Anke Meyer

Upregulation of HMGA2 in thyroid carcinomas: a novel molecular marker to distinguish between benign and malignant follicular neoplasias.

The identification of molecular markers allowing to differentiate between benign and malignant thyroid tumors remains a diagnostic challenge. Herein, we have used the expression of the high mobility group protein gene HMGA2 and its protein, respectively, as a possible marker detecting malignant growth of thyroid tumors. HMGA2 belongs to the high mobility group proteins, i.e. small, highly charged DNA‐binding proteins. While HMGA2 is highly expressed in most embryonic tissues, its expression in adult tissues is very low. However, a reactivation of HMGA2 expression has been described for various malignant tumors and often correlates with the aggressiveness of the tumors. The aim of this study was to investigate whether the HMGA2 expression can be used to detect malignant thyroid tumors. RNA from 64 formalin‐fixed paraffin‐embedded thyroid tissues including normal tissue (n = 3), thyroiditis (n = 2), and follicular adenomas (n = 19) as well as follicular (n = 9), papillary (n = 28), and anaplastic (n = 3) carcinomas was reverse transcribed. Finally, real‐time quantitative RT‐PCR was performed. Expression differences of up to 400‐fold were detected between benign and malignant thyroid tumors. Based on HMGA2 expression alone, it was possible to distinguish between benign and malignant thyroid tissues with a sensitivity of 95.9% and a specificity of 93.9%. There was a highly significant (P < 0.001) difference with histology of the tumors being the gold standard between the benign lesions and malignant tumors. Our results show that even as a stand‐alone marker HMGA2 expression has a high potential to improve diagnoses of follicular neoplasms of the thyroid.

Overexpression of HMGA2 in uterine leiomyomas points to its general role for the pathogenesis of the disease

An overexpression of HMGA2 is supposed to be a key event in the genesis of leiomyoma with chromosomal rearrangements affecting the region 12q14‐15 targeting the HMGA2 gene, but gene expression data regarding differences between uterine leiomyomas with and those without 12q14‐15 aberrations are insufficient. To address the question whether HMGA2 is only upregulated in the 12q14‐15 subgroup, the expression of HMGA2 was analyzed in a comprehensive set of leiomyomas (n = 180) including tumors with 12q14‐15 chromosomal aberrations (n = 13) and matching myometrial tissues (n = 51) by quantitative RT‐PCR. The highest expression levels for HMGA2 were observed in tumors with rearrangements affecting the region 12q14‐15, but although HMGA2 is expressed at lower levels in leiomyomas without such aberrations, the comparison between the expression in myomas and matching myometrial tissues indicates a general upregulation of HMGA2 regardless of the presence or absence of such chromosomal abnormalities. The significant (P < 0.05) overexpression of HMGA2 also in the group of fibroids without chromosomal aberrations of the 12q14‐15 region suggests a general role of HMGA2 in the development of the disease.

Non-Hodgkin lymphoma expressing high levels of the danger-signalling protein HMGB1

HMGB1 is a high mobility group protein that can act either as a DNA binding protein or extracellularly as a cytokine-like danger signal. Extracellular HMGB1, either actively secreted or passively released by necrotic cells, is linked to inflammation and cancer. Herein, the results of a study to quantify the expression of HMGB1 in lymphomas by quantitative real-time RT-PCR are presented. HMGB1 expression was analysed in 18 non-Hodgkin lymphomas and two lymphoma cell lines. 11/18 primary lymphomas expressed HMGB1 mRNA at a level exceeding the average of normal lymph nodes. Immunohistochemistry showed that HMGB1 positivity is confined to the lymphoma cells. No correlation between HMGB1 expression and grading was found. However, a high percentage of lymphomas is overexpressing a danger-signalling protein. This protein can support the growth and angiogenesis of lymphoma cells in a paracrine way when released e.g. due to necrosis. Thus it constitutes an interesting therapeutic target as well.

Chromatin Immunoprecipitation to Analyze DNA Binding Sites of HMGA2

Background HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified. Methodology/Principal Findings In this investigation chromatin immunoprecipitation (ChIP) experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. Conclusion After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp) of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

6p21 rearrangements in uterine leiomyomas targeting HMGA1

To quantify the expression of HMGA1 mRNA in uterine leiomyomas, the expression of HMGA1 was analyzed in a series including tumors with aberrations of chromosome 6 (n = 7) and cytogenetically normal tumors (n = 8) as a control group by quantitative reverse transcriptase-polymerase chain reaction. The average expression level in the 6p21 group was found to be 5.6 times higher than that in the control group, and with one exception, all cases with 6p21 alteration revealed a high expression of HMGA1 mRNA than cytogenetically normal tumors. Nevertheless, compared to fibroids with a normal karyotype, the upregulation of the HMGA1 mRNA in these cases was much less strong than that of HMGA2 mRNA in case of 12q14∼15 aberrations identified in previous studies.

