The Methylation of the TSC2 Promoter Underlies the Abnormal Growth of TSC2 Angiomyolipoma-Derived Smooth Muscle Cells
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080799
ISSN1525-2191
AutoresElena Lesma, Silvia Maria Sirchia, Silvia Ancona, Stephana Carelli, Silvano Bòsari, Filippo Ghelma, E. Montanari, Anna Maria Di Giulio, Alfredo Gorio,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoTuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2−/meth ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2−/− ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2−/meth ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis. Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that is caused by mutations in either the TSC1 or TSC2 gene. Smooth muscle-like cells (ASMs) were isolated from an angiomyolipoma of a patient with TSC. These cells lacked tuberin, were labeled by both HMB45 and CD44v6 antibodies, and had constitutive S6 phosphorylation. The cells bear a germline TSC2 intron 8-exon 9 junction mutation, but DNA analysis and polymerase chain reaction amplification failed to demonstrate loss of heterozygosity. Testing for an epigenetic alteration, we detected methylation of the TSC2 promoter. Its biological relevance was confirmed by tuberin expression and a reduction in HMB45 labeling and S6 constitutive phosphorylation after exposure to the chromatin-remodeling agents, trichostatin A and 5-azacytidine. These cells were named TSC2−/meth ASMs. Their proliferation required epidermal growth factor in the medium as previously described for TSC2−/− ASMs. Blockade of epidermal growth factor with monoclonal antibodies caused the death of TSC2−/meth ASMs. In addition, rapamycin effectively blocked the proliferation of these cells. Our data show for the first time that methylation of the TSC2 promoter might cause a complete loss of tuberin in TSC2 cells, and that the pathogenesis of angiomyolipomas might also originate from epigenetic defects in smooth muscle cells. Additionally, the effect of chromatin-remodeling agents in these cells suggests a further avenue for the treatment of TSC as well as lymphangioleiomyomatosis. Tuberous sclerosis complex (TSC) is an autosomal-dominant disease characterized by hamartomas, in a wide array of tissues and organs, such as brain, kidney, skin, heart, and lungs.1Young J Povey S The genetic basis of tuberous sclerosis.Mol Med Today. 1998; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Abdominal angiomyolipomas are often present in TSC patients; they may cause life-threatening hemorrhages and in such conditions their surgical resection is required.2Neumann HP Schwarzkopf G Henske EP Renal angiomyolipomas, cysts, and cancer in tuberous sclerosis complex.Semin Pediatr Neurol. 1998; 5: 269-275Abstract Full Text PDF PubMed Scopus (57) Google Scholar The tumor suppressor genes, TSC1 and TSC2, are associated with the development of TSC, and mutations in either gene are responsible for familial and sporadic forms of the disease.1Young J Povey S The genetic basis of tuberous sclerosis.Mol Med Today. 1998; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar The TSC2 gene is located on chromosome 16p13 whereas TSC1 on chromosome 9q34.3European Chromosome 16 Tuberous Sclerosis Consortium Identification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1511) Google Scholar, 4van Slegtenhorst M de Hoogt R Hermans C Nellist M Jansen B Verhoef S Lindhout D van den Ouweland A Halley D Young J Burley M Jeremiah S Woodward K Nahmias J Fox M Ekong R Osborne J Wolfe J Povey S Snell RG Cheadle JP Jones AC Tachataki M Ravine D Sampson JR Reeve MP Richardson P Wilmer F Munro C Hawkins TL Sepp T Ali JB Ward S Green AJ Yates JR Kwiatkowska J Henske EP Short MP Haines JH Jozwiak S Kwiatkowski DJ Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1397) Google Scholar Hamartin, the TSC1 gene product, stabilizes tuberin, the TSC2 gene product, through binding with it, thereby preventing tuberin from ubiquitination and degradation.5Benvenuto G Li S Brown SJ Braverman R Vass WC Cheadle JP Halley DJ Sampson JR Wienecke R DeClue JE The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.Oncogene. 