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• W1989398321 abstract "TMS1/ASC is an intracellular signaling molecule with proposed roles in the regulation of apoptosis, nuclear factor-κB activation, and cytokine maturation. Previous studies have shown that TMS1/ASC is silenced by epigenetic means in human breast tumors. In this study, we examined methylation and expression of TMS1/ASC in glioblastoma multiforme (GBM). Whereas normal brain tissue was unmethylated at the TMS1 locus and expressed TMS1 message, 11 of 23 human GBM cell lines exhibited reduced or absent expression of TMS1 that was associated with aberrant methylation of a CpG island in the promoter of the TMS1 gene. Quantitative analysis showed that there was an inverse correlation between the degree of methylation and level of TMS1 expression. Treatment of GBM cell lines lacking TMS1 expression with the methyltransferase inhibitor 5-aza-2′deoxycytidine resulted in partial demethylation and re-expression of TMS1. Analysis of primary tissues indicated that the TMS1 gene is unmethylated and expressed in normal brain, where its expression is restricted to astrocytes. In contrast, TMS1 was aberrantly methylated in 43% (10 of 23) primary GBM specimens. Tumors that exhibited aberrant methylation of TMS1 generally expressed reduced or absent expression of TMS1 as compared to unmethylated cases. Methylation of TMS1 was not associated with patient age, gender, or treatment status. Although the relationship did not reach statistical significance, there was a trend toward increased overall survival for patients with unmethylated tumors. For one patient, disease progression from astrocytic astrocytoma (World Health Organization grade III) to GBM (World Health Organization grade IV) was associated with selective expansion of TMS1-negative cells. The data suggest a role for the epigenetic silencing of TMS1 in the pathogenesis of human GBM. Methylation of TMS1 may prove to be a useful prognostic marker and/or predictor of patient survival and tumor malignancy. TMS1/ASC is an intracellular signaling molecule with proposed roles in the regulation of apoptosis, nuclear factor-κB activation, and cytokine maturation. Previous studies have shown that TMS1/ASC is silenced by epigenetic means in human breast tumors. In this study, we examined methylation and expression of TMS1/ASC in glioblastoma multiforme (GBM). Whereas normal brain tissue was unmethylated at the TMS1 locus and expressed TMS1 message, 11 of 23 human GBM cell lines exhibited reduced or absent expression of TMS1 that was associated with aberrant methylation of a CpG island in the promoter of the TMS1 gene. Quantitative analysis showed that there was an inverse correlation between the degree of methylation and level of TMS1 expression. Treatment of GBM cell lines lacking TMS1 expression with the methyltransferase inhibitor 5-aza-2′deoxycytidine resulted in partial demethylation and re-expression of TMS1. Analysis of primary tissues indicated that the TMS1 gene is unmethylated and expressed in normal brain, where its expression is restricted to astrocytes. In contrast, TMS1 was aberrantly methylated in 43% (10 of 23) primary GBM specimens. Tumors that exhibited aberrant methylation of TMS1 generally expressed reduced or absent expression of TMS1 as compared to unmethylated cases. Methylation of TMS1 was not associated with patient age, gender, or treatment status. Although the relationship did not reach statistical significance, there was a trend toward increased overall survival for patients with unmethylated tumors. For one patient, disease progression from astrocytic astrocytoma (World Health Organization grade III) to GBM (World Health Organization grade IV) was associated with selective expansion of TMS1-negative cells. The data suggest a role for the epigenetic silencing of TMS1 in the pathogenesis of human GBM. Methylation of TMS1 may prove to be a useful prognostic marker and/or predictor of patient survival and tumor malignancy. Glioblastoma multiforme (GBM) is the most prevalent and lethal of human gliomas and accounts for 50 to 60% of primary brain tumors. Despite intensive research throughout the past 2 decades, there has been little progress in the treatment of GBM; the median survival after diagnosis remains at 1 year, although patient outcome can vary considerably from a few months to 5% of patients surviving more than 2 years.1Fine HA The basis for current treatment recommendations for malignant gliomas.J Neurooncol. 