A Link Between the Expression of the Stem Cell Marker HMGA2, Grading, and the Fusion CRTC1-MAML2 in Mucoepidermoid Carcinoma

Recently, the concept of cancer stem cells and their expression of embryonic stem cell markers has gained considerable experimental support. In this study, we examined the expression of one such marker, the high‐mobility group AT‐hook 2 gene (HMGA2) mRNA, in 53 formalin‐fixed, paraffin‐embedded mucoepidermoid carcinomas (MEC) and four normal parotid tissues using quantitative real‐time RT‐PCR (qPCR). MECs are often characterized by the fusion gene CRTC1‐MAML2, the detection of which is an important tool for the diagnosis and prognosis of MEC. For detection of the CRTC1‐MAML2 fusion transcript, we performed RT‐PCR. The mean expression level of HMGA2 was higher in fusion negative (302.8 ± 124.4; n = 14) than in positive tumors (67.3 ± 13.1; n = 39). Furthermore, the fusion‐negative tumors were often high‐grade tumors and the HMGA2 expression level rose with the tumor grade (low: 43.7 ± 11.0, intermediate: 126.2 ± 28.3, and high: 271.2 ± 126.5). A significant difference was found in the HMGA2 expression levels between the different grading groups (one‐way ANOVA, P = 0.04) and among the fusion‐negative and ‐positive tumors (t‐test, P = 0.05), indicating that the expression level of HMGA2 was closely linked to grading, the presence/absence of the CRTC1‐MAML2 fusion, and the tumor behavior of MECs. These findings offer further evidence for the theory that the MEC group comprises two subgroups: one group with the CRTC1‐MAML2 fusion, which is a group with a moderate aggressiveness and prognosis, and the other group lacking that fusion corresponding to an increased stemness, and thus, higher aggressiveness and worse prognosis.

Manganese-Mediated MRI Signals Correlate With Functional β-Cell Mass During Diabetes Progression

Diabetes diagnostic therapy and research would strongly benefit from noninvasive accurate imaging of the functional β-cells in the pancreas. Here, we developed an analysis of functional β-cell mass (BCM) by measuring manganese (Mn2+) uptake kinetics into glucose-stimulated β-cells by T1-weighted in vivo Mn2+-mediated MRI (MnMRI) in C57Bl/6J mice. Weekly MRI analysis during the diabetes progression in mice fed a high-fat/high-sucrose diet (HFD) showed increased Mn2+-signals in the pancreas of the HFD-fed mice during the compensation phase, when glucose tolerance and glucose-stimulated insulin secretion (GSIS) were improved and BCM was increased compared with normal diet–fed mice. The increased signal was only transient; from the 4th week on, MRI signals decreased significantly in the HFD group, and the reduced MRI signal in HFD mice persisted over the whole 12-week experimental period, which again correlated with both impaired glucose tolerance and GSIS, although BCM remained unchanged. Rapid and significantly decreased MRI signals were confirmed in diabetic mice after streptozotocin (STZ) injection. No long-term effects of Mn2+ on glucose tolerance were observed. Our optimized MnMRI protocol fulfills the requirements of noninvasive MRI analysis and detects already small changes in the functional BCM.

Evaluation of Existing Methods for Human Blood mRNA Isolation and Analysis for Large Studies

Aims Prior to implementing gene expression analyses from blood to a larger cohort study, an evaluation to set up a reliable and reproducible method is mandatory but challenging due to the specific characteristics of the samples as well as their collection methods. In this pilot study we optimized a combination of blood sampling and RNA isolation methods and present reproducible gene expression results from human blood samples. Methods The established PAXgeneTM blood collection method (Qiagen) was compared with the more recent TempusTM collection and storing system. RNA from blood samples collected by both systems was extracted on columns with the corresponding Norgen and PAX RNA extraction Kits. RNA quantity and quality was compared photometrically, with Ribogreen and by Real-Time PCR analyses of various reference genes (PPIA, β-ACTIN and TUBULIN) and exemplary of SIGLEC-7. Results Combining different sampling methods and extraction kits caused strong variations in gene expression. The use of PAXgeneTM and TempusTM collection systems resulted in RNA of good quality and quantity for the respective RNA isolation system. No large inter-donor variations could be detected for both systems. However, it was not possible to extract sufficient RNA of good quality with the PAXgeneTM RNA extraction system from samples collected by TempusTM collection tubes. Comparing only the Norgen RNA extraction methods, RNA from blood collected either by the TempusTM or PAXgeneTM collection system delivered sufficient amount and quality of RNA, but the TempusTM collection delivered higher RNA concentration compared to the PAXTM collection system. The established Pre-analytix PAXgeneTM RNA extraction system together with the PAXgeneTM blood collection system showed lowest CT-values, i.e. highest RNA concentration of good quality. Expression levels of all tested genes were stable and reproducible. Conclusions This study confirms that it is not possible to mix or change sampling or extraction strategies during the same study because of large variations of RNA yield and expression levels.

β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype

Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow−derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes. Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment. With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP. After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.