- Email: [email protected]
Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques
Cancer Genetics and Cytogenetics 141 (2003) 138–142
Short communication
Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques A. Martínez-Ramírez*, S. Rodríguez-Perales, B. Meléndez, B. Martínez-Delgado, M. Urioste, J.C. Cigudosa, J. Benítez Department of Human Genetics, Molecular Pathology Program, Spanish National Cancer Center (CNIO), Instituto de Salud Carlos III, Madrid, Spain Received 18 April 2002; received in revised form 27 June 2002; accepted 28 June 2002
Abstract
The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Cell lines are useful tools in the field of carcinogenesis for studying biologic, biochemical, and molecular aspects of malignant transformation. Many cell lines have been characterized using molecular cytogenetic methods such as fluorescence in situ hybridization (FISH), multicolor FISH, and spectral karyotyping (SKY), and the results have contributed to a better knowledge of neoplasia [1–6]. The A673 cell line was established from a patient with a possible primary rhabdomyosarcoma (RMS) [7]. This cell line has been widely used as an important tool for improving our knowledge of tumor biology. These cells are known to produce several growth factors with oncogenic potential, as well as cell growth-inhibitory factors [8]. These cells can also induce tumors in nude mice [9,10] and contain some cancer-related genes with hypermethylated promoters [11,12]. The A673 cell line is referred to in the literature both as Ewing tumor (ET) or sarcoma (ES) and as RMS. Although both these tumor types have specific and well-characterized translocations, no cytogenetic report describing the complete karyotype has been published. Ewing tumor presents a t(11;22)(q24;q12) recurrent chromosomal translocation in about 85% of the cases. At the molecular level, this translocation shows that the EWS and FLI1 genes are
* Corresponding author. Fax: 34-91-224-69-23. E-mail address: [email protected] (A. Martínez-Ramírez).
fused [13–15]. The most frequent chromosome rearrangements identified in RMS are t(2;13)(q35;q14) and its variant t(1;13)(p36;q14), which give rise to two fusion genes, PAX3/FKHR and PAX7/FKHR, respectively [16–18]. At the molecular level, A673 displays fusion of EWS and FLI1 genes as a consequence of the t(11;22) translocation, but does not present the fusions of PAX3 and PAX7 with FKHR resulting from t(2;13) and t(1;13), respectively [19,20]. These discrepancies prompted us to characterize the A673 cell line by conventional and molecular cytogenetic techniques. The combination of SKY, FISH, and reverse transcriptase polymerase chain reaction (RT-PCR) has revealed only the presence of a complex translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes. Both these events are specific to ET. 2. Materials and methods 2.1. Cell line The A673 cell line (American Type Culture Collection [ATCC], Manassas, VA, USA) and the alveolar rhabdomyosarcoma cell line RC2 (a gift from a Dr. Lollini, Cancer Institute, University of Bologna, Italy), with the t(1;13) [20], were maintained in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine and 13% fetal bovine serum. The alveolar rhabdomyosarcoma cell line Rh30 [19], which contains the t(2;13), was maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% fe-
0165-4608/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S0165-4608(02)00 6 7 0 - 2
A. Martínez-Ramírez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142
tal bovine serum. All media and supplements for cell cultures were obtained from Gibco (Gaithersburg, MD, USA). 2.2. Cytogenetic studies 2.2.1. Conventional cytogenetics Cells of the cell line were exposed to colcemid (0.1 g/mL) for 1.5 hours at 37C and harvested routinely. Metaphase chromosomes were GTG-banded by a conventional trypsin– Giemsa technique and karyotyped according to the International System for Human Cytogenetic Nomenclature, 1995 revision [21]. 2.2.2. SKY study Slide preparation and hybridization, using commercial probes, were carried out according to the manufacturer’s instructions (Applied Spectral Imaging [ASI], Migdal Ha’Emek, Israel). Images were acquired using an ASI SD300 Spectracube mounted on a Zeiss Axioplan 2 microscope. 2.2.3. FISH study Chromosome spreads, prepared directly from the A673 cell line, were used for conventional analysis and were left overnight at room temperature. Slides were placed on a plate at 90C for 10 minutes, dehydrated through a series of ethanol washes, and denatured in the presence of a probe on a plate at 75C for 1 minute. Specific bacterial artificial chro-
139
mosome (BAC) (RPCI-11 Human BAC Clone Library, Children’s Hospital Oakland Research Institute [CHORI]) for the EWS, FLI1, PAX3, PAX7, and FKHR genes were labeled with different colors by nick translation (Vysis, London, UK). These BACs were used to detect the EWS/FLI1, PAX3/ FKHR, and PAX7/FKHR rearrangements. Cell images were captured using a charge-coupled device (CCD) camera (Photometrics SenSys camera, Tucson, AZ, USA) connected to a microcomputer running the Chromofluor image analysis system (Applied Imaging, Newcastle-upon-Tyne, UK).
2.3. Molecular studies After cDNA synthesis, PCR was performed to detect chimeric transcripts derived from the t(11;22), t(2;13), and t(1; 13). All samples were analyzed according to standardized primers, protocols, and criteria [18,22–24]. To verify the integrity of the isolated RNA and the correct synthesis of the cDNA, the ubiquitously expressed ABL gene was amplified in a separate PCR reaction. The PCR was performed in two steps: the first round was performed with the external primers and the second with reverse internal primers. Amplification was performed with a 9700 Perkin Elmer Thermocycler (Perkin Elmer, Germany). Twenty microliters of the final PCR products were analyzed on a 3% agarose gel (MetaPhor agarose; BMA, Rockland, ME, USA) and visualized by ethidium bromide staining.
Fig. 1. Representative complex karyotype from Giemsa-banding of the A673 cell line showing multiple rearrangements and two der(13):der(13)t(1;13) and der(13)t(11;13)t(11;22) (arrows) and der(22)t(11;22).
140
A. Martınez-Ramırez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142
3. Results 3.1. ET studies A conventional cytogenetic study was carried out on the A673 cell line. This revealed a complex karyotype with multiple rearrangements affecting chromosomes 3, 5, 8, 9, and 16. Additional unbalanced translocations were found [der(1)t(1;19), t(5;8), der(16)t(3;16), der(9)t(9;13), and der(11)t(11;13)] and loss of material was observed in chromosomes 3 and 4 (Fig. 1). Neither the t(11;22) nor its variants, characteristic of ET, were detected in the karyotype. By means of SKY analysis we established the karyotype of the A673 cell line (Fig. 2a) to be 4647,XX,der(1)t(1;9) (p36;q22),der(3)del(3)(p21)del(3)(q21),del(4)(q21q31), t(5;8)(q33;q21),der(9)t(9;13)(q22;q14),der(11)t(11;13) (p13;q14),der(13)t(1;13)(p36;q14),der(13)t(11;13)(q13;q14) t(11;22)(q24;q12),der(16)t(3;16)(q21?;q22),der(22)t(11;22) (q24;q12) [cp15]. There was a specific translocation of ET, although this was located on a derivative chromosome 13 with a complex translocation between chromosomes 13, 11, and 22: der (13)t(11;13)(q13;q14)t(11;22)(q24;q12) (Fig. 2a). The expected chromosome der(22) generated by the t(11;22) was also detected as a small chromosome 22.
The FISH study with EWS and FLI1 BAC probes yielded one yellow signal on the der(22) generated by the t(11;22) and the EWS/FLI1 fusion gene, one green signal on the der(13)t(13;11;22), and two red signals belonging to the normal chromosomes 11 (Fig. 2b). An RT-PCR specific for the amplification of the EWS/FLI1 fusion revealed a molecular rearrangement compatible with the translocation. 3.2. RMS studies With regard to RMS, the most consistent chromosome rearrangements are the t(2;13)(q35;q14) and its variant t(1;13)(p36;q14). We did not observe the t(2;13), but we did detect a t(1;13) with similar breakpoints on chromosome 13. FISH analysis using specific BAC of PAX3/FKHR and PAX7/FKHR fusion genes was negative (Fig. 2c and 2d), suggesting that other breakpoints are involved (Fig. 2d). Molecular analysis by RT-PCR confirmed the absence of the PAX3/FKHR and PAX7/FKHR fusion genes. These results are consistent with those we obtained by FISH.
4. Discussion Since its establishment, the A673 cell line, which was derived from a possible primary RMS [7], has been widely
Fig. 2. (a) SKY of the A673 cell line. Arrows point to the rearrangements of chromosomes 13 and 22. (b) FISH of a metaphase spread and interphase nucleus of the A673 cell line. Arrows point to one yellow fusion signal on the der(22), generated by the t(11;22), and one green signal on the der(13)t(13;11;22). The two red signals correspond to the normal chromosomes 11. (c and d) FISH analysis using specific BAC for PAX3, PAX7, and FKHR. Neither PAX3/FKHR nor PAX7/FKHR fusion genes were detected (panels c and d, respectively).
A. Martínez-Ramírez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142
used to extend our knowledge of tumor biology in RMS. This cell line is defined as one of RMS in the ATCC and has been used in different studies during the 1980s to obtain and purify tumor growth inhibitory factors [25,26] or to investigate the mechanisms of human peripheral blood monocytemediated cytotoxicity in tumor cells [27]. It is currently being used as an RMS to evaluate tumor growth inhibition by administration of the antihuman vascular endothelial growth factor (VEGF) [9,28,29]. A673 is also referred to in the literature as an ET cell line, however, and during the 1990s it was used to characterize growth-inhibitory activity and to extend our knowledge of the role of hypermethylation [8, 11,12,14]. As no cytogenetic or molecular report describing the A673 cell line had been previously published, we undertook the characterization of the cell line A673 using a combination of G-banding analysis and molecular cytogenetic methods. Giemsa-banding revealed a complex karyotype with multiple chromosomal rearrangements but without an apparent ET-specific t(11;22). Tumors of the Ewing sarcoma family are characterized by the t(11;22) or t(21;22) in about 85% and 15% of cases, respectively [14]. Our SKY analysis revealed a complex translocation among chromosomes 13, 11, and 22 and a der(22) chromosome that originated through the t(11;22). The t(11;22) was confirmed with FISH and molecular analysis using probes and primers specific for the detection of the EWS/FLI1 fusion gene. Furthermore, we observed with G-banding, and confirmed with SKY, a t(1;13) but no t(2;13). This t(1:13) is about five times less frequent than the t(2;13) in RMS and produces the fusion between the PAX7 gene, located at 1p36, and the FKHR gene, located at 13q14 [12]. The FISH and molecular studies of the PAX3/FKHR fusion genes of the t(2;13) gave negative results. In the case of the t(1;13), FISH probes indicated that the breakpoints involved in this particular aberration did not affect the PAX7 or FKHR genes. This finding was subsequently confirmed by molecular studies of the PAX7 and FKHR genes. In summary, we have conducted a complete molecular cytogenetic characterization of the A673 cell line, originally described as a RMS origin, and studied the typical translocations in ET and RMS from cytogenetic and molecular points of view. The results indicate that this cell line should be classified as an Ewing-related tumor and used as a model for this kind of tumor. In addition, these results show the need to perform exhaustive analyses of the more commonly used cell lines to fully understand the role of their abnormalities and their consequences in tumor biology. Acknowledgments We would like to thank Blanca Fernández Martínez, Teresa López Jiménez, and MaCarmen Guijarro Martín for their technical assistance; and to Angel Pestaña for providing the cell line. Angel Martínez-Ramírez is a fellow of the Instituto de Salud Carlos III. Bárbara Meléndez and Sandra Ro-
141
dríguez-Perales are fellows of the Comunidad Autónoma de Madrid and Centro Nacional de Investigaciones Oncológicas, respectively.
References [1] Turc-Carel C, Philip I, Berger MP, Philip T, Lenoir GM. Chromosome study of Ewing’s sarcoma (ES) cell lines: consistency of a reciprocal translocation t(11;22)(q24;q12). Cancer Genet Cytogenet 1984; 12:1–19. [2] Pang E, Wong N, Lai PBS, To KF, Lau KWY, Johnson PJ. A comprehensive karyotypic analysis of a newly developed hepatocellular carcinoma cell line, HKCI-1, by spectral karyotyping and comparative genomic hybridization. Cancer Genet Cytogenet 2000;121:9–16. [3] Masramon L, Ribas M, Cifuentes P, Arribas R, García F, Egozcue J, Peinado MA, Miró R. Cytogenetic characterization of two colon cell lines by using conventional G-banding, comparative genomic hybridization, and whole chromosome painting. Cancer Genet Cytogenet 2000;121:17–21. [4] Odero MD, Zeleznick-Le NJ, Chinwalla V, Rowley JD. Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an HLL-AF9 translocation. Genes Chromosomes Cancer 2000;29(4):333–8. [5] Strefford JC, Foot NJ, Chaplin T, Neat MJ, Oliver RTD, Young BD, Jones LK. The characterization of the lymphoma cell line U937, using comparative genomic hybridisation and multiplex FISH. Cytogenet Cell Genet 2001;94:9–14. [6] Micci F, Teixeira MR, Heim S. Complete cytogenetic characterization of human breast cancer cell line MA11 combining G-banding, comparative genomic hybridization, multicolor fluorescence in situ hybridization, RxFISH, and chromosome-specific painting. Cancer Genet Cytogenet 2001;131:25–30. [7] Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 1973; 51:1417–23. [8] Stam K, Stewart AA, Qu GY, Iwata KK, Fenyo D, Chait BT, Marshak DR, Haley JD. Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673. Biochem J 1995;305:87–92. [9] Borgstrom P, Hillan KJ, Sriramarao P, Ferrara N. Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy. Cancer Res 1996; 56:4032–9. [10] Engbrecht BW, Menon C, Kachur AV, Hahn SM, Fraker DL. Photofrin-mediated photodynamic therapy induces vascular occlusion and apoptosis in a human sarcoma xenograft model. Cancer Res 1999;59: 4334–42. [11] Chen B, He L, Savell VH, Jenkins JJ, Parham DM. Inhibition of interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region. Cancer Res 2000;60:3290–8. [12] Archey WB, Sweet MP, Alig GC, Arrick BA. Methylation of CpGs as a determinant of transcriptional activation at alternative promoters for transforming growth factor-beta3. Cancer Res 1999;59:2292–6. [13] Aryee DNT, Sommergruber W, Muehlbacher K, Dochhorn-Dworniczak B, Zoubek A, Kovar H. Variability in gene expression patterns of Ewing tumor cell lines differing in EWS-FLI1 fusion type. Lab Invest 2000;80:1833–44. [14] Lessnick SL, Braun BS, Denny CT, May WA. Multiple domains mediate transformation by Ewing’s sarcoma EWS/FLI1 fusion gene. Oncogene 1995;10:423–31. [15] May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O, Zucman J, Thomas G, Denny CT. Ewing sarcoma 11;22
142
[16]
[17]
[18]
[19]
[20]
[21] [22]
A. Martınez-Ramırez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A 1993;90:5752–6. Anderson J, Gordon A, Pritchard-Jones K, Shipley J. Genes, chromosomes, and rhabdomyosarcoma. Genes Chromosomes Cancer 1999; 26:275–85. Davis RJ, Barr FG. Fusion genes resulting from alternative chromosomal translocations are overexpressed by gene-specific mechanisms in alveolar rhabdomyosarcoma. Proc Natl Acad Sci U S A 1997;94: 8047–57. Davis RJ, D’Cruz CM, Lovell MA, Biegel JA, Barr FG. Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res 1994;54:2869–72. Fredericks WJ, Galili N, Mukhopadhyay S, Rovera G, Bennicelli J, Barr FG, Rauscher FJ 3rd. The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol Cell Biol 1995;15: 1522–35. Frascella E, Lenzini E, Schafer BW, Brecevic L, Dorigo E, Toffolatti L, Nanni P, De Giovanni C, Rosolen A. Concomitant amplification and expression of PAX7-FKHR and MYCN in a human rhabdomyosarcoma cell line carrying a cryptic t(1;13)(p36;q14). Cancer Genet Cytogenet 2000;121:139–45. ISCN. An international system for human cytogenetic nomenclature. F Mitelman, editor. Basel: S. Karger, 1995. Kelly KM, Womer RB, Barr FG. Minimal disease detection in pa-
[23]
[24]
[25]
[26]
[27]
[28]
[29]
tients with alveolar rhabdomyosarcoma using a reverse transcriptasepolymerase chain reaction method. Cancer 1996;78:1320–7. Schlott T, Nagel H, Ruschenburg I, Reimer S, Droese M. Reverse transcriptase polymerase chain reaction for detecting Ewing’s sarcoma in archival fine needle aspiration biopsies. Acta Cytol 1997;41:795–801. Galili N, Davis RJ, Fredericks WJ, Mukhopadhyay S, Rauscher FJ 3rd, Emanuel BS, Rovera G, Barr FG. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet 1993;5:230–5. Romeo DS, Mizel SB. Partial purification of an immunosuppressive protein from a human tumor cell line and analysis of its relationship to transforming growth factor beta. Cell Immunol 1989;122:483–92. Fryling C, Dombalagian M, Burgess W, Hollander N, Shereider AB, Haimovich J. Purification and characterization of tumor inhibitory factor-2: its identity to interleukin 1. Cancer Res 1989;49:3333–7. Feinman R, Henriksen-DeStefano D, Tsujimoto M, Vilcek J. Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes. J Immunol 1987;138:635–40. Borgstrom P, Bourdon MA, Hillan KJ, Sriramarao P, Ferrara N. Neutralizing anti-vascular endothelial growth factor antibody completely inhibits angiogenesis and growth of human prostate carcinoma micro tumors in vivo. Prostate 1998;35:1–10. Gerber HP, Kowalski J, Sherman D, Eberhard DA, Ferrara N. Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor. Cancer Res 2000;60:6253–8.
Short communication
Characterization of the A673 cell line (Ewing tumor) by molecular cytogenetic techniques A. Martínez-Ramírez*, S. Rodríguez-Perales, B. Meléndez, B. Martínez-Delgado, M. Urioste, J.C. Cigudosa, J. Benítez Department of Human Genetics, Molecular Pathology Program, Spanish National Cancer Center (CNIO), Instituto de Salud Carlos III, Madrid, Spain Received 18 April 2002; received in revised form 27 June 2002; accepted 28 June 2002
Abstract
The A673 cell line was established from a patient with a primary rhabdomyosarcoma (RMS), which is referred to in the literature either as a Ewing tumor (ET) or as RMS. Although the two tumoral types are associated with specific and well-characterized translocations, no cytogenetic report on this cell line has been published. We characterized the A673 cell line using a combination of spectral karyotyping (SKY), fluorescence in situ hybridization (FISH), and reverse transcriptase polymerase chain reaction (RT-PCR), which revealed the presence of a complex karyotype and a translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes, both events being specific to ET. Neither cytogenetics nor molecular alterations specific to RMS were found. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Cell lines are useful tools in the field of carcinogenesis for studying biologic, biochemical, and molecular aspects of malignant transformation. Many cell lines have been characterized using molecular cytogenetic methods such as fluorescence in situ hybridization (FISH), multicolor FISH, and spectral karyotyping (SKY), and the results have contributed to a better knowledge of neoplasia [1–6]. The A673 cell line was established from a patient with a possible primary rhabdomyosarcoma (RMS) [7]. This cell line has been widely used as an important tool for improving our knowledge of tumor biology. These cells are known to produce several growth factors with oncogenic potential, as well as cell growth-inhibitory factors [8]. These cells can also induce tumors in nude mice [9,10] and contain some cancer-related genes with hypermethylated promoters [11,12]. The A673 cell line is referred to in the literature both as Ewing tumor (ET) or sarcoma (ES) and as RMS. Although both these tumor types have specific and well-characterized translocations, no cytogenetic report describing the complete karyotype has been published. Ewing tumor presents a t(11;22)(q24;q12) recurrent chromosomal translocation in about 85% of the cases. At the molecular level, this translocation shows that the EWS and FLI1 genes are
* Corresponding author. Fax: 34-91-224-69-23. E-mail address: [email protected] (A. Martínez-Ramírez).
fused [13–15]. The most frequent chromosome rearrangements identified in RMS are t(2;13)(q35;q14) and its variant t(1;13)(p36;q14), which give rise to two fusion genes, PAX3/FKHR and PAX7/FKHR, respectively [16–18]. At the molecular level, A673 displays fusion of EWS and FLI1 genes as a consequence of the t(11;22) translocation, but does not present the fusions of PAX3 and PAX7 with FKHR resulting from t(2;13) and t(1;13), respectively [19,20]. These discrepancies prompted us to characterize the A673 cell line by conventional and molecular cytogenetic techniques. The combination of SKY, FISH, and reverse transcriptase polymerase chain reaction (RT-PCR) has revealed only the presence of a complex translocation involving chromosomes 11 and 22 and the fusion of EWS and FLI1 genes. Both these events are specific to ET. 2. Materials and methods 2.1. Cell line The A673 cell line (American Type Culture Collection [ATCC], Manassas, VA, USA) and the alveolar rhabdomyosarcoma cell line RC2 (a gift from a Dr. Lollini, Cancer Institute, University of Bologna, Italy), with the t(1;13) [20], were maintained in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine and 13% fetal bovine serum. The alveolar rhabdomyosarcoma cell line Rh30 [19], which contains the t(2;13), was maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10% fe-
0165-4608/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S0165-4608(02)00 6 7 0 - 2
A. Martínez-Ramírez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142
tal bovine serum. All media and supplements for cell cultures were obtained from Gibco (Gaithersburg, MD, USA). 2.2. Cytogenetic studies 2.2.1. Conventional cytogenetics Cells of the cell line were exposed to colcemid (0.1 g/mL) for 1.5 hours at 37C and harvested routinely. Metaphase chromosomes were GTG-banded by a conventional trypsin– Giemsa technique and karyotyped according to the International System for Human Cytogenetic Nomenclature, 1995 revision [21]. 2.2.2. SKY study Slide preparation and hybridization, using commercial probes, were carried out according to the manufacturer’s instructions (Applied Spectral Imaging [ASI], Migdal Ha’Emek, Israel). Images were acquired using an ASI SD300 Spectracube mounted on a Zeiss Axioplan 2 microscope. 2.2.3. FISH study Chromosome spreads, prepared directly from the A673 cell line, were used for conventional analysis and were left overnight at room temperature. Slides were placed on a plate at 90C for 10 minutes, dehydrated through a series of ethanol washes, and denatured in the presence of a probe on a plate at 75C for 1 minute. Specific bacterial artificial chro-
139
mosome (BAC) (RPCI-11 Human BAC Clone Library, Children’s Hospital Oakland Research Institute [CHORI]) for the EWS, FLI1, PAX3, PAX7, and FKHR genes were labeled with different colors by nick translation (Vysis, London, UK). These BACs were used to detect the EWS/FLI1, PAX3/ FKHR, and PAX7/FKHR rearrangements. Cell images were captured using a charge-coupled device (CCD) camera (Photometrics SenSys camera, Tucson, AZ, USA) connected to a microcomputer running the Chromofluor image analysis system (Applied Imaging, Newcastle-upon-Tyne, UK).
2.3. Molecular studies After cDNA synthesis, PCR was performed to detect chimeric transcripts derived from the t(11;22), t(2;13), and t(1; 13). All samples were analyzed according to standardized primers, protocols, and criteria [18,22–24]. To verify the integrity of the isolated RNA and the correct synthesis of the cDNA, the ubiquitously expressed ABL gene was amplified in a separate PCR reaction. The PCR was performed in two steps: the first round was performed with the external primers and the second with reverse internal primers. Amplification was performed with a 9700 Perkin Elmer Thermocycler (Perkin Elmer, Germany). Twenty microliters of the final PCR products were analyzed on a 3% agarose gel (MetaPhor agarose; BMA, Rockland, ME, USA) and visualized by ethidium bromide staining.
Fig. 1. Representative complex karyotype from Giemsa-banding of the A673 cell line showing multiple rearrangements and two der(13):der(13)t(1;13) and der(13)t(11;13)t(11;22) (arrows) and der(22)t(11;22).
140
A. Martınez-Ramırez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142
3. Results 3.1. ET studies A conventional cytogenetic study was carried out on the A673 cell line. This revealed a complex karyotype with multiple rearrangements affecting chromosomes 3, 5, 8, 9, and 16. Additional unbalanced translocations were found [der(1)t(1;19), t(5;8), der(16)t(3;16), der(9)t(9;13), and der(11)t(11;13)] and loss of material was observed in chromosomes 3 and 4 (Fig. 1). Neither the t(11;22) nor its variants, characteristic of ET, were detected in the karyotype. By means of SKY analysis we established the karyotype of the A673 cell line (Fig. 2a) to be 4647,XX,der(1)t(1;9) (p36;q22),der(3)del(3)(p21)del(3)(q21),del(4)(q21q31), t(5;8)(q33;q21),der(9)t(9;13)(q22;q14),der(11)t(11;13) (p13;q14),der(13)t(1;13)(p36;q14),der(13)t(11;13)(q13;q14) t(11;22)(q24;q12),der(16)t(3;16)(q21?;q22),der(22)t(11;22) (q24;q12) [cp15]. There was a specific translocation of ET, although this was located on a derivative chromosome 13 with a complex translocation between chromosomes 13, 11, and 22: der (13)t(11;13)(q13;q14)t(11;22)(q24;q12) (Fig. 2a). The expected chromosome der(22) generated by the t(11;22) was also detected as a small chromosome 22.
The FISH study with EWS and FLI1 BAC probes yielded one yellow signal on the der(22) generated by the t(11;22) and the EWS/FLI1 fusion gene, one green signal on the der(13)t(13;11;22), and two red signals belonging to the normal chromosomes 11 (Fig. 2b). An RT-PCR specific for the amplification of the EWS/FLI1 fusion revealed a molecular rearrangement compatible with the translocation. 3.2. RMS studies With regard to RMS, the most consistent chromosome rearrangements are the t(2;13)(q35;q14) and its variant t(1;13)(p36;q14). We did not observe the t(2;13), but we did detect a t(1;13) with similar breakpoints on chromosome 13. FISH analysis using specific BAC of PAX3/FKHR and PAX7/FKHR fusion genes was negative (Fig. 2c and 2d), suggesting that other breakpoints are involved (Fig. 2d). Molecular analysis by RT-PCR confirmed the absence of the PAX3/FKHR and PAX7/FKHR fusion genes. These results are consistent with those we obtained by FISH.
4. Discussion Since its establishment, the A673 cell line, which was derived from a possible primary RMS [7], has been widely
Fig. 2. (a) SKY of the A673 cell line. Arrows point to the rearrangements of chromosomes 13 and 22. (b) FISH of a metaphase spread and interphase nucleus of the A673 cell line. Arrows point to one yellow fusion signal on the der(22), generated by the t(11;22), and one green signal on the der(13)t(13;11;22). The two red signals correspond to the normal chromosomes 11. (c and d) FISH analysis using specific BAC for PAX3, PAX7, and FKHR. Neither PAX3/FKHR nor PAX7/FKHR fusion genes were detected (panels c and d, respectively).
A. Martínez-Ramírez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142
used to extend our knowledge of tumor biology in RMS. This cell line is defined as one of RMS in the ATCC and has been used in different studies during the 1980s to obtain and purify tumor growth inhibitory factors [25,26] or to investigate the mechanisms of human peripheral blood monocytemediated cytotoxicity in tumor cells [27]. It is currently being used as an RMS to evaluate tumor growth inhibition by administration of the antihuman vascular endothelial growth factor (VEGF) [9,28,29]. A673 is also referred to in the literature as an ET cell line, however, and during the 1990s it was used to characterize growth-inhibitory activity and to extend our knowledge of the role of hypermethylation [8, 11,12,14]. As no cytogenetic or molecular report describing the A673 cell line had been previously published, we undertook the characterization of the cell line A673 using a combination of G-banding analysis and molecular cytogenetic methods. Giemsa-banding revealed a complex karyotype with multiple chromosomal rearrangements but without an apparent ET-specific t(11;22). Tumors of the Ewing sarcoma family are characterized by the t(11;22) or t(21;22) in about 85% and 15% of cases, respectively [14]. Our SKY analysis revealed a complex translocation among chromosomes 13, 11, and 22 and a der(22) chromosome that originated through the t(11;22). The t(11;22) was confirmed with FISH and molecular analysis using probes and primers specific for the detection of the EWS/FLI1 fusion gene. Furthermore, we observed with G-banding, and confirmed with SKY, a t(1;13) but no t(2;13). This t(1:13) is about five times less frequent than the t(2;13) in RMS and produces the fusion between the PAX7 gene, located at 1p36, and the FKHR gene, located at 13q14 [12]. The FISH and molecular studies of the PAX3/FKHR fusion genes of the t(2;13) gave negative results. In the case of the t(1;13), FISH probes indicated that the breakpoints involved in this particular aberration did not affect the PAX7 or FKHR genes. This finding was subsequently confirmed by molecular studies of the PAX7 and FKHR genes. In summary, we have conducted a complete molecular cytogenetic characterization of the A673 cell line, originally described as a RMS origin, and studied the typical translocations in ET and RMS from cytogenetic and molecular points of view. The results indicate that this cell line should be classified as an Ewing-related tumor and used as a model for this kind of tumor. In addition, these results show the need to perform exhaustive analyses of the more commonly used cell lines to fully understand the role of their abnormalities and their consequences in tumor biology. Acknowledgments We would like to thank Blanca Fernández Martínez, Teresa López Jiménez, and MaCarmen Guijarro Martín for their technical assistance; and to Angel Pestaña for providing the cell line. Angel Martínez-Ramírez is a fellow of the Instituto de Salud Carlos III. Bárbara Meléndez and Sandra Ro-
141
dríguez-Perales are fellows of the Comunidad Autónoma de Madrid and Centro Nacional de Investigaciones Oncológicas, respectively.
References [1] Turc-Carel C, Philip I, Berger MP, Philip T, Lenoir GM. Chromosome study of Ewing’s sarcoma (ES) cell lines: consistency of a reciprocal translocation t(11;22)(q24;q12). Cancer Genet Cytogenet 1984; 12:1–19. [2] Pang E, Wong N, Lai PBS, To KF, Lau KWY, Johnson PJ. A comprehensive karyotypic analysis of a newly developed hepatocellular carcinoma cell line, HKCI-1, by spectral karyotyping and comparative genomic hybridization. Cancer Genet Cytogenet 2000;121:9–16. [3] Masramon L, Ribas M, Cifuentes P, Arribas R, García F, Egozcue J, Peinado MA, Miró R. Cytogenetic characterization of two colon cell lines by using conventional G-banding, comparative genomic hybridization, and whole chromosome painting. Cancer Genet Cytogenet 2000;121:17–21. [4] Odero MD, Zeleznick-Le NJ, Chinwalla V, Rowley JD. Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an HLL-AF9 translocation. Genes Chromosomes Cancer 2000;29(4):333–8. [5] Strefford JC, Foot NJ, Chaplin T, Neat MJ, Oliver RTD, Young BD, Jones LK. The characterization of the lymphoma cell line U937, using comparative genomic hybridisation and multiplex FISH. Cytogenet Cell Genet 2001;94:9–14. [6] Micci F, Teixeira MR, Heim S. Complete cytogenetic characterization of human breast cancer cell line MA11 combining G-banding, comparative genomic hybridization, multicolor fluorescence in situ hybridization, RxFISH, and chromosome-specific painting. Cancer Genet Cytogenet 2001;131:25–30. [7] Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 1973; 51:1417–23. [8] Stam K, Stewart AA, Qu GY, Iwata KK, Fenyo D, Chait BT, Marshak DR, Haley JD. Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673. Biochem J 1995;305:87–92. [9] Borgstrom P, Hillan KJ, Sriramarao P, Ferrara N. Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy. Cancer Res 1996; 56:4032–9. [10] Engbrecht BW, Menon C, Kachur AV, Hahn SM, Fraker DL. Photofrin-mediated photodynamic therapy induces vascular occlusion and apoptosis in a human sarcoma xenograft model. Cancer Res 1999;59: 4334–42. [11] Chen B, He L, Savell VH, Jenkins JJ, Parham DM. Inhibition of interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region. Cancer Res 2000;60:3290–8. [12] Archey WB, Sweet MP, Alig GC, Arrick BA. Methylation of CpGs as a determinant of transcriptional activation at alternative promoters for transforming growth factor-beta3. Cancer Res 1999;59:2292–6. [13] Aryee DNT, Sommergruber W, Muehlbacher K, Dochhorn-Dworniczak B, Zoubek A, Kovar H. Variability in gene expression patterns of Ewing tumor cell lines differing in EWS-FLI1 fusion type. Lab Invest 2000;80:1833–44. [14] Lessnick SL, Braun BS, Denny CT, May WA. Multiple domains mediate transformation by Ewing’s sarcoma EWS/FLI1 fusion gene. Oncogene 1995;10:423–31. [15] May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O, Zucman J, Thomas G, Denny CT. Ewing sarcoma 11;22
142
[16]
[17]
[18]
[19]
[20]
[21] [22]
A. Martınez-Ramırez et al. / Cancer Genetics and Cytogenetics 141 (2003) 138–142 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A 1993;90:5752–6. Anderson J, Gordon A, Pritchard-Jones K, Shipley J. Genes, chromosomes, and rhabdomyosarcoma. Genes Chromosomes Cancer 1999; 26:275–85. Davis RJ, Barr FG. Fusion genes resulting from alternative chromosomal translocations are overexpressed by gene-specific mechanisms in alveolar rhabdomyosarcoma. Proc Natl Acad Sci U S A 1997;94: 8047–57. Davis RJ, D’Cruz CM, Lovell MA, Biegel JA, Barr FG. Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. Cancer Res 1994;54:2869–72. Fredericks WJ, Galili N, Mukhopadhyay S, Rovera G, Bennicelli J, Barr FG, Rauscher FJ 3rd. The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3. Mol Cell Biol 1995;15: 1522–35. Frascella E, Lenzini E, Schafer BW, Brecevic L, Dorigo E, Toffolatti L, Nanni P, De Giovanni C, Rosolen A. Concomitant amplification and expression of PAX7-FKHR and MYCN in a human rhabdomyosarcoma cell line carrying a cryptic t(1;13)(p36;q14). Cancer Genet Cytogenet 2000;121:139–45. ISCN. An international system for human cytogenetic nomenclature. F Mitelman, editor. Basel: S. Karger, 1995. Kelly KM, Womer RB, Barr FG. Minimal disease detection in pa-
[23]
[24]
[25]
[26]
[27]
[28]
[29]
tients with alveolar rhabdomyosarcoma using a reverse transcriptasepolymerase chain reaction method. Cancer 1996;78:1320–7. Schlott T, Nagel H, Ruschenburg I, Reimer S, Droese M. Reverse transcriptase polymerase chain reaction for detecting Ewing’s sarcoma in archival fine needle aspiration biopsies. Acta Cytol 1997;41:795–801. Galili N, Davis RJ, Fredericks WJ, Mukhopadhyay S, Rauscher FJ 3rd, Emanuel BS, Rovera G, Barr FG. Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma. Nat Genet 1993;5:230–5. Romeo DS, Mizel SB. Partial purification of an immunosuppressive protein from a human tumor cell line and analysis of its relationship to transforming growth factor beta. Cell Immunol 1989;122:483–92. Fryling C, Dombalagian M, Burgess W, Hollander N, Shereider AB, Haimovich J. Purification and characterization of tumor inhibitory factor-2: its identity to interleukin 1. Cancer Res 1989;49:3333–7. Feinman R, Henriksen-DeStefano D, Tsujimoto M, Vilcek J. Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes. J Immunol 1987;138:635–40. Borgstrom P, Bourdon MA, Hillan KJ, Sriramarao P, Ferrara N. Neutralizing anti-vascular endothelial growth factor antibody completely inhibits angiogenesis and growth of human prostate carcinoma micro tumors in vivo. Prostate 1998;35:1–10. Gerber HP, Kowalski J, Sherman D, Eberhard DA, Ferrara N. Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor. Cancer Res 2000;60:6253–8.