RESUMO
DNA methylation and histone modifications are two major epigenetic events regulating gene expression and chromatin structure, and their alterations are linked to human carcinogenesis. DNA methylation plays an important role in tumor suppressor gene inactivation, and can be revised by DNA methylation inhibitors. The reversible nature of DNA methylation forms the basis of epigenetic cancer therapy. However, it has been reported that DNA re-methylation and gene re-silencing could occur after removal of demethylation treatment and this may significantly hamper the therapeutic value of DNA methylation inhibitors. In this study we have provided detailed evidence demonstrating that mammalian cells possess a bona fide DNA methylation recovery system. We have also shown that DNA methylation recovery was mediated by the major human DNA methyltransferase, DNMT1. In addition, we found that H3K9-tri-methylation and H3K27-tri-methylation were closely associated with this DNA methylation recovery. These persistent transcriptional repressive histone modifications may have a crucial role in regulating DNMT1-mediated DNA methylation recovery. Our findings may have important implications towards a better understanding of epigenetic regulation and future development of epigenetic therapeutic intervention.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Inativação Gênica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Transcrição Gênica/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/efeitos dos fármacos , Decitabina , Proteínas Ativadoras de GTPase/genética , Inativação Gênica/efeitos dos fármacos , Loci Gênicos/genética , Células HCT116 , Células HeLa , Humanos , Processos Estocásticos , Transcrição Gênica/efeitos dos fármacos , Proteínas Supressoras de Tumor/genéticaRESUMO
Co-deletion of chromosomes 1p and 19q is a common event in oligodendroglial tumors (OTs), suggesting the presence of OT-related genes. The aim of this study was to identify the target genes residing in the minimally deleted regions on chromosome 1p36.31-p36.32 that might be involved in OTs. A novel gene KIAA0495/p53-dependent apoptosis modulator (PDAM) was found frequently deregulated, with 37 of 58 (63.8%) OTs examined showing reduced expression compared with normal brain. Chromosome 1p loss and epigenetic modifications were the major mechanisms contributing to PDAM downregulation. The role of PDAM in chemosensitivity was also evaluated. PDAM knockdown had no effect on sensitivity to vincristine, lomustine, temozolomide and paclitaxel, but could induce cisplatin resistance in glioma cells harboring wild-type p53. B-cell CCL/lymphoma 2 (BCL2)-like 1 (BCL2L1) exhibited significant upregulation, while BCL2 showed partial derepression in PDAM-silenced cells after cisplatin treatment, suggesting that alteration of anti-apoptotic genes contributed in part to cisplatin resistance. Knockdown of BCL2L1 abrogated the induced cisplatin-resistant phenotype. Moreover, our data suggested that PDAM might function as a non-protein-coding RNA. Collectively, these findings suggest that PDAM deregulation may play a role in OT development and that PDAM may possess the capacity to modulate apoptosis via regulation of p53-dependent anti-apoptotic genes.
Assuntos
Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Oligodendroglioma/metabolismo , RNA Longo não Codificante/fisiologia , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Criança , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Interações Medicamentosas , Feminino , Glioma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Sensação Térmica/genética , Transfecção , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
The epithelial membrane protein 3 (EMP3) gene located on chromosome 19q13 has been implicated as a candidate tumor suppressor gene (TSG) in neuroblastomas and gliomas. The aim of this study was to investigate whether EMP3 is involved in oligodendroglial tumors (OTs), which frequently carry combined chromosomes 1p and 19q deletion. We first investigated the transcript level of EMP3 in a cohort of 57 OTs by quantitative real-time RT-PCR. Our results showed that 10 (18%) tumors had reduced EMP3 expression level compared to normal brains. Six of these tumors carried chromosome 19q13 deletion but no statistical correlation was found between the 2 parameters. Intriguingly, a similar proportion (11 of 57, 19%) of tumors displayed EMP3 overexpression, with 8 of them having transcript level >10-fold higher than normal brain. All 11 OTs retained chromosomes 1p36 and 19q13, and a significant association was found between EMP3 overexpression and balanced chromosomes 1p36 and 19q13 (p = 0.004). The methylation status of EMP3 was evaluated by bisulfite sequencing in 29 OTs with diverse expression levels. All tumors except 3 showed aberrant methylation of EMP3 and no correlation was observed between transcript level and methylation status, suggesting that methylation alone does not mediate transcriptional down-regulation of EMP3 in OTs. In conclusion, our study demonstrates that EMP3 overexpression is involved in OTs retaining chromosomes 1p and 19q and does not support EMP3 as the target TSG on chromosome 19q13 in OTs.
Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 1/genética , Metilação de DNA , Glicoproteínas de Membrana/genética , Oligodendroglioma/genética , Adolescente , Adulto , Idoso , Astrocitoma/genética , Astrocitoma/metabolismo , Astrocitoma/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Criança , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Oligodendroglioma/metabolismo , Oligodendroglioma/patologia , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
UNLABELLED: In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. CONCLUSION: Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.