Germ line mutations of the HMGA2 gene are rare among the general population.

Proteins encoded by the HMGA family are architectural transcription factors, which induce conformational changes in the DNA and thus influence gene expression. Despite the obvious association of the expression of high mobility group protein genes with human cancer, very little is known about the variation of the HMGA proteins within human populations. Therefore, the coding regions of HMGA2 from 87 normal healthy donors were sequenced with the aim of detecting single nucleotide polymorphisms. There was only one sequence divergence leading to an amino acid change in coding regions of HMGA2. Thus, HMGA2 is not only well conserved between species but there is also a high intra-individual conservation of HMGA2, further supporting the important role of HMGA proteins in cellular processes. This analysis clearly demonstrates that as a rule, germ line mutations of HMGA2 are not the cause for benign tumors, e.g. uterine leiomyomas, or human malignant solid tumors.

Expression patterns of the LPP-HMGA2 fusion transcript in pulmonary chondroid hamartomas with t(3;12)(q27 approximately 28;q14 approximately 15).

The high frequency of the t(3;12)(q27 approximately 28; q14 approximately 15) in lipomas and pulmonary chondroid hamartomas (PCHs) makes the HMGA2-LPP fusion gene the most frequent fusion gene in human tumors. We analyzed 11 PCHs with a t(3;12)(q27 approximately 28;q14 approximately 15) for the expression of the LPP-HMGA2 fusion transcript. In a previous study, all of these tumors were shown to express the HMGA2-LPP fusion transcript, composed of exons 1-3 of HMGA2 and exons 9-11 of LPP. In the present study, reverse transcriptase-polymerase chain reaction revealed the expression of the reciprocal fusion transcript in 8 of 11 cases. In all positive tumors, the reciprocal fusion transcripts had the same structure, namely, exons 1-8 of LPP and exons 4-5 of HMGA2 encoding a protein composed of the proline-rich region and the first LIM-domain of LPP and the acidic tail of HMGA2. To our knowledge, this is the first report of the expression of the LPP-HMGA2 fusion transcript in a series of PCHs. Its frequent occurrence in PCHs indicates the absence of a larger deletion of the LPP locus accompanying the translocation, such as has been described in a lipoma. Thus, based on this one finding, a role of LPP-HMGA2 in PCH should be considered.

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.

Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule.

The initiation of angiogenesis, called the angiogenetic switch, is a crucial early step in tumor progression and propagation, ensuring an adequate oxygen supply. The rapid growth of tumors is accompanied by a reduced microvessel density, resulting in chronic hypoxia that often leads to necrotic areas within the tumor. These hypoxic and necrotic regions exhibit increased expression of angiogenetic growth factors, eg, vascular endothelial growth factor, and may also attract macrophages, which are known to produce a number of potent angiogenetic cytokines and growth factors. A group of molecules that may act as mediators of angiogenesis are the so-called high-mobility group proteins. Recent studies showed that HMGB1, known as an architectural chromatin-binding protein, can be extracellularly released by passive diffusion from necrotic cells and activated macrophages. To examine the angiogenetic effects of HMGB1 on endothelial cells an in vitro spheroid model was used. The results of the endothelial-sprouting assay clearly show that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner. Thus, this is the first report showing strong evidence for HMGB1-induced sprouting of endothelial cells.

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.

High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity.

High mobility group A2 (HMGA2) chromosomal non-histone protein and its derivatives play an important role in development and progression of benign and malignant tumors, obesity and arteriosclerosis, although the underlying mechanisms of these conditions are poorly understood. Therefore, we tried to identify target genes for this transcriptional regulator and to provide insights in the mechanism of interaction to its target. Multiple genes have been identified by microarray experiments as being transcriptionally regulated by HMGA2. Among these we chose the ERCC1 gene, encoding a DNA repair protein, for this study. DNA-binding studies were performed using HMGA2 and C-terminally truncated DeltaHMGA2, a derivative that is frequently observed in a variety of tumors. A unique high affinity HMGA2 binding site was mapped to a specific AT-rich region located -323 to -298 upstream of the ERCC1 transcription start site, distinguishing it from other potential AT-rich binding sites. The observed 1:1 stoichiometry for the binding of wild-type HMGA2 to this region was altered to 1:2 upon binding of truncated DeltaHMGA2, causing DNA bending. Furthermore, the regulatory effect of HMGA2 was confirmed by luciferase promoter assays showing that ERCC1 promoter activity is down-regulated by all investigated HMGA2 forms, with the most striking effect exerted by DeltaHMGA2. Our results provide the first insights into how HMGA2 and its aberrant forms bind and regulate the ERCC1 promoter.

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.

HMGA2 expression in uterine leiomyomata and myometrium: quantitative analysis and tissue culture studies.

The high mobility group gene, HMGA2, is frequently expressed in uterine leiomyomata (UL) with chromosomal rearrangements of 12q15. In contrast, HMGA2 expression has not been detected in karyotypically normal UL or in myometrium, but has been detected in these tissues after culture. To characterize further the expression pattern of HMGA2, we assessed HMGA2 expression by RT-PCR followed by Southern blot hybridization, and by real-time PCR in three tissue panels: (1) primary myometrial cultures, (2) uncultured tissue from 15 karyotypically normal samples consisting of eleven 46,XX UL and four matched myometrial specimens, and (3) uncultured tissue from ten UL with 12q15 rearrangements and three matched myometrial specimens. HMGA2 expression was detected in all samples from the three panels. The level of HMGA2 expression in karyotypically normal UL was similar to the level of expression in myometrium; however, it was significantly less than the level measured in UL with 12q15 rearrangements. This expression analysis by use of detection methods of different sensitivities underscores the importance of studies of HMGA2 expression in uncultured tissues and of careful interpretation of results from experiments on cultured cells. Moreover, detection of HMGA2 expression in myometrium and in UL without 12q15 rearrangements, tissues previously thought not to express HMGA2, suggests that HMGA2 expression is required in normal adult myometrial physiology.

Expression of the HMGA2-LPP fusion transcript in only 1 of 61 karyotypically normal pulmonary chondroid hamartomas.

The HMGA2 gene has been found to be rearranged in a variety of benign solid tumors. However, in all tumor entities where aberrations of the corresponding chromosomal region have been found, a large percentage of tumors do not show any detectable cytogenetic deviation. Thus, the question arises whether or not in some of these cases, small subpopulations of tumor cells characterized by HMGA2 rearrangements exist. The existence of these populations would strongly suggest a secondary nature of the chromosomal aberrations. Herein, we have addressed this question by RT-PCR analyses of the HMGA2-LPP fusion resulting from t(3;12)(q27 approximately q28;q14 approximately q15) in a series of 61 pulmonary chondroid hamartomas (PCH) with an apparently normal karyotype. As a result, the HMGA2-LPP fusion transcript was amplified in only one of 61 PCH with a normal karyotype. In this case, fluorescence in situ hybridization analysis revealed a hidden chromosomal aberration. The absence of the HMGA2-LPP fusion in small populations of tumors with a normal karyotype suggests the primary nature of chromosomal rearrangements in the development of PCH affected by those aberrations.

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.

Absence of HMGIC-LHFP fusion in pulmonary chondroid hamartomas with aberrations involving chromosomal regions 12q13 through 15 and 13q12 through q14.

In a variety of benign solid human tumors the high mobility group protein gene HMGIC is affected by aberrations involving the chromosomal region 12q14 through q15. Beside the two predominant alterations t(3;12) (q27 through 28;q14 through q15) and t(12;14)(q14 through q15;q23 through q24), the t(12;13)(q14 through q15;q12 through q14) is another aberration observed recurrently in these tumors. Very recently, an HMGIC-LHFP (lipoma HMGIC fusion partner) fusion gene has been detected in a lipoma with a t(12;13). The results of the present study demonstrated the absence of the HMGIC-LHFP fusion in three pulmonary chondroid hamartomas (PCH) with complex aberrations involving chromosomal regions 12q13 through q15 and 13q12q through q14 and one PCH with a simple t(12;13)(q14 through 15;q13) by reverse transcription-polymerase chain reaction. Thus, intragenic rearrangements within the LHFP gene leading to its fusion to HMGIC are not a consistent finding in mesenchymal tumors with clonal aberrations of both chromosomal regions 12q13 through q15 and 13q12 through q14.