Extensive expression studies revealed a complex alternative splicing pattern of the HMGA2 gene.

Chromosomal rearrangements of the HMGA2 locus belong to the most common aberrations in human benign tumors. HMGA2 rearrangements often result in chimeric genes expressing transcripts consisting of the first three exons of HMGA2 followed by ectopic sequences derived from intron 3 of that gene. RT-PCR-based expression studies of 4 of these HMGA2 transcripts revealed a co-expression with the "wild-type" HMGA2a in tumor samples as well as in normal tissues. Northern blot hybridizations of the lipoma cell line Li-14 revealed the expression of five additional HMGA2 transcripts consisting of exons 1 to 3 but not exons 4 to 5 besides the full-length HMGA2a transcript. In silico analyses have been performed showing a high homology to well-established consensus sequences for the 3' splice acceptor site, the branch site, and poly(A) signal. Thus, it is quite obvious that the HMGA2 transcripts described herein are alternative, not aberrant, splice-products of the HMGA2 gene. It is hypothesized that HMGA2-dependent tumorigenesis is caused by a disturbed equilibrium in the co-expression of the HMGA2 splice variants leading to aberrant cell proliferation and/or malignant transformation of cells.

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Expression pattern of the HMGB1 gene in sarcomas of the dog.

BACKGROUND: The human high mobility group protein B1 (HMGB1) has attracted considerable interest among oncologists because it sensitises cancer cells to the anticancer drug cisplatin by shielding cisplatin-DNA adducts from nucleotide excision repair. MATERIALS AND METHODS: Since cisplatin is the cornerstone of adjuvant systemic therapy for osteosarcomas, in both humans and dogs, the expression pattern of the HMGB1 gene in seven canine sarcomas was investigated by Northern blot analysis and semi-quantitative RT-PCR. RESULTS: A strong intertumoural variation of HMGB1 expression was detected by Northern blot analysis and confirmed by the semi-quantitative RT-PCR established herein. CONCLUSION: The observed variations of HMGB1 expression in canine sarcomas emphasises the role of HMGB1 as a potential marker of clinical interest as its expression level may predict the clinical outcome of therapies based on cisplatin. The semi-quantitative RT-PCR established allows a quick and convenient determination of the HMGB1 expression level as necessary for clinical applications.

The canine HMGA1.

Due to the emerging advantages of numerous canine diseases as a genetic model for their human orthologs, the dog could join the mouse as the species of choice to unravel genetic mechanisms, e.g. of cancer predisposition, development and progression. However, precondition for such studies is the characterisation of the corresponding canine genes. Human and murine HMGA1 non-histone proteins participate in a wide variety of cellular processes including regulation of inducible gene transcription, integration of retroviruses into chromosomes, and the induction of neoplastic transformation and promotion of metastatic progression of cancer cells. Chromosomal aberrations affecting the human HMGA1 gene at 6p21 were described in several tumours like pulmonary chondroid hamartomas, uterine leiomyomas, follicular thyroid adenomas and others. Over-expression of the proteins of HMGA1 is characteristic for various malignant tumours suggesting a relation between high titer of the protein and the neoplastic phenotype. In this study, we characterised the molecular structure of the canine HMGA1 cDNA, its splice variants and predicted proteins HMGA1a and HMGA1b. Furthermore, we compared the coding sequence(s) (CDS) of both splice variants for 12 different breeds, screened them for single nucleotide polymorphisms (SNPs) and characterised a basic expression pattern.

Tissue-specific expression patterns of the RAGE receptor and its soluble forms--a result of regulated alternative splicing?

The receptor for advanced glycation end products (RAGE) is known to be causally involved in a variety of pathophysiological processes, e.g. immune/inflammatory disorders, Alzheimer disease, tumors, and abnormalities associated with diabetes as arteriosclerosis or disordered wound healing. So far, human cDNAs have been characterized encoding for the RAGE receptor and a truncated soluble form lacking the transmembrane and the cytosolic domain. The latter form represents a naturally occurring competitive inhibitor of signalling pathways induced by the membrane-standing RAGE receptor. In order to perform a relative expression analysis of both RAGE forms, an RT-PCR experiment was designed allowing the simultaneous amplification of corresponding transcripts. We were able to identify three novel human RAGE transcripts all encoding truncated soluble forms of RAGE. The relative expression ratios for the full-length RAGE transcript to the sum of its splice-variants encoding the soluble variants varied strongly among the tissues tested. Therefore, the pre-mRNA of RAGE must be subject to regulated alternative splicing activated by extracellular cues of yet unknown cellular signalling pathways. Thus, as deduced from the occurrence at the RNA level, it can be hypothesized that there is a complex RAGE regulation network involving isoforms competing for the binding of ligands.

Expression of the high-mobility group protein HMGI(Y) in human trophoblast: potential role in trophoblast invasion of maternal tissue.

The high-mobility group protein HMGI(Y) is a member of a family of non-histone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. In the present study, we analyzed the expression of HMGI(Y) in the human placenta using immunohistochemistry. We found expression of HMGI(Y), with nuclear localization, in the villous cytotrophoblast (vCT), which is a highly proliferative cell type. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. Interestingly, expression of HMGI(Y) was strongest in anchoring villi at the implantation site and in extravillous (intermediate) trophoblast (EVT) invading the maternal decidua. As vCT cells differentiate to become EVT, the HMGI(Y) protein appears to switch from a nuclear to a cytoplasmic localization. Expression of HMGI(Y) in isolated trophoblast populations in primary cell culture was also confirmed using Western-blot analysis. This study shows for the first time expression and localization of HMGI(Y) in the subpopulations of placental tissue.

Molecular-cytogenetic analysis of fragmentation of chromosome 17 in the breast cancer cell line EFM-19.

Because a previous study by conventional cytogenetics had revealed a nullisomy 17 in the breast cancer cell line EFM-19, we analysed that cell line by SKY-FISH and by FISH using different probes derived from chromosome 17. A bicolor FISH using a HER2-specific probe and a chromosome 17 centromeric probe showed five HER2 and six centromeric signals all appearing on different chromosomes A further bicolor FISH using a chromosome 17-specific painting probe and a HER2-specific probe revealed that the HER2 signals were always localized within chromosome 17 segments constituting part of structurally altered chromosomes as deduced from their G-banding. Further FISH analyses using single-locus probes of chromosome 17, i.e., for MDS, p53, SMS and RARA, showed that all five chromosome 17 painting segments contained material from the long arm but only two painting segments had additional material from the short arm. A SKY-FISH confirmed the results of the chromosome 17 painting by FISH, except for one structurally altered chromosome showing additional chromosome 17 material detected by the SKY experiment. These results allow us to conclude that, in this cell line, polysomy 17 has preceeded the fragmentation of chromosome 17 leading to amplification of small parts of that chromosome as well as to extended losses. As to a general mechanism, polysomy 17 and a fragility of this, chromosome in breast cancer cells may not only account for part of the cases with HER2 amplification but, at the same time, may further support malignant progression due to the loss of tumor suppressor genes as e.g. p53.

Sequencing of intron 3 of HMGA2 uncovers the existence of a novel exon.

Aberrations affecting the gene encoding the high mobility group protein HMGA2 (formerly HMGIC) have been found in a variety of human tumors, e.g., uterine leiomyomas, lipomas, and pulmonary chondroid hamartomas. These aberrations lead to fusion genes, transcriptional up-regulation, or aberrant transcripts of HMGA2. In the latter case, truncated transcripts consisting of exons 1 to 3 of HMGA2, encoding the three DNA-binding domains, and ectopic sequences derived from chromosome 12 are frequent. There are several lines of evidence indicating that the biological and tumorigenic features of truncated HMGA2 derivatives, i.e., those composed of the DNA-binding domains and a shortened acidic tail, clearly differ from those of the normal protein consisting of three DNA-binding domains and one large acidic tail. By sequencing the complete 112 kb third intron of HMGA2, we were able to detect several of the ectopic sequences, known as fused to HMGA2. Expression studies revealed co-expression of one of these transcripts with the normal transcript in tumors with 12q14-15 aberrations as well as in other tumors, and in normal tissues. Thus, this transcript (HMGA2b) is flanked by an alternative terminal exon of HMGA2. Due to the loss of the part encoding the acidic tail, the expression of the latter transcript may have more striking effects than the "wild type" HMGA2 (HMGA2a) in terms of tumorigenesis. This finding clearly indicates that functional studies also should address the role of the HMGA2b transcript.