Sequential cytogenetic and molecular cytogenetic characterization of an SV40T-immortalized nasopharyngeal cell line transformed by Epstein-Barr virus latent membrane protein-1 gene

Cancer Genetics and Cytogenetics 150 (2004) 144–152

Sequential cytogenetic and molecular cytogenetic characterization of an SV40T-immortalized nasopharyngeal cell line transformed by Epstein-Barr virus latent membrane protein-1 gene Hao Zhanga,b, Sai Wah Tsaoc, Charlotte Jind, Bodil Stro¨mbeckd, Po Wing Yuena, Yok-Lam Kwonge, Yuesheng Jina,d,e,* a

Department of Surgery, Queen Mary Hospital, University of Hong Kong, Medical Centre, L843, 102 Pok Fu Lam Road, Hong Kong, China b Department of Otolaryngology, Ruijin Hospital, Shanghai Second Medical University, Ruijin Road, Shanghai, China c Department of Anatomy, Faculty of Medicine, University of Hong Kong, 21, Sassoon Road, Hong Kong, China d Department of Clinical Genetics, University Hospital, Lund, Sweden e Department of Medicine, Queen Mary Hospital, University of Hong Kong, Medical Centre, L843, 102 Pok Fu Lam Road, Hong Kong, China Received 5 March 2003; received in revised form 26 August 2003; accepted 4 September 2003

Abstract

Cytogenetic and molecular cytogenetic analyses were performed on four sublines derived from a newly established, SV40T-immortalized nasopharyngeal (NP) cell line, NP69, with two of the sublines expressing LMP1, an Epstein-Barr virus–encoded gene. A total of seven cytogenetically related subclones were identified, all having highly complex karyotypes with massive numerical and structural rearrangements. Centromeric rearrangements in the form of isochromosomes and whole-arm translocations were prevalent. A cytogenetic sign of gene amplification [i.e., homogeneously staining region (HSR)] was detected at 1q25 in all metaphase cells analyzed. Multicolor combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) was used to confirm the karyotypic interpretations. Furthermore, multicolor COBRA-FISH also showed that part of the HSR contained chromosome 20 material. Extensive clonal evolution could be observed by the assessment of karyotypic variation among different subclones and individual metaphase cells. The evaluation of clonal evolution enabled the identification of the temporal order of chromosome aberrations during cell immortalization and malignant transformation. A striking karyotypic similarity was found between sublines expressing LMP1 and an NP carcinoma cell line, with loss of genetic material from chromosome arm 3p being an important recurrent observation. More interestingly, the karyotypic features of NP69 were also similar to those of many epithelial malignancies. Our observations suggest that serial transformation of NP cell lines might provide a useful in vitro model for the study of the multistep neoplastic transformation of NP cells. 쑖 2004 Elsevier Inc. All rights reserved.

1. Introduction Nasopharyngeal carcinoma (NPC) is a common cancer in the south of China and is the fourth most common cancer in Hong Kong [1]. Epidemiological studies have shown that NPC is closely associated with Epstein-Barr virus (EBV) infection, with the geographic incidence of NPC correlating

* Corresponding author. Tel.: ⫹852-28199752; fax: ⫹852-28162095. E-mail address: [email protected] (Y. Jin). 0165-4608/04/$ – see front matter 쑖 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2003.09.007

strongly with the prevalence of EBV carriers [2,3]. However, most of the evidence supporting the important role of EBV in the development of NPC comes from studies of EBV transformation on cellular systems other than nasopharyngeal (NP) cells, including mouse embryonic fibroblasts, human keratinocytes, and gastric carcinoma cells [4–6]. This might result from difficulties in obtaining NPC specimens and in establishing experimental models from normal NP cells. Like most other tumor types, the genetic mechanisms underlying the initiation and malignant transformation of NPC is far from clear.

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We have successfully established an in vitro cell culture system of NP cells through transfection with viral oncogenes [7]. These immortalized NP cell lines could serve as a useful model system for studying cellular biologic characteristics and genotypic features in different stages of NP cells during malignant transformation, as well as the role of EBV in the tumorigenesis of NPC. In this study, a newly established NP cell line, NP69-SV40T, was thoroughly characterized by cytogenetic and molecular cytogenetic techniques. This line was first immortalized by the transfection of SV40T antigen at passage 1 and then an EBV-encoded gene, latent membrane protein 1 (LMP1), was introduced at passage 21. The aim of this study was to identify the genetic events that might be critical for cell immortalization and the cytogenetic aberrations associated with LMP1 transfection. We also intended to find out whether the karyotypic profile in LMP1transduced NP cells might be similar to that of NPC cell lines. For these purposes, sequential cytogenetic analyses on different passages of NP69-SV40T and sublines with or without the expression of LMP1 were undertaken to assess changes during clonal evolution. In addition, an NPC cell line, HONE1 was also characterized by cytogenetic techniques.

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2. Materials and methods 2.1. Immortalization of normal NP cells The initiation of primary cultures and establishment of NP epithelial cell lines have been described in detail [7]. Briefly, a fresh mucosal biopsy from the nasopharynx of a normal donor was obtained after informed consent, minced into approximately 1-mm3 pieces, and placed onto 6-cm culture dishes (Bacto Laboratories, Liverpool, NSW, Australia) containing 2 mL of RPMI-1640 medium supplemented with 1% dialyzed fetal bovine serum. The resulting monolayer epithelial cells were then cultured in chemically defined MCDB 151 medium with a low calcium concentration (0.1 mmol/L) to stimulate epithelial cell proliferation and inhibit fibroblast outgrowth. The nasopharyngeal epithelial cells at passage one were transfected with the SV40T by adding 0.1 µg PX8 plasmid DNA containing an insert of SV40 antigen suspended in 100 µL serum-free culture medium with 6 µL Fugene 6 solution (Roche; Fig. 1). 2.2. LMP1 gene transfection For transfection of the LMP1 gene, a 1.95-Kb fragment containing an NPC-derived full-length LMP1 gene was

Fig. 1. A schematic diagram illustrating the process by which the NP69 cell line was initiated and immortalized, as well as the establishment of various sublines.

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cloned into the retroviral vector pLNSX to generate pLNSXLMP1. At passage 21, NP69 cells were transfected with the vector pLNSX-LMP1. A control subline was transfected with the empty retroviral vector pLNSX at the same passage. 2.3. Cytogenetic analysis Cytogenetic analysis was performed on NP69 cell cultures at passages 12 and 21 before LMP1 transfection. For sublines obtained after the LMP1 transfection (i.e., sublines NP69-LMP1, NP69-LMP1-agar, and the control subline NP69-pLNSX), chromosome analysis was carried out on passages 43, 57, and 54, respectively. The cell cultures were harvested by conventional cytogenetic techniques. Briefly, cells were blocked at the metaphase stage by exposure to colchicine (0.01 µg/mL) for 12–16 hours. The cells were then subjected to hypotonic treatment by 0.06 mol/L potassium chloride for 35 minutes, followed by fixation in methanol/glacial acetic acid (3:1). The cell suspension was spread on slides, and metaphase chromosomes were Gbanded with Wright stain. The criteria of clonality and karyotypic description followed the International System for Human Cytogenetic Nomenclature (ISCN 1995) [8]. 2.4. Fluoresence in situ hybridization (FISH) Multicolor combined binary ratio labeling (COBRA) FISH was performed as described by Tanke et al. [9], with minor modifications. Whole chromosome painting (WCP) probes used for COBRA-FISH were supplied by Cytocell (Adderbury, Banbury, UK). Briefly, slides with metaphase chromosomes were pretreated with RNase A and pepsin according to Wiegant et al. [10]. Eight microliters of probe mix comprising 24 human chromosomes labeled with 5 distinct fluorochromes by COBRA was applied to 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 with TNT (0.1 mol/L Tris, 0.15 mol/L NaCl, 0.05% Tween 20 in 100 mL) plus 4′,6-diamidinophenylindole (2–10 µL, 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 (Zeiss, Oberkochen, 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) according to the principles outlined in Szuhai et al. [11]. To verify the COBRA results, WCP probes for chromosomes 1, 2, 3, 5, 11, 12, and 20 were also used. All DNA probes were labeled with biotin-dUTP or digoxygenin-dUTP (Boehringer Mannheim, Mannheim, Germany) using random hexanucleotides (Amersham, Buckinghamshire, UK). FISH and detection were performed as described by Ho¨glund et al. [12].

3. Results 3.1. General feature of the immortalized NP cell line, NP69SV40T Transfection of the normal NP line NP69 with SV40T led to immortalization and long-term proliferation. The cells entered crisis (3–6 months) at passage 2, and most of the cells had ceased proliferation. However, a few cells overcame the crisis and became immortalized. These cells had been further subcultured over 100 population doublings without signs of senescence. To test the hypothesis that LMP1 might play an important role in the oncogenic transformation of nasopharyngeal epithelial cells, the NP69SV40T cells were further transfected by LMP1 at passage 21. By Western blotting analysis [13], it was shown that LMP1 protein was present in the LMP1-transduced cells but absent in the control cells (NP69-pLNSX) that were infected by the empty vector. The successfully infected cells were selected for further culturing and the subline was designated as NP69-LMP1. The expression of LMP1 led to more aggressive phenotypic changes than the parental NP69-SV40T cells, including morphological transformation, a significant increase in cell proliferation resistant to serum-free induced cell death, enhanced cell migration, and invasion. By cDNA array hybridization, 28 genes were identified to be differentially expressed in NP69 cells expressing LMP1 when compared with vector control NP69 cells. The majority of the differentially expressed genes were associated with cell growth, differentiation, cell shape, migration, invasion, and angiogenesis (data not shown). Furthermore, the NP69-LMP1 cells have a potential of anchorage-independent growth in that they are capable of growing on semisolid medium, soft agar. The clone isolated from soft agar was designated as NP69-LMP1-agar. The tumorigenicity of NP69-LMP1 cells was also tested in nude mice, but no tumors were observed after injection of these cells for 4 months. 3.2. Karyotypic profiles of NP69-SV40T cells and NPC cell line, HONE1 Detailed karyotypes from different passages or sublines derived from NP69 were summarized in Table 1. All metaphase cells analyzed were cytogenetically related, indicating that they originated from a single cell progenitor. A total of seven cytogenetically related subclones were identified. All clones displayed highly complex karyotypes with multiple numerical and structural rearrangements (Figs. 2 and 3). All structural rearrangements were unbalanced, leading to losses or gains of chromosomal segments. The centromeric region (i.e., centromeric bands p10 and q10, or justa-centromeric bands p11 and q11) were affected very frequently, leading to the formation of isochromosomes [e.g., i(6p) and i(9q)] and whole-arm translocations, such as der(1;21)(p10;q10), der(2;17)(p10;q10), der(2;21)(q10;q10), der(10;12)(q10), and der(11;17)(q10;q10). The aberrations der(2;17), der(3)

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Table 1 Karyotypic profiles of NP69 cells and the NPC cell line HONE1 Lines Nasopharyngeal cell line NP69 NP69 P12 subclone A

NP69 P12 subclone B

NP69 P21

NP69-LMP1 P43

NP69-pLNSX P54

NP69-LMP1-agar P57 subclone A

NP69-LMP1-agar P57 subclone B

NPC cell line HONE1 P38

Karyotypes 101~109,XXXYY,der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),⫺2,der(2)t(2;5)(q37;q13)[1],⫺4, ⫹5,⫺6,add(6)(q11)[1],del(6)(q21)[2],i(6)(p10)[1],i(9)(q10),⫺10,der(10;12)(q10;q10)[2],⫺11,⫺11, der(12)t(6;12)(q11;p13)[3],⫺14,del(14)(q24q32)[1],⫺15,⫺16,⫺17,⫺18,⫺19,⫺19,⫺20,⫺21,⫺22[cp4] 101~109,XXXYY,der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),⫺2,der(2)t(2;5)(q37;q13)[1],⫺4, ⫹5,⫺6,add(6)(q11)[4],del(6)(q21)[3],i(6)(p10)[2],der(8)t(8;17)(p11;?),i(9)(q10)[5], ⫺10,der(10;12) (q10;q10)[2],⫺11,⫺11,der(11;21)(q10;q10)[2],der(12)t(6;12)(q11;p13)[4],⫺14,del(14)(q24q32)[1], ⫺15,⫺16,⫺17,der(17;21)(q10;q10)[6],⫺18,⫺19,⫺19,⫺20,⫺21,⫺22[cp6] 81~100,XXYY,add(1)(p11),del(1)(p22),der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),⫹2,⫺3,⫺4, ⫹5,⫹5,⫺6,add(6)(q11),i(6)(p10),⫹7,⫺8,der(8)t(8;17)(p11;?),i(9)(q10),⫹der(10;12)(q10;q10),der(11;17) (q10;q10),der(12)t(6;12)(q11;p13)[2],der(12;13)(q10;q10)[2],⫺14,⫺15,⫺16,der(17;21)(q10;q10),⫺19, ⫺19,⫺21,der(?)hsr(?),der(?)t(?;17)(?;q21)[cp5] 85~97,X,⫺X,YY,⫹Y,der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),⫺2,der(2;17)(p10;q10)[2],der(3) t(3;12)(p11;?),⫺4,add(4)(p11)[2],⫹5,⫹5,⫹5[3],der(5)t(5;14)(q13;q13)[4],del(6)(q21)[3],der(6;21) (p10;q10)[2],add(7)(q36)[2],⫹8,der(8)t(8;14)(q24;q11)[6],i(9)(q10)[6],der(10;12)(q10;q10)[5],der(11) t(9;11)(q13;q23)[6],der(11;17)(q10;q10)[5],der(11;21)(q10;q10)[5],der(12)t(6;12)(q11;p13)[6],⫺13, ⫺14,del(14)(q24q32)[4],⫺16,⫺17,⫺18,⫺19,⫹20[4],add(21)(p11)[2],⫺22,der(?)t(?;1)(?;q25),der(?) t(?;1)(?;q25)[cp7] 75~94,X,⫺X,YY,⫹add(1)(p11)[3],der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),⫺4,add(4)(p11)[1], add(4)(p16)[2],⫹5,⫺6,del(6)(q21)[4],i(6)(p10)[4],add(7)(p22)[1],add(7)(q11)[2],⫹8,der(8)t(8;17)(p11;?) [3],i(9)(q10)×1~2[6],⫺10,der(10,12)(q10;q10)[2],⫺11,der(11)t(9;11)(q13;q23)[2],der(11;17)(q10;q10) [5],der(12)t(6;12)(q11;p13)[6],⫺13,⫺14,⫺15,⫺16,⫺18,⫺19,⫺22,inc[cp7] 74~84,X,der(X)t(X;2)(p22;q31)[3],YY,⫹Y,der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),der(1;21) (p10;q10),der(1;21)(q10;q10)[3],⫺2,der (2;17)(p10;q10)[4],der(2;21)(q10;q10)[5],der(3)t(3;12)(p11;?)[5], ⫺4,add(4)(p11)[3],add(4)(p11)[6],⫹5[4],der(5)t(5;14)(q13;q13)t(8;14)(q13;q32)[7],⫺6,del(6)(q13)[4], del(6)(q21)[2],der(6;21)(p10;q10)[5],del(7)(q11)[5],der(7)t(1;7)(q22;p22)[2],der(8)t(8;14)(q24;q11)[8], der(9;21)(q10;q10)[7],der(10;12)(q10;q10),⫺11,der(11)t(9;11)(q13;q23)[6],der(11;17)(q10;q10)[7], der(12)t(6;12)(q11;p13),⫺13,⫺13,⫺14,del(14)(q24q32)[6],⫺15,⫺16,⫺18,⫺19,⫺20,⫺22[cp9] 78~82,X,der(X)t(X;2)(p22;q31),YY,⫹Y,der(1)dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?),der(1;21)(p10;q10), der(2;17)(p10;q10),der(2;21)(q10;q10)[5],der(3)t(3;12)(p11;?),⫺4,add(4)(p11),der(4;14)(q10;q10),⫹5, der(5)t(5;14)(q13;q13)t(8;14)(q13;q32),⫺6[4],del(6)(q13)[4],der(7;22)(q10;q10)[3],del(7)(q11),der(7)t(1;7) (q22;p22),⫺8[3],der(8)t(8;17)(p11;?)[2],der(8)t(8;14)(q24,q11),der(9;21)(q10;q10)[4],⫺10[3],der(10;12) (q10;q10),der(11)t(9;11)(q13;q23),der(11;17)(q10;q10),der(11;21)(q10;q10)[5],der(12)t(6;12)(q11;p13), ⫺13,⫺13,⫺14,del(14)(q24q32),⫺15,⫺16[3],⫺18,i(18)(p10)[3],⫺19,⫺19,⫺20,⫺20,⫺22,⫺22,der(?)t(?;1) (?;q25)[3],der(?)t(?;1)(?;q25)[4][cp6] 84~88,X,⫺X,del(X)(q22),i(X)(p10),⫹add(1)(p11)[4],del(1)(p36),der(1)del(1)(p22p34)ins(1;?)(p22;?)del(1) (q32),der(1;9)(q10;q10),⫺2,add(2)(p21),⫺3,add(3) (p11),add(3)(q29),ins(3;?)(q21;?),⫺4,add(4)(p11), i(5)(p10)×2,⫺6,⫺6,⫹del(7)(q22),der(7)t(1;7)(q21;q32),⫺8,del(8)(p21),⫺9,⫺10,⫺10,⫺10,add(12)(p11), add(13)(p11),i(13)(q10),⫺14,i(14)(q10)[3],add(15)(p11),add(15)(p11)×1~3,⫺18,add(19)(p11),i(19)(q10), der(21;22)(q10;q10),i(21)(q10),⫺22,⫺22,⫺22,⫹mar1,⫹mar2,⫹mar3[cp5]

t(3;12), der(5)t(5;14), and der(8)t(8;14) were found only in sublines expressing LMP1 (Figs. 3 and 4). Homogeneously staining regions (hsr) (i.e., cytogenetic evidence of highlevel gene amplification) was found in all metaphase cells karyotyped. hsr was located at chromosomal band 1q25. In addition, in sublines NP69-P21 (the NP69 cells at passage 21), an hsr located in a marker chromosome was also detected. Interestingly, pronounced karyotypic similarities were found among the sublines NP69-LMP1, NP69-LMP1-agar, and the NPC cell line HONE1. These cell lines displayed highly complex karyotypes. A number of chromosomal bands were commonly involved, including 1q21, 3p11, 4p11, 8p11, 9q10, 14q10, and 21q10. An unbalanced aberration, add(3)(p11), leading to net loss of the entire chromosome

arm 3p, was also found NPC cell line. Furthermore, an assessment of genomic imbalances showed that NP and NPC cell lines shared many similar chromosomal gains and losses, including over-representation of 1q25→q32 and 5p, and losses of 3p11→pter, 4p11→pter, 8p21→pter, 9p, 10p, 12p11→pter, 13p11→pter, 14p, 14q24→qter, 15p11→pter, 18q11→qter, 19p, 21p, and 22p. 3.3. COBRA-FISH and FISH To identify the origin of hsr, COBRA-FISH, and FISH with specific chromosome painting probes were applied on metaphase cells from the subline NP69-LMP1-agar (Fig. 5). It was shown that part of the hsr in chromosome 1 contained DNA materials from chromosome 20. The derivative

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Fig. 2. Representative karyotype from NP69, passage 12, showing complex karyotype.

chromosome 1 harboring hsr was accordingly reinterpreted as der(1) dup(1)(q11q25)ins(1;20)(q25;?)hsr(20)(?). Furthermore, COBRA-FISH has confirmed all cytogenetic interpretations and identified a number of additional aberrations that could not be interpreted at the cytogenetic level. 3.4. Cytogenetic heterogeneity and evaluation of clonal evolution A high degree of cytogenetic heterogeneity was found among the NP69 sublines. The karyotypic variation from cell to cell enabled the assessment of clonal evolution pathways of the NP69 subline (Fig. 6) and the identification of the temporal order of the chromosomal aberrations occurring during cell immortalization. As shown in Fig. 6, some of the structural rearrangements, including the derivative chromosome 1 harboring hsr, der(10;12), der(12)t(6;12), and numerical changes ⫺4, ⫹5, ⫺14, ⫺16, and ⫺19 could be identified

in each metaphase cell, suggesting that these changes represented early genetic events. On the other hand, some other aberrations, such as der(2;17), der(3)t(3;12), der(5)t(5;14), and der(8) t(8;14), were detected only in sublines expressing LMP1 and absent in the vector control subline, suggesting that these changes might be secondary and represent genetic evolution changes. 4. Discussion Carcinogenesis is generally viewed as a multistep process in which a normal cell undergoes immortalization and oncogenesis to become a fully transformed malignant cell [14]. Several lines of research, all of them taking as their starting point the central tenet of the somatic mutation theory of cancer, have provided substantial evidence that genetic mechanisms are crucial to this transition [15–17]. The study of stepwise genetic events associated with the various stages of

Fig. 3. Representative karyotype from the subline NP69-LMP1-agar, passage 57. Inset, rearranged chromosomes from another metaphase cell, showing der(1;21), der(3)t(3;12), der(7), and der(6;21). See Table 1 for karyotype description.

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Fig. 4. Partial karyotype from subline NP69-LMP1, passage 54, showing structural rearrangements der(3)t(3;12), der(5)t(5;14), and der(8)t(8;14).

neoplastic transformation, however, requires longitudinal monitoring of the neoplastic process with multiple sampling at several points in time, which is hardly feasible in most human neoplasia. The establishment of long-term cell lines from normal cells provides a useful model system for such studies. Sequential genetic investigations are easily undertaken in established lines during their immortalization and transformation, which may yield useful clues regarding the immortalization process and the genetic events that characterize the early stages of tumorigenesis. Therefore, the establishment of an NP cell line, NP69-SV40T, provided for the first

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time an experimental system for studying the genetic events associated with NP cell transformation. To identify chromosomal aberrations that may be associated with cell immortalization, the chromosome changes of cells in the pre-crisis stage ideally should be compared with thosein post-crisis.Inthis study,however,itwasnotpossibleto examine cytogenetic changes of the cells during pre-crisis because the NP69-SV40T cells entered crisis at passage 2 and most of the cells ceased to proliferate at this stage. Also, only few cells survived and continued to proliferate in early passages (P3–P7) after crisis. We therefore revived cells in the earliest available passage (P9), and they were subcultured three times before cytogenetic analysis. In this study, we tried to define the temporal order of chromosomal aberrations by comparing the karyotypic profiles in seven subclones identified from different passages and sublines, and by assessing the karyotypic variation in each individual metaphase cell, assuming that the chromosomal anomalies shared by all cells should represent relatively early genetic events. The fact that the structural rearrangements der(1) with hsr, der(10;12), der(12)t(6;12), and numerical changes ⫺4, ⫺14, ⫺16 and ⫺19 could be identified in all metaphase cells suggested that these aberrations occur early and may be associated with cell immortalization. On the contrary, chromosome rearrangements der(2;17), der(3) t(3;12), der(5)t(5;14), and

Fig. 5. (A) Representative COBRA FISH karyotype from subline NP69LMP1-agar, showing that the hsr in derivative chromosome 1 contains material from chromosome 20. (B) FISH with whole chromosome–specific probes for chromosomes 1 (WCP1 in red) and 20 (WCP20 in green).

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Fig. 6. Schematic presentation of presence (filled boxes) and absence of structurally rearranged chromosomes in different sublines. For designation of sublines and complete karyotypes, see Table 1.

der(8)t(8;14) present only in sublines with the expression of LMP1 (i.e., NP69-LMP1 and NP69-LMP1-agar) might be chromosomal changes secondary to LMP-1 transfection, and might thus contribute more to the phenotype of LMP1-transformed lines. It has been suggested that LMP-1, an EBV-encoded gene, is critically involved in the pathogenesis of NPC. The establishment of immortalized NP cell lines with and without the expression of LMP-1 offered an opportunity to correlate karyotypic and phenotypic features during NP cell transformation by LMP-1. A number of chromosome aberrations were

identified only in sublines with the expression of LMP-1. Among these aberrations, der(3)t(3;12), leading to the loss of chromosome arm 3p material, is of particular interest. A derivative chromosome 3 [i.e., add(3)(p11)] was found in an NPC cell line, HONE1, investigated in the present study and in another NPC cell line (NPC-BM1) derived from a bone marrow metastasis [18]. Although it is not known whether loss of chromosome 3p material resulting from the der(3)t(3;12) was induced directly by LMP-1, the outcome of this imbalance could contribute to the further transformation of the NP cell lines. Indeed, evidence from several lines of research supports this contention. First, a number of previous cytogenetic studies on immortalized human normal epithelial cell lines have shown that loss of chromosome 3p material through various unbalanced translocations of chromosome 3 confers a tumorigenic capacity on these cell lines [19–23]. Second, rearrangement of chromosome arm 3p, often in the form of deletions, is a common chromosome abnormality observed in a broad spectrum of tumor types [24]. Finally, loss of genetic material of chromosome 3p has been repeatedly demonstrated in primary NPC or cell lines derived from NPC by comparative genomic hybridization [25–28] and loss of heterozygosity experiments [29–31]. These observations thus suggest that loss of chromosome 3p might be a genetic event of general importance in neoplastic transformation of epithelial cells. An intriguing finding in this study is the occurrence of hsr, a cytogenetic sign of gene amplification, observed in all metaphase cells analyzed, indicating that it is an early genetic event associated with cell immortalization. HSR is a cancer-related aberration observed in a broad spectrum of tumor types. This aberration has never been described in normal cells. The use of COBRA-FISH and FISH with WCP20 disclosed that the hsr was composed of chromosome 20 material. A review of the literature revealed that gain of copy number or the amplification of 20q was a consistent finding reported in E7transformeduroepithelialcelllines[32],further supportingthe notion that amplification of gene/genes in chromosome 20 is a nonrandom genetic event critical for cell immortalization. More importantly, in this context, the amplification of 20q was repeatedly detected in a number of epithelial cancers, such as breast cancer and ovarian cancer [33,34]. It remains to be defined whether the molecular targets involved in the NP69 line are also those frequently amplified in cancer cells with amplification of 20q. Furthermore, the karyotypic features in the NP69-SV40T line are similar to the pattern observed in many types of epithelial malignancies: the karyotype being highly complex with numerous karyotypic aberrations, leading to large-scale genomic imbalances. A peculiar cytogenetic feature of NP69 is the preferential involvement of the centromeric region in the form of isochromosomes and whole-arm translocations. It is noteworthy that some of the structural rearrangements found in NP69 cells, notably isochromosomes i(6p) and i(9q), are wellknown cancer-associated aberrations [35] identified in many different types of malignancies. These cytogenetic aberrations

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may hence be viewed as pathogenetically important events in cell transformation. The fundamental pathogenetic impact of centromeric rearrangements remains to be defined. Finally, little is known about the mechanisms underlying the nonrandom formation of centromeric rearrangements in cancer cells. Some clastogenic agents, such as mitomycin C, have a strong predilection for centromeric DNA, inducing centromeric breaks on chromosomes 1, 9, and 16 [36]. A potential mechanism for the extensive clonal evolution and the generation of massive centromeric rearrangements in the NP69 cell lines might be directly attributed to centromeric instability or indirectly induced by persistent interaction of viral genes SV40T and LMP1 on centromeric DNA, which needs to be verified by further studies.

[12]

[13]

[14] [15] [16] [17]

Acknowledgments

[18]

This work was supported by the Kadoorie Charitable Foundation, research grants from the University of Hong Kong, and grants from Swedish Cancer Society. Dr. Zhang’s stay at Queen Mary Hospital is sponsored by the Youth fund from Ruijin Hospital, Shanghai Second Medical University, Shanghai, China.

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