2000; 19: 6306-6316Crossref PubMed Scopus (208) Google Scholar Tuberin acts as a GTPase-activating protein to regulate Rheb function through the conversion of Rheb from the active GTP-bound form to the inactive GDP-bound form.6Garami A Zwartkruis FJ Nobukuni T Joaquin M Roccio M Stocker H Kozma SC Hafen E Bos JL Thomas G Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.Mol Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar, 7Inoki K Li Y Xu T Guan KL Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1424) Google Scholar Active Rheb activates mTOR, and the up-regulation of the TSC/mTOR signaling pathway leads to increased protein synthesis, cell proliferation, and ultimately to tumorigenesis.8Inoki K Corradetti MN Guan KL Dysregulation of the TSC-mTOR pathway in human disease.Nat Genet. 2005; 37: 19-24Crossref PubMed Scopus (842) Google Scholar TSC occurs because of a germline mutation in either TSC1 or TSC2. In most hamartomas, TSC follows a second hit inactivating the wild-type allele. The loss of heterozygosity (LOH) in TSC1 or TSC2 has been documented in angiomyolipomas (AMLs), cardiac rabdomiomas, and lymphangioleiomyomatosis (LAM) cells, but it has only rarely been found in cerebral cortical tubers and skin lesions.9Chan JA Zhang H Roberts PS Jozwiak S Wieslawa G Lewin-Kowalik J Kotulska K Kwiatkowski DJ Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation.J Neuropathol Exp Neurol. 2004; 63: 1236-1242Crossref PubMed Scopus (226) Google Scholar, 10Henske EP Scheithauer BW Short MP Wollmann R Nahmias J Hornigold N van Slegtenhorst M Welsh CT Kwiatkowski DJ Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions.Am J Hum Genet. 1996; 59: 400-406PubMed Google Scholar, 11Niida Y Stemer-Rachamimow AO Logrip M Tapon D Perez R Kwiatkowski DJ Sims K MacCollin M Louis DN Ramesh V Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions.Am J Hum Genet. 2001; 69: 493-503Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Therefore, it is not clear whether inactivation of both alleles is the necessary step for hamartoma pathogenesis. Various explanations have been raised to define the inability to find a second somatic event in TSC lesions, and the failure to demonstrate such events has been attributed to either different genetic and epigenetic deficits in TSC genes or cell heterogeneity in TSC hamartomas.12Au KS Hebert AA Roach ES Northrup H Complete inactivation of the TSC2 gene leads to formation of hamartomas.Am J Hum Genet. 1999; 65: 1790-1795Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Cheadle J Reeve M Sampson J Kwiatkowski D Molecular genetic advances in tuberous sclerosis complex.Hum Genet. 2000; 107: 97-104Crossref PubMed Scopus (283) Google Scholar DNA methylation is an epigenetic change that induces chromatin modifications and repression of transcription via a methyl CpG binding protein MeCP2, and recruitment of a Sin3A/HDAC co-repressor complex.14Ng HH Bird A DNA methylation and chromatin modification.Curr Opin Genet Dev. 1999; 9: 158-163Crossref PubMed Scopus (537) Google Scholar, 15Razin A CpG methylation, chromatin structure and gene silencing-a three-way connection.EMBO J. 1998; 17: 4905-4908Crossref PubMed Scopus (661) Google Scholar Twenty-four hamartomas from 10 patients were analyzed by Niida and colleagues11Niida Y Stemer-Rachamimow AO Logrip M Tapon D Perez R Kwiatkowski DJ Sims K MacCollin M Louis DN Ramesh V Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions.Am J Hum Genet. 2001; 69: 493-503Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar for second-hit mutations by promoter methylation of TSC2, but no evidence of such inactivation of the second allele was detected. Here we report the isolation and characterization of a homogenous population of smooth muscle-like cells from AML cells (ASM cells) of a TSC2 patient. The ASM cells were mostly positive for HMB4516El-Hashemite N Walker V Kwiatkowski DJ Estrogen enhances whereas tamoxifen retards development of Tsc mouse liver hemangioma: a tumor related to renal angiomyolipoma and pulmonary lymphangioleiomyomatosis.Cancer Res. 2005; 65: 2474-2481Crossref PubMed Scopus (43) Google Scholar and CD44v6,17Pacheco-Rodriguez G Steagall WK Crooks DM Stevens LA Hashimoto H Li S Wang JA Darling TN Moss J TSC2 loss in lymphangioleiomyomatosis cells correlated with expression of CD44v6, a molecular determinant of metastasis.Cancer Res. 2007; 67: 10573-10581Crossref PubMed Scopus (52) Google Scholar that are markers of TSC and LAM cells, and showed a germline TSC2 intron 8-exon 9 junction mutation with no LOH. However, tuberin was undetectable by immunochemistry and Western blotting. We found that these cells were methylated in the TSC2 promoter, and the involvement of methylation in the inhibition of TSC2 gene was confirmed by the cellular expression of tuberin after exposure to the chromatin remodeling agent, trichostatin A. Thus, ASM cells were named TSC2−/meth ASM cells. The proliferative, morphological, and biochemical characteristics of TSC2−/meth ASM cells were very similar to TSC2−/− smooth muscle cells with LOH that we previously isolated from an AML of a female TSC2 patient (TSC2−/− ASM cells).18Lesma E Grande V Lesma E Carelli S Brancaccio D Canevini MP Alfano RM Coggi G Di Giulio AM Gorio A Isolation and growth of smooth muscle-like cells derived from tuberous sclerosis complex-2 human renal angiomyolipoma: EGF is the required growth factor.Am J Pathol. 2005; 167: 1093-1103Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 19Carelli S Lesma E Paratore S Grande V Zadra G Di Giulio AM Gorio A Survivin expression in tuberous sclerosis complex cells.Mol Med. 2007; 13: 166-177Crossref PubMed Scopus (19) Google Scholar The growth of TSC2−/meth ASM cells requires the addition of epidermal growth factor (EGF) to the culture medium, whereas the exposure to specific monoclonal antibody raised against EGFR causes the blockade of proliferation and their death. Our data show for the first time that the methylation of the TSC2 promoter might cause loss of tuberin in TSC2 cells, and that such epigenetic alteration of smooth muscle cell function may underlie their abnormal growth and likely lead to AML development. The renal angiomyolipoma sample was obtained during total nephrectomy from a 36-year-old man with a history of TSC who had given his informed consent according to the Declaration of Helsinki. The cells were obtained as shown in Lesma and colleagues.18Lesma E Grande V Lesma E Carelli S Brancaccio D Canevini MP Alfano RM Coggi G Di Giulio AM Gorio A Isolation and growth of smooth muscle-like cells derived from tuberous sclerosis complex-2 human renal angiomyolipoma: EGF is the required growth factor.Am J Pathol. 2005; 167: 1093-1103Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Briefly, the tumor tissue was manually dissociated using collagenase type II (Sigma, St. Louis, MO) by means of repetitive pipetting. The collagenase was neutralized with a serum-containing medium (50:50 mixture of Dulbecco's Eagle's medium/Ham F12; Euroclone, Paignton, UK) supplemented with hydrocortisone (2 × 10−7mol/L) (Sigma-Aldrich, St. Louis, MO), EGF (10 ng/ml) (Sigma-Aldrich), sodium selenite (5 × 10−8 mol/L) (Sigma-Aldrich), insulin (25 μg/ml) (Sigma-Aldrich), transferrin (10 μg/ml) (Sigma-Aldrich), ferrous sulfate (1.6 × 10−6 mol/L) (Sigma-Aldrich), and 15% fetal bovine serum (Euroclone) as indicated by Arbiser and colleagues.20Arbiser JL Yeung R Weiss SH Arbiser ZK Amin MB Cohen C Frank D Mahajan S Herron GS Yang J Onda H Zhang HB Bai X Uhlmann E Loehr A Northrup H Au P Davis I Fisher DE Gutmann DH The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma.Am J Pathol. 2001; 159: 483-491Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Vascular smooth muscle-like cells (VSMCs) and A549 cells were grown in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum 10%. The cells were cultured on glass slides, permeabilized with Cytoskelfix (Cytoskeleton, Denver, CO) and dried in air. The primary antibody against α-actin (1:100, Sigma-Aldrich), vimentin (1:70; Santa Cruz Biotechnology, Santa Cruz, CA), S100 (1:8000; DAKO, Carpinteria, CA) keratin 8/18 (1:100; New Marker, Fremont, CA), HMB45 (1:100, DAKO), CD44v6 (1:100; Invitrogen, Carlsbad, CA), hamartin (1:100, Santa Cruz Biotechnology), tuberin (C-20) (1:100, Santa Cruz Biotechnology) were applied overnight at 4°C. The samples were incubated for 3 hours at room temperature with fluorescein isothiocyanate-conjugated anti-mouse antibody (Alexa, Eugene, OR) for α-actin, HMB45, and keratin 8/18, with fluorescein isothiocyanate-conjugated anti-goat antibody (Alexa) for vimentin and hamartin, with rhodamine-conjugated anti-rabbit antibody (Alexa) for tuberin. HMB45 positivity was evaluated by immunofluorescence. Cells were incubated for 72 hours with trichostatin (3.3 mmol/L). HMB45 labeling was defined