Antoaneta Mincheva

Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high‐level amplifications

Distinction of adrenocortical carcinoma from benign adrenocortical lesions by standard criteria is often difficult. In order to search for additional diagnostic parameters, a series of 25 adrenocortical tumors, 8 adenomas, 14 primary carcinomas, 1 metastasis, and the 2 adrenocortical carcinoma cell lines SW13 and NCI‐H295 were analyzed by the approach of comparative genomic hybridization (CGH). Except for the two smallest adenomas, all tumors showed chromosomal imbalances with a high incidence of chromosomal gains, most frequently involving chromosomes or chromosome arms 5, 7, 8, 9q, 11q, 12q, 14q, 16, 17q, 19, 20, and 22q. The only significant loss of material concerned the distal part of 9p. Furthermore, 21 high‐level amplifications were identified in 15 different regions of the genome. The consensus regions of recurrent gains and the focal high‐level amplifications allowed identification of a series of chromosomal subregions containing candidate proto‐oncogenes of potential pathogenic function in adrenocortical tumors: 1p34.3–pter, 1q22–q25, 3p24–pter, 3q29, 7p11.2–p14, 9q34, 11q12–11q13, 12q13, 12q24.3, 13q34, 14q11.2–q12, 14q32, 16p, 17q24–q25, 19p13.3, 19q13.4, and 22q11.2–q12. A subset of the CGH data was independently confirmed by interphase cytogenetics. Interestingly, the adenomas larger than 4 cm contained gained material of regions also overrepresented in carcinomas. In addition, several chromosomal gains, in particular the high‐level amplifications, were exclusive for the malignant status of the tumors. These data indicate that the larger adrenal lesions need to be carefully considered in the diagnosis of adrenocortical tumors, and that genetic aberrations might provide useful markers for a better diagnostic differentiation. Genes Chromosomes Cancer 28:145–152, 2000.

Comprehensive genomic analysis of desmoplastic medulloblastomas : identification of novel amplified genes and separate evaluation of the different histological components

Desmoplastic medulloblastoma (DMB) is a malignant cerebellar tumour composed of two distinct tissue components, pale islands and desmoplastic areas. Previous studies revealed mutations in genes encoding members of the sonic hedgehog pathway, including PTCH, SMOH and SUFUH in DMBs. However, little is known about other genomic aberrations. We performed comparative genomic hybridization (CGH) analysis of 22 sporadic DMBs and identified chromosomal imbalances in 20 tumours (91%; mean, 4.9 imbalances/tumour). Recurrent chromosomal gains were found on chromosomes 3, 9 (six tumours each), 20, 22 (five tumours each), 2, 6, 7, 17 (four tumours each) and 1 (three tumours). Recurrent losses involved chromosomes X (eight tumours), Y (six of eleven tumours from male patients), 9, 12 (four tumours each), as well as 10, 13 and 17 (three tumours each). Four tumours demonstrated high‐level amplifications involving sequences from 1p22, 5p15, 9p, 12p13, 13q33‐q34 and 17q22‐q24, respectively. Further analysis of the 9p and 17q22‐q24 amplicons by array‐based CGH (matrix‐CGH) and candidate gene analyses revealed amplification of JMJD2C at 9p24 in one DMB and amplification of RPS6KB1, APPBP2, PPM1D and BCAS3 from 17q23 in three DMBs. Among the 17q23 genes, RPS6KB1 showed markedly elevated transcript levels as compared to normal cerebellum in five of six DMBs and four of five classic medulloblastomas investigated. Finally, CGH analysis of microdissected pale islands and desmoplastic areas showed common chromosomal imbalances in five of six informative tumours. In summary, we have identified several novel genetic alterations in DMBs and provide genetic evidence for a monoclonal origin of their different tissue components. Copyright

Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridization

Metaphase chromosomes of three cervical cancer cell lines (HeLa, CasKi, SiHa) were subjected to in situ hybridizations with the DNA of human papillomaviruses (HPV) types 16 and 18, respectively. Previous studies have demonstrated multiple copies of HPV 18 DNA in HeLa and of HPV 16 DNA in CasKi cells, but only 1–2 HPV 16 copies in cells of the SiHa line. The viral DNA persists in an integrated state (Schwarz et al 1985). Analysis of the integration sites revealed at least 11 chromosomal sites of HPV 16 integration in CasKi cells. SiHa cells contain integrated HPV 16 DNA in the region q21–q31 of chromosome No. 13. In HeLa cells integration of HPV 18 occurred in chromosome No. 8, band q24. Thus, no evidence was obtained for the existence of preferential chromosomal regions for HPV integration. The data indirectly support a trans-acting function of HPV-mediated cell transformation.

Pbx4, a new Pbx family member on mouse chromosome 8, is expressed during spermatogenesis.

Members of the Pbx family are involved in a diverse range of developmental processes including axial patterning and organogenesis. Pbx functions are in part mediated by the interaction of Pbx proteins with members of the Hox and Meis/Prep families. We have identified a fourth mammalian Pbx family member. Pbx4 in the mouse and PBX4 in humans are located on chromosome 8 and chromosome 19, respectively. Pbx4 expression is confined to the testis, especially to spermatocytes in the pachytene stage of the first meiotic prophase.

