Abstract
Human immortal cells maintain their telomeres either by telomerase or by alternative lengthening of telomeres (ALT) that is based on homologous telomeric recombination. Previous studies showed that the ALT mechanism is activated in non-ALT cells when heterochromatic features are reduced. In this study, we examined the ALT phenotypes of ALT cells after treatment with trichostatin-A (TSA), which is an inhibitor of histone deacetylases and causes global chromatin decondensation. The ALT cells remained telomerase-negative after TSA treatment. ALT-associated promyelocytic leukemia (PML) nuclear bodies and telomere sister chromatid exchanges, typical ALT phenotypes, markedly increased in the TSA-treated cells, while the telomere length remained unchanged. In addition, telomerase expression in the ALT cells suppressed TSA-mediated ALT phenotype enhancement. Our results show that certain ALT phenotypes become more pronounced when chromatin is decondensed, and also suggest that the ALT mechanism may compete with telomerase for telomere maintenance in cells that lack heterochromatin.
Most tumor cells utilize telomerase for the purpose of telomere maintenance to gain cellular immortality (1). The other mechanism that maintains telomere length is the alternative lengthening of telomeres (ALT). In fact, some tumor cells maintain their telomere length in the absence of detectable telomerase activity and have the capacity to undergo infinite division (2). These so-called ALT cells are characterized by heterogeneous telomere lengths that range from 3 to 50 kb (2), the formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs) (3), and frequent post-replicative exchanges between telomeric repeats, as shown by telomere sister chromatid exchange (T-SCE) (4). It has been suggested that the telomere length of ALT cells is maintained by a mechanism that is based on homologous telomeric recombination (5).
Heterochromatic features, such as a high level of DNA methylation, the tri-methylation of histone 3 Lys 9 (H3K9) and histone 4 Lys 20 (H4K20), are found in telomeric and sub-telomeric chromatins (6). When the chromatin structure of a telomere is dysregulated, regulation of the telomere length seems to be affected. For instance, cells that were derived from a histone methyltransferase-knockout mouse exhibited lower H3K9 and H4K20 tri-methylation at the telomeres, frequent telomeric recombination, and aberrant telomere elongation (7, 8). Similar phenotypes were also observed in DNA methyltransferase-deficient cells (9). In addition, the treatment of human cancer cells that were telomerase-positive with DNA-demethylating drugs increased their telomere recombination, thereby facilitating telomere elongation (10). These studies showed that the loss of heterochromatic features resulted in the emergence of an ALT mechanism in telomerase-positive cells and mouse somatic cells.
The question may arise as to whether or not the epigenetic modifications have any influence on the ALT mechanism in ALT cells. In this study, we investigated the ALT phenotypes of ALT cells whose chromatin structures were relaxed. To bring about chromatin decondensation, the ALT cells were treated with trichostatin-A (TSA), which is a histone de-acetylase inhibitor, thereby leading to global histone hyperacetylation (11, 12). We then examined the changes that occurred in the ALT phenotypes such as APBs, T-SCEs, and telomere length upon TSA treatment of the ALT cells. Our results showed that certain ALT phenotypes became more evident in the TSA-treated ALT cells and that the introduction of telomerase into the ALT cells reduced TSA-mediated ALT phenotype enhancement.
Materials and Methods
Cell culture and establishment of GM847 stably-expressing human telomerase reverse transcriptase (hTERT). WI38VA13 and GM847 cells, which were derived from lung and skin fibroblasts, respectively, were purchased from the Korean Cell Line Bank (Seoul, Korea) and the Coriell Institute (Camden, NJ, USA), respectively. GM847/hTERT and GM847/vector cells were established by infection with lenti-hTERT and lenti-enhanced green fluorescence protein (EGFP) viral supernatant, respectively, which were purchased from Applied Biological Materials Inc. (Cat. G200; ABM, BC, Canada).
Western blot analysis. For the detection of acetyl histones, cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer (50 mM Tris-Cl [pH 7.5], 1% SDS, and 10% glycerol) and sonicated to fractionate the genomic DNA. The protein concentration was determined using a DC-protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins (15 μg) were separated on 12.5% native polyacrylamide gels and then transferred to membranes. The membranes were incubated with primary antibodies against H3K9 (1:500; Millipore, Billerica, MA, USA) for 2 h, H4K8 (1:1,000; Cell Signaling, Danvers, MA, USA) for 2 h, and glyceraldehydes-3-phosphate dehydrogenase (1:20,000; GAPDH; Abcam, Cambridge, MA, USA) for 1 h. For the detection of hTERT, the cells were lysed with RIPA lysis buffer, and the hTERT protein was detected using a rabbit monoclonal antibody to hTERT (1:1,000; Epitomics, Burlingame, CA, USA).
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) for hTERT. Real-time RT-PCR for hTERT mRNA was performed with 1× Power SYBR® green PCR master mix (Applied Biosystems, Foster City, CA, USA). The sequences of the hTERT primers have been described previously (13). GAPDH amplification was performed using a Taqman GAPDH gene expression assay® (Applied Biosystems).
Detection of APBs. APBs were detected by fluorescence in situ hybridization (FISH) combined with immunofluorescence as described in a previous report (14). Briefly, cells that were treated with TSA (Sigma, St. Louis, MO, USA) at a final concentration of 100 nM or 200 nM for 48 h were permeated, fixed, and permeated once again. The hybridization was performed with Cy3 conjugated-(C3TA2)3 peptide nucleic acid (PNA) probe (Panagene, Daejeon, Korea), and then the cells were incubated with a PML antibody (1:500; Abcam) at 4°C overnight. After incubation with a mixture of Alexa Fluor 488 goat anti-rabbit IgG (1:400; Invitrogen, Carlsbad, CA, USA) and 4’,6-diamidino-2-phenylindole (DAPI), the cells were then mounted in anti-fading medium (Invitrogen). Images were captured by using a confocal microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with Image-Pro® Plus (Media Cybernetics, Bethesda, MD, USA).
Detection of telomere length. Telomere length was determined using a quantitative telomeric fluorescence in situ hybridization (Q-FISH) method, as previously described (15). Briefly, cells were treated with TSA at a final concentration of 100 nM or 200 nM, and colcemid was added to the culture after 41 h (2 μg/ml for GM847, GM847/vector, and GM847/hTERT cells, and 10 μg/ml for WI38VA13 cells). The cells were harvested after 7 h, swelled in hypotonic buffer, and fixed in fixative solution. The cell suspensions were dropped onto coated slides and fixed twice. Then, hybridization was performed, as described in the APB methods. After extensive washing, the slides were stained with DAPI and covered with antifade solution (Invitrogen). Images of metaphases were captured by using a fluorescence microscope (Olympus, Tokyo, Japan), and the fluorescence intensities of the telomeres were quantified using Image-Pro® Plus (Media Cybernetics).
Chromosome orientation-FISH. The T-SCE frequency was measured by chromosome orientation FISH (CO-FISH) by a previously described method, with a slight modification (16). Cells were treated with TSA, and 24 h later, 5-bromo-2-deoxyuridine (BrdU; Sigma) was added to the culture at a final concentration of 10 μM for 24 h, which allowed a single cell-cycle to occur; colcemid was then added, as described in the Q-FISH analysis. Cells were harvested, and metaphases were prepared on poly-D lysine coated coverslips, as described in the Q-FISH methods. Coverslips were incubated with 0.5 mg/ml RNase A (Sigma) at 37°C for 10 min, and then stained with 10 μg/ml Hoechst 33258 in 2× saline-sodium citrate (SSC), at room temperature for 10 min. Coverslips were then mounted with 1× phosphate buffered saline (PBS) and exposed to UV light for 30 min. This was followed by fixation, washing, and dehydration. Hybridization was then performed as described in the FISH analysis procedure without the denaturation step. Images were captured on a fluorescence microscope (Olympus) and T-SCEs were quantified using Image-Pro® Plus (Media Cybernetics).
Statistical analysis. Statistical analyses were performed using the SPSS 13.0 software (SPSS, Chicago, IL, USA) and assessed by using the Student's t-test and the χ2-test. Significance was defined as a p-value of less than 0.05.
Results
TSA induces histone acetylation but hTERT expression remains silenced in GM847 cells. To examine histone acetylation in ALT cells, GM847 cells were initially treated with TSA at different concentrations and durations. Immunoblotting showed that histone acetylation at H3K9 and H4K8 was induced at 100 nM TSA and that the level of histone acetylation persisted without a significant decrease for at least 48 h (Figure 1A). Next, we examined the mRNA of hTERT, which is a catalytic component of telomerase, to find out if TSA induces hTERT expression in GM847 cells. Cells that were treated with 100 nM TSA for 48 h were subjected to quantitative RT-PCR. hTERT mRNA remained silenced in both the TSA-treated and untreated cells (Figure 2), suggesting that TSA-induced histone acetylation exerts no influence on hTERT gene expression in the GM847 cells. These results suggest that TSA treatment induced histone acetylation; however, the GM847 cells remained telomerase- negative.
TSA increases APB formation in GM847 cells. To investigate whether TSA influences the ALT phenotype of GM847 cells, APBs detected by merging the telomere and PML protein staining were examined in the cells treated with 100 nM TSA for 48 h. We analyzed 100 cells from two independent experiments. To minimize the inclusion of non-specific spots, APBs of ≥0.1 μm3 in size were counted using Image-Pro® Plus (Media Cybernetics). The number of APBs increased markedly in the TSA-treated cells (p<0.001; Figures 1B and C). Out of 100 TSA-treated cells, 63 cells were APB-positive and seven cells contained ≥6 APBs/cell. Some telomere spots were apparently large with strong intensities, while PML signals seemed to remain unaffected by the treatment of TSA, and strong telomere spots were mostly co-localized with PML bodies (Figure 1B). On the other hand, in the untreated cells, a smaller fraction of the cells were APB-positive (30/100) and none of those contained more than six APBs per cell. These results show that TSA treatment increases the formation of APBs and enlarges some APBs in GM847 cells.
TSA treatment increases T-SCE frequency in GM847 cells but telomere length remains unchanged. CO-FISH analysis was used to detect SCE events at telomeres. Metaphases were prepared from cells treated with TSA, followed by telomere-FISH analysis, and chromosomes with T-SCEs were counted (Figure 1D). T-SCEs were more frequently observed in TSA-treated cells; T-SCEs were detected in 20% of the chromosomes (129/634) in the TSA-treated cells, whereas the control cells had T-SCEs only in 11% of their chromosomes (50/459; p<0.001). These results show that TSA induces SCE events at telomeres in GM847 cells (Figures 1D and E).
We further investigated whether TSA treatment leads to any changes in telomere length. Telomere length was examined by performing Q-FISH analysis (Figures 1F and G). There was no noticeable difference in the telomere length between the TSA-treated and untreated cells; the mean telomere intensity, which is proportional to the length, was 111±58.9 in the TSA-treated cells and 112±59.4 in the untreated cells (Figure 1G; p=0.593). We also looked at the distribution of telomere intensity and found a similar pattern in both the TSA-treated and untreated cells (Figure 1G). It is not clear why the telomere length was not affected, although APBs and T-SCEs were obviously increased after treatment with TSA. In general, long-term monitoring is required for the detection of changes in the telomere length from ALT cells in which the telomere maintenance mechanism is disrupted (17, 18). We, thus, speculated that 48 h of TSA treatment might not have been sufficient to show a difference in the telomere length. Taken together, APBs and T-SCEs seem to be more prevalent in ALT cells when chromatin structure is less condensed. The results of APB and quantitative FISH analyses showed that the emergence of strong telomere spots at PML bodies in the TSA-treated cells were unlikely to be due to lengthened telomeres. It is thought that some telomeres in different chromosomes seem to be assembled at PML bodies upon TSA treatment.
Characteristics of WI38VA13 cells upon TSA treatment. To analyze whether the phenotypes that were observed in GM847 cells upon TSA treatment are the general features of ALT cells, we performed additional experiments with another well-characterized ALT cell line, WI38VA13, which is an SV40-immortalized lung fibroblast cell line. The TSA treatment was set at 200 nM for 48 h because histone acetylation was strongly induced under this condition (Figure 3A). WI38VA13 cells, which contained a tiny amount of hTERT mRNA in the untreated cells, showed no increase in hTERT mRNA upon TSA treatment (Figure 2). APBs were detected more frequently in the TSA-treated cells (38/100) than in untreated cells (18/100; p<0.001; Figures 3B and C). Large size APBs were also frequently seen in the TSA-treated WI38VA13 cells (Figure 3B), as in the case of the GM847 cells. On the other hand, in the untreated cells, a smaller fraction of the cells were APB-positive (18/100) and most APB-positive cells (17/18) contained fewer than five APBs per cell (Figure 3B and C). T-SCE events were also found to be more frequent in the TSA-treated cells (233/682, 34%), unlike in untreated cells (102/721, 14%; Figures 3D and E). The telomere length remained unchanged after 48 h of TSA treatment (Figures 3F and G). Our results for the WI38VA13 cells upon TSA treatment were quite similar to those for the GM847 cells, revealing that TSA induced the formation of APBs and T-SCEs in the ALT cells, but hTERT mRNA and telomere length remained unaffected.
Introduction of telomerase expression suppresses TSA-mediated ALT phenotype enhancement. One question that may arise is whether telomerase expression in ALT cells can cause changes in ALT phenotypes after TSA treatment. GM847 cells that stably expressed hTERT were established and confirmed by detecting hTERT protein expression, strong telomerase activity, and telomere elongation in GM847/hTERT cells (data not shown). In the GM847/hTERT cells, the induction of histone acetylation started at 100 nM TSA, and the induction level was maintained at 200 nM (Figure 4A). In the GM847/vector cells, however, histone acetylation was actually induced at 200 nM. Thus, the condition for TSA treatment was set to 200 nM for 48 h (Figure 4A). With TSA treatment, the hTERT mRNA remained undetected in the GM847/vector cells, but increased in the GM847/hTERT cells (Figure 2), showing that TSA induced exogenous hTERT gene expression while GM847/vector cells remained telomerase-negative.
Analysis of FISH, combined with immunostaining revealed that TSA treatment increased APBs in both the GM847/hTERT and GM847/vector cells; however, the increase was smaller in the GM847/hTERT cells (Figures 4B and C). In the case of the GM847/vector cells, TSA treatment increased the number of APB-positive cells by 2.1-fold; APB-positive cells were found to compose 71% with TSA treatment and 34% without TSA treatment. In the case of the GM847/hTERT cells, the number of APB-positive cells increased only by 1.3-fold with TSA treatment (Figure 4C).
Incidence of T-SCEs was increased slightly, by 1.3-fold in GM847/hTERT cells after TSA treatment, which was statistically insignificant (p=0.155; Figure 5A). In the GM847/vector cells, TSA treatment yielded no significant change in T-SCEs (p=0.444; Figure 5A), which differed from the results for the parental GM847 cells where T-SCEs increased by nearly 2-fold (Figure 1E). It is not clear why different results were observed in the T-SCE analysis with the parental GM847 cells and the GM847/vector cells. Characteristics of GM847/vector cells might be slightly changed. In fact, the condition of TSA treatment for histone acetylation differed: 100 nM of TSA for the parental GM847 cells and 200 nM of TSA for the GM847/vector cells. Nevertheless, if we compare the data only from parental GM847 and GM847/hTERT cells, hTERT expression seemed to suppress TSA-mediated T-SCE events in the GM847/hTERT cells.
We also measured the telomere length of the GM847/hTERT and GM847/vector cells through Q-FISH analysis and found no noticeable changes in length under TSA treatment (Figure 5B). Although hTERT mRNA expression was up-regulated in the TSA-treated GM847/hTERT cells, changes in the telomere length were not noticed. Consistent with the results for the GM847 and WI38VA13 cells, noticeable changes in the telomere length may not have occurred during 48 h of TSA treatment.
Discussion
The state of chromatin is closely associated with telomere length regulation in telomerase-positive cells. However, the relationship between the state of chromatin and ALT phenotype has not been investigated in ALT cells. In this study, we observed that the treatment of ALT cell lines with TSA increased the number of APBs, enlarged some APBs, and enhanced the frequency of T-SCEs.
Previous reports showed that treatment of human cancerous cells with TSA results in histone hyperacetylation, which leads to global chromatin relaxation (11, 12). Although telomeric chromatin status was not examined in this study, TSA-induced histone acetylation supports the possibility of telomeres existing in a less compact structure. The results of the telomere length analysis and APB formation suggested that strong telomere spots that were found at the PML bodies of the TSA-treated cells were unlikely to be due to lengthened telomeres. Given the emergence of strong telomere spots in the PML bodies and increased T-SCEs upon TSA treatment, we expected that TSA-induced chromatin relaxation facilitated telomere assembly at PML bodies. In particular, telomeres with a less compact structure might have been preferentially recruited to PML bodies by the proteins that are required for telomeric recombination (Figure 5C). Consistent with previous reports (7-10), our results suggested a close relationship between telomeric recombination and chromatin relaxation.
GM847, GM847/vector, and GM847/hTERT cells that were not treated with TSA exhibited similar levels of APBs and T-SCEs, although a slight increase in APBs and T-SCEs was found in GM847/hTERT cells compared to GM847/vector, which was, however, statistically insignificant (Figures 1, 4, and 5). These results indicate that the introduction of telomerase into the GM847 cells did not alter the ALT phenotype, which is consistent with a previous report (19). Interestingly, telomerase expression in the GM847 cells seemed to suppress the TSA-mediated increase in APBs and T-SCEs. This may have been achieved by inhibiting telomere assembly at PML bodies. Previous reports suggest that telomerase prefers telomeres with less compact structures (20, 21). Our results also showed that the ALT mechanism favors telomeres with less compact structures. Therefore, in TSA-treated ALT cells with telomerase expression, the ALT mechanism may compete with telomerase for telomere maintenance.
In this study, we demonstrated that certain ALT phenotypes become more pronounced in TSA-treated ALT cells. Our findings provide important insights into telomere regulation in telomerase-negative cancerous cells. In future, it would be interesting to study this molecular mechanism in greater depth and to clarify how TSA-mediated chromatin relaxation facilitates telomeric recombination.
Acknowledgements
This research was supported by the Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF), for which funding by the Ministry of Education, Science and Technology (2010-0020841) was given to S.Y.R. It was also funded by grants from the NRF (2010-0008254 and 2011-0015638) that were directed to B.K.O. and partly by the Priority Research Centers Program through the NRF (2009-0093824), which were also directed to B.K.O.
- Received December 9, 2012.
- Revision received January 21, 2013.
- Accepted January 22, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved