[A clinical Case-of Icenko-Cushing Syndrome in a Female Patient with Morbit Obesity after Bariatric Surgery].

The efficacy of morbid obesity therapy at present remains rather low. Bariatric surgery is one of the most efficient methods for the treatment. Safety, efficacy and favourable results of the surgery are provided only after thorough examination of the patients.

Association between the rat homologue of CO-029, a metastasis-associated tetraspanin molecule and consumption coagulopathy.

Recently, we have described a panel of metastasis-associated antigens in the rat, i.e., of molecules expressed on metastasizing, but not on nonmetastasizing tumor lines. One of these molecules, recognized by the monoclonal antibody D6.1 and named accordingly D6. 1A, was found to be abundantly expressed predominantly on mesenchyme-derived cells. The DNA of the antigen has been isolated and cloned. Surprisingly, the gene product proved to interfere strongly with coagulation. The 1.182-kb cDNA codes for a 235-amino acid long molecule with a 74.2% homology in the nucleotide and a 70% homology in the amino acid sequence to CO-029, a human tumor-associated molecule. According to the distribution of hydrophobic and hydrophilic amino acids, D6.1A belongs to the tetraspanin superfamily. Western blotting of D6.1A-positive metastasizing tumor lines revealed that the D6.1A, like many tetraspanin molecules, is linked to further membrane molecules, one of which could be identified as alpha6beta1 integrin. Transfection of a low-metastasizing tumor cell line with D6.1A cDNA resulted in increased metastatic potential and provided a clue as to the functional role of D6.1A. We noted massive bleeding around the metastases and, possibly as a consequence, local infarctions predominantly in the mesenteric region and all signs of a consumption coagulopathy. By application of the D6.1 antibody the coagulopathy was counterregulated, though not prevented. It has been known for many years that tumor growth and progression is frequently accompanied by thrombotic disorders. Our data suggest that the phenomenon could well be associated with the expression of tetraspanin molecules.

A highly amplified mouse gene is homologous to the human interferon-responsive Sp100 gene encoding an autoantigen associated with nuclear dots.

In human cells, three proteins are currently known to colocalize in di screte nuclear domains (designated nuclear dots): Sp100, a transcription-activating protein autoantigenic primarily in patients with primary biliary cirrhosis; PML, a tumor suppressor protein involved in development of acute promyelocytic leukemia; and NDP52, a protein of unknown function. Here we report sequence similarities between the Sp100 protein and a putative protein encoded by a highly amplified mouse gene which is visible as an inherited homogeneously staining region (HSR) on chromosome 1 of some mouse populations. By in situ hybridization, the Sp100 gene was mapped to locus 2q37, the syntenic region of the HSR on mouse chromosome 1. Unlike the highly amplified mouse gene, Sp100 was found to be a single-copy gene and showed no restriction fragment length polymorphisms. Sequence similarities in the promoter regions and similar exon-intron organizations of the two genes were revealed. As for Sp100, steady-state levels of the mRNAs of the HSR-encoded genes could be greatly increased by interferon (IFN) treatment. As in human cells, IFN treatment led to an enlargement in both size and number of nuclear dots in mouse cells as visualized by immunofluorescence staining with autoimmune sera from patients with primary biliary cirrhosis. These data indicate that a gene located in the inherited HSR of mice, designated mSp100, is homologous to the human Sp100 gene, has a similar gene organization, and responds similarly to IFN treatment.

Duplication of N-MYC at its resident site 2p24 may be a mechanism of activation alternative to amplification in human neuroblastoma cells.

Amplification of the human N-MYC proto-oncogene is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions of aggressively growing neuroblastomas. N-MYC maps to chromosome 2 band p23-24, but homogeneously staining regions have never been observed at this band, suggesting transposition of N-MYC during amplification. Previous studies had suggested that in cells with amplified N-MYC the chromosomes 2 appear to be unaltered and to carry one apparently normal copy of N-MYC each. In contrast, the contribution of N-MYC to tumors which lack amplification has been unclear. We here show, by fluorescence in situ hybridization, that N-MYC is occasionally duplicated at its resident site in neuroblastoma cell lines previously thought to have a single copy gene. Additionally, we detected duplication in a neuroblastoma cell line carrying amplification. Our results raise the possibility that duplication may, in some neuroblastomas, either be a prelude to amplification or an alternative pathway by which N-MYC becomes activated.

