Abstract

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.

Highlights

  • According to recent global cancer statistics, prostate cancer is the second most frequent diagnosed cancer and sixth leading cause of death among males in economically developed countries [1]

  • CT258 cells transfected with the non-recombinant pEGFP-C1 expression vector showed green fluorescence all over the cytoplasm due to enhanced green fluorescent protein (EGFP) expression (Fig. 1B) whereas unmodified native CT1258 cells showed no EGFP fluorescence (Fig. 1A)

  • Transfection of CT1258 with pEGFPC1-High-Mobility-Group Protein A2 (HMGA2) resulted in the expression of a recombinant canine EGFP-HMGA2 fusion protein which could solely be detected in the nucleus of the transfected cells (Fig. 1C)

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Summary

Introduction

According to recent global cancer statistics, prostate cancer is the second most frequent diagnosed cancer and sixth leading cause of death among males in economically developed countries [1]. The dog is the only known domesticated mammalian species developing spontaneous prostate cancer with considerable interest [2]. Unlike the situation in men, the incidence of canine prostate carcinomas is low accounting for 0.2 to 0.6% of canine neoplasias [3]. The mean age at diagnosis in dogs is ten years and predominantly affecting elder individuals as it is reported in men [5,6,7]. Considering the physiologic age at prostate cancer diagnosis, the respective life span is similar between the two species showing increased incidence with age [6]

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