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Cytogenetic and molecular cytogenetic characterization of the stable ovarian carcinoma cell line (OvBH-1)
Cancer Genetics and Cytogenetics 164 (2006) 10–15
Cytogenetic and molecular cytogenetic characterization of the stable ovarian carcinoma cell line (OvBH-1) Kamila Schlade-Bartusiaka, Maria M. Sasiadeka,*, Julia K. Barb, Steffi Urbschatc, Nikolaus Blind, Mathias Montenarhe, Antonina Har1ozin´ska-Szmyrkab a
Departament of Genetics, bDepartment of Clinical Immunology, Wroclaw Medical University, Marcinkowskiego 1, Wroclaw 50-368, Poland c Departament of Human Genetics, University of the Saarland, Gebaude 44, D-66424 Homburg, Germany d Division of Molecular Genetics, Tuebingen University, Wilhelmstr 27, D-72074 Tuebingen, Germany e Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg, Germany Received 3 January 2005; received in revised form 8 April 2005; accepted 11 April 2005
Abstract
Detailed characterization and identification of cancer cell lines is the basis for the credibility of experimental studies. Therefore, chromosomal analysis should be routinely included in the protocol of cell line characterization and in the protocols of experimental studies performed on cell lines. In 2000, our group established and characterized cytomorphologically and immunophenotypically a new cell line, OvBH-1, which was derived from the ascitic fluid cells of an untreated patient with ovarian clear-cell adenocarcinoma. The aim of the current study was to characterize OvBH-1 cytogenetically and to monitor its stability by comparison of morphologic, immunohistochemical, and cytogenetic features between the early (135) and late (385) passages. Conventional and molecular cytogenetic analyses (fluorescence in situ hybridization and spectral karyotyping) of OvBH-1 revealed the following hypotriploid karyotype with random translocations: der(2)t(2;13), der(4)t(4;22), der(5)t(2;5). Complex rearrangements involving chromosomes 3, 15, and 20 were also found. FISH analysis with a p53 probe indicated the deletion of this region in two out of three copies of chromosome 17. The morphologic and immunophenotypic features, as well as the karyotypes observed in OvBH-1 in passages 135 and 385, were comparable. The monoclonality of the cell line was confirmed in a single cell cloning experiment. Our study indicated that OvBH-1 is characterized by a distinct karyotype and remains stable over 250 passages. Taking into account its thermosensitivity, its unusual karyotype, and its stability, this line can be considered as a valuable model for various experimental studies. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Biologically well-characterized cell lines represent powerful tools to define the cellular and molecular events involved in the process of cancer transformation and development. The biologic characteristics of cell lines usually comprise the description of morphologic and immunophenotypic features, including the expression of suppressor gene and oncogene products [1,2]. DNA fingerprinting and chromosomal analysis revealed that 18% of 298 human cell lines obtained directly by the investigators were misidentified before being delivered to the Deutsche Sammlung
* Corresponding author. Tel.: 148-71-784-12-55; fax: 148-71-78400-63. E-mail address: [email protected] (M.M. Sasiadek). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.04.011
von Mikroorganismen Zellkulturen despite their detailed biologic characterization [3]. Moreover, Wilson et al. [1] reported that DNA fingerprinting and cytogenetic analyses showed that two cell lines that had been previously described as separate lines appeared to be identical. Similarly, Drexler et al. [4] noted that about 15% of human leukemia– lymphoma cell lines were cross-contaminated. The authors concluded that only genetic analyses allowed the detection of misclassified cell lines and, therefore, these techniques should be applied in the case of routine cell line identification [1,5,6]. Chromosomal analysis appears highly valuable in cases in which chromosomal aberrations are typical for primary tumors and in cases with specific chromosomal aberrations [6]. Ovarian cancer is the leading cause of mortality from gynecological malignancies [7]. Studies on the etiology
K. Schlade-Bartusiak et al. / Cancer Genetics and Cytogenetics 164 (2006) 10–15
and progression of ovarian cancer have revealed a high degree of biologic heterogeneity. Up to now, almost 80 ovarian tumor cell lines have been described. The majority of them, however, were derived from solid ovarian carcinomas of treated patients. To our knowledge, only a few reports have been published on the establishment and characterization of cell lines derived from ascitic fluid cells, mainly from serous ovarian carcinoma of untreated patients. Individual data have been also published regarding cell lines obtained from ovarian clear-cell adenocarcinoma [7–9]. These carcinomas are not very often recognized, but they are associated with a poor prognosis and short survival time [7]. A new cell line (OvBH-1) was derived from the ascitic fluid cells of an untreated patient diagnosed with ovarian clear-cell adenocarcinoma in 2000 [10]. Detailed cytomorphologic and immunophenotypic characteristics of this cell line confirmed our data published earlier, indicating the clinical and biologic heterogeneity of ovarian carcinoma [10]. A correct identification of ovarian cancer cell lines is the basis for the credibility of experimental studies. The aim of the current study was to cytogenetically characterize OvBH-1 and to assess its stability by comparing the morphologic, immunohistochemical, and cytogenetic features of OvBH-1 for early (135) and late (385) passages. 