Reliable quantification of plasma HDV RNA is of paramount importance for treatment monitoring: A European multicenter study.

OBJECTIVES: Quantification of plasma hepatitis D virus (HDV) RNA is the essential tool for patient management under antiviral therapy. The aim of this European multicenter study was to improve the comparability of quantitative results reported by different laboratories using the CE/IVD-labeled RoboGene HDV RNA Quantification Kit 2.0 (Roboscreen GmbH) with different manual or automated nucleic acid extraction protocols/platforms and amplification/detection devices. METHODS: For harmonization of HDV RNA concentrations obtained by different protocols, correction factors (CF) were determined using the 1st WHO International Standard for HDV RNA. The limit of detection (LOD) and accuracy were determined for each protocol by using reference material. Furthermore, clinical samples were analyzed and results compared. RESULTS: The CF ranged from 20 to 1,870 depending on the protocol used. The LOD was found between 4 and 450 IU/ml. When accuracy was tested, external quality control (EQC) samples containing low HDV RNA concentrations were not detected by those protocols with higher LODs. For EQC samples, the maximum standard deviation of HDV RNA concentrations was found to be 0.53 log10 IU/ml, for clinical samples 0.87 log10 IU/mL. CONCLUSION: To ensure reliability in quantification of HDV RNA, any modification of the extraction and amplification/detection protocol validated by the manufacturer requires revalidation. With the 1st WHO International Standard for HDV RNA, the CF could easily be calculated leading to harmonization of quantitative results. This warrants both accurate monitoring of response to existing anti-HDV treatment and comparability of study results investigating novel anti-HDV drugs.

Adipocytes induce distinct gene expression profiles in mammary tumor cells and enhance inflammatory signaling in invasive breast cancer cells.

Obesity is a known risk factor for breast cancer. Since obesity rates are constantly rising worldwide, understanding the molecular details of the interaction between adipose tissue and breast tumors becomes an urgent task. To investigate potential molecular changes in breast cancer cells induced by co-existing adipocytes, we used a co-culture system of different breast cancer cell lines (MCF-7 and T47D: ER+/PR+/HER2- and MDA-MB-231: ER-/PR-/HER2-) and murine 3T3-L1 adipocytes. Here, we report that co-culture with adipocytes revealed distinct changes in global gene expression pattern in the different breast cancer cell lines. Our microarray data revealed that in both ER+ cell lines, top upregulated genes showed significant enrichment for hormone receptor target genes. In triple-negative MDA-MB-231 cells, co-culture with adipocytes led to the induction of pro-inflammatory genes, mainly involving genes of the Nf-κB signaling pathway. Moreover, co-cultured MDA-MB-231 cells showed increased secretion of the pro-inflammatory interleukins IL-6 and IL-8. Using a specific NF-κB inhibitor, these effects were significantly decreased. Finally, migratory capacities were significantly increased in triple-negative breast cancer cells upon co-culture with adipocytes, indicating an enhanced aggressive cell phenotype. Together, our studies illustrate that factors secreted by adipocytes have a significant impact on the molecular biology of breast cancer cells.

Indirect p53-dependent transcriptional repression of Survivin, CDC25C, and PLK1 genes requires the cyclin-dependent kinase inhibitor p21/CDKN1A and CDE/CHR promoter sites binding the DREAM complex.

The transcription factor p53 is central to cell cycle control by downregulation of cell cycle-promoting genes upon cell stress such as DNA damage. Survivin (BIRC5), CDC25C, and PLK1 encode important cell cycle regulators that are repressed following p53 activation. Here, we provide evidence that p53-dependent repression of these genes requires activation of p21 (CDKN1A, WAF1, CIP1). Chromatin immunoprecipitation (ChIP) data indicate that promoter binding of B-MYB switches to binding of E2F4 and p130 resulting in a replacement of the MMB (Myb-MuvB) by the DREAM complex. We demonstrate that this replacement depends on p21. Furthermore, transcriptional repression by p53 requires intact DREAM binding sites in the target promoters. The CDE and CHR cell cycle promoter elements are the sites for DREAM binding. These elements as well as the p53 response of Survivin, CDC25C, and PLK1 are evolutionarily conserved. No binding of p53 to these genes is detected by ChIP and mutation of proposed p53 binding sites does not alter the p53 response. Thus, a mechanism for direct p53-dependent transcriptional repression is not supported by the data. In contrast, repression by DREAM is consistent with most previous findings and unifies models based on p21-, E2F4-, p130-, and CDE/CHR-dependent repression by p53. In conclusion, the presented data suggest that the p53-p21-DREAM-CDE/CHR pathway regulates p53-dependent repression of Survivin, CDC25C, and PLK1.

Role of epigenetic mechanisms in epithelial-to-mesenchymal transition of breast cancer cells.

The epithelial-to-mesenchymal transition (EMT) is a crucial process during normal development that allows dynamic and reversible shifts between epithelial and mesenchymal cell states. Cancer cells take advantage of the complex, interrelated cellular networks that regulate EMT to promote their migratory and invasive capabilities. During the past few years, evidence has accumulated that indicates that genetic mutations and changes to epigenetic mechanisms are key drivers of EMT in cancer cells. Recent studies have begun to shed light on the epigenetic reprogramming in cancer cells that enables them to switch from a noninvasive form to an invasive, metastatic form. The authors review the current knowledge of alterations of epigenetic machinery, including DNA methylation, histone modifications, nucleosome remodeling and expression of microRNAs, associated with EMT and tumor progression of breast cancer cells. Last, existing and upcoming drug therapies targeting epigenetic regulators and their potential benefit for developing novel treatment strategies are discussed.