Affiliations of authors: Department of Pathology, Unit of Molecular Pathology (RDMS, DK, CJLMM, PJFS), Department of Otolaryngology/Head and Neck Surgery (BJMB), and Department of Obstetrics and Gynecology (RHMV), Vrije Universiteit Medical Center, Amsterdam, The Netherlands; Cancer Research UK Immunology Group, Paterson Institute for Cancer Research, Manchester, UK (PLS).
Correspondence to: Renske D. M. Steenbergen, PhD, Department of Pathology, Unit of Molecular Pathology, Vrije Universiteit Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands (e-mail: r.steenbergen{at}vumc.nl)
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
However, genetic alterations are also required for a high-risk HPVinfected cell to progress to invasive carcinoma because HPV infections alone do not result in malignant transformation. For example, results of a cell fusion study (7) suggested that HPV-infected cells must acquire immortal and anchorage-independent phenotypes to become tumorigenic. All of these phenotypes are recessive traits, suggesting the involvement of tumor suppressor genes. The E6 and E7 oncogene products encoded by the high-risk HPV types inactivate the p53 and Rb tumor suppressor gene pathways, respectively, initially leading to genetic instability and an extended lifespan (8). However, HPV-transformed cells occasionally produce immortal subclones that, after prolonged culturing, can successively produce anchorage-independent and tumorigenic descendants (9,10). Results of microcell-mediated chromosome-transfer studies (1113) revealed that HPV-mediated immortalization and the concomitant activation of telomerase results from the additional loss of yet-unknown tumor suppressor genes on chromosomes 3, 4, 6, and/or 10.
We (Steenbergen RD, Kramer D, Braakhuis BJ, Meijer CJ, Snijders PJ: unpublished data) and others (14,15) have found that microcell-mediated transfer of chromosome 11 reverses the tumorigenic phenotype of HPV DNAcontaining cervical cancer cells, without having an effect on the immortal phenotype (12), suggesting that the loss of a tumor suppressor gene on chromosome 11 is involved in the acquisition of the tumorigenic phenotype. Results of allelotype studies in cervical carcinomas suggest that two loci on chromosome 11 [i.e., 11q13 (1618) and 11q2223 (1921)] undergo high-frequency loss of heterozygosity and thus might contain such a tumor suppressor gene. In particular, loss of heterozygosity at 11q23 is associated with vaso-invasive and metastatic cervical squamous cell carcinomas (22). Results of a functional complementation study in lung cancer A549 cells (23) suggest that this chromosomal region contains a candidate tumor suppressor gene, which was named tumor suppressor in lung cancer 1 (TSLC1) (23). The TSLC1 gene encodes a member of the immunoglobulin superfamily with structural homology to the neural cell adhesion molecules NCAM1 and NCAM2. Tslc1 is a 75-kd amino-terminal-glycosylated protein that exists as a homodimer at the cell membrane and is involved in intracellular adhesion through Ca2+- and Mg2+-independent homophilic trans-interactions (24).
Reexpression of the TSLC1 gene in A549 cells (which normally do not express TSLC1) suppressed their tumorigenicity in nude mice, and absent or reduced TSLC1 gene expression in other lung cancer cell lines was associated with tumorigenicity and metastasis (23). Silencing of TSLC1 mRNA expression in these cell lines was associated with hypermethylation of the TSLC1 promoter. In addition, TSLC1 promoter hypermethylation, with or without allelic loss, occurred in approximately 40% of the nonsmall-cell lung cancers examined, whereas inactivating mutations were rare (23). We examined TSLC1 gene expression and promoter hypermethylation in HPV-immortalized cell lines, cervical carcinoma cell lines, and cervical tissue specimens.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary human keratinocytes were isolated from the foreskins of four donors and cultured as described previously (25). Cultured primary human foreskin keratinocytes of one of these donors (i.e., EK942 cells) transfected with the entire genome of HPV16 or HPV18 were used to establish cell lines FK16A and FK16B or FK18A and FK18B, respectively, which were cultured and characterized as described previously (25). Human cervical carcinoma SiHa, HeLa, and CaSki cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle Medium (Life Technologies, Breda, The Netherlands) supplemented with 10% fetal calf serum, penicillin at 100 U/mL, streptomycin at 100 µg/mL, and L-glutamine at 2 mmol/L (all from Life Technologies). In some experiments, SiHa, HeLa, and CaSki cells were incubated with 5 µM 5-aza-2'-deoxycytidine (Sigma Chemical Co., St. Louis, MO) diluted in phosphate-buffered saline (PBS) or PBS alone for 1.5, 3, 5, or 7 days to analyze the effect of methylation inhibition on TSLC1 mRNA expression. All incubations were performed in duplicate wells, and cells were directly harvested for RNA isolation at the indicated times. The eight human cervical carcinoma cell lines (i.e., 778, 808, 866, 873, 877, 878, 879, and 915) were cultured as described previously (26). All cell lines were harvested by trypsinization of exponentially growing cells. DNA and RNA were isolated from cell lines with the use of a Puregene DNA isolation kit (Biozym, Landgraaf, The Netherlands) and RNAzolB reagent (Tel-Test, Friendswood, TX), respectively.
Tissue Specimens
We obtained formalin-fixed, paraffin-embedded specimens of normal cervix (n = 9); CIN I (n = 12), CIN II (n = 6), and CIN III (n = 14) lesions; and cervical carcinomas (n = 52), all collected during the course of routine clinical practice from women who were undergoing biopsy or surgery at the Department of Obstetrics and Gynecology at the Vrije Universiteit Medical Center (Amsterdam). All cervical carcinomas were squamous cell carcinomas; 29 (56%) were classified as poorly differentiated, 21 (40%) were classified as moderately differentiated, and two (4%) were classified as well-differentiated. The formalin-fixed tissue specimens were HPV typed using the general primer GP5+/6+ polymerase chain reaction (PCR) enzyme immunoassay, as described by Jacobs et al. (27).
We also used conventional archival cervical smears and cervical cancer biopsy samples from cervical cancer patients that were obtained in a retrospective casecontrol study designed to examine the value of high-risk HPV testing to detect false-negative cervical smears in women who developed cervical cancer and to assess whether high-risk HPV DNA is present in cervical smears of women before they are diagnosed with cervical cancer (28). The DNA integrity of all archival samples was assessed by -globin PCR, as described previously (28), and all samples included in this study tested positive for
-globin DNA.
We also used conventional cervical smears that were classified as low-grade SILs (LSILs), contained HPV16 DNA, and were freshly collected from women participating in a Dutch population-based cervical cancer screening program (29) as control samples for the bisulfite genomic sequencing method. We extracted DNA from these clinical specimens by incubating them in proteinase K at 0.1 mg/mL and 0.5% Tween 20 at 37 °C overnight and purified the DNA using a High Pure PCR Template Preparation kit (Roche Diagnostics, Almere, The Netherlands).
Three snap-frozen biopsy samples of normal cervical epithelium, all of which were collected during the course of routine clinical practice from women who were undergoing biopsy or surgery at the Department of Obstetrics and Gynecology at the Vrije Universiteit Medical Center, were microdissected with the use of a laser capture microdissection microscope (Leica, Heidelberg, Germany). RNA was isolated from the microdissected tissue with the use of Trizol Reagent (Life Technologies). We also isolated RNA from snap-frozen cervical smears that were freshly collected from two women participating in a Dutch population-based cervical cancer screening program and that were classified as having normal cytology. Written informed consent was obtained from the women who supplied samples.
Real-Time Quantitative Reverse TranscriptionPolymerase Chain Reaction
TSLC1 mRNA levels were quantified by using real-time quantitative reverse transcriptionpolymerase chain reaction (RTPCR) and LightCycler technology (Roche Diagnostics). Each reaction mixture contained 50 ng of total RNA, 3.25 mM Mn(OAc)2, oligonucleotide primers at 0.3 µM (forward primer: 5'-CCCCAGCCTGTGATGGTAA-3'; reverse primer: 5'- GGATAGTTGTGGGGGGATCGTA-3'), hybridization probes 1 and 2 at 0.2 µM (TIB MolBiol, Berlin, Germany) (probe 1: 5'-ATAATGGTACATACCGCTGTGAAGCTTC-fluorescein-3' and probe 2: 5'-LC Red640-AACATAGTGGGGAAAGCTCACTCG G-3'), and 7.5 µL of RNA LightCycler RNA Master Hybridization Probes mixture (Roche Diagnostics) in a total volume of 10 µL. Reverse transcription was performed by incubating the reaction mixtures at 61 °C for 20 minutes, followed by 30 seconds at 95 °C, 50 cycles of PCR amplification (95 °C for 1 second, 55 °C for 15 seconds, and 72 °C for 13 seconds), and a final incubation at 40 °C for 30 seconds. We also used a dilution series of primary human keratinocyte RNA ranging from 0.1 ng to 200 ng as templates in PCR reactions to serve as a standard curve. We also performed RTPCR for the housekeeping gene porphobilinogen deaminase (PBGD) as a reference for quantitative assessment of TSLC1 mRNA levels as described previously (30). To prevent amplification of residual genomic DNA, the RNA samples were pretreated with RQ1-DNase, according to the manufacturer's directions (Promega, Leiden, The Netherlands). We normalized mRNA expression levels in cell lines and cervical tissues to the levels measured in primary foreskin keratinocytes according to the following formula: intensity ratio (TSLC1/PBGD) of analyzed cells/intensity ratio (TSLC1/PBGD) of primary keratinocytes x 100%.
Sequencing of Sodium BisulfiteModified Genomic DNA
We analyzed TSLC1 promoter methylation by sequencing sodium bisulfitemodified genomic DNA isolated from primary foreskin keratinocytes, cell lines, and cervical tissue specimens and smears. Sodium bisulfite modification of DNA converts unmethylated cytosine residues to uracil residues but does not affect methylated cytosine residues. Subsequent PCR amplification and sequencing of sodium bisulfitemodified DNA reveals whether cytosine residues in CpG islands (i.e., regions of the genome that are primarily associated with the 5' ends of most housekeeping genes and many regulated genes) were methylated (and thus remain as cytosine residues) or unmethylated (in which case PCR amplification replaces a uracil residue with a thymine residue) (31). We used a CpGenome DNA modification kit (Intergene, Purchase, NY) to modify genomic DNA. PCR amplification of the TSLC1 promoter was performed using the forward (5'-GTGAGTGACGGAAATTTGTAATGTTTGGTT- 3'; forward primer nucleotide -487 to -458 with respect to ATG) and reverse (5'-AATCTAACTTCTTATACACCTTTAT TAAAA-3'; reverse primer nucleotide -333 to -364 with respect to ATG) primers described by Kuramochi et al. (23). Both primers efficiently anneal to unmethylated and methylated DNA. Although the forward primer covers two CpG sites, the change of one CpG site to a TpG site and the retention of cytosine at the other CpG site ensured proper amplification of both unmethylated and methylated DNA. The reverse primer does not anneal to any potential methylation sites and therefore also reacts with both methylated and unmethylated DNA.
The PCR mixtures contained 20 ng of modified genomic DNA, primers at 0.5 µM, each of the four deoxynucleotide triphosphates at 200 µM, 1.5 mM MgCl2, and 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). Amplification conditions were 95 °C for 7 minutes; 60 cycles of 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 45 seconds; and a final elongation step at 72 °C for 4 minutes. PCR products were purified using a QIAgen PCR purification kit (Westburg, Leusden, The Netherlands), and 93 base pairs (bp) of the TSLC1 promoter region (i.e., nucleotide -457 to -365 with respect to ATG) were sequenced directly using the forward or reverse primers labeled with [-33P]ATP (Amersham Life Sciences, Cleveland, OH) and an AmpliCycle Sequencing Kit (Applied Biosystems) (32). We performed two separate sequencing reactions with each primer to analyze either the thymine or the cytosine residues using the forward primer or the adenine or guanine residues using the reverse primer. The sequencing products were separated on a 6% denaturing acryl/ bis-acrylamide sequencing gel using a Genomyx LR Sequencer (Beckman Coulter, Fullerton, CA). Gels were dried directly in the Genomyx LR Sequencer for 1 hour and exposed overnight at room temperature to Biomax Imaging Film (Kodak, Labsystems, Breda, The Netherlands).
Microsatellite Analysis
We used the following microsatellite markers, all located at chromosome 11q2223, to analyze loss of heterozygosity: D11S35, D11S1885, D11S2077, D11S4111, and D11S29. We obtained the DNA sequences of these microsatellite markers from the Genome Database (The Johns Hopkins University, Baltimore, MD) and used them to make forward and reverse oligonucleotide primers for each marker. Forward primers were labeled with one of two fluorescent dyes, fluorescein (6-FAM) or 6-carboxy-4',5'dichloro-2',7'-dimethoxy fluorescein (JOE) (Eurogentec, Seraing, Belgium). PCR amplifications were performed in 50-µL reaction mixtures that contained 10 ng of genomic DNA, forward and reverse primers for each marker at 0.2 µM, four deoxynucleotide triphosphates at 200 µM, 1.5 mM MgCl2, and 1 U of Taq DNA polymerase (AmpliTaq; Applied Biosystems) according to the following conditions: 95 °C for 5 minutes; 30 cycles of 95 °C for 1 minute, 5558 °C (depending on the primer set) for 1 minute, and 72 °C for 1 minute; and a final elongation step at 72 °C for 5 minutes. We mixed 1 µL of each PCR reaction product with 12 µL of deionized formamide and 0.5 µL of GeneScan-350 [ROX] size standards (Applied Biosystems), loaded them onto an automated ABI PRISM sequencer (3100 Genetic Analyzer; Applied Biosystems), and analyzed the data with GeneScan Analysis software (Applied Biosystems). The GeneScan Analysis data are presented as electropherograms, in which allele sizes on the horizontal axis are plotted against the signal intensities on the vertical axis. Allele losses were identified by the absence of peaks in DNA isolated from carcinoma cell lines compared with DNA isolated from normal cells of the same patient. Microsatellite instability was identified by a change in peak size in DNA isolated from carcinoma cell lines compared with DNA isolated from normal cells of the same patient. For control DNA, we used DNA isolated from the primary donor keratinocytes to analyze the HPV-immortalized cell lines, and we used DNA isolated from either lymphoblasts or fibroblasts of the same patients to analyze the eight cervical carcinoma cell lines.
Cloning and Transfection of TSLC1 Complementary DNA
We PCR-amplified the full-length TSLC1 complementary DNA (cDNA) from a primary human keratinocyte poly A+ mRNA library by using the following primers: forward primer, 5'-ACATGGCGAGTGTAGTGCTG-3' and reverse primer, 5'-CTAGATGAAGTACTCTTTCTTTTCTTCG-3'. PCR amplification was performed in a 50-µL reaction mixture that contained each primer at 1 µM, each deoxynucleotide triphosphate at 200 µM, 1x Native Plus Pfu buffer, and 2.5 U of Native Pfu DNA polymerase (Stratagene, La Jolla, CA) according to the following conditions: 45 cycles of 94 °C for 1 minute, 59 °C for 1 minute and 30 seconds, and 72 °C for 2 minutes. The PCR product containing the TSLC1 cDNA was cloned into the SrfI site of a PCR-Script vector (Stratagene). The resulting plasmid was then digested with KpnI and NotI (Roche Diagnostics), and the 1411-bp fragment containing the full-length TSLC1 cDNA was recloned into the KpnI and NotI sites of pcDNA3.1hygro (+) (Invitrogen, Carlsbad, CA). The sequence of the cloned TSLC1 cDNA was verified by sequence analysis using a ThermoSequenase Dye Terminator Cycle Sequencing Kit (Amersham Life Sciences) and an ABI373A/xl DNA automated sequencer (Applied Biosystems). pcDNA3.1hygro (+)/TSLC1 and pcDNA3.1hygro (+) (i.e., without the TSLC1 cDNA) were used (separately) to stably transfect SiHa cells by using DAC-30 transfection reagent (Eurogentec). The transfection efficiency was approximately 5%, as determined by dividing the number of colonies by the total number of cells used for transfection; cells stably transfected with pcDNA3.1hygro (+)/TSLC1 (i.e., SiHa/TSLC1 cells) or pcDNA3.1hygro (+) (i.e., SiHa/hygro cells) were selected by continuous culturing of the transfected SiHa cells in the presence of hygromycin B at 300 µg/mL (Roche Diagnostics) and by using cloning cylinders to pick single colonies.
Cell Proliferation and Anchorage-Independence Assays
Cell proliferation was measured in duplicate experiments with the use of WST-1 Reagent (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3 benzene disulfonate) according to the manufacturer's instructions (Roche Diagnostics). Anchorage-independent cell growth was analyzed by plating 0.35% top agarose (Seaplague agarose; FMC Bioproducts, Rockland, ME) containing 5000 cells on a surface of 0.6% bottom agarose in 6-cm dishes and incubating at 37 °C. Cells were fed weekly by overlaying the agarose with fresh medium. Colonies were photographed and counted after 3 weeks of incubation.
Tumorigenicity Assay
The tumorigenicity of SiHa, SiHa/TSLC1, and SiHa/hygro cells was assessed by subcutaneously injecting 3 x 106 to 5 x 106 cells in 150 µL Dulbecco's modified Eagle Medium per site into 8-week-old athymic female Hsd: nu/nu mice (five mice for SiHa cell injections, two mice for SiHa/hygro injections, and two mice for each of four different SiHa/TSLC1 transfectant injections) obtained from Harlan (Horst, The Netherlands). Tumorigenicity of 778, 808, 866, and 879 cervical carcinoma cells was assessed by injecting 1 x 106 cells mixed 1 : 1 with Matrigel Basement Membrane Matrix (BD Biosciences, Bedford, MA) per site in a total volume of 200 µL subcutaneously into female CB17 scid/scid mice (68 weeks old, four mice per cell line; Harlan). Each cell line was injected two to five times. Tumor volume (in mm3) was measured once a week with vernier calipers and was calculated according to the following formula: length x width x height x 0.5 (33). The mice were killed by cervical dislocation when the tumors had reached a volume greater than 250 mm3, when infiltrative growth of the tumor into the skin was observed, or at 3 months after injection, whichever came first. All tumors were excised and examined histopathologically. Tumor growth was scored as positive when tumor volume exceeded 50 mm3. This volume was based on our experience in previous studies in which 50 mm3 was found to correspond to a true tumor (33). All animal experiments were performed according to Dutch law and approved by the authorities of Vrije Universiteit.
Statistical Analysis
Fisher's exact tests (two-sided) and Wilcoxon signed rank tests (two-sided) were used to assess the statistical significance of differences between the various cell lines and tissue specimens. Differences were considered to be statistically significant at P<.05. Student's t test or a normal reference distribution was used to construct 95% confidence intervals for the differences in the various cell lines and tissue specimens.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We measured TSLC1 mRNA levels in normal cervical epithelial cells that were derived from biopsy samples and cervical smears and in 11 cervical carcinoma cell lines that contained high-risk HPV DNA. The mRNA levels were measured by real-time quantitative RTPCR and compared with the average TSLC1 mRNA level measured in three isolates of primary keratinocytes, which was set at 100%. In normal cervical epithelial cells, the TSLC1 mRNA levels were comparable to those observed in the primary keratinocytes (data not shown). By contrast, TSLC1 mRNA was undetectable in seven of the 11 cervical carcinoma cell lines (i.e., SiHa, CaSki, 778, 808, 873, 877, and 879 cells), and HeLa, 866, and 915 cells had only 3%, 5%, and 3%, respectively, of the TSLC1 mRNA level of primary keratinocytes. The only cervical carcinoma cell line that had a TSLC1 mRNA level comparable to that observed in the primary keratinocytes was 878 (Table 1 and Fig. 1, A). Thus, we observed either a complete loss of or a marked decrease in TSLC1 mRNA expression in 10 of the 11 (91%, 95% confidence interval [CI] = 59% to 100%) cervical carcinoma cell lines we analyzed.
|
|
In summary, TSLC1 gene silencing was observed in 91% of the cervical carcinoma cell lines we analyzed but in 0% of the non-tumorigenic HPV-immortalized cell lines (difference = 91%, 95% CI = 74% to 100%; P = .004). These data suggest that TSLC1 silencing is likely to be associated with the transition from an immortal to a tumorigenic phenotype.
TSLC1 Promoter Hypermethylation and Loss of Heterozygosity at 11q23
We next examined the possible mechanism(s) underlying the reduced levels of TSLC1 mRNA observed among the cervical cancer cell lines. We specifically examined the role of DNA methylation because hypermethylation of CpG islands in the promoter regions of genes is associated with the inhibition of gene transcription and because hypermethylation of the TSLC1 gene promoter is associated with TSLC1 gene silencing in human lung cancers (23). We treated SiHa, HeLa, and CaSki cells with the DNA methylation inhibitor 5-aza-2'-deoxycytidine for various times and compared the levels of TSLC1 mRNA with that in primary keratinocytes, which was set to 100%. After 57 days of treatment with 5-aza-2'-deoxycytidine, TSLC1 mRNA levels in SiHa cells increased from 0% to 26% (difference = 26%, 95% CI = 21% to 31%; P = .18) of that in primary keratinocytes, TSLC1 mRNA levels in CaSki cells increased from 0% to 33% (difference = 33%, 95% CI = 23% to 43%, P = .18) of that in primary keratinocytes, and TSLC1 mRNA levels in HeLa cells increased from 4% to 58% (difference = 54%, 95% CI = 34% to 74%; P = .18) of that in primary keratinocytes (Fig. 1, B). These data suggest that, in these three cervical carcinoma cell lines, TSLC1 gene silencing could be due, at least in part, to methylation of the TSLC1 promoter itself or to methylation of the promoter of a gene that encodes a protein that regulates TSLC1 transcription.
We directly examined the methylation status of the TSLC1 promoter by sequencing sodium bisulfitemodified genomic DNA isolated from the 11 cervical carcinoma cell lines. We observed three distinct patterns of DNA methylation at the six CpG sites within the TSLC1 promoter (Fig. 2, A; Table 1). In the first pattern, which was displayed by SiHa, HeLa, CaSki, 778, 873, and 879 cells, we detected no unmethylated sites; this methylation pattern suggested that the TSLC1 promoter was methylated at both alleles in all cells. The second pattern, which was observed in 808, 877, and 878 cells, was characterized by heterogeneity in promoter methylation, suggesting that the TSLC1 promoter was not completely methylated in all cells or that methylation involved only one allele. In the third pattern, which was displayed by 866 and 915 cells, none of the CpG sites were methylated. Hypermethylation of the TSLC1 promoter was associated with reduced or undetectable TSLC1 mRNA levels in all cell lines tested except for the 878 cells. This cell line had both methylated and unmethylated CpG sites, leading us to hypothesize that TSLC1 mRNA expression was attributable to the unmethylated allele. We detected no TSLC1 promoter methylation among four isolates of primary human keratinocytes (one of which is shown in Fig. 2, A) or among the four non-tumorigenic HPV-immortalized cells (i.e., FK16A, FK16B, FK18A, or FK18B cells), all of which were assayed at multiple passages after immortalization (Fig. 2, A).
|
|
Effects of TSLC1 Expression on Anchorage-Independent Cell Growth and Tumorigenicity
Anchorage-independent cell growth is thought to represent a phenotype that is expressed by cells as they progress from immortality to tumorigenicity (7). We tested whether TSLC1 gene silencing was associated with anchorage-independent cell growth by comparing the growth in soft agarose of TSLC1-expressing HPV-immortalized FK16A, FK16B, FK18A, and FK18B cells with that of the TSLC1 mRNA-negative cervical cancer SiHa cells. We found that the HPV-immortalized cells cultured in soft agarose produced either no colonies or a small number of colonies (i.e., 0100 colonies per 5000 cells) (Table 1). By contrast, tumorigenic cervical carcinoma SiHa cells gave rise to approximately 700800 colonies per 5000 cells when cultured in soft agarose.
To test the hypothesis that TSLC1 gene silencing was associated with the acquisition of the anchorage-independent growth phenotype, we transfected SiHa cells with a TSLC1 expression vector or an empty control vector, selected for cells stably transfected with each plasmid, and examined their TSLC1 mRNA expression, proliferation rate, and ability to grow in soft agarose. All eight (100%, 95% CI = 63% to 100%) of the SiHa/TSLC1 transfectants tested expressed TSLC1 mRNA and did not show an altered proliferation rate compared with parental cells. However, seven (88%, 95% CI = 47% to 100%) of the eight SiHa/TSLC1 transfectants displayed a marked reduction in anchorage-independent growth (i.e., 0100 colonies per 5000 cells; Table 1) compared with none of the four (0%, 95% CI = 0% to 60%) SiHa transfectants bearing the empty vector (i.e., SiHa/hygro transfectants; difference = 88%, 95% CI = 65% to 100%; P = .01) or untransfected SiHa cells; Fig. 4, AD. We picked 17 colonies derived from the SiHa/TSLC1 transfectants that were growing in soft agarose and grew them under standard culture conditions on plastic. Subsequent RTPCR analysis revealed that seven of the 17 transfectants no longer expressed detectable TSLC1 mRNA, suggesting that anchorage-independent growth in those colonies was the result of suppression of mRNA expression from the ectopic TSLC1 gene. Colonies isolated from SiHa and SiHa/hygro control transfectants that were growing in soft agarose had no detectable TSLC1 mRNA expression.
|
Methylation Status of the TSLC1 Promoter During Cervical Carcinogenesis
To determine whether and when the TSLC1 promoter becomes hypermethylated during cervical carcinogenesis in vivo, we analyzed the methylation status of the TSLC1 promoter in DNA isolated from cervical tissue specimens. We analyzed all tissue samples for the presence of high-risk HPV DNA and found detectable levels in 56% (5/9) of normal cervical epithelium specimens, most of which were distant tissue biopsy samples derived from women with cervical cancer or high-grade CIN lesions: 25% (3/12) of CIN I lesions, 50% (3/6) of CIN II lesions, 93% (13/14) of CIN III lesions, and 96% (50/52) of cervical carcinomas.
Because tissue specimens contain stromal cells with unmethylated DNA as well as abnormal cells with methylated DNA, we first determined the sensitivity of our assay to detect methylated CpG sites in a background of unmethylated DNA by analyzing a dilution series of SiHa DNA (methylated TSLC1 promoter) in DNA derived from primary keratinocytes (unmethylated TSLC1 promoter). We found that we could detect as little as 5%10% methylated DNA in a background of unmethylated DNA with high reliability (Fig. 2, B).
None (0%, 95% CI = 0% to 34%) of the nine normal cervical epithelial biopsy samples tested had detectable TSLC1 promoter methylation (Fig. 2, C). Similarly, none (0%, 95% CI = 0% to 22%) of the 12 CIN I lesions tested had detectable levels of TSLC1 promoter methylation. By contrast, 35% (7/20; 95% CI = 15% to 59%) of high-grade CIN lesions (i.e., CIN II and III) had detectable levels of TSLC1 promoter methylation (Fig. 2, C). In addition, we analyzed tissue sections of 52 cervical squamous cell carcinomas for TSLC1 promoter methylation status and found that 58% (30/52; 95% CI = 43% to 71%) had detectable TSLC1 promoter methylation (Fig. 2, C). There was a statistically significant difference between the percentage of high-grade CIN lesions with detectable TSLC1 promoter methylation (7/20; 35%, 95% CI = 15% to 59%) and the percentage of normal cervix samples (0/9) and low-grade CIN lesions with detectable TSLC1 promoter methylation (0/12; 0/23 combined, 0%; difference = 35%, 95% CI = 14% to 56%; P = .003). Moreover, there was a statistically significant difference between the percentage of cervical carcinomas with detectable TSLC1 promoter methylation (30/52; 58%) and the percentage of normal cervix samples (0/9) and low-grade CIN lesions with detectable TSLC1 promoter methylation (0/12; 0/23 combined, 0%; difference = 58%, 95% CI = 44% to 71%; P<.001). In all samples with detectable TSLC1 promoter methylation, all CpG islands sequenced were found to be methylated, and no partial methylation was seen.
Finally, we assessed whether TSLC1 promoter methylation was detectable in cervical smears. We first analyzed TSLC1 promoter methylation in freshly collected cervical smears (n = 15), all of which were classified as LSILs and contained HPV16 DNA, and found that 0% (0/15; 95% CI = 0% to 18%) had detectable levels of TSLC1 promoter methylation. We also analyzed TSLC1 promoter methylation in archival index cervical smears taken from 11 women at the time of cervical cancer diagnosis (28) as well as in the corresponding cervical cancer biopsy samples for 10 of those women (no biopsy sample was available for patient 4). Six (6/11; 55%, 95% CI = 23% to 83%) of the index smears (i.e., from patients 1, 4, 5, 7, 10, and 11) tested positive for TSLC1 promoter hypermethylation, as did the corresponding cancer biopsy samples for those patients with an available biopsy sample (Fig. 2, D; Table 2). Five of these six patients had archival cervical smears that had been taken as long as 19 years before cervical cancer was diagnosed and which were classified as either LSILs or high-grade SILs (HSILs). We found that smears that were taken up to 7 years prior to cervical cancer diagnosis had detectable levels of TSLC1 promoter hypermethylation (i.e., those from patients 1, 4, and 5), whereas smears taken more than 7 years prior to cervical cancer diagnosis had no detectable TSLC1 promoter hypermethylation (i.e., those from patients 7, 10, and 11) (Fig. 2, D; Table 2).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We also showed that TSLC1 gene expression is associated with the suppression of anchorage-independent growth, a phenotypic alteration that is thought to precede tumorigenicity (7). This finding suggests that TSLC1 gene silencing is involved in the early phases of tumorigenic growth induction. Our detection of TSLC1 gene silencing in nearly all cervical cancer cell lines and TSLC1 promoter hypermethylation in the majority of cervical carcinomas (58%) suggests an association between TSLC1 gene silencing and cervical cancer invasion. However, because genetic alterations (e.g., allelic loss and inactivating mutations) as well as epigenetic events (e.g., DNA methylation) can lead to the inactivation of a tumor suppressor gene, we expected that the actual percentage of cervical carcinomas in which the TSLC1 gene is silenced would be even higher than the percentage of carcinomas with TSLC1 promoter hypermethylation. Indeed, we found that a higher percentage of the cervical cancer cell lines had TSLC1 gene silencing than TSLC1 promoter hypermethylation (i.e., 91% versus 82%), suggesting the existence of alternative silencing mechanisms. Moreover, two of the cervical carcinoma cell lines we tested, cell lines 808 and 877, displayed markedly reduced levels of TSLC1 mRNA and a heterogeneous pattern of promoter methylation. This finding may indicate that one allele is methylated and the other allele is not. The unmethylated allele could, however, be inactivated by other mechanisms, e.g., an inactivating mutation or methylation of promoter regions outside the 93-bp region analyzed in this study [i.e., the same region as studied in lung cancers (23)], which might also result in TSLC1 gene silencing. However, we also found that a heterogeneous pattern of TSLC1 promoter hypermethylation was associated with unaltered TSLC1 mRNA expression in one of the cell lines (cell line 878). It is possible that in this cell line, Tslc1 protein was expressed but not functional, perhaps because of an inactivating gene mutation. Alternatively, our finding that 878 was the only cell line that expressed TSLC1 might be related to the presence of HPV45 DNA in this cell line; all of the other cell lines contained the more common high-risk types of HPV, i.e., HPV16 and HPV18.
The frequency of TSLC1 promoter hypermethylation among the cervical carcinomas we analyzed (58%) was substantially higher than the frequency of TSLC1 promoter hypermethylation that has been reported for other human malignancies. TSLC1 promoter hypermethylation has been detected in 40% of lung cancers (23), 32% of prostate cancers (38), 27% of primary pancreatic adenocarcinomas (39), 16% of primary gastric cancers (40), and in 33% of breast cancers showing loss of heterozygosity at 11q (41). However, only 10% of the latter tumor type displayed complete, or nearly complete, hypermethylation (41). We analyzed the same promoter region and found that all of the CpG islands of the TSLC1 promoter were fully methylated in all cervical carcinomas and CIN lesions showing TSLC1 promoter hypermethylation. This finding indicates that, in cervical cancer, both the frequency and the extent of TSLC1 promoter methylation are high.
Nearly all of the CIN lesions and cervical carcinomas in our study that had TSLC1 promoter methylation were positive for high-risk HPV DNA. However, we have no evidence that TSLC1 gene silencing is linked to the functions of the E6 and E7 oncoproteins expressed by high-risk HPV types. The TSLC1 gene was not silenced in the HPV-immortalized cell lines or in low-grade CIN lesions that were positive for high-risk HPV DNA. Considering the substantially higher frequency of TSLC1 gene silencing in cervical carcinomas compared with the other, nonHPV-associated human cancers analyzed so far (23,3941), we cannot exclude the possibility that infection with high-risk HPV predisposes cells to a disruption of TSLC1-mediated cell adhesion rather than to other mechanisms that induce expression of an invasive phenotype.
Finally, we detected TSLC1 promoter hypermethylation in archival cervical smears derived from women with cervical cancer. Interestingly, smears taken at the time of cervical cancer diagnosis as well as smears that were taken up to 7 years before cervical cancer diagnosis tested positive for TSLC1 promoter hypermethylation.
Our findings raise the possibility that TSLC1 promoter methylation analysis could have future applications in population-based cervical screening programs. However, such an application would require a more sensitive and less laborious method to detect TSLC1 promoter methylation. We recommend that methylation-specific PCR for TSLC1 should be applied in larger (nested) casecontrol studies (42) to determine the sensitivity and specificity of TSLC1 promoter hypermethylation to predict invasive cervical cancer.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Machteld van Dompselaar for excellent technical assistance with RTPCR analysis, Arie Kegel and Marco Schreurs for their help with the tumorigenicity studies, and Johannes Berkhof for his help with statistical analysis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1 Pisani P, Bray F, Parkin DM. Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 2002;97:7281.[CrossRef][ISI][Medline]
2 Richart RM. Causes and management of cervical intraepithelial neoplasia. Cancer 1987;60:19519.[ISI][Medline]
3 Solomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287:21149.
4 zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2:34250.[CrossRef][ISI][Medline]
5 Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:129.[CrossRef][ISI][Medline]
6 Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:51827.
7 Chen TM, Pecoraro G, Defendi V. Genetic analysis of in vitro progression of human papillomavirus-transfected human cervical cells. Cancer Res 1993;53:116771.[Abstract]
8 Scheffner M, Romanczuk H, Münger K, Huibregtse JM, Mietz JA, Howley PM. Functions of human papillomavirus proteins. In: Zur Hausen H, editor. Human pathogenic papillomaviruses-current topics in microbiology and immunology. Vol 186. Berlin (Germany): Springer-Verlag; 1994. p. 8399.
9 Hurlin PJ, Smith PP, Perez-Reyes N, Blanton RA, McDougall JK. Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Proc Natl Acad Sci U S A 1991;88:5704.[Abstract]
10 Pecoraro G, Lee M, Morgan D, Defendi V. Evolution of in vitro transformation and tumorigenesis of HPV16 and HPV18 immortalized primary cervical epithelial cells. Am J Pathol 1991;138:18.[Abstract]
11 Backsch C, Wagenbach N, Nonn M, Leistritz S, Stanbridge E, Schneider A, et al. Microcell-mediated transfer of chromosome 4 into HeLa cells suppresses telomerase activity. Genes Chromosomes Cancer 2001;31:1968.[CrossRef][ISI][Medline]
12 Steenbergen RD, Kramer D, Meijer CJ, Walboomers JM, Trott DA, Cuthbert AP, et al. Telomerase suppression by chromosome 6 in a human papillomavirus type 16-immortalized keratinocyte cell line and in a cervical cancer cell line. J Natl Cancer Inst 2001;93:86572.
13 Poignee M, Backsch C, Beer K, Jansen L, Wagenbach N, Stanbridge EJ, et al. Evidence for a putative senescence gene locus within the chromosomal region 10p14-p15. Cancer Res 2001;61:711821.
14 Saxon PJ, Srivatsan ES, Stanbridge EJ. Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells. EMBO J 1986;5:34616.[Abstract]
15 Koi M, Morita H, Yamada H, Saboh H, Barrett JC, Oshimura M. Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa. Mol Carcinog 1989;2:1221.[ISI][Medline]
16 Jesudasan RA, Rahman RA, Chandrashekharappa S, Evans GA, Srivatsan ES. Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines. Am J Hum Genet 1995;56:70515.[ISI][Medline]
17 Srivatsan ES, Misra BC, Venugopalan M, Wilczynski SP. Loss of alleles on chromosome 11 in cervical carcinoma. Am J Hum Genet 1991;49:86877.[ISI][Medline]
18 Srivatsan ES, Chakrabarti R, Zainabadi K, Pack SD, Benyamini P, Mendonca MS, et al. Localization of deletion to a 300 Kb interval of chromosome 11q13 in cervical cancer. Oncogene 2002;21:563142.[CrossRef][ISI][Medline]
19 Hampton GM, Penny LA, Baergen RN, Larson A, Brewer C, Liao S, et al. Loss of heterozygosity in cervical carcinoma: subchromosomal localization of a putative tumor-suppressor gene to chromosome 11q22-q24. Proc Natl Acad Sci U S A 1994;91:69537.[Abstract]
20 Bethwaite PB, Koreth J, Herrington CS, McGee JO. Loss of heterozygosity occurs at the D11S29 locus on chromosome 11q23 in invasive cervical carcinoma. Br J Cancer 1995;71:8148.[ISI][Medline]
21 Pulido HA, Fakruddin MJ, Chatterjee A, Esplin ED, Beleno N, Martinez G, et al. Identification of a 6-cM minimal deletion at 11q23.123.2 and exclusion of PPP2R1B gene as a deletion target in cervical cancer. Cancer Res 2000;60:667782.
22 O'Sullivan MJ, Rader JS, Gerhard DS, Li Y, Trinkaus KM, Gersell DJ, et al. Loss of heterozygosity at 11q23.3 in vasculoinvasive and metastatic squamous cell carcinoma of the cervix. Hum Pathol 2001;32:4758.[CrossRef][ISI][Medline]
23 Kuramochi M, Fukuhara H, Nobukuni T, Kanbe T, Maruyama T, Ghosh HP, et al. TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer. Nat Genet 2001;27:42730.[CrossRef][ISI][Medline]
24 Masuda M, Yageta M, Fukuhara H, Kuramochi M, Maruyama T, Nomoto A, et al. The tumor suppressor protein TSLC1 is involved in cell-cell adhesion. J Biol Chem 2002;277:310149.
25 Steenbergen RD, Walboomers JM, Meijer CJ, van der Raaij-Helmer EM, Parker JN, Chow LT, et al. Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q. Oncogene 1996;13:124957.[ISI][Medline]
26 Brady CS, Bartholomew JS, Burt DJ, Duggan-Keen MF, Glenville S, Telford N, et al. Multiple mechanisms underlie HLA dysregulation in cervical cancer. Tissue Antigens 2000;55:4011.[CrossRef][ISI][Medline]
27 Jacobs MV, Snijders PJ, van den Brule AJ, Helmerhorst TJ, Meijer CJ, Walboomers JM. A general primer GP5/GP6 mediated PCR immunoassay method for rapid detection of 14 high risk and 6 low risk human papillomavirus genotypes in cervical scrapes. J Clin Microbiol 1997;35:7915.[Abstract]
28 Zielinski GD, Snijders PJ, Rozendaal L, Voorhorst FJ, van der Linden HC, Runsink AP, et al. HPV presence precedes abnormal cytology in women developing cervical cancer and signals false negative smears. Br J Cancer 2001;85:398404.[CrossRef][ISI][Medline]
29 Bulkmans NW, Rozendaal L, Snijders PJ, Voorhorst FJ, Boeke AJ, Zandwijken GR, et al. POBASCAM, a population-based randomised controlled trial for implementation of high-risk HPV testing in cervical screening; Design, methods and baseline data of 44,102 women. Int J Cancer. In press 2004.
30 Westerman BA, Neijenhuis S, Poutsma A, Steenbergen RD, Breuer RH, Egging M, et al. Quantitative reverse transcription-polymerase chain reaction measurement of HASH1 (ASCL1), a marker for small cell lung carcinomas with neuroendocrine features. Clin Cancer Res 2002;8:10826.
31 Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 1992;89:182731.[Abstract]
32 Sidransky D, Von Eschenbach A, Tsai YC, Jones P, Summerhayes I, Marshall F, et al. Identification of p53 gene mutations in bladder cancers and urine samples. Science 1991;252:7069.[ISI][Medline]
33 Braakhuis BJ, Ruiz van Haperen VW, Welters MJ, Peters GJ. Schedule-dependent therapeutic efficacy of the combination of gemcitabine and cisplatin in head and neck cancer xenografts. Eur J Cancer 1995;31A:233540.[CrossRef][ISI][Medline]
34 Steenbergen RD, Parker JN, Isern S, Snijders PJ, Walboomers JM, Meijer CJ, et al. Viral E6-E7 transcription in the basal layer of organotypic cultures without apparent p21cip1 protein precedes immortalization of human papillomavirus type 16- and 18-transfected human keratinocytes. J Virol 1998;72:74957.
35 Snijders PJ, van Duin M, Walboomers JM, Steenbergen RD, Risse EK, Helmerhorst TJ, et al. Telomerase activity exclusively in cervical carcinomas and a subset of cervical intraepithelial neoplasia grade III lesions: strong association with elevated messenger RNA levels of its catalytic subunit and high-risk human papillomavirus DNA. Cancer Res 1998;58:38128.[Abstract]
36 Wisman GB, De Jong S, Meersma GJ, Helder MN, Hollema H, de Vries EG, et al. Telomerase in (pre)neoplastic cervical disease. Hum Pathol 2000;31:130412.[CrossRef][ISI][Medline]
37 van Duin M, Steenbergen RD, De Wilde J, Helmerhorst TJ, Verheijen RH, Risse EK, et al. Telomerase activity in high-grade cervical lesions is associated with allelic imbalance at 6Q1422. Int J Cancer 2003;105:57782.[CrossRef][ISI][Medline]
38 Fukuhara H, Kuramochi M, Fukami T, Kasahara K, Furuhata M, Nobukuni T, et al. Promoter methylation of TSLC1 and tumor suppression by its gene product in human prostate cancer. Jpn J Cancer Res 2002;93:6059.[ISI][Medline]
39 Jansen M, Fukushima N, Rosty C, Walter K, Altink R, Heek TV, et al. Aberrant methylation of the 5' CpG island of TSLC1 is common in pancreatic ductal adenocarcinoma and is first manifest in high-grade PanlNs. Cancer Biol Ther 2002;1:2936.[ISI][Medline]
40 Honda T, Tamura G, Waki T, Jin Z, Sato K, Motoyama T, et al. Hypermethylation of the TSLC1 gene promoter in primary gastric cancers and gastric cancer cell lines. Jpn J Cancer Res 2002;93:85760.[ISI][Medline]
41 Allinen M, Peri L, Kujala S, Lahti-Domenici J, Outila K, Karppinen SM, et al. Analysis of 11q2124 loss of heterozygosity candidate target genes in breast cancer: indications of TSLC1 promoter hypermethylation. Genes Chromosomes Cancer 2002;34:3849.[CrossRef][ISI][Medline]
42 Baker SG, Kramer BS, Srivastava S. Markers for early detection of cancer: statistical guidelines for nested case-control studies. BMC Med Res Methodol 2002;2:4.[CrossRef][Medline]
Manuscript received June 27, 2003; revised December 2, 2003; accepted December 15, 2003.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |