Merkel cell carcinoma (MCC), a skin tumour with neuroendocrine features, was recently found to be associated with a new type of human polyomavirus, called Merkel cell virus (MCV). We investigated the specificity of this association as well as a causal role of MCV in oncogenesis. DNA and RNA from ten cases of MCC were analysed using PCR and RT-PCR. DNA from 1241 specimens of a wide range of human tumours was also analysed. The DIPS technique was used to identify the integration locus of viral DNA sequences. Array CGH was performed to analyse structural alterations of the cell genome. MCV DNA sequences were found in all ten cases of MCC and in none of the 1241 specimens of other tumour types. Clonal integration of MCV into the host genome was seen in all MCC cases and was checked by FISH in one case. A recurrent pattern of conserved viral sequences which encompassed the replication origin, the small tumour (ST), and the 5' part of the large tumour (LT) antigen DNA sequences was observed. Both ST and LT viral sequences were found to be significantly expressed in all MCCs. Neither recurrent site of integration nor alteration of cellular genes located near the viral sequences was observed. The tight association of MCV with MCC, the clonal pattern of MCV integration, and the expression of the viral oncoproteins strongly support a causative role for MCV in the tumour process. This information will help the development of novel approaches for the assessment and therapy of MCC and biologically related tumours.
A modified comparative genomic hybridization (mCGH) technique was applied to a series of 17 primary breast carcinomas in which cytogenetic study (CG) demonstrated the presence of homogeneously staining region(s), suggesting the occurrence of DNA amplification. mCGH demonstrated recurrent amplifications of the whole chromosome arms 8q (9 times) and 1q (7 times) and of DNA loci in the following bands: 11q13 (6 times), 9p13 and 17q21.1 (4 times), 1q21.1 and 16p11.2 (3 times), and 8q22, 8q24.1, 10q22, 15q26, 17q23, and 20q13.3 (twice). Amplification of whole chromosome arms is likely to have resulted from unbalanced translocations or isochromosomes, whereas amplifications of smaller chromosomal segments probably arose through real DNA amplification processes. In all tumors but one, more than one amplified locus was detected. The fact that many chromosomal sites were involved suggests that the process of amplification is complex and that many genes are potential targets.
We have used two-dimensional electrophoresis of enzyme-digested genomic DNA to identify a novel gene GAC1, which maps at 1q32.1 and which is overexpressed in malignant gliomas in which it is ampli®ed. GAC1 encodes a protein which belongs to the leucine-rich repeat superfamily. Ampli®cation and overexpression of GAC1 was demonstrated in two of eight tumors where ampli®cations were previously evidenced by comparative genomic hybridization (one glioblastoma multiforme and one anaplastic astrocytoma), and in one of eight unselected glioblastomas multiforme. GAC1 exhibits sequence homology with other proteins which function as cell-adhesion molecules or as signal transduction receptor and is a likely candidate for the target gene in the 1q32.1 amplicon in malignant gliomas.
Azacytidine (ACR) is known to induce uncoiling and somatic association involving the constitutive heterochromatin of human chromosomes 1, 9, 15, and 16 and the Y. These regions are composed of alphoid and classical satellite DNA sequences. Using specific probes for chromosomes 1 and 16, we have performed two-color fluorescence in situ hybridization on human lymphocytes cultured in the presence of ACR. We demonstrate that for these two chromosomes (1) uncoiling and association specifically occur in classical satellite-containing regions at the first cell generation, (2) breakages also affect these regions, and (3) somatic recombinations occur between these regions and lead to translocations at the next cell generation. These results suggest that changes in methylation of repetitive DNA sequences are related to chromosomal instability occurring during cell transformation and tumorigenesis.
To determine possible relationships between DNA hypomethylation and chromosome instability, human lymphoblastoid cell lines from different genetic constitutions were studied with regard to 1) uncoiling and rearrangements, which preferentially affect the heterochromatic segments of chromosomes 1 and 16; 2) the methylation status of the tandemly repetitive sequences (classical satellite and alphoid DNAs) from chromosomes 1 and 16, and of the L1Hs interspersed repetitive sequences. The methylation status largely varied from cell line to cell line, but for a given cell line, the degree of methylation was similar for all the repetitive DNAs studied. Two cell lines, one obtained from a Fanconi anemia patient and the other from an ataxia telangiectasia patient were found to be heavily hypomethylated. The heterochromatic segments of their chromosomes 1 and 16 were more frequently elongated and rearranged than those from other cell lines, which were found to be less hypomethylated. Thus, in these lymphoblastoid cell lines, alterations characterized by uncoiling and rearrangements of heterochromatic segments from chromosomes 1 and 16 seem to correlate with the hypomethylation of their repetitive DNAs. Two-color in situ hybridizations demonstrated that these elongations and rearrangements involved only classical satellite-DNA-containing heterochromatin. This specificity may be related to the excess of breakages affecting the chromosomes carrying these structures in a variety of pathological conditions.
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