Journal of Heredity Advance Access originally published online on June 30, 2005
Journal of Heredity 2005 96(7):782-785; doi:10.1093/jhered/esi085
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Establishment of a Cell Line Derived from a Canine Prostate Carcinoma with a Highly Rearranged Karyotype
From the Centre for Human Genetics, University of Bremen, Leobener Strasse ZHG, 28359 Bremen, Germany (Winkler, Reimann-Berg, Bullerdiek) and Small Animal Clinic, School of Veterinary Medicine, Bischofsholer Damm 15, 30173 Hanover, Germany (Murua Escobar, Eberle, Nolte)
Address correspondence to Jörn Bullerdiek at the address above, or e-mail: bullerd{at}uni-bremen.de.
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Abstract |
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Akin to the situation in humans, dogs are frequently affected by tumors of the prostate. The malignancies share many similarities between both species, for example, median age at the onset of the disease and metastatic behavior. In human prostatic tumor samples, investigations of prepared metaphase spreads showed a variety of chromosomal aberrations, with trisomies of chromosomes 7, 8, and 17 as the leading cytogenetic abnormalities. In this article we present one case of a canine adenocarcinoma of the prostate, including clinical examination and establishment of a cell line from a tumor sample obtained from the affected 10-year-old male Briard. Searching for similarities between both species in respect to chromosomal changes within the tumor samples, we investigated prepared metaphases of the canine cell line cytogenetically. These investigations presented a highly rearranged karyotype showing a large biarmed marker consisting of material from chromosomes 1 and 2 in addition to centromeric fusions between dog chromosomes 1 and 5 that both could be identified in every metaphase investigated, while centric fusions of chromosomes 4 and 5 occurred in up to 50% of the metaphases. The cell line grew very well and showed evidence of being spontaneously immortalized when it crossed the 20th passage.
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Introduction |
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Cytogenetic investigations of human malignancies look back on a long history. They revealed different chromosomal aberrations including mono- or trisomies, translocations, and isochromosomes. Some of them are known to be characteristic, for example, trisomy 12 in chronic lymphatic leukaemia (Anastasi et al. 1992) or the Philadelphia chromosome and/or isochromosome 17 in chronic myeloid leukaemia (Fioretos et al. 1999; Reid et al. 2003). However, centric fusions have only very rarely been described in human neoplasias. In most cases, the altered expression or fusion transcripts of tumor-associated genes are associated with tumor formation and progression. Thus chromosome analysis of solid tumors as well as hematopoietic malignancies has become an important tool to help establish a correct diagnosis and/or to decide between benign or malignant tumors of various origins (Mitelman 1998; Mitelman et al. 1997).
Over the past few years, the dog has become an increasingly important model for genetic diseases. Dogs and humans share the same environment, have access to qualified medical care, and last but not least share a variety of genetic diseases, including cancer (Withrow and MacEwen 1989, 2001). Compared to the situation in humans, cytogenetic studies in tumors of the dog are rare. This is certainly due to the difficult karyotype of the dog with its 76 small acrocentric autosomes and metacentric X- and Y-chromosomes (Reimann et al. 1996a). The existing reports showed that, akin to human tumors, malignant as well as benign canine tumors show an apparently higher incidence of clonal aberrations in mesenchymal tumors (lipomas and sarcomas) compared to epithelial neoplasms (Reimann et al. 1999a). As for the type of aberrations in canine tumors, numerical changes were found most frequently (Mayr et al. 1994; Reimann et al. 1996b, 1998, 1999a), followed by centric fusions (Mayr et al. 1991a,b, 1992).
Another step forward in cytogenetic investigations of canine tumors was taken when fluorescence in situ hybridization (FISH) was used for assignment of canine cancer-related genes and for comparative genomic hybridization (CGH) (Murua Escobar et al. 2001; Richter et al. 2004; Thomas et al. 2003a,b; Winkler et al. 2004). By the use of CGH analysis Thomas et al. (2003b) were able to show that in canine multicentric lymphoma gains of chromosome material were significantly more common than losses.
Carcinomas of the prostate are the third leading cause of death in human male patients, with an incidence of 193,000 deaths in 1990 and an expected number of cancer-related deaths of 393,000 by 2020 (Brundtland 2001). Besides humans, the dog is the only mammalian species that spontaneously develops tumors of the prostate (Boutemmine et al. 2002). Even though they show a lower incidence than their human counterparts, there is an increasing number of dogs developing tumors of the prostate. These tumors may present different histology, but adenocarcinomas predominate, and all of them are likely to metastasize (Nolte and Nolte 2000). In this article we present a case of prostate carcinoma in a dog, including clinical examination, establishment of a cell line, and cytogenetic analyses, the latter revealing a highly rearranged karyotype.
Case Report
In February 2003, a 10-year-old male Briard was presented at the Clinic for Small Animals Hanover with the following symptoms. The dog had troubles of defecation and gain of abdominal girth with reduced ingestion and increased drinking. Clinical examination showed no abnormalities in heart, lung, testicles, and lymph nodes; the body temperature was about 38.7°C. The abdomen was strained; X-ray examination revealed only reduced perceptibility of details. On diagnostic laparatomy, enlargement of the prostate and several small metastases in the mesentery were visible. The tumor was removed surgically, and pathohistological examination revealed a highly malignant adenocarcinoma of the prostate. Because of poor prognosis the dog was euthanized during surgical treatment.
