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[Cancer Research 65, 2778-2786, April 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Reversible Conversion of Immortal Human Cells from Telomerase-Positive to Telomerase-Negative Cells

Shin-ichi Kumakura1, Takeo W. Tsutsui1, Junko Yagisawa1, J. Carl Barrett2 and Takeki Tsutsui1

1 Department of Pharmacology, Nippon Dental University, School of Dentistry at Tokyo, Tokyo, Japan and 2 Laboratory of Biosystems and Cancer, National Cancer Institute, NIH, Bethesda, Maryland

Requests for reprints: J. Carl Barrett, Center for Cancer Research, National Cancer Institute, Building 31, Room 3A11, 31 Center Drive, MSC-2440, Bethesda, MD 20892-2240. Phone: 301-496-4345; Fax: 301-496-0775; E-mail: barrett{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel+) to telomerase-negative (Tel–) cells. Tel+ cells converted spontaneously to Tel– cells that reverted to Tel+ cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel– E6 Cl 6 cells. Tel– E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel– E6 Cl 6 cells and ALT cell lines were greater than those in Tel+ cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

Key Words: immortal human cells • telomerase • alternative lengthening of telomeres • DNA methylation • chromatin structure


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Telomeres, tracts of repetitive DNA at the end of linear chromosomes, shorten in the majority of normal somatic cells with each cell division because of incomplete replication of telomeric DNA by DNA polymerase (1). In contrast to normal cells, however, in vitro immortalized and tumor-derived cells do not exhibit telomere shortening during DNA replication, suggesting that activation of a telomere maintenance mechanism is required for cellular immortalization and for tumorigenesis (2).

Most human tumors and immortal cell lines maintain their telomere lengths via the activity of a specialized reverse transcriptase, telomerase (i.e., via the telomerase pathway; ref. 3), whereas some human tumors and immortal cell lines maintain their telomeres via a telomerase-independent mechanism, referred to as alternative lengthening of telomeres (ALT; ref. 4). The regulation of telomerase occurs at different levels through multiple pathways. It has been recently found that at least three tumor suppressor pathways, transforming growth factor-ß, MAD 1, and menin, negatively regulate the telomerase pathway (5). Meanwhile, it has been suggested that telomeres in cells using the ALT pathway are maintained via homologous recombination and copy switching between telomeric tracts (6). However, the mechanism of how the telomerase and ALT pathways are activated in human tumors and immortal cell lines has not been fully elucidated.

Epigenetic modification by DNA methylation may contribute to the regulation of the hTERT gene. In human immortal cell lines without expression of hTERT transcript and telomerase activity, a DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZC) induces demethylation of the hTERT promoter and expression of the hTERT transcript (7, 8). Because methylation of CpG islands is an important biological phenomenon associated with transcriptional repression as well as regulation of chromatin structure (9), modification of nucleosomal histones can play a role in the regulation of hTERT expression.

Inhibition of the telomere maintenance pathways is considered as a potential target for cancer therapy; therefore, it is important to determine whether the telomerase and ALT pathways coexist within the same cells. Ectopic expression of telomerase in in vitro immortalized human cells with the ALT pathway suggests that the telomerase and ALT pathways can coexist in the same cells (10–12). There is, however, no clear evidence demonstrating that tumor cells possess both the telomerase and ALT pathways under nonexperimental circumstances. Furthermore, it is not known whether tumor cells with either the telomerase or ALT pathways are derived from cells that acquired one of the corresponding pathways, or from cells that converted the pathways. Moreover, it has not been clearly shown that tumor cells with either pathway reversibly convert to cells with the other pathway, although Bechter et al. (13) has recently presented evidence that ALT-like telomere elongation can occur in a telomerase-positive human colon cancer cell line expressing a dominant-negative hTERT construct without reappearance of telomerase activity. In the present study, we report the first evidence of the reversible conversion of human fibroblasts immortalized by introduction of the human papillomavirus type 16 (HPV-16) E6 gene from telomerase-positive (Tel+) to telomerase-negative (Tel–) cells. Chromatin structure-mediated regulation of hTERT expression can be involved in this conversion. This has important implications for cancer therapy by inhibition of the telomere maintenance pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells and culture medium. A normal human embryonic fibroblast strain WHE 7 (designated simply as WHE) was established as described previously (14). WHE at 9 PD was infected with a retrovirus vector encoding HPV-16 E6 or HPV-16 E7 or both (E6/E7), resulting in the creation of the clones E6 Cl 4– Cl 7, E7 Cl 1-Cl 4, and E6/E7 Cl 1-Cl 5 as described previously (15, 16). KMST-6 (17) immortalized by irradiation of KMS-6 (human embryonic fibroblasts) with 60Co, and OUMS-24F (18) immortalized by treatment of OUMS-24 (human embryonic fibroblasts) with 4-nitroquinoline 1-oxide were generously provided by Dr. M. Namba (Okayama University, Okayama, Japan). WI-38 VA13 (19), a cell line immortalized by infection of WI-38 (human embryonic fibroblasts) with SV40 virus, was obtained from the American Type Culture Collection (Rockville, MD). HT1080 (20), a human fibrosarcoma cell line, was generously provided by Dr. B. Weissman (University of North Carolina). WHE, E6 Cl 4, E6 Cl 5, E6 Cl 7, E6/E7 Cl 1, and E6/E7 Cl 4 are mortal cell strains lacking both telomerase activity and the ALT mechanism (15, 16). E7 Cl 1-Cl 4 in the precrisis period is also negative for both telomere maintenance mechanisms (15, 16) . On the other hand, E6 Cl 6, E7 Cl 1-Cl 4 in the postcrisis period, E6/E7 Cl 2, E6/E7 Cl 3, E6/E7 Cl 5, KMST-6, OUMS-24F, WI-38 VA13, and HT1080 are positive for either telomerase activity or ALT mechanism (15–19). The culture medium used for WHE, KMST-6, OUMS-24F, WI-38 VA13, and HT1080 was Eagle's MEM containing 10% fetal bovine serum, 0.2 mmol/L serine, 0.1 mmol/L aspartic acid, 1.0 mmol/L pyruvate, and 0.22% NaHCO3 (complete medium). The other cells were cultured with complete medium supplemented with 50 µg mL–1 G418.

Analysis of telomere restriction fragment length. A 2-µg sample of genomic DNA extracted from cells was digested with HinfI and subjected to 0.6% agarose gel electrophoresis. The gel was blotted onto nitrocellulose membranes, which were prehybridized at 65°C and hybridized overnight at 50°C with a 32P-end-labeled (TTAGGG)4 telomeric probe as described previously (16). After being washed, the membranes were autoradiographed on X-ray film (SR-H, Konica Co., Tokyo, Japan) at –80°C. The peak TRF length was estimated from the peak migration distance of each lane autoradiographed and analyzed by densitometry, although the peak migration was not the rigorous calculation of average telomere length as has been done previously (21).

Telomerase activity. Telomerase activity in cells was detected as described previously (22). According to the telomeric repeat amplification protocol (TRAP) developed by Wright et al. (23), cell lysates equivalent to 104 cells were assayed in 50 µL of reaction mixture containing 50 µmol/L of each deoxynucleotide triphosphate, 344 nmol/L of the deoxyoligonucleotide primer TS (5'-AATCCGTCGAGCAGAGTT-3'), 0.5 µmol/L T4 gene 32 protein (Boehringer Mannheim, Mannheim, Germany), 4 µCi ({alpha}-32P) dCTP (specific activity of ~3,000 Ci/mmol, Amersham, Tokyo, Japan), 2 units of Taq DNA polymerase (Takara, Tokyo, Japan), and 5 µL of 10 x PCR buffer in a 0.5 mL tube that contained 344 nmol/L of the deoxyoligonucleotide primer CX (5'-CCTTACCCTTACCCTTACCCTAA-3'). The reaction mixture was amplified by PCR for 31 cycles in the presence of 5 ag of an internal TRAP assay standard (ITAS; generously provided by Dr. E. Hiyama, Hiroshima University, Japan). The PCR products were subjected to electrophoresis on 10% polyacrylamide gels.

RNA expression. Total cellular RNA (2 µg) was reverse transcribed (RT) with random and oligo(dT) primers using RTG You-Prime First-Standard Beads (Amersham) and the RT reaction product was amplified by PCR with sense and antisense primers for hTERC, hTEP1, hTERT, HPV-16 E6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described previously (15, 22). After the PCR products were subjected to agarose gel electrophoresis, the gels were stained with SYBR Green I (Bio Whittaker Molecular Applications, Rockland, ME) and analyzed using a fluorescence imaging analyzer.

Western blot analysis. Western blots were done as described previously (15). Total cellular protein (50 µg) was run on a 12% SDS-PAGE gel and then transferred to nitrocellulose membranes (Millipore, Tokyo, Japan). The membranes were hybridized with polyclonal antibodies against HPV-16 E6 (Santa Cruz Biotechnology, Santa Cruz, CA), K9-dimethylated H3 (Upstate Biotechnology, Lake Placid, NY), K9-acetylated H3 (Upstate Biotechnology), K4-dimethylated H3 (Upstate Biotechnology), and K14-acetylated H3 (Upstate Biotechnology). Secondary antibodies (Santa Cruz Biotechnology) were anti-goat-IgG-horseradish peroxidase for HPV-16 E6, and anti-rabbit-IgG-horseradish peroxidase for methylated and acetylated histones.

