Large CTG Repeats Trigger p16-Dependent Premature Senescence in Myotonic Dystrophy Type 1 Muscle Precursor Cells
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080560
ISSN1525-2191
AutoresAnne Bigot, Arnaud F. Klein, E. Gasnier, Virginie Jacquemin, Philippe Ravassard, Gillian Butler‐Browne, Vincent Mouly, Denis Furling,
Tópico(s)Telomeres, Telomerase, and Senescence
ResumoA CTG repeat amplification is responsible for the dominantly inherited neuromuscular disorder, myotonic dystrophy type 1 (DM1), which is characterized by progressive muscle wasting and weakness. The expanded (CTG)n tract not only alters the myogenic differentiation of the DM1 muscle precursor cells but also reduces their proliferative capacity. In this report, we show that these muscle precursor cells containing large CTG expansion sequences have not exhausted their proliferative capacity, but have entered into premature senescence. We demonstrate that an abnormal accumulation of p16 is responsible for this defect because the abolition of p16 activity overcomes early growth arrest and restores an extended proliferative capacity. Our results suggest that the accelerated telomere shortening measured in DM1 cells does not contribute to the aberrant induction of p16. We propose that a cellular stress related to the amplified CTG repeat promotes premature senescence mediated by a p16-dependent pathway in DM1 muscle precursor cells. This mechanism is responsible for the reduced proliferative capacity of the DM1 muscle precursor cells and could participate in both the impaired regeneration and atrophy observed in the DM1 muscles containing large CTG expansions. A CTG repeat amplification is responsible for the dominantly inherited neuromuscular disorder, myotonic dystrophy type 1 (DM1), which is characterized by progressive muscle wasting and weakness. The expanded (CTG)n tract not only alters the myogenic differentiation of the DM1 muscle precursor cells but also reduces their proliferative capacity. In this report, we show that these muscle precursor cells containing large CTG expansion sequences have not exhausted their proliferative capacity, but have entered into premature senescence. We demonstrate that an abnormal accumulation of p16 is responsible for this defect because the abolition of p16 activity overcomes early growth arrest and restores an extended proliferative capacity. Our results suggest that the accelerated telomere shortening measured in DM1 cells does not contribute to the aberrant induction of p16. We propose that a cellular stress related to the amplified CTG repeat promotes premature senescence mediated by a p16-dependent pathway in DM1 muscle precursor cells. This mechanism is responsible for the reduced proliferative capacity of the DM1 muscle precursor cells and could participate in both the impaired regeneration and atrophy observed in the DM1 muscles containing large CTG expansions. Myotonic dystrophy type 1 (DM1) is the most prevalent form of adult neuromuscular disorder, characterized by myotonia, muscle wasting, and weakness as well as other multisystemic defects.1Harper PS Myotonic Dystrophy. ed 3. W.B. Saunders, London2004: 17-45Google Scholar DM1 is an autosomal dominant disease caused by the expansion of a CTG repeat in the 3′ untranslated region (3′UTR) of the DMPK gene.2Aslanidis C Jansen G Amemiya C Shutler G Mahadevan M Tsilfidis C Chen C Alleman J Wormskamp NG Vooijs M Buxton J Johnson K Smeets JM Lennon G Carrano AV Korneluk R Wieringa B de Jong P Cloning of the essential myotonic dystrophy region and mapping of the putative defect.Nature. 1992; 355: 548-551Crossref PubMed Scopus (462) Google Scholar, 3Brook JD McCurrach ME Harley HG Buckler AJ Church D Aburatani H Hunter K Stanton VP Thirion JP Hudson T Sohn R Zemelman B Snell RG Rundle SA Crow S Davies J Shelbourne P Buxton J Jones C Juvonen V Johnson K Harper P Shaw DJ Housman D Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member.Cell. 1992; 68: 799-808Abstract Full Text PDF PubMed Scopus (2085) Google Scholar, 4Fu YH Pizzuti A Fenwick Jr, RG King J Rajnarayan S Dunne PW Dubel J Nasser GA Ashizawa T de Jong P Wieringa B Korneluk R Perryman MB Epstein HF Caskey CT An unstable triplet repeat in a gene related to myotonic muscular dystrophy.Science. 1992; 255: 1256-1258Crossref PubMed Scopus (1289) Google Scholar, 5Mahadevan M Tsilfidis C Sabourin L Shutler G Amemiya C Jansen G Neville C Narang M Barcelo J O'Hoy K Leblond S Earl-Macdonald J de Jong P Wieringa B Korneluk R Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene.Science. 1992; 255: 1253-1255Crossref PubMed Scopus (1444) Google Scholar Unaffected individuals have fewer than 38 CTG repeats whereas DM1 patients have from 100 to several thousand repeats in the most severe cases. In general, the size of the expansion correlates with the age of onset and the severity of the disease.6Hunter A Tsilfidis C Mettler G Jacob P Mahadevan M Surh L Korneluk R The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy.J Med Genet. 1992; 29: 774-779Crossref PubMed Scopus (200) Google Scholar, 7Tsilfidis C MacKenzie AE Mettler G Barcelo J Korneluk RG Correlation between CTG trinucleotide repeat length and frequency of severe congenital myotonic dystrophy.Nat Genet. 1992; 1: 192-195Crossref PubMed Scopus (311) Google Scholar An increasing amount of evidence supports a RNA gain-of-function mechanism in DM1 disease. The mutant transcripts containing the CUG expansion fold into RNA hairpins that are not exported to the cytoplasm but accumulate in the nuclei as discrete foci. These mutant RNAs interfere with the activities of proteins involved in RNA processing such as MBNL, CUG-BP1, hnRNP H, leading to specific misregulated splicing events.8Paul S Dansithong W Kim D Rossi J Webster NJ Comai L Reddy S Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing.EMBO J. 2006; 25: 4271-4283Crossref PubMed Scopus (121) Google Scholar As an example, the misregulation of alternative splicing of the chloride channel ClC-1 mRNAs found in DM1 patients has been directly linked to myotonia, a characteristic feature of the disease.9Mankodi A Takahashi MP Jiang H Beck CL Bowers WJ Moxley RT Cannon SC Thornton CA Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy.Mol Cell. 2002; 10: 35-44Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar The CTG mutation is very unstable and its amplification occurs both over generations and in somatic tissues. Intergenerational instability of the expanded microsatellite provides the molecular basis for the anticipation phenomenon described in DM1 disease: the size of the CTG expansion progressively increases in successive generations of DM1 families, and correlates with the severity of the disease.1Harper PS Myotonic Dystrophy. ed 3. W.B. Saunders, London2004: 17-45Google Scholar After several generations, the patients often develop the severe DM1 congenital form associated with a large CTG expansion (>1000) and characterized by delayed muscle maturation and atrophy. Somatic instability is also measured throughout the life of a DM1 patient with a gradual increase in the average repeat size. Moreover, variable repeat sizes are detected in different tissues of the same patient with the largest size being found in the skeletal muscle.10Anvret M Ahlberg G Grandell U Hedberg B Johnson K Edstrom L Larger expansions of the CTG repeat in muscle compared with lymphocytes from patients with myotonic dystrophy.Hum Mol Genet. 1993; 2: 1397-1400Crossref PubMed Scopus (176) Google Scholar, 11Thornton CA Johnson K Moxley III, RT Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes.Ann Neurol. 1994; 35: 104-107Crossref PubMed Scopus (249) Google Scholar In a previous study, we have shown that the size of the CTG expansion increases progressively at each cellular division during the proliferative lifespan of DM1 muscle precursor cells, also called satellite cells in vivo or myoblasts in culture, indicating a replication-dependent somatic instability of the expanded microsatellite.12Furling D Coiffier L Mouly V Barbet JP St Guily JL Taneja K Gourdon G Junien C Butler-Browne GS Defective satellite cells in congenital myotonic dystrophy.Hum Mol Genet. 2001; 10: 2079-2087Crossref PubMed Scopus (97) Google Scholar In addition to myogenic differentiation defects, we showed that the proliferative capacity of the satellite cells isolated from congenital DM1 patients with large CTG repeats was significantly reduced as compared with age-matched controls. The proliferative capacity of human satellite cells, like the majority of human diploid somatic cells, is limited by cellular senescence. One major mechanism implicated in the replicative senescence of human cells is the progressive erosion of their telomeres at each cell division. Once a critically short telomere length is reached, replicative senescence is triggered through a p53-dependent pathway.13Vaziri H West MD Allsopp RC Davison TS Wu YS Arrowsmith CH Poirier GG Benchimol S ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.EMBO J. 1997; 16: 6018-6033Crossref PubMed Scopus (334) Google Scholar The introduction of the catalytic subunit of the telomerase (hTERT) gene into human fibroblasts is sufficient to block telomere shortening and prevent replicative senescence in these cells, and lead to their immortalization.14Bodnar AG Ouellette M Frolkis M Holt SE Chiu CP Morin GB Harley CB Shay JW Lichtsteiner S Wright WE Extension of life-span by introduction of telomerase into normal human cells.Science. 1998; 279: 349-352Crossref PubMed Scopus (4147) Google Scholar However, expression of hTERT is not sufficient to confer immortality to several types of human cells including satellite cells, indicating the existence of cell-type-specific differences in the regulation of the proliferative capacity.15Di Donna S Mamchaoui K Cooper RN Seigneurin-Venin S Tremblay J Butler-Browne GS Mouly V Telomerase can extend the proliferative capacity of human myoblasts, but does not lead to their immortalization.Mol Cancer Res. 2003; 1: 643-653PubMed Google Scholar The p16-dependent pathway has been shown to provoke proliferative arrest before telomeres reach their critical value, and inhibition of p16 in addition to hTERT activity results in the immortalization of keratinocytes, epithelial cells, and satellite cells.16Ramirez RD Herbert BS Vaughan MB Zou Y Gandia K Morales CP Wright WE Shay JW Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells.Oncogene. 2003; 22: 433-444Crossref PubMed Scopus (77) Google Scholar, 17Zhu CH Mouly V Cooper RN Mamchaoui K Bigot A Shay JW Di Santo JP Butler-Browne GS Wright WE Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies.Aging Cell. 2007; 6: 515-523Crossref PubMed Scopus (211) Google Scholar p16 is up-regulated in response to several telomere-independent stress mechanisms including DNA damage, oncogenic signals, and oxidative stress,18Kim WY Sharpless NE The regulation of INK4/ARF in cancer and aging.Cell. 2006; 127: 265-275Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar but has also been proposed to be involved in response to telomere damage.19Jacobs JJ de Lange T Significant role for p16INK4a in p53-independent telomere-directed senescence.Curr Biol. 2004; 14: 2302-2308Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar Recently several reports have indicated that an increase in p16 expression in the brain, bone marrow, and pancreas progenitor cells during aging contributes to stem cell decline, senescence, and aging.20Campisi J d'Adda di Fagagna F Cellular senescence: when bad things happen to good cells.Nat Rev Mol Cell Biol. 2007; 8: 729-740Crossref PubMed Scopus (3061) Google Scholar In this report, we have analyzed the premature growth arrest of DM1 satellite cells carrying large CTG expansions. Our results indicate that a mechanism of premature senescence limits the proliferative capacity of the DM1 satellite cells. We demonstrate that the p16 pathway is responsible for this premature growth arrest in response to a CTG-related stress. In addition our results indicate that the accelerated telomere shortening measured in DM1 cells is not involved in the early accumulation of p16 but is rather a consequence of the cellular stress induced by the CTG mutation. The human satellite cells were isolated from muscle biopsies obtained from the Bank of Tissues for Research (a partner in the European Union network EuroBioBank) in accordance with European recommendations and French legislation. Three DM1 fetuses (28-, 34-, and 37-weeks old) showing clinical symptoms of the congenital form with varus feet, arthrogryposis, muscular hypotrophy, and carrying more than 2000 CTG were included in this study. Three individuals (2 fetuses of 29 and 37 weeks of age, and one 5-day-old infant) showing no sign of neuromuscular disease were used as controls. The muscle biopsies were finely minced and explants were plated onto noncoated Petri dishes in drops of fetal calf serum (Invitrogen, Carlsbad, CA). The cells were cultivated at 37°C in a humid atmosphere containing 5% CO2. Once mononucleated cells had migrated out from the explants, growth medium consisting of Ham's F10 medium (Invitrogen) supplemented with 20% fetal calf serum and 5 μg/ml gentamicin was added. At the time of cell isolation, all cell populations were considered to be at 1 mean population doubling. Cell populations were trypsinized when they reached 80% of confluence. At each passage, the number of divisions was calculated according to the formula: log (N/n)/log 2 where N is the number of cells counted and n is the number of cells initially plated. The cultures were considered to be in an irreversible growth arrest when they failed to divide during 3 weeks in proliferative conditions. Viability was controlled by trypan blue (Sigma, St. Louis, MO) exclusion. The myogenic purity of the populations was monitored by immunocytochemistry using desmin as a marker because it is only expressed in myogenic cells. All cell populations used in this study had a myogenic purity greater than 75%. For 5-aza-2′deoxycytidine experiment, cells were treated with 3 μmol/L 5-aza-2′deoxycytidine (Sigma) for 7 days as described by Darbro and colleagues.21Darbro BW Lee KM Nguyen NK Domann FE Klingelhutz AJ Methylation of the p16(INK4a) promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells.Oncogene. 2006; 25: 7421-7433Crossref PubMed Scopus (24) Google Scholar hTERT and Cdk4 cDNA were cloned into distinct pBABE retroviral vectors containing, respectively, the puromycin selection marker and a neomycin resistance gene. Transduced cell cultures were submitted to selection in the presence of puromycin (0.5 μg/ml) and/or neomycin (0.3 mg/ml) for 10 days. For bromodeoxyuridine (BrdU) labeling, cell cultures were grown for 72 hours in the presence of 10 μg/ml BrdU and then fixed for 10 minutes with ethanol. Cells that had incorporated BrdU were detected using the monoclonal antibody Bu20a (DAKO, Glostrup, Denmark), revealed by a specific secondary antibody directly coupled to Alexa Fluor 488 (Molecular Probes, Eugene, OR). DAPI (Calbiochem, San Diego, CA) was used to visualize the nuclei. All images were digitalized using the MetaView image analysis system (Universal Imaging Corp., Downington, PA). To determine the percentage of positive cells, at least 500 cells were counted. The expression of desmin was revealed using the antibody D33 (1/50, DAKO) and specific antibody binding was revealed with peroxidase (Vectastain; Vector Laboratories, Burlingame, CA). SA-ß-galactosidase activity was revealed as described by Dimri and colleagues.22Dimri GP Lee X Basile G Acosta M Scott G Roskelley C Medrano EE Linskens M Rubelj I Pereira-Smith O Peacocke M Campisi J A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc Natl Acad Sci USA. 1995; 92: 9363-9367Crossref PubMed Scopus (5788) Google Scholar Two μg of genomic DNA were digested with Hinf1 (Biolabs, Santa Ana, CA) to generate telomere restriction fragments. The samples were resolved by electrophoresis in a 0.7% agarose gel that was hybridized to a 32P-(TTAGGG)4 probe. The signal responses were revealed using the Personal-Molecular-Imager (Bio-Rad, Hercules, CA) and analyzed by a computer-assisted system using National Institutes of Health (Bethesda, MD) Image 1.62 and ProFit software. The mean of telomere lengths was determined from three independent gels. Two μg of total RNA isolated using Trizol (Invitrogen) were reverse-transcribed into cDNA according to the manufacturer's instructions. Equal amounts of the reverse transcription products were subjected to PCR amplification (ABGene, Rochester, NY). Amplification was initiated by 4 minutes of denaturation at 94°C, followed by 20 (GAPDH) or 35 cycles (p16) of amplification. Each cycle consists of 60 seconds at 94°C, 60 seconds at 55°C (GAPDH), or 62°C (p16) and 60 seconds at 72°C. A final step of extension was performed for 10 minutes at 72°C. After amplification, 15 μl of each PCR reaction product were separated on a 1% agarose gel containing ethidium bromide. The following primers, synthesized by Sigma-Proligo were used: GAPDH, forward, 5′-GATGACAAGCTTCCCGTTCTCAGCC-3′; GAPDH, reverse, 5′TGAAGGTCGGAGTCAACGGATTTGGT-3′; p16, forward, 5′-TGGAGCCTTCGGCTGACTGGCTGGC-3′; and p16, reverse, 5′-CTACGAAAGCGGGGTGGGTTGT-3′. Thirty μg of total protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and incubated with different antibodies directed against Rb (Pharmingen, San Diego, CA), p16 (Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Santa Cruz Biotechnology), cyclin D (Cell Signaling, Beverly, MA) and Emerin (Novocastra, Newcastle, UK). Secondary antibodies coupled to horseradish peroxidase were revealed using the ECL kit (Pierce, Rockford, IL). The signal was detected on film (Fuji Film, Fuji, Tokyo, Japan) and quantified by densitometry. Prism software was used to calculate the statistical significance and the SEM. Significance was tested by Student's unpaired t-test or Mann-Whitney test. For all tests, the groups were considered statistically different for P value <0.05 (*P < 0.05, **P < 0.01, ***P < 0.001). The proliferative capacity of satellite cells isolated from muscles of three congenital DM1 patients with large CTG repeats (>2000) was compared in vitro to that of satellite cells isolated from three age-matched and nonaffected individuals. The cells were grown under identical culture conditions until they ceased to respond to mitogenic stimuli for 3 weeks and entered into an irreversible cell-cycle arrest. As shown in Figure 1A, the average proliferative lifespan of the DM1 satellite cells was significantly reduced by 40% as compared with that of control cells. When they stopped dividing both DM1 and control satellite cells displayed a flattened and enlarged morphology characteristic of senescent cells (Figure 1B). Senescent-associated β-galactosidase activity was detected in growth-arrested DM1 cells, as well as high levels of cyclin D1, which is a reliable marker of replicative senescence and was detected in both DM1 and control cells at the end of their lifespan (Figure 1C). Analysis of the phosphorylation status of Rb showed that arrested DM1 and control satellite cells contained only the hypophosphorylated form, in contrast to the proliferating cells at earlier passages, which expressed in addition the hyperphosphorylated form essential to overcome the G1 checkpoint (Figure 1D). Cell-cycle fluorescence-activated cell sorting analysis confirmed that the majority of DM1 and control cells were arrested in G1 (data not shown). These results indicate that a mechanism similar to senescence is responsible for the early proliferative growth arrest of DM1 satellite cells because these cells expressed biomarkers usually observed in senescent cells, although much earlier in their replicative lifespan as compared with control cultures. To evaluate if the premature growth arrest observed in satellite cells isolated from DM1 patients was attributable to an excessive proliferation in vivo before their isolation, we measured the size of the telomeres in these cells. Telomere length represents a predictive marker of the regenerative capacity of the human satellite cells as shown in Duchenne muscular dystrophy in which the extensive proliferation of the satellite cells after continuous cycles of degeneration and regeneration leads to reduced telomere length.23Decary S Hamida CB Mouly V Barbet JP Hentati F Butler-Browne GS Shorter telomeres in dystrophic muscle consistent with extensive regeneration in young children.Neuromuscul Disord. 2000; 10: 113-120Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar As shown in Figure 1E, DM1 satellite cells stop dividing with telomeres significantly longer than those of control cells (respectively, 8.7 kb versus 7.4 kb), indicating that telomeres of DM1 cells had not reached their critical size and consequently that these cells had not exhausted their proliferative capacity. This confirms that a mechanism of premature senescence alters and limits in vitro the replicative lifespan of DM1 satellite cells carrying large CTG expansions. As demonstrated recently, the p16 cyclin-dependent kinase inhibitor is a key regulator of the replicative senescence of human satellite cells.17Zhu CH Mouly V Cooper RN Mamchaoui K Bigot A Shay JW Di Santo JP Butler-Browne GS Wright WE Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies.Aging Cell. 2007; 6: 515-523Crossref PubMed Scopus (211) Google Scholar To determine whether the p16/Rb pathway is involved in the premature senescence of DM1 cells in vitro, we have measured the expression of p16 protein at the end of their lifespan. In these experiments we found that a similar level of p16 was detected in both DM1 and control cultures once they had entered proliferative arrest (Figure 2A). Moreover, it should be noted that DM1 cells made significantly fewer divisions than control cells (Figure 2B). The accumulated p16 will bind Cdk4 thus inhibiting its activity and block cell-cycle progression. To assess the role of p16 in DM1 premature senescence, we stably overexpressed Cdk4 to neutralize p16 activity. As seen in Figure 2B, DM1-Cdk4 cells were able to bypass the premature growth arrest observed in DM1 cells. Their lifespan was extended by 59% to 51 divisions, which is very close to the 52 divisions observed in control cells. In addition, overexpression of p16 in young control cells resulted in a premature proliferative arrest, as evidenced by absence of BrdU incorporation after incubation for 72 hours (data not shown). These results demonstrate that p16 is responsible for triggering in vitro the premature senescence of DM1 satellite cells. However, we also abolished p16 activity by overexpression of Cdk4 in control cells and observed that the DM1-Cdk4 cells still make fewer divisions than the control-Cdk4 age-matched cells (51 versus 70 divisions, respectively) before entering into replicative senescence (Figure 2B). To assess if a residual activity of p16 could be responsible for the proliferative arrest of the Cdk4-overexpressing cells, RNA interference directed to p16 was induced in these cells, but no further expansion of their lifespan was observed (data not shown). Measurements of telomere lengths showed that DM1-Cdk4 and control-Cdk4 satellite cells both stop growing with telomere lengths of 6.2 kb and 6.1 kb, respectively. These values are almost identical to the 6 kb measured in senescent human fibroblasts in which a telomere-dependent senescence is induced by short telomeres. Both the DM1- and control Cdk4-transduced cells stopped growing with short telomeres but the DM1-Cdk4 cells reached this minimum value after fewer divisions than the control-Cdk4 cells, suggesting that large CTG expansions may interfere with the telomere homeostasis machinery in DM1 cells. Telomere loss per division was calculated from measurements made at several time points during the lifespan of DM1 and control cells, and a significant 59% increase in the amount of telomeric DNA lost per division was measured in DM1 satellite cell cultures (171.9 ± 17 bp) as compared with control cultures (108.1 ± 10 bp) (Figure 3A). To determine whether or not the increase in the amount of telomeric DNA lost per division in DM1 satellite cells was a consequence of an increased cell death in DM1 cultures, which would result in an increase in the number of divisions made by the surviving cells and thus in an increase in mean telomere shortening, we assessed both cell death and proliferation in control and DM1 populations. No significant difference was observed in the number of proliferating cells, as demonstrated by BrdU incorporation in the middle of their lifespan: 77 ± 3% cells in control populations versus 75 ± 4% cells in DM1 populations were BrdU-positive, indicating that both populations have similar growth rates. Viability of the cells was assessed by trypan blue exclusion, and only very low levels of cell death were detected in both cultures (3.7 ± 0.8% in control and 4.6 ± 