as strong (+), and negative (−). Quantitation of HMB45 intensity was achieved with a confocal microscopy (TCS-SP2; Leica, Wetzlar, Germany) and analyzed by Leica confocal software using the profile through stack/series methods as follows: 30 equal polygonal areas covering the cells were randomly selected on each image. The fluorescence intensity values of the selected cells were quantified and the mean and the SD of the data were calculated by GraphPad software (La Jolla, CA). The level of statistical significance was determined by Student's t-test. The cells were lysed in lysis buffer (5 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L deoxycholic acid, 3% sodium dodecyl sulfate), boiled, electrophoretically run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL). After being blocked at room temperature for 3 hours with 5% dry milk (Merck, Darmstadt, Germany), the membranes were incubated overnight at 4°C with antibodies against tuberin, phospho-tuberin (Thr1462) (1:1000; Cell Signaling, Beverly, MA), hamartin (a gift from Dr. Mark Nellist and Dr. Dicky Halley, Erasmus University, Rotterdam, The Netherlands), phospho-Akt (Ser473) (1:1000, Cell Signaling), phospho-S6 ribosomal protein (Ser235/236) (1:1000, Cell Signaling), S6 ribosomal protein (1:1000, Cell Signaling), phospho-extracellular signal-regulated kinase (Erk) (Thr202/Tyr204) (1:1000, Cell Signaling), Erk (1:1000; Cell Signaling), EGF receptor (1:200; Santa Cruz Biotechnology), β-actin (1:10,000, Sigma-Aldrich), p53 (1:100, Santa Cruz Biotechnology), p21 (1:1000, Cell Signaling). The membranes were washed and incubated for 1 hour with anti-rabbit antibody (1:10,000; Chemicon, Temecula, CA). The reaction was revealed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Densitometric analysis was performed by Kodak MJ project program (Eastman-Kodak, Rochester, NY). Data were expressed as optical density. The DNAs were extracted from peripheral blood, angiomyolipoma, and isolated cultured cells using the Wizard Genomic DNA purification kit (Promega, Madison, WI). All of the exons and intron-exons junctions of TSC1 and TSC2 from the genomic DNAs were amplified by means of standard polymerase chain reaction (PCR) and described primers.21Jones AC Sampson JR Hoogendoorn B Cohen D Cheadle JP Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis.Hum Genet. 2000; 106: 663-668Crossref PubMed Scopus (52) Google Scholar The sequencing reactions were performed as previously reported by Lesma and colleagues18Lesma E Grande V Lesma E Carelli S Brancaccio D Canevini MP Alfano RM Coggi G Di Giulio AM Gorio A Isolation and growth of smooth muscle-like cells derived from tuberous sclerosis complex-2 human renal angiomyolipoma: EGF is the required growth factor.Am J Pathol. 2005; 167: 1093-1103Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar and repeated at least twice. The panel of microsatellite markers near the TSC2 locus on chromosome 16p13.3 consisted of D16S525, D16S3024, D16S3394, D16S291, D16S664, and D16S663. The 5′ sense primers were labeled with 6-FAM fluorescent dyes (M-Medical, Cornaredo, Italy). The primer sequences were obtained from the Genome Database (http://www.ncbi.nlm.nih.gov/). Microsatellite orientation and position were obtained from UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway). LOH was analyzed as previously described in Lesma and colleagues.18Lesma E Grande V Lesma E Carelli S Brancaccio D Canevini MP Alfano RM Coggi G Di Giulio AM Gorio A Isolation and growth of smooth muscle-like cells derived from tuberous sclerosis complex-2 human renal angiomyolipoma: EGF is the required growth factor.Am J Pathol. 2005; 167: 1093-1103Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar The methylation status of the TSC2 and ZFX promoter regions was analyzed by PCR using as template the genomic DNA previously digested with the methylation-sensitive enzyme HpaII and with its isoschizomer MspI, insensitive to methylation (New England Biolabs, Beverly, MA). The primers used for amplifications spanned several methylation-sensitive restriction sites (seven for TSC2 gene) in the 5′ of the tested genes. The UCSC Browser (http://genome.ucsc.edu/cgi-bin/hgGateway) and the CpG island searcher (http://cpgislands.usc.edu/) were consulted to identify TSC2 promoter region and CpG island. The primers used to TSC2 