Immunofluorescence. Cells were grown on coverslips and fixed in 3.7% formaldehyde in PBS for 10 minutes. After being permeabilized in 0.5% Triton X-100 in PBS, cells were blocked with 0.5% bovine serum albumin in PBS for 1 hour. For detecting colocalization of APBs and the telomere-specific binding protein TRF2, cells were incubated for 1 hour at room temperature with a goat-polyclonal antibody against promyelocytic leukemia (PML, Santa Cruz Biotechnology) and a mouse-monoclonal antibody against TRF2 (Upstate Biotechnology). Primary antibodies were detected by incubation for 1 hour at room temperature with FITC-conjugated rabbit anti-goat IgG (Sigma, St. Louis, MO) and TRITC-conjugated goat anti-mouse IgG (Sigma), respectively, diluted at 1:200. Image acquisition and overlay analysis were done using a Leica confocal microscope (Leica Camera AG, Solms, Germany).

DNA fingerprinting. DNA fingerprint analysis using the multilocus probes 33.15 and 33.6 after digestion with HinfI was done as described previously (16).

Methylation-specific PCR-based assay. The extracted DNA (1 µg) underwent a bisulfite modification using a CpGenomeTM DNA modification kit (Intergen Co., Purchase, NY). The primers used for amplification of the hTERT CpG island which is from 654 bp upstream of the putative transcription start site to 510 bp downstream of the transcription start site were the same as those described by Dessain et al. (8). The primer pair used analyzes DNA sequences from 211 bp upstream of the transcription start site to 30 bp downstream of the transcription start site (8). PCR was done using published conditions (8) in the reaction mixture (25 µL) containing 1 µL of the DNA, 0.025 units of Taq polymerase (Amplitaq Gold, Applied Biosystems, New York, NY), 200 µmol/L deoxynucleotide triphosphate, 15 mmol/L Tris-HCl (pH 8.0), 50 mmol/L KCl, 2.5 mmol/L MgCl2, and 20 pmol of primers. Methylation-specific PCR products were purified using the MinElute PCR purification kit (Qiagen GmbH, Hilden, Germany), and then sequenced by Espec Co. (Tsukuba, Japan).

Chromatin immunoprecipitation assay. The chromatin immunoprecipitation (ChIP) assay was done with a ChIP assay kit (Upstate) with anti-K9-dimethylated H3, anti-K9-acetylated H3, anti-K14-acetylated H3, or anti-K4-dimethylated H3. All of the antibodies were purchased from Upstate. Immunoprecipitated DNA was amplified with an advantage-GC genomic polymerase mix (Clontech, Palo Alto, CA). The PCR conditions were as follows: 95°C for 9 minutes, 33 cycles at 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and 72°C for 7 minutes. The primers used were as follows: hTERT promoter sense 5'-GGCCGATTCGACCTCTCT-3' and hTERT promoter antisense 5'-AAGAAGCGGAACTGGAAGGT-3'. PCR products were subjected to 2% agarose gel electrophoresis. The gels were stained with SYBR Green I and analyzed using a fluorescence imaging analyzer.

Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A. Cells were treated with either 3 µmol/L 5-AZC (Sigma) for 96 hours or 500 nmol/L trichostatin A (TSA; Sigma) for 24 hours. In cultures cotreated with 5-AZC and TSA, cells were initially treated with 3 µmol/L 5-AZC for 72 hours and subsequently cotreated with 3 µmol/L 5-AZC and 500 nmol/L TSA for an additional 24 hours.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Telomere restriction fragment length dynamics of E6-immortalized cells. E6 Cl 6, one of the clones isolated from WHE infected with HPV-16 E6, grew rapidly for the first 36 PD after infection and then decreased in growth rate (M1 phase). The growth recovered but decreased again at 79 to 81 PDs (M2 phase). Then the clone escaped from crisis and continued to grow with no lag phase (15). The cells grew for >500 PD. The parental WHE at 10 PD had the peak telomere restriction fragment (TRF) length of 12.0 kb. The peak TRF lengths of E6 Cl 6 shortened progressively until 88 PD (15) and subsequently stabilized at 3.5 kb up to 153 PD [Fig. 1A, lane (1)]. However, the TRF length in the cells elongated to 4.8 kb at 206 PD and further elongated to 8.5 kb at 256 to 349 PDs with little variation. When assayed at 401 PD, E6 Cl 6 had a peak TRF length of 10.7 kb accompanied by long telomeres. The TRF length shortened to 9.7 kb at 441 PD. The patterns of TRF lengths in the cells at 401 and 441 PDs were different from those observed in ALT cell lines such as E7 Cl 1 (PD 285) and KMST-6 [Fig. 1A, lane (1)]. The size and density of TRF signals were the same between HinfI-digested and RsaI-digested DNAs from E6 Cl 6 at 428 PD and decreased after digestion of the cellular DNA with exonuclease Bal 31, although E6 Cl 6 was more resistant to Bal 31 than KMST-6 [Fig. 1A, lanes (2-4)]. This shows that the TTAGGG-hybridizing fragments observed in E6 Cl 6 are telomeric DNA.



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  		Figure 1. Telomere maintenance mechanism in E6 Cl 6. A, autoradiograph of radiolabeled telomere probe (TTAGGG)4. Lane (1), HinfI-digested genomic DNA either from WHE at 10 PD or from E6 Cl 6 with different levels of PDs. E7 Cl 1 (PD 285) and KMST-6 were used as positive controls of ALT cell lines. Lane (2), HinfI- or RsaI-digested genomic DNAs from E6 Cl 6 at 428 PD. Lanes (3) or (4), genomic DNAs from E6 Cl 6 at 428 PD or KMST-6, respectively, digested with Bal 31 for 0 to 60 minutes followed by HinfI. *, a 3-fold excess of Bal 31 was used for digestion. B, telomerase activity in WHE at 30 PD and E6 Cl 6 with different levels of PDs. Cells were harvested at the indicated levels of PDs and counted by a hemocytometer. Cell lysates were prepared from 106 cells using the CHAPS detergent lysis method, and the equivalent of 104 cell lysate was analyzed by the TRAP assay with ITAS used to verify the efficiency of amplification. Position of the ITAS. HT1080 as a positive control. C, expressions of genes encoding three components of the human telomerase enzyme in WHE at 15 PD and E6 Cl 6 with different levels of PDs detected by RT-PCR analysis. GAPDH as an amplification control.

 
Telomerase activity of E6-immortalized cells. No telomerase activity was detected in WHE at 30 PD (Fig. 1B). E6 Cl 6 at 55 and 68 PDs was also telomerase negative in the TRAP assay. Telomerase activity in the cells was observed at 83 to 206 PDs but decreased markedly at 256 PD, and became undetectable at 298 to 441 PDs. Telomerase activity was undetectable in E6 Cl 6 at 415 PD even when a 10-fold excess of cell lysate was examined in the TRAP assay (data not shown).

To determine which telomerase components were involved in the loss of telomerase activity, we assayed with reverse transcription-PCR the expressions of an RNA component (hTERC), a telomerase-associated protein (hTEP1), and a telomerase catalytic subunit (hTERT) in E6 Cl 6 at 83 to 441 PDs. The parental WHE at 15 PD and E6 Cl 6 at any levels of population doublings expressed both hTERC RNA and hTEP1 mRNA. Conversely, hTERT mRNA was expressed only in E6 Cl 6 with telomerase activity (Fig. 1C).

Because telomerase activity in normal human keratinocytes is induced by the expression of E6 protein that up-regulates the hTERT promoter activity in the cells (24), we next examined the correlation between the expression levels of E6 and the telomerase activity in E6 Cl 6 with different levels of population doublings. E6 Cl 6 at 83 to 441 PDs expressed E6 mRNA, corresponding to the viral oncogene introduced into the cells (Fig. 2A). E6 Cl 6 also expressed similar levels of E6 protein over all of the levels of population doublings examined (Fig. 2B).



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  		Figure 2. mRNA and protein expressions of HPV-16 E6 in WHE at 15 PD and E6 Cl 6 with different levels of PDs. A, mRNA expression detected by RT-PCR analysis. B, protein expression detected by Western blot analysis. The expressions of GAPDH and extracellular-signal related kinase 1 (ERK 1) were used as internal controls.

 
Alternative lengthening of telomere–associated promyelocytic leukemia nuclear bodies. Although observed in up to 5% of interphase cells, ALT cell lines have ALT-associated PML nuclear bodies (APB) that contain telomeric DNA, telomere-specific binding proteins hTRF1 and hTRF2, and proteins involved in DNA synthesis and recombination such as RAD 51 and RAD 52 (25). We next examined whether Tel– E6 Cl 6 had APBs. APBs were observed in ALT cell lines, including E7 Cl 1, E6/E7 Cl 3, and KMST-6, used as positive controls in which immunofluorescence of PML protein and TRF 2 merged but not in Tel– E6 Cl 6 at 262 to 456 PDs (Fig. 3A). There were no significant differences in the level of RAD 52 expression among the parental WHE at 15 PD, Tel+ or Tel– E6 Cl 6, and ALT cell lines as detected by Western blot analysis (Fig. 3B and C), suggesting that RAD 52 is not important for the conversion from Tel+ to Tel– cells. Our findings together with the TRF length pattern shown in Fig. 1A indicate that Tel– E6 Cl 6 lacks the phenotypes characteristic of ALT cell lines.



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  		Figure 3. APBs and expression of RAD 52 protein. A, immunofluorescence for detecting APBs in E6 Cl 6 with different levels of PDs. WHE at 15 PD and E6 Cl 6 at 262-456 PDs were telomerase-negative, while E6 Cl 6 at 97 and 194 PDs were telomerase-positive. E7 Cl 1 (PD 395), E6/E7 Cl 3 (PD 295), and KMST-6 were used as APBs-positive cells. Right, quantitation of the total number of cells containing APBs. The staining with antibodies against PML protein and TRF 2 in WHEand E6 Cl 6 was very weak because the exposure was similar to that for the ALT cells where the APBs are so bright that the other PML protein and TRF 2 are hardly seen. B, expression of RAD 52 protein in E6 Cl 6 with different levels of PDs detected by Western blot analysis. ß-Actin as an internal control. C, relative levels of RAD 52 expression quantified by densitometry with Software LabWorks version 4.0 (UVP, Inc., Upland, CA). WHE (PD 15) was the parental cells of E6 Cl 6, and E7 Cl 1 (PD 320) and KMST-6 were used as positive controls of ALT cell lines.

 
Conversion from Tel+ to Tel– cells. DNA fingerprints from E6 Cl 6 with different levels of population doublings were compared with those from the parental WHE at 14 PD. DNA fingerprint analysis using the multilocus probes 33.15 and 33.6 after digestion with HinfI showed that DNA fingerprint profiles of the parental WHE and E6 Cl 6 at 48 to 347 PDs were mostly identical to each other (data not shown). In addition, the DNA fingerprints of E6 Cl 6 at 181 PD were different from those of the other Tel+ or ALT cell lines established from the same parental WHE infected with HPV-16 E6, E7, or E6/E7, indicating that E6 Cl 6 is not contaminated with the other Tel+ or ALT cell lines derived from WHE.

To examine whether Tel– cells preexisting in the Tel+ cell population were involved in the conversion to the Tel– cell population, E6 Cl 6 at 99 PD was continuously cultured with medium containing the telomerase inhibitor epigallocatechin gallate (EGCG). Treatment of E6 Cl 6 at 99 PD for 72 hours with 50 or 100 µmol/L inhibited 67 or 90%, respectively, of telomerase activity in the cells. Because 100 µmol/L EGCG were highly toxic, we used 50 µmol/L EGCG for the experiment. As shown in Fig. 4A, cumulative growth of E6 Cl 6 was weakly decreased by the treatment. When analyzed at 112 and 131 PDs, the peak TRF lengths of EGCG-treated cells were ~3.3 kb similar to those of untreated cells with telomerase activity (Fig. 4B). Very long and heterogeneous TRF lengths characteristic of ALT cell lines were not observed in the treated cells. To examine further the possibility of the preexistence of Tel– cells, we plated E6 Cl 6 at 246 PD at the clonal density on 60-mm dishes and isolated many colonies. The colony-forming efficiency of the cells was less than 0.5%. Five of 10 colonies formed on the dishes were not able to be isolated because of poor growth. All of the other five colonies isolated ceased growing 3 to 15 passages after cloning (Fig. 5A). TRAP assay showed that the five clones had little telomerase activity (Fig. 5B). The peak TRF lengths of the five clones were between 3.4 and 5.0 kb which were not characteristic of ALT cell lines (Fig. 5C). Furthermore, the colony-forming efficiency of Tel– E6 Cl 6 at 416 PD was 12.8 % ± 0.4 (SD), which was much different from that of E6 Cl 6 at 246 PD.



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  		Figure 4. Cumulative growth (A) and radiolabeled signals of TRF lengths (B) in E6 Cl 6 treated with or without EGCG. A, E6 Cl 6 at 99 PD was continuously cultured with medium containing (solid line) or not containing (broken line) 50 µmol/L EGCG. B, E6 Cl 6 was continuously cultured with medium containing or not containing 50 µmol/L EGCG was analyzed at the indicated levels of PDs for determining TRF lengths.

 


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  		Figure 5. Cumulative growth (A) of E6 Cl 6 and its five clones isolated from E6 Cl 6 at 246 PD and telomerase activity (B) and radiolabeled signals showing TRF lengths (C) in these cells. Four hundred E6 Cl 6 at 246 PD were plated on 60-mm dishes, and cultured to form colonies for isolating clones. A, cumulative growths of (solid lines) isolated five clones (E6 Cl 6-1 to E6 Cl 6-5) and their (broken line) parental E6 Cl 6, respectively.

 
Comparing telomerase activity in E6 Cl 6 at 256 PD (Fig. 1B) with that in the cells at 246 PD (Fig. 5B), there was a big difference in terms of robust activity at 246 PD and negligible at 256 PD. The range of TRF lengths in E6 Cl 6 at 246 PD was from 3.3 to 7.3 kb (Fig. 5C), whereas only 10 PD later at 256 PD, the range for this clone was from 4 to 11 kb (Fig. 1A), an elongation rate of at least 300 bp/population doubling. The range for E6 Cl 6 at 246 PD was nearly identical to the cells at 206 PD shown in Fig. 1A. The kinetics of telomere elongation seem to be rapid during conversion to a telomerase-negative phenotype, strongly suggesting that there is a clonal outgrowth of cells between PDs at 246 and 256 for telomeres to elongate so quickly and the telomerase-positive and telomerase-negative phenotypes can coexist in this population. It raises curiosity that none of the clones isolated from E6 Cl 6 at 246 PD had telomerase and none had longer telomeres, but only 10 clones were observed and only five of those survived the cloning procedure.

Tel– E6 Cl 6 lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and APBs. There are three alternatives for the mechanism of telomere maintenance in Tel– E6 Cl 6. (a) The cells use a telomere maintenance pathway that does not involve telomerase, in which case it is by definition a new ALT mechanism and could be referred to as a putative variant ALT mechanism. (b) It is possible that the cells use the previously described ALT mechanism, but the hallmarks of this telomere maintenance pathway have somehow been masked. (c) The cells may have no telomere maintenance pathway but survive somehow for a prolonged period. It was shown previously that cells within strongly telomerase-positive cell lines spontaneously generate subclones that have switched off telomerase activity (26). The telomerase-negative subclones regained activity within 18 PD. It has also been shown that lengthening of telomeres by a brief burst of exogenous telomerase can result in a 50% increase in the life span of normal human fibroblasts lacking telomerase activity (27). Our data show that regardless of whether Tel– E6 Cl 6 uses ALT or not, long-term survival (for >240 PD) can occur in the absence of telomerase. Tel– E6 Cl 6 may be overtaken successively by at least two subclones, the first of which dominated the culture between 256 and 349 PDs and maintained moderately long telomeres with a low level of telomerase activity. Later the culture had longer telomeres but had essentially switched off its telomerase activity. Between 401 and 441 PDs, there was a decrease in telomere length of 1.0 kb. This equates with a shortening rate of 25 bp/population doubling, which is within the range seen in normal/mortal telomerase-negative cells.

Methylation status of the hTERT promoter. Using a methylation-specific PCR (MSP)-based assay, we next examined the methylation status of the CpG islands in the promoter region of the hTERT gene in E6 Cl 6 with different levels of population doublings (Fig. 6A). The hTERT CpG island was unmethylated in the parental WHE at 15 PD and E6 Cl 6 at 34 and 68 PDs, all of which lacked hTERT mRNA expression and telomerase activity. Weak and strong signals showing the methylated or unmethylated hTERT CpG island, respectively, became detectable in E6 Cl 6 at 83 PD with both hTERT mRNA expression and telomerase activity. In E6 Cl 6 at 108 to 206 PDs, which also exhibited hTERT mRNA expression and telomerase activity, the intensity of the signals showing the methylated CpG island became comparable to that of the signals showing the unmethylated CpG island. These results indicate that the CpG island of the hTERT promoter in Tel+ E6 Cl 6 was partially methylated. Conversely, Tel– E6 Cl 6 at 256 to 441 PDs with neither hTERT mRNA expression nor telomerase activity had complete methylation of the hTERT CpG island. The states of methylation of the CpG island were confirmed by the sequencing of MSP products where the CpG cytosines which lie between –64 and –212 bp downstream from the hTERT transcription start site were largely unmethylated or methylated in WHE at 15 PD or E6 Cl 6 at 257 PD, respectively, and partially methylated in E6 Cl 6 at 208 PD (Fig. 6B). These findings indicate the association of the methylation status of the hTERT promoter in E6 Cl 6 with activation or inactivation of the telomerase pathway, which is supported strongly by others (7, 8).



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  		Figure 6. Methylation status of the hTERT promoter. A, methylation status of the CpG island within the promoter region of hTERT in WHE at 15 PD and E6 Cl 6 with different levels of PDs. Unmethylated (U) or methylated (M) DNA within the promoter region of hTERT was detected by methylation-specific PCR analysis. ALT–, ALT-negative cells. B, methylation status of the hTERT promoter in WHE at 15 PD and E6 Cl 6 at 208 and 257 PDs as detected by DNA sequencing of the methylation-specific PCR products. The locations of the CpG sites ({circ}, {bullet}, and {blacktriangleup}) are given in reference to the transcription start site. {circ}, unmethylated CpG island; {bullet}, methylated CpG island; {blacktriangleup}, unmethylated or methylated CpG island. C, methylation status of the hTERT promoter in various human fibroblasts with either telomerase activity or an ALT mechanism or with neither of them. U or M DNA within the promoter region of hTERT as detected by MSP analysis. ALT-, ALT-negative cells.

 
Next, we compared the methylation status of the hTERT CpG island between mortal or precrisis cells and immortal cells and between Tel+ cells and ALT cell lines. As shown in Fig. 6C, all of the human mortal and precrisis cell strains lacking both the telomerase and ALT pathways had an unmethylated hTERT CpG island, and all of the immortal cell lines with the telomerase pathway had a partially methylated hTERT CpG island. Conversely, all ALT cell lines examined with neither hTERT mRNA expression nor telomerase activity had complete methylation of the hTERT CpG island. Our findings indicate that during the immortalization process, WHE infected with E6 first acquired the telomerase pathway, and the resultant E6 Cl 6 exhibited an extended life span, during which the hTERT promoter was partially methylated. The E6 Cl 6 then lacked telomerase activity by complete methylation of the hTERT promoter, which led toTel– E6 Cl 6. Some E6 Cl 6 lacking telomerase activity might become senescent but the cumulative growth of E6 Cl 6 during the conversion from Tel+ to Tel– cells had no lag phase (Fig. 5A). Our findings also suggest that the hTERT promoter and ALT-associated gene(s) or promoter(s) postulated by Perrem et al. (28) are adjacent to each other and that activation of either the telomerase or ALT pathway is regulated by a common repressor in a manner dependent on the methylation status of their promoters or genes.

To determine whether the inhibition of cytosine methylation and/or histone deacetylation could induce hTERT mRNA expression and/or telomerase activity in the immortal cell lines with complete methylation of the hTERT CpG island, we treated Tel– E6 Cl 6 at 420 PD and ALT cell lines with the cytosine methylation inhibitor 5-AZC and/or the histone deacetylase inhibitor TSA (Fig. 7). Treatment of the parental WHE at 15 PD with either TSA or 5-AZC alone or both TSA and 5-AZC neither affected the methylation status of the hTERT CpG island nor induced either hTERT mRNA expression or telomerase activity. Treatment of Tel+ E6 Cl 6 at 120 PD, in which the hTERT CpG island was partially methylated, with TSA and/or 5-AZC increased the level of unmethylated CpG islands and decreased the level of methylated CpG islands. These treatments, however, caused little effects both on the expression of telomerase components and telomerase activity in the cells. Similar results were observed in HT1080 with telomerase activity where hTERT mRNA expression was decreased by 5-AZC alone or together with TSA as described by Guilleret and Benhatter (29). Conversely, treatment with TSA and/or 5-AZC induced partial methylation of the hTERT CpG island and expression of hTERT mRNA in Tel– E6 Cl 6 and an ALT cell line E7 Cl 1. Treatment with 5-AZC either alone or in combination with TSA also induced telomerase activity in both cells, although the activity was weak in E7 Cl 1. Telomerase activity was undetectable in both cells by TSA alone probably due to the sensitivity of the TRAP assay. Telomerase activity induced by cotreatment of Tel– E6 Cl 6 with TSA and 5-AZC was transient because the activity was undetectable in analysis of the treated cells grown for 5 to 10 PDs in medium not containing TSA and 5-AZC. In the other ALT cell line WI-38 VA 13, both partial methylation of the hTERT CpG island and expressions of hTERC RNA and hTERT mRNA were induced by treatment with 5-AZC either alone or in combination with TSA. The same treatment also induced telomerase activity in WI-38 VA13, although the inducibility was weaker than that of E7 Cl 1, suggesting that induction of telomerase activity also requires hTERC demethylation (30). Treatment with TSA alone did not affect any of these effects. Partial methylation of the hTERT CpG island was not observed even when WI-38 VA 13 was treated with 2- to 4-fold concentrations of TSA. Telomerase activity induced in WI-38 VA13 was not enhanced in the cells treated with 2- to 4-fold concentrations of TSA and/or 5-AZC (data not shown). These findings suggest that although partial methylation of the hTERT CpG island is associated with expressions of hTERT mRNA and telomerase, some differential mechanisms for exhibiting resistance to TSA or 5-AZC exist between Tel– E6 Cl 6 and ALT cell lines, including E7 Cl 1 and WI-38 VA13.



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  		Figure 7. Effect of TSA and/or 5-AZC on the methylation status of the hTERT promoter and the mRNA expression of telomerase subunits (hTERC, hTEP1, and hTERT) as well as telomerase activity in various cells. A, methylation status of the hTERT promoter as detected by MSP analysis. U or M, unmethylated or methylated CpG island, respectively. B, mRNA expressions of hTERC, hTEP1, and hTERT detected by RT-PCR analysis. C and D, telomerase activity detected by the TRAP assay. The equivalent of 104 (C) or 105 (D) cell lysate was analyzed for the TRAP assay with ITAS used to verify the efficiency of amplification. Position of the ITAS.

 
Expressions of hTERC RNA and hTEP1 mRNA in the parental WHE at 15 PD, both E6 Cl 6 (Tel+ and Tel–), and HT1080 were not affected by treatment with TSA and/or 5-AZC. Expressions of hTERC RNA in ALT cell lines were also not affected by treatment with TSA alone but were enhanced by treatment with 5-AZC either alone or in combination with TSA (Fig. 7B), suggesting that hTERC expression is dependent on demethylation of the hTERC promoter in ALT cell lines as reported by Hoare et al. (30).

Chromatin immunoprecipitation assay at the hTERT promoter. Because a difference in the methylation status of the hTERT CpG island was found between Tel+ E6 Cl 6 and the other cells, including Tel– E6 Cl 6, E7 Cl 1, and WI-38 VA13, we did ChIP assay of the patterns of methylations of histone H3 lysine 9 (H3-K9) and histone H3 lysine K4 (H3-K4), as well as acetylations of histone H3 lysine 9 (H3-K9) and histone H3 lysine14 (H3-K14) to examine the possible involvement of chromatin states in the transcriptional regulation of the hTERT promoter and telomerase activity. Acetylation of H3-K9 and methylation of H3-K4 are well known to be associated with an open chromatin configuration such as that found at transcriptionally active promoters. In contrast, methylation of H3-K9 is a marker of condensed, inactive chromatin (31, 32). As shown in Fig. 8A, the hTERT CpG island in the parental WHE at 15 PD exhibited H3-K9 hypomethylation, H3-K4 hypermethylation, and acetylations of H3-K9 and H3-K14. The hTERT CpG island in Tel+ E6 Cl 6 at 184 PD had similar levels of H3-K9 and H3-K4 methylations and H3-K9 and H3-K14 acetylations to those in Tel– E6 Cl 6 at 438 PD. Conversely, in E7 Cl 1 (PD 377) and WI-38 VA13, both of which were ALT-positive, the hTERT CpG island displayed high levels of H3-K9 methylation and low levels of both H3-K4 methylation and H3-K9 and H3-K14 acetylations. In HT1080 (Tel+), the levels of both H3-K4 methylation and H3-K9 and H3-K14 acetylations were higher than the level of H3-K9 methylation. To compare the levels of H3-K9 methylation with those of H3-K4 methylation or H3-K9 or H3-K14 acetylation, ChIP PCR signals were quantified by densitometry (Fig. 8B). The ratios of H3-K9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation in Tel+ E6 Cl 6 were similar to those in the parental WHE. The ratios of these four chromatin markers in Tel– E6 Cl 6 were slightly greater than those in Tel+ E6 Cl 6 but much smaller than those in the ALT cell lines, including E7 Cl 1 and WI-38 VA13. These ratios in E7 Cl 1 and WI-38 VA13 were decreased by cotreatment with TSA and 5-AZC, inversely corresponding to their induced-telomerase activities. Treatment with either TSA or 5-AZC alone also decreased the ratio of H3-K9 methylation to H3-K4 methylation in Tel– E6 Cl 6 and E7 Cl 1. 5-AZC decreased the ratios more than TSA in these cells (Fig. 8C), indicating that either demethylation or acetylation of histone H3 alone can induce transcriptional activation of hTERT in these cells. To examine whether cotreatment with TSA and 5-AZC affected the expression levels of H3-K9 and H3-K4 methylation and H3-K9 and H3-K14 acetylation, we did Western blot analysis using Tel– E6 Cl 6 at 448 PD. The expression levels of any methylated or acetylated H3 hardly changed after cotreatment with TSA and 5-AZC, suggesting that alterations in H3 methylation and acetylation detected by ChIP assay were regional specific (Fig. 8D). Our findings suggest that the chromatin containing the hTERT promoter is slightly heterochromatic in Tel– E6 Cl 6 more than in Tel+ E6 Cl 6, and is much more heterochromatic in E7 Cl 1 and WI-38 VA13. Treatment of these cell lines with TSA and/or 5-AZC could induce euchromatinization of the heterochromatic domains of the hTERT promoters, leading to telomerase activity with different levels dependent on the methylation and acetylation levels of histone H3. More tightly packed chromatin may render the hTERT promoter more invulnerable to TSA or 5-AZC.



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  		Figure 8. Methylation or acetylation of histone H3. A, methylation or acetylation status of lysine 4, 9, or 14 of histone H3 detected by ChIP assay in WHE at 15 PD and E6 Cl 6 at 184 and 438 PDs treated with or without TSA and 5-AZC. E7 Cl 1, WI-38 VA13, or HT1080 were used as controls of ALT or Tel+ cell lines. B, quantitation of ChIP PCR signals of methylation or acetylation of histone H3 in various cells treated with or without TSA and 5-AZC. ChIP PCR products were subjected to 2% agarose gel electrophoresis. The gels were stained with SYBR Green I, the UV signals of which were quantified by densitometry with Software LabWorks version 4.0 (UVP). K9 Me, histone H3 lysine 9 methylation; K4 Me, histone H3 lysine 4 methylation; K9 Ac, histone H3 lysine 9 acetylation; K14 Ac, histone H3 lysine 14 acetylation. C, ratio of histone H3 lysine 9 (K9) methylation to histone H3 lysine 4 (K4) methylation in Tel– E6 Cl 6 (PD 420) and E7 Cl 1 (PD 363) after treatment with either TSA or 5-AZC alone. D, Western blot analysis showing the expression levels of histone H3 protein in Tel– E6 Cl 6 (PD 448) which was treated with or without TSA and 5-AZC. ERK 1 as an internal control.

 
In the present study, we showed the reversible conversion of HPV-16 E6-induced immortal human fibroblasts from Tel+ to Tel– cells. Tel– cells lacked the phenotypes characteristic of ALT cells such as very long and heterogenous telomeres and APBs, but grew for >240 PD after they became telomerase negative. The mechanism of the reversible conversion was associated with the chromatin structure-mediated regulation of hTERT expression. Our findings suggest that human tumors could reversibly interconvert their telomere maintenance phenotypes. This has important implications for the proposed use of telomerase inhibitors as anticancer therapies.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Izumi Horikawa for his helpful discussions.


   Footnotes
 
Note: S. Kumakura and T. Tsutsui contributed equally to this work.

Received 6/24/04. Revised 12/16/04. Accepted 1/12/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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