1% in DM1 cultures), with no significant difference being observed between them. These results confirm that the higher rate of telomere loss in DM1 cells is not a consequence of an increased cell death, which would result in an increase number of divisions made by the surviving cells. To determine whether a higher rate of telomere shortening may induce elevated levels of p53 and thus participate to the premature proliferative arrest, p53 protein levels were measured by Western blot in DM1 cells presenting a higher rate of telomere loss and control cells, both at the end of their lifespan. A similar level of p53 was detected in both cultures (Figure 3B), indicating that an elevation in the level of expression of p53 is not triggered by increased telomere loss. The increased loss of telomeric DNA per division will reduce the number of divisions that DM1 cells can make before reaching telomere-driven senescence and explains why the critical telomere size of 6 kb is reached by the DM1-Cdk4 cells after having made fewer divisions than control-Cdk4 cells. To determine whether such an accelerated telomere loss could act as a signal or could be responsible for the premature senescence of the DM1 cells, we expressed the catalytic subunit of telomerase hTERT because the activity of this enzyme should compensate for the increased telomere erosion. As seen in Figure 4A, DM1 cells expressing hTERT were not able to make more divisions than DM1 cells before entering into premature growth arrest suggesting that the higher rate of telomere erosion may not contribute to the process of premature senescence observed in vitro in DM1 satellite cells. However, among these arrested cells, a clone of proliferating cells emerged after 2 months of culture (Figure 4B), which overcame the arrested population (Figure 4B, lane S) and presented an abolition of p16 expression, as demonstrated by the decline in the level of p16 detected at two successive time points (Figure 4, B and C, lanes 1 and 2) in culture. The promoter region of the p16 gene contains a 5′CpG island that has been found to exhibit increased levels of methylation in various tumors as well as in telomerase immortalized human keratinocytes.21Darbro BW Lee KM Nguyen NK Domann FE Klingelhutz AJ Methylation of the p16(INK4a) promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells.Oncogene. 2006; 25: 7421-7433Crossref PubMed Scopus (24) Google Scholar The abolition of p16 expression in this clone was confirmed at the RNA level and a re-expression of p16 was measured when these cells were treated with 5-aza-2′ deoxycytidine (Figure 4D, lane 2*). This result indicates that methylation of the p16 promoter had occurred in this DM1-hTERT clone, leading to its immortalization because these cells have now made more than 200 divisions. Immortalization of the DM1 satellite cells was also obtained by the dual overexpression of hTERT and Cdk4, and these cells have been maintained for more than 150 divisions (Figure 4E). Measurement of the telomere lengths showed that the expression of hTERT in DM1-Cdk4 satellite cells stabilized the telomere length to ∼10 kb, a value similar to that observed in control hTERT/Cdk4-cells (Figure 4E, inset). We conclude that blocking both the p16 and the telomere pathways leads to the immortalization of DM1 satellite cells. In this report, we provide new evidence that large CTG expansions trigger in vitro a mechanism of premature senescence through a p16-dependent pathway, which reduces significantly the proliferative capacity of DM1 muscle precursor cells. Measurements of telomere lengths at the end of their lifespan indicate that DM1 cells had not exhausted their proliferative capacity confirming that a premature growth arrest, independent from an excessive in vivo turnover, occurred in satellite cells isolated from DM1 biopsies. Analysis of several biomarkers suggests that a mechanism similar to cellular senescence is responsible for the premature and irreversible cell-cycle arrest of DM1 muscle precursor cells containing large CTG expansions. Our data demonstrated that the Rb regulator p16 triggers in vitro this premature senescence becau
Referência(s)