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Cytogenetic aberrations in immortalization of esophageal epithelial cells
Cancer Genetics and Cytogenetics 165 (2006) 25–35
Cytogenetic aberrations in immortalization of esophageal epithelial cells Hao Zhanga, Yuesheng Jinb, Xuehua Chena, Charlotte Jinb, Simon Lawc, Sai-Wah Tsaod, Yok-Lam Kwonga,* a
Department of Medicine, University of Hong Kong, Professorial Block, Pockfulam Road, Hong Kong, China b Department of Clinical Genetics, University Hospital, 22 Klinikgatan, S-22185, Lund, Sweden c d Department of Surgery, Department of Anatomy, University of Hong Kong, Hong Kong, Professorial Block, Pockfulam Road, Hong Kong, China Received 8 June 2005; received in revised form 8 July 2005; accepted 20 July 2005
Abstract
To define the early cytogenetic events important in esophageal carcinogenesis, we immortalized normal esophageal epithelial cells by overexpression of human papillomavirus type 16 E6/E7 (HPV16E6/E7) and human telomerase reverse transcriptase (hTERT), and characterized the chromosomal abnormalities serially before and after cellular immortalizaiton. During crisis, most cells had simple nonclonal karyotypic changes with cytogenetic divergence. Mitotically unstable chromosomes (i.e., telomere association and dicentric chromosomes) were the most common aberrations. After crisis, the karyotypic patterns were more convergent with nonrandom clonal changes. A few clones dominated the culture. Gain of chromosome 20q was consistently observed in four HPVE6/E7 immortalized esophageal lines, whereas amplification of chromosome 5q was preferentially found in hTERT immortalized cells. In addition, chromosomal aberrations of immortalized cells, including del(3p) and centromere rearrangements, were similar to those observed in esophageal cancer. Furthermore, in E6/E7-expressing cells, the frequency of negative telomere termini and anaphase bridges were high during crisis and low after crisis. These findings suggested that telomere dysfunction might be an important cause of cellular crisis, and the resultant chromosomal aberrations, mainly amplification of chromosome 20q or 5q, might be early genetic events required in esophageal cell immortalization. These alterations might be valuable models for further study of molecular mechanisms contributing to esophageal carcinogenesis. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Esophageal carcinoma is the ninth most common cancer worldwide and the second in China [1]. In Hong Kong, approximately 90% of esophageal carcinoma is squamous cell carcinoma (SCC), and the overall five-year survival rate remains low [2]. In Western countries, the incidence of esophageal adenocarcinoma has increased dramatically in recent years [3], with more than 85% of patients dying within 2 years of diagnosis [4]. Although esophageal cancer is an important malignancy, the genetic mechanisms underlying carcinogenesis have not been well defined, partly due to the difficulties in culturing esophageal tumor cells in vitro and the lack of suitable cell models. Cytogenetic information is available in 42 cases only [5]. Most of these tumors displayed complicated cytogenetic aberrations. Also, a few esophageal cancer cell
* Corresponding author. Tel.: 1852-2-855-4597; fax: 1852-2-9741165. E-mail address: [email protected] (Y.-L. Kwong). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.07.016
lines available were mostly derived from advanced esophageal cancer and were characterized by highly complex karyotypes [6–9]. The relative importance of these complex aberrations in esophageal carcinogenesis remains undefined. Recent studies have suggested that infection with the high-risk human papillomavirus type 16 E6/E7 (HPV16E6/E7) might play an important pathogenetic role in esophageal SCC in Chinese patients [10]. Progressive up-regulation of telomerase, however, appears to be the important feature of adenocarcinoma in the west [11]. Esophageal cells can be immortalized in vitro by the overexpression of the oncoprotein HPV16 E6/E7 or the catalytic subunit of human telomerase reverse transcriptase (hTERT) [12]. As immortalization may be an initial step of carcinogenesis, the study of cytogenetic changes associated with immortalization may help to define the genetic events critical for neoplastic transformation. In this study, esophageal cell lines obtained by immortalization with transfection of the HPV16E6/E7 and hTERT genes were characterized serially before and after
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H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
immortalization. The karyotypic profiles of these cell lines were also compared with those of esophageal carcinoma cell lines. Finally, the relationship between the telomere status and chromosomal instability was evaluated in one of the HPV16E6/E7-expressing cell lines.
2. Materials and methods 2.1. Immortalization of normal esophageal epithelial cells The method of primary culture and immortalization of normal epithelial cell lines [13] and the establishment and maintenance of the esophageal cancer cell lines HKESC1 [6], KYSE-140 [8], SLMT-1 [9], EC-1, and EC-18 were as described previously. Five cell lines, four with the expression of HPV16E6/E7, NE108/E6E7, NE083/E6E7, NE3/E6E7, and NECA6/E6E7, and one with hTERT and NE083/hTERT, were newly immortalized. 2.2. Cytogenetic analysis The cell cultures were harvested by conventional cytogenetic techniques. Briefly, cells were blocked at metaphase by exposure to colcemid (0.025 m/mL; GIBCO, Invitrogen Corp., Paisley, UK) for 14 hours. After hypotonic and fixative treatment, the cell suspension was spread on slides, and the metaphase chromosomes were G-banded with Wright stain. The clonality and karyotypes were described according to the International System for Human Cytogenetic Nomenclature (1995) [14]. 2.3. Fluorescence in situ hybridization (FISH) Multicolor combined binary ratio labeling (COBRA) FISH was performed as described [15]. Briefly, slides with metaphase chromosomes were pretreated with pepsin and formaldehyde. Probe mix (8 mL) containing 24 human chromosomes labeled with four distinct fluorochromes by the combination of binary and ratio labeling was applied on the slide. The slide and probe were denatured simultaneously by incubation at 72 C on a hot plate. After hybridization for 48–72 hours at 37 C in a humid chamber, the slides were washed in 74 C 0.4 standard saline citrate (SSC) for 2 minutes. Chromosomes were counterstained by TNT (0.1 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20 in 100 mL of phosphate buffered saline) and 4’,6-diamidino phenylindole (DAPI) (0.5 mg/mL) for 10 minutes. The slides were embedded in Citifluor (Ted Pella, Redding, CA) before microscopic evaluation. For analysis, an Axioplan-2 microscope (Carl Zeiss GmbH, Jena, Germany) coupled to a cooled charge-coupled device camera and a 12-position filter wheel was used. Acquired images were evaluated with the CytoVision ChromoFluor System (Applied Imaging, Newcastle, UK) [15].
Based on cytogenetic findings, specific bacterial artificial chromosomes (BAC) were selected for further FISH analysis (http://www.ncbi.nlm.nih.gov/cgi-bin/Etreez/mapsearch, 2005). The labeled probes were purified, precipitated, and dissolved in standard hybridization solution. The slide and probe were denatured simultaneously by incubation at 72 C on a hot plate. After hybridization overnight at 37 C in a humid chamber, the slides were washed in 74 C 0.4 SSC for 2 minutes, DAPI-stained, and analyzed. Telomeric TTAGGG repeats were detected using telomeric FISH with a fluorescein-conjugated (CCCTAA)3 peptide nucleic acid (PNA) probe. The number of negative chromosome termini for a metaphase cell was recorded. At least 20 cells were evaluated per case. 2.4. Mitotic cell morphology For analysis of mitoses, cells cultured on chamber slides were washed in phosphate-buffered saline (PBS) for 5 minutes, fixed in methanol/acetic acid (3:1) at 220 C for 30 minutes, air-dried, and stained with hematoxylin (Accustain; Sigma, St. Louis, MO) and erythrosine (BDH, Poole, England). At least 50 anaphase cells were examined per population doublings (PD). An anaphase cell showing at least one string of chromatin connecting the poles was defined as harboring an anaphase bridge. 3. Results 3.1. Sequential cytogenetic characterization of immortalized normal esophageal cells To identify cytogenetic changes potentially relevant to NE cell immortalization, sequential karyotyping was performed in four HPV16E6/E7 and one hTERT immortalized cell line. The cytogenetic profiles of the five NE cell lines are summarized in Table 1. 3.2. NE108/E6E7 This line was thoroughly studied during immortalization. At PD21 (before crisis), most cells were karyotypically normal, although a few nonclonal chromosomal changes began to appear in 10.4% (5/48) of metaphase cells. At PD28–PD34 (crisis), cells with normal chromosome complement dropped to less than 50%, whereas cells harboring chromosome aberrations increased. At this stage, a considerable portion of metaphase cells showed nonclonal aberrations, but a few cytogenetically unrelated clones emerged. Chromosomal changes were highly heterogeneous, with nearly 30% of cells displaying mitotically unstable chromosomes [e.g., telomere association (tas), dicentric (dic), and ring (r) chromosomes]. Centromeric rearrangements (e.g., isochromosomes and whole-arm translocations) were also common. During immortalization (PD41–PD64), cells with related clonal chromosomal changes expanded, whereas
Table 1 Sequential cytogenetic analysis of five immortalized esophageal epithelial cell lines Cells containing mitotically unstable chromosome
48 44 25 27 28
0(0) 10(22.7) 8(32.0) 12(44.4) 21(75.0)
5(10.4) 16(36.4) 9(36.0) 11(40.7) 5(17.9)
0 11 5 4 3
0 2 1 3 1
0 0 1 0 1
0(0) 13(29.5) 7(28.0) 7(25.9) 5(17.9)
4(4) 3(3) 3(3) 6(4)
54 A 54 B
17 21
13(76.5) 18(85.7)
4(23.5) 3(14.3)
1 2
1 1
1 0
3(17.6) 3(14.3)
4(1) 7(3)
54 C 64 A 64 B
16 15 19
13(81.2) 13(86.7) 17(89.5)
3(18.8) 2(13.3) 2(10.5)
1 1 2
2 0 0
0 0 0
3(18.8) 1(6.7) 2(10.5)
3(1) 3(3) 5(5)
64 C
14
12(85.7)
2(14.3)
1
0
0
1(7.1)
6(3)
94 A
38
37(97.4)
1(2.6)
1
1
0
2(5.3)
8(0)
94 B
33
32(97.0)
1(3.0)
0
0
0
0(0)
94 C
40
39(97.5)
1(2.5)
1
4
0
5(12.5)
5(0)
37 46
0 32(86.5) 43(93.5)
0 3(8.1) 3(6.5)
0 0 4
0 0 4
0 0 0
0 0(0) 8(17.4)
4(2) 6(2)
59
59(100)
0(0)
1
1
2(1.7)
4(1)
46 77 50
0 49(63.6) 36(72.0)
0 6(7.8) 0(0)
0 1 0
0 0 0
0 0 0
0 1(1.3) 0(0)
2(2) 2(2)
15 24 15 16
0 18(75.0) 11(73.3) 12(75.0)
0 4(16.7) 4(26.7) 4(25.0)
0 1 1 0
0 2 0 9
0 1 0 0
0 4(16.7) 1(6.7) 9(56.2)
1(0) 3(0) 1(0) 1(0)
Clones Cells (unrelated Karyotypes analyzed Clonal(%) Nonclonal(%) Tas Dic R Total(%) clones)
NE108/E6E7 21 28 34 40 47
NE083/E6E7 18 33 46 59 NE083/hTERT 37 60 70 NE3/E6E7 12 24 40 86
12(1)
46,XY[43] 47,XY,120[cp4]/46,XY,i(8)(q10)[2]/45,XY,tas(12;13)(q24;p13)[2]/45,XY,der(13;16)(q10;q10)[2] 47,XY,120[cp5]/43,XY,der(8;16)(q10;q10)[cp2]/46,XY,i(8)(q10)[1] 47,XY,120[6]/43~45,XY,222[cp4]/42~43,XY,i(8)(q10)[2] 45~46,XY,i(8)(q10)[cp5]/46~47,XY,120[cp4]/47,XY,1i(20)(q10)[3]/44~45, XY,222[3]/ 42~43,XY,der(8;11)(q10;q10),222[cp3]/42~44,XY,der(8;13) (q10;q10)[cp3] 47,XY,120[6]/47,XY,1i(20)(q10)[5]/48,XY,120,1i(20)(q10)[1]/47,XY,1i(8)(q10)[1] 47,XY,1i(20)(q10)[3]/48,XY,1i(20)(q10)2[5]/48,XY,120,1i(20)(q10)[2]/ 47,XY,120[4]/45,XY,222[2]/ 46,XY,i(8)(q10)[1]/46,XY,der(8;11)(q10;q10) [1] 47,XY,1i(20)(q10)[10]/48,XY,120,1i(20)(q10)[2]/47,XY,120[1] 47,XY,120[6]/45~47,XY,1i(20)(q10)[cp6]/46,XY,der(8;11)(q10;q10)[1] 46~47,1i(20)(q10)[cp6]/43~44,XY,der(8;11)(q10;q10),222[cp5]/46,XY,i(8) (q10)[3]/47,XY,120[2]/ 46,XY,der(8;13)(q10;q10)[1] 45~47,XY,1i(20)(q10)[cp3]/45~46,XY,1i(20)(q10),add(11)(q11)[cp3]/48,XY,120,1i(20)(q10),add(12)(q24)[1]/ 48,XY,1i(20)(q10)2[3]/48~49,XY,11,120,add(13)(p11)[1]/43,XY,der(8;11)(q10;q10),222[1] 45~46,XY,1i(20)(q10),222[2]/46,idem,del(11)(q13)[21]/46,idem,del(3)(p11),del(11)(q13)[4]/45~48, XY,1i(20)(q10)[cp6]/48,XY,17,1i(20)(q10) [1]/48,XY,116,1i(20)(q10)[1]/48,XY,120,1i(20)(q10)[1]/ 48,XY,1i(20) (q10)2[1] 47,XY,1i(20)(q10)[2]/47,idem,del(11)(q13)[1]/46,idem,del(11)(q13),222[2]/46,idem,del(3)(p11), del(11)(q13),222[11]/46,idem,der(3;7)(q10;q10),del(11)(q13),222[2]/48~49,XY,15,1i(20)(q10)[cp5]/47~49, XY,15,der(12)t(Y;12)(q11;q24),1i(20)(q10),120[cp5]/49,XY,1i(20)(q10)2,add(3)(p11)[1]/ 47,XY,del(3)(p11),1i(20)(q10)[1]/46,XY,i(20)(q10)[1]/48,XY,1i(20)(q10),120 [1]/47,XY,120[1] 46,XY,1i(20)(q10),222[2]/46,idem,del(11)(q13)[16]/46,idem,del(3)(p11),del(11)(q13)[13]/46~48, XY,1i(20)(q10)[cp7]/47,XY,120[1] 46,XY[25] 47,XY,120[23]/48,XY,19,120[3]/47~48,XY,18[cp3]/47~48,XY,19[cp3]46,XY[2] 47,XY,120[27]/48,XY,19,120[6]/47,XY,del(3)(p21),120[4]/48,XY,del(3)(p21),19,120[1]/47,XY, 19,tas(9;22)[3]/46,XY,add(19)(q13),add(21) (p11)[2] 47,XY,120[11]/48,XY,19,120[7]/46,XY,add(19)(q13),add(21)(p11)[11]/ 88~91,XXYY,19,210,211,213,215,120,120,221,der(21)t(13;21)(q14,p11)[30]
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
Cells having chromosome changes
Lines/ Populations doublings
46,XY[46] 46,XY,dup(5)(q31q33)[46],46,XY,der(16)t(5;16)[3],46,XY[22] 46,XY,dup(5)(q31q33)[32],46,XY,der(16)t(5;16)[4],46,XY[14] 46,XY[15] 47~49,XY,15,120[cp13]/47,XY,120[3]/47,dic(X;21)(p22;q22),Y,15,120[2] 47,dic(X;21)(p22;q22),Y,15,der(8;14)(q10;q10),120[cp11] 46~47,dic(X;21)(p22;q22),Y,15,der(8;14)(q10;q10),der(15;21)(q10;q10), 1der(20)ins(20;?)(q13;?)t(1;20)(p13;q13)[cp12] 27
(Continued)
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
cells with nonclonal changes decreased drastically, parallel with a reduction of mitotically unstable chromosomes (i.e., tas and dic). In early crisis (PD28), a few cytogenetically unrelated, pseudo-, or near-diploid clones with simple numerical and structural aberrations, such as 120, i(8q), der(13;16), and tas(12;13), could be identified. Some of the clones, such as the clone with i(8q), survived in PD64 but were eventually subdued as a result of selection pressure. However, the clones carrying 120 survived. This clone expanded progressively and eventually took over the cultures. The clone with i(20q) also existed throughout the entire postcrisis culturing time (PD47–PD94), increased in frequency, and eventually dominated the cultures. At PD94, all clones identified were related by gain of chromosome 20 and/or isochromosome 20q, leading to an increased copy number of the long arm of chromosome 20, in addition to one to three other numerical and/or structural aberrations. At PD50, NE108/E6E7 cells were subcultured independently into three sublines and were cytogenetically analyzed to define genetic convergence or divergence during immortalization. An obvious decrease of cytogenetic divergence was found, with the initial polyclonality reduced to a monoclonality, with one clone making up to 97% of the cells. On the other hand, by PD94, owing to clonal evolution, the karyotypes became more complex in each clone, giving rise to several cytogenetic related clones. This was shown in all three sublines d A, B, and C d which were cultured independently. The dominant clone in all three sublines contained i(20q) with more changes, giving rise to a relatively complex karyotype [i.e., 46, XY, del(3)(p11), del(11)(q13), 1i(20)(q10), 222]. Abbreviations: Tas, Telomere association; Dic, Dicentric chromosome; R, ring.
2(0) 8(1) 0 0 0 0 0 0 0 0 0(0) 0(0) 22 37 92 110
22(100) 37(100)
0 30 60
28(93.3)
2(6.7)
0
0
0
6(0)
46,XY[27]/46,XY,120[1] 47,XY,120[cp17]/48,XY,120,120[cp6]/~58,XY,der(11;19)(q10,q10)[2] 48,XY,15,120[5]/48,XY,17,120[3]/49,XY,15,19,120[1]/45,XY,tas(12;14)(q24;p13),120[1]/ 43~46,XY,15[cp4]/ 47,XY,15,19,215 [1] 48,XY,120,120[9]/48,XY,19,120[6]/47,XY,120[5]/48,XY,15,120[4]/ 49,XY,15,19,120[2]/ 50,XY,15,19,120,120[cp2] 48,XY,15,t(7;8)(q22;q23),120[19]/46~47,XY,15,der(8;13)(q10;q10),120[cp3] 47,XY,120[10]/47,XY,t(7;8)(q22;q23),120[7]/48,XY,120,120[6]/48,XY,11,120[4]/47,XY, der(8;13)(q10;q10),120[3]/46,XY,t(7;8)(q22;q23)[3]/49,XY,17, 120,120[2]/49,XY,11,120,120[2] 1(0) 3(1) 6(2) 3(9.1) 2(5.3) 1(5.6) 0 0 0 1 2 1 1(3.0) 25(65.8) 15(83.3) 33 38 18
5(15.2) 5(13.2) 3(16.7)
2 0 0
22(78.6) 1(0) 13(72.2) 1(0) 0 0 22 13 0 0 4(14.3) 1(6.0) 24(85.7) 17(94.0)
114 130 NECA6/E6E7 18 24 40
28 18
0 25 100
19(76.0)
6(24.0)
16
0
16(64.0) 3(0)
46~47,Y,dic(X;21)(p22;q22),Y,15,17,der(8;14)(q10;q10),der(15;21)(q10;q10),120[2]/46~47,idem, 1dup(20)(q13q11)[4]/46~47,idem,-(17),111,1der(20)ins(20;?)(q13;?)t(1;20)(p13;q13)[7] 47~48,Y,dic(X;21)(p22;q22),Y,15,der(8;14)(q10;q10),der(15;21)(q10;q10),120[cp10]/ 47~48,idem,der(4)t(4;4)(p16;q27)[cp5]/47~48,idem,der(4)t(4;4)(p16;q27),17[cp4] 47,Y,dic(X;21)(p22;q22),Y,15,17,der(8;14)(q10;q10),der(15;21)(q10;q10),1dup(20)(q13q11)[cp24] 47,Y,dic(X;21)(p22;q22),Y,15,17,der(8;14)(q10;q10),der(15;21)(q10;q10),1dup(20)(q13q11)[cp17] 13(81.3) 3(0) 0 13 0 3(18.7) 13(81.3) 16 96
R Dic
Lines/ Populations doublings
Table 1 Continued
Cells having chromosome changes
Cells analyzed Clonal(%) Nonclonal(%) Tas
Cells containing mitotically unstable chromosome
Clones (unrelated Total(%) clones) Karyotypes
28
3.3. NE083/E6E7 At early PDs before crisis, the cells had normal karyotypes. At PD33, two out of four subclones related by an extra copy of chromosome 20 were observed. Clonal chromosomal changes accounted for 86.5% of post-crisis cells, with the percentage increasing with the culture propagation. Cells tended to be more homogenous with complex karyotypes, suggesting that the majority of cells were derived from the same progenitor. 3.4. NE3/E6E7 The NE3/E6E7 parental cells (PD12) showed a normal karyotype. At PD24, three clones related by an extra copy of chromosome 20 were detected, accounting for approximately 75% of cells analyzed. Nonclonal changes, including tas and dic, were found in about 25% of the cells. During crisis, genetic divergence led to cytogenetic heterogeneity in the form of several subclones developing from a clone in PD24. After immortalization, only one subclone dominated the culture. Apart from sharing several numerical and structural aberrations with the relevant subclone at
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
earlier passages, an extra derivative chromosome 20 with duplicate of 20q material was found, resulting effectively in a total of four copies of 20q.
29
E6E7 cells, where the duplicated fragment was inverted as dup(20)(q13q11) (Fig. 2C). 3.9. Mitotic abnormalities
3.5. NECA6/E6E7 In pre-crisis (PD18), the majority of NECA6/E6E7 cells had normal karyotypes, whereas 5 of 33 cells had nonclonal chromosome changes. In one of the cells, an extra copy of chromosome 20 was detected as the sole anomaly. Surprisingly, this cell expanded rapidly, becoming the dominating clone during crisis and the only clone surviving the crisis. More subclones deriving from this clone appeared after immortalization. 3.6. NE083/hTERT The overexpression of hTERT in esophageal epithelial cells was attempted several times with materials from different individuals. However, only one cell line overcame the crisis and was immortalized successfully. In contrast to the cells immortalized by HPV16E6/E7, this line was genomically stable during the immortalization process. At PD37, all metaphases analyzed had normal karyotypes. At PD70, approximately 60% of cells acquired a structural rearrangement, dup(5)(q31q33), and in a small clone der(16). No nonclonal aberrations, telomere association, or dicentric chromosomes were observed. 3.7. Clonal evolution observed in esophageal cell lines expressing E6/E7 Clonal evolution could be traced in cell lines immortalized by HPV16E6/E7. The potential evolutionary pathways are depicted in Fig. 1. Apparently, gain of chromosome 20 or chromosomal 20q, occurring in the early passages as the sole anomaly and present in all cells surviving the crisis, was an early nonrandom genetic event critical for cell immortalization. 3.8. FISH analysis COBRA-FISH analysis confirmed the cytogenetic interpretation of all structural and numerical rearrangements in NE108/E6E7 (Fig. 2A). The cytogenetic interpretation of dup(5)(q31q33) in NE083/hTERT was also confirmed. Further FISH analyses with the use of BAC clones spanning 20q were performed in NE108/E6E7 and NE3/E6E7 cells to verify i(20q) and dup(20q), and to define if submicroscopic amplifications of 20q13 might be present. The following BAC clones were used: RP4-724E16, mapping to 20q13.12~20q13.32 and directly labeled with FITC; and RP11-445H22, mapping to 20q13.1 and directly labeled with Cy3. FISH results confirmed the presence of i(20q) in the NE108/E6E7 cells (Fig. 2B), and dup(20) in NE3/
Two events related to chromosomal instability (e.g., TTAGGG-negative telomere termini and anaphase bridges) were investigated in NE108/E6E7. Both peaked in frequency during crisis. The TTAGGG-negative chromosomal termini increased to 6.0 6 1.2 and 4.7 6 1.5 per cell, accounting for 6.7 and 5.3% of total chromosome ends at PD28 and PD34, respectively (Fig. 2D). The number was decreased to 2.6 6 0.7 per cell (3 6 0.7%) at PD 94 (P!0.001; Table 3). At PD28 and PD34, the frequency of anaphase bridges was up to 28–31% of mitotic cells (Fig. 2E), but decreased to 3.6% at PD 94 (P!0.001). These findings correlated well with the frequencies of telomere association, dicentric chromosomes, and nonclonal chromosomal changes observed, which were increased during crisis but decreased after immortalization. 3.10. Cytogenetic profiles of esophageal cancer cell lines Cytogenetic analyses were performed in five esophageal squamous cell carcinoma lines. All five lines showed highly complex karyotypes with numerous numerical and structural aberrations (Table 2). The aberrations were apparently nonrandomly distributed (Fig. 3). Losses of chromosome material occurred more often than gains. The bands or regions preferentially involved in losses were 1p11~1pter, 3p11~3pter, 4pter~4qter, 8p21~8pter, 9q11~9qter, 11q13~11qter, 13pter~13qter, 15q11~qter, 18p11~18pter, 21pter~21qter, 22pter~22qter, and Xp11~Xpter. The bands or regions of gains were frequently found in 3q21~3qter, 6p21~6pter, 7p11~7pter, and 8q10~8qter. Homogeneously staining regions (hsr), the cytogenetic sign of high-level gene amplification, were found in chromosome 11q in two lines and in chromosome 17p in one line. The hsr was interpreted as der(11)del(11)(q13)hsr(11)(q13) in HKESC-1, der(11)add(11)(p15)dup(11)(q13;q21) hsr(11)(q13) in KYSE140, and der(17)(p11)hsr(17) (p11) in SLMT-1. FISH with the use of a CCND1 probe showed the amplification of CCND1 in HKESC-1 with a der(11)del(11)(q13)hsr(11)(q13) (Fig. 2F) and KYSE-140 with a der(11)add(11)(p15) dup(11)(q13;q21)hsr(11)(q13). 3.11. Cytogenetic similarities and differences between immortalized esophageal cell lines and esophageal carcinoma cell lines Karyotypes of esophageal cell lines were compared with those of esophageal carcinoma cell lines investigated in this study and the primary esophageal carcinomas we had investigated previously [16]. Karyotypic complexity was far more pronounced in carcinoma than in immortalized
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
30
A
PD21
PD28, 34, 40
46,XY
PD47 47,XY,+20
47,XY,+20
PD54A,B,C
Chromosomal instability
PD54A,B,C
PD64C, PD94 A,B
47,XY,+i(20)(q10)
PD94A 48,XX,+7,+i(20)(q10)
PD54, 64, 94 A,B,C
PD94A 48,XX,+16,+i(20)(q10)
PD94B
48,XY,+i(20)(q10)×2
47,XY,+20
PD64C
48,XY,+20,+i(20)(q10)
PD47
PD64A,B
47,XY,+20
48,XY,+20,+i(20),add(12)(q24) PD94B 49,XY,+i(20)(q10)×2,del(3)(p11)
PD94A,C 46,XY,+i(20)(q10),-22
47,XY,+i(20)(q10) PD94B
48~49,XY,+5,+i(20)(q10)
47,XY,del(11)(q13),+i(20)(q10)
PD94B 49,XY,+5,der(12)t(Y;12),+ i(20)(q10),+20
PD94A,B,C 46,XY,del(11)(q13),+i(20)(q10), -22 PD94A,B,C 46,XY,del(3)(p11),del(11)(q13),+ i(20)(q10),-22
B
PD12
PD24
46,XY
47,XY,+20
PD100 PD24 47~49,XY,+5,+20
PD24 47,Y,dic(X;21)(p22;q22), Y,+5,+20 PD40 47,Y,dic(X;21)(p22;q22),Y,+5 der(8;14)(q10;q10),+20
PD86, PD 96 47~48,Y,dic(X;21)(p22;q22),Y, +5,der(8;14)(q10;q10),der(15;21) (q10;q10),+der(20)ins(20;?) (q13;?)t(1;20)(p13;q13),+20
47~48,Y,dic(X;21)(p22;q22),Y,der(4) t(4;4)(p16;q27),+5,der(8;14)(q10; q10),der(15;21)(q10;q10),+20
PD100 47~48,Y,dic(X;21)(p22;q22),Y,der(4) t(4;4)(p16;q27),+5,+7,der(8;14) (q10;q10),der(15;21)(q10;q10),+20
PD96 47~48,Y,dic(X;21)(p22;q22),Y,+5, +7,der(8;14)(q10;q10),der(15;21) (q10;q10),+20 PD96, PD114 PD100 47~48,Y,dic(X;21)(p22;q22),Y,+5, der(8;14)(q10;q10),der(15;21) (q10;q10),+20
47,Y,dic(X;21)(p22;q22),Y,+5,+7, der(8;14)(q10;q10),der(15;21) (q10;q10),+dup(20)(q13q11)
PD130 47,Y,dic(X;21)(p22;q22),Y,+5,+7, der(8;14)(q10;q10),der(15;21) (q10;q10),+dup(20)(q13q11) Fig. 1. Proposed scenario of the presumed karyotypic evolution. (A) NE108/E6E7 cell line. (B) NE3/E6E7 cell line.
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Fig. 2. FISH and telomere dysfunction of immortalized esophageal cells. (A) Representative COBRA FISH karyogram of NE108/E6E7, showing the del(3)(p11), del(11)(q13), 1i(20)(q10) and 222. (B) Metaphase of NE108/E6E7 (PD 54) hybridized with specific 20q probes, RP4-724E16 (FITC, green), and RP11-445H22 (Cy3, red). Chromosomes were stained with DAPI. Two sets of 20q signals could be identified at both arms of one chromosome 20, indicating isochromosomes 20q. The other two chromosomes 20 were normal. (C) Metaphase of NE3/E6E7 (PD 114) hybridized with the same specific 20q probes. Arrow indicated the inverted duplication of 20q11~q13 material. (D) Metaphase of NE108/E6E7 (PD28, crisis) hybridized with FITC-conjugated PNA probe. Telomeric TTAGGG repeats (green) were visualized at the ends of chromosomes. Chromosomal ends without green color represent the negative telomere termini. (E) Anaphase bridge in NE108/E6E7, PD28. (F) Metaphase of HKESC-1 hybridized with a CCND probe. The arrow indicated the hsr in 11q13.
esophageal cells. Some karyotypic similarities, however, could be identified. Both immortalized esophageal cell lines and cancer cell lines displayed numerical and unbalanced structural rearrangements that affected the centromeric regions in particular. The common rearrangements identified in esophageal carcinomas (i.e., deletion of 3p or loss of 3p material, i(8q), and structural rearrangements affecting the centromere of chromosome 8, 13, 14, and 21) were found in NE108/E6E7 and NE3/E6E7 cells.
4. Discussion In this study, we characterized four HPV16E6/E7 and one hTERT immortalized esophageal cell lines by cytogenetic and molecular cytogenetic techniques. To our knowledge, it is the first study on the dynamic cytogenetic changes in immortalized esophageal cell lines. Our results showed that in all cell lines with the expression of E6/E7, there was a progressive increase of chromosomal
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6
8
64
102
KYSE140
SLMT-1
EC-1
EC-18
58~66,X,2X,2Y,del(1)(p32),add(1)(p36),del(2)(q33),23,add(3)(p11),24,24,24,add(5)(p15),der(7)t(3;7)(p13;p22),add(7)(q36),28,29,der(11)del(11)(q13) hsr(11)(q13), 212,213, add(13)(p11),add(14)(p11),215,216,217,218,dic(19;20)(p13;q13),221,add(21)(p11),222,inc[6]/62~65,idem,add(X)(p11)[cp2]/58~63,idem,2add(5)(p15),add(10)(p11)[cp3]/ 60~66,idem,2add(5)(p15),add(10)(p11),del(11)(p11)[cp3]/58~60,idem,2add(5)(p15),add(10) (p11),der(10)t(3;10)(q21;p15),del(11)(p11)[cp3]/62~63,X,add(X)(p11),2Y,del(1)(p32), add(1)(p36),del(2)(q33),add(3)(p11)2,24,24,24, 1add(6)(p23),der(7)t(3;7)(p13;p22),add(7)(q36),28,29,der(11)del(11)(q13) hsr(11)(q13),212,add(13)(p11), add(14)(p11),215,216,217,dic(19;20)(p13;q13), add(19)(p13),221,222,inc[cp2] 57~66,X,2X,Y,der(1)t(1;8)(q25;q22),add(1)(q42),22,der(3;13)(q10;q10),del(3)(q13),1der(3;5)(q10;p10),24,der(4)t(4;15)(p16;q22),26,add(6) (q13), der(7)t(1;7)(q32;q22), add(7)(q36),28,add(9)(q34),del(9)(p21),del(9)(p11),1add(9)(q11),dic(11;?)(p11;?),der(11)add(11)(p15)dup(11)(q13;q21) hsr(11)(q13),212,add(12)(q15),213,213, add(13)(p11),214,215,der(15)t(6;15)(p11;p11),1add(16)(p11),1add(17)(q24),218,add(18)(q21),219,add(19) (q13),220,221,222,222,inc[cp20] 72~81,XX,2X or Y,der(X;1)(q10;q10),der(1;14)(q10;q10),i(3)(q10),add(3)(p11),del(3)(q11),dup(4)(q21q31)2,1del(4)(q21q31),der(5;14)(q10;q10) 2,1i(5p)1~2, 1der(6;7)(p10;p10),i(6)(p10),17,der(8)t(3;8)(q21;p23)2,18,i(9)(p10)2,del(9)(q11),add(10)(p11)2,110,del(11)(q14),1del(11)(q11)2,1der(12;14)(q10;q10),213,213, add(13)(p13),der(?5;15)(q10;q10)2,116,217,218,tri(18)(q11q23),der(18)tri(18)(q11q23)t(1;18)(p31;p11),der(18) t(1;18)(p31;p11),219,add(?20)(p11)2,221,221, 122,1mar1,1mar2[cp6]/73~74,idem,2der(6;7)(q10;q10),1i(7)(p10),2der(18)tri(18)t(1;18) [cp2]/75~79,idem,2der(X,1),add(1)(p11),2i(6)(p10),2der(6;7), 2der(8)t(3;8), add(8)(p23),1i(8)(q10),del(10)(q22),inv(11)(p15q13),2der(12;14), add(13)(p11),2der(18)tri(18)t(1;18),1tri(18)[cp5]/69~72,idem,2der(X;1),2der(1;14),add(1)(p11)2, 2der(6;7),der(17)(p11)hsr(17)(p11),2der(18)tri(18)t(1;18),1tri(18)[cp4] 84~90,XX,2X or Y,der(1;3)(q10;q10),23,23,24,16,del(7)(q32)2,i(8)(q10)2,19,add(10)(p11),213,214,215,215,1add(16)(q11)1~2,217,218,120,120,221,221, add(?21)(p11)2,222,222,1mar1~2,cp[16] 50~51,X,2X or Y,del(1)(q42),dup(2)(p21;p23),add(3)(q11),add(3)(q21),1add(3)(q21),del(4)(p14),ins(5;?)(q13;?),1del(5)(p11),add(6)(p23),1der(6;15)(p10;q10),1del(7)(q22), add(13)(p11),add(14)(p11),add(15)(p11),add(21)(p13),add(21)(p13),t(4;22)(q21;p11)[cp8]/48~50,idem,add(16)(q24)[5]/50~51,idem,2add (15)(p11)[3] 70 HKESC-1
Karyotypes Passages Lines
Table 2 Cytogenetic analyses of five esophageal cancer cell lines
32
aberrations during the early stage of crisis, with greater genetic instability and cytogenetic divergence. Telomere associations and dicentric chromosomes were the major aberrations affecting many chromosomes. In the later stage of crisis, all cells carried aberrations, but the karyotypes were more convergent with an increased karyotypic complexity. All cell lines surviving crisis had nonrandom clonal chromosomal changes that dominated the immortalized cells. A possible interpretation is that most of the chromosomal rearrangements generated during the crisis are random and have little or no impact on cell immortalization. However, some chromosomal aberrations may promote cellular proliferation, so that cells that acquire these aberrations are able to overcome the crisis. Such aberrations are therefore likely to persist after cellular immortalization. In HPV16E6/E7-immortalized esophageal cells, the most consistent finding was over-representation of a part or the whole of chromosome 20q, either as gain of the entire chromosome 20 (NE083/E6E7 and NECA6/E6E7), or as isochromosome 20q or duplication of 20q (NE108/E6E7 and NE3/E6E7). The nonrandom occurrence of chromosome 20q gain strongly suggests that this may contribute to esophageal cell immortalization. We have recently detected a high-level amplification of chromosome 20, in the form of a homogeneously staining region (hsr), in an SV-40 immortalized normal nasopharyngeal cell line [17]. Furthermore, using FISH and cytogenetic studies of five immortalized human ovarian surface epithelial cell lines, we have also identified high-level amplification of chromosomal material from band 20q13 in two of the lines [18]. Copy number increase or amplification of 20q was also a consistent observation in HPV16E6/E7 immortalized human bronchial epithelial cells and uroepithelial cell lines [19,20]. Taken together, these investigations provided strong evidence supporting the important role of chromosome 20 amplification as a nonrandom genetic event in cellular immortalization. In hTERT-transfected NE cells, genetic stability was demonstrated throughout the immortalization process. Amplification of chromosome 5q material was the main abnormality observed after immortalization. Gain of chromosome 5 is a common finding in immortalized cell lines and tumors [21,22]. This aberration may involve another mechanism that facilitates immortalization. We further investigated five esophageal cancer cell lines in this study and reviewed aberrations in previously reported primary esophageal carcinomas [16], and compared the aberrations in immortalized esophageal cell lines with those in esophageal cancer cell lines and primary carcinomas. The karyotypic profiles of immortalized esophageal cells lines differed to some extent from that of esophageal carcinomas. The karyotypic complexity was more pronounced in carcinoma than in the immortalized esophageal cells. These findings suggested that during multistage tumorigenesis, multiple genetic changes were necessary for malignant transformation and tumor progression. In spite
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Table 3 Telomeric status and anaphase bridges of NE108/E6E7 Negative telomere termini Population doublings
Chromosomes in metaphase
21 28 34 40 47 54 64 94
40.4 44.7 44.8 42.7 42.1 44.2 42.7 44.3 a b
6 6 6 6 6 6 6 6
3.5 1.0 1.4 3.2 2.0 2.2 4.2 2.3
Anaphase bridges Negative termini 1.5 6.0 4.7 4.0 3.7 2.6 2.7 2.6
6 6 6 6 6 6 6 6
Frequency
0.4 1.2 1.5 1.3 0.6 0.9 0.9 0.7
1.8 6.7 5.3 4.7 4.4 2.9 3.2 3.0
6 6 6 6 6 6 6 6
a
0.4% 1.4% 1.7% 1.5% 0.8% 1.1% 0.9% 0.7%
Total cells analyzed
Bridges
Frequencyb
50 55 50 60 60 55 50 55
1 17 14 16 14 9 6 2
2.00% 30.9% 28.0% 26.7% 23.3% 16.4% 12.0% 3.6%
Student-Newman-Keuls Test, P ! 0.001. Pearson Chi-square, c2 5 28.921, P ! 0.001.
chromosomes 8, 13, 14, and 21, would presumably contribute to the early step of esophageal carcinogenesis. Telomere dysfunction and defective DNA damage response may induce chromosomal instability [23]. In the absence of DNA repair response, unstable telomere repeats may lead to fusions of chromosome ends that, in turn, trigger the breakage–fusion–bridge (BFB) cycle. The model of the BFB cycle, originally described by McClintock in 1938, is speculated to be one of the mechanisms behind genetic instability observed in tumor cells and has been supported recently by some evidence [24]. The parallel assessment
of the karyotypic complexities, some similarities were observed. The most common imbalance detected in esophageal carcinomas (e.g., loss of 3p material through deletion or unbalanced translocations) was also found in later passages of NE108/E6E7 as del(3p11) and in NE083/E6E7 as del(3p21). Also, structural rearrangements involving the centromere (e.g., isochromosomes and whole-arm translocations), a characteristic feature of cancer cells, were also present in some of the esophageal cell lines with the expression of E6E7. These genetic hits, such as del(3p), structural rearrangements involving the centromeric regions of
1
2
3
6
7
8
13
14
15
19
20
9
4
5
10
11
12
16
17
18
2
21
22 X
Y
Fig. 3. Distribution of karyotypic imbalances caused by numerical or structural changes in five esophageal cancer cell lines. Vertical lines on the left of chromosome indicated loss of chromosome region(s) and lines on the right gain of chromosome region(s).
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20q11 20q13
Chromosome 20
Aberration
Breakage
Replication
Bridge
Fusion
Cytokinesis
Fig. 4. A schematic illustration of the BFB cycle leading to inverted duplication of 20q11~20q13 in the NE3/E6E7 line. An initial break distal to 20q13 led to sister chromotid fusion after replication. The two centromeres were pulled to opposite directions, generating a bridge between one centromere and an adhered segment. The centromere was cleaved finally at telophase, giving rise to the novel aberrant chromosome, which contained the duplicated material of q11~q13.
of telomere status, BFB events, and cells with anaphase bridges, as well as tas and dic chromosomes, were performed in NE108/E6E7 cells. Our results supported the telomere and BFB hypotheses of immortalization. During morphologic cell crisis, a high frequency of negative termini was recorded, accompanied by a significant increase of cells with tas and dic chromosomes and anaphase bridges. After crisis, the mean number of negative telomeres decreased, leading to a drop in cells with tas and dic chromosomes and anaphase bridges in post-crisis and immortalized cells. At the same time, there was a shift of the chromosomal aberration pattern from nonclonal chromosome changes and unrelated clones to nonrandom and more complex clonal changes. These stable clonal chromosome aberrations persisted after crisis. The preferential occurrence of centromeric aberrations (i.e., whole-arm translocations and isochromosomes) might also be caused by BFB events. A possible interpretation is that the centromeres of some chromosomes are more vulnerable than the others and may be broken during the separation of the two different centromeres toward opposite spindle poles. The broken centromeres may engage in fusions, giving rise to isochromosomes if centromeres of two sister chromatids are involved, or whole-arm translocations if two different chromosomes are involved. An inverted duplication dup(20)(q13q11) in the NE3/E6E7 line was another strong piece of evidence supporting the BFB model. In this model, an initial break distal to 20q13 led to sister chromatid fusion after replication. The two centromeres were pulled to opposite directions, generating a bridge between one centromere and an adhered segment. The centromere was finally cleaved at telophase, giving rise
to the novel aberrant chromosome (Fig. 4). A possible consequence of repetition of this cycle may be the formation of hsr, which is frequently observed in cancer cells. Our observations further support the notion that chromosomal instability resulting from BFB cycles contributes to the formation of some genetic aberrations that are essential for cell immortalization and malignant transformation.
Acknowledgments This study was supported by the Kadoorie Charitable Foundation, research grants from the University of Hong Kong, and grants from the Swedish Cancer society. References [1] Blot WJ. Esophageal cancer trends and risk factors. Semin Oncol 1994;21:403–10. [2] Lam KY, Ma L. Pathology of esophageal cancers: local experience and current insights. Chin Med J (Engl) 1997;110:459–64. [3] Pohl H, Welch HG. The role of overdiagnosis and reclassification in the marked increase of esophageal adenocarcinoma incidence. J Natl Cancer Inst 2005;97:142–6. [4] Munro AJ. Oesophageal cancer: a view over overviews. Lancet 2004;364:566–8. [5] Mitelman F, Johansson B, Mertens F. Mitelman Database of Chromosome Aberrations in Cancer. Available at: http://www.cgap.nci.nih. gov/Chromosomes/Mitelman. Accessed in May 2005. [6] Hu YC, Lam KY, Wang TS, Fang W, Ma ES, Chan LC, Srivastava G. Establishment and characterization of HKESC-1, a new cancer cell line from human esophageal squamous cell carcinoma. Cancer Genet Cytogenet 2000;118:112–20. [7] Hu YC, Lam KY, Law SY, Wan TS, Ma ES, Kwong YL, Chan LC, Wong J, Srivastava G. Establishment, characterization, karyotyping,
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[17] Zhang H, Tsao SW, Jin C, Strombeck B, Yuen PW, Kwong YL, Jin Y. Sequential cytogenetic and molecular cytogenetic characterization of an SV40T-immortalized nasopharyngeal cell line transformed by Epstein-Barr virus latent membrane protein-1 gene. Cancer Genet Cytogenet 2004;150:144–52. [18] Jin Y, Zhang H, Tsao SW, Jin C, Lv M, Strombeck B, Wiegant J, Wan TS, Yuen PW, Kwong YL. Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20. Gynecol Oncol 2004;92:183–91. [19] Coursen JD, Bennett WP, Gollahon L, Shay JW, Harris CC. Genomic instability and telomerase activity in human bronchial epithelial cells during immortalization by human papillomavirus-16 E6 and E7 genes. Exp Cell Res 1997;235:245–53. [20] Savelieva E, Belair CD, Newton MA, DeVries S, Gray JW, Waldman F, Reznikoff CA. 20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells. Oncogene 1997;14:551–60. [21] Dowen SE, Neutze DM, Pett MR, Cottage A, Stern P, Coleman N, Stanley MA. Amplification of chromosome 5p correlates with increased expression of Skp2 in HPV-immortalized keratinocytes. Oncogene 2003;22:2531–40. [22] Ueno T, Tangoku A, Yoshino S, Abe T, Toshimitsu H, Furuya T, Kawauchi S, Oga A, Oka M, Sasaki K. Gain of 5p15 detected by comparative genomic hybridization as an independent marker of poor prognosis in patients with esophageal squamous cell carcinoma. Clin Cancer Res 2002;8:526–33. [23] Gollin SM. Chromosomal instability. Curr Opin Oncol 2004;16: 25–31. [24] Gisselsson D, Jonson T, Petersen A, Strombeck B, Dal CP, Hoglund M, Mitelman F, Mertens F, Mandahl N. Telomere dysfunction triggers extensive DNA fragmentation and evolution of complex chromosome abnormalities in human malignant tumors. Proc Natl Acad Sci USA 2001;98:12683–8.
Cytogenetic aberrations in immortalization of esophageal epithelial cells Hao Zhanga, Yuesheng Jinb, Xuehua Chena, Charlotte Jinb, Simon Lawc, Sai-Wah Tsaod, Yok-Lam Kwonga,* a
Department of Medicine, University of Hong Kong, Professorial Block, Pockfulam Road, Hong Kong, China b Department of Clinical Genetics, University Hospital, 22 Klinikgatan, S-22185, Lund, Sweden c d Department of Surgery, Department of Anatomy, University of Hong Kong, Hong Kong, Professorial Block, Pockfulam Road, Hong Kong, China Received 8 June 2005; received in revised form 8 July 2005; accepted 20 July 2005
Abstract
To define the early cytogenetic events important in esophageal carcinogenesis, we immortalized normal esophageal epithelial cells by overexpression of human papillomavirus type 16 E6/E7 (HPV16E6/E7) and human telomerase reverse transcriptase (hTERT), and characterized the chromosomal abnormalities serially before and after cellular immortalizaiton. During crisis, most cells had simple nonclonal karyotypic changes with cytogenetic divergence. Mitotically unstable chromosomes (i.e., telomere association and dicentric chromosomes) were the most common aberrations. After crisis, the karyotypic patterns were more convergent with nonrandom clonal changes. A few clones dominated the culture. Gain of chromosome 20q was consistently observed in four HPVE6/E7 immortalized esophageal lines, whereas amplification of chromosome 5q was preferentially found in hTERT immortalized cells. In addition, chromosomal aberrations of immortalized cells, including del(3p) and centromere rearrangements, were similar to those observed in esophageal cancer. Furthermore, in E6/E7-expressing cells, the frequency of negative telomere termini and anaphase bridges were high during crisis and low after crisis. These findings suggested that telomere dysfunction might be an important cause of cellular crisis, and the resultant chromosomal aberrations, mainly amplification of chromosome 20q or 5q, might be early genetic events required in esophageal cell immortalization. These alterations might be valuable models for further study of molecular mechanisms contributing to esophageal carcinogenesis. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Esophageal carcinoma is the ninth most common cancer worldwide and the second in China [1]. In Hong Kong, approximately 90% of esophageal carcinoma is squamous cell carcinoma (SCC), and the overall five-year survival rate remains low [2]. In Western countries, the incidence of esophageal adenocarcinoma has increased dramatically in recent years [3], with more than 85% of patients dying within 2 years of diagnosis [4]. Although esophageal cancer is an important malignancy, the genetic mechanisms underlying carcinogenesis have not been well defined, partly due to the difficulties in culturing esophageal tumor cells in vitro and the lack of suitable cell models. Cytogenetic information is available in 42 cases only [5]. Most of these tumors displayed complicated cytogenetic aberrations. Also, a few esophageal cancer cell
* Corresponding author. Tel.: 1852-2-855-4597; fax: 1852-2-9741165. E-mail address: [email protected] (Y.-L. Kwong). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.07.016
lines available were mostly derived from advanced esophageal cancer and were characterized by highly complex karyotypes [6–9]. The relative importance of these complex aberrations in esophageal carcinogenesis remains undefined. Recent studies have suggested that infection with the high-risk human papillomavirus type 16 E6/E7 (HPV16E6/E7) might play an important pathogenetic role in esophageal SCC in Chinese patients [10]. Progressive up-regulation of telomerase, however, appears to be the important feature of adenocarcinoma in the west [11]. Esophageal cells can be immortalized in vitro by the overexpression of the oncoprotein HPV16 E6/E7 or the catalytic subunit of human telomerase reverse transcriptase (hTERT) [12]. As immortalization may be an initial step of carcinogenesis, the study of cytogenetic changes associated with immortalization may help to define the genetic events critical for neoplastic transformation. In this study, esophageal cell lines obtained by immortalization with transfection of the HPV16E6/E7 and hTERT genes were characterized serially before and after
26
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immortalization. The karyotypic profiles of these cell lines were also compared with those of esophageal carcinoma cell lines. Finally, the relationship between the telomere status and chromosomal instability was evaluated in one of the HPV16E6/E7-expressing cell lines.
2. Materials and methods 2.1. Immortalization of normal esophageal epithelial cells The method of primary culture and immortalization of normal epithelial cell lines [13] and the establishment and maintenance of the esophageal cancer cell lines HKESC1 [6], KYSE-140 [8], SLMT-1 [9], EC-1, and EC-18 were as described previously. Five cell lines, four with the expression of HPV16E6/E7, NE108/E6E7, NE083/E6E7, NE3/E6E7, and NECA6/E6E7, and one with hTERT and NE083/hTERT, were newly immortalized. 2.2. Cytogenetic analysis The cell cultures were harvested by conventional cytogenetic techniques. Briefly, cells were blocked at metaphase by exposure to colcemid (0.025 m/mL; GIBCO, Invitrogen Corp., Paisley, UK) for 14 hours. After hypotonic and fixative treatment, the cell suspension was spread on slides, and the metaphase chromosomes were G-banded with Wright stain. The clonality and karyotypes were described according to the International System for Human Cytogenetic Nomenclature (1995) [14]. 2.3. Fluorescence in situ hybridization (FISH) Multicolor combined binary ratio labeling (COBRA) FISH was performed as described [15]. Briefly, slides with metaphase chromosomes were pretreated with pepsin and formaldehyde. Probe mix (8 mL) containing 24 human chromosomes labeled with four distinct fluorochromes by the combination of binary and ratio labeling was applied on the slide. The slide and probe were denatured simultaneously by incubation at 72 C on a hot plate. After hybridization for 48–72 hours at 37 C in a humid chamber, the slides were washed in 74 C 0.4 standard saline citrate (SSC) for 2 minutes. Chromosomes were counterstained by TNT (0.1 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20 in 100 mL of phosphate buffered saline) and 4’,6-diamidino phenylindole (DAPI) (0.5 mg/mL) for 10 minutes. The slides were embedded in Citifluor (Ted Pella, Redding, CA) before microscopic evaluation. For analysis, an Axioplan-2 microscope (Carl Zeiss GmbH, Jena, Germany) coupled to a cooled charge-coupled device camera and a 12-position filter wheel was used. Acquired images were evaluated with the CytoVision ChromoFluor System (Applied Imaging, Newcastle, UK) [15].
Based on cytogenetic findings, specific bacterial artificial chromosomes (BAC) were selected for further FISH analysis (http://www.ncbi.nlm.nih.gov/cgi-bin/Etreez/mapsearch, 2005). The labeled probes were purified, precipitated, and dissolved in standard hybridization solution. The slide and probe were denatured simultaneously by incubation at 72 C on a hot plate. After hybridization overnight at 37 C in a humid chamber, the slides were washed in 74 C 0.4 SSC for 2 minutes, DAPI-stained, and analyzed. Telomeric TTAGGG repeats were detected using telomeric FISH with a fluorescein-conjugated (CCCTAA)3 peptide nucleic acid (PNA) probe. The number of negative chromosome termini for a metaphase cell was recorded. At least 20 cells were evaluated per case. 2.4. Mitotic cell morphology For analysis of mitoses, cells cultured on chamber slides were washed in phosphate-buffered saline (PBS) for 5 minutes, fixed in methanol/acetic acid (3:1) at 220 C for 30 minutes, air-dried, and stained with hematoxylin (Accustain; Sigma, St. Louis, MO) and erythrosine (BDH, Poole, England). At least 50 anaphase cells were examined per population doublings (PD). An anaphase cell showing at least one string of chromatin connecting the poles was defined as harboring an anaphase bridge. 3. Results 3.1. Sequential cytogenetic characterization of immortalized normal esophageal cells To identify cytogenetic changes potentially relevant to NE cell immortalization, sequential karyotyping was performed in four HPV16E6/E7 and one hTERT immortalized cell line. The cytogenetic profiles of the five NE cell lines are summarized in Table 1. 3.2. NE108/E6E7 This line was thoroughly studied during immortalization. At PD21 (before crisis), most cells were karyotypically normal, although a few nonclonal chromosomal changes began to appear in 10.4% (5/48) of metaphase cells. At PD28–PD34 (crisis), cells with normal chromosome complement dropped to less than 50%, whereas cells harboring chromosome aberrations increased. At this stage, a considerable portion of metaphase cells showed nonclonal aberrations, but a few cytogenetically unrelated clones emerged. Chromosomal changes were highly heterogeneous, with nearly 30% of cells displaying mitotically unstable chromosomes [e.g., telomere association (tas), dicentric (dic), and ring (r) chromosomes]. Centromeric rearrangements (e.g., isochromosomes and whole-arm translocations) were also common. During immortalization (PD41–PD64), cells with related clonal chromosomal changes expanded, whereas
Table 1 Sequential cytogenetic analysis of five immortalized esophageal epithelial cell lines Cells containing mitotically unstable chromosome
48 44 25 27 28
0(0) 10(22.7) 8(32.0) 12(44.4) 21(75.0)
5(10.4) 16(36.4) 9(36.0) 11(40.7) 5(17.9)
0 11 5 4 3
0 2 1 3 1
0 0 1 0 1
0(0) 13(29.5) 7(28.0) 7(25.9) 5(17.9)
4(4) 3(3) 3(3) 6(4)
54 A 54 B
17 21
13(76.5) 18(85.7)
4(23.5) 3(14.3)
1 2
1 1
1 0
3(17.6) 3(14.3)
4(1) 7(3)
54 C 64 A 64 B
16 15 19
13(81.2) 13(86.7) 17(89.5)
3(18.8) 2(13.3) 2(10.5)
1 1 2
2 0 0
0 0 0
3(18.8) 1(6.7) 2(10.5)
3(1) 3(3) 5(5)
64 C
14
12(85.7)
2(14.3)
1
0
0
1(7.1)
6(3)
94 A
38
37(97.4)
1(2.6)
1
1
0
2(5.3)
8(0)
94 B
33
32(97.0)
1(3.0)
0
0
0
0(0)
94 C
40
39(97.5)
1(2.5)
1
4
0
5(12.5)
5(0)
37 46
0 32(86.5) 43(93.5)
0 3(8.1) 3(6.5)
0 0 4
0 0 4
0 0 0
0 0(0) 8(17.4)
4(2) 6(2)
59
59(100)
0(0)
1
1
2(1.7)
4(1)
46 77 50
0 49(63.6) 36(72.0)
0 6(7.8) 0(0)
0 1 0
0 0 0
0 0 0
0 1(1.3) 0(0)
2(2) 2(2)
15 24 15 16
0 18(75.0) 11(73.3) 12(75.0)
0 4(16.7) 4(26.7) 4(25.0)
0 1 1 0
0 2 0 9
0 1 0 0
0 4(16.7) 1(6.7) 9(56.2)
1(0) 3(0) 1(0) 1(0)
Clones Cells (unrelated Karyotypes analyzed Clonal(%) Nonclonal(%) Tas Dic R Total(%) clones)
NE108/E6E7 21 28 34 40 47
NE083/E6E7 18 33 46 59 NE083/hTERT 37 60 70 NE3/E6E7 12 24 40 86
12(1)
46,XY[43] 47,XY,120[cp4]/46,XY,i(8)(q10)[2]/45,XY,tas(12;13)(q24;p13)[2]/45,XY,der(13;16)(q10;q10)[2] 47,XY,120[cp5]/43,XY,der(8;16)(q10;q10)[cp2]/46,XY,i(8)(q10)[1] 47,XY,120[6]/43~45,XY,222[cp4]/42~43,XY,i(8)(q10)[2] 45~46,XY,i(8)(q10)[cp5]/46~47,XY,120[cp4]/47,XY,1i(20)(q10)[3]/44~45, XY,222[3]/ 42~43,XY,der(8;11)(q10;q10),222[cp3]/42~44,XY,der(8;13) (q10;q10)[cp3] 47,XY,120[6]/47,XY,1i(20)(q10)[5]/48,XY,120,1i(20)(q10)[1]/47,XY,1i(8)(q10)[1] 47,XY,1i(20)(q10)[3]/48,XY,1i(20)(q10)2[5]/48,XY,120,1i(20)(q10)[2]/ 47,XY,120[4]/45,XY,222[2]/ 46,XY,i(8)(q10)[1]/46,XY,der(8;11)(q10;q10) [1] 47,XY,1i(20)(q10)[10]/48,XY,120,1i(20)(q10)[2]/47,XY,120[1] 47,XY,120[6]/45~47,XY,1i(20)(q10)[cp6]/46,XY,der(8;11)(q10;q10)[1] 46~47,1i(20)(q10)[cp6]/43~44,XY,der(8;11)(q10;q10),222[cp5]/46,XY,i(8) (q10)[3]/47,XY,120[2]/ 46,XY,der(8;13)(q10;q10)[1] 45~47,XY,1i(20)(q10)[cp3]/45~46,XY,1i(20)(q10),add(11)(q11)[cp3]/48,XY,120,1i(20)(q10),add(12)(q24)[1]/ 48,XY,1i(20)(q10)2[3]/48~49,XY,11,120,add(13)(p11)[1]/43,XY,der(8;11)(q10;q10),222[1] 45~46,XY,1i(20)(q10),222[2]/46,idem,del(11)(q13)[21]/46,idem,del(3)(p11),del(11)(q13)[4]/45~48, XY,1i(20)(q10)[cp6]/48,XY,17,1i(20)(q10) [1]/48,XY,116,1i(20)(q10)[1]/48,XY,120,1i(20)(q10)[1]/ 48,XY,1i(20) (q10)2[1] 47,XY,1i(20)(q10)[2]/47,idem,del(11)(q13)[1]/46,idem,del(11)(q13),222[2]/46,idem,del(3)(p11), del(11)(q13),222[11]/46,idem,der(3;7)(q10;q10),del(11)(q13),222[2]/48~49,XY,15,1i(20)(q10)[cp5]/47~49, XY,15,der(12)t(Y;12)(q11;q24),1i(20)(q10),120[cp5]/49,XY,1i(20)(q10)2,add(3)(p11)[1]/ 47,XY,del(3)(p11),1i(20)(q10)[1]/46,XY,i(20)(q10)[1]/48,XY,1i(20)(q10),120 [1]/47,XY,120[1] 46,XY,1i(20)(q10),222[2]/46,idem,del(11)(q13)[16]/46,idem,del(3)(p11),del(11)(q13)[13]/46~48, XY,1i(20)(q10)[cp7]/47,XY,120[1] 46,XY[25] 47,XY,120[23]/48,XY,19,120[3]/47~48,XY,18[cp3]/47~48,XY,19[cp3]46,XY[2] 47,XY,120[27]/48,XY,19,120[6]/47,XY,del(3)(p21),120[4]/48,XY,del(3)(p21),19,120[1]/47,XY, 19,tas(9;22)[3]/46,XY,add(19)(q13),add(21) (p11)[2] 47,XY,120[11]/48,XY,19,120[7]/46,XY,add(19)(q13),add(21)(p11)[11]/ 88~91,XXYY,19,210,211,213,215,120,120,221,der(21)t(13;21)(q14,p11)[30]
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
Cells having chromosome changes
Lines/ Populations doublings
46,XY[46] 46,XY,dup(5)(q31q33)[46],46,XY,der(16)t(5;16)[3],46,XY[22] 46,XY,dup(5)(q31q33)[32],46,XY,der(16)t(5;16)[4],46,XY[14] 46,XY[15] 47~49,XY,15,120[cp13]/47,XY,120[3]/47,dic(X;21)(p22;q22),Y,15,120[2] 47,dic(X;21)(p22;q22),Y,15,der(8;14)(q10;q10),120[cp11] 46~47,dic(X;21)(p22;q22),Y,15,der(8;14)(q10;q10),der(15;21)(q10;q10), 1der(20)ins(20;?)(q13;?)t(1;20)(p13;q13)[cp12] 27
(Continued)
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
cells with nonclonal changes decreased drastically, parallel with a reduction of mitotically unstable chromosomes (i.e., tas and dic). In early crisis (PD28), a few cytogenetically unrelated, pseudo-, or near-diploid clones with simple numerical and structural aberrations, such as 120, i(8q), der(13;16), and tas(12;13), could be identified. Some of the clones, such as the clone with i(8q), survived in PD64 but were eventually subdued as a result of selection pressure. However, the clones carrying 120 survived. This clone expanded progressively and eventually took over the cultures. The clone with i(20q) also existed throughout the entire postcrisis culturing time (PD47–PD94), increased in frequency, and eventually dominated the cultures. At PD94, all clones identified were related by gain of chromosome 20 and/or isochromosome 20q, leading to an increased copy number of the long arm of chromosome 20, in addition to one to three other numerical and/or structural aberrations. At PD50, NE108/E6E7 cells were subcultured independently into three sublines and were cytogenetically analyzed to define genetic convergence or divergence during immortalization. An obvious decrease of cytogenetic divergence was found, with the initial polyclonality reduced to a monoclonality, with one clone making up to 97% of the cells. On the other hand, by PD94, owing to clonal evolution, the karyotypes became more complex in each clone, giving rise to several cytogenetic related clones. This was shown in all three sublines d A, B, and C d which were cultured independently. The dominant clone in all three sublines contained i(20q) with more changes, giving rise to a relatively complex karyotype [i.e., 46, XY, del(3)(p11), del(11)(q13), 1i(20)(q10), 222]. Abbreviations: Tas, Telomere association; Dic, Dicentric chromosome; R, ring.
2(0) 8(1) 0 0 0 0 0 0 0 0 0(0) 0(0) 22 37 92 110
22(100) 37(100)
0 30 60
28(93.3)
2(6.7)
0
0
0
6(0)
46,XY[27]/46,XY,120[1] 47,XY,120[cp17]/48,XY,120,120[cp6]/~58,XY,der(11;19)(q10,q10)[2] 48,XY,15,120[5]/48,XY,17,120[3]/49,XY,15,19,120[1]/45,XY,tas(12;14)(q24;p13),120[1]/ 43~46,XY,15[cp4]/ 47,XY,15,19,215 [1] 48,XY,120,120[9]/48,XY,19,120[6]/47,XY,120[5]/48,XY,15,120[4]/ 49,XY,15,19,120[2]/ 50,XY,15,19,120,120[cp2] 48,XY,15,t(7;8)(q22;q23),120[19]/46~47,XY,15,der(8;13)(q10;q10),120[cp3] 47,XY,120[10]/47,XY,t(7;8)(q22;q23),120[7]/48,XY,120,120[6]/48,XY,11,120[4]/47,XY, der(8;13)(q10;q10),120[3]/46,XY,t(7;8)(q22;q23)[3]/49,XY,17, 120,120[2]/49,XY,11,120,120[2] 1(0) 3(1) 6(2) 3(9.1) 2(5.3) 1(5.6) 0 0 0 1 2 1 1(3.0) 25(65.8) 15(83.3) 33 38 18
5(15.2) 5(13.2) 3(16.7)
2 0 0
22(78.6) 1(0) 13(72.2) 1(0) 0 0 22 13 0 0 4(14.3) 1(6.0) 24(85.7) 17(94.0)
114 130 NECA6/E6E7 18 24 40
28 18
0 25 100
19(76.0)
6(24.0)
16
0
16(64.0) 3(0)
46~47,Y,dic(X;21)(p22;q22),Y,15,17,der(8;14)(q10;q10),der(15;21)(q10;q10),120[2]/46~47,idem, 1dup(20)(q13q11)[4]/46~47,idem,-(17),111,1der(20)ins(20;?)(q13;?)t(1;20)(p13;q13)[7] 47~48,Y,dic(X;21)(p22;q22),Y,15,der(8;14)(q10;q10),der(15;21)(q10;q10),120[cp10]/ 47~48,idem,der(4)t(4;4)(p16;q27)[cp5]/47~48,idem,der(4)t(4;4)(p16;q27),17[cp4] 47,Y,dic(X;21)(p22;q22),Y,15,17,der(8;14)(q10;q10),der(15;21)(q10;q10),1dup(20)(q13q11)[cp24] 47,Y,dic(X;21)(p22;q22),Y,15,17,der(8;14)(q10;q10),der(15;21)(q10;q10),1dup(20)(q13q11)[cp17] 13(81.3) 3(0) 0 13 0 3(18.7) 13(81.3) 16 96
R Dic
Lines/ Populations doublings
Table 1 Continued
Cells having chromosome changes
Cells analyzed Clonal(%) Nonclonal(%) Tas
Cells containing mitotically unstable chromosome
Clones (unrelated Total(%) clones) Karyotypes
28
3.3. NE083/E6E7 At early PDs before crisis, the cells had normal karyotypes. At PD33, two out of four subclones related by an extra copy of chromosome 20 were observed. Clonal chromosomal changes accounted for 86.5% of post-crisis cells, with the percentage increasing with the culture propagation. Cells tended to be more homogenous with complex karyotypes, suggesting that the majority of cells were derived from the same progenitor. 3.4. NE3/E6E7 The NE3/E6E7 parental cells (PD12) showed a normal karyotype. At PD24, three clones related by an extra copy of chromosome 20 were detected, accounting for approximately 75% of cells analyzed. Nonclonal changes, including tas and dic, were found in about 25% of the cells. During crisis, genetic divergence led to cytogenetic heterogeneity in the form of several subclones developing from a clone in PD24. After immortalization, only one subclone dominated the culture. Apart from sharing several numerical and structural aberrations with the relevant subclone at
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
earlier passages, an extra derivative chromosome 20 with duplicate of 20q material was found, resulting effectively in a total of four copies of 20q.
29
E6E7 cells, where the duplicated fragment was inverted as dup(20)(q13q11) (Fig. 2C). 3.9. Mitotic abnormalities
3.5. NECA6/E6E7 In pre-crisis (PD18), the majority of NECA6/E6E7 cells had normal karyotypes, whereas 5 of 33 cells had nonclonal chromosome changes. In one of the cells, an extra copy of chromosome 20 was detected as the sole anomaly. Surprisingly, this cell expanded rapidly, becoming the dominating clone during crisis and the only clone surviving the crisis. More subclones deriving from this clone appeared after immortalization. 3.6. NE083/hTERT The overexpression of hTERT in esophageal epithelial cells was attempted several times with materials from different individuals. However, only one cell line overcame the crisis and was immortalized successfully. In contrast to the cells immortalized by HPV16E6/E7, this line was genomically stable during the immortalization process. At PD37, all metaphases analyzed had normal karyotypes. At PD70, approximately 60% of cells acquired a structural rearrangement, dup(5)(q31q33), and in a small clone der(16). No nonclonal aberrations, telomere association, or dicentric chromosomes were observed. 3.7. Clonal evolution observed in esophageal cell lines expressing E6/E7 Clonal evolution could be traced in cell lines immortalized by HPV16E6/E7. The potential evolutionary pathways are depicted in Fig. 1. Apparently, gain of chromosome 20 or chromosomal 20q, occurring in the early passages as the sole anomaly and present in all cells surviving the crisis, was an early nonrandom genetic event critical for cell immortalization. 3.8. FISH analysis COBRA-FISH analysis confirmed the cytogenetic interpretation of all structural and numerical rearrangements in NE108/E6E7 (Fig. 2A). The cytogenetic interpretation of dup(5)(q31q33) in NE083/hTERT was also confirmed. Further FISH analyses with the use of BAC clones spanning 20q were performed in NE108/E6E7 and NE3/E6E7 cells to verify i(20q) and dup(20q), and to define if submicroscopic amplifications of 20q13 might be present. The following BAC clones were used: RP4-724E16, mapping to 20q13.12~20q13.32 and directly labeled with FITC; and RP11-445H22, mapping to 20q13.1 and directly labeled with Cy3. FISH results confirmed the presence of i(20q) in the NE108/E6E7 cells (Fig. 2B), and dup(20) in NE3/
Two events related to chromosomal instability (e.g., TTAGGG-negative telomere termini and anaphase bridges) were investigated in NE108/E6E7. Both peaked in frequency during crisis. The TTAGGG-negative chromosomal termini increased to 6.0 6 1.2 and 4.7 6 1.5 per cell, accounting for 6.7 and 5.3% of total chromosome ends at PD28 and PD34, respectively (Fig. 2D). The number was decreased to 2.6 6 0.7 per cell (3 6 0.7%) at PD 94 (P!0.001; Table 3). At PD28 and PD34, the frequency of anaphase bridges was up to 28–31% of mitotic cells (Fig. 2E), but decreased to 3.6% at PD 94 (P!0.001). These findings correlated well with the frequencies of telomere association, dicentric chromosomes, and nonclonal chromosomal changes observed, which were increased during crisis but decreased after immortalization. 3.10. Cytogenetic profiles of esophageal cancer cell lines Cytogenetic analyses were performed in five esophageal squamous cell carcinoma lines. All five lines showed highly complex karyotypes with numerous numerical and structural aberrations (Table 2). The aberrations were apparently nonrandomly distributed (Fig. 3). Losses of chromosome material occurred more often than gains. The bands or regions preferentially involved in losses were 1p11~1pter, 3p11~3pter, 4pter~4qter, 8p21~8pter, 9q11~9qter, 11q13~11qter, 13pter~13qter, 15q11~qter, 18p11~18pter, 21pter~21qter, 22pter~22qter, and Xp11~Xpter. The bands or regions of gains were frequently found in 3q21~3qter, 6p21~6pter, 7p11~7pter, and 8q10~8qter. Homogeneously staining regions (hsr), the cytogenetic sign of high-level gene amplification, were found in chromosome 11q in two lines and in chromosome 17p in one line. The hsr was interpreted as der(11)del(11)(q13)hsr(11)(q13) in HKESC-1, der(11)add(11)(p15)dup(11)(q13;q21) hsr(11)(q13) in KYSE140, and der(17)(p11)hsr(17) (p11) in SLMT-1. FISH with the use of a CCND1 probe showed the amplification of CCND1 in HKESC-1 with a der(11)del(11)(q13)hsr(11)(q13) (Fig. 2F) and KYSE-140 with a der(11)add(11)(p15) dup(11)(q13;q21)hsr(11)(q13). 3.11. Cytogenetic similarities and differences between immortalized esophageal cell lines and esophageal carcinoma cell lines Karyotypes of esophageal cell lines were compared with those of esophageal carcinoma cell lines investigated in this study and the primary esophageal carcinomas we had investigated previously [16]. Karyotypic complexity was far more pronounced in carcinoma than in immortalized
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
30
A
PD21
PD28, 34, 40
46,XY
PD47 47,XY,+20
47,XY,+20
PD54A,B,C
Chromosomal instability
PD54A,B,C
PD64C, PD94 A,B
47,XY,+i(20)(q10)
PD94A 48,XX,+7,+i(20)(q10)
PD54, 64, 94 A,B,C
PD94A 48,XX,+16,+i(20)(q10)
PD94B
48,XY,+i(20)(q10)×2
47,XY,+20
PD64C
48,XY,+20,+i(20)(q10)
PD47
PD64A,B
47,XY,+20
48,XY,+20,+i(20),add(12)(q24) PD94B 49,XY,+i(20)(q10)×2,del(3)(p11)
PD94A,C 46,XY,+i(20)(q10),-22
47,XY,+i(20)(q10) PD94B
48~49,XY,+5,+i(20)(q10)
47,XY,del(11)(q13),+i(20)(q10)
PD94B 49,XY,+5,der(12)t(Y;12),+ i(20)(q10),+20
PD94A,B,C 46,XY,del(11)(q13),+i(20)(q10), -22 PD94A,B,C 46,XY,del(3)(p11),del(11)(q13),+ i(20)(q10),-22
B
PD12
PD24
46,XY
47,XY,+20
PD100 PD24 47~49,XY,+5,+20
PD24 47,Y,dic(X;21)(p22;q22), Y,+5,+20 PD40 47,Y,dic(X;21)(p22;q22),Y,+5 der(8;14)(q10;q10),+20
PD86, PD 96 47~48,Y,dic(X;21)(p22;q22),Y, +5,der(8;14)(q10;q10),der(15;21) (q10;q10),+der(20)ins(20;?) (q13;?)t(1;20)(p13;q13),+20
47~48,Y,dic(X;21)(p22;q22),Y,der(4) t(4;4)(p16;q27),+5,der(8;14)(q10; q10),der(15;21)(q10;q10),+20
PD100 47~48,Y,dic(X;21)(p22;q22),Y,der(4) t(4;4)(p16;q27),+5,+7,der(8;14) (q10;q10),der(15;21)(q10;q10),+20
PD96 47~48,Y,dic(X;21)(p22;q22),Y,+5, +7,der(8;14)(q10;q10),der(15;21) (q10;q10),+20 PD96, PD114 PD100 47~48,Y,dic(X;21)(p22;q22),Y,+5, der(8;14)(q10;q10),der(15;21) (q10;q10),+20
47,Y,dic(X;21)(p22;q22),Y,+5,+7, der(8;14)(q10;q10),der(15;21) (q10;q10),+dup(20)(q13q11)
PD130 47,Y,dic(X;21)(p22;q22),Y,+5,+7, der(8;14)(q10;q10),der(15;21) (q10;q10),+dup(20)(q13q11) Fig. 1. Proposed scenario of the presumed karyotypic evolution. (A) NE108/E6E7 cell line. (B) NE3/E6E7 cell line.
H. Zhang et al. / Cancer Genetics and Cytogenetics 165 (2006) 25–35
31
Fig. 2. FISH and telomere dysfunction of immortalized esophageal cells. (A) Representative COBRA FISH karyogram of NE108/E6E7, showing the del(3)(p11), del(11)(q13), 1i(20)(q10) and 222. (B) Metaphase of NE108/E6E7 (PD 54) hybridized with specific 20q probes, RP4-724E16 (FITC, green), and RP11-445H22 (Cy3, red). Chromosomes were stained with DAPI. Two sets of 20q signals could be identified at both arms of one chromosome 20, indicating isochromosomes 20q. The other two chromosomes 20 were normal. (C) Metaphase of NE3/E6E7 (PD 114) hybridized with the same specific 20q probes. Arrow indicated the inverted duplication of 20q11~q13 material. (D) Metaphase of NE108/E6E7 (PD28, crisis) hybridized with FITC-conjugated PNA probe. Telomeric TTAGGG repeats (green) were visualized at the ends of chromosomes. Chromosomal ends without green color represent the negative telomere termini. (E) Anaphase bridge in NE108/E6E7, PD28. (F) Metaphase of HKESC-1 hybridized with a CCND probe. The arrow indicated the hsr in 11q13.
esophageal cells. Some karyotypic similarities, however, could be identified. Both immortalized esophageal cell lines and cancer cell lines displayed numerical and unbalanced structural rearrangements that affected the centromeric regions in particular. The common rearrangements identified in esophageal carcinomas (i.e., deletion of 3p or loss of 3p material, i(8q), and structural rearrangements affecting the centromere of chromosome 8, 13, 14, and 21) were found in NE108/E6E7 and NE3/E6E7 cells.
4. Discussion In this study, we characterized four HPV16E6/E7 and one hTERT immortalized esophageal cell lines by cytogenetic and molecular cytogenetic techniques. To our knowledge, it is the first study on the dynamic cytogenetic changes in immortalized esophageal cell lines. Our results showed that in all cell lines with the expression of E6/E7, there was a progressive increase of chromosomal
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6
8
64
102
KYSE140
SLMT-1
EC-1
EC-18
58~66,X,2X,2Y,del(1)(p32),add(1)(p36),del(2)(q33),23,add(3)(p11),24,24,24,add(5)(p15),der(7)t(3;7)(p13;p22),add(7)(q36),28,29,der(11)del(11)(q13) hsr(11)(q13), 212,213, add(13)(p11),add(14)(p11),215,216,217,218,dic(19;20)(p13;q13),221,add(21)(p11),222,inc[6]/62~65,idem,add(X)(p11)[cp2]/58~63,idem,2add(5)(p15),add(10)(p11)[cp3]/ 60~66,idem,2add(5)(p15),add(10)(p11),del(11)(p11)[cp3]/58~60,idem,2add(5)(p15),add(10) (p11),der(10)t(3;10)(q21;p15),del(11)(p11)[cp3]/62~63,X,add(X)(p11),2Y,del(1)(p32), add(1)(p36),del(2)(q33),add(3)(p11)2,24,24,24, 1add(6)(p23),der(7)t(3;7)(p13;p22),add(7)(q36),28,29,der(11)del(11)(q13) hsr(11)(q13),212,add(13)(p11), add(14)(p11),215,216,217,dic(19;20)(p13;q13), add(19)(p13),221,222,inc[cp2] 57~66,X,2X,Y,der(1)t(1;8)(q25;q22),add(1)(q42),22,der(3;13)(q10;q10),del(3)(q13),1der(3;5)(q10;p10),24,der(4)t(4;15)(p16;q22),26,add(6) (q13), der(7)t(1;7)(q32;q22), add(7)(q36),28,add(9)(q34),del(9)(p21),del(9)(p11),1add(9)(q11),dic(11;?)(p11;?),der(11)add(11)(p15)dup(11)(q13;q21) hsr(11)(q13),212,add(12)(q15),213,213, add(13)(p11),214,215,der(15)t(6;15)(p11;p11),1add(16)(p11),1add(17)(q24),218,add(18)(q21),219,add(19) (q13),220,221,222,222,inc[cp20] 72~81,XX,2X or Y,der(X;1)(q10;q10),der(1;14)(q10;q10),i(3)(q10),add(3)(p11),del(3)(q11),dup(4)(q21q31)2,1del(4)(q21q31),der(5;14)(q10;q10) 2,1i(5p)1~2, 1der(6;7)(p10;p10),i(6)(p10),17,der(8)t(3;8)(q21;p23)2,18,i(9)(p10)2,del(9)(q11),add(10)(p11)2,110,del(11)(q14),1del(11)(q11)2,1der(12;14)(q10;q10),213,213, add(13)(p13),der(?5;15)(q10;q10)2,116,217,218,tri(18)(q11q23),der(18)tri(18)(q11q23)t(1;18)(p31;p11),der(18) t(1;18)(p31;p11),219,add(?20)(p11)2,221,221, 122,1mar1,1mar2[cp6]/73~74,idem,2der(6;7)(q10;q10),1i(7)(p10),2der(18)tri(18)t(1;18) [cp2]/75~79,idem,2der(X,1),add(1)(p11),2i(6)(p10),2der(6;7), 2der(8)t(3;8), add(8)(p23),1i(8)(q10),del(10)(q22),inv(11)(p15q13),2der(12;14), add(13)(p11),2der(18)tri(18)t(1;18),1tri(18)[cp5]/69~72,idem,2der(X;1),2der(1;14),add(1)(p11)2, 2der(6;7),der(17)(p11)hsr(17)(p11),2der(18)tri(18)t(1;18),1tri(18)[cp4] 84~90,XX,2X or Y,der(1;3)(q10;q10),23,23,24,16,del(7)(q32)2,i(8)(q10)2,19,add(10)(p11),213,214,215,215,1add(16)(q11)1~2,217,218,120,120,221,221, add(?21)(p11)2,222,222,1mar1~2,cp[16] 50~51,X,2X or Y,del(1)(q42),dup(2)(p21;p23),add(3)(q11),add(3)(q21),1add(3)(q21),del(4)(p14),ins(5;?)(q13;?),1del(5)(p11),add(6)(p23),1der(6;15)(p10;q10),1del(7)(q22), add(13)(p11),add(14)(p11),add(15)(p11),add(21)(p13),add(21)(p13),t(4;22)(q21;p11)[cp8]/48~50,idem,add(16)(q24)[5]/50~51,idem,2add (15)(p11)[3] 70 HKESC-1
Karyotypes Passages Lines
Table 2 Cytogenetic analyses of five esophageal cancer cell lines
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aberrations during the early stage of crisis, with greater genetic instability and cytogenetic divergence. Telomere associations and dicentric chromosomes were the major aberrations affecting many chromosomes. In the later stage of crisis, all cells carried aberrations, but the karyotypes were more convergent with an increased karyotypic complexity. All cell lines surviving crisis had nonrandom clonal chromosomal changes that dominated the immortalized cells. A possible interpretation is that most of the chromosomal rearrangements generated during the crisis are random and have little or no impact on cell immortalization. However, some chromosomal aberrations may promote cellular proliferation, so that cells that acquire these aberrations are able to overcome the crisis. Such aberrations are therefore likely to persist after cellular immortalization. In HPV16E6/E7-immortalized esophageal cells, the most consistent finding was over-representation of a part or the whole of chromosome 20q, either as gain of the entire chromosome 20 (NE083/E6E7 and NECA6/E6E7), or as isochromosome 20q or duplication of 20q (NE108/E6E7 and NE3/E6E7). The nonrandom occurrence of chromosome 20q gain strongly suggests that this may contribute to esophageal cell immortalization. We have recently detected a high-level amplification of chromosome 20, in the form of a homogeneously staining region (hsr), in an SV-40 immortalized normal nasopharyngeal cell line [17]. Furthermore, using FISH and cytogenetic studies of five immortalized human ovarian surface epithelial cell lines, we have also identified high-level amplification of chromosomal material from band 20q13 in two of the lines [18]. Copy number increase or amplification of 20q was also a consistent observation in HPV16E6/E7 immortalized human bronchial epithelial cells and uroepithelial cell lines [19,20]. Taken together, these investigations provided strong evidence supporting the important role of chromosome 20 amplification as a nonrandom genetic event in cellular immortalization. In hTERT-transfected NE cells, genetic stability was demonstrated throughout the immortalization process. Amplification of chromosome 5q material was the main abnormality observed after immortalization. Gain of chromosome 5 is a common finding in immortalized cell lines and tumors [21,22]. This aberration may involve another mechanism that facilitates immortalization. We further investigated five esophageal cancer cell lines in this study and reviewed aberrations in previously reported primary esophageal carcinomas [16], and compared the aberrations in immortalized esophageal cell lines with those in esophageal cancer cell lines and primary carcinomas. The karyotypic profiles of immortalized esophageal cells lines differed to some extent from that of esophageal carcinomas. The karyotypic complexity was more pronounced in carcinoma than in the immortalized esophageal cells. These findings suggested that during multistage tumorigenesis, multiple genetic changes were necessary for malignant transformation and tumor progression. In spite
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Table 3 Telomeric status and anaphase bridges of NE108/E6E7 Negative telomere termini Population doublings
Chromosomes in metaphase
21 28 34 40 47 54 64 94
40.4 44.7 44.8 42.7 42.1 44.2 42.7 44.3 a b
6 6 6 6 6 6 6 6
3.5 1.0 1.4 3.2 2.0 2.2 4.2 2.3
Anaphase bridges Negative termini 1.5 6.0 4.7 4.0 3.7 2.6 2.7 2.6
6 6 6 6 6 6 6 6
Frequency
0.4 1.2 1.5 1.3 0.6 0.9 0.9 0.7
1.8 6.7 5.3 4.7 4.4 2.9 3.2 3.0
6 6 6 6 6 6 6 6
a
0.4% 1.4% 1.7% 1.5% 0.8% 1.1% 0.9% 0.7%
Total cells analyzed
Bridges
Frequencyb
50 55 50 60 60 55 50 55
1 17 14 16 14 9 6 2
2.00% 30.9% 28.0% 26.7% 23.3% 16.4% 12.0% 3.6%
Student-Newman-Keuls Test, P ! 0.001. Pearson Chi-square, c2 5 28.921, P ! 0.001.
chromosomes 8, 13, 14, and 21, would presumably contribute to the early step of esophageal carcinogenesis. Telomere dysfunction and defective DNA damage response may induce chromosomal instability [23]. In the absence of DNA repair response, unstable telomere repeats may lead to fusions of chromosome ends that, in turn, trigger the breakage–fusion–bridge (BFB) cycle. The model of the BFB cycle, originally described by McClintock in 1938, is speculated to be one of the mechanisms behind genetic instability observed in tumor cells and has been supported recently by some evidence [24]. The parallel assessment
of the karyotypic complexities, some similarities were observed. The most common imbalance detected in esophageal carcinomas (e.g., loss of 3p material through deletion or unbalanced translocations) was also found in later passages of NE108/E6E7 as del(3p11) and in NE083/E6E7 as del(3p21). Also, structural rearrangements involving the centromere (e.g., isochromosomes and whole-arm translocations), a characteristic feature of cancer cells, were also present in some of the esophageal cell lines with the expression of E6E7. These genetic hits, such as del(3p), structural rearrangements involving the centromeric regions of
1
2
3
6
7
8
13
14
15
19
20
9
4
5
10
11
12
16
17
18
2
21
22 X
Y
Fig. 3. Distribution of karyotypic imbalances caused by numerical or structural changes in five esophageal cancer cell lines. Vertical lines on the left of chromosome indicated loss of chromosome region(s) and lines on the right gain of chromosome region(s).
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20q11 20q13
Chromosome 20
Aberration
Breakage
Replication
Bridge
Fusion
Cytokinesis
Fig. 4. A schematic illustration of the BFB cycle leading to inverted duplication of 20q11~20q13 in the NE3/E6E7 line. An initial break distal to 20q13 led to sister chromotid fusion after replication. The two centromeres were pulled to opposite directions, generating a bridge between one centromere and an adhered segment. The centromere was cleaved finally at telophase, giving rise to the novel aberrant chromosome, which contained the duplicated material of q11~q13.
of telomere status, BFB events, and cells with anaphase bridges, as well as tas and dic chromosomes, were performed in NE108/E6E7 cells. Our results supported the telomere and BFB hypotheses of immortalization. During morphologic cell crisis, a high frequency of negative termini was recorded, accompanied by a significant increase of cells with tas and dic chromosomes and anaphase bridges. After crisis, the mean number of negative telomeres decreased, leading to a drop in cells with tas and dic chromosomes and anaphase bridges in post-crisis and immortalized cells. At the same time, there was a shift of the chromosomal aberration pattern from nonclonal chromosome changes and unrelated clones to nonrandom and more complex clonal changes. These stable clonal chromosome aberrations persisted after crisis. The preferential occurrence of centromeric aberrations (i.e., whole-arm translocations and isochromosomes) might also be caused by BFB events. A possible interpretation is that the centromeres of some chromosomes are more vulnerable than the others and may be broken during the separation of the two different centromeres toward opposite spindle poles. The broken centromeres may engage in fusions, giving rise to isochromosomes if centromeres of two sister chromatids are involved, or whole-arm translocations if two different chromosomes are involved. An inverted duplication dup(20)(q13q11) in the NE3/E6E7 line was another strong piece of evidence supporting the BFB model. In this model, an initial break distal to 20q13 led to sister chromatid fusion after replication. The two centromeres were pulled to opposite directions, generating a bridge between one centromere and an adhered segment. The centromere was finally cleaved at telophase, giving rise
to the novel aberrant chromosome (Fig. 4). A possible consequence of repetition of this cycle may be the formation of hsr, which is frequently observed in cancer cells. Our observations further support the notion that chromosomal instability resulting from BFB cycles contributes to the formation of some genetic aberrations that are essential for cell immortalization and malignant transformation.
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