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Molecular cytogenetic characterization of human papillomavirus16-transformed foreskin keratinocyte cell line 16-MT
Cancer Genetics and Cytogenetics 168 (2006) 36–43
Molecular cytogenetic characterization of human papillomavirus 16-transformed foreskin keratinocyte cell line 16-MT Eva M. McGheea,b,*, Philip D. Cotterc,d, Jingly F. Weiere, Jennifer W. Berlinef, Mary A. Turnerg, Mathew Gormleye, Joel M. Palefskyb,f a
Department of Community Health Systems, University of California, San Francisco (UCSF), Room N505, Box 0608 San Francisco, CA 94143-0608 b Comprehensive Cancer Center, UCSF, San Francisco, CA 94143 c Department of Pediatrics, Medical Genetics, UCSF, San Francisco, CA 94143 d Department of Pathology, Children’s Hospital and Research Center at Oakland, 747 52nd street, Oakland, CA 94609 e Department of Obstetrics, Gynecology and Reproductive Services, UCSF, San Francisco, CA 94143 f Department of Medicine, UCSF, San Francisco, CA 94143 g Department of Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 Received 28 September 2005; received in revised form 15 December 2005; accepted 30 December 2005
Abstract
Anogenital cancers are closely associated with human papillomavirus (HPV), and HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancers. Although genomic instability has been documented in HPV-infected keratinocytes, the full spectrum of genetic changes in HPV-associated lesions has not been fully defined. To address this, we examined an HPV16-transformed foreskin keratinocyte cell line, 16-MT, by GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (array CGH); these analyses revealed multiple numerical, complex, and cryptic chromosome rearrangements. Based on GTGbanding, the 16-MT karyotype was interpreted as 78~83,XXY,1add(1)(p36.3),13,14,15, 15,17,18,1i(8)(q10)2,110,?der(12),der(13;14)(q10;q10),115,116,add(19)(q13.3),121,121, 222[cp20]. Multicolor analysis by SKY confirmed and further characterized the anomalies identified by GTG banding. The add(1) was identified as a der(1)(1qter/1q25::1p36.1/1qter), the add(19) as a dup(19), and the der(12) interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement with chromosome 19. In addition, previously unidentified der(9)t(9;22), der(3)t(3;19), and der(4)t(4;9) were noted. The 16-MT cell line showed losses and gains of DNA due to unbalanced translocations and complex rearrangements of regions containing known tumor suppressor genes. Chromosomal changes in these regions might explain the increased risk of cancer associated with HPV. Also, array CGH detected copy-number gains or amplifications of chromosomes 2, 8, 10, and 11 and deletions of chromosomes 3, 4, 11, and 15. These results provide the basis for the identification of candidate oncogenes responsible for cervical and anal cancer in amplified regions, and for putative tumor suppressor genes in commonly deleted regions like 11q22~23. Furthermore, these data represent the first full characterization of the HPV-positive cell line 16-MT. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Anogenital cancers are closely associated with human papillomavirus (HPV) infection [1–4]. HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancer [1,2]. Genomic instability has been previously documented in human HPV-infected keratinocytes [5–11]. The development of genomic instability is an early and crucial event during * Corresponding author. Tel.: (415) 476-3931; fax: (415) 476-6042. E-mail address: [email protected] (E.M. McGhee). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.12.015
HPV-associated carcinogenesis [12]; however, the full spectrum of genomic changes in HPV-infected keratinocytes has not been fully defined. The identification of chromosomal alterations with pathologic implications in HPVrelated squamous cell carcinomas is of major importance, given that the carcinomas account for substantial mortality and morbidity [6]. Although a substantial number of HPVrelated cancers have been examined, specific chromosomal alterations or changes associated with initiation of tumor progression have not been fully characterized. The mechanisms of chromosomal rearrangement and other genomic changes in HPV-infected cells are not well
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understood. Rare HPV-negative carcinoma cell lines do exist [9,13,14], but almost all cervical carcinoma cell lines contain HPV DNA, and most cervical carcinoma cell lines have been shown to have single or multiple copies of integrated HPV [15]. Integration of HPV in rearranged chromosomes in cervical carcinomas or HPV-immortalized epithelial cells implicates the process of viral integration as at least one mechanism of HPV-induced genomic reorganization [16]. In addition, HPV gene products, such as the E6 and E7 oncoproteins, are also potential contributing factors to the generation of chromosomal rearrangements [17]. These oncoproteins target tumor suppressor signaling pathways that are critical for control of cellular growth. The HPV E6 protein of oncogenic HPV types such as HPV16 induces accelerated proteasomal degradation of p53 protein. In addition, the HPV E7 protein of oncogenic HPV types binds and degrades the pRB retinoblastoma tumor suppressor protein and interacts with the cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1, resulting in dysfunction of the cell cycle checkpoints and causing genomic instability [11]. To better characterize genomic instability induced by HPV 16, we studied the 16-MT cell line. 16-MT cells were derived from a neonatal human foreskin and then transformed with whole genomic HPV16 DNA. We characterized this cell line using several different cytogenetic techniques including GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (CGH). We also explored the relationship between telomerase expression and chromosomal instability in 16-MT cells. Regulation of telomerase activity is dependent on the reverse-transcriptase component of telomerase (hTERT) [18]. hTERT and low level of telomerase activity have been shown to correlate with increased chromosomal instability and an increase in chromosome copy number [19]. HPV16 E6 can upregulate hTERT transcription. Here we report the G-banded composite, SKY characterization, and array CGH analysis of the HPV-positive 16MT cell line. Our data show that 16-MT cells demonstrate chromosomal instability, an increase in chromosomal copy number, and low-level telomerase activity.
2. Materials and methods 2.1. Cell line and growth conditions Primary keratinocyte cells were generated from a neonatal human foreskin and the 16-MT cell line was generated by transfecting the cells with HPV16 using calcium phosphate methodology. Stable transfection was confirmed by dot blot analysis [20,21]. The 16-MT cell line was grown in complete keratinocyte growth media (KGM CC-4131; Clonetics, San Diego, CA) and complete Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (DMEM) (3:1) and incubated at 37 C in a 5% CO2
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incubator. 16-MT cells from passage 13 were studied. The nontransformed parental cell line was also grown in complete KGM medium under the same conditions as 16MT. Cell lines were tested for mycoplasma using a MycoAlert mycoplasma detection kit (Cambrex, East Rutherford, NJ); the results were negative. 2.2. GTG-banding analysis Metaphase chromosomes were prepared from 16-MT cells and analyzed by GTG-banding using standard protocols. Clonal chromosomal abnormalities identified by means of GTG-banding were described according to ISCN 1995 [22]. 2.3. SKY SKY analysis was performed on the 16-MT cells using aged metaphases on unstained slides. The slides were aged in 2 saline sodium citrate (SSC) at 37 C for 30 min. Cells were pretreated with pepsin (30 mg/mL pepsin in 0.01 mol/ L HCl) at 37 C for 5 min, 1 phosphate buffered saline (PBS) at room temperature for 5 min, then postfixed in 1% formaldehyde in 1 PBS/MgCl2 at room temperature for 5 min and washed in PBS. Finally, the slides were dehydrated in 70, 80, and 100% ethanol for 2 min. The SKY probes (Vial 1, SKY kit; Spectral Imaging, Carlsbad, CA) were denatured at 76 C for 7 min, then allowed to preanneal at 37 C for 60 min. The slides were denatured at 76 C for 5 min in 70% formamide–2 SSC, pH 7.0, then were dehydrated in 70, 80, and 100% ethanol for 2 min and allowed to air dry. The hybridization mixture was applied to the slides, covered with a glass coverslip, and sealed with rubber cement. The slides were hybridized at 37 C for 40–48 h in a moisture chamber. Following hybridization, the slides were washed at 43 C three times in 50% formamide–2 SSC for 10 min each, followed by two washes in 2 SSC for 10 min each, one wash in 0.4 SSC for 5 min, and a final wash in 0.1% Tween 20–4 SSC at room temperature for 2 min. Next, 80 mL of blocking reagent (Vial 2, SKY kit; Applied Spectral Imaging) was applied to each slide; the slides were then covered with a plastic coverslip and incubated at 37 C for 45 min. Spectral images were acquired using an SD200 SpectraCube spectral imaging system (Applied Spectral Imaging, Migdal Ha’emek, Israel) [23]. The SD200 imaging system attached to a Nikon E600 microscope consisted of an optical head (a Sagnac interferometer) coupled to a multiline charge-coupled device CCD camera (Hamamatsu, Bridgewater, NJ) to capture images at discrete interferometric steps. The images were stored as a stack in a Pentium 586/300 MHz computer. 2.4. Array CGH To further analyze the 16-MT cell line, array CGH analysis was performed as previously described [24,25]. The
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array consisted of 2,464 genomic clones printed in triplicate with an average resolution of 1.4 Mb across the genome. 16-MT cell DNA and normal reference DNA (from whole blood) were labeled by random priming with Cy3- or Cy5-labeled nucleotides and hybridized for 2 days to the array. Sixteen-bit 1024 1024 pixel images, with 40 ,6-diamidino-2-phenylindole (DAPI) Cy3 and Cy5 staining, were collected using a custom CCD camera system [26]. The data were analyzed using UCSF SPOT computer software [27] to automatically segment the array spots and to calculate the log2 ratios of the total Cy3 and Cy5 intensities for each spot after background subtraction. A second computer program, SPROC, was used to calculate averaged ratios of the triplicate spots for each clone, standard deviations of the triplicates, and plotting position for each clone on the array on the July 2003 freeze of the draft human genome sequence (http://genome.ucsc.edu). SPROC also implements a filtering procedure to reject data based on a number of criteria, including low reference-to-DAPI signal intensity and low correlation of the Cy3 and Cy5 intensities with a spot. The data files were edited to remove ratios on clones for which only one of the triplicates remained after SPROC analysis and/or the standard deviation of the log2 ratios of the triplicates was O 0.2 [25]. 2.5. Telomerase activity Telomerase activity was determined by using the TRAPeze XL Kit (Chemicon, Temecula, CA). The TRAPeze XL Kit uses fluorescence energy transfer (ET) primers to
generate fluorescently labeled TRAP products, which permit nonisotopic quantitative analysis of telomerase activity [28].
3. Results 3.1. Cytogenetic characterization of the 16-MT and parental cell lines by GTG- banding Cytogenetic analysis of 16-MT cells by GTG-banding showed that all metaphases had a complex hypertriploid karyotype with multiple structural and numerical abnormalities. The 16-MT cells displayed a karyotype of 78~83, XXY,1add(1)(p36.3),13,14,15,15,17,18,1i(8)(q10)2, 110,?der(12),der(13;14)(q10;q10),115,116,add(19)(q13.3), 121,121,222[cp20]. Figure 1 shows a representative karyotype of a GTG-banded metaphase for 16-MT cell line. The 16-MT karyotype interpreted from the GTG banding is summarized in Table 1. The parental human foreskin keratinocyte cells had a 46,XY karyotype (Table 1). 3.2. SKY characterization of the 16-MT cell line Multicolor SKY analysis confirmed the presence of the i(8)(q10) and der(13;14) rearrangements and further characterized the add(1), ?der(12), and add(19) rearrangements noted with GTG banding. The add(1) was identified as a der(1)(1qter/1q25::1p36.1/1qter) and the add(19) as a dup(19); the der(12) was interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement
Fig. 1. Representative GTG-banded karyotype composite of the 16-MT keratinocyte cell line. The karyotype for the cells shown is interpreted as 78~83,XXY, indicating a hypertriploid cell line.
E.M. McGhee et al. / Cancer Genetics and Cytogenetics 168 (2006) 36–43 Table 1 Representative karyotypes of keratinocyte cell line 16-MT as determined by conventional cytogenetic analysis and by spectral karyotyping Analysis
Karyotype
GTG-banding
78~83,XXY,1add(1)(p36.3),13,14,15,15,17, 18,1i(8)(q10)2,110,?der(12),der(13;14)(q10;q10), 115,116,add(19)(q13.3),121,121,222[cp20] 78~83,XXY,1der(1)(1qter/1q25::1p36.1/1qter), 13,der(3)t(3;19),14,der(4)t(4;9),15,15,17,18, 1i(8)(q10)2,der(9)t(9;22),110,211,add(11)(p15), der(12)t(11;12)(q24.3;q13),der(13;14)(q10;q10), 115,116,der(17)t(4;17),dup(19),121,121,222, 1mar[cp20]
SKY
The karyotype for the control parental line was a normal 46,XY.
with chromosome 19 (Fig. 2). Abnormalities identified by SKY, not revealed by GTG banding, included der(3)t(3;19), der(4)t(4;9), der(9)t(9;22), and der(17)t(4;17) (Fig. 2). The composite karyotype based on SKY analysis is given in Table 1. 3.3. Numerical and aneuploid abnormalities characterization of the 16-MT cell line Both GTG-banding and SKY analysis identified and confirmed numerical and aneuploid abnormalities for the 16-MT cell line. Figure 3 shows the mean for aneuploid abnormalities. The profile for chromosome 8 showed the highest copy number for 16-MT, with at least six copies of chromosome 8 appearing in each metaphase counted (Fig. 3). 3.4. Array CGH characterization of the 16-MT cell line Array CGH experiments were used to detect and map chromosomal imbalances. Array CGH data provided precise measurement (standard deviation of log2 ratios 5 0.05–0.10) of numerical chromosome changes. We detected copy number gains and losses as well as amplifications in the 16-MT cell line (Fig. 4). These included cryptic deletions on chromosomes 3, 4, 6, 11, 12, 18, 19, and X and amplifications on chromosomes 2, 4, 8, 11, 13, and 14. The array CGH analysis also revealed whole chromosome gain (chromosomes 4, 5, 6, and 8) in the 16-MT cells, as well as a del(X) (Fig. 4). A deletion on chromosome 11q22~23 was also noted, with this region containing the locus for the ATM gene as well as other tumor suppressor genes (Fig. 4). 3.5. Telomerase activity of the 16-MT cell line Telomerase activity was measured in 16-MT cells. These cells exhibited low telomerase activity (Fig. 5). In contrast, an immortalized breast cancer cell line (MDA-MB-231) used for control showed high telomerase activity, as expected (Fig. 5).
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4. Discussion Cancer has been characterized as a disease of genomic instability and the malignant transformation of human epithelial cells in vitro by the action of HPV is paralleled by cytogenetic changes. We developed a new HPV16-positive cell line, 16-MT, by HPV 16 transfection of normal human foreskin keratinocyte cells as a model to study HPV-associated cancers. The 16-MT cell line showed specific derivative chromosomal rearrangements involving several chromosomes, and some of these chromosomes are noted in the literature to be associated with cervical carcinoma and leukemiadspecifically, chromosomes 1, 3q, 5, 8, 10, and 11q [8]. Previous reports of in vitro systems developed to study genomic instability steps of HPV-induced chromosomal instability of transformed keratinocytes already postulated a multistep process for cancer development [7,9,29]. The primary untransformed human foreskin parental cells of the 16-MT cell line showed no cytogenetic alterations, which confirms that HPV-induced transformation led to complex chromosomal rearrangements. G-banding analysis of 16-MT cells showed a hypertriploid karyotype containing multiple structural and numerical abnormalities. This suggests that induced chromosomal instability came at least in part from the induction of HPV integration into common fragile sites of the host DNA, and produced specific chromosomal translocations and numerical changes [30]. Karyotypic instability after HPV integration preceding neoplastic transformation has been demonstrated for human fibroblasts [31]. Characterization by multicolor SKY analysis and array CGH revealed and confirmed numerous chromosomal imbalances in 16-MT cells (Figs. 2 and 4). Note in particular the finding of the amplified regions on 3q and 11q. Chromosome 11q is considered a strong candidate region for protooncogenes playing a key role in immortalization of keratinocytes upon transfection with HPV [32–34], and 3q is one of the most frequent abnormalities in invasive carcinoma and the gain of 3q accompanies progression from high grade squamous intraepithelial lesions (HGSIL) to invasion [35]. In our in vitro model system, chromosomes 3, 4, 5, 8, 9, 11, 13, 14, 16, 17, 18, 19, and 22 were found to be frequently affected by translocations and derivative rearrangements, as well as by numerical imbalances (Figs. 2 and 3). Inactivation of checkpoint components due to chromosomal alterations of genes controlling cellular damage can activate the process of immortalization [29,36–38]. Our findings from SKY analysis greatly improved the conventional cytogenetic description of the complex chromosomal rearrangements by revealing small interstitial changes and previously unrecognized abnormalities. Combined, the GTG-banding and SKY analyses revealed complex and recurrent translocations between chromosomes: (3;19), (4;9), (4;17), (9;22), (11;19), (14;13), (5;16;5), (18;5), (13;19), i(4), i(8),
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Fig. 2. Representative karyotype from SKY analysis composite of the 16-MT keratinocyte cell line. (A) Red–green–magenta image, (B) DAPI image, (C) classified image, (D) red–green–blue metaphase image indicating structural changes (E–Q) and additional structural changes confirmed by SKY analysis.
i(13), and i(16) (Figs. 2E–2Q). Several of these alterations have been reported in HPV-related cancers cell lines, including SiHa, Caski, and CamCell3 [12]. In 16-MT cells, chromosome 1 was involved in a substantial portion of the abnormal metaphases scored. Chromosome 1 is among those undergoing the most alterations in cervical cancers [12], frequently involving several genes, such as TP73 and TP53, located on chromosome 1p36.3. Loss of chromosome arm 3p and gain of 3q are also known to be common events in HPV immortalized cell lines, cervical and anal cancer [39]. In a recent study involving a small
number of cases, gain of 3q was reported as the most commonly found genetic change in anal intraepithelial neoplasia (AIN) of HIV-positive men [40], and gain of 3q was reported as defining the transition from severe dysplasia to invasive carcinoma of the uterine cervix [41]. In the 16-MT cells, however, our array CGH analysis showed both loss and gain on chromosome 3. Previous reports show that structural rearrangement of chromosome 8 particularly i(8q) derivatives, were commonly found in HPV-infected cancer cells [42]. In our study of 16-MT cells, i(8q) was found in several metaphases, as
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Fig. 3. Representative profile for numerical abnormalities and aneuploid chromosomal number analyzed by SKY in 16-MT keratinocyte cells. Analysis from composite showed chromosome 8 to be the most common aneuploid chromosome in the 20 metaphases scored.
well as chromosomes i(5) and i(10). The 11;19 rearrangement in 16-MT was detected in >85% of the metaphases analyzed. Deletion of genes on the long arm of chromosome 11 in the 16-MT cell line is likely to be important, given that multiple tumor suppressor genes are localized at region 11q.
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Our array CGH data showed that chromosomes 4, 8, 11, and X have distinct regions of focal gain and loss (4q gain, 8p gain, 8q gain, 11q loss and gain; and, Xp telomeric loss). The array CGH results revealed cryptic deletions and gains involving chromosomes whose localization was cytogenetically compatible with the aberrant regions identified by SKY. The high level of amplifications of chromosome 8 may reflect later events, thus exhibiting focal amplifications superimposed upon the background of whole chromosome gain. A high level of telomerase activity has been shown to stabilize telomere length and reduce chromosomal rearrangements [19]. In contrast, low telomerase activity correlated with increased chromosomal instability [19]. Consistent with these findings, we showed high frequencies of chromosomal structural aberrations (Figs. 1 and 2) associated with low telomerase activity (Fig. 5) in the 16-MT cell line. These data further support the correlations between telomerase activity and chromosome or genomic instability.
Fig. 4. Representative array CGH profile of the 16-MT keratinocyte cell line. Array CGH analysis was used to detect and map chromosomal gains and losses in the 16-MT cells by hybridizing targets of genomic DNA from a test and a reference sample to sequences immobilized on prepared slides. These probes are genomic DNA sequences (i.e., bacterial artificial chromosomes, or BACs, that are mapped on the genome). The array CGH data show specific chromosomes of interest that indicated gains (3, 4, 5, 8, 9, 11, 19) and losses (3, 4, 6, 11, 12, 13, 14, 18, 19, 22).
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Acknowledgments The authors would like to thank Ms. Maria Da Costa, Gloria Tung and Ms. Inna Govberg for their technical contributions to these studies, and the Blackburn laboratory (Dr. Jue Lin for her help with the telomerase detection). This work was supported by a grant from the National Institutes of Health, National Cancer Institute, R01 CA088739-05S1. References
Fig. 5. Representative telomerase activity in 16-MT keratinocyte cell line. Telomerase activity was shown to be at a low level in the 16-MT cells. Lane 1: 1,000 cells. Lane 2: 500 cells. Lane 3: 100 cells. Lane 4: 16MT cells plus RNase 1,000 cells. Lane 5: 16-MT cells1RNase, 500 cells. Lane 6: MDA-MB-231 breast cancer, 200 cells. Lane 7: breast cancer, 100 cells. Lanes 8, 200 cells, and lane 9, 100 cells, show MDA-MD-231 breast cancer cells treated with RNase.
In summary, we found specific chromosomal aberrations such as t(3;19), t(11;19), and del(X) involving chromosome gains and loss of chromosomes (from the 16-MT composite karyotype) associated with HPV-16 transformation and induced genomic instability of human HPV16-transformed keratinocytes. The data presented here are consistent with previous reports describing chromosomal instability in HPV-infected epithelial cells, but also indicate newly identified chromosomal aberrations such as der(3;19), der(11;19), and der (13;14) originating from this process. The identification of HPV-induced chromosomal changes in the tumorigenic cell lines using advanced molecular cytogenetic techniques such as SKY and array CGH supports the usefulness of the 16-MT cell line as an in vitro model for studying important steps during HPV-induced related malignant transformation in human keratinocytes.
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Molecular cytogenetic characterization of human papillomavirus 16-transformed foreskin keratinocyte cell line 16-MT Eva M. McGheea,b,*, Philip D. Cotterc,d, Jingly F. Weiere, Jennifer W. Berlinef, Mary A. Turnerg, Mathew Gormleye, Joel M. Palefskyb,f a
Department of Community Health Systems, University of California, San Francisco (UCSF), Room N505, Box 0608 San Francisco, CA 94143-0608 b Comprehensive Cancer Center, UCSF, San Francisco, CA 94143 c Department of Pediatrics, Medical Genetics, UCSF, San Francisco, CA 94143 d Department of Pathology, Children’s Hospital and Research Center at Oakland, 747 52nd street, Oakland, CA 94609 e Department of Obstetrics, Gynecology and Reproductive Services, UCSF, San Francisco, CA 94143 f Department of Medicine, UCSF, San Francisco, CA 94143 g Department of Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 Received 28 September 2005; received in revised form 15 December 2005; accepted 30 December 2005
Abstract
Anogenital cancers are closely associated with human papillomavirus (HPV), and HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancers. Although genomic instability has been documented in HPV-infected keratinocytes, the full spectrum of genetic changes in HPV-associated lesions has not been fully defined. To address this, we examined an HPV16-transformed foreskin keratinocyte cell line, 16-MT, by GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (array CGH); these analyses revealed multiple numerical, complex, and cryptic chromosome rearrangements. Based on GTGbanding, the 16-MT karyotype was interpreted as 78~83,XXY,1add(1)(p36.3),13,14,15, 15,17,18,1i(8)(q10)2,110,?der(12),der(13;14)(q10;q10),115,116,add(19)(q13.3),121,121, 222[cp20]. Multicolor analysis by SKY confirmed and further characterized the anomalies identified by GTG banding. The add(1) was identified as a der(1)(1qter/1q25::1p36.1/1qter), the add(19) as a dup(19), and the der(12) interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement with chromosome 19. In addition, previously unidentified der(9)t(9;22), der(3)t(3;19), and der(4)t(4;9) were noted. The 16-MT cell line showed losses and gains of DNA due to unbalanced translocations and complex rearrangements of regions containing known tumor suppressor genes. Chromosomal changes in these regions might explain the increased risk of cancer associated with HPV. Also, array CGH detected copy-number gains or amplifications of chromosomes 2, 8, 10, and 11 and deletions of chromosomes 3, 4, 11, and 15. These results provide the basis for the identification of candidate oncogenes responsible for cervical and anal cancer in amplified regions, and for putative tumor suppressor genes in commonly deleted regions like 11q22~23. Furthermore, these data represent the first full characterization of the HPV-positive cell line 16-MT. Ó 2006 Elsevier Inc. All rights reserved.
1. Introduction Anogenital cancers are closely associated with human papillomavirus (HPV) infection [1–4]. HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancer [1,2]. Genomic instability has been previously documented in human HPV-infected keratinocytes [5–11]. The development of genomic instability is an early and crucial event during * Corresponding author. Tel.: (415) 476-3931; fax: (415) 476-6042. E-mail address: [email protected] (E.M. McGhee). 0165-4608/06/$ – see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.12.015
HPV-associated carcinogenesis [12]; however, the full spectrum of genomic changes in HPV-infected keratinocytes has not been fully defined. The identification of chromosomal alterations with pathologic implications in HPVrelated squamous cell carcinomas is of major importance, given that the carcinomas account for substantial mortality and morbidity [6]. Although a substantial number of HPVrelated cancers have been examined, specific chromosomal alterations or changes associated with initiation of tumor progression have not been fully characterized. The mechanisms of chromosomal rearrangement and other genomic changes in HPV-infected cells are not well
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understood. Rare HPV-negative carcinoma cell lines do exist [9,13,14], but almost all cervical carcinoma cell lines contain HPV DNA, and most cervical carcinoma cell lines have been shown to have single or multiple copies of integrated HPV [15]. Integration of HPV in rearranged chromosomes in cervical carcinomas or HPV-immortalized epithelial cells implicates the process of viral integration as at least one mechanism of HPV-induced genomic reorganization [16]. In addition, HPV gene products, such as the E6 and E7 oncoproteins, are also potential contributing factors to the generation of chromosomal rearrangements [17]. These oncoproteins target tumor suppressor signaling pathways that are critical for control of cellular growth. The HPV E6 protein of oncogenic HPV types such as HPV16 induces accelerated proteasomal degradation of p53 protein. In addition, the HPV E7 protein of oncogenic HPV types binds and degrades the pRB retinoblastoma tumor suppressor protein and interacts with the cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1, resulting in dysfunction of the cell cycle checkpoints and causing genomic instability [11]. To better characterize genomic instability induced by HPV 16, we studied the 16-MT cell line. 16-MT cells were derived from a neonatal human foreskin and then transformed with whole genomic HPV16 DNA. We characterized this cell line using several different cytogenetic techniques including GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (CGH). We also explored the relationship between telomerase expression and chromosomal instability in 16-MT cells. Regulation of telomerase activity is dependent on the reverse-transcriptase component of telomerase (hTERT) [18]. hTERT and low level of telomerase activity have been shown to correlate with increased chromosomal instability and an increase in chromosome copy number [19]. HPV16 E6 can upregulate hTERT transcription. Here we report the G-banded composite, SKY characterization, and array CGH analysis of the HPV-positive 16MT cell line. Our data show that 16-MT cells demonstrate chromosomal instability, an increase in chromosomal copy number, and low-level telomerase activity.
2. Materials and methods 2.1. Cell line and growth conditions Primary keratinocyte cells were generated from a neonatal human foreskin and the 16-MT cell line was generated by transfecting the cells with HPV16 using calcium phosphate methodology. Stable transfection was confirmed by dot blot analysis [20,21]. The 16-MT cell line was grown in complete keratinocyte growth media (KGM CC-4131; Clonetics, San Diego, CA) and complete Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (DMEM) (3:1) and incubated at 37 C in a 5% CO2
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incubator. 16-MT cells from passage 13 were studied. The nontransformed parental cell line was also grown in complete KGM medium under the same conditions as 16MT. Cell lines were tested for mycoplasma using a MycoAlert mycoplasma detection kit (Cambrex, East Rutherford, NJ); the results were negative. 2.2. GTG-banding analysis Metaphase chromosomes were prepared from 16-MT cells and analyzed by GTG-banding using standard protocols. Clonal chromosomal abnormalities identified by means of GTG-banding were described according to ISCN 1995 [22]. 2.3. SKY SKY analysis was performed on the 16-MT cells using aged metaphases on unstained slides. The slides were aged in 2 saline sodium citrate (SSC) at 37 C for 30 min. Cells were pretreated with pepsin (30 mg/mL pepsin in 0.01 mol/ L HCl) at 37 C for 5 min, 1 phosphate buffered saline (PBS) at room temperature for 5 min, then postfixed in 1% formaldehyde in 1 PBS/MgCl2 at room temperature for 5 min and washed in PBS. Finally, the slides were dehydrated in 70, 80, and 100% ethanol for 2 min. The SKY probes (Vial 1, SKY kit; Spectral Imaging, Carlsbad, CA) were denatured at 76 C for 7 min, then allowed to preanneal at 37 C for 60 min. The slides were denatured at 76 C for 5 min in 70% formamide–2 SSC, pH 7.0, then were dehydrated in 70, 80, and 100% ethanol for 2 min and allowed to air dry. The hybridization mixture was applied to the slides, covered with a glass coverslip, and sealed with rubber cement. The slides were hybridized at 37 C for 40–48 h in a moisture chamber. Following hybridization, the slides were washed at 43 C three times in 50% formamide–2 SSC for 10 min each, followed by two washes in 2 SSC for 10 min each, one wash in 0.4 SSC for 5 min, and a final wash in 0.1% Tween 20–4 SSC at room temperature for 2 min. Next, 80 mL of blocking reagent (Vial 2, SKY kit; Applied Spectral Imaging) was applied to each slide; the slides were then covered with a plastic coverslip and incubated at 37 C for 45 min. Spectral images were acquired using an SD200 SpectraCube spectral imaging system (Applied Spectral Imaging, Migdal Ha’emek, Israel) [23]. The SD200 imaging system attached to a Nikon E600 microscope consisted of an optical head (a Sagnac interferometer) coupled to a multiline charge-coupled device CCD camera (Hamamatsu, Bridgewater, NJ) to capture images at discrete interferometric steps. The images were stored as a stack in a Pentium 586/300 MHz computer. 2.4. Array CGH To further analyze the 16-MT cell line, array CGH analysis was performed as previously described [24,25]. The
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array consisted of 2,464 genomic clones printed in triplicate with an average resolution of 1.4 Mb across the genome. 16-MT cell DNA and normal reference DNA (from whole blood) were labeled by random priming with Cy3- or Cy5-labeled nucleotides and hybridized for 2 days to the array. Sixteen-bit 1024 1024 pixel images, with 40 ,6-diamidino-2-phenylindole (DAPI) Cy3 and Cy5 staining, were collected using a custom CCD camera system [26]. The data were analyzed using UCSF SPOT computer software [27] to automatically segment the array spots and to calculate the log2 ratios of the total Cy3 and Cy5 intensities for each spot after background subtraction. A second computer program, SPROC, was used to calculate averaged ratios of the triplicate spots for each clone, standard deviations of the triplicates, and plotting position for each clone on the array on the July 2003 freeze of the draft human genome sequence (http://genome.ucsc.edu). SPROC also implements a filtering procedure to reject data based on a number of criteria, including low reference-to-DAPI signal intensity and low correlation of the Cy3 and Cy5 intensities with a spot. The data files were edited to remove ratios on clones for which only one of the triplicates remained after SPROC analysis and/or the standard deviation of the log2 ratios of the triplicates was O 0.2 [25]. 2.5. Telomerase activity Telomerase activity was determined by using the TRAPeze XL Kit (Chemicon, Temecula, CA). The TRAPeze XL Kit uses fluorescence energy transfer (ET) primers to
generate fluorescently labeled TRAP products, which permit nonisotopic quantitative analysis of telomerase activity [28].
3. Results 3.1. Cytogenetic characterization of the 16-MT and parental cell lines by GTG- banding Cytogenetic analysis of 16-MT cells by GTG-banding showed that all metaphases had a complex hypertriploid karyotype with multiple structural and numerical abnormalities. The 16-MT cells displayed a karyotype of 78~83, XXY,1add(1)(p36.3),13,14,15,15,17,18,1i(8)(q10)2, 110,?der(12),der(13;14)(q10;q10),115,116,add(19)(q13.3), 121,121,222[cp20]. Figure 1 shows a representative karyotype of a GTG-banded metaphase for 16-MT cell line. The 16-MT karyotype interpreted from the GTG banding is summarized in Table 1. The parental human foreskin keratinocyte cells had a 46,XY karyotype (Table 1). 3.2. SKY characterization of the 16-MT cell line Multicolor SKY analysis confirmed the presence of the i(8)(q10) and der(13;14) rearrangements and further characterized the add(1), ?der(12), and add(19) rearrangements noted with GTG banding. The add(1) was identified as a der(1)(1qter/1q25::1p36.1/1qter) and the add(19) as a dup(19); the der(12) was interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement
Fig. 1. Representative GTG-banded karyotype composite of the 16-MT keratinocyte cell line. The karyotype for the cells shown is interpreted as 78~83,XXY, indicating a hypertriploid cell line.
E.M. McGhee et al. / Cancer Genetics and Cytogenetics 168 (2006) 36–43 Table 1 Representative karyotypes of keratinocyte cell line 16-MT as determined by conventional cytogenetic analysis and by spectral karyotyping Analysis
Karyotype
GTG-banding
78~83,XXY,1add(1)(p36.3),13,14,15,15,17, 18,1i(8)(q10)2,110,?der(12),der(13;14)(q10;q10), 115,116,add(19)(q13.3),121,121,222[cp20] 78~83,XXY,1der(1)(1qter/1q25::1p36.1/1qter), 13,der(3)t(3;19),14,der(4)t(4;9),15,15,17,18, 1i(8)(q10)2,der(9)t(9;22),110,211,add(11)(p15), der(12)t(11;12)(q24.3;q13),der(13;14)(q10;q10), 115,116,der(17)t(4;17),dup(19),121,121,222, 1mar[cp20]
SKY
The karyotype for the control parental line was a normal 46,XY.
with chromosome 19 (Fig. 2). Abnormalities identified by SKY, not revealed by GTG banding, included der(3)t(3;19), der(4)t(4;9), der(9)t(9;22), and der(17)t(4;17) (Fig. 2). The composite karyotype based on SKY analysis is given in Table 1. 3.3. Numerical and aneuploid abnormalities characterization of the 16-MT cell line Both GTG-banding and SKY analysis identified and confirmed numerical and aneuploid abnormalities for the 16-MT cell line. Figure 3 shows the mean for aneuploid abnormalities. The profile for chromosome 8 showed the highest copy number for 16-MT, with at least six copies of chromosome 8 appearing in each metaphase counted (Fig. 3). 3.4. Array CGH characterization of the 16-MT cell line Array CGH experiments were used to detect and map chromosomal imbalances. Array CGH data provided precise measurement (standard deviation of log2 ratios 5 0.05–0.10) of numerical chromosome changes. We detected copy number gains and losses as well as amplifications in the 16-MT cell line (Fig. 4). These included cryptic deletions on chromosomes 3, 4, 6, 11, 12, 18, 19, and X and amplifications on chromosomes 2, 4, 8, 11, 13, and 14. The array CGH analysis also revealed whole chromosome gain (chromosomes 4, 5, 6, and 8) in the 16-MT cells, as well as a del(X) (Fig. 4). A deletion on chromosome 11q22~23 was also noted, with this region containing the locus for the ATM gene as well as other tumor suppressor genes (Fig. 4). 3.5. Telomerase activity of the 16-MT cell line Telomerase activity was measured in 16-MT cells. These cells exhibited low telomerase activity (Fig. 5). In contrast, an immortalized breast cancer cell line (MDA-MB-231) used for control showed high telomerase activity, as expected (Fig. 5).
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4. Discussion Cancer has been characterized as a disease of genomic instability and the malignant transformation of human epithelial cells in vitro by the action of HPV is paralleled by cytogenetic changes. We developed a new HPV16-positive cell line, 16-MT, by HPV 16 transfection of normal human foreskin keratinocyte cells as a model to study HPV-associated cancers. The 16-MT cell line showed specific derivative chromosomal rearrangements involving several chromosomes, and some of these chromosomes are noted in the literature to be associated with cervical carcinoma and leukemiadspecifically, chromosomes 1, 3q, 5, 8, 10, and 11q [8]. Previous reports of in vitro systems developed to study genomic instability steps of HPV-induced chromosomal instability of transformed keratinocytes already postulated a multistep process for cancer development [7,9,29]. The primary untransformed human foreskin parental cells of the 16-MT cell line showed no cytogenetic alterations, which confirms that HPV-induced transformation led to complex chromosomal rearrangements. G-banding analysis of 16-MT cells showed a hypertriploid karyotype containing multiple structural and numerical abnormalities. This suggests that induced chromosomal instability came at least in part from the induction of HPV integration into common fragile sites of the host DNA, and produced specific chromosomal translocations and numerical changes [30]. Karyotypic instability after HPV integration preceding neoplastic transformation has been demonstrated for human fibroblasts [31]. Characterization by multicolor SKY analysis and array CGH revealed and confirmed numerous chromosomal imbalances in 16-MT cells (Figs. 2 and 4). Note in particular the finding of the amplified regions on 3q and 11q. Chromosome 11q is considered a strong candidate region for protooncogenes playing a key role in immortalization of keratinocytes upon transfection with HPV [32–34], and 3q is one of the most frequent abnormalities in invasive carcinoma and the gain of 3q accompanies progression from high grade squamous intraepithelial lesions (HGSIL) to invasion [35]. In our in vitro model system, chromosomes 3, 4, 5, 8, 9, 11, 13, 14, 16, 17, 18, 19, and 22 were found to be frequently affected by translocations and derivative rearrangements, as well as by numerical imbalances (Figs. 2 and 3). Inactivation of checkpoint components due to chromosomal alterations of genes controlling cellular damage can activate the process of immortalization [29,36–38]. Our findings from SKY analysis greatly improved the conventional cytogenetic description of the complex chromosomal rearrangements by revealing small interstitial changes and previously unrecognized abnormalities. Combined, the GTG-banding and SKY analyses revealed complex and recurrent translocations between chromosomes: (3;19), (4;9), (4;17), (9;22), (11;19), (14;13), (5;16;5), (18;5), (13;19), i(4), i(8),
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Fig. 2. Representative karyotype from SKY analysis composite of the 16-MT keratinocyte cell line. (A) Red–green–magenta image, (B) DAPI image, (C) classified image, (D) red–green–blue metaphase image indicating structural changes (E–Q) and additional structural changes confirmed by SKY analysis.
i(13), and i(16) (Figs. 2E–2Q). Several of these alterations have been reported in HPV-related cancers cell lines, including SiHa, Caski, and CamCell3 [12]. In 16-MT cells, chromosome 1 was involved in a substantial portion of the abnormal metaphases scored. Chromosome 1 is among those undergoing the most alterations in cervical cancers [12], frequently involving several genes, such as TP73 and TP53, located on chromosome 1p36.3. Loss of chromosome arm 3p and gain of 3q are also known to be common events in HPV immortalized cell lines, cervical and anal cancer [39]. In a recent study involving a small
number of cases, gain of 3q was reported as the most commonly found genetic change in anal intraepithelial neoplasia (AIN) of HIV-positive men [40], and gain of 3q was reported as defining the transition from severe dysplasia to invasive carcinoma of the uterine cervix [41]. In the 16-MT cells, however, our array CGH analysis showed both loss and gain on chromosome 3. Previous reports show that structural rearrangement of chromosome 8 particularly i(8q) derivatives, were commonly found in HPV-infected cancer cells [42]. In our study of 16-MT cells, i(8q) was found in several metaphases, as
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Fig. 3. Representative profile for numerical abnormalities and aneuploid chromosomal number analyzed by SKY in 16-MT keratinocyte cells. Analysis from composite showed chromosome 8 to be the most common aneuploid chromosome in the 20 metaphases scored.
well as chromosomes i(5) and i(10). The 11;19 rearrangement in 16-MT was detected in >85% of the metaphases analyzed. Deletion of genes on the long arm of chromosome 11 in the 16-MT cell line is likely to be important, given that multiple tumor suppressor genes are localized at region 11q.
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Our array CGH data showed that chromosomes 4, 8, 11, and X have distinct regions of focal gain and loss (4q gain, 8p gain, 8q gain, 11q loss and gain; and, Xp telomeric loss). The array CGH results revealed cryptic deletions and gains involving chromosomes whose localization was cytogenetically compatible with the aberrant regions identified by SKY. The high level of amplifications of chromosome 8 may reflect later events, thus exhibiting focal amplifications superimposed upon the background of whole chromosome gain. A high level of telomerase activity has been shown to stabilize telomere length and reduce chromosomal rearrangements [19]. In contrast, low telomerase activity correlated with increased chromosomal instability [19]. Consistent with these findings, we showed high frequencies of chromosomal structural aberrations (Figs. 1 and 2) associated with low telomerase activity (Fig. 5) in the 16-MT cell line. These data further support the correlations between telomerase activity and chromosome or genomic instability.
Fig. 4. Representative array CGH profile of the 16-MT keratinocyte cell line. Array CGH analysis was used to detect and map chromosomal gains and losses in the 16-MT cells by hybridizing targets of genomic DNA from a test and a reference sample to sequences immobilized on prepared slides. These probes are genomic DNA sequences (i.e., bacterial artificial chromosomes, or BACs, that are mapped on the genome). The array CGH data show specific chromosomes of interest that indicated gains (3, 4, 5, 8, 9, 11, 19) and losses (3, 4, 6, 11, 12, 13, 14, 18, 19, 22).
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Acknowledgments The authors would like to thank Ms. Maria Da Costa, Gloria Tung and Ms. Inna Govberg for their technical contributions to these studies, and the Blackburn laboratory (Dr. Jue Lin for her help with the telomerase detection). This work was supported by a grant from the National Institutes of Health, National Cancer Institute, R01 CA088739-05S1. References
Fig. 5. Representative telomerase activity in 16-MT keratinocyte cell line. Telomerase activity was shown to be at a low level in the 16-MT cells. Lane 1: 1,000 cells. Lane 2: 500 cells. Lane 3: 100 cells. Lane 4: 16MT cells plus RNase 1,000 cells. Lane 5: 16-MT cells1RNase, 500 cells. Lane 6: MDA-MB-231 breast cancer, 200 cells. Lane 7: breast cancer, 100 cells. Lanes 8, 200 cells, and lane 9, 100 cells, show MDA-MD-231 breast cancer cells treated with RNase.
In summary, we found specific chromosomal aberrations such as t(3;19), t(11;19), and del(X) involving chromosome gains and loss of chromosomes (from the 16-MT composite karyotype) associated with HPV-16 transformation and induced genomic instability of human HPV16-transformed keratinocytes. The data presented here are consistent with previous reports describing chromosomal instability in HPV-infected epithelial cells, but also indicate newly identified chromosomal aberrations such as der(3;19), der(11;19), and der (13;14) originating from this process. The identification of HPV-induced chromosomal changes in the tumorigenic cell lines using advanced molecular cytogenetic techniques such as SKY and array CGH supports the usefulness of the 16-MT cell line as an in vitro model for studying important steps during HPV-induced related malignant transformation in human keratinocytes.
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