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Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells.

Mooney LM, Al-Sakkaf KA, Brown BL, Dobson PR - Br. J. Cancer (2002)

Bottom