2. Materials and methods All analyses were performed on the ovarian cell line OvBH-1, applying passages 135 and 385. This cell line was established by the authors in 2000 from the ascitic fluid cells of a 54-year-old patient diagnosed with ovarian clearcell adenocarcinoma. The tumor was poorly differentiated (G3) and the disease was staged International Federation of Gynecology and Obstetrics (FIGO) IV. Detailed morphologic and immunologic characteristics of the cell line were continuously performed from the 1st to the 135th passages and described in detail elsewhere [10]. Morphologic and immunohistochemical studies of the cell line, passage 135, were performed as described earlier [10]. Briefly, morphologic analysis was performed on the cytospin preparations fixed in cold acetone for 10 minutes. After staining with hematoxylin-eosin, the cytomorphologic features were evaluated. Immunophenotypic characterization of cells was performed by peroxidase-antiperoxidase testing using the monoclonal antibodies against intermediated cellular filaments (cytokeratin and vimentin; Dako, Copenhagen, Denmark); ovarian tumor associated antigens (e.g., CA125 and OV632; Dako); and CEA, p53, and HER-2/neu (Novocastra, Newcastle, UK). To confirm the monoclonality of the OvBH-1 cell line, the single-cell cloning experiment was performed on passages 135 and 385. Ten single cells were selected and their morphologic, immunological, and genetic characteristics were established. Briefly, the morphology of cells was established by the analysis of their nucleus and cytoplasmic features, as
11
described above. Immunohistochemical characteristics of subcloned cells were determined by using monoclonal antibodies, the same as those that were used for the characterization of the parental cell line. Classical cytogenetic analysis was performed according to standard procedures [11]. Karyotypes were prepared from Giemsa–trypsin-G (GTG)-banded chromosomes according to the International System for Human Cytogenetic Nomenclature (1995) [12]. Fluorescence in situ hybridization (FISH) was performed with Chromoprobe Multiprobe-M Octochrome (Cytocell Ltd., Cambridge, UK), following the procedure provided by the manufacturer. Briefly, cells in the suspension were spotted onto the template slides. The slides were denatured at 75 C for 5 minutes. After overnight hybridization at 37 C, the slides were washed at 72 C in 0.4 standard saline citrate (SSC) and then at room temperature in ST buffer (2 SSC/0.05% Tween-20). The slides were counterstained in 4’,6-diamidino-phenylindole (DAPI)-antifade solution and left in the dark for 10 minutes before viewing. The microscopic evaluation was performed using a standard DAPI/ FITC/Texas red triple filter. Twenty-four differently labeled chromosome-specific painting probes (SKY kit; Applied Spectral Imaging, Mannheim, Germany) were denatured and hybridized to denatured tumor metaphases according to the manufacturer’s protocol for 2 days at 37 C. Chromosomes were counterstained with a DAPI-antifade solution. Images were acquired with an SD 200 Spectra Cube (Applied Spectral Imaging) mounted on a Zeiss Axioskop II microscope using a custom-designed optical filter (SKY-1; Chroma Technology, Rockingham, VT) that allows for simultaneous excitation of all dyes and measurement of their emission spectra. Using a Sagnac interferometer in the optical head, an interferogram was generated at all image points, which was deduced from the optical path difference of the light, which, in turn, depended on the wavelength of the emitted fluorescence. The spectrum was recovered by Fourier transformation [13]. The spectral data were displayed by assigning red, green, or blue colors to certain ranges of the spectrum. The red, green, and blue (RGB) colors displayed a similar color to chromosomes that are labeled with spectrally overlapping fluorochromes. Based on the measurement of the spectrum of each chromosome, a spectral classification algorithm was applied that allowed the assignment of a pseudocolor to all points in the image within the same spectrum. This algorithm formed the basis for chromosome identification by SKY. DAPI images were acquired from all metaphases analyzed using a DAPI-specific optical filter. 3. Results Analysis by light microscopy of the cell line OvBH-1 performed on passages 135 and 385 (separated by 3 years of culturing) revealed that cells from passage 135 possess
12
K. Schlade-Bartusiak et al. / Cancer Genetics and Cytogenetics 164 (2006) 10–15
Fig. 1. Morphologic features of OvBH-1 cells. (A) Passage 385. (B) Subclone (hematoxylin and eosin staining; scale bar, 20 mm).
morphologic features of malignancy comparable to cells from passage 385. Independent of culturing time, the morphologic analyses of the cell lines revealed the presence of large, round cells with indistinct cell borders. Their nuclei are atypically or centrally located, differing in size and shape. The chromatin is granular and irregular. A second type of cell, which is moderate in size with indistinct cell borders and cytoplasm containing small vacuoles, was also observed. Immunophenotypic analysis of OvBH-1 cells (passage 385) showed their similarity to passage 135. The single-cell cloning experiment revealed that the morphologic features of 10 subclones are comparable to those observed in the parental cell line (see Fig. 1, A and B). Moreover, the expression of the studied proteins is similar to that of the parental line and 10 subclones. The results of immunohistochemical analysis for the parental cell line and four representative subclones are presented in Table 1, confirming the monoclonality of our OvBH-1 cell line. Cytogenetic analysis of OvBH-1 revealed a complex karyotype with a hypotriploid set of chromosomes that show numeric and structural aberrations (Fig. 2). Multiprobe FISH and SKY analyses confirmed these cytogenetic data and further identified the derivative chromosome as der (2) and der (4). These are unbalanced translocations, as described below: der(2)t(2;13)(q35;q14) and der(4)t(4;22)(q33;q11) (Figs. 3, A and B, and 4, A and B). Additional derivative chromosomes could not be resolved because of the complex rearrangements in these chromosomes. The composite karyotype, as derived from G-banding, FISH, and SKY, is as follows: 56~60,XX,2X,der(2)t(2;13)(q35;q14)2,23,der(4) t(4;22)(q33;q11),2,add(5q),der(5q)2,1der(5)t(2;5) (?;?)2,add(7),29,der(10)t(10;17)(p11;?),der(12)
t(12;20)(?;?),213,213,der(13)t(13;14)(p12;?),der(14;15) (q10;q10),del(15)(q15),del(17)(q24),218,219,der(19) t(17;19)(?;qter),220, der(20)t(20;21)(?;?),221,der(21) t(3;21)(?;?),222,222,222,1mar This karyotype is identical to the karyotype observed in cells from passage 135. 4. Discussion It has been well documented that patients with ovarian clear-cell carcinoma have a significantly worse prognosis than patients with other histologic types of ovarian carcinoma. Thus, biologically establishing well-characterized cell lines that originate from this type of ovarian carcinoma is important for a variety of experimental analyses
Table 1 Immunohistochemical characteristics of OvBH-1 parental cell line and four subclones Subclones
Antibodies Cytokeratin 1, 5, 6, 8, 10, 14, 18 Cytokeratin CK7 Cytokeratin CK 16/18 Vimentin OC125 OV-TL3 CEA p53 HER-2
Parental cell line OvBH-1
1
2
3
4
Percentage of positive cells (mean values) (%)
90
90
95
90
95
80 90 80 10 90 0 90 40
80 90 90 20 90 0 90 40
90 90 80 10 90 0 90 50
80 80 90 20 80 0 90 40
85 95 80 10 90 0 90 40
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Fig. 2. Karyotype of OvBH-1 cell line, 56~60,XX,2X,der(2)t(2;13)(q35;q14)2,23,der(4)t(4;22)(q33;q11),2,add(5q),der(5q)2,1der(5)t(2;5)(?;?) 2,add(7),29,der(10)t(10;17)(p11;?),der(12)t(12;20)(?;?),213,213,der(13)t(13;14)(p12;?),der(14;15)(q10;q10),del(15)(q15),del(17)(q24),218,219,der(19)t(17;19) (?, qter),220,der(20)t(20;21)(?;?),221,der(21)t(3;21)(?;?),222,222,222,1mar.
[5,10,14]. The difficulties in obtaining permanent cell lines from primary ovarian carcinomas are well known [1,3,14,15]. It has been shown that the phenotype of cancer cells can change in long-term cultures and, due to extensive subcloning, many of the established cell lines often display phenotypic and genotypic instability, with eventual loss of some features of the tumors from which they originated
[7,14]. In the majority of studies describing the morphologic and immunophenotypic features of cell lines, some differences between early and late passages were observed [7,14]. Current studies performed on the OvBH-1 ovarian clear-cell carcinoma line, passages 135 and 385, revealed that the morphology and expression of all studied markers were comparable for both the early and late passages,
Fig. 3. Derivative chromosome 4. (A) SKY analysis. (B) G-banding and schematic presentation.
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Fig. 4. Derivative chromosome 2. (A) SKY analysis. (B) G-banding and schematic presentation.
