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.

Multi-body simulation of a canine hind limb: model development, experimental validation and calculation of ground reaction forces

Background: Among other causes the long-term result of hip prostheses in dogs is determined by aseptic loosening. A prevention of prosthesis complications can be achieved by an optimization of the tribological system which finally results in improved implant duration. In this context a computerized model for the calculation of hip joint loadings during different motions would be of benefit. In a first step in the development of such an inverse dynamic multi-body simulation (MBS-) model we here present the setup of a canine hind limb model applicable for the calculation of ground reaction forces. Methods: The anatomical geometries of the MBS-model have been established using computer tomography- (CT-) and magnetic resonance imaging- (MRI-) data. The CT-data were collected from the pelvis, femora, tibiae and pads of a mixed-breed adult dog. Geometric information about 22 muscles of the pelvic extremity of 4 mixed-breed adult dogs was determined using MRI. Kinematic and kinetic data obtained by motion analysis of a clinically healthy dog during a gait cycle (1 m/s) on an instrumented treadmill were used to drive the model in the multi-body simulation. Results and Discussion: As a result the vertical ground reaction forces (z-direction) calculated by the MBS-system show a maximum deviation of 1.75%BW for the left and 4.65%BW for the right hind limb from the treadmill measurements. The calculated peak ground reaction forces in z- and y-direction were found to be comparable to the treadmill measurements, whereas the curve characteristics of the forces in y-direction were not in complete alignment. Conclusion: In conclusion, it could be demonstrated that the developed MBS-model is suitable for simulating ground reaction forces of dogs during walking. In forthcoming investigations the model will be developed further for the calculation of forces and moments acting on the hip joint during different movements, which can be of help in context with the in silico development and testing of hip prostheses.