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Materials and Methods |
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For establishment of the cell line, tumor samples were minced into small fragments followed by collagenase treatment (0.35%) for 2 h at 37°C. The dissociated cells were transferred into sterile flasks containing 5 ml medium 199. The cultures were incubated in 5% CO2/air at 37°C for 3 days. Well-grown culture flasks were subcultivated. For chromosome preparation colcemid was added at a final concentration of 0.1 µg/ml for 1.5 h before harvesting. The preparation of cell cultures for chromosome analyses followed routine methods (Bartnitzke et al. 1992). The cell suspension was dropped onto ice-cold slides, which were then allowed to age for 7 days at 37°C followed by GTG-banding according to a modification (Bartnitzke et al. 1992) of the protocol described by Seabright (1971). Karyotype description followed the nomenclature proposed by Reimann et al. (1996a).
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Results |
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Cell culture resulted in well growing cells with a high mitotic rate. The cells were subcultivated about once every 10 days. Cytogenetic investigation of 30 metaphases revealed the presence of a hyperdiploid karyotype (Figure 1). The chromosome number ranged between 81 and 131, with various centromeric fusions and several biarmed markers. Centromeric fusions between dog chromosomes 1 and 5 were observed in every metaphase investigated, whereas centric fusions of chromosomes 4 and 5 occurred in up to 50%. Additionally, a large biarmed marker was found in every metaphase investigated, consisting of material from chromosomes 1 and 2. It is thus likely to assume that chromosome material deriving from chromosome 1 is overrepresented in the presented cell line.
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Discussion |
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In human prostate cancers, a variety of genetic aberrations is known to occur. Aneuploidia in the form of trisomies has frequently been described, in most cases trisomies of chromosomes 7, 8, and 17 (Liu et al. 2001; Mark et al. 1999; Skacel et al. 2001).
In the present case, canine chromosome 1 is the chromosome affected in several ways, leading to the overrepresentation of chromosome material deriving from CFA 1. Previous publications dealing with canine tumors showed interesting cytogenetic abnormalities, including centric fusions involving chromosome 1 (Mayr et al. 1990, 1991a; Nolte et al. 1993), a third copy of chromosome 4 involved in tandem translocation (Mayr et al. 1994), or derivative chromosomes 4 and 7 (Reimann et al. 1999b). In a cytogenetic study investigating 270 canine solid tumors, it was shown that chromosomes 1, 2, 4, 5, and 25 are frequently involved in numerical changes as well as in structural aberrations (Reimann et al. 1999a). Horsting et al. (1999) speculated that chromosome 1 might contain a gene responsible for tumor development and that chromosome fusions involving chromosome 1 might be an initiating factor leading to karyotype instability and complex karyotype changes, respectively. In fact, all metaphases investigated in our study showed a highly rearranged karyotype with several centric fusions. This finding corresponds to the assumption that aneuploidy caused by failures of accuracy of chromosome disjunction is common in tumor cells and is assumed to be a general feature (Holliday 1989).
The appearance of biarmed chromosomes is also a frequent event during tumorigenesis in the dog (Reimann et al. 1994). They can be divided into two categories: isochromosomes, consisting of two arms of the same chromosome and translocation chromosomes, consisting of two acrocentric chromosomes. Both of them are present in the metaphase spreads of the canine prostatic cell line. Compared to each other, all results together strengthen the speculation that humans and dog often share the same genetic pathways in generation of cancer.
To the best of our knowledge, up to now there are only three well-known human prostate carcinoma cell lines and their various sublines, which are frequently used for prostate cancer research: DU-145, established from the tumor tissue removed from the metastatic central nervous system lesion of a 69-year-old man with prostate carcinoma in 1975 (Mickey 1980); PC-3, established from the bone marrow metastasis isolated postmortem from a 62-year-old Caucasian man with grade IV prostate cancer (poorly differentiated adenocarcinoma) after androgen suppression therapy (Kaighn et al. 1979); and LNCaP, established from the left supraclavicular lymph node metastasis from a 50-year-old man with prostate carcinoma in 1977 (Horoszewicz 1981). These cell lines are used in cancer research and drug discovery to test the effects of various agents on, for example, gene expression, cell proliferation, apoptosis, and metastatic behavior. With the newly established canine prostate carcinoma cell line presented herein, there is now another tool available for the research in prostate cancer. Due to the above-mentioned similarities seen in canine and human cancer genetics, the availability of the new canine prostate carcinoma cell line could open new fields in terms of comparison of this kind of neoplasia in both species.
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Acknowledgments |
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This paper was delivered at the 2nd International Conference on the "Advances in Canine and Feline Genomics: Comparative Genome Anatomy and Genetic Disease," Universiteit Utrecht, Utrecht, The Netherlands, October 14–16, 2004.
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Footnotes |
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Corresponding Editor: Elaine Ostrander
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