showing only negligible changes in the percentage of positive cells in immunohistochemistry. Moreover, the monoclonality of our cell line was confirmed in a single-cell cloning experiment and, therefore, it became obvious that the small differences observed in cell morphology could reflect only various stages of cell differentiation. Similarly, Mo¨buse et al. [14] showed variations in the percentage of positive cells with expression of cellular filaments and ovarian tumor–associated antigens in long-term cultures of histologically different ovarian carcinomas. It is also in concordance with the results of Gorai et al. [7], who found some variability in cell size and shape during the cultivation of ovarian clear-cell carcinoma. In the OvBH-1 cell line, a complex karyotype was detected. The first cytogenetic analysis of this cell line was performed in 2000 on passage 135, using GTG, SKY, and FISH. These analyses revealed a hypotriploid karyotype with multiple chromosomal aberrations such as random translocations der(2)t(2;13), der(4)t(4;22), and der(5)t(2;5), as well as complex translocations involving chromosomes 3, 15, and 20. FISH analysis with a p53 probe indicated the deletion of this region in two out of three copies of chromosome 17 (data not shown). In this study, chromosomal analysis was repeated on passage 385. The classic cytogenetic study was followed by molecular cytogenetic analyses (multiprobe FISH and SKY), which allowed us to determine the origin of most of the numerous marker chromosomes. Although not all aberrations of the complex karyotype were fully delineated, the results of this chromosomal analysis provide valuable information regarding the cytogenetic characteristics of this ovarian clear cell adenocarcinoma line. Bayani et al. [16] analyzed 13 sporadic ovarian carcinomas using SKY, CGH, and expression microarrays. They observed the highest level of aberrations in chromosomes 3, 8, 11, 17, and 21. Conventional cytogenetic analysis of 244 primary ovarian cancer cases revealed a statistically significant nonrandom occurrence of
chromosomal aberrations at regions 1p1*, 1q1*, 1p2*, 1q2*, 1p3*, 1q3, 3p1*, 1q4*, 6q1*, 6p2, 6q2, 7p1*, 7q1, 7p2*, 11p1*, 11q1, 11q2*, 12p1, 12q2*, 13p1, and 19q1. The authors disclosed that 13 commonly involved regions (denoted by an asterisk) are associated with both tumor progression and course of the disease and, therefore, could be considered as predictors of patient survival [17]. Comparison of the karyotype of OvBH-1 with the karyotypes of different primary ovarian carcinomas, which have been described by other authors [16,17], revealed that OvBH-1 is characterized by a unique, stable karyotype. The observation that three types of aberrations occurred in two out of three copies of chromosomes 2, 4, and 5 [der(2) t(2;13)(q35;q14), der(4)t(4;22)(q33;q11), der(5)t(2;5)(?;?)] may suggest that the hypotriploid karyotype observed in OvBH-1 resulted from the partial duplication of a haploid set of chromosomes bearing the translocations mentioned above. From our study it can be concluded that OvBH-1 remained stable over 250 passages (3 years of culturing), and that it is also characterized by a distinct karyotype. It is worth emphasizing that, as described elsewhere [18], OvBH-1 is the first human ovarian carcinoma cell line showing temperature sensitivity and a wild-type primary structure of the p53 protein. Therefore, OvBH-1 is a valuable model for investigating cell cycle regulators and p53 status. Taking into account the unique biologic properties of OvBH-1, the unusual karyotype, and the stability of the cell line, this line can be considered an interesting, valuable model for experimental studies, including the molecular pharmacology of anticancer drugs [19].
Acknowledgment This work was supported by grants from the Polish Scientific and Technological Cooperation Joint Project (PPPD/04/25552), the ‘‘Deutsche Krebshilfe’’ (W77/93/MO2),
K. Schlade-Bartusiak et al. / Cancer Genetics and Cytogenetics 164 (2006) 10–15
the ‘‘Founds der Chemischen Industrie,’’ and NATO Collaborative Linkage Grant no. 979488.
[10]
References [11] [1] Wilson AP, Dent M, Pejovic T, Hubbold L, Radford H. Characterization of seven human ovarian tumour cell lines. Br J Cancer 1996;74: 722–7. [2] Mo¨bus VJ, Moll R, Gerharz CD, Kieback DG, Weikel W, Hoffmann G, Kreienberg R. Establishment of new ovarian and colon carcinoma cell lines: differentiation is only possible by cytokeratin analysis. Br J Cancer 1994;69:422–8. [3] Quentmeier H, Osborn M, Reinhardt J, Zaborski M, Drexler HG. Immunocytochemical analysis of cell lines derived from solid tumors. J Histochem Cytochem 2001;49:1369–78. [4] Drexler HG, Dirks WG, Matsuo Y, MacLeod RA. False leukemialymphoma cell lines: an update on over 500 cell lines. Leukemia 2003;17:416–26. [5] MacLeod RAF, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG. Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int J Cancer 1999;83: 555–63. [6] Mro´zek K, Tanner SM, Heinonen K, Bloomfield CD. Molecular cytogenetic characterization of the KG-1 and KG-1a acute myeloid leukemia cell lines by use of spectral karyotyping and fluorescence in situ hybridization. Genes Chromosomes Cancer 2003;38:249–52. [7] Gorai I, Nakazawa T, Miyagi E, Hirahara F, Nagashima Y, Minaguchi H. Establishment and chatacterization of two human ovarian clear cell adenocarcinoma cell lines from metastatic lesions with different properties. Gynecol Oncol 1995;57:33–46. [8] Wong WSF, Wong WF, Ng YTA, Huang PD, Chew EC, Ho TH, Chang A. Establishment and characterization of a new human cell line derived from ovarian clear cell carcinoma. Gynecol Oncol 1990;38:37–45. [9] Ramakrishna V, Negri DRM, Brusic V, Fontanelli R, Canevari S, Bolis G, Castelli Ch, Parmiani G. Generation and phenotypic characterization of new human ovarian cancer cell lines with the
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identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells. Int J Cancer 1997;73:143–50. Bar JK, Har1ozin´ska A. Morphological and phenotypic characterization of a new established ovarian carcinoma cell line (OvBH-1). Anticancer Res 2000;20:2975–80. Limon J, Dal Cin P, Sandberg AA. Application of long-term collagenase disaggregation for the cytogenetic analysis of human solid tumors. Cancer Genet Cytogenet 1986;23:305–13. Mitelman F. ISCN guidelines for cancer cytogenetics. Supplement to an international system for cytogenetic nomenclature. Basel: S. Karger, 1995. Malik Z, Dishi M, Garini Y. Fourier transform multipixel spectroscopy and spectral imaging of protoporphyrin in single melanoma cells. Photochem Photobiol 1996;63:608–14. Mo¨bus V, Gerharz D, Press U, Moll R, Beck T, Mellin W, Pollow K, Knastein PG, Kreinberg R. Morphological, immunohistochemical and biochemical characterization of 6 newly established human ovarian carcinoma cell lines. Int J Cancer 1992;52(1):76–84. Siwek B, Larsimont D, Lacroix M, Body JJ. Establishment and characterization of three new breast cancer cell lines. Int J Cancer 1998;76:677–83. Bayani J, Brenton JD, MacGregor PF, Beheshti B, Albert M, Nallainathan D, Karaskova J, Rosen B, Murphy J, Laframboise S, Zanke B, Squire JA. Parallel analysis of sporadic primary ovarian carcinomas by spectral karyotyping, comparative genomic hybridization and expression microarrays. Cancer Res 2002;62:3466–76. Taetle R, Aickin M, Yang JM, Emerson J, Roe D, Adair L, Thompson F, Liu Y, Wisner L, Davis JR, Trent J, Alberts DS. Chromosome abnormalities in ovarian adenocarcinoma: I. Nonrandom chromosome abnormalities from 244 cases. Genes Chromosomes Cancer 1999;25:290–300. Bar JK, Har1ozin´ska S, Kartarius S, Montenarh M, Wyrodek E, Rodriguez Parkitna JM, Kochman M, O_zychar A. Temperature-sensitive ovarian carcinoma cell line (OvBH-1). Jpn J Cancer Res 2002;93:976–85. Yamori T. Panel of human cancer cell lines provides valuable database for drug discovery and bioinformatics. Cancer Chemother Pharmacol 2003;52(Suppl. 1):74–9.
Cytogenetic and molecular cytogenetic characterization of the stable ovarian carcinoma cell line (OvBH-1) Kamila Schlade-Bartusiaka, Maria M. Sasiadeka,*, Julia K. Barb, Steffi Urbschatc, Nikolaus Blind, Mathias Montenarhe, Antonina Har1ozin´ska-Szmyrkab a
Departament of Genetics, bDepartment of Clinical Immunology, Wroclaw Medical University, Marcinkowskiego 1, Wroclaw 50-368, Poland c Departament of Human Genetics, University of the Saarland, Gebaude 44, D-66424 Homburg, Germany d Division of Molecular Genetics, Tuebingen University, Wilhelmstr 27, D-72074 Tuebingen, Germany e Medical Biochemistry and Molecular Biology, University of the Saarland, Homburg, Germany Received 3 January 2005; received in revised form 8 April 2005; accepted 11 April 2005
Abstract
Detailed characterization and identification of cancer cell lines is the basis for the credibility of experimental studies. Therefore, chromosomal analysis should be routinely included in the protocol of cell line characterization and in the protocols of experimental studies performed on cell lines. In 2000, our group established and characterized cytomorphologically and immunophenotypically a new cell line, OvBH-1, which was derived from the ascitic fluid cells of an untreated patient with ovarian clear-cell adenocarcinoma. The aim of the current study was to characterize OvBH-1 cytogenetically and to monitor its stability by comparison of morphologic, immunohistochemical, and cytogenetic features between the early (135) and late (385) passages. Conventional and molecular cytogenetic analyses (fluorescence in situ hybridization and spectral karyotyping) of OvBH-1 revealed the following hypotriploid karyotype with random translocations: der(2)t(2;13), der(4)t(4;22), der(5)t(2;5). Complex rearrangements involving chromosomes 3, 15, and 20 were also found. FISH analysis with a p53 probe indicated the deletion of this region in two out of three copies of chromosome 17. The morphologic and immunophenotypic features, as well as the karyotypes observed in OvBH-1 in passages 135 and 385, were comparable. The monoclonality of the cell line was confirmed in a single cell cloning experiment. Our study indicated that OvBH-1 is characterized by a distinct karyotype and remains stable over 250 passages. Taking into account its thermosensitivity, its unusual karyotype, and its stability, this line can be considered as a valuable model for various experimental studies. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Biologically well-characterized cell lines represent powerful tools to define the cellular and molecular events involved in the process of cancer transformation and development. The biologic characteristics of cell lines usually comprise the description of morphologic and immunophenotypic features, including the expression of suppressor gene and oncogene products [1,2]. DNA fingerprinting and chromosomal analysis revealed that 18% of 298 human cell lines obtained directly by the investigators were misidentified before being delivered to the Deutsche Sammlung
* Corresponding author. Tel.: 148-71-784-12-55; fax: 148-71-78400-63. E-mail address: [email protected] (M.M. Sasiadek). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.04.011
von Mikroorganismen Zellkulturen despite their detailed biologic characterization [3]. Moreover, Wilson et al. [1] reported that DNA fingerprinting and cytogenetic analyses showed that two cell lines that had been previously described as separate lines appeared to be identical. Similarly, Drexler et al. [4] noted that about 15% of human leukemia– lymphoma cell lines were cross-contaminated. The authors concluded that only genetic analyses allowed the detection of misclassified cell lines and, therefore, these techniques should be applied in the case of routine cell line identification [1,5,6]. Chromosomal analysis appears highly valuable in cases in which chromosomal aberrations are typical for primary tumors and in cases with specific chromosomal aberrations [6]. Ovarian cancer is the leading cause of mortality from gynecological malignancies [7]. Studies on the etiology
K. Schlade-Bartusiak et al. / Cancer Genetics and Cytogenetics 164 (2006) 10–15
and progression of ovarian cancer have revealed a high degree of biologic heterogeneity. Up to now, almost 80 ovarian tumor cell lines have been described. The majority of them, however, were derived from solid ovarian carcinomas of treated patients. To our knowledge, only a few reports have been published on the establishment and characterization of cell lines derived from ascitic fluid cells, mainly from serous ovarian carcinoma of untreated patients. Individual data have been also published regarding cell lines obtained from ovarian clear-cell adenocarcinoma [7–9]. These carcinomas are not very often recognized, but they are associated with a poor prognosis and short survival time [7]. A new cell line (OvBH-1) was derived from the ascitic fluid cells of an untreated patient diagnosed with ovarian clear-cell adenocarcinoma in 2000 [10]. Detailed cytomorphologic and immunophenotypic characteristics of this cell line confirmed our data published earlier, indicating the clinical and biologic heterogeneity of ovarian carcinoma [10]. A correct identification of ovarian cancer cell lines is the basis for the credibility of experimental studies. The aim of the current study was to cytogenetically characterize OvBH-1 and to assess its stability by comparing the morphologic, immunohistochemical, and cytogenetic features of OvBH-1 for early (135) and late (385) passages. 2. Materials and methods All analyses were performed on the ovarian cell line OvBH-1, applying passages 135 and 385. This cell line was established by the authors in 2000 from the ascitic fluid cells of a 54-year-old patient diagnosed with ovarian clearcell adenocarcinoma. The tumor was poorly differentiated (G3) and the disease was staged International Federation of Gynecology and Obstetrics (FIGO) IV. Detailed morphologic and immunologic characteristics of the cell line were continuously performed from the 1st to the 135th passages and described in detail elsewhere [10]. Morphologic and immunohistochemical studies of the cell line, passage 135, were performed as described earlier [10]. Briefly, morphologic analysis was performed on the cytospin preparations fixed in cold acetone for 10 minutes. After staining with hematoxylin-eosin, the cytomorphologic features were evaluated. Immunophenotypic characterization of cells was performed by peroxidase-antiperoxidase testing using the monoclonal antibodies against intermediated cellular filaments (cytokeratin and vimentin; Dako, Copenhagen, Denmark); ovarian tumor associated antigens (e.g., CA125 and OV632; Dako); and CEA, p53, and HER-2/neu (Novocastra, Newcastle, UK). To confirm the monoclonality of the OvBH-1 cell line, the single-cell cloning experiment was performed on passages 135 and 385. Ten single cells were selected and their morphologic, immunological, and genetic characteristics were established. Briefly, the morphology of cells was established by the analysis of their nucleus and cytoplasmic features, as
11
described above. Immunohistochemical characteristics of subcloned cells were determined by using monoclonal antibodies, the same as those that were used for the characterization of the parental cell line. Classical cytogenetic analysis was performed according to standard procedures [11]. Karyotypes were prepared from Giemsa–trypsin-G (GTG)-banded chromosomes according to the International System for Human Cytogenetic Nomenclature (1995) [12]. Fluorescence in situ hybridization (FISH) was performed with Chromoprobe Multiprobe-M Octochrome (Cytocell Ltd., Cambridge, UK), following the procedure provided by the manufacturer. Briefly, cells in the suspension were spotted onto the template slides. The slides were denatured at 75 C for 5 minutes. After overnight hybridization at 37 C, the slides were washed at 72 C in 0.4 standard saline citrate (SSC) and then at room temperature in ST buffer (2 SSC/0.05% Tween-20). The slides were counterstained in 4’,6-diamidino-phenylindole (DAPI)-antifade solution and left in the dark for 10 minutes before viewing. The microscopic evaluation was performed using a standard DAPI/ FITC/Texas red triple filter. Twenty-four differently labeled chromosome-specific painting probes (SKY kit; Applied Spectral Imaging, Mannheim, Germany) were denatured and hybridized to denatured tumor metaphases according to the manufacturer’s protocol for 2 days at 37 C. Chromosomes were counterstained with a DAPI-antifade solution. Images were acquired with an SD 200 Spectra Cube (Applied Spectral Imaging) mounted on a Zeiss Axioskop II microscope using a custom-designed optical filter (SKY-1; Chroma Technology, Rockingham, VT) that allows for simultaneous excitation of all dyes and measurement of their emission spectra. Using a Sagnac interferometer in the optical head, an interferogram was generated at all image points, which was deduced from the optical path difference of the light, which, in turn, depended on the wavelength of the emitted fluorescence. The spectrum was recovered by Fourier transformation [13]. The spectral data were displayed by assigning red, green, or blue colors to certain ranges of the spectrum. The red, green, and blue (RGB) colors displayed a similar color to chromosomes that are labeled with spectrally overlapping fluorochromes. Based on the measurement of the spectrum of each chromosome, a spectral classification algorithm was applied that allowed the assignment of a pseudocolor to all points in the image within the same spectrum. This algorithm formed the basis for chromosome identification by SKY. DAPI images were acquired from all metaphases analyzed using a DAPI-specific optical filter. 3. Results Analysis by light microscopy of the cell line OvBH-1 performed on passages 135 and 385 (separated by 3 years of culturing) revealed that cells from passage 135 possess
12
K. Schlade-Bartusiak et al. / Cancer Genetics and Cytogenetics 164 (2006) 10–15
Fig. 1. Morphologic features of OvBH-1 cells. (A) Passage 385. (B) Subclone (hematoxylin and eosin staining; scale bar, 20 mm).
morphologic features of malignancy comparable to cells from passage 385. Independent of culturing time, the morphologic analyses of the cell lines revealed the presence of large, round cells with indistinct cell borders. Their nuclei are atypically or centrally located, differing in size and shape. The chromatin is granular and irregular. A second type of cell, which is moderate in size with indistinct cell borders and cytoplasm containing small vacuoles, was also observed. Immunophenotypic analysis of OvBH-1 cells (passage 385) showed their similarity to passage 135. The single-cell cloning experiment revealed that the morphologic features of 10 subclones are comparable to those observed in the parental cell line (see Fig. 1, A and B). Moreover, the expression of the studied proteins is similar to that of the parental line and 10 subclones. The results of immunohistochemical analysis for the parental cell line and four representative subclones are presented in Table 1, confirming the monoclonality of our OvBH-1 cell line. Cytogenetic analysis of OvBH-1 revealed a complex karyotype with a hypotriploid set of chromosomes that show numeric and structural aberrations (Fig. 2). Multiprobe FISH and SKY analyses confirmed these cytogenetic data and further identified the derivative chromosome as der (2) and der (4). These are unbalanced translocations, as described below: der(2)t(2;13)(q35;q14) and der(4)t(4;22)(q33;q11) (Figs. 3, A and B, and 4, A and B). Additional derivative chromosomes could not be resolved because of the complex rearrangements in these chromosomes. The composite karyotype, as derived from G-banding, FISH, and SKY, is as follows: 56~60,XX,2X,der(2)t(2;13)(q35;q14)2,23,der(4) t(4;22)(q33;q11),2,add(5q),der(5q)2,1der(5)t(2;5) (?;?)2,add(7),29,der(10)t(10;17)(p11;?),der(12)
t(12;20)(?;?),213,213,der(13)t(13;14)(p12;?),der(14;15) (q10;q10),del(15)(q15),del(17)(q24),218,219,der(19) t(17;19)(?;qter),220, der(20)t(20;21)(?;?),221,der(21) t(3;21)(?;?),222,222,222,1mar This karyotype is identical to the karyotype observed in cells from passage 135. 4. Discussion It has been well documented that patients with ovarian clear-cell carcinoma have a significantly worse prognosis than patients with other histologic types of ovarian carcinoma. Thus, biologically establishing well-characterized cell lines that originate from this type of ovarian carcinoma is important for a variety of experimental analyses
Table 1 Immunohistochemical characteristics of OvBH-1 parental cell line and four subclones Subclones
Antibodies Cytokeratin 1, 5, 6, 8, 10, 14, 18 Cytokeratin CK7 Cytokeratin CK 16/18 Vimentin OC125 OV-TL3 CEA p53 HER-2
Parental cell line OvBH-1
1
2
3
4
Percentage of positive cells (mean values) (%)
90
90
95
90
95
80 90 80 10 90 0 90 40
80 90 90 20 90 0 90 40
90 90 80 10 90 0 90 50
80 80 90 20 80 0 90 40
85 95 80 10 90 0 90 40
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Fig. 2. Karyotype of OvBH-1 cell line, 56~60,XX,2X,der(2)t(2;13)(q35;q14)2,23,der(4)t(4;22)(q33;q11),2,add(5q),der(5q)2,1der(5)t(2;5)(?;?) 2,add(7),29,der(10)t(10;17)(p11;?),der(12)t(12;20)(?;?),213,213,der(13)t(13;14)(p12;?),der(14;15)(q10;q10),del(15)(q15),del(17)(q24),218,219,der(19)t(17;19) (?, qter),220,der(20)t(20;21)(?;?),221,der(21)t(3;21)(?;?),222,222,222,1mar.
[5,10,14]. The difficulties in obtaining permanent cell lines from primary ovarian carcinomas are well known [1,3,14,15]. It has been shown that the phenotype of cancer cells can change in long-term cultures and, due to extensive subcloning, many of the established cell lines often display phenotypic and genotypic instability, with eventual loss of some features of the tumors from which they originated
[7,14]. In the majority of studies describing the morphologic and immunophenotypic features of cell lines, some differences between early and late passages were observed [7,14]. Current studies performed on the OvBH-1 ovarian clear-cell carcinoma line, passages 135 and 385, revealed that the morphology and expression of all studied markers were comparable for both the early and late passages,
Fig. 3. Derivative chromosome 4. (A) SKY analysis. (B) G-banding and schematic presentation.
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Fig. 4. Derivative chromosome 2. (A) SKY analysis. (B) G-banding and schematic presentation.
showing only negligible changes in the percentage of positive cells in immunohistochemistry. Moreover, the monoclonality of our cell line was confirmed in a single-cell cloning experiment and, therefore, it became obvious that the small differences observed in cell morphology could reflect only various stages of cell differentiation. Similarly, Mo¨buse et al. [14] showed variations in the percentage of positive cells with expression of cellular filaments and ovarian tumor–associated antigens in long-term cultures of histologically different ovarian carcinomas. It is also in concordance with the results of Gorai et al. [7], who found some variability in cell size and shape during the cultivation of ovarian clear-cell carcinoma. In the OvBH-1 cell line, a complex karyotype was detected. The first cytogenetic analysis of this cell line was performed in 2000 on passage 135, using GTG, SKY, and FISH. These analyses revealed a hypotriploid karyotype with multiple chromosomal aberrations such as random translocations der(2)t(2;13), der(4)t(4;22), and der(5)t(2;5), as well as complex translocations involving chromosomes 3, 15, and 20. FISH analysis with a p53 probe indicated the deletion of this region in two out of three copies of chromosome 17 (data not shown). In this study, chromosomal analysis was repeated on passage 385. The classic cytogenetic study was followed by molecular cytogenetic analyses (multiprobe FISH and SKY), which allowed us to determine the origin of most of the numerous marker chromosomes. Although not all aberrations of the complex karyotype were fully delineated, the results of this chromosomal analysis provide valuable information regarding the cytogenetic characteristics of this ovarian clear cell adenocarcinoma line. Bayani et al. [16] analyzed 13 sporadic ovarian carcinomas using SKY, CGH, and expression microarrays. They observed the highest level of aberrations in chromosomes 3, 8, 11, 17, and 21. Conventional cytogenetic analysis of 244 primary ovarian cancer cases revealed a statistically significant nonrandom occurrence of
chromosomal aberrations at regions 1p1*, 1q1*, 1p2*, 1q2*, 1p3*, 1q3, 3p1*, 1q4*, 6q1*, 6p2, 6q2, 7p1*, 7q1, 7p2*, 11p1*, 11q1, 11q2*, 12p1, 12q2*, 13p1, and 19q1. The authors disclosed that 13 commonly involved regions (denoted by an asterisk) are associated with both tumor progression and course of the disease and, therefore, could be considered as predictors of patient survival [17]. Comparison of the karyotype of OvBH-1 with the karyotypes of different primary ovarian carcinomas, which have been described by other authors [16,17], revealed that OvBH-1 is characterized by a unique, stable karyotype. The observation that three types of aberrations occurred in two out of three copies of chromosomes 2, 4, and 5 [der(2) t(2;13)(q35;q14), der(4)t(4;22)(q33;q11), der(5)t(2;5)(?;?)] may suggest that the hypotriploid karyotype observed in OvBH-1 resulted from the partial duplication of a haploid set of chromosomes bearing the translocations mentioned above. From our study it can be concluded that OvBH-1 remained stable over 250 passages (3 years of culturing), and that it is also characterized by a distinct karyotype. It is worth emphasizing that, as described elsewhere [18], OvBH-1 is the first human ovarian carcinoma cell line showing temperature sensitivity and a wild-type primary structure of the p53 protein. Therefore, OvBH-1 is a valuable model for investigating cell cycle regulators and p53 status. Taking into account the unique biologic properties of OvBH-1, the unusual karyotype, and the stability of the cell line, this line can be considered an interesting, valuable model for experimental studies, including the molecular pharmacology of anticancer drugs [19].
Acknowledgment This work was supported by grants from the Polish Scientific and Technological Cooperation Joint Project (PPPD/04/25552), the ‘‘Deutsche Krebshilfe’’ (W77/93/MO2),
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the ‘‘Founds der Chemischen Industrie,’’ and NATO Collaborative Linkage Grant no. 979488.
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