title: Generation and characterisation of a canine EGFP-HMGA2 prostate cancer in vitro model.
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.
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Saskia
authors.initials: S
authors.lastname: Willenbrock
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany; Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.
authors.forename: Siegfried
authors.initials: S
authors.lastname: Wagner
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Nicola
authors.initials: N
authors.lastname: Reimann-Berg
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moulay
authors.affiliation: Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Hewicker-Trautwein
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Nolte
authors.affiliation: Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany; Division of Medicine, Haematology, Oncology and Palliative Medicine, University of Rostock, Rostock, Germany.
authors.forename: Hugo
authors.initials: H
authors.lastname: Murua Escobar
mesh_terms: D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002869:Chromosome Aberrations; D004285:Dogs; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D025743:HMGA2 Protein / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D015820:Cadherins; D025743:HMGA2 Protein; D035683:MicroRNAs; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors