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

Chromosomal assignment of canine THADA gene to CFA 10q25

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<i>HMGA1</i> real-time PCR analyses.

<p>Relative <i>HMGA1/HPRT1</i> and <i>HMGA1/GUSB</i> expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. *p≤0.05 indicates a statistical significant increased expression of <i>HMGA1</i> in CT1258-HMGA2-EGFP cells when compared to native CT1258 and CT1258-EGFP.</p

<i>Let-7a</i> real-time PCR analyses.

<p>Relative <i>let-7a</i>/RNU6B expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. No statistical significant expression deregulation of <i>let-7a</i> in CT1258-EGFP and CT1258-HMGA2-EGFP was detected when compared to native CT1258 cells. Statistical significant p value was defined as ≤0.05.</p

<i>HMGA2</i> real-time PCR analyses.

<p>Relative <i>HMGA2/HPRT1</i> and <i>HMGA2/GUSB</i> expression in native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. Error bars are standard deviations. *p≤0.05 indicates a statistical significant expression deregulation of <i>HMGA2</i> in CT1258-HMGA2-EGFP cells when compared to native CT1258.</p

BrdU cell proliferation assay.

<p>Measured proliferation of native CT1258, CT1258-EGFP and CT1258-HMGA2-EGFP cells. A statistical significant increased proliferation was detected for CT1258 cells expressing the EGFP-HMGA2 fusion protein in comparison to native CT1258 and EGFP expressing CT1258 cells. Each bar represents a mean ± SD, *p≤0.05, ***p≤0.001.</p

Cytogenetic analyses.

<p>Metaphase spreads derived from CT1258 (A), CT1258-EGFP (B) and CT1258-EGFP-HMGA2 (C) cells after GTG-banding. The arrows indicate the centromeric fusions between the canine chromosomes 1 and 5 (der(1;5)) and a large bi-armed marker (mar) consisting of material from chromosomes 1 and 2 being characteristic for the CT1258 cell line.</p

Details of CT1258 chromosomal aberrations.

<p>Detailed presentation of chromosomal aberrations found in metaphases of CT1258, CT1258-EGFP and CT1258-EGFP-HMGA2. Two derivative chromosomes (der(1;5)) and the large bi-armed marker chromosome (mar) were found in each cell line.</p

Immunocytochemical staining.

<p>A: Native CT1258 cells, B: CT1258-EGFP cells, C: CT1258-EGFP-HMGA2 cells. Approximately 50% of the native CT1258 cell line and of CT1258-EGFP cells showed a HMGA2-positive nuclear labelling. In approximately 70–80% of CT1258-EGFP-HMGA2 cells, a strong and exclusively nuclear labelling for HMGA2 was detectable.</p