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PDCD4 gene silencing in gliomas is associated with 5'CpG island methylation and unfavourable prognosis.

Gao F, Wang X, Zhu F, Wang Q, Zhang X, Guo C, Zhou C, Ma C, Sun W, Zhang Y, Chen YH, Zhang L - J. Cell. Mol. Med. (2009)

Bottom Line: Loss of PDCD4 expression has been found in several types of human cancers including the most common cancer of the brain, the gliomas.Blocking methylation in glioma cells using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the PDCD4 gene expression, inhibited their proliferation and reduced their colony formation capacity.These results also indicate that PDCD4 reactivation might be an effective new strategy for the treatment of gliomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, School of Medicine, Shandong University, Jinan, Shandong, China.

ABSTRACT
Programmed cell death 4 (PDCD4) is a newly described tumour suppressor that inhibits oncogenesis by suppressing gene transcription and translation. Loss of PDCD4 expression has been found in several types of human cancers including the most common cancer of the brain, the gliomas. However, the molecular mechanisms responsible for PDCD4 gene silencing in tumour cells remain unclear. Here we report the identification of 5'CpG island methylation as the predominant cause of PDCD4 mRNA silencing in gliomas. The methylation of the PDCD4 5'CpG island was found in 47% (14/30) of glioma tissues, which was significantly associated with the loss of PDCD4 mRNA expression (gamma=-1.000, P < 0.0001). Blocking methylation in glioma cells using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the PDCD4 gene expression, inhibited their proliferation and reduced their colony formation capacity. Longitudinal studies of a cohort of 84 patients with gliomas revealed that poor prognosis of patients with high-grade tumours were significantly associated with loss of PDCD4 expression. Thus, our current study suggests, for the first time, that PDCD4 5'CpG island methylation blocks PDCD4 expression at mRNA levels in gliomas. These results also indicate that PDCD4 reactivation might be an effective new strategy for the treatment of gliomas.

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Methylation of PDCD4 5′CpG island in glioma cell lines, non-tumour brain and glioma tissues. (A) Schematic representation of the CpG island of human PDCD4 gene. (B) Representative figure of methylation. T: tumour tissue; N: non-tumour brain tissue. Arrows, the methylated CpGs. (C)–(F). PDCD4 mRNA (C) and protein (D) expression and PDCD4 gene methylation (E) as determined by RT-PCR, Western blot and methylation-specific PCR, respectively. U251 and U87, two glioma cell lines; normal, non-tumour brain tissue; M, methylated; U, unmethylated. (F) Methylation status of the PDCD4 cytosine island in non-tumour brain and primary glioma tissues. 1–30, primary glioma tissues from 30 patients; N, non-tumour brain tissues; H2O, untransfected control
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fig01: Methylation of PDCD4 5′CpG island in glioma cell lines, non-tumour brain and glioma tissues. (A) Schematic representation of the CpG island of human PDCD4 gene. (B) Representative figure of methylation. T: tumour tissue; N: non-tumour brain tissue. Arrows, the methylated CpGs. (C)–(F). PDCD4 mRNA (C) and protein (D) expression and PDCD4 gene methylation (E) as determined by RT-PCR, Western blot and methylation-specific PCR, respectively. U251 and U87, two glioma cell lines; normal, non-tumour brain tissue; M, methylated; U, unmethylated. (F) Methylation status of the PDCD4 cytosine island in non-tumour brain and primary glioma tissues. 1–30, primary glioma tissues from 30 patients; N, non-tumour brain tissues; H2O, untransfected control

Mentions: Of the 30 frozen glioma samples examined previously, 47% (14/30) of them lacked the PDCD4 mRNA expression [4]. However, the mechanisms of the PDCD4 silencing at mRNA levels are unclear. 5′CpG island methylation is a common epigenetic mechanism implicated in the silencing of tumour suppressor genes in human cancers [24]. To determine whether 5′CpG island methylation is responsible for PDCD4 silencing in gliomas, we first searched for 5′CpG islands in the PDCD4 gene as described in the section ‘Materials and methods’. A typical CpG island (−251 to +918 bp) was found around the transcription start site of the PDCD4 gene including the first exon and part of the upstream transcription start site sequence (Fig. 1A). This suggests that PDCD4 may be vulnerable to methylation-mediated silencing. To test this theory, genomic DNAs from glioma cell lines and primary gliomas as well non-tumour brain specimens were purified and used as templates for BSP and PCR (MSP) after bisulphite modification. As shown in Fig. 1B–F and Table 2, the methylation of PDCD4 5′CpG island was detected in both PDCD4– cell lines (U251 and U87) and 14 specimens of PDCD4 mRNA– primary gliomas whereas no methylation was found in non-tumour brain tissues. Among methylated glioma samples, 5 (nos. 3, 7, 17, 19, 21) showed complete methylation (M: + and U: −) and the rest (nos. 2, 9, 15, 25, 26, 27, 28, 29 and 30) as well as two cell lines, showed incomplete methylation (M: + and U: +). Both complete and incomplete methylations of 5′CpG island were able to cause the silencing of PDCD4 at mRNA level.


PDCD4 gene silencing in gliomas is associated with 5'CpG island methylation and unfavourable prognosis.

Gao F, Wang X, Zhu F, Wang Q, Zhang X, Guo C, Zhou C, Ma C, Sun W, Zhang Y, Chen YH, Zhang L - J. Cell. Mol. Med. (2009)

Methylation of PDCD4 5′CpG island in glioma cell lines, non-tumour brain and glioma tissues. (A) Schematic representation of the CpG island of human PDCD4 gene. (B) Representative figure of methylation. T: tumour tissue; N: non-tumour brain tissue. Arrows, the methylated CpGs. (C)–(F). PDCD4 mRNA (C) and protein (D) expression and PDCD4 gene methylation (E) as determined by RT-PCR, Western blot and methylation-specific PCR, respectively. U251 and U87, two glioma cell lines; normal, non-tumour brain tissue; M, methylated; U, unmethylated. (F) Methylation status of the PDCD4 cytosine island in non-tumour brain and primary glioma tissues. 1–30, primary glioma tissues from 30 patients; N, non-tumour brain tissues; H2O, untransfected control
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496131&req=5

fig01: Methylation of PDCD4 5′CpG island in glioma cell lines, non-tumour brain and glioma tissues. (A) Schematic representation of the CpG island of human PDCD4 gene. (B) Representative figure of methylation. T: tumour tissue; N: non-tumour brain tissue. Arrows, the methylated CpGs. (C)–(F). PDCD4 mRNA (C) and protein (D) expression and PDCD4 gene methylation (E) as determined by RT-PCR, Western blot and methylation-specific PCR, respectively. U251 and U87, two glioma cell lines; normal, non-tumour brain tissue; M, methylated; U, unmethylated. (F) Methylation status of the PDCD4 cytosine island in non-tumour brain and primary glioma tissues. 1–30, primary glioma tissues from 30 patients; N, non-tumour brain tissues; H2O, untransfected control
Mentions: Of the 30 frozen glioma samples examined previously, 47% (14/30) of them lacked the PDCD4 mRNA expression [4]. However, the mechanisms of the PDCD4 silencing at mRNA levels are unclear. 5′CpG island methylation is a common epigenetic mechanism implicated in the silencing of tumour suppressor genes in human cancers [24]. To determine whether 5′CpG island methylation is responsible for PDCD4 silencing in gliomas, we first searched for 5′CpG islands in the PDCD4 gene as described in the section ‘Materials and methods’. A typical CpG island (−251 to +918 bp) was found around the transcription start site of the PDCD4 gene including the first exon and part of the upstream transcription start site sequence (Fig. 1A). This suggests that PDCD4 may be vulnerable to methylation-mediated silencing. To test this theory, genomic DNAs from glioma cell lines and primary gliomas as well non-tumour brain specimens were purified and used as templates for BSP and PCR (MSP) after bisulphite modification. As shown in Fig. 1B–F and Table 2, the methylation of PDCD4 5′CpG island was detected in both PDCD4– cell lines (U251 and U87) and 14 specimens of PDCD4 mRNA– primary gliomas whereas no methylation was found in non-tumour brain tissues. Among methylated glioma samples, 5 (nos. 3, 7, 17, 19, 21) showed complete methylation (M: + and U: −) and the rest (nos. 2, 9, 15, 25, 26, 27, 28, 29 and 30) as well as two cell lines, showed incomplete methylation (M: + and U: +). Both complete and incomplete methylations of 5′CpG island were able to cause the silencing of PDCD4 at mRNA level.

Bottom Line: Loss of PDCD4 expression has been found in several types of human cancers including the most common cancer of the brain, the gliomas.Blocking methylation in glioma cells using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the PDCD4 gene expression, inhibited their proliferation and reduced their colony formation capacity.These results also indicate that PDCD4 reactivation might be an effective new strategy for the treatment of gliomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, School of Medicine, Shandong University, Jinan, Shandong, China.

ABSTRACT
Programmed cell death 4 (PDCD4) is a newly described tumour suppressor that inhibits oncogenesis by suppressing gene transcription and translation. Loss of PDCD4 expression has been found in several types of human cancers including the most common cancer of the brain, the gliomas. However, the molecular mechanisms responsible for PDCD4 gene silencing in tumour cells remain unclear. Here we report the identification of 5'CpG island methylation as the predominant cause of PDCD4 mRNA silencing in gliomas. The methylation of the PDCD4 5'CpG island was found in 47% (14/30) of glioma tissues, which was significantly associated with the loss of PDCD4 mRNA expression (gamma=-1.000, P < 0.0001). Blocking methylation in glioma cells using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the PDCD4 gene expression, inhibited their proliferation and reduced their colony formation capacity. Longitudinal studies of a cohort of 84 patients with gliomas revealed that poor prognosis of patients with high-grade tumours were significantly associated with loss of PDCD4 expression. Thus, our current study suggests, for the first time, that PDCD4 5'CpG island methylation blocks PDCD4 expression at mRNA levels in gliomas. These results also indicate that PDCD4 reactivation might be an effective new strategy for the treatment of gliomas.

Show MeSH
Related in: MedlinePlus