promoter analysis were sense 5′-atgccgctaccggaagtgc-3′ (chr16: 2037,961-2037,979, UCSC Genome Browser) and antisense 5′-tggtctagagagctctctact-3′ (chr16:2038,391-2038,371, UCSC Genome Browser). After denaturation at 94°C for 5 minutes, 35 cycles of PCR were done; each cycle consisted of 45 seconds at 95°C, 45 seconds at 54°C, and 45 seconds at 72°C. ZFX primers and methods are fully described in Gilbert and Sharp.22Gilbert SL Sharp PA Promoter-specific hypoacetylation of X-inactivated genes.Proc Natl Acad Sci USA. 1999; 96: 13825-13830Crossref PubMed Scopus (81) Google Scholar To quantify the methylation levels of TSC2 promoter, pyrosequencing technology was used to analyze the same region studied by methylation-sensitive digestion assay. The bisulphite conversion of genomic DNA (1 μg) was obtained using EZ DNA methylation kit (Zymo Research, Orange, CA). After bisulphite treatment, PCR was performed in a final volume of 50 μl with 2.5 U of Promega Go-Taq Hot Start Polymerase (Promega). The primer sequences for modified sequences are: forward primer 5′-ttygttagagggyggtatagaat-3′ (chr16: 2037,899-2037,921, UCSC Genome Browser) and biotinylated reverse primer 5′-acactacraaatccrcctctc-3′ (chr16: 2038,141-2038,121, UCSC Genome Browser). The PCR conditions were: step 1: 95°C, 2 minutes; step 2: 95°C, 30 seconds; step 3: 61.2°C decrease 0.5°C per cycle, 30 seconds; step 4: 72°C, 30 seconds; step 5: repeat steps 2 to 4 14 more times; step 6: 95°C, 30 seconds; step 7: 54°C, 30 seconds; step 8: 72°C, 30 seconds; step 9: repeat steps 6 to 8 31 more times; step 10: 72°C, 5 minutes. Forty μl of PCR product were used for pyrosequencing assay using the sequencing primers 5′-atyggaagtgygggt-3′ (chr16: 2037,969-2037,983, UCSC Genome Browser). Pyrosequencing reactions were performed in the PSQ HS 96 system (Biotage, Uppsala, Sweden) by using Pyro Gold reagent kits (Biotage). Methylation was quantified using Pyro Q-CpG Software (Biotage), which calculates the ratio of converted Cs (Ts) to unconverted Cs at each CpG and expresses this as a percentage of methylation. To assess the methylation pattern in normal condition we analyzed different normal DNA from peripheral blood lymphocytes. The proliferation growth factor dependence of the smooth muscle-like cells and VSMCs were assayed in the presence or absence of EGF (10 ng/ml) or by replacing EGF with insulin-like growth factor 1 (IGF-1) (50 ng/ml), by counting the cells after 4, 7, 17, 21, and 25 days of culture in a Neubauer chamber. Anti-EGFR antibody (clone 225; Calbiochem, Darmstadt, Germany), and IGF-1R (clone αIR3, Calbiochem) were added at a concentration of 5 μg/ml to the complete medium, and cell proliferations were evaluated after 4, 7, 17, 21, and 25 days of culture. Each data point of the proliferation experiments is the mean of four independent experiments. Trichostatin A (Sigma-Aldrich) was incubated for 72 hours at the concentration of 3.3 mmol/L. 5-Azacytidine (Sigma-Aldrich) was incubated for 96 hours at the concentration of 1 or 10 μmol/L. In the Western blotting experiments to study S6 and Erk phosphorylation, cells were incubated with rapamycin (1 ng/ml) or anti-EGFR antibody (5 μg/ml) for 24 or 48 hours. Cells were incubated for 2 hours with IGF-1 (50 ng/ml) with or without LY294002 (20 μmol/L) (Sigma). The action of rapamycin (Rapamune-Sirulimus; Wyeth Europa, Maidenhead, UK) was evaluated by adding 1 ng/ml and 5 ng/ml to the TSC2−/meth cells at plating time and 3 hours after plating, and measuring cell proliferation after 5, 8, and 10 days or evaluating the apoptotic effect by terminal dUTP nick-end labeling (TUNEL) analysis after a 10-day incubation. For TUNEL analysis VSMCs were incubated with staurosporin (100 mmol/L) for 1 hour 30 minutes. Apoptotic cells were examined by TUNEL analysis using an in situ cell death DeadEnd colorimetric TUNEL system (Promega) following the manufacturer's instruction. Briefly, cells were fixed for 25 minutes in 4% paraformaldehyde, washed twice in PBS for 5 minutes, permeabilized in 0.2% Triton X-100 for 5 minutes, and washed twice in PBS for 5 minutes. Slides were incubated with rTdT reaction mix for 1 hour at 37°C, then endogenous peroxidases were blocked with 0.3% hydrogen peroxide for 5 minutes at room temperature. After incubation with streptavidin horseradish peroxidase, apoptotic cells