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Centrosome amplification induced by survivin suppression enhances both chromosome instability and radiosensitivity in glioma cells.

Saito T, Hama S, Izumi H, Yamasaki F, Kajiwara Y, Matsuura S, Morishima K, Hidaka T, Shrestha P, Sugiyama K, Kurisu K - Br. J. Cancer (2008)

Bottom Line: In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN).As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells.TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. taiichi@hiroshima-u.ac.jp

ABSTRACT
Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.

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Apoptosis was assessed by TUNEL assay. The TUNEL assay was used for the morphological detection of any apoptotic changes. U251MG or D54MG cells with siRNA/control or survivin transfection, nonirradiated or irradiated at a dose of 4 Gy, were cultured for 3 (72 h) or 5 days (120 h), and were stained according to the manufacturer's instructions after fixation with 4% formaldehyde. The AI was defined as the percentage of TUNEL-positive U251 (A) or D54MG cells (B). (C) TUNEL signals were detected by fluorescence microscopy in the nuclei of some irradiated and siRNA/control-transfected U251MG cells.
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fig8: Apoptosis was assessed by TUNEL assay. The TUNEL assay was used for the morphological detection of any apoptotic changes. U251MG or D54MG cells with siRNA/control or survivin transfection, nonirradiated or irradiated at a dose of 4 Gy, were cultured for 3 (72 h) or 5 days (120 h), and were stained according to the manufacturer's instructions after fixation with 4% formaldehyde. The AI was defined as the percentage of TUNEL-positive U251 (A) or D54MG cells (B). (C) TUNEL signals were detected by fluorescence microscopy in the nuclei of some irradiated and siRNA/control-transfected U251MG cells.

Mentions: To determine whether the death of siRNA/survivin-transfected U251MG and D54MG cells after irradiation was due to apoptosis, we performed a TUNEL assay and measured the level of apoptosis based on the TUNEL positivity on days 3 (72 h) and 5 (120 h) either with or without irradiation. Figure 8A and B show the percentage of TUNEL-positive cells. Irradiated cells that were transfected with siRNA/control had a higher proportion of TUNEL-positive cells than the corresponding nonirradiated cells in both the cell lines on days 3 and 5. However, irradiated cells that were transfected with siRNA/survivin had the same proportion of TUNEL-positive cells as the corresponding nonirradiated cells in both the cell lines on days 3 and 5. As shown in Figure 8C, the irradiated siRNA/control-transfected cells were identified as scattered clusters of fluorescent staining, suggesting the death of these cells to be due to apoptosis in both the cell lines. In contrast, siRNA/survivin-transfected cells did not emit fluorescent signals even after irradiation in both the cell lines. Therefore, the cell death of the siRNA/survivin-transfected cells following irradiation is not considered to be due to apoptosis.


Centrosome amplification induced by survivin suppression enhances both chromosome instability and radiosensitivity in glioma cells.

Saito T, Hama S, Izumi H, Yamasaki F, Kajiwara Y, Matsuura S, Morishima K, Hidaka T, Shrestha P, Sugiyama K, Kurisu K - Br. J. Cancer (2008)

Apoptosis was assessed by TUNEL assay. The TUNEL assay was used for the morphological detection of any apoptotic changes. U251MG or D54MG cells with siRNA/control or survivin transfection, nonirradiated or irradiated at a dose of 4 Gy, were cultured for 3 (72 h) or 5 days (120 h), and were stained according to the manufacturer's instructions after fixation with 4% formaldehyde. The AI was defined as the percentage of TUNEL-positive U251 (A) or D54MG cells (B). (C) TUNEL signals were detected by fluorescence microscopy in the nuclei of some irradiated and siRNA/control-transfected U251MG cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361434&req=5

fig8: Apoptosis was assessed by TUNEL assay. The TUNEL assay was used for the morphological detection of any apoptotic changes. U251MG or D54MG cells with siRNA/control or survivin transfection, nonirradiated or irradiated at a dose of 4 Gy, were cultured for 3 (72 h) or 5 days (120 h), and were stained according to the manufacturer's instructions after fixation with 4% formaldehyde. The AI was defined as the percentage of TUNEL-positive U251 (A) or D54MG cells (B). (C) TUNEL signals were detected by fluorescence microscopy in the nuclei of some irradiated and siRNA/control-transfected U251MG cells.
Mentions: To determine whether the death of siRNA/survivin-transfected U251MG and D54MG cells after irradiation was due to apoptosis, we performed a TUNEL assay and measured the level of apoptosis based on the TUNEL positivity on days 3 (72 h) and 5 (120 h) either with or without irradiation. Figure 8A and B show the percentage of TUNEL-positive cells. Irradiated cells that were transfected with siRNA/control had a higher proportion of TUNEL-positive cells than the corresponding nonirradiated cells in both the cell lines on days 3 and 5. However, irradiated cells that were transfected with siRNA/survivin had the same proportion of TUNEL-positive cells as the corresponding nonirradiated cells in both the cell lines on days 3 and 5. As shown in Figure 8C, the irradiated siRNA/control-transfected cells were identified as scattered clusters of fluorescent staining, suggesting the death of these cells to be due to apoptosis in both the cell lines. In contrast, siRNA/survivin-transfected cells did not emit fluorescent signals even after irradiation in both the cell lines. Therefore, the cell death of the siRNA/survivin-transfected cells following irradiation is not considered to be due to apoptosis.

Bottom Line: In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN).As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells.TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. taiichi@hiroshima-u.ac.jp

ABSTRACT
Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.

Show MeSH
Related in: MedlinePlus