Short communication Cell culture and senescence in uterine fibroids Dominique Nadine Markowski a , Sabine Bartnitzke a , Gazanfer Belge a , Norbert Drieschner a , Burkhard Maria Helmke b , Jo ¨rn Bullerdiek a,c, * a Center of Human Genetics, University of Bremen, Leobener Strasse ZHG, 28359 Bremen, Germany b Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany c Small Animal Clinic, University of Veterinary Medicine/Research Clusterof Excellence “REBIRTH,” Bischofsholer Damm 15, 30173 Hannover, Germany Received 18 February 2010; received in revised form 7 June 2010; accepted 14 June 2010 Abstract The in vitro growth of cells from uterine fibroids is characterized by an early onset of senescence. Often, an even lower growth potential than that of matching myometrial cells is noted. Also, the tremendous differences in the expression of the high mobility group protein HMGA2 seen when comparing fibroids of different genetic subtypes are surprisingly not reflected by significant differ- ences in their growth potential in vitro. We aimed to evaluate possible changes of the HMGA2 expression level between the native tissue and cell cultures, so we performed quantitative real- time polymerase chain reaction studies that revealed a marked decrease of the HMGA2 mRNA in culture in those cases with overexpression of HMGA2. In the two cases initially showing the highest expression, it decreased by approximately 97%. Associated with the decrease of HMGA2 was a clearly increased expression of the senescence-associated p19 Arf . Together, these findings explain the similar behavior of cell cultures from fibroids of different genetic subgroups and may also offer an explanation for the early onset of in vitro senescence in these cell cultures. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Despite their high growth potential in vivo, uterine leio- myomas show a strictly limited in vitro growth that is even exceeded by that of matching myometrial samples. This holds true for karyotypically normal leiomyomas as well as those characterized by the various types of recurrent chromosomal abnormalities such as deletions of the long arm of chromosome 7 or rearrangements of 12q14w15 [1,2]. The latter group of fibroids is characterized by a high expression of the gene-encoding high-mobility-group protein HMGA2 [3,4], which is targeted by these chromo- somal rearrangements [5]. Recently, the abundant expres- sion of that protein has been associated with an impaired DNA repair, which might also contribute to the high genomic instability seen in many malignant epithelial tumors in vivo [6] and in corresponding cell lines in vitro. On the other hand, regarding uterine fibroids, the problem of possible genomic instability has not been addressed in detail, but most, if not all, of them show a low tendency to undergo malignant transformation, which argues in favor of a relatively high genomic stability. Likewise, cell cultures from these tumors have not been reported to immortalize spontaneously and do not show gross differ- ences in their in vitro behavior when compared to cytoge- netically normal cases [7]. However, to our knowledge, no data allowing a direct comparison of the expression of HMGA2 in malignant epithelial vs. benign mesenchymal tumors and addressing changes of HMGA2 expression of leiomyomas of the 12q14w15 group when setting up cell cultures have been published so far. Thus, we have compared the HMGA2 expression levels of several leiomyomas with and without 12q14w15 rearrangements in native tumor samples as well as in the corresponding cell cultures. In addition, the expression has been compared to that seen in lung cancer samples and in a colon cancer cell line expressing compa- rably high levels of HMGA2. 2. Materials and methods 2.1. Tissue samples Tumor samples from fibroids were taken during surgery, immediately frozen in liquid nitrogen, and stored at 80  C for RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) analysis or transferred to Hanks * Corresponding author. Tel.: 49-421-218-2589; fax: 49-421-218- 4239. E-mail address: bullerdiek@uni-bremen.de (J. Bullerdiek). 0165-4608/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2010.06.010 Cancer Genetics and Cytogenetics 202 (2010) 53e57