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Results for “DNA Modification Methylases”

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PubMed2006

[New therapeutic approaches for solid tumors: histone deacetylase, methyltransferase and proteasome inhibitors].

Recent results from basic and translational research on tumor genesis and progression establish the basis for future therapeutic approaches. Targeted therapeutics are tailored toward the molecular abnormalities that cause tumor progression and could potentially provide an effective, non-toxic therapeutic approach in a broad range of cancers including melanoma. Cancer is as much a (cyto)genetic disease as it is an epigenetic disease. Indeed, the fate of the cell depends on a delicate balance between expression and repression of genes. The notion that drastic changes in DNA methylation and histone modifications are present in a variety of human tumors has prompted the development and characterization of epigenetic drugs. Inhibitors of histone deacetylases and methyltransferases as well as of the proteasome are covered in this review.

Antineoplastic Agents↗

PubMed2004

Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine.

It remains unclear to what extent drugs targeting transcriptional repressor complexes affect global gene expression in cells derived from target and nontarget human tissues. To address this question, we used genome-wide expression analysis using microarrays to analyze the response of three tumor and one normal epithelial cell line to treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR). Notably, we found that 5-aza-CdR treatment induced a limited number of genes (mean, 0.67%; range, 0.17-1.8% of 25,940 genes screened) in each cell line tested. The majority of the gene expression changes that followed 5-aza-CdR treatment were conserved in tumor and normal cells, including genes that function in cell proliferation, differentiation, immune presentation, and cytokine signaling. In contrast, 5-aza-CdR treatment induced the expression of cancer-testis class tumor antigens only in tumor cell lines. To explain this tissue-specific response, we analyzed the mechanism of transcriptional regulation of the prototype member of this tumor antigen gene family, MAGE-1. Taken from our analysis of MAGE-1 gene regulation, we propose that 5-aza-CdR-mediated gene activation has two distinct requirements: 1) the reversal of promoter hypermethylation, and 2) the presence of transcriptional activators competent for the activation of hypomethylated target promoters. This latter requirement for gene activation by 5-aza-CdR is probably mediated by sequence-specific transcription factors and may account for the limited number of human genes induced by 5-aza-CdR treatment. This revised model for gene activation by 5-aza-CdR has important implications for the use of DNA methyltransferase inhibitors in clinical settings.

Antigens, Neoplasm↗

PubMed1987

Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis in Escherichia coli.

We have developed a new research tool for the study of S-adenosylmethionine (AdoMet) metabolism by cloning the coliphage T3 AdoMet hydrolase (AdoMetase; EC 3.3.1.2) gene into the M13mp8 expression vector. The recombinant bacteriophage clones expressed an AdoMetase activity in Escherichia coli like that found in T3-infected cells. High levels of AdoMetase expression impaired AdoMet-mediated activities such as dam and dcm methylase-directed DNA modifications and the synthesis of spermidine from putrescine. Expression vectors containing the cloned AdoMetase gene thus provide an alternate approach to the use of chemical inhibitors or mutants defective in AdoMet biosynthesis to probe the effect of AdoMet limitation.

Cloning, Molecular↗

PubMed2004

The methyltransferase from the LlaDII restriction-modification system influences the level of expression of its own gene.

The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (P(llaDIIM)) up to 1,000-fold, whereas the activity of the llaDIIR promoter (P(llaDIIR)) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within P(llaDIIM) are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR, which appears to be constitutively expressed.

Base Sequence↗

PubMed1998

Interactions between the structural domains of the RNA replication proteins of plant-infecting RNA viruses.

Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

Animals↗

PubMed2001

Infection of lymphoid cells by integration-defective human immunodeficiency virus type 1 increases de novo methylation.

DNA methylation, by regulating the transcription of genes, is a major modifier of the eukaryotic genome. DNA methyltransferases (DNMTs) are responsible for both maintenance and de novo methylation. We have reported that human immunodeficiency virus type 1 (HIV-1) infection increases DNMT1 expression and de novo methylation of genes such as the gamma interferon gene in CD4(+) cells. Here, we examined the mechanism(s) by which HIV-1 infection increases the cellular capacity to methylate genes. While the RNAs and proteins of all three DNMTs (1, 3a, and 3b) were detected in Hut 78 lymphoid cells, only the expression of DNMT1 was significantly increased 3 to 5 days postinfection. This increase was observed with either wild-type HIV-1 or an integrase (IN) mutant, which renders HIV replication defective, due to the inability of the provirus to integrate into the host genome. Unintegrated viral DNA is a common feature of many retroviral infections and is thought to play a role in pathogenesis. These results indicate another mechanism by which unintegrated viral DNA affects the host. In addition to the increase in overall genomic methylation, hypermethylation and reduced expression of the p16(INK4A) gene, one of the most commonly altered genes in human cancer, were seen in cells infected with both wild-type and IN-defective HIV-1. Thus, infection of lymphoid cells with integration-defective HIV-1 can increase the methylation of CpG islands in the promoters of genes such as the p16(INK4A) gene, silencing their expression.