were localized using 3′,3′-diaminobenzidine tetrahydrochloride. The number of TUNEL-positive cells was counted and the data presented are mean of the percentage of TUNEL-positive cells. The data are expressed as mean values ± SEM, and were statistically analyzed using Student's t-test; significance is indicated for P values of *<0.05, **<0.01, and *** G) was detected by DNA sequencing in patient blood and angiomyolipoma (data not shown) and in angiomyolipoma-derived ASM cells (Figure 2A). The LOH of these ASM cells was tested by means of PCR amplification, using a panel of microsatellite markers near the TSC2 locus on chromosome 16p13.3, but we failed to detect LOH (data not shown). On the other hand, these newly isolated ASM cells lacked tuberin as immunofluorescence and Western blotting failed to reveal any positive reactivity to specific antibodies. This negative result can be compared with what observed in TSC2−/− ASM cells, whereas VSMCs and A549 cells, used as control, expressed tuberin, as expected (Figure 2B). Differently, hamartin was present at comparable levels in all tested cells and β-actin was evaluated as protein control (Figure 2B). The absence of LOH and the lack of tuberin expression in ASM cells led us into assessing whether the Knudson second hits could be epigenetic. We analyzed the DNA methylation of the CpG island within the TSC2 promoter region, using methylation-sensitive digestion with HpaII restriction enzyme. Methylation (presence of PCR amplification after HpaII digestion) of the promoter in ASM cells was found while normal VSMCs cells were unmethylated (absence of PCR amplification after HpaII digestion) (Figure 3A). To control the enzyme cleavage efficiency we chose two different approaches. In the first case, it was used the digestion with MspI, an isoschizomer of HpaII that, unlike HpaII, can cleave the sequence when the internal C of the restriction site CCGG is methylated. In the second case the cleavage efficiency was tested with the amplification of ZFX promoter, which is always unmethylated. As expected, we found a complete DNA digestion in both controls, and this resulted in no PCR amplification in all samples (Figure 3A). In addition we observed the TSC2 promoter methylation in the original AML tissue, although there was a reduced extent probably because of the heterogeneity of AML (Figure 3A). To confirm the above reported qualitative methylation data we performed the pyrosequencing quantitative assay on the same promoter regions analyzed with methylation-sensitive digestion. This method allows us to reveal quantitatively the methylation of all CpGs position. In normal samples TSC2 resulted demethylated in all CpGs analyzed (Figure 3B). In the TSC2−/meth ASM cells, consistently with our previous results, we found a promoter methylation pattern indicating an epigenetic silencing. The low methylation levels (29%, 6%, 6%) obtained with the pyrosequencing analysis were indicative of methylation of only one allele. In the isolated ASM cells the epigenetic silencing of TSC2 was confirmed by transcriptional reactivation with consequent tuberin expression after treatment with the histone deacetylase inhibitor trichostatin A for 72 hours or with the DNA methylase inhibitor 5-azacytidine for 96 hours (Figure 3C). Trichostatin A and 5-azacytidine are chromatin-remodeling agents able to reactivate genes epigenetically silenced.23Sirchia SM Ferguson AT Sironi E Subramanyan S Orlandi R Sukumar S Sacchi N Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor β2 promoter in breast cancer cells.Oncogene. 2000; 19: 1556-1563Crossref PubMed Scopus (193) Google Scholar, 24Sirchia SM Ren M Pili R Sironi E Somenzi G Ghidoni R Toma S Nicolò G Sacchi N Endogenous reactivation of the RAR beta2 tumor suppressor gene epigenetically silenced in breast cancer.Cancer Res. 2002; 62: 2455-2461PubMed Google Scholar, 25Sirchia SM Ramoscelli L Grati FR Barbera F Cordini D Rossella F Porta G Lesma E Ruggeri A Radice P Simoni G Miozzo M Loss of the inactive X chromosome and replication of the active X in BRCA1 effective and wild-type breast cancer cells.Cancer Res. 2005; 65: 2139-2146Crossref PubMed Scopus (78) Google Scholar After the demonstration of the DNA methylation of the CpG island, the angiomyolipoma-derived ASM cells were named TSC2−/meth ASM cells, and the
Referência(s)