1994; 20: 111-120Crossref PubMed Scopus (120) Google Scholar This clinical variability is likely because GBMs exhibit considerable heterogeneity at both the genetic and biological levels. GBMs can be classified into two groups based on their clinical phenotype: primary, which occur de novo (ie, without having progressed from a less malignant precursor) in older patients;2Kleihues P Burger PC Scheithauer BW The new WHO classification of brain tumours.Brain Pathol. 1993; 3: 255-268Crossref PubMed Scopus (1466) Google Scholar and secondary, which are much less frequent (∼10% of cases) and tend to develop from a low-grade or anaplastic astrocytomas in younger patients.2Kleihues P Burger PC Scheithauer BW The new WHO classification of brain tumours.Brain Pathol. 1993; 3: 255-268Crossref PubMed Scopus (1466) Google Scholar, 3Daumas-Duport C Scheithauer B O'Fallon J Kelly P Grading of astrocytomas. A simple and reproducible method.Cancer. 1988; 62: 2152-2165Crossref PubMed Scopus (773) Google Scholar Although these two groups are histologically indistinguishable, they appear to have arisen along distinct genetic pathways.4Hunter SB Brat DJ Olson JJ Von Deimling A Zhou W Van Meir EG Alterations in molecular pathways of diffusely infiltrating glial neoplasms: application to tumor classification and anti-tumor therapy.Int J Oncol. 2003; 23: 857-869PubMed Google Scholar Typically, primary GBMs show frequent amplification of EGFR, while only rarely harboring TP53 mutations, whereas secondary GBMs show frequent mutations in TP53 in the absence of EGFR alterations. These relatively few genetic and clinical distinctions provide only limited information beyond the traditional histological classification of GBM and thus far have not significantly improved our ability to predict patient prognosis or response to therapy. Thus, the impetus remains to seek out additional genetic and epigenetic alterations that might provide insight into mechanisms that contribute to the malignancy of GBMs. Aberrant methylation of CpG island-associated genes is a frequent epigenetic alteration associated with the inactivation of tumor suppressor and other genes in human cancers.5Baylin SB Herman JG DNA hypermethylation in tumorigenesis: epigenetics joins genetics.Trends Genet. 2000; 16: 168-174Abstract Full Text Full Text PDF PubMed Scopus (1398) Google Scholar, 6Jones PA Baylin SB The fundamental role of epigenetic events in cancer.Nat Rev Genet. 2002; 3: 415-428Crossref PubMed Google Scholar, 7Costello JF Plass C Methylation matters.J Med Genet. 2001; 38: 285-303Crossref PubMed Scopus (468) Google Scholar Approximately one-half of human genes contain CpG islands; short stretches of CpG-dense DNA typically associated with the 5′ ends of genes.8Bird AP CpG-rich islands and the function of DNA methylation.Nature. 1986; 321: 209-213Crossref PubMed Scopus (2998) Google Scholar Unmethylated in normal tissues, these regions can become methylated de novo in cancer cells. This change is accompanied by alterations in histone modification and chromatin conformation rendering the CpG island and its embedded promoter transcriptionally inert.9Bird AP Wolffe AP Methylation-induced repression—belts, braces, and chromatin.Cell. 1999; 99: 451-454Abstract Full Text Full Text PDF PubMed Scopus (1545) Google Scholar In human gliomas, such epigenetic mechanisms have been implicated in the silencing of several key regulators of the cell cycle (RB, p16INK4A, p73), DNA repair (O6MGMT), apoptosis (DAP kinase), angiogenesis (THBS1), and invasion (TIMP3).10Costello JF DNA methylation in brain development and gliomagenesis.Front Biosci. 2003; 8: s175-s184Crossref PubMed Scopus (39) Google Scholar CpG island methylation is frequent in low-grade gliomas, preceding many of the aforementioned genetic alterations, and the number of events increase with tumor progression.11Costello JF Plass C Cavenee WK Aberrant methylation of genes in low-grade astrocytomas.Brain Tumor Pathol. 