Cell Line↗

PubMed1998

Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production.

The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-gamma), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-gamma, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-gamma gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-gamma gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-gamma gene, and increased IFN-gamma production, demonstrating a direct link between methyltransferase and IFN-gamma gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-gamma gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

CD4-Positive T-Lymphocytes↗

PubMed2006

High-frequency promoter hypermethylation of the deleted in liver cancer-1 gene in multiple myeloma.

BACKGROUND: Deleted in liver cancer-1 (DLC-1) is a tumour suppressor gene that is inactive in liver carcinogenesis. It encodes a rho-guanosine triphosphatase-activating protein (rho-GAP) and maps to one of the deleted regions (8p21.3-22). Little is known, however, about the methylation status of the DLC-1 promoter in myeloma cells. AIM: To identify whether methylation of DLC-1 was associated in pathogenesis of multiple myeloma. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect DLC-1 transcripts in RPMI 8226, U266, OPM-2 and XG-2 cell lines. The methylation status was determined by methylation-specific PCR followed by bisulphite DNA sequencing in these four cell lines and in the bone marrow of 14 patients with multiple myeloma and 4 normal patients. DLC-1 mRNA expression in cells with or without treatment with 5-aza-deoxycytidine (5-aza-CdR) or trichostatin A (TSA) was investigated by real-time RT-PCR. RESULTS: RPMI 8226 and U266 showed complete methylation and XG-2 showed partial methylation. DLC-1 was expressed only in OPM-2 cell lines that showed no methylation. DLC-1 methylation was shown in 11 of 14 (78%) patients with multiple myeloma and none of the normal controls. The exposure of cell lines to 5-aza-CdR or TSA resulted in the up regulation of DLC-1 gene expression. CONCLUSIONS: DLC-1 methylation is often present in multiple myeloma and has a key role in DLC-1 silencing.

Azacitidine↗

PubMed1996

The role of DNA methylation in cancer genetic and epigenetics.

The past few years have seen a wider acceptance of a role for DNA methylation in cancer. This can be attributed to three developments. First, the documentation of the over-representation of mutations at CpG dinucleotides has convincingly implicated DNA methylation in the generation of oncogenic point mutations. The second important advance has been the demonstration of epigenetic silencing of tumor suppressor genes by DNA methylation. The third development has been the utilization of experimental methods to manipulate DNA methylation levels. These studies demonstrate that DNA methylation changes in cancer cells are not mere by-products of malignant transformation, but can play an instrumental role in the cancer process. It seems clear that DNA methylation plays a variety of roles in different cancer types and probably at different stages of oncogenesis. DNA methylation is intricately involved in a wide diversity of cellular processes. Likewise, it appears to exert its influence on the cancer process through a diverse array of mechanisms. It is our task not only to identify these mechanisms, but to determine their relative importance for each stage and type of cancer. Our hope then will be to translate that knowledge into clinical applications.

Animals↗

PubMed2003

Uteroplacental insufficiency increases apoptosis and alters p53 gene methylation in the full-term IUGR rat kidney.

Uteroplacental insufficiency causes intrauterine growth retardation (IUGR), which is associated with adult onset diseases such as hypertension. Previous studies demonstrate that growth retardation in humans and rats decreases glomeruli number; however, the molecular mechanisms responsible for this reduction are unknown. Apoptosis plays a key role in renal organogenesis. We therefore hypothesized that the in utero deprivation associated with uteroplacental insufficiency decreases glomeruli, increases apoptosis, and alters the mRNA levels of key apoptosis-related proteins in full-term IUGR kidneys. To prove this hypothesis, we induced asymmetric IUGR through bilateral uterine artery ligation of the pregnant rat. We found that uteroplacental insufficiency significantly reduced glomeruli number while increasing TUNEL staining and caspase-3 activity in the IUGR kidney. A significant decrease in Bcl-2 mRNA and a significant increase in Bax and p53 mRNA further characterized the IUGR kidney. Because altered p53 CpG methylation affects p53 expression, we analyzed p53 promoter CpG methylation using methylation-sensitive restriction enzymes and real-time PCR. Uteroplacental insufficiency specifically decreased CpG methylation of the renal p53 BstU I site promoter without affecting the Hha I or the Aci I sites. Uteroplacental insufficiency also induced a relative hypomethylation from exon 5 to exon 8, which was associated with deceased mRNA levels of DNMT1. We conclude that uteroplacental insufficiency alters p53 DNA CpG methylation, affects mRNA levels of key apoptosis-related proteins, increases renal apoptosis, and reduces glomeruli number in the IUGR kidney. We speculate that these changes represent mechanisms that contribute to the fetal origins of adult disease.

Animals↗

PubMed2005

Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases.

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.

Binding Sites↗

PubMed2005

Epigenetic up-regulation of C-C chemokine receptor 7 and C-X-C chemokine receptor 4 expression in melanoma cells.

Histone deacetylation and DNA methylation establish epigenetic modifications, which through chromatin remodeling may result in gene silencing. We hypothesized that chemokine receptors C-C chemokine receptor 7 (CCR7) and C-X-C chemokine receptor 4 (CXCR4) on melanoma cells undergo epigenetic regulation. We investigated whether a histone deacetylase inhibitor and a demethylating agent influence CCR7 and CXCR4 expression on melanoma cells. Initially, microarray analysis was done to screen changes in chemokine receptor expression on melanoma cells after treatment with trichostatin A (TSA) and 5-Aza-2-deoxycytidine (5-Aza). CCR7 and CXCR4 mRNA expression were uniformly altered and selected for further investigation. Quantitative real-time reverse transcription-PCR assay, immunohistochemistry, and Western blot analysis were used to assess changes in mRNA and protein expression induced by TSA and 5-Aza in melanoma lines. Cell migration assays were conducted to assess the effects of altered CCR7 and CXCR4 expression on cell function. Treatment with TSA or 5-Aza increased gene expression of both CCR7 and CXCR4 in melanoma lines. TSA was the strongest enhancer. With combined treatment, CCR7 and CXCR4 mRNA expression was also up-regulated. Immunohistochemistry after combined treatment showed enhanced staining of both CCR7 and CXCR4 compared with control cells. Melanoma cell migration in TSA- and 5-Aza-treated cells was 7- and 2-fold higher than control cells for CCR7 and CXCR4, respectively. In summary, a histone deacetylase inhibitor and a demethylating agent up-regulated CCR7 and CXCR4 expression on melanoma cells. This increase in chemokine receptor expression correlated with functional activity. Most importantly, we have identified an epigenetic mechanism that may endogenously regulate chemokine receptor expression on melanoma cells.

Acetylation↗

PubMed2005

Inhibition of DNA methylation sensitizes glioblastoma for tumor necrosis factor-related apoptosis-inducing ligand-mediated destruction.

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments.

Adult↗

PubMed2006

Epigenetic regulation of tumor endothelial cell anergy: silencing of intercellular adhesion molecule-1 by histone modifications.

Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel wall interactions, thereby attenuating infiltration of leukocytes into the tumor. This report describes a novel mechanism of endothelial cell anergy regulation. We recently reported that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have angiostatic activity. Here, we studied whether epigenetic mechanisms regulate this angiogenesis-mediated escape from immunity. We found that DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine, as well as HDAC inhibitor trichostatin A, reexpressed intercellular adhesion molecule-1 (ICAM-1) on tumor-conditioned endothelial cells in vitro, resulting in restored leukocyte-endothelial cell adhesion. In addition, treatment with DNMT or HDAC inhibitors in vivo also restored ICAM-1 expression on tumor endothelial cells from two different mouse tumor models. Furthermore, leukocyte-vessel wall interactions in mouse tumors were increased by these compounds, as measured by intravital microscopy, resulting in enhanced leukocyte infiltration. We show that ICAM-1 down-regulation in tumor endothelial cells is associated with ICAM-1 promoter histone H3 deacetylation and loss of histone H3 Lys(4) methylation but not with DNA hypermethylation. In conclusion, our data show that ICAM-1 is epigenetically silenced in tumor endothelial cells by promoter histone modifications, which can be overcome by DNMT and HDAC inhibitors, suggesting a new molecular mechanism based on which novel therapeutic approaches for cancer can be pursued.

Acetylation↗

PubMed2004

Favorable neuroblastoma genes and molecular therapeutics of neuroblastoma.

