Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

JOURNAL OF

GENETICS AND GENOMICS J. Genet. Genomics 35 (2008) 1116

www.jgenetgenomics.org

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization Yiling Yang a, b, Jiayou Chu a, *, Yupeng Wu b, Manli Luo b, Xin Xu b, Yaling Han b, Yan Cai b, Qimin Zhanb, Mingrong Wang b, * a

Department of Genetics, Institute of Medical Biology, Peking Union Medical College and Chinese Academy of Medical Sciences, Kunming 650118, China b State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China Received for publication 15 June 2007; revised 15 August 2007; accepted 15 August 2007

Abstract Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors. Keywords: multicolor fluorescence in situ hybridization; KYSE 410-4; karyotype; esophageal squamous cell carcinoma

Introduction Chromosome aberrations in human solid tumors are hallmarks of gene deregulation and genome instability (Albertson et al., 2003). As most solid tumors exhibit complex abnormalities, traditional banding methods are difficult to detect and describe cryptic translocations and complex marker chromosomes. Multicolor fluorescence in situ hybridization (M-FISH), such as, spectral karyotype (SKY) (Schrock et al., 1996) and multiplex-FISH (Speicher et al., 1996), bridge the gap between traditional banding analysis and molecular cytogenetic tools. How* Corresponding author. Fax: +86-10-8777 8651. E-mail address: [email protected] (M. Wang); [email protected] (J. Chu).

ever, the cost of these techniques is too high to be used in common laboratories. Several groups have shown various repetitive hybridization protocols on one slice (Wang et al., 1995; Zhen et al., 1998; Muller et al., 2002). These protocols show that the same metaphase spread can be used to perform hybridization several times, using various DNA probes. Esophageal squamous cell carcinoma (ESCC) ranks among the 10 most common malignant tumors in the world (Ando et al., 2000). Despite recent reports have revealed multiple genetic abnormalities in ESCC (Kuwano et al., 2005), the molecular mechanism of esophageal carcinogenesis remains ill defined. Analysis of chromosomal changes will contribute to the understanding of molecular mechanisms underlying the development and progression of esophageal cancer. However, it is very difficult to obtain

12

Yiling Yang et al. / Journal of Genetics and Genomics 35 (2008) 1116

a good quality of chromosome morphology from surgical samples of ESCCs. Cell lines, as important experimental materials, are a good choice for cancer research. Studying the genetic background of cell lines is a foundation for their appropriate use. In the current study, we established 6-color FISH and used repetitive M-FISH/reversing DAPI banding technique to characterize molecular cytogenetics of the ESCC cell line KYSE 410-4. Multiple novel chromosome aberrations have been identified, including gains, deletions, and translocations. The results here have indicated that repetitive M-FISH in combination with inverted banding technique is not only useful for analyzing complex karyotypes, but also cheap for popularizing the foreground.

Materials and methods Cell line The cell line KYSE 410 was derived from the poorly differentiated invasive esophageal squamous cell carcinoma, resected from the cervical esophagus of a 51-year-old Japanese man. The KYSE 410 cell line was kindly provided by Yutaka Shimada, (Shimada et al., 1992) and its monoclone KYSE 410-4, was established in our laboratory. Cell cultures were grown in 90% RPMI (Roswell Park Memorial Institute) 1640 with 10% FBS (Fetal Bovine Serum) at 37°C and 5% CO2. Metaphase preparation Metaphase spreads for FISH experiments were prepared according to standard protocols. One hour before harvesting, colchicine (10 ȝmol/L, Sigma, St. Louis, MO, USA) was added to each culture. And then, cells were treated with hypotonic solution (0.075 mol/L KCl) for 20 min, and fixed in three changes of fixative solution, and stored at 20°C overnight. Cell suspensions were dropped onto cold, humidified standard microscope slides and dried overnight before M-FISH. Probe labeling Whole-chromosome painting (WCP) probes subjected to M-FISH analysis had been described in our previous study (Wu et al., 2006). The 24 probes were labeled utilizing degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) with fluorescein isothiocyanate-12-dUTP (FITC) (Perkin-Elmer, Waltham, MA, USA), Cy3 (Amersham Biosciences, Piscataway, NJ, USA) and Cy5 (Amersham Biosciences, Piscataway, NJ, USA). The pools of combinatorial probes were designed as shown in Table 1.