2000; 17: 49-56Crossref PubMed Scopus (55) Google Scholar Indeed, methylation of the promoter of the DNA repair gene O6MGMT in gliomas has shown promise as an independent predictor of response to treatment with alkylating agents, and of disease-free survival.12Esteller M Garcia-Foncillas J Andion E Goodman SN Hidalgo OF Vanaclocha V Baylin SB Herman JG Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents.N Engl J Med. 2000; 343: 1350-1354Crossref PubMed Scopus (1829) Google Scholar, 13Balana C Ramirez JL Taron M Roussos Y Ariza A Ballester R Sarries C Mendez P Sanchez JJ Rosell R O6-methyl-guanine-DNA methyltransferase methylation in serum and tumor DNA predicts response to 1,3-bis(2-chloroethyl)-1-nitrosourea but not to temozolamide plus cisplatin in glioblastoma multiforme.Clin Cancer Res. 2003; 9: 1461-1468PubMed Google Scholar, 14Komine C Watanabe T Katayama Y Yoshino A Yokoyama T Fukushima T Promoter hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase is an independent predictor of shortened progression free survival in patients with low-grade diffuse astrocytomas.Brain Pathol. 2003; 13: 176-184Crossref PubMed Scopus (127) Google Scholar Thus aberrant methylation events have become critical to our understanding of the initiation and progression of human brain malignancies and are showing promise as prognostic tools. Recently, we identified a novel CpG island-associated gene, TMS1 (for target of methylation-associated silencing), that is aberrantly methylated and silenced in a significant proportion of human breast cancers.15Conway KE McConnell BB Bowring CE Donald CD Warren ST Vertino PM TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.Cancer Res. 2000; 60: 6236-6242PubMed Google Scholar Also known as ASC,16Masumoto J Taniguchi S Ayukawa K Sarvotham H Kishino T Niikawa N Hidaka E Katsuyama T Higuchi T Sagara J ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.J Biol Chem. 1999; 274: 33835-33838Crossref PubMed Scopus (427) Google Scholar TMS1 encodes a bipartite adaptor molecule composed of an N-terminal Pyrin domain and a C-terminal CARD domain, and functions as a mediator of intracellular signaling from apoptotic and inflammatory stimuli.17McConnell BB Vertino PM TMS1/ASC: the cancer connection.Apoptosis. 2004; 9: 5-25Crossref PubMed Scopus (83) Google Scholar In previous studies, we showed that overexpression of TMS1 induces apoptosis and inhibits the growth of breast cancer cells, consistent with a putative tumor suppressor role.15Conway KE McConnell BB Bowring CE Donald CD Warren ST Vertino PM TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.Cancer Res. 2000; 60: 6236-6242PubMed Google Scholar Recent studies showing that TMS1 modulates the activity of caspase-1 and can block the downstream activation of nuclear factor-κB suggest that methylation-mediated silencing of TMS1 could promote tumorigenesis by allowing cells to bypass apoptosis, to evade a local immune response, and by allowing nuclear factor-κB-dependent survival signals to go unchecked.18Srinivasula SM Poyet JL Razmara M Datta P Zhang Z Alnemri ES The PYRIN-CARD protein ASC is an activating adaptor for caspase-1.J Biol Chem. 2002; 277: 21119-21122Crossref PubMed Scopus (439) Google Scholar, 19Martinon F Burns K Tschopp J The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.Mol Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4100) Google Scholar, 20Stehlik C Lee SH Dorfleutner A Stassinopoulos A Sagara J Reed JC Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation.J Immunol. 