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Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization
Cancer Genetics and Cytogenetics 170 (2006) 102e107
Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization Yu-Peng Wua,b, Yi-Ling Yanga, Guang-Zhi Yanga, Xue-Ying Wangb, Man-Li Luoa, Yu Zhanga, Yan-Bin Fenga, Xin Xua, Ya-Ling Hana, Yan Caia, Qi-Min Zhana, Min Wua, Jin-Tang Dongc, Ming-Rong Wanga,* a
National Laboratory of Molecular Oncology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences and Peking Union Medical College, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China b Biological Science & Technology College, Shenyang Agricultural University, 120 Dangling Road, Shenyang 110161, China c Winship Cancer Institute, Emory University School of Medicine, 1365-C Clifton Road, Atlanta, GA 30322 Received 14 February 2006; received in revised form 3 May 2006; accepted 15 May 2006
Abstract
Analysis of chromosomal changes in esophageal squamous cell carcinoma (ESCC) can illuminate the molecular mechanisms underlying the development and progression of this cancer, which is among the 10 most common malignant tumors. Cell lines are better suited than surgical samples for chromosome analysis in this cancer. This study used multicolor fluorescence in situ hybridization (M-FISH) to characterize the molecular cytogenetics of ESCC in cell line KYSE180. Two pools of 12-color whole-chromosome painting probes were designed, and two rounds of FISH were performed on the same metaphase spreads. Loss of DNA copy number was observed at 4p, 5q, 6q, 9, 10p, 12p, 13, 14p, 15p, 18p, 18q, 20, 22, and Y. Chromosomal gains and translocations occurred at the entire or part of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q, 11, 12q, 14q, 16, 17q, 19, and Xp. Seven derivative chromosomes (5, 8, 12, 14, 14, 14, and 17) presented complex translocations, each involving three or four chromosomes. No chromosomes 9, 13, or Y were detected. These results add significant information to the existing karyotype description of KYSE180 and provide detailed cytogenetic background data for appropriate use of the cell line. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Esophageal squamous cell carcinoma (ESCC) is among the 10 most common malignant tumors. Analysis of chromosomal changes will contribute to the understanding of the molecular mechanisms underlying the development and progression of esophageal cancer; however, it is very difficult to perform chromosome analysis on operative samples of esophageal squamous cell carcinomas. An important experimental material for cancer research, cell lines have been used for studying the function of genes associated with human cancer, the mechanisms of carcinogenesis and tumor evolution, targets for cancer gene therapy, and the screening of cancer drugs. Before using a given cell line, one should know the genetic background of the cell
line; otherwise, inappropriate choice of cell lines can lead to misleading results or wrong conclusion. KYSE180 is a cell line of esophageal squamous cell carcinoma established by Shimada et al. [1]. The cell line has been used in studies on the molecular biology of esophageal cancer by investigators worldwide [2e4], but its cytogenetic background still remains unclear. Our primary objective was to contribute to a fuller characterization of cell line KYSE180 by analyzing it with multicolor fluorescence in situ hybridization (M-FISH). Multiple novel chromosome aberrations were identified, including gains, deletions, and translocations.
2. Materials and methods 2.1. Cell line and metaphase preparation
* Corresponding author. Tel.: þ86-10-87788425; fax: þ86-1087778651. E-mail address: [email protected] (M.-R. Wang). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.05.006
The esophageal squamous cell carcinoma KYSE180 cell line was kindly provided by Yutaka Shimada (Kyoto University, Kyoto, Japan). The cell line was derived from
Y.-P. Wu et al. / Cancer Genetics and Cytogenetics 170 (2006) 102e107
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a primary esophageal tumor developed in a male patient [1]. The cells were maintained in M199 medium with 15% fetal calf serum and cultured at 37 C, 5% CO2. Metaphase spreads for FISH experiments were prepared according to standard protocols. 2.2. Probe and labeling Whole-chromosome painting (WCP) probes were a gift from Professor Hong Chen (Basic Medical Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China). The probes were labeled using degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) with diethylaminocoumarin-5-dUTP (DEAC) (PerkinElmer, Norwalk, CT), fluorescein isothiocyanate-12-dUTP (FITC) (PerkinElmer), Cy3-dUTP (Amersham Biosciences, Piscataway, NJ), and Cy5-dUTP (Amersham Biosciences) fluorochromes.
Fig. 1. Representative image of the first-round 12-color FISH with the probes of pool 1 on KYSE180 cell line.
2.3. First-round FISH
2.4. Second-round FISH on the same metaphases
The slides with metaphase chromosomes were pretreated with RNase (100 mg/mL in 2 saline sodium citrate [SSC]) and pepsin (50 mg/mL in 0.01 mol/L HCl). Denaturation was done in 70% formamidee2 SSC at 72 C for 3 min, quickly cooled with two rinses of cold 2 SSC at 4 C, dehydrated in an ethanol series (75, 85, and 100%), and air dried. The labeled probes were precipitated with ethanolesodium acetic acid, added to a hybridization mixture (50% formamide, 10% dextran sulfate, 2 SSC), denatured at 75 C for 8 min, and quick-chilled on ice for 3 min. Hybridization was performed in a humid chamber at 37 C for 48 h. Posthybridization washes were done in 50% formamidee2 SSC for 15 min at 43 C and twice for 3 min each in 2 SSC at room temperature, dehydrated in an ethanol series (75, 85, and 100%), and air dried. The slides were counterstained with 20 mL 40 ,6-diamidino-2phenylindole (DAPI) (1 mg/mL, 2% 1,4-diazabicyclo[2.2.2]octane [DABCO]) and covered with coverslips.
After digital fluorescence image acquisition (see section 2.5), coverslips on the slides were removed by dipping in 100% ethanol for 30 min, then were washed twice in 100% ethanol for 3 min each time, and air dried. The previous M-FISH signals were eliminated by denaturing the slides in 70% formamidee2 SSC at 72 C for 3 min. The slides were then cooled quickly with two rinses of cold 2 SSC at 4 C, dehydrated in an ethanol series (75, 85, and 100%) and air dried. Second-round hybridization was performed as in the first-round FISH, except with different probes. 2.5. Digital fluorescence image acquisition Gray-level images were captured with a cooled chargedcoupled device (CCD) camera (Princeton Instruments,
Table 1 Two pools of probes labeled by distinct fluorochromes Pool 1 Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr
3 4 6 7 9 11 13 15 17 19 21 X
Pool 2 DEAC Cy3 FITC Cy5 Cy3:DEAC Cy3: FITC DEAC: FITC Cy3:Cy5 Cy5: FITC Cy3:Cy5:FITC Cy3:Cy5:DEAC Cy5:DEAC
Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr
1 2 5 8 10 12 14 16 18 20 22 Y
Cy3 Cy5 Cy3:Cy5 DEAC Cy3:Cy5:DEAC Cy3:FITC FITC Cy5:FITC Cy3:Cy5:FITC Cy5:DEAC DEAC:FITC Cy3:DEAC
Abbreviations: Chr, chromosome; DEAC, diethylaminocoumarin-5dUTP; FITC, fluorescein isothiocyanate-12-dUTP.
Fig. 2. Results of the second-round 12-color FISH with the probes of pool 2 on the same metaphase as in Fig. 1.
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Fig. 3. Reverse DAPI banding image of the same metaphase as in Fig. 1.
Trenton, NJ) equipped with a Zeiss Opton fluorescence microscope, a computer-controlled filter wheel with excitation filters, and a manually controlled slipper block with multiple emission filters for visualization of DAPI, DEAC, FITC, Cy3, and Cy5 fluorochromes. The images were analyzed using the MetaMorph Imaging System (Universal Imaging Corporation, Downingtown, PA).
same metaphase spreads. The karyotype was analyzed through a combination of reverse DAPI banding and MFISH (Figs. 1e3). The chromosome count in KYSE180 ranged from 51 to 55, with a modal number of 54. Except for chromosome 21, all the other chromosomes presented structural aberrations or numerical abnormalities. There were three normal chromosomes 11, two normal chromosomes each for 1, 2, 8, 16, and 19, and only one normal chromosome each for 4, 5, 7, 10, 12, 17, and 18. Monosomy 20 and 22 were observed without any structural aberrations involving these two chromosomes. No chromosomes 9, 13, or Y were detected. Other DNA copy number losses were seen at 4p, 5q, 6q, 10p, 12p, 14p, 15p, 18p, and 18q. Chromosomal gains and translocations occurred at the entire or part of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q, 11, 12q, 14q, 16, 17q, 19, and Xp. Chromosome 3 displayed highlevel amplification of multiple bands. There were four derivative chromosomes involving intact chromosomes 14, 15, 17, and 19. Complex translocations were observed in seven derivative chromosomes (5, 8, 12, 14, 14, 14, and 17), each involving three or four chromosomes. The chromosome aberrations are summarized in Tables 2 and 3 and in Fig. 4.
4. Discussion 3. Results Two pools of 12-color WCP probes (Table 1) were designed, and two rounds of FISH were performed on the
Chromosome aberrations are distinctive features of human malignant tumors. Most solid tumors present complex cytogenetic abnormalities. Multiple chromosome aberrations have been reported for ESCC, including numerical
Table 2 Chromosome aberrations in the KYSE180 cell line Chr 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 Y
Aberrations þder(1)(6pter/6p10::1q10/1qter), þder(1)(1q31/1q10::3q21/3q26) dup(3)(q24/q26), der(3)(:p21/q27:), þder(3)(:p21/q27:), þder(3)(3qter/3q10::5q14/5q23), þder(3)(6q24/6q16::3p12/3qter), þder(3)(3pter/3q10::6q11/6qter) der(4)(4p15/4qter::8q23) der(5)(5pter/5q23::14q21/14q31::3q28/3q24), þder(5)(5pter/5p10::8q24/8q23), þder(5)(5pter/5p10::6p12/6p22) 6, der(6)(6pter/6q14::3q13/3q22) del(7)(pter/q32:), þder(7)(pter/q32:), þder(7)(10q21/10q10::7q10/7qter) þder(8)(8p12/8q11::16p13/16p12::16q12/16q24::2p24/2p22) 9, 9 der(10)(14qter/14q10::10q10/10qter) þ11 del(12)(:q10/qter) þder(12)(4p15::12p12/12q12::3q21/3q24::2p12/2p24) 13, 13 der(14)(5p14::14q10/14qter::1p11/1p21), der(14)(3qter/3q13::14pter/14qter::5p14/5p11), þder(14)(14qter/14q10::Xp10/ Xp22::3q13/3q26) der(15)(14q12/14q21::15pter/15qter), der(15)(18q22/18q10::15q10/15qter) þder(16)(:p12/q22:) der(17)(17pter/17qter::3p14/3p24), þder(17)(3pter/3p14::11p14/11p11::17q10/17q24::8p22/8p12) 18 þder(19)(10p12/10p11::19pter/19qter) 20 22 Y
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Table 3 Summary of chromosome gains and losses in KYSE180 cell line Chr
Normal
Gain
1
2
2
2
3
0
1p11~p21 1q10~qter 1q10~q31 2p12~p24 2p22~p24 3p12~q10 3q24~q26 3p24~p14 3p21~q27 3pter~p14 3p21~q27 3q13~q26 3q13~q22 3q21~q24 3q21~q26 3q24~q28 3q10~qter 3q13~qter 4p15 5p14 5p14~p11 5pter~p10 5pter~p10 5q14~q23 6p22~p12
4 5
6
7
1 1
0
1
Loss
Chr
Normal
Gain
8
2
8p12~q11 8p22~p12 8q23 8q23~q24
9 10 11
0 1 2
12 13 14
1 0 0
15 16
0 2
4pter~p16 5q31~qter
17 18
1 1
6q10 6q15 6q25~qter
19 20 21 22 X Y
2 1 2 1 1 0
10q21~q10 11 11p14~p11 12q10~q12 14q12~q21 14q21~q31 14q10~qter 14q10~qter
Loss
9 10p13~pter
12p13 13 14pter~p10
15pter~p10 16p12~q22 16p13~p12 16q12~q24 17q10~q24 18pter~p10 18q23~qter 19pter~qter 20 22 Xp22~p10 Y
7pter~q32 7q10~q32
and structural aberrations [5e7]; however, accurate karyotype analysis of ESCC is frequently hampered by the small number of recognizable metaphases and low quality of chromosome morphology. M-FISH allows simultaneous analysis of multiple chromosomes in a single experiment. Diverse cytogenetic abnormalities occurring in the whole genome can be observed by M-FISH, such as chromosomal gains, deletions, inversions, insertions, and translocations; the technique is also useful for identifying the origin of structural aberrations and marker chromosomes [8,9]. M-FISH is therefore a rapid and effective technique for chromosome characterization of solid tumors, especially for the karyotyping analysis of cancer cell lines. KYSE180, a commercial cell line of ESCC, has been widely used in cancer research. In the international cell line bank, the original cytogenetic description of the KYSE180 cell line was recorded as hyperdiploid karyotype: 50~57,XX,þ1,þ1,þ3,þ7,þ7,þ8,14,þ16,17,17,18,18,þ5~8 mar,del(X)(q23),add(1)(q32),der(1;6)(q10; p10),der(1;13)(q10;q10),add(3)(p26),add(5)(q25),add(6)(p11), del(6)(q23),add(7)(p11),del(7)(p11),add(7)(q35),i(7q),del(8) (p22),add(10)(p11),add(11)(q11),del(11)(q11q14),add(13) (p11),add(13)(q33),add(14)(p11),add(15)(p11),add(21)(p11) (http://www.dsmz.de/human_and_animal_cell_lines/info. php?dsmz_nr5379&term5KYSE-180&highlight5). It is
obvious that most of these aberrant chromosomes have not been well identified. The present study provides detailed karyotype information from two-round multicolor FISH. Our results give significant additional information on chromosome aberrations in KYSE180 cells. DNA gains were found at the part or the entire chromosome of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q 11, 12q, 14q, 16, 17q, 19, and Xp. DNA copy number losses were observed at 4p, 5q, 6q, 10p, 12p, 14p, 15p, 18p, 18q, 20, and 22. No chromosomes 9, 13, or Y were detected. Many of the findings are consistent with those seen under comparative genomic hybridization (CGH) in ESCC in populations of East Asia [7,10,11]. The chromosomal regions 1q, 8q, 11q, and 17q with DNA gains in KYSE180 cells contain protooncogenes and the genes promoting cell growth or suppressing cell apoptosis, such as CENPF (1q32), MYC (8q24), CCND1 (11q13), BIRC2 (alias cIAP) (11q21~q23), and ERBB2 (17q12~q21) and its aliases HER-2 and neu (17q21). Some tumor suppressor genes and genes regulating cell proliferation locate in the chromosomes or bands deleted in KYSE180; these include IRF1 (5q31.1), CDKN2B (alias p15) (9p21), CDKN2A (alias p16) (9p21), RB1 (13q14), BRCA2 (13q12.3), DCC (18q23), and SMAD4 (18q21.1). Alteration of these genes might be implicated in the
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passage. As we have mentioned, however, many chromosome aberrations in the KYSE180 cell line were consistent with those reported in primary squamous cell carcinomas of the esophagus. The cell line is therefore able to simulate, to a degree, the biological behavior of some esophageal squamous cell carcinomas. In summary, our results provide detailed data on chromosome aberrations in the cell line KYSE180, data that can contribute to appropriate applications for this esophageal cancer cell line.
Acknowledgments This work was supported by State Key Basic Research Grant of China (2004CB518705), National Natural Science Foundation (30470969, 30328024, 30400207), and Specialized Research Fund of Beijing Municipal Science & Technology Commission (D0905001040331). Fig. 4. Karyogram of the KYSE180 cell line. The top panel for each chromosome gives results of first-round FISH; the middle panel, second-round FISH; and the bottom panel, reverse DAPI banding image of the same chromosome.
development of ESCC or in the malignant behavior of KYSE180 cells. Their copy number changes can be further assessed by array CGH [12], digital karyotyping [13], or molecular biology techniques. The breakpoints of some chromosome translocations occurred around centromere regions 1q10, 3q10, 5p10, 6p10, 7q10, 10q10, 14q10, 15q10, 17q10, 18q10, and Xp10. Among them, breakage at 7q10, 14q10, and 15q10 were also observed in ESCC from Taiwan [7]. However, the translocation breakpoints on 2p, 3q, 6p, 6q, 8q, 14q, 16p, 16q, and 17q are mostly located in noncentromere regions, including 2p24~p12, 2p24~p22, 3q13~q22, 3q21~q24, 3q21~q26, 3q24~q28, 6pter~q14, 6p12~p22, 6q11~qter, 6q16~q24, 8p12~q11, 8q23~q24, 8q23, 14q12~q21, 14q21~q31, 16p12~q22, 16p13~p12, 16q12~q24, and 17q10~q24. These chromosomal regions harbor protooncogenes and genes promoting cell proliferation or growth, for example, MYCN (2p23~p24), PIK3CA (3q26), VEGF (6p12), FYN (6q21), MYC (8q24), TGFB3 (14q24), FOS (14q24.3), and ERBB2 (17q12~q21). The long arm of chromosome 3 (3q) was the region with the highest gain frequency in KYSE180. Gains or amplifications of 3q were often observed in other solid tumors, such as ovarian cancer or squamous cell carcinomas of lung, head, and neck [14,15], suggesting that 3q encompasses one or more protooncogenes associated with multiple types of solid tumors. We note that, due to the differences between the culture condition and the microenvironment in which primary tumor growth in vivo, some greater or lesser acquired molecular changes probably occurred during in vitro culture and
References [1] Shimada Y, Imamura M, Wagata T, Yamaguchi N, Tobe T. Characterization of 21 newly established esophageal cancer cell lines. Cancer 1992;69:277e84 [Erratum in: Cancer 1992;70:206]. [2] Tong T, Zhong Y, Kong J, Dong L, Song Y, Fu M, Liu Z, Wang M, Guo L, Lu S, Wu M, Zhan Q. Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma. Clin Cancer Res 2004;10:7304e10 [Erratum in: Clin Cancer Res. 2005;11:4635]. [3] Zhou C, Liu S, Zhou X, Xue L, Quan L, Lu N, Zhang G, Bai J, Wang Y, Liu Z, Zhan Q, Zhu H, Xu N. Overexpression of human pituitary tumor transforming gene (hPTTG), is regulated by b-catenin/TCF pathway in human esophageal squamous cell carcinoma. Int J Cancer 2005;113:891e8. [4] Ma X, Sheng T, Zhang Y, Zhang X, He J, Huang S, Chen K, Sultz J, Adegboyega PA, Zhang H, Xie J. Hedgehog signaling is activated in subsets of esophageal cancers. Int J Cancer 2006;118:139e48. [5] Xiao S, Feng XL, Geng JS, Yan FC, Liu QZ, Li P. Cytogenetic studies of five primary esophageal cancers. Cancer Genet Cytogenet 1991;55:197e205. [6] Rosenblum-Vos LS, Meltzer SJ, Leana-Cox J, Schwartz S. Cytogenetic studies of primary cultures of esophageal squamous cell carcinoma. Cancer Genet Cytogenet 1993;70:127e31. [7] Yen CC, Chen YJ, Lu KH, Hsia JY, Chen JT, Hu CP, Chen PM, Liu JH, Chiou TJ, Wang WS, Yang MH, Chao TC, Lin CH. Genotypic analysis of esophageal squamous cell carcinoma by molecular cytogenetics and real-time quantitative polymerase chain reaction. Int J Oncol 2003;23:871e81. [8] Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996;12: 368e75. [9] Schro¨ck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494e7. [10] Yen CC, Chen YJ, Chen JT, Hsia JY, Chen PM, Liu JH, Fan FS, Chiou TJ, Wang WS, Lin CH. Comparative genomic hybridization of esophageal squamous cell carcinoma: correlations between chromosomal aberrations and disease progression/prognosis. Cancer 2001;92:2769e77. [11] Obara K, Ghazizadeh M, Shimizu H, Arai R, Tenjin T, Suzuki S, Moriyama Y, Kawanami O. Comparative genomic hybridization
Y.-P. Wu et al. / Cancer Genetics and Cytogenetics 170 (2006) 102e107 study of genetic changes associated with vindesine resistance in esophageal carcinoma. Int J Oncol 2002;20:255e60. [12] Pinkel D, Albertson DG. Array comparative genomic hybridization and its applications in cancer. Nat Genet 2005;37(Suppl): S11e7. [13] Wang TL, Maierhofer C, Speicher MR, Lengauer C, Vogelstein B, Kinzler KW, Velculescu VE. Digital karyotyping. Proc Natl Acad Sci U S A 2002;99:16156e61.
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[14] Sonoda G, Palazzo J, du Manoir S, Godwin AK, Feder M, Yakushiji M, Testa JR. Comparative genomic hybridization detects frequent overrepresentation of chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas. Genes Chromosomes Cancer 1997;20:320e8. [15] Chen YK, Lin LM. Placental glutathione S-transferase isoenzyme expression in polycyclic aromatic hydrocarbon-induced hamster buccal pouch mucosa. Oral Oncol 1998;34:180e5.
Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization Yu-Peng Wua,b, Yi-Ling Yanga, Guang-Zhi Yanga, Xue-Ying Wangb, Man-Li Luoa, Yu Zhanga, Yan-Bin Fenga, Xin Xua, Ya-Ling Hana, Yan Caia, Qi-Min Zhana, Min Wua, Jin-Tang Dongc, Ming-Rong Wanga,* a
National Laboratory of Molecular Oncology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences and Peking Union Medical College, 17 Panjiayuan Nanli, Chaoyang District, Beijing 100021, China b Biological Science & Technology College, Shenyang Agricultural University, 120 Dangling Road, Shenyang 110161, China c Winship Cancer Institute, Emory University School of Medicine, 1365-C Clifton Road, Atlanta, GA 30322 Received 14 February 2006; received in revised form 3 May 2006; accepted 15 May 2006
Abstract
Analysis of chromosomal changes in esophageal squamous cell carcinoma (ESCC) can illuminate the molecular mechanisms underlying the development and progression of this cancer, which is among the 10 most common malignant tumors. Cell lines are better suited than surgical samples for chromosome analysis in this cancer. This study used multicolor fluorescence in situ hybridization (M-FISH) to characterize the molecular cytogenetics of ESCC in cell line KYSE180. Two pools of 12-color whole-chromosome painting probes were designed, and two rounds of FISH were performed on the same metaphase spreads. Loss of DNA copy number was observed at 4p, 5q, 6q, 9, 10p, 12p, 13, 14p, 15p, 18p, 18q, 20, 22, and Y. Chromosomal gains and translocations occurred at the entire or part of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q, 11, 12q, 14q, 16, 17q, 19, and Xp. Seven derivative chromosomes (5, 8, 12, 14, 14, 14, and 17) presented complex translocations, each involving three or four chromosomes. No chromosomes 9, 13, or Y were detected. These results add significant information to the existing karyotype description of KYSE180 and provide detailed cytogenetic background data for appropriate use of the cell line. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Esophageal squamous cell carcinoma (ESCC) is among the 10 most common malignant tumors. Analysis of chromosomal changes will contribute to the understanding of the molecular mechanisms underlying the development and progression of esophageal cancer; however, it is very difficult to perform chromosome analysis on operative samples of esophageal squamous cell carcinomas. An important experimental material for cancer research, cell lines have been used for studying the function of genes associated with human cancer, the mechanisms of carcinogenesis and tumor evolution, targets for cancer gene therapy, and the screening of cancer drugs. Before using a given cell line, one should know the genetic background of the cell
line; otherwise, inappropriate choice of cell lines can lead to misleading results or wrong conclusion. KYSE180 is a cell line of esophageal squamous cell carcinoma established by Shimada et al. [1]. The cell line has been used in studies on the molecular biology of esophageal cancer by investigators worldwide [2e4], but its cytogenetic background still remains unclear. Our primary objective was to contribute to a fuller characterization of cell line KYSE180 by analyzing it with multicolor fluorescence in situ hybridization (M-FISH). Multiple novel chromosome aberrations were identified, including gains, deletions, and translocations.
2. Materials and methods 2.1. Cell line and metaphase preparation
* Corresponding author. Tel.: þ86-10-87788425; fax: þ86-1087778651. E-mail address: [email protected] (M.-R. Wang). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.05.006
The esophageal squamous cell carcinoma KYSE180 cell line was kindly provided by Yutaka Shimada (Kyoto University, Kyoto, Japan). The cell line was derived from
Y.-P. Wu et al. / Cancer Genetics and Cytogenetics 170 (2006) 102e107
103
a primary esophageal tumor developed in a male patient [1]. The cells were maintained in M199 medium with 15% fetal calf serum and cultured at 37 C, 5% CO2. Metaphase spreads for FISH experiments were prepared according to standard protocols. 2.2. Probe and labeling Whole-chromosome painting (WCP) probes were a gift from Professor Hong Chen (Basic Medical Institute, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China). The probes were labeled using degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) with diethylaminocoumarin-5-dUTP (DEAC) (PerkinElmer, Norwalk, CT), fluorescein isothiocyanate-12-dUTP (FITC) (PerkinElmer), Cy3-dUTP (Amersham Biosciences, Piscataway, NJ), and Cy5-dUTP (Amersham Biosciences) fluorochromes.
Fig. 1. Representative image of the first-round 12-color FISH with the probes of pool 1 on KYSE180 cell line.
2.3. First-round FISH
2.4. Second-round FISH on the same metaphases
The slides with metaphase chromosomes were pretreated with RNase (100 mg/mL in 2 saline sodium citrate [SSC]) and pepsin (50 mg/mL in 0.01 mol/L HCl). Denaturation was done in 70% formamidee2 SSC at 72 C for 3 min, quickly cooled with two rinses of cold 2 SSC at 4 C, dehydrated in an ethanol series (75, 85, and 100%), and air dried. The labeled probes were precipitated with ethanolesodium acetic acid, added to a hybridization mixture (50% formamide, 10% dextran sulfate, 2 SSC), denatured at 75 C for 8 min, and quick-chilled on ice for 3 min. Hybridization was performed in a humid chamber at 37 C for 48 h. Posthybridization washes were done in 50% formamidee2 SSC for 15 min at 43 C and twice for 3 min each in 2 SSC at room temperature, dehydrated in an ethanol series (75, 85, and 100%), and air dried. The slides were counterstained with 20 mL 40 ,6-diamidino-2phenylindole (DAPI) (1 mg/mL, 2% 1,4-diazabicyclo[2.2.2]octane [DABCO]) and covered with coverslips.
After digital fluorescence image acquisition (see section 2.5), coverslips on the slides were removed by dipping in 100% ethanol for 30 min, then were washed twice in 100% ethanol for 3 min each time, and air dried. The previous M-FISH signals were eliminated by denaturing the slides in 70% formamidee2 SSC at 72 C for 3 min. The slides were then cooled quickly with two rinses of cold 2 SSC at 4 C, dehydrated in an ethanol series (75, 85, and 100%) and air dried. Second-round hybridization was performed as in the first-round FISH, except with different probes. 2.5. Digital fluorescence image acquisition Gray-level images were captured with a cooled chargedcoupled device (CCD) camera (Princeton Instruments,
Table 1 Two pools of probes labeled by distinct fluorochromes Pool 1 Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr
3 4 6 7 9 11 13 15 17 19 21 X
Pool 2 DEAC Cy3 FITC Cy5 Cy3:DEAC Cy3: FITC DEAC: FITC Cy3:Cy5 Cy5: FITC Cy3:Cy5:FITC Cy3:Cy5:DEAC Cy5:DEAC
Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr
1 2 5 8 10 12 14 16 18 20 22 Y
Cy3 Cy5 Cy3:Cy5 DEAC Cy3:Cy5:DEAC Cy3:FITC FITC Cy5:FITC Cy3:Cy5:FITC Cy5:DEAC DEAC:FITC Cy3:DEAC
Abbreviations: Chr, chromosome; DEAC, diethylaminocoumarin-5dUTP; FITC, fluorescein isothiocyanate-12-dUTP.
Fig. 2. Results of the second-round 12-color FISH with the probes of pool 2 on the same metaphase as in Fig. 1.
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Fig. 3. Reverse DAPI banding image of the same metaphase as in Fig. 1.
Trenton, NJ) equipped with a Zeiss Opton fluorescence microscope, a computer-controlled filter wheel with excitation filters, and a manually controlled slipper block with multiple emission filters for visualization of DAPI, DEAC, FITC, Cy3, and Cy5 fluorochromes. The images were analyzed using the MetaMorph Imaging System (Universal Imaging Corporation, Downingtown, PA).
same metaphase spreads. The karyotype was analyzed through a combination of reverse DAPI banding and MFISH (Figs. 1e3). The chromosome count in KYSE180 ranged from 51 to 55, with a modal number of 54. Except for chromosome 21, all the other chromosomes presented structural aberrations or numerical abnormalities. There were three normal chromosomes 11, two normal chromosomes each for 1, 2, 8, 16, and 19, and only one normal chromosome each for 4, 5, 7, 10, 12, 17, and 18. Monosomy 20 and 22 were observed without any structural aberrations involving these two chromosomes. No chromosomes 9, 13, or Y were detected. Other DNA copy number losses were seen at 4p, 5q, 6q, 10p, 12p, 14p, 15p, 18p, and 18q. Chromosomal gains and translocations occurred at the entire or part of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q, 11, 12q, 14q, 16, 17q, 19, and Xp. Chromosome 3 displayed highlevel amplification of multiple bands. There were four derivative chromosomes involving intact chromosomes 14, 15, 17, and 19. Complex translocations were observed in seven derivative chromosomes (5, 8, 12, 14, 14, 14, and 17), each involving three or four chromosomes. The chromosome aberrations are summarized in Tables 2 and 3 and in Fig. 4.
4. Discussion 3. Results Two pools of 12-color WCP probes (Table 1) were designed, and two rounds of FISH were performed on the
Chromosome aberrations are distinctive features of human malignant tumors. Most solid tumors present complex cytogenetic abnormalities. Multiple chromosome aberrations have been reported for ESCC, including numerical
Table 2 Chromosome aberrations in the KYSE180 cell line Chr 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 22 Y
Aberrations þder(1)(6pter/6p10::1q10/1qter), þder(1)(1q31/1q10::3q21/3q26) dup(3)(q24/q26), der(3)(:p21/q27:), þder(3)(:p21/q27:), þder(3)(3qter/3q10::5q14/5q23), þder(3)(6q24/6q16::3p12/3qter), þder(3)(3pter/3q10::6q11/6qter) der(4)(4p15/4qter::8q23) der(5)(5pter/5q23::14q21/14q31::3q28/3q24), þder(5)(5pter/5p10::8q24/8q23), þder(5)(5pter/5p10::6p12/6p22) 6, der(6)(6pter/6q14::3q13/3q22) del(7)(pter/q32:), þder(7)(pter/q32:), þder(7)(10q21/10q10::7q10/7qter) þder(8)(8p12/8q11::16p13/16p12::16q12/16q24::2p24/2p22) 9, 9 der(10)(14qter/14q10::10q10/10qter) þ11 del(12)(:q10/qter) þder(12)(4p15::12p12/12q12::3q21/3q24::2p12/2p24) 13, 13 der(14)(5p14::14q10/14qter::1p11/1p21), der(14)(3qter/3q13::14pter/14qter::5p14/5p11), þder(14)(14qter/14q10::Xp10/ Xp22::3q13/3q26) der(15)(14q12/14q21::15pter/15qter), der(15)(18q22/18q10::15q10/15qter) þder(16)(:p12/q22:) der(17)(17pter/17qter::3p14/3p24), þder(17)(3pter/3p14::11p14/11p11::17q10/17q24::8p22/8p12) 18 þder(19)(10p12/10p11::19pter/19qter) 20 22 Y
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Table 3 Summary of chromosome gains and losses in KYSE180 cell line Chr
Normal
Gain
1
2
2
2
3
0
1p11~p21 1q10~qter 1q10~q31 2p12~p24 2p22~p24 3p12~q10 3q24~q26 3p24~p14 3p21~q27 3pter~p14 3p21~q27 3q13~q26 3q13~q22 3q21~q24 3q21~q26 3q24~q28 3q10~qter 3q13~qter 4p15 5p14 5p14~p11 5pter~p10 5pter~p10 5q14~q23 6p22~p12
4 5
6
7
1 1
0
1
Loss
Chr
Normal
Gain
8
2
8p12~q11 8p22~p12 8q23 8q23~q24
9 10 11
0 1 2
12 13 14
1 0 0
15 16
0 2
4pter~p16 5q31~qter
17 18
1 1
6q10 6q15 6q25~qter
19 20 21 22 X Y
2 1 2 1 1 0
10q21~q10 11 11p14~p11 12q10~q12 14q12~q21 14q21~q31 14q10~qter 14q10~qter
Loss
9 10p13~pter
12p13 13 14pter~p10
15pter~p10 16p12~q22 16p13~p12 16q12~q24 17q10~q24 18pter~p10 18q23~qter 19pter~qter 20 22 Xp22~p10 Y
7pter~q32 7q10~q32
and structural aberrations [5e7]; however, accurate karyotype analysis of ESCC is frequently hampered by the small number of recognizable metaphases and low quality of chromosome morphology. M-FISH allows simultaneous analysis of multiple chromosomes in a single experiment. Diverse cytogenetic abnormalities occurring in the whole genome can be observed by M-FISH, such as chromosomal gains, deletions, inversions, insertions, and translocations; the technique is also useful for identifying the origin of structural aberrations and marker chromosomes [8,9]. M-FISH is therefore a rapid and effective technique for chromosome characterization of solid tumors, especially for the karyotyping analysis of cancer cell lines. KYSE180, a commercial cell line of ESCC, has been widely used in cancer research. In the international cell line bank, the original cytogenetic description of the KYSE180 cell line was recorded as hyperdiploid karyotype: 50~57,XX,þ1,þ1,þ3,þ7,þ7,þ8,14,þ16,17,17,18,18,þ5~8 mar,del(X)(q23),add(1)(q32),der(1;6)(q10; p10),der(1;13)(q10;q10),add(3)(p26),add(5)(q25),add(6)(p11), del(6)(q23),add(7)(p11),del(7)(p11),add(7)(q35),i(7q),del(8) (p22),add(10)(p11),add(11)(q11),del(11)(q11q14),add(13) (p11),add(13)(q33),add(14)(p11),add(15)(p11),add(21)(p11) (http://www.dsmz.de/human_and_animal_cell_lines/info. php?dsmz_nr5379&term5KYSE-180&highlight5). It is
obvious that most of these aberrant chromosomes have not been well identified. The present study provides detailed karyotype information from two-round multicolor FISH. Our results give significant additional information on chromosome aberrations in KYSE180 cells. DNA gains were found at the part or the entire chromosome of 1, 2p, 3, 4p, 5p, 5q, 6p, 7, 8, 10q 11, 12q, 14q, 16, 17q, 19, and Xp. DNA copy number losses were observed at 4p, 5q, 6q, 10p, 12p, 14p, 15p, 18p, 18q, 20, and 22. No chromosomes 9, 13, or Y were detected. Many of the findings are consistent with those seen under comparative genomic hybridization (CGH) in ESCC in populations of East Asia [7,10,11]. The chromosomal regions 1q, 8q, 11q, and 17q with DNA gains in KYSE180 cells contain protooncogenes and the genes promoting cell growth or suppressing cell apoptosis, such as CENPF (1q32), MYC (8q24), CCND1 (11q13), BIRC2 (alias cIAP) (11q21~q23), and ERBB2 (17q12~q21) and its aliases HER-2 and neu (17q21). Some tumor suppressor genes and genes regulating cell proliferation locate in the chromosomes or bands deleted in KYSE180; these include IRF1 (5q31.1), CDKN2B (alias p15) (9p21), CDKN2A (alias p16) (9p21), RB1 (13q14), BRCA2 (13q12.3), DCC (18q23), and SMAD4 (18q21.1). Alteration of these genes might be implicated in the
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passage. As we have mentioned, however, many chromosome aberrations in the KYSE180 cell line were consistent with those reported in primary squamous cell carcinomas of the esophagus. The cell line is therefore able to simulate, to a degree, the biological behavior of some esophageal squamous cell carcinomas. In summary, our results provide detailed data on chromosome aberrations in the cell line KYSE180, data that can contribute to appropriate applications for this esophageal cancer cell line.
Acknowledgments This work was supported by State Key Basic Research Grant of China (2004CB518705), National Natural Science Foundation (30470969, 30328024, 30400207), and Specialized Research Fund of Beijing Municipal Science & Technology Commission (D0905001040331). Fig. 4. Karyogram of the KYSE180 cell line. The top panel for each chromosome gives results of first-round FISH; the middle panel, second-round FISH; and the bottom panel, reverse DAPI banding image of the same chromosome.
development of ESCC or in the malignant behavior of KYSE180 cells. Their copy number changes can be further assessed by array CGH [12], digital karyotyping [13], or molecular biology techniques. The breakpoints of some chromosome translocations occurred around centromere regions 1q10, 3q10, 5p10, 6p10, 7q10, 10q10, 14q10, 15q10, 17q10, 18q10, and Xp10. Among them, breakage at 7q10, 14q10, and 15q10 were also observed in ESCC from Taiwan [7]. However, the translocation breakpoints on 2p, 3q, 6p, 6q, 8q, 14q, 16p, 16q, and 17q are mostly located in noncentromere regions, including 2p24~p12, 2p24~p22, 3q13~q22, 3q21~q24, 3q21~q26, 3q24~q28, 6pter~q14, 6p12~p22, 6q11~qter, 6q16~q24, 8p12~q11, 8q23~q24, 8q23, 14q12~q21, 14q21~q31, 16p12~q22, 16p13~p12, 16q12~q24, and 17q10~q24. These chromosomal regions harbor protooncogenes and genes promoting cell proliferation or growth, for example, MYCN (2p23~p24), PIK3CA (3q26), VEGF (6p12), FYN (6q21), MYC (8q24), TGFB3 (14q24), FOS (14q24.3), and ERBB2 (17q12~q21). The long arm of chromosome 3 (3q) was the region with the highest gain frequency in KYSE180. Gains or amplifications of 3q were often observed in other solid tumors, such as ovarian cancer or squamous cell carcinomas of lung, head, and neck [14,15], suggesting that 3q encompasses one or more protooncogenes associated with multiple types of solid tumors. We note that, due to the differences between the culture condition and the microenvironment in which primary tumor growth in vivo, some greater or lesser acquired molecular changes probably occurred during in vitro culture and
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