Cloning of the human aflatoxin B1-aldehyde reductase gene at 1p35-1p36.1 in a region frequently altered in human tumor cells.

Alterations of the distal portion of the short arm of chromosome 1 (1p) are among the earliest abnormalities of human colorectal tumors. Loss of heterozygosity analysis has previously revealed a smallest region of overlapping deletion (SRO) B, at 1p35-36.1, deleted in 48% of sporadic tumors. From this region we have now cloned a gene encoding a protein of 330 amino acids that is 78% identical with the Rattus norvegicus aflatoxin B1 aldehyde reductase (Afar) and, therefore, likely represents its human homologue. In rat liver, Afar is strongly inducible by the antioxidants ethoxyquin and butylated hydroxyanisole, which protect the rat against aflatoxin B1-induced liver tumorigenesis by detoxifying its genotoxic and cytotoxic dialdehyde. Human AFAR is expressed in a broad range of tissues and, therefore, is likely involved in endogenous detoxication pathways. Impaired detoxication of genotoxic aldehydes and ketones, which are involved in tumorigenesis of the colon and breast, may be a crucial factor both for tumor initiation and progression. We here provide a detailed contig of 1.5-2 Mbp/2.7 cM encompassing part of SRO B, including known genes and previously unmapped expressed sequence tags. PLA2G2A (secretory type II phospholipase A2), described previously as a candidate, is localized outside SRO B.

Constitutional genomic instability with inversions, duplications, and amplifications in 9p23-24 in BRCA2 mutation carriers.

Germ-line mutations of the BRCA2 gene (13q12-13) account for a large proportion of familial breast cancer cases in females and the majority of familial breast cancers in males. Recent studies provide evidence for a role of the BRCA2 protein in the maintenance of genomic integrity by involvement in DNA repair and recombination. In pursuit of identifying in humans genetic damage resulting from mutated BRCA2, we have analyzed constitutional karyotypes of BRCA2 mutation carriers. The present study establishes that constitutional distal 9p rearrangements without obvious additional gross chromosomal alterations are a recurrent feature of independently ascertained families. From our cytogenetic analyses we have no indication of additional gross rearrangements, but we cannot exclude more subtle recombinations in other genomic regions. We also show that the topography of the 9p rearrangements can differ among family members, even within an individual that can have cell populations with different 9p rearrangements. Collectively these results raise point to an association of mutant BRCA2 with genomic instability and gene alteration in 9p23-24 in at least a subset of BRCA2 mutation carriers.

An interstitial tandem duplication of 9p23-24 coexists with a mutation in the BRCA2 gene in the germ line of three brothers with breast cancer.

Germ-line mutations of the BRCA2 gene account for the majority of families with both male and female breast cancer. However, among independently ascertained families with the same mutation, cases of male breast cancer often appear to cluster in a single family or in a particular branch of one family. This suggests that the risk of male breast cancer conferred by BRCA2 mutations may be modified by other genetic or environmental factors. We report a family in which three brothers with breast cancer carry in their germ line two genetic abnormalities: an insertion A at nucleotide 2041 in exon 10 of BRCA2, which leads to premature termination of the encoded protein at codon 615, and a tandem interstitial duplication involving chromosome bands 9p23-24. We propose that the coexistence of this rare chromosomal abnormality with BRCA2 mutation may be augmenting the risk of male breast cancer conferred by the BRCA2 mutation.

Two regions of deletion in 9p23-24 in sporadic breast cancer.

Allelic deletions of 9p including band 21-22 are common in various types of human carcinomas including breast cancer. Our previous cytogenetic studies had identified constitutional chromosomal changes in 9p23-24 in patients of a male-breast-cancer family and 9p23-24 alterations in a cell line established from a sporadic female breast cancer. To find out whether this genomic region is involved more frequently in alterations in sporadic breast cancers, we have surveyed 80 microdissected tumor samples for both loss of heterozygosity (LOH) and homozygous deletion at 22 microsatellite loci spanning 9p22 to 9p24 using fluorescent multiplex PCR. LOH at one or more loci was observed in 32 (40%) of these tumors. Homozygous deletion was detected in four cases. Eleven tumors had LOH at all of the informative loci analyzed, whereas 21 tumors showed partial-terminal or interstitial allelic loss of 9p. Deletion mapping identified two common regions of deletion: (a) 4 cM including D9S281 to D9S286; and (b) 1 cM including D9S1808 to D9S268.

Human homologue of a candidate for the Mom1 locus, the secretory type II phospholipase A2 (PLA2S-II), maps to 1p35-36.1/D1S199.

Mice heterozygous for the dominant Min mutation in their Apc gene develop multiple intestinal neoplasia. Analogously, family members from familial adenomatous polyposis kindreds inheriting mutations in their human APC homologue develop a similar phenotype. Quantitative trait loci studies have identified the Mom1 locus (for modifier of Min-1), which is responsible for part of the genetic variability in polyp number found among inbred mouse strains. The secretory type II phospholipase [nonpancreatic Pla2s (type II Pla2s or Pla2s-II)] has been demonstrated to be a candidate for Mom1, and a mutation in Pla2s-II in mice carrying the Min mutation has been proposed to account for an increased polyp number compared to mice without the Pla2s-II mutation. In this study, we have mapped the chromosomal position of the human homologue of Pla2s-II. We have identified 3 mega-yeast artificial chromosomes that carry PLA2S-II and localized one of them by fluorescence in situ hybridization to the border between 1p35 and 1p36.1. The presence of the microsatellite marker D1S199 in all three clones integrates PLA2S-II into different genetic maps. This highly polymorphic CA repeat D1S199 has previously been shown by us to identify loss of heterozygosity in 48% of sporadic colorectal tumors, indicating that the human homologue of the Pla2s-II/Mom1 locus might be related to human colorectal cancer.

Translocation junctions cluster at the distal short arm of chromosome 1 (1p36.1-2) in human neuroblastoma cells.

Human neuroblastoma cells show at high frequency structural changes of the distal short arm of chromosome 1 (1p). The commonly altered region has been identified in previous loss-of-heterozygosity (LOH) studies to involve deletion of 1p36.1-2. These bands are also the site of constitutional alterations in patients with neuroblastoma. In an approach to define the 1p36.1-2 alterations in more detail we here employ four neuroblastoma cell lines to map translocation breaks involving 1p36.1-2 by fluorescence in situ hybridization (FISH). A chromosomal interval flanked by loci DIS96 and DIS98 contained translocation junctions in each of four lines. This analysis identifies in 1p a restricted genomic region as involved in chromosomal rearrangement in different neuroblastomas. The specificity of neuroblastoma translocation junctions at the molecular level implicates this genomic region in tumor development.

A reciprocal translocation (1;15) (36.2;q24) in a neuroblastoma cell line is accompanied by DNA duplication and may signal the site of a putative tumor suppressor-gene.

Cytogenetic analyses and molecular deletion studies of human neuroblastomas have indicated the chromosomal bands 1p36.1-1p36.2 as a location of genetic information which may be involved in tumorigenesis. To define this putative neuroblastoma locus in more detail we have analysed cell lines with alterations of distal 1p. Here we show, by fluorescence in situ hybridization (FISH), that cell line NGP has a reciprocal 1;15 translocation. Loci D1S214/D1S96 could be shown to map telomeric/distal, D1S228 centromeric/proximal to the break. We have identified yeast artificial chromosomes (YACs) that cover the break and map to D1S160 and D1S244. This chromosomal position is within the smallest region of overlap (SRO) found in neuroblastoma tumors (Weith et al., 1989; Caron et al., 1993; Schleiermacher et al., 1994) and within the region of a constitutional interstitial deletion of a neuroblastoma patient (Biegel et al., 1993). Mapping studies with FISH revealed that the translocation is associated with duplication of DNA. It appears, as if the subchromosomal region we describe here is a good candidate for harboring the postulated neuroblastoma suppressor-gene.

Non-syntenic amplification of MDM2 and MYCN in human neuroblastoma.