PURPOSE AND EXPERIMENTAL DESIGN: Neuroblastoma (NB) is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. Favorable NB genes (EPHB6, EFNB2, EFNB3, NTRK1, and CD44) are genes whose high-level expression predicts favorable NB outcome, and forced expression of these genes inhibits growth of unfavorable NB cells. In this study, we investigated whether favorable NB gene expression could be augmented in unfavorable NB cells by chemical compounds and whether an increased expression of these genes was associated with suppression of NB growth and metastasis. RESULTS: We found that inhibitors of DNA methylation [5-aza-2'-deoxycytidine (5AdC)], histone deacetylase (HDAC) [4-phenylbutyrate (4PB)], and proteasome (MG262) enhanced the expression of favorable NB genes in NB cell lines and inhibited the growth of these cells in vitro (P < 0.0005). The growth-inhibitory effects of 5AdC and 4PB in vitro were in part due to caspase-dependent cell death and inhibition of DNA synthesis. Administration of 5AdC and/or 4PB also suppressed growth of subcutaneous NB xenografts in nude mice (P < 0.001), which was accompanied by enhanced favorable NB gene expression and an increase in apoptosis. Moreover, 4PB suppressed bone marrow and liver metastases of NB cells in severe combined immunodeficient/Beige mice (P = 0.007 and P = 0.008, respectively). The growth-suppressive activity of HDAC inhibitors on NB was further confirmed by the efficacy of trichostatin A, a potent and specific HDAC inhibitor. CONCLUSIONS: Collectively, these observations further emphasize the link between the elevated favorable NB gene expression and a benign phenotype of NB.

Animals↗

PubMed2005

Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine.

PURPOSE: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation. EXPERIMENTAL DESIGN: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys. RESULTS: In mice, plasma zebularine concentrations declined with terminal half-lives (t(1/2)) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 mug/mL min, respectively, corresponding to a total body clearance (CL(tb)) of 13.65 mL/min/kg, apparent total body clearance (CL(app)) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t(1/2) of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 mug/mL min, respectively, corresponding to a CL(tb) of 3.99 mL/min/kg for 50 mg/kg i.v. and CL(app) of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t(1/2) was 70 and 150 minutes, CL(tb) was 3.55 and 10.85 mL/min/kg after i.v. administration, and CL(app) was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CL(tb) = 6.46(weight(0.9)). CONCLUSIONS: Zebularine has limited oral bioavailability. Interspecies scaling projects a CL(tb) of 296 mL/min in humans.

Administration, Oral↗

PubMed2005

The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors.

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2'-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2'-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Antigens, Differentiation↗

PubMed2006

Epigenetic inactivation of Wnt inhibitory factor-1 plays an important role in bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway.

PURPOSE: Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the pathogenesis of several human cancers. Wnt inhibitory factor-1 (Wif-1) was identified as one of the secreted antagonists that can bind Wnt protein. We hypothesize that Wif-1 plays an important role in bladder cancer pathogenesis. EXPERIMENTAL DESIGN: To test this hypothesis, epigenetic and genetic pathways involved in the Wif-1 gene modulation and expression of Wnt/beta-catenin-related genes were analyzed in 4 bladder tumor cell lines and 54 bladder tumor and matched normal bladder mucosa. RESULTS: Wif-1 mRNA expression was significantly enhanced after 5-aza-2'-deoxycytidine treatment in bladder tumor cell lines. Wif-1 promoter methylation level was significantly higher and Wif-1 mRNA expression was significantly lower in bladder tumor samples than in bladder mucosa samples. In the total bladder tumor and bladder mucosa samples, an inverse correlation was found between promoter methylation and Wif-1 mRNA transcript levels. However, loss-of-heterozygosity at chromosome 12q14.3 close to the Wif-1 gene loci was a rare event (3.7%). Nuclear accumulation of beta-catenin was significantly more frequent in bladder tumor than in bladder mucosa and inversely correlated with Wif-1 expression. In addition, known targets of the canonical Wnt/beta-catenin signaling pathway, such as c-myc and cyclin D1, were up-regulated in bladder tumor compared with bladder mucosa, and this up-regulation was associated with reduced Wif-1 expression at both mRNA and protein levels. Furthermore, transfection of Wif-1 small interfering RNA into bladder tumor cells expressing Wif-1 mRNA transcripts had increased levels of c-myc and cyclin D1 and accelerated cell growth. CONCLUSION: This is the first report showing that CpG hypermethylation of the Wif-1 promoter is a frequent event in bladder tumor and may contribute to pathogenesis of bladder cancer through aberrant canonical Wnt/beta-catenin signaling pathway. The present study elucidates novel pathways that are involved in the pathogenesis of bladder cancer.

Adaptor Proteins, Signal Transducing↗