scholarly journals Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Jorge Muñoz ◽  
María del Mar Inda ◽  
Paula Lázcoz ◽  
Idoya Zazpe ◽  
Xing Fan ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Promoter Hypermethylation ◽  
Suppressor Genes ◽  
Primary Tumors ◽  
Specific Pcr ◽  
Inactivation Mechanism ◽  
Altered Gene ◽  
Homozygous Deletions

While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent.

scholarly journals Homozygous deletions localize novel tumor suppressor genes in B-cell lymphomas

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Cinta Mestre-Escorihuela ◽  
Fanny Rubio-Moscardo ◽  
Jose A. Richter ◽  
Reiner Siebert ◽  
Joan Climent ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Tumor Suppressor Genes ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Promoter Hypermethylation ◽  
Homozygous Deletion ◽  
Suppressor Genes ◽  
Mantle Cell ◽  
Homozygous Deletions

Abstract Integrative genomic and gene-expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene-expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated 20 homozygous deletions at 7 chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms. Notably, the pattern of genetic and epigenetic inactivation differed among B-NHL subtypes. Thus, the P53-inducible PIG7/LITAF was silenced by homozygous deletion in primary mediastinal B-cell lymphoma and by promoter hypermethylation in germinal center lymphoma, the proapoptotic BIM gene presented homozygous deletion in mantle cell lymphoma and promoter hypermethylation in Burkitt lymphoma, the proapoptotic BH3-only NOXA was mutated and preferentially silenced in diffuse large B-cell lymphoma, and INK4c/P18 was silenced by biallelic mutation in mantle-cell lymphoma. Our microarray strategy has identified novel candidate tumor suppressor genes inactivated by genetic and epigenetic mechanisms that substantially vary among the B-NHL subtypes.

Global Methylation Analysis Reveals Novel Candidate Tumor Suppressor Genes In Natural Killer Cell Lymphomas

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1262-1262
Author(s):  
Can Kucuk ◽  
Xiaozhou Hu ◽  
Bei Jiang ◽  
Philippe Gaulard ◽  
David Klinkebiel ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Nk Cells ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Nk Cell ◽  
Killer Cell ◽  
Promoter Hypermethylation ◽  
Suppressor Genes ◽  
Candidate Tumor Suppressor

Abstract Background Natural killer cell lymphomas (NKCLs) are aggressive neoplasms with poor prognosis. A few candidate tumor suppressor genes were identified with locus specific methylation analysis. However, no genome-wide study has been performed on NKCL cases to identify the spectrum of epigenetically silenced candidate genes contributing to the neoplastic transformation of NK cells. Methods Here, we analyzed genome-wide methylation profile in NKCL cases (n=12) and NK cell lines (n=2) by performing methyl-sensitive cut counting (MSCC). The methylation profile of malignant NK samples (n=14) were compared with IL2 activated normal NK cells (n=3) to identify genes with promoter hypermethylation. HG U133+ II DNA Microarray was performed on 6 NKCL cases, 2 NK cell lines and resting or activated normal NK cells (n=4).q-MSP was performed on NK cell lines (n=7) or NKCL cases (n=8) for selected genes to validate the promoter methylation identified by MSSC. q-RT-PCR was performed on NK cell lines to validate epigenetic silencing. Decitabine treatment and q-RT-PCR was performed on NK cell lines to assess reactivation of genes with promoter methylation. Apoptosis assay was performed by Annexin V-PE staining. Cell survival assay was performed on Asparaginase treated NK cell lines to show the correlation of Asparagine synthetase (ASNS) mRNA expression and sensitivity to the asparaginase treatment. Results We observed significant global promoter hypermethylation in malignant NK samples compared with activated NK cells. Using stringent criteria which included significant correlation between promoter methylation and gene expression, we identified 152 genes showing recurrent promoter hypermethylation and transcriptional repression in NKCL cases compared with normal NK cells. 34.4% (51 of 152) of the silenced genes have been previously shown to be tumor suppressor gene in at least one cancer type. Gene Ontology and pathway analysis of the methylated/downregulated genes showed enrichment of the cell cycle inhibitors, pro-apoptotic genes such as BIM and DAPK1, DNA methylation modifiers such as TET2 and DNMT3A, metabolic pathway genes, transcription factors, negative regulators of JAK/STAT signaling such as SOCS6 and IL12Rb2, WNT pathway and NK-cell activation pathways. Decitabine treatment of NK cell lines reactivated expression of all 10 selected genes. Ectopic expression of BIM in two BIM-null NK cell lines led to increased apoptosis and eventual elimination of BIM-transduced cells. Reintroduction of BIM also sensitized NK cell lines to chemotherapy-induced apoptosis. NK cell lines with ASNS methylation and repression showed increased sensitivity to treatment with L-asparaginase, an agent highly effective in the treatment of NKCLs. Conclusions We identified novel candidate tumor suppressor genes epigenetically silenced in NKCLs which may contribute to the pathogenesis of NKCLs. Reactivation of these silenced genes may be therapeutically effective in NKCL patients. In addition, we showed that epigenetic inactivation of ASNS has an association with response to Asparaginase treatment. Promoter methylation status of ASNS in NKCL patients may be used in the decision of which patients are more likely to respond to asparaginase containing chemotherapeutic regimens. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Consistent DNA Hypermethylation Patterns in Laryngeal Papillomas