Transcriptional and translational downregulation of H-REV107, a class II tumour suppressor gene located on human chromosome 11q11-12

The H-rev107 tumour suppressor was isolated as a gene specifically expressed in rat fibroblasts resistant toward malignant transformation by the activated HRAS gene (; ). Here we describe the human homologue of the rat H-rev107 gene. The predicted rat and human proteins are highly conserved exhibiting an overall amino acid identity of 83%. The H-REV107-1 gene is ubiquitously expressed with the exception of haematopoetic cells and tissues. In contrast, H-REV107-1 mRNA was found only in eight of 27 cell lines derived from mammary carcinoma, lung carcinoma, gastric carcinoma, kidney carcinoma, melanoma, neuroblastoma and other tumours. The H-REV107-1 protein was not detectable in any of these tumour cells. Loss of H-REV107-1 expression was not restricted to cultured human tumour cell lines, but also found in primary squamous cell carcinomas. Gross structural aberrations of the H-REV107-1 gene were absent in tumorigenic cell lines. Thus, the block to H-REV107-1 expression is achieved both at the level of transcription and translation. By fluorescence in situ hybridisation the human H-REV107-1 gene was localised to chromosome 11q11-12.

Murine epidermal lipoxygenase (Aloxe) encodes a 12-lipoxygenase isoform

Using a combination of conventional screening procedures and polymerase chain reaction cloning, we have isolated a cDNA encoding an epidermis‐type 12‐lipoxygenase (e12‐lipoxygenase) from mouse epidermis. The open reading frame corresponds to a protein of 662 amino acids and was found to be 99.8% identical to the ORF of an epidermal lipoxygenase gene Aloxe, described recently [Van Dijk et al. (1995) Biochim. Biophys. Acta 1259, 4–8]. When expressed in human embryonic kidney cells the recombinant protein could be shown to synthesize 12(S)‐HETE from arachidonic acid. By fluorescence in situ hybridization the e12‐lipoxygenase gene was localized to chromosome band 11 B1–B3.

Human homeodomain-interacting protein kinase-2 (HIPK2) is a member of the DYRK family of protein kinases and maps to chromosome 7q32-q34

Here we identified the human serine/threonine kinase HIPK2 as a novel member of the DYRK kinase subfamily. Alignment of several DYRK family proteins including the kinases minibrain, MJAK, PKY, the Dictyostelium kinase YakA and Saccharomyces YAK1 allowed the identification of several evolutionary conserved DYRK consensus motifs within the kinase domain. A lysine residue conserved between all DYRK kinase family members was found to be essential for the kinase function of HIPK2. Human HIPK2 was mapped to chromosome 7q32-q34 and murine HIPK2 to chromosome 6B, the homologue to human chromosome 7.

Spleen-specific Expression of the Malaria-inducible Intronless Mouse Gene imap38

We characterize the mouse gene imap38and its inducibility by Plasmodium chabaudi malaria among different lymphoid tissues and mouse strains of differentH-2 complex and non-H-2 background.Imap38 is a single copy gene assigned to chromosome 6B. It consists of only one exon of 1900 base pairs encoding a highly basic 25.8-kDa protein. Confocal laser scanning microscopy localizes differently tagged IMAP38 proteins in nuclei of transfected cells. Reporter gene assays reveal that the 1730-base pair 5′-flanking region, containing an RSINE1 repeat immediately adjacent to initiation site +1, exhibits promoter activity in nonmurine cells, while it is largely repressed in diverse mouse cell lines, which corresponds to the situation in mouse tissues. P. chabaudi malaria inducesimap38 expression almost exclusively in the spleen but not in other lymphoid organs. Parasite lysates are able to induceimap38 in the spleen, but not in spleen cells ex vivo. Activation of spleen cells by LPS and other stimuli is not sufficient to induce imap38. Inducibility ofimap38 requires signals from both parasites and the intact spleen, and it is controlled by genes of that non-H-2background, which also controls development of protective immunity against P. chabaudi malaria.

Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma

A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well‐controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild‐type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi‐strategy mediates specific knock‐down of gene expression. Sitespecific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down‐regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site‐specific shRNA‐strategy circumvents interference by IFN‐response induced when using other RNAi gene knock‐down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL‐derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease‐related genetic defects.— Pscherer, A., Schliwka, J., Wildenberger, K., Mincheva, A., Schwaenen, C., Döhner, H., Stilgenbauer, S., Lichter, P. Antagonizing inactivated tumor suppressor genes and activated oncogenes by a versatile transgenesis system: application in mantle cell lymphoma. FASEB J. 20, E315—E323 (2006)

Genomic organization, splice products and mouse chromosomal localization of genes for transcription factor Nuclear Factor One

Transcription factor Nuclear Factor One (NFI) proteins are derived from a small family of four vertebrate genes (NFIA, B, C and X), all of which produce a fair number of protein variants by alternative splicing. In order to ultimately locate RNA signal sequences around exon/intron borders for the production of regulated splice variants, we have determined the exon structure of the chicken NFIB gene as the last of the four vertebrate genes for which the gene structure was not yet elucidated. This made it possible to compile nine newly isolated and sequenced mouse NFI cDNA sequences together with all previously available ones and to deduce corresponding splicing patterns for the orthologous vertebrate genes of all four paralogous gene types. Results from the analysis of alternative splicing and of NFI gene mapping in the genome of human and mouse argue for a phylogenetic route in which the four vertebrate NFI genes result from a single duplication of a genomic segment containing two NFI intermediate genes rather than from two independent duplications of two separated single ancestor genes.