Amplification of the MYCN gene is a well documented genetic alteration of aggressively growing human neuroblastomas. Through cytogenetic studies we have identified neuroblastoma cell lines which, in addition to amplified MYCN, carry amplified DNA not harbouring MYCN. In situ hybridization of biotinylated total genomic DNA to metaphase chromosomes of normal human lymphocytes by reverse genomic hybridization revealed the amplified DNA to be derived from chromosome 12 band q13-14. Subsequent filter analyses showed a 20- to 40-fold amplification of the MDM2 gene, located at 12q13-14, both in three cell lines and in an original tumor, in addition to amplified MYCN. As the apparent consequence of amplification abundant MDM2 protein was present, a part of which was complexed with p53.

Cloning of the human homologue of the metastasis-associated rat C4.4A.

We have previously described a rat metastasis-associated molecule, C4.4A, which has some common features with the uPAR. Because of its restricted expression in non-transformed tissues a search for the human homologue became of interest. Human C4.4A was cloned from a placental cDNA library. As in the rat, the human uPAR and the human C4.4A genes appear to belong to the same family. Both genes are located on chromosome 19q13.1-q13.2 and both molecules have a glycolipid anchor site and are composed of three extracellular domains. Only domains one and two of the human C4.4A and the uPAR protein show a significant degree of identity. Expression of the human C4.4A was observed by RT-PCR and Northern blotting in placental tissue, skin, esophagus and peripheral blood leukocytes, but not in brain, lung, liver, kidney, stomach, colon and lymphoid organs. Yet, tumors derived from the latter tissues frequently contained C4.4A mRNA. As demonstrated for malignant melanoma, C4.4A mRNA expression correlated with tumor progression. While nevi were negative and only a minority of primary malignant melanoma expressed C4.4A, all metastases were C4.4A-positive. Taking into account the high degree of homology between rat and human C4.4A, the conformity of the expression profiles and the association of rat C4.4A with tumor progression, human C4.4A might well become a prognostic marker and possibly a target of therapy.

Cytogenetic evolution of MYCN and MDM2 amplification in the neuroblastoma LS tumour and its cell line.

Amplification of the MYCN gene is frequently seen either in extrachromosomal double minutes (DMs) or in homogeneously staining regions (HSRs) of aggressively growing neuroblastomas. Total genomic DNA from cell line LS, from early passages of the same line and from original tumour material was biotinylated and hybridised to metaphase chromosomes of normal human lymphocytes. The reverse genomic hybridisation revealed the amplified DNA to be derived both from chromosome 2p23-24, which is the position of MYCN, and from chromosome 12 band q13-14. The MDM2 gene, located at 12q13-14, was found amplified both in early and late passages of LS, in addition to amplified MYCN. Amplification units of MYCN and MDM2 appear first to develop within DMs, which then integrate into different chromosomes to develop to HSRs.

Reciprocal translocation at 1p36.2/D1S160 in a neuroblastoma cell line: isolation of a YAC clone at the break.

Band 1p36.1-1p36.2 is frequently involved in chromosomal aberrations of neuroblastoma cells, and therefore thought to harbour genetic information which may be involved in tumorigenesis. To map this putative neuroblastoma locus, we screened neuroblastoma cell lines for reciprocal translocations at 1p36.1-2 which may signal the site of an affected gene. We identified a reciprocal 1;15 translocation in cell line NGP by fluorescence in situ hybridisation (FISH). As a strategy to clone the translocation breakpoint, we isolated yeast artificial chromosomes (YACs) specific for loci at 1p36. Screening of cell line NGP by FISH identified a YAC, 1050 kbp in size, which hybridised to both derivative 1;15 and 15;1 chromosomes. We conclude that this YAC, which maps to D1S160, covers the break. This chromosomal position is within the smallest region of overlap (SRO) found in neuroblastoma tumours and within the region of a constitutional interstitial deletion of a neuroblastoma patient. The YAC we describe here should serve as a DNA source for gene cloning approaches towards the isolation of candidates for the putative neuroblastoma suppressor gene.

Amplification of oncogenes revisited: from expression profiling to clinical application.