2010 ◽  
Vol 1 (2) ◽  
pp. 69-77 ◽  
Author(s):  
Josena K Stephen ◽  
Kang Mei Chen ◽  
Veena Shah ◽  
Vanessa G Schweitzer ◽  
Glendon Gardner ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Methylation Status ◽  
Promoter Hypermethylation ◽  
Aberrant Methylation ◽  
Dna Hypermethylation ◽  
Suppressor Genes ◽  
Multiple Tumor ◽  
Specific Pcr

Abstract Introduction This study examined the contribution of promoter hypermethylation to the pathogenesis of respiratory papillomatosis (RP), including recurrences (RRP) and progression to squamous cell carcinoma (SSC). Materials and methods A retrospective cohort of 25 laryngeal papilloma cases included 21 RRP, two of which progressed to SCC. Aberrant methylation status was determined using the multigene (22 tumor suppressor genes) methylation-specific multiplex ligationdependent probe amplification assay and confirmed using methylation specific PCR. Results Twenty genes had altered DNA methylation in 22 of 25 cases. Aberrant methylation of CDKN2B and TIMP3 was most frequent. Promoter hypermethylation of BRCA2, APC, CDKN2A and CDKN2B was detected in 2 RRP cases with subsequent progression to SCC. Of the 25 cases, 22 were positive for HPV-6, 2 for HPV-11 and 1 for HPV-16 and 33. Conclusion Consistent aberrant methylation of multiple tumor suppressor genes contributes to the pathogenesis of laryngeal papillomas. Persistent aberrant DNA methylation events in 2 RRP cases that progressed to cancer indicate an epigenetic monoclonal progression continuum to SCC.

Inactivation of tumor suppressor genes and deregulation of the c-myc gene in urothelial cancer cell lines

1995 ◽  
Vol 23 (5) ◽  
pp. 293-300 ◽  
Author(s):  
M.-O. Grimm ◽  
B. J�rgens ◽  
W. A. Schulz ◽  
K. Decken ◽  
D. Makri ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Cancer Cell ◽  
Tumor Suppressor Genes ◽  
Urothelial Cancer ◽  
Cancer Cell Lines ◽  
Suppressor Genes ◽  
Myc Gene ◽  
Urothelial Cancer Cell

Strategies to Re-Express Epigenetically-Silenced Tumor Suppressor Genes Converge on the Requirement for Inhibition of the Histone Methyltransferase SUV39H1.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3357-3357
Author(s):  
Asha Lakshmikuttyamma ◽  
Stuart Scott ◽  
David P. Sheridan ◽  
John DeCoteau ◽  
Ron Geyer
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Fold Increase ◽  
Dna Demethylation ◽  
Dna Hypermethylation ◽  
Suppressor Genes ◽  
Promoter Dna Methylation ◽  
Promoter Dna ◽  
E Cadherin

Abstract Gene silencing mediated by aberrant promoter DNA hypermethylation represents a key mechanism by which tumor suppressor gene expression is silenced in cancer and it is associated with multiple repressive histone modifications. Histone H3 lysine 9 (H3K9) methylation is a key repressive chromatin modification with important implications for regulating cell proliferation, differentiation, and gene expression. SUV39H1 is a methyltransferase that catalyzes the addition of trimethyl groups to H3K9. SUV39H1 is associated with regions of hypermethylated CpG islands, with repressive complexes, such as RB/E2F, and with DNA-binding proteins involved in leukemogenesis, such as AML1 and PML-RAR, where its H3K9 trimethylation activity promotes heterochromatin formation and gene silencing. We studied the requirement of SUV39H1 in the epigenetic silencing of heavily methylated tumor suppressor genes p15INK4B and E-cadherin in acute myeloid leukemia (AML). Treatment of AML cell lines AML193, KG1a, and Kasumi with the DNA methyltransferase (DNMT) inhibitor 5-Aza-2’-deoxycytidine (5-Aza-dC) induces p15INK4B and E-cadeherin re-expression in association with dramatic decreases in p15INK4B and E-cadherin promoter DNA methylation and marked reductions in the levels of SUV39H1 and H3K9 trimethylation at these promoters. Interestingly, treatment of these cell lines with SUV39H1 shRNA, or the SUV39H1 inhibitor chaetocin, also induces p15INK4B and E-cadherin re-expression and H3K9 demethylation, without affecting promoter DNA methylation. Thus, re-expression of hypermethylated tumor suppressors requires histone H3K9 demethylation, which can be achieved indirectly by decreasing the amount of SUV39H1 associated with the promoter using 5-Aza-dC, or directly by inhibiting SUV39H1 expression or activity without requiring promoter DNA demethylation. Furthermore, we found that SUV39H1 shRNA or chaetocin in combination with 5-Aza-dC acts synergistically to re-express epigenetically silenced p15INK4B and E-cadherin in AML cell lines. Treatment of primary human AML blasts obtained from two patients with combinations of 5-Aza-C and chaetocin also results in synergistic re-expression of p15INK4B and E-cadherin (2–6 fold increase with 5-Aza-C or chaetocin treatment vs. 11–14 fold increase with co-treatment). Our study has important implications for developing novel epigenetic therapies of relevance to AML as it suggests that the re-expression of tumor suppressor genes silenced by aberrant promoter DNA hypermethylation converges on the requirement for SUV39H1 and H3K9 methylation inhibition but not promoter DNA demethylation. Our finding that SUV39H1 inhibition may function synergistically with DNMT inhibitors to enhance gene reactivation and chromatin changes also highlights the needs for developing more inhibitors of histone methyltransferases and for performing detailed drug interaction studies to identify the best drug combinations for optimal epigenetic therapies.

Antitumor Activity of Cda-2, An Extract from Unine, in a High-Risk Myelodysplastic Syndrome Cells in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5099-5099
Author(s):  
Lin Qiu ◽  
Xiao-dan Wang ◽  
Bing-hong Han ◽  
Zhao-min Zhan ◽  
Zhang Bo-long ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
High Risk ◽  
Tumor Suppressor ◽  
Myelodysplastic Syndrome ◽  
Tumor Suppressor Genes ◽  
Dna Methyltransferase ◽  
Dependent Manner ◽  
Suppressor Genes ◽  
Specific Pcr

Abstract DNA methyltransferase inhibitors (DNMTI), including 5-azacytidine and 5-aza-2′- deoxycytidine, are a new class of epigenetic drug, which exhibit higher response rates in myelodysplastic syndrome (MDS) patients. Cell differentiation agent (CDA-2) is a kind of urine extracts, which contains several DNMTIs. A phase IV clinical trials for MDS showed total response rate is 69.22%. In the present study, we investigated the mechanism of CDA-2 on MDS using high-risk MDS cell line namely MuTz-1. MTT assay results showed that CDA-2 significantly inhibit the cell growth at a dose and time-dependent manner. Flow cytometer anlyasis showed that this growth inhibition was remarkblely associated with cycle arrest in G1-phase, but not associated with apoptosis. In addition, CDA-2 increased the expression of CD11b/CD14, a pair markers representing cell differentiation. we found the spectrum of hypermethylated tumor suppressor genes (TIMP3, CDKN2B, CHFR, CD44, RASSF1, TP73, IGSF4, CDH13 and DAPK) in MuTz-1 cells by Methylation-Specific Multiplex ligation-dependent Probe amplification (MS-MLPA), but the hypermethylation of these genes were remarkable decreased, as well as the expressions of DNA methyltransferase genes DNMT1 and DNMT3B at mRNA and protein level were downregulated in the treatment for 3 days with CDA-2. Also, we used CDA-2 for treatment of three MDS patients, whose several tumor suppressor genes are hypermethylated. After tour weeks of treatment, all the hypermethylation genes were undetected, part of this result was verified by methylation specific PCR (MSP) and bisulphite sequencing. In conclusion, our results demonstrated that CDA-2 may be an effective agent targeting hepermethylated tumor suppressor genes on MDS.

Genome-Wide Methylation Analysis of Primary Mantle Cell Lymphomas Identifies Novel Gene Targets for Epigenetic Drug Therapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 673-673
Author(s):  
Violetta Leshchenko ◽  
Pei-Yu Kuo ◽  
Rita Shaknovich ◽  
Tobias Gellen ◽  
Yvonne Remache ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Gene Promoter ◽  
Fold Increase ◽  
Therapeutic Benefit ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Suppressor Genes ◽  
Epigenetic Drug

Abstract Abstract 673 Mantle Cell Lymphoma (MCL) is an aggressive and incurable malignancy arising from naïve B cells (NBC) in the mantle zone of lymph node follicles. Murine models over-expressing Cyclin D1, the putative oncogene implicated in the majority of MCL, do not fully recapitulate the disease phenotype. We therefore hypothesize that there are additional mechanisms contributing to MCL pathogenesis and undertook an integrative approach by studying genome-wide DNA methylation and gene expression in primary MCL to uncover additional genes and pathways involved in MCL pathogenesis. We therefore compared and contrasted the DNA methylation levels of 14,000 gene promoters in MCL patients and normal tonsillar NBC controls using the HELP (HPA II tiny fragment Enrichment by Ligation mediated PCR) assay. All patient samples were obtained prior to any treatment from peripheral blood or apheresis specimens from newly diagnosed patients with histologically confirmed MCL. We found significant hypo-methylation of gene promoters in the MCL patients as compared to normal NBCs. Integrating genomic methylation data from HELP and gene expression data from Affymetrix U133 arrays, we determined a signature of differentially methylated genes with reciprocal changes in mRNA levels. Using pathway analysis and gene ontogeny, we selected genes for validation by choosing loci that were differentially methylated and fulfilling the following characteristics (i) demonstrating a clear methylation state change (from hypomethylated in normal B cells to methylated in MCL or vice-versa) using a threshold of 0.0 on the log ratio scale, (ii) Genes that function as tumor suppressors and were hypermethylated and suppressed in MCLs in our data (iii) Overexpressed/ hypomethylated genes with existing therapeutic options available or in clinical trial (iv) involved in pathways controlling biological processes with known relevance to MCL i.e. cell cycle control, apoptosis. Our panel included four differentially hypermethylated genes CDKN2B, MLF-1, PCDH8, HOXD8 and four differentially hypomethylated genes CD37, HDAC1, NOTCH1 and CDK5. MassArray Epityper analysis confirmed the presence of differential methylation at the promoter region of these genes, which was consistent between MCL patients and cell lines in all 8 genes studied. Remarkably, PCDH8 and CDKN2B have previously been shown to be silenced by methylation at their gene promoters and transfection of PCDH8 and CDKN2B have been shown to reduce tumor growth in breast cancer and MCL cell line models respectively. Based on these data, we hypothesized that the aberrantly hypermethylated and thereby silenced tumor suppressor genes in MCL could be pharmacologically induced by DNA hypomethylating agents and HDAC inhibitors for therapeutic benefit. We therefore next treated MCL cell lines MINO and Z138 with the DNA methyltransferase inhibitor Decitabine alone and in combination with the HDAC inhibitor SAHA. HELP analysis of MINO and Z138 cells treated with hypomethylating doses of decitabine (0.5uM × 3days) showed widespread reversal of aberrant gene promoter hypermethylation. Hypomethylation in these cell lines was accompanied with 3-7 fold increase in mRNA levels of tumor suppressor genes CDKN2B, MLF-1, PCDH8 and HOXD8. Concurrent treatment with SAHA (1 uM x 1 dose) synergized with Decitabine leading to 5-15 fold increase in mRNA of these tumor suppressor genes in Z138 cells. Importantly, treatment with Decitabine and SAHA as single agents decreased MCL cell viability by 60% and 40% respectively and the combination synergised in anti-MCL cytotoxicity with > 90% decrease in cell viability. In conclusion, our analysis shows prominent aberrant gene promoter methylation patterns in MCL genome and identifies novel differentially methylated and expressed genes in MCL cell lines and primary MCLs. Furthermore, we demonstrate that reversal of aberrant hypermethylated and silenced genes can be targeted for therapeutic benefit using epigenetic drugs in MCL. We are currently modeling our combination epigenetic drug therapy in primary MCL cells in culture and murine xenograft systems. We expect these results to support the development of clinical trials investigating the prospective use of combination epigenetic therapy in MCL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss of Heterozygosity at the Ink4a/ArfLocus Facilitates Abelson Virus Transformation of Pre-B Cells

2000 ◽  
Vol 74 (20) ◽  
pp. 9479-9487 ◽  
Author(s):  
Justin Mostecki ◽  
Anne Halgren ◽  
Arash Radfar ◽  
Zohar Sachs ◽  
James Ravitz ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Suppressor ◽  
Loss Of Heterozygosity ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Single Copy ◽  
Transformation Process ◽  
Suppressor Genes ◽  
Abelson Virus

ABSTRACT In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including theInk4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived fromInk4a/Arf +/− mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.

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