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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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Re-constitution of PDCD4– glioma cells with PDCD4 inhibited their growth and induced their apoptosis. (A) After transient transfection with PDCD4 for 48 hrs, the U251 cells were examined by fluorescent microscopy. Transfected cells express the red fluorescent protein, DsRed2, which are shown in red. (B) PDCD4 expression in U251 glioma cells was examined by RT-PCR after transient transfection (24, 48 and 72 hrs) with PDCD4 plasmids (PDCD4), or empty vector (Mock). Untransfected PDCD4– glioma cells were used as a control. (C) The morphology of U251 cells with or without PDCD4 expression. (D) Total number of U251 cells per millilitre of culture medium 24 and 48 hrs after the transfection. *, P < 0.05. Data shown are representative of three independent experiments. (E) Cell cycle analysis. (F) Apoptosis analysis. Data shown are representative of three independent experiments.
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fig03: Re-constitution of PDCD4– glioma cells with PDCD4 inhibited their growth and induced their apoptosis. (A) After transient transfection with PDCD4 for 48 hrs, the U251 cells were examined by fluorescent microscopy. Transfected cells express the red fluorescent protein, DsRed2, which are shown in red. (B) PDCD4 expression in U251 glioma cells was examined by RT-PCR after transient transfection (24, 48 and 72 hrs) with PDCD4 plasmids (PDCD4), or empty vector (Mock). Untransfected PDCD4– glioma cells were used as a control. (C) The morphology of U251 cells with or without PDCD4 expression. (D) Total number of U251 cells per millilitre of culture medium 24 and 48 hrs after the transfection. *, P < 0.05. Data shown are representative of three independent experiments. (E) Cell cycle analysis. (F) Apoptosis analysis. Data shown are representative of three independent experiments.

Mentions: The frequent loss of PDCD4 expression in glioma cells suggests that PDCD4 may inhibit the proliferation of gliomas. To determine the effect of PDCD4 on the growth and survival of glioma cells, a recombinant pDsRed plasmid carrying the full-length PDCD4 cDNA was transfected into PDCD4– U251 and U87 glioma cells. Forty-eight hours later, both the fluorescent marker and PDCD4 were detected in the glioma cells (Fig. 3A and B). Importantly, PDCD4 expression in these cells significantly inhibited their growth, compared with untransfected control and mock-transfected groups (Fig. 3C and D). Cell cycle analysis further revealed that PDCD4 expression significantly increased the number of cells in the S-phase of the cell cycles (38.47%± 1.39 in mock versus 50.83%± 2.27 in the PDCD4 group) (Fig. 3E). Furthermore, annexin V-FITC/PI analysis clearly showed that PDCD4-transfected glioma cells underwent more apoptosis than the controls (4.96%± 1.04 in mock versus 11.62%± 0.95 in the PDCD4 group) (Fig. 3F).


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)

Re-constitution of PDCD4– glioma cells with PDCD4 inhibited their growth and induced their apoptosis. (A) After transient transfection with PDCD4 for 48 hrs, the U251 cells were examined by fluorescent microscopy. Transfected cells express the red fluorescent protein, DsRed2, which are shown in red. (B) PDCD4 expression in U251 glioma cells was examined by RT-PCR after transient transfection (24, 48 and 72 hrs) with PDCD4 plasmids (PDCD4), or empty vector (Mock). Untransfected PDCD4– glioma cells were used as a control. (C) The morphology of U251 cells with or without PDCD4 expression. (D) Total number of U251 cells per millilitre of culture medium 24 and 48 hrs after the transfection. *, P < 0.05. Data shown are representative of three independent experiments. (E) Cell cycle analysis. (F) Apoptosis analysis. Data shown are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496131&req=5

fig03: Re-constitution of PDCD4– glioma cells with PDCD4 inhibited their growth and induced their apoptosis. (A) After transient transfection with PDCD4 for 48 hrs, the U251 cells were examined by fluorescent microscopy. Transfected cells express the red fluorescent protein, DsRed2, which are shown in red. (B) PDCD4 expression in U251 glioma cells was examined by RT-PCR after transient transfection (24, 48 and 72 hrs) with PDCD4 plasmids (PDCD4), or empty vector (Mock). Untransfected PDCD4– glioma cells were used as a control. (C) The morphology of U251 cells with or without PDCD4 expression. (D) Total number of U251 cells per millilitre of culture medium 24 and 48 hrs after the transfection. *, P < 0.05. Data shown are representative of three independent experiments. (E) Cell cycle analysis. (F) Apoptosis analysis. Data shown are representative of three independent experiments.
Mentions: The frequent loss of PDCD4 expression in glioma cells suggests that PDCD4 may inhibit the proliferation of gliomas. To determine the effect of PDCD4 on the growth and survival of glioma cells, a recombinant pDsRed plasmid carrying the full-length PDCD4 cDNA was transfected into PDCD4– U251 and U87 glioma cells. Forty-eight hours later, both the fluorescent marker and PDCD4 were detected in the glioma cells (Fig. 3A and B). Importantly, PDCD4 expression in these cells significantly inhibited their growth, compared with untransfected control and mock-transfected groups (Fig. 3C and D). Cell cycle analysis further revealed that PDCD4 expression significantly increased the number of cells in the S-phase of the cell cycles (38.47%± 1.39 in mock versus 50.83%± 2.27 in the PDCD4 group) (Fig. 3E). Furthermore, annexin V-FITC/PI analysis clearly showed that PDCD4-transfected glioma cells underwent more apoptosis than the controls (4.96%± 1.04 in mock versus 11.62%± 0.95 in the PDCD4 group) (Fig. 3F).

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