Regulatory or structural alterations of cellular oncogenes have been implicated in the causation of cancers. Amplification represents one of the major molecular pathways by which gene expression is constitutively enhanced above the level of physiologically normal variation. Consequently, the significance of oncogene amplification in tumorigenesis originally had emerged from expression profiling of tumor cells by oncogene arrays. Amplified oncogenes have been found associated with more aggressive tumor variants and in selected settings are clinical markers to determine patient prognosis.

Chromosomal mapping of human genes by radioactive hybridization of cDNAs to CEPH-YAC high density gridded filter sets.

Chromosomal assignment of human transcribed sequences has been done mainly by high throughput genome analysis in specialized genome centres and, in a more classical fashion, by fluorescence in-situ hybridization (FISH) analysis. Not every laboratory has the ability to map cDNAs by FISH analysis. We here report a rapid mapping approach that is based on the hybridization of cDNA probes to high density gridded CEPH-YAC filters followed by subsequent computational analysis by database searches in the internet. Not only transcribed sequences but also genomic DNA could be subjected to this mapping approach. The presented approach allows to map human transcribed and genomic DNAs within 1-3 days and with a high level of resolution that will constantly increase in line with the incorporation of data deriving from high throughput genome mapping.

Chromosomal mapping of human genes by radioactive hybridization of cDNAs to Centre d'Etude du Polymorphisme humain high density gridded filter sets.

Chromosomal assignment of human transcribed sequences has been done mainly by high throughput genome analysis in specialized genome centres and, in a more classical fashion, by fluorescence in-site hybridization (FISH) analysis. Not every laboratory has the ability to map cDNAs by FISH analysis. We here report a rapid mapping approach that is based on the hybridization of cDNA probes to high density gridded Centre d'Etude du Polymorphisme Humain filters followed by subsequent computational analysis by database searches in the internet. Not only transcribed sequences but also genomic DNA could be subjected to this mapping approach. The presented approach allows to map human transcribed and genomic DNAs within 1-3 days and with a high level of resolution that will constantly increase in line with the incorporation of data deriving from high throughput genome mapping.

Amplification of satellite DNA at 16q11.2 in the germ-line of a patient with breast-cancer.

Cytogenetic analyses of peripheral blood cells of breast cancer patients unveiled in one case a grossly enlarged G-band in one copy of chromosome 16 (16qh+), which scored also strongly positive in C-banding. Fluorescence in situ hybridization with both a total chromosome 16 library and with probe pHuR195 detecting locus D16Z3 further documented the increase of the copy number of the corresponding satellite DNA at 16q-11.2. Slot-blot analyses with pHuR195 revealed an approximately 10-fold amplification compared to a random selection of normal chromosomes 16. The chromosome 16 carrying amplified DNA is passed on to one of 3 children, who shows no obvious anomaly. Previous studies (Kokalj-Vokac N, Alemeida A, Gerbault-Seureau M, Malfoy B, Dutrillaux B: Gene Chromosome Cancer 7: 8-14, 1993) had revealed that satellite DNA in chromosome 16 often participates in interchromosomal recombinations, preferentially with chromosome 1, in breast cancer cells. Although the increased copy number of satellite DNA could represent a polymorphism, it might provide an enlarged target for recombination events and therefore could be a determinant for cancer predisposition.

Heterogeneity and balance of chromosomes in human cell line M-HeLa-76: analysis of 100 karyotypes.

The cell subline M-HeLa-76 with the modal chromosome number 49 was used to define the level of karyotypic variability. By means of a G-banding technique, 100 metaphase plates were analyzed. Among them, 11 different karyotypes were found to range from 2% to 34%. Most probable evolutionary pathways of cell karyotypes within the cell subline were demonstrated. Determination of the origin of all marker chromosomes (32 in all) and unique structural rearrangements (16) revealed a nonrandom distribution of chromosome breaks. These are primarily centromeric regions of chromosomes 3 and 5. Identification of the overall chromosome material in the cells of each karyotype via karyotype reconstruction showed their close similarity in spite of differences in the sets of normal and marker chromosomes. On the basis of reconstructed karyotypes (RK) of each cell group, a generalized reconstructed karyotype (GRK) of the cell line was obtained. This permitted the conclusion of a balanced chromosome set in a cell population as a whole and obligatory retention of diploidy in all chromosomes of a normal set. The role of variability and selection in the evolution of cell populations in vitro is discussed.