2003; 171: 6154-6163PubMed Google Scholar, 21Stehlik C Fiorentino L Dorfleutner A Bruey JM Ariza EM Sagara J Reed JC The PAAD/PYRIN-family protein ASC is a dual regulator of a conserved step in nuclear factor kappaB activation pathways.J Exp Med. 2002; 196: 1605-1615Crossref PubMed Scopus (141) Google Scholar The goal of the present study was to determine whether methylation-mediated silencing of TMS1 plays a role in the pathogenesis of human gliomas. We find that TMS1 is aberrantly methylated in a significant proportion of GBM cell lines and primary tumors, and that methylation of the TMS1 gene is correlated with the tumor-specific down-regulation of TMS1 in primary GBMs. Primary GBM tumor samples used in this study were obtained from 25 patients who underwent neurosurgical resection at Emory University Hospital between 1996 and 2001. Patients ranged in age from 27 to 81 years. Tumor specimens were frozen at −80°C immediately after resection. All cases were reviewed by a single pathologist (D.B.) for histological confirmation of GBM (World Health Organization grade IV astrocytoma) before being included in this study. For DNA isolations, frozen tumor specimens were macrodissected at the time of histological verification. For comparison, brain tissue from patients without cancer (n = 5) were obtained at the time of autopsy, frozen at −80°C, and dissected into white and gray matter before DNA and RNA isolation. Human GBM cell lines were maintained as previously described.22Ishii N Maier D Merlo A Tada M Sawamura Y Diserens AC Van Meir EG Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines.Brain Pathol. 1999; 9: 469-479Crossref PubMed Scopus (482) Google Scholar For 5-aza-2′-deoxycytidine experiments, U251 MG and LN 229 cells were seeded at 106 cells per 75-cm2 flask, allowed to attach overnight, and treated with 0.5 μmol/L 5-aza-2′-deoxycytidine. Cells were harvested after 48 hours treatment, and DNA and RNA were extracted. DNA and RNA from all GBM cell lines and primary tumors were extracted using the DNeasy tissue and RNeasy kits (Qiagen, Valencia, CA) according to the manufacturer's protocol. DNA was subjected to bisulfite modification and methylation-specific PCR as previously described.15Conway KE McConnell BB Bowring CE Donald CD Warren ST Vertino PM TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.Cancer Res. 2000; 60: 6236-6242PubMed Google Scholar, 23Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5208) Google Scholar Approximately 50 ng of bisulfite-modified DNA was amplified by PCR with the following reaction conditions: 67 mmol/L Tris-HCl (pH 8.8), 16.6 mmol/L NH4SO4, 6.7 μmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L β-mercaptoethanol, 4.7 mmol/L MgCl2, and 1 μmol/L of each primer in a 25-μl reaction. A hot start was performed (95°C, 5 minutes), followed by the addition of 0.5 U of Taq polymerase (Life Technologies, Inc., Grand Island, NY) and 35 cycles of PCR (95°C, 30 seconds; 58°C, 30 seconds; and 72°C, 30 seconds). Reaction products were separated by electrophoresis on a 6% polyacrylamide/Tris-borate-EDTA gel, stained with ethidium bromide, and photographed. Primers were designed such that they overlay three potential methylation (ie, CpG) sites. Primers used were 5′-GGT TGT AGT GGG GTG AGT GGT-3′ and 5′-CAA AAC ATC CAT AAA CAA CAA CAC A-3′ for unmethylated reaction, and 5′-TTG TAG CGG GGT GAG CGG C-3′ and 5′-AAC GTC CAT AAA CAA CAA CGC G-3′ for the methylated reaction. COBRA was performed essentially as described.24Xiong Z Laird PW COBRA: a sensitive and quantitative DNA methylation assay.Nucleic Acids Res. 1997; 25: 2532-2534Crossref PubMed Scopus (1037) Google Scholar Briefly, DNA from GBM cell lines was bisulfite modified and amplified by PCR using the same conditions described above for methylation-specific PCR (MSP) except, in this case, primers were designed to avoid potential methylation sites (CpGs) so that unmethylated and methylated sequences would be amplified equally. DNA from the amplification reaction was purified using a Wizard PCR Clean-Up kit (Promega, Madison, WI) and digested with 10 U of ClaI for 4 hours at 37°C. Reaction products were separated by electrophoresis on a 6% polyacrylamide/Tris-borate-EDTA gel, stained with ethidium bromide, and digitally photographed. If TMS1 is unmethylated, the 557-bp PCR product remains intact. If methylated, this band is digested to 453- and 104-bp products. Percent methylation was determined as the ratio of intensities of the intact band to that of the digested bands using Imagequant v5.2 software (Molecular Dynamics, Sunnyvale, CA). DNA from breast cancer cell lines with well-characterized methylation patterns at the TMS1 locus [MCF7 (unmethylated), T47D (∼50% methylated), and MDA MB231 (100% methylated)]15Conway KE McConnell BB Bowring CE Donald CD Warren ST Vertino PM TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.Cancer Res. 2000; 60: 6236-6242PubMed Google Scholar, 25Levine JJ Stimson-Crider KM Vertino PM Effects of methylation on expression of TMS1/ASC in human breast cancer cells.Oncogene. 2003; 22: 3475-3488Crossref PubMed Scopus (55) Google Scholar were included to verify the quantitative nature of the technique. For bisulfite sequencing, the bisulfite modification and PCR reaction were performed as described above again using primers that avoid CpG sites. In this case, the resulting amplicon was subcloned using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA). Eight to twelve individual subclones were isolated per PCR reaction and sequenced by the University of Michigan Sequencing Facility. Primers used for COBRA and bisulfite sequencing were: 5′-TTG GTG TAA GTT TAG AGA TAA GT-3′ and 5′-ACC ATC TCC TAC AAA CCC ATA-3′. Total RNA (6 μg) was pretreated with DNase I (Life Technologies, Inc.) and reverse-transcribed using random hexamer primers and MMLV reverse transcriptase (Life Technologies, Inc.). One-thirtieth of the reverse transcription reaction (200 ng of starting RNA) was used in a PCR reaction. The PCR reaction conditions were: 67 mmol/L Tris-HCl (pH 8.8), 16.6 mmol/L NH4SO4, 6.7 μmol/L EDTA, 10 mmol/L β-mercaptoethanol, 4.7 mmol/L MgCl2, 10% dimethyl sulfoxide, and 400 nmol/L of each primer in a 25-μl reaction. A hot start was performed (5 minutes, 95°C), followed by the addition of 0.5 U of Taq polymerase (Life Technologies, Inc.) and 35 cycles of PCR (95°C, 30 seconds; 50 to 55°C, 60 seconds; and 72°C, 60 seconds). TMS1 primers (5′-TGG GCC TGC AGG AGA TG-3′ and 5′-ATT TGG TGG GAT TGC CAG-3′) were used at an annealing temperature of 50°C. β-Actin primers (5′-CCT TCC TGG GCA TGG AGT CCT G-3′ and 5′-GGA GCA ATG ATC TTG ATC TTC-3′) were used at an annealing temperature of 55°C. Reaction products were separated by electrophoresis on a 6% polyacrylamide/Tris-borate-EDTA gel, stained with ethidium bromide, and photographed. For real-time analysis, 1 μl of a 50-fold dilution of the reverse transcriptase reaction (10 ng of starting RNA) was amplified per reaction using the iQ SYBR Green Supermix kit (Bio-Rad, Hercules, CA) and the MyIQ real-time detection system. Reaction conditions included a hot start (3 minutes, 95°C), followed by 50 cycles of 95°C, 10 seconds and 55°C, 60 seconds). Melt curve analysis was performed to ensure a single product species. Parallel reactions were performed using primers to 18S rRNA as an internal control. Starting quantities were calculated by comparison to a common standard curve of MCF10A cell cDNA that was included in each run. Primers for real-time PCR analysis were for TMS1, 5′-TCC AGC AGC CAC TCA ACG-3′ and 5′-GCA CTT TAT AGA CCA GCA-′; and for 18S, 5′-GAG GGA GCC TGA GAA ACG G-3′ and 5′-GTC GGG AGT GGG TAA TTT GC-3′. An affinity-purified rabbit polyclonal antibody was developed against the C-terminus of human TMS1/ASC. A peptide corresponding to amino acids 182 to 195 of human TMS1/ASC was synthesized by the Microchemical Core Facility of Emory University, coupled to KLH via an N-terminal cysteine, and used to immunize New Zealand White rabbits (Cocalico Biologicals, Lancaster, PA). Crude antiserum (EU107) was affinity purified by selection over a column consisting of the same peptide coupled to a solid support (Sulfo-link; Pierce, Rockford, IL) and elution with 100 mmol/L glycine, pH 2.0. Fractions were neutralized with Tris-base, dialyzed against PBS, and analyzed for specificity by Western blot analysis using a partially purified recombinant GST-TMS1 fusion protein. Immunohistochemistry was performed on archived formalin-fixed and paraffin-embedded human GBM resection specimens. Tissue sections used for routine histological examination and for immunohistochemistry contained both regions that were diagnostic of GBM as well as adjacent nonneoplastic brain. For immunohistochemical studies, sections were deparaffinized and subjected to antigen retrieval by steaming (20 minutes, 80°C). Slides were then incubated at room temperature with a 1:320 dilution of affinity-purified anti-human TMS1 antibody (EU107). Negative controls included normal saline and irrelevant IgG substitution for the primary antibody. Antibodies were detected using the avidin-biotin complex method, using diaminobenzidine as the chromogen (Envision System; DAKO, Carpinteria, CA). Sections were counterstained with hematoxylin. Staining for human CD3 and CD68 was performed using a CD3 polyclonal antibody (1:80 dilution, DAKO) and the KP-1 monoclonal antibody (1:320 dilution, DAKO), respectively. To study the potential role of TMS silencing in pathogenesis of GBM, we first established the expression and the methylation status of the TMS1 gene in normal human brain tissue. Brain tissue derived from cancer-free patients at autopsy was analyzed for the methylation of the TMS1 CpG island by MSP and for TMS1 expression by reverse transcriptase (RT)-PCR (Figure 1). TMS1 was found to be expressed in normal cerebral cortex and white matter (n = 2), brain and the TMS1 CpG island was predominately unmethylated in five of five normal brain specimens. We next examined methylation and expression of TMS1 in 22 GBM cell lines. All but one of the cell lines examined showed some degree of aberrant methylation of the TMS1 CpG island (Figure 2A). One cell line was devoid of methylation at the TMS1 locus; 13 of 22 (59%) GBM cell lines examined were partially methylated and 8 of 22 (36%) cell lines showed only methylated DNA at the TMS1 CpG island. Quantitation of the degree of methylation in a subset of GBM cell lines using a COBRA24Xiong Z Laird PW COBRA: a sensitive and quantitative DNA methylation assay.Nucleic Acids Res. 1997; 25: 2532-2534Crossref PubMed Scopus (1037) Google Scholar assay confirmed that methylation was indeed extensive in many cases (Table 1). Eleven of twenty-three cell lines examined showed reduced or absent expression of TMS1 by RT-PCR (Figure 2B). Cell lines that were predominantly or completely methylated at the TMS1 promoter expressed little or no TMS1 message, whereas cell lines that were completely unmethylated or exhibited only low levels of methylation expressed TMS1 (Table 1). Quantitative analysis of percent methylation (by COBRA assay) and TMS1 expression by real-time PCR confirmed an inverse correlation between the degree of methylation and the level of gene expression in the GBM cell lines (Table 1 and Figure 2C).Figure 2Methylation and expression of TMS1 in glioblastoma cell lines. A: DNA from the indicated GBM cell line was bisulfite modified and analyzed for methylation of the TMS1 CpG island by methylation-specific PCR analysis. Parallel amplification reactions were performed using primers specific to the unmethylated (U) or methylated (M) DNA. B: Total RNA isolated from 20 GBM cell lines was reverse transcribed (+RT) and amplified with primers specific for the TMS1 transcript (top) or for the human β-actin transcript (bottom). Control reactions (−RT) were amplified under the same conditions for both TMS1 and human β-actin. C: DNA methylation levels were quantified using a COBRA assay and TMS1 expression by reverse transcription and real-time PCR analysis. The relative expression of TMS1 was determined by comparison to a common human cell cDNA standard included in each run. Shown are the relative levels of TMS1 expression normalized to the levels of 18S rRNA. Each data point represents a different GBM cell line. Dotted line, linear regression; R2 = 0.76.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Methylation and Expression of TMS1 in Human Glioblastoma Cell LinesCell lineCpG island methylation*Methylation of the TMS1 gene was analyzed by methylation-specific PCR. U, completely unmethylated; U/M, both unmethylated and methylated copies present; M, completely methylated.% Methylation (COBRA)†The percent methylation was determined by COBRA analysis.mRNA expression‡Expression of TMS1 was determined by RT-PCR. Expression was scored as strong (++); weak (+/−); or undetectable (−−) after 35 cycles of PCR.LN 464U0++LN 827U/Mnd¶nd, not determined.