Table 1 Four pools of probes labeled by distinct fluorochromes Pools

Cy3

Cy5

FITC

Cy3: Cy5

Cy3: FITC

Cy5: FITC

G1

Chr 10

Chr 17

Chr 4

Chr 5

Chr 2

Chr Y

G2

Chr 13

Chr 12

Chr 6

Chr 20

Chr 7

Chr 18

G3

Chr 19

Chr 8

Chr 11

Chr 15

Chr 1

Chr 9

G4

Chr 16

Chr 21

Chr 22

Chr X

Chr 3

Chr 14

Repetitive M-FISH For the first FISH, the metaphase slides were pretreated with the routine procedure and denatured with 70% formamide/2 u sodium saline citrate (SSC) at 75°C for 2 min. The probe was denatured at 73°C for 8 min, incubated at 37°C for 30 min and subsequently applied to the slides. Following hybridization for 24 h, the slides were washed in 50% formamide/2 u SSC (pH 7) at 43°C for 15 min, followed by three washes in 2 u SSC and counterstaining with 4, 6-diamidino-2-phenylindole (DAPI). After digital fluorescence image acquisition, the slides were immersed in cool 100% ethanol dehydration for 4 u 5 min, to remove the covership and hybridization solution. A second or sequential hybridization was performed as the first-round FISH in order, but with different pools. Image analysis The images were analyzed according to the protocols described previously (Wang et al., 1995; Wu et al., 2006). During each round of denaturation, a hybridized probe was removed from the slide. Images of metaphase spreads were captured with a cooled charged-coupled device (CCD) camera (Princeton Instruments, Trenton, NJ, USA) equipped with a Zeiss Opton fluorescence microscope (Carl Zeiss, Gottingen, Germany), for visualization of DAPI, FITC, Cy3, and Cy5 fluorochromes. Color composite images were constructed from separate monochrome images and processed using the MetaMorph Imaging System (Universal Imaging Corporation, Downingtown, PA, USA). Ten metaphases were analyzed by M-FISH, and the localization of each metaphase was well recorded. Karyotypes compiled from the combination of the inverted DAPI banding and M-FISH analyses were described according to the International System for Human Cytogenetic Nomenclature (ISCN, 1995).

Results We designed four pools of 6-color WCP probes, which were sequentially hybridized to the same metaphase preparations from KYSE 410-4 (Table 1). After each round

Yiling Yang et al. / Journal of Genetics and Genomics 35 (2008) 1116

of hybridization, the representative metaphase spread in M-FISH classification colors, is illustrated in Fig. 1. Repetitive 6-color FISH was successfully established in this way. M-FISH combed with DAPI reversing banding was applied to the metaphase chromosome preparations, to analyze the karyotypes (Table 2). As a result, the numerous complex alterations and cryptic aberrations were defined in the karyotypes of KYSE 410-4. The number of chromosomes ranged from 52 to 58, with a modal number of 55. Apart from chromosomes 6, 10, 13, and 16, all chromosomes were affected by either structural aberrations or numerical abnormalities. Chromosome changes in 1, 7, and 11 were the most frequent. The absence of chromosome Y and monosomy 19 without any aberration was observed. Chromosomal gains occurred at 2q, 3, 8, 17p, and X. Moreover, an isochromosome 3q was visualized in

13

the cell line. In addition, there were 16 structural breakpoints (Fig. 2). Four complex translocations were observed in the derivative chromosomes 7, 8, 14, and 20, of which derivative chromosome 8 involved chromosomes 6, 9, 12, and 17.

Discussion Chromosome change is one mechanism by which cells might tiptoe toward cancer (Albertson et al., 2003). There are a broad range of chromosomal abnormalities in solid tumors, including altered ploidy, gain or loss of individual chromosomes or portions thereof, and structural rearrangements. However, conventional cytogenetic banding methods are extremely different to clarify the numer-

Fig. 1. Representative metaphase after four rounds of repetitive M-FISH. Repetitive M-FISH with the probes of pool 1 (G1), pool 2 (G2), pool 3 (G3), and pool 4 (G4) on the KYSE 410-4 cell line (see Table 1 for probe composition and color assignment).

14

Yiling Yang et al. / Journal of Genetics and Genomics 35 (2008) 1116

Fig. 2. Sixteen structural arrangements in KYSE 410-4. The top panel gives reversing DAPI banding image for each translocation chromosome; and the bottom panel, results of M-FISH. Table 2 Chromosome aberrations in the KYSE 410-4 cell line after multicolor fluorescence in situ hybridization analyses Chromosome

M-FISH nomenclature

1

-1, +der(1)t(1;19)(p10;?), del(1)(q32), +der(1)t(1;9)(q10;?),

2

2q+

3

+der(3)del(3)(p10)t(7;3)(p10;p10), i(3q),

4

del(4)(q21)

5

der(5)t(5;16)(q35;q12)

7

-7×2, der(7)t(7;1)(q10;?)t(19;1)(?;?), +der(7)t(11;7)(?;q10), +der(7)t(7;9)(q33;q10)

8

+der(8)ins(8)(8;6)t(8;17)(p12;?)t(8;9)(?;?)

9

del(9)(q31), der(9)del(p21)t(12;9)(?;q22)

11

der(11)t(11;5)(qter;p15), der(11)del(11)(p12)t(11;7)(p10;p14), 11q1?+, 11q+

12

der(12)t(12;6)(p10;?)

14

der(14)del(14)(q23)t(9;14)(q23;?)t(9;14)(q31;p10)

15

der(15)t(18;15)(?;p10)

17

17p11+

18

?18q-, ?18q+

19

-19

20

?der(20)t(20;4)(q23;q22-24)t(8;4)(?;q24)

21 22

der(22)t(22;5)(p13;?)

X

+X

Y

-Y

M

Marker

ous complex alterations and uncover cryptic aberrations. Recent development of molecular cytogenetic techniques proves to be valuable in solving some of these problems (Schulten et al., 2002; Stamouli et al., 2004). A number of different multicolor-FISH strategies have been introduced to delineate all 24 human chromosomes in different colors, termed 24-color FISH karyotyping or genome painting, such as, spectral karyotype (SKY) and multiplex-FISH (Schrock et al., 1996; Speicher et al., 1996). These tech-

niques require several narrow band pass fluorescence filters or an interferometer, which are limited in some small laboratories. Here, we designed four, 6-colour probe pools, which were repetitively hybridized onto the same specimen to describe 24 human chromosomes. In this strategy, they only needed an epifluorescent microscope equipped with the four-filter wheel. In addition, they simplified the pre-rehybridized method, which proved to be effective in removing the covership and hybridization solution through

Yiling Yang et al. / Journal of Genetics and Genomics 35 (2008) 1116

100% ethanol dehydration. There are some noticeable things in repetitive M-FISH, which are as follows: 1) During detection of the first hybridizing signal, one should record all localizations of the appropriate morphology chromosomes. Through the coordinates, a series of hybridization images can be easily captured. 2) To obtain high quality pictures, one can appropriately prolong the hybridizing time, to ensure that the signals were satisfactory. 3) All manipulations should be careful and gentle, especially in the washing processes. Using the repetitive M-FISH principle, the authors have detailed the characterization of complex karyotypes with massive genomic alterations in KYSE 410-4. KYSE 410, a commercial cell line of ESCC, has been widely used in cancer research (Sebastian et al., 2000; Wang et al., 2006). The original cytogenetic description of the KYSE 410 is available online in DSMZ (http://www.dsmz.de/human_and_animal_cell_lines/info.p hp?dsmz_nr=381&term=KYSE%20410&highlight=). In the database, numerical aberrations such as gains and deletions are the main changes that are reported. There are only two structural breakpoints mentioned in DSMZ, one of which is confirmed in our data. Particularly, we found 15 other structural arrangements in KYSE 410-4. It is obvious that most of these aberrant chromosomes have not been well identified. In this study, the karyotype description of KYSE 410-4 is refined from four rounds of multicolor FISH. The main advantage of M-FISH is to identify the origin of the chromosomal material. These results give additional information on chromosome aberrations in KYSE 410-4 cells. Chromosomal gains have been found at the part or the entire of 2q, 3q, 8, 17p, and X. Chromosomal losses have been observed at 19 and Y. Many of the findings are consistent with those observed under the comparative genomic hybridization (CGH) in ESCC (Wei et al., 2002; Wu et al., 2007). This study also reveals significant structural aberrations of chromosomes, in addition to numerical changes. The breakpoints of some chromosome translocations occur around regions of 1p10, 1q10, 3p10, 4q22-24, 4q24, 5p15, 5q23, 16q12, 7p10, 7p14, 7q10, 7q33, 8p12, 9q10, 9q22, 9q23, 9q31, 11p10, 12p10, 14p10, 15p10, 20q23, and 22p13. In particular, aberrations that frequently involve the 4q22-24 and 9q22-31 regions are the most common breakpoints found in KYSE 410-4. The preferential involvement of the centromeric regions, accounts for approximately 44% of the total rearrangements. This finding is in good agreement with the previous observation in ESCC from Hong Kong (Jin et al., 2004). This may be the reason for the formation of isochromosome 3q, which results in 3q gain and 3p loss. The i(3q) induces a 'cut' phenotype, abnormal centrosome amplification, aneuploidy, and loss of G1 arrest following gamma-irradiation (Smith et al., 1998). The amplification of 3q has been observed in solid tumors, such as, ovarian cancer (Nanjundan et al.,

15

2007) or squamous cell carcinomas of lung (Yokoi et al., 2003), head and neck (Lin et al., 2006), and esophagus (Yen et al., 2005), suggesting that 3q might harbor genes upregulated through DNA amplification and is involved in the development and progression of solid tumors. The gain of 3q is an earlier event in the tumorgenesis of ESCC (Jin et al., 2004). We hypothesized that these potential proto-oncogenes in the region, for example, CCNL1, MDS1, TERC, PKCȚ, ECT2, and PIK3CA, could amplified targets in the region. Their chromosomal changes can be further assessed by array CGH and single-probe based FISH. Investigations to identify specific genes involved in chromosome aberrations are underway. In conclusion, we report that M-FISH/inverted DAPI banding analysis of KYSE 410-1, for a detailed characterization of complex chromosomal abnormalities, could contribute to appropriate applications for this esophageal cancer cell line. The repetitive 6-color FISH providing cytogenetic information of the whole genome is a reliable complementary technique to resolve complex karyotypes.

Acknowledgements This study was supported by the National Science Foundation (No. 30630067), the State Key Basic Research Grant of China (No. 2004CB518705) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0416). References Albertson, D.G., Collins, C., McCormick, F., and Gray, J.W. (2003). Chromosome aberrations in solid tumors. Nat. Genet. 34: 369376. Ando, N., Ozawa, S., Kitagawa, Y., Shinozawa, Y., and Kitajima, M. (2000). Improvement in the results of surgical treatment of advanced squamous esophageal carcinoma during 15 consecutive years. Ann. Surg. 232: 225232. ISCN. (1995). An International System for Human Cytogenetic Nomenclature. 1st edn. Jin, Y., Jin, C., Law, S., Chu, K.M., Zhang, H., Strombeck, B., Yuen, A.P., and Kwong, Y.L. (2004). Cytogenetic and fluorescence in situ hybridization characterization of clonal chromosomal aberrations and CCND1 amplification in esophageal carcinomas. Cancer Genet. Cytogenet. 148: 2128. Kuwano, H., Kato, H., Miyazaki, T., Fukuchi, M., Masuda, N., Nakajima, M., Fukai, Y., Sohda, M., Kimura, H., and Faried, A. (2005). Genetic alterations in esophageal cancer. Surg. Today 35: 718. Lin, M., Smith, L.T., Smiraglia, D.J., Kazhiyur-Mannar, R., Lang, J.C., Schuller, D.E., Kornacker, K., Wenger, R., and Plass, C. (2006). DNA copy number gains in head and neck squamous cell carcinoma. Oncogene 25: 14241433. Muller, S., Neusser, M., and Wienberg, J. (2002). Towards unlimited colors for fluorescence in situ hybridization (FISH). Chromosome Res. 10: 223232. Nanjundan, M., Nakayama, Y., Cheng, K.W., Lahad, J., Liu, J., Lu, K., Kuo, W.L., Smith-McCune, K., Fishman, D., Gray, J.W., and Mills, G.B. (2007). Amplification of MDS1/EVI1 and EVI1, located in the 3q26.2 amplicon, is associated with favorable patient prognosis in ovarian cancer. Cancer Res. 67: 30743084.

16

Yiling Yang et al. / Journal of Genetics and Genomics 35 (2008) 1116

Schrock, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., Ferguson-Smith, M.A., Ning, Y., Ledbetter, D.H., Bar-Am, I., Soenksen, D., Garini, Y., and Ried, T. (1996). Multicolor spectral karyotyping of human chromosomes. Science 273: 494497. Schulten, H.J., Gunawan, B., Otto, F., Hassmann, R., Hallermann, C., Noebel, A., and Fuzesi, L. (2002). Cytogenetic characterization of complex karyotypes in seven established melanoma cell lines by multiplex fluorescence in situ hybridization and DAPI banding. Cancer Genet. Cytogenet. 133: 134141. Sebastian, J.L., Rigberg, D.A., Shrivatsan, E., Revasova, E., McFadden, D.M., and Levingston, E.H. (2000). The use of large-scale cDNA analysis to profile differential gene expression in KYSE 410 human esophageal cancer cells after irradiation. Bull Exp Biol Med. 130: 882885. Shimada, Y., Imamura, M., Wagata, T., Yamaguchi, N., and Tobe, T. (1992). Characterization of 21 newly established esophageal cancer cell lines. Cancer 69: 277284. Smith, L., Liu, S.J., Goodrich, L., Jacobson, D., Degnin, C., Bentley, N., Carr, A., Flaggs, G., Keegan, K., Hoekstra, M., and Thayer, M.J. (1998). Duplication of ATR inhibits MyoD, induces aneuploidy and eliminates radiation-induced G1 arrest. Nat. Genet. 19: 3946. Speicher, M.R., Gwyn Ballard, S., and Ward, D.C. (1996). Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12: 368375. Stamouli, M.I., Panani, A.D., Ferti, A.D., Petraki, C., Oliver, R.T., Raptis, S.A., and Young, B.D. (2004). Detection of genetic alterations in primary bladder carcinoma with dual-color and multiplex fluorescence in situ hybridization. Cancer Genet. Cytogenet. 149: 107113. Wang, H., Qian, H., Yu, J., Zhang, X., Zhang, L., Fu, M., Liang, X., Zhan, Q., and Lin, C. (2006). Administration of PUMA adenovirus increases the sensitivity of esophageal cancer cells to anticancer drugs. Cancer Biol. Ther. 5: 380385.

Wang, M.R., Perissel, B., and Malet, P. (1995). Rehybridization on metaphases studied previously by FISH. An approach to analyze chromosome aberrations. Cancer Genet. Cytogenet. 85: 5860. Wei, F., Ni, J., Wu, S.S., Liu, H., Xu, X., Han, Y.L., Cai, Y., Zhang, J.W., Chen, X.J., Pang, H., Lu, N., Ji, L., Wu, M., and Wang, M.R. (2002). Cytogenetic studies of esophageal squamous cell carcinomas in the northern Chinese population by comparative genomic hybridization. Cancer Genet. Cytogenet. 138: 3843. Wu, S.S., Liu, J.F., and Wang, M.R. (2007). Analyzing chromosomal abnormality in primary esophageal cancer by comparative genomic hybridization. Ai zheng 26: 132136 (in Chinese with an English abstract). Wu, Y.P., Yang, Y.L., Yang, G.Z., Wang, X.Y., Luo, M.L., Zhang, Y., Feng, Y.B., Xu, X., Han, Y.L., Cai, Y., Zhan, Q.M., Wu, M., Dong, J.T., and Wang, M.R. (2006). Identification of chromosome aberrations in esophageal cancer cell line KYSE180 by multicolor fluorescence in situ hybridization. Cancer Genet. Cytogenet. 170: 102107. Yen, C.C., Chen, Y.J., Pan, C.C., Lu, K.H., Chen, P.C., Hsia, J.Y., Chen, J.T., Wu, Y.C., Hsu, W.H., Wang, L.S., Huang, M.H., Huang, B.S., Hu, C.P., Chen, P.M., and Lin, C.H. (2005). Copy number changes of target genes in chromosome 3q25.3-qter of esophageal squamous cell carcinoma: TP63 is amplified in early carcinogenesis but down-regulated as disease progressed. World J. Gastroenterol. 11: 12671272. Yokoi, S., Yasui, K., Iizasa, T., Imoto, I., Fujisawa, T., and Inazawa, J. (2003). TERC identified as a probable target within the 3q26 amplicon that is detected frequently in non-small cell lung cancers. Clin. Cancer Res. 9; 47054713. Zhen, D.K., Wang, J.Y., Falco, V.M., Weber, W., Delli-Bovi, L., and Bianchi, D.W. (1998). Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood. Prenat. Diagn. 18: 11811185.