Untersuchung der subzellulären Lokalisation der Rezeptortyrosinkinase HER-3

Receptors tyrosine kinases play a pivotal role in tumour development and progression, and its altered expression has been observed in breast cancer, ovarian cancer and various other malignant tumour diseases. For example, approximately 20-30 % of breast cancer patients show an overexpression of the human epidermal growth factor receptor 2, HER-2. Therefore to counterpart this overexpression of HER-2, a therapeutic monoclonal humanized antibody (Herceptin®) directed against the extracellular domain of HER-2 was developed and is currently approved for clinical therapy. Receptors are normally localized to the cell membrane where they are activated by ligandbinding to trigger an intracellular signalling network. In the last decade several nuclear receptors were identified, but the function of many of these receptors remains unclear. To determine the subcellular distribution of the HER-2 and HER-3 an immunhistochemical analysis was conducted in ovarian and breast cancer tissues. In ovarian cancer HER-3 appear to have prognostic relevance in the patient’s survival. Patients with high HER-3 expression have a poorer prognosis than patients with low HER-3 expression. Additionally, breast cancer patients have a significant association between HER-2 expression and overall survival. Interestingly, 43.2 % of breast cancer patients showed a nuclear distribution of HER-3 with little or no HER-3 in cytoplasm. Reverse is also true where patients with cytoplasmic HER-3 had little or none localized to the nucleus. Thus, there appears to be "make-or-break" situation in the tumour samples but neither the mechanism nor the driving force behind the phenomenon is currently known. A further noteworthy observation is that cultured tumour cells have noticeably lower nuclear HER-3 expression (3-9%) compared to the patients’ specimens, providing a potential explanation as to why this observation has been neglected in the past. To investigate this phenomenon in more detail, cultured tumour cells were injected subcutaneously in nude mice and the nuclear HER-3 expression was analysed. Immunohistochemical analysis revealed high expression of HER-3 in the newly formed tumour tissue leading to the assumption that the nuclear HER-3 expression might be associated with the three dimensional feature of solid tumours. A main characteristic of solid tumours is the altered morphology which causes an undersupply of nutrients, oxygen and energy within the tumour. To determine whether challenging cells ( with nutrient depletion or drug treatment) altered HER-3 localization, different cell lines were depleted of ATP, glucose or treated with the drug cisplatin and the expression of HER-3 analysed. ATP depletion was carried out using Oligomycin B and 2 deoxyglucose, resulting in an accumulation of HER-3 in the nucleus to all cells examined. In contrast, the response to glucose depletion was cell line-dependent. Under glucose depletion the cervical cancer cell line HeLa showed an elevated nuclear HER-3 expression, whereas the breast cancer cell line MCF-7 did not show increased nuclear HER-3 localization. Dittmann et al. showed translocation of human epidermal growth factor (EGFR) into the nucleus after cisplatin exposure. Based on this observation, HER-3 localization was also analyzed after cisplatin exposure in MCF-7 cells. The result showed a clear nuclear translocation of HER-3. Although this effect was not comparable to the depletion of ATP and glucose, this finding provided further evidence that subcellular distribution of HER-3 is influenced by genotoxic stress. An additional factor which was found to influence the cellular distribution of HER-3 was confluency. A subconfluent growing cell culture shows higher amount of nuclear HER-3 compared to cell culture with a cell density of 100 %. Therefore an association between proliferation and the nuclear HER-3 expression may exist. Two main factors are linked to the signalling of HER-3: the dimerization partner of HER-3 (HER-2) and the ligand (HRGb1). Both factors were investigated to determine if they influence the subcellular distribution of HER-3. A Doxycycline-inducible HER-2 overexpressing cell line showed no increase in nuclear HER-3 expression with increased expression of HER-2. Furthermore, stimulation with HRGb1 did not have any effect on the nuclear HER-3 expression. Overall, the present results suggest that the translocation of HER-3 to the nucleus may be an important step in its signalling process. Depleting cells of ATP or glucose, or treating with a genotoxic chemical such as cisplatin, resulted in nuclear HER-3 expression. The current study is also the first to show that HER-3 nuclear location in tumour samples are more abundant compared to cell lines, tools commonly used to study receptor signalling. Although the functional role of nuclear HER-3 remains unclear, this work provides some key initial factors that can be used to further elucidate its function. Additionally, the significant increase in nuclear HER-3 expression in solid tumours may be in some way linked to resistance of such tumours to irradiation and chemotherapy

Differentiation of In Vitro–Modified Human Peripheral Blood Monocytes Into Hepatocyte–like and Pancreatic Islet-like Cells

BACKGROUND & AIMS: Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. METHODS: Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions. RESULTS: In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed beta cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1. CONCLUSIONS: The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer

Introduction: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. Methods: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. Results: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. Conclusions: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner

Comparison of scores for bimodality of gene expression distributions and genome-wide evaluation of the prognostic relevance of high-scoring genes

<p>Abstract</p> <p>Background</p> <p>A major goal of the analysis of high-dimensional RNA expression data from tumor tissue is to identify prognostic signatures for discriminating patient subgroups. For this purpose genome-wide identification of bimodally expressed genes from gene array data is relevant because distinguishability of high and low expression groups is easier compared to genes with unimodal expression distributions.</p> <p>Recently, several methods for the identification of genes with bimodal distributions have been introduced. A straightforward approach is to cluster the expression values and score the distance between the two distributions. Other scores directly measure properties of the distribution. The kurtosis, e.g., measures divergence from a normal distribution. An alternative is the outlier-sum statistic that identifies genes with extremely high or low expression values in a subset of the samples.</p> <p>Results</p> <p>We compare and discuss scores for bimodality for expression data. For the genome-wide identification of bimodal genes we apply all scores to expression data from 194 patients with node-negative breast cancer. Further, we present the first comprehensive genome-wide evaluation of the prognostic relevance of bimodal genes. We first rank genes according to bimodality scores and define two patient subgroups based on expression values. Then we assess the prognostic significance of the top ranking bimodal genes by comparing the survival functions of the two patient subgroups. We also evaluate the global association between the bimodal shape of expression distributions and survival times with an enrichment type analysis.</p> <p>Various cluster-based methods lead to a significant overrepresentation of prognostic genes. A striking result is obtained with the outlier-sum statistic (<it>p </it>< 10^-12). Many genes with heavy tails generate subgroups of patients with different prognosis.</p> <p>Conclusions</p> <p>Genes with high bimodality scores are promising candidates for defining prognostic patient subgroups from expression data. We discuss advantages and disadvantages of the different scores for prognostic purposes. The outlier-sum statistic may be particularly valuable for the identification of genes to be included in prognostic signatures. Among the genes identified as bimodal in the breast cancer data set several have not yet previously been recognized to be prognostic and bimodally expressed in breast cancer.</p

Hepatitis B Virus Particle Formation in the Absence of Pregenomic RNA and Reverse Transcriptase

Cytoplasmic hepatitis B virus (HBV) capsids are not enveloped and secreted unless the packaged RNA pregenome is reverse transcribed. The expression of the capsid protein C, together with envelope proteins in the absence of pregenomic RNA, produced normal amounts of intracellular capsids, but the secretion of virion-like particles was greatly reduced. The I97L C protein mutant, allowing immature nucleocapsid envelopment in the background of an HBV genome, did not promote the envelopment of capsids lacking a pregenome, suggesting that this mutation is not sufficient to induce secretion competence independently of the pregenome

Genome-wide analysis of Homo sapiens, Arabidopsis thaliana, and Saccharomyces cerevisiae reveals novel attributes of tail-anchored membrane proteins

Abstract Background Tail-anchored membrane proteins (TAMPs) differ from other integral membrane proteins, because they contain a single transmembrane domain at the extreme carboxyl-terminus and are therefore obliged to target to membranes post-translationally. Although 3–5% of all transmembrane proteins are predicted to be TAMPs only a small number are well characterized. Results To identify novel putative TAMPs across different species, we used TAMPfinder software to identify 859, 657 and 119 putative TAMPs in human (Homo sapiens), plant (Arabidopsis thaliana), and yeast (Saccharomyces cerevisiae), respectively. Bioinformatics analyses of these putative TAMP sequences suggest that the list is highly enriched for authentic TAMPs. To experimentally validate the software predictions several human and plant proteins identified by TAMPfinder that were previously uncharacterized were expressed in cells and visualized at subcellular membranes by fluorescence microscopy and further analyzed by carbonate extraction or by bimolecular fluorescence complementation. With the exception of the pro-apoptotic protein harakiri, which is, peripherally bound to the membrane this subset of novel proteins behave like genuine TAMPs. Comprehensive bioinformatics analysis of the generated TAMP datasets revealed previously unappreciated common and species-specific features such as the unusual size distribution of and the propensity of TAMP proteins to be part of larger complexes. Additionally, novel features of the amino acid sequences that anchor TAMPs to membranes were also revealed. Conclusions The findings in this study more than double the number of predicted annotated TAMPs and provide new insights into the common and species-specific features of TAMPs. Furthermore, the list of TAMPs and annotations provide a resource for further investigation

Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 mu m. The diffusive limit of competition for media occurred with a pitch of &gt;= 1250 mu m and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research