++LN 71U/Mnd++LN 215U/M71++LN 401U/Mnd++LN 308U/Mnd++LN 992U/M24++U 87 MGU/M80++LN 235U/Mnd+/−LN 405U/Mnd−LN 427U/M100−LN 428Mnd+/−LN 18Mnd+/−U 138 MGM89−LN 229Mnd−U 343 MGM70−U 373 MGM80−U 251 MGMnd−U 118 MGM95−MCF7§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.U§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.0++§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.MB231§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.M§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.95−§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.T47D§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.U/M§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.51+/−§Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from references 15 and 25. DNA from these cell lines were used as controls for the COBRA analysis.* Methylation of the TMS1 gene was analyzed by methylation-specific PCR. U, completely unmethylated; U/M, both unmethylated and methylated copies present; M, completely methylated.† The percent methylation was determined by COBRA analysis.‡ Expression of TMS1 was determined by RT-PCR. Expression was scored as strong (++); weak (+/−); or undetectable (−−) after 35 cycles of PCR.§ Methylation-specific PCR and expression data for the breast cancer cell lines MCF7, MDA MB231, and T47D is derived from 15Conway KE McConnell BB Bowring CE Donald CD Warren ST Vertino PM TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers.Cancer Res. 2000; 60: 6236-6242PubMed Google Scholar, 25Levine JJ Stimson-Crider KM Vertino PM Effects of methylation on expression of TMS1/ASC in human breast cancer cells.Oncogene. 2003; 22: 3475-3488Crossref PubMed Scopus (55) Google Scholar. DNA from these cell lines were used as controls for the COBRA analysis.¶ nd, not determined. Open table in a new tab If methylation plays a role in the silencing of TMS1, demethylation of the locus should result in the reactivation of gene expression. Treatment of two GBM cell lines, U251 and LN 229, with the DNA methyltransferase inhibitor 5′-aza-2′-deoxycytidine for 48 hours resulted in the partial demethylation and re-expression of TMS1 (Figure 3). These data indicate that methylation is the only impediment to TMS1 expression and suggest that methylation plays a direct role in the repression of TMS1 in GBM cell lines. To determine whether aberrant methylation of the TMS1 in GBM cell lines reflects an epigenetic event occurring in primary GBM, we next examined 23 primary GBMs for TMS1 methylation. As mentioned above, brain ti" @default.
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• W1989398321 title "Aberrant Methylation and Down-Regulation of TMS1/ASC in Human Glioblastoma" @default.
• W1989398321 cites W1242607330 @default.
• W1989398321 cites W1504303645 @default.
• W1989398321 cites W1529674879 @default.
• W1989398321 cites W1969810368 @default.
• W1989398321 cites W1972011430 @default.
• W1989398321 cites W1980442505 @default.
• W1989398321 cites W1990933277 @default.
• W1989398321 cites W1994465394 @default.
• W1989398321 cites W1995070143 @default.
• W1989398321 cites W2000580034 @default.
• W1989398321 cites W2014602273 @default.
• W1989398321 cites W2015565066 @default.
• W1989398321 cites W2015660037 @default.
• W1989398321 cites W2020206240 @default.
• W1989398321 cites W2028719556 @default.
• W1989398321 cites W2030780359 @default.
• W1989398321 cites W2049183025 @default.
• W1989398321 cites W2050549183 @default.
• W1989398321 cites W2054823635 @default.
• W1989398321 cites W2063110349 @default.
• W1989398321 cites W2063739042 @default.
• W1989398321 cites W2070177926 @default.
• W1989398321 cites W2076036454 @default.
• W1989398321 cites W2084118479 @default.
• W1989398321 cites W2084654188 @default.
• W1989398321 cites W2085006185 @default.
• W1989398321 cites W2085277801 @default.
• W1989398321 cites W2091658754 @default.
• W1989398321 cites W2095429655 @default.
• W1989398321 cites W2134075860 @default.
• W1989398321 cites W2143244707 @default.
• W1989398321 cites W2147943691 @default.
• W1989398321 cites W2154964951 @default.
• W1989398321 cites W2155140015 @default.
• W1989398321 cites W2166865493 @default.
• W1989398321 cites W2168002000 @default.
• W1989398321 cites W2328946792 @default.
• W1989398321 cites W2409852939 @default.
• W1989398321 cites W4233749076 @default.
• W1989398321 doi "https://doi.org/10.1016/s0002-9440(10)63376-7" @default.
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