Generation and Characterisation of a Canine EGFP-HMGA2 Prostate Cancer In Vitro Model

The architectural transcription factor HMGA2 is abundantly expressed during embryonic development. In several malignant neoplasias including prostate cancer, high re-expression of HMGA2 is correlated with malignancy and poor prognosis. The let-7 miRNA family is described to regulate HMGA2 negatively. The balance of let-7 and HMGA2 is discussed to play a major role in tumour aetiology. To further analyse the role of HMGA2 in prostate cancer a stable and highly reproducible in vitro model system is precondition. Herein we established a canine CT1258-EGFP-HMGA2 prostate cancer cell line stably overexpressing HMGA2 linked to EGFP and in addition the reference cell line CT1258-EGFP expressing solely EGFP to exclude EGFP-induced effects. Both recombinant cell lines were characterised by fluorescence microscopy, flow cytometry and immunocytochemistry. The proliferative effect of ectopically overexpressed HMGA2 was determined via BrdU assays. Comparative karyotyping of the derived and the initial CT1258 cell lines was performed to analyse chromosome consistency. The impact of the ectopic HMGA2 expression on its regulator let-7a was analysed by quantitative real-time PCR. Fluorescence microscopy and immunocytochemistry detected successful expression of the EGFP-HMGA2 fusion protein exclusively accumulating in the nucleus. Gene expression analyses confirmed HMGA2 overexpression in CT1258-EGFP-HMGA2 in comparison to CT1258-EGFP and native cells. Significantly higher let-7a expression levels were found in CT1258-EGFP-HMGA2 and CT1258-EGFP. The BrdU assays detected an increased proliferation of CT1258-HMGA2-EGFP cells compared to CT1258-EGFP and native CT1258. The cytogenetic analyses of CT1258-EGFP and CT1258-EGFP-HMGA2 resulted in a comparable hyperdiploid karyotype as described for native CT1258 cells. To further investigate the impact of recombinant overexpressed HMGA2 on CT1258 cells, other selected targets described to underlie HMGA2 regulation were screened in addition. The new fluorescent CT1258-EGFP-HMGA2 cell line is a stable tool enabling in vitro and in vivo analyses of the HMGA2-mediated effects on cells and the development and pathogenesis of prostate cancer.

Towards a gravitational wave observatory designer: sensitivity limits of spaceborne detectors

The most promising concept for low frequency (millihertz to hertz) gravitational wave observatories are laser interferometric detectors in space. It is usually assumed that the noise floor for such a detector is dominated by optical shot noise in the signal readout. For this to be true, a careful balance of mission parameters is crucial to keep all other parasitic disturbances below shot noise. We developed a web application that uses over 30 input parameters and considers many important technical noise sources and noise suppression techniques to derive a realistic position noise budget. It optimizes free parameters automatically and generates a detailed report on all individual noise contributions. Thus one can easily explore the entire parameter space and design a realistic gravitational wave observatory. In this document we describe the different parameters, present all underlying calculations, and compare the final observatory's sensitivity with astrophysical sources of gravitational waves. We use as an example parameters currently assumed to be likely applied to a space mission proposed to be launched in 2034 by the European Space Agency. The web application itself is publicly available on the Internet at http://spacegravity.org/designer. Future versions of the web application will incorporate the frequency dependence of different noise sources and include a more detailed model of the observatory's residual acceleration noise.