedded material was processed for flow cytometry, and the survival rates of the patients with DNA diploid, aneuploid, and tetraploid tumors were compared using analysis of variance. In addition, the mitotic index was correlated with the results of flow cytometry. Finally, the results of flow cytometry, histopathologic grading, and counting mitoses were tested for dependency. Thirty-one percent of the tumors were diploid, 39% were tetraploid, and 31% were aneuploid. The results of the DNA flow cytometry did not correlate with the survival times (P = 0.798) or with tumor degree. In contrast, the number of mitoses (P less than 0.05), and the grades of the grading system of Smith (P less than 0.003) had relevance for the prognosis. No correlation between flow cytometry, histopathologic grading, and mitotic index was found. It is concluded that flow cytometry has no value in predicting the biologic behavior of oligodendrogliomas, whereas the number of mitoses is a valuable prognostic parameter and thus is considered to be incorporated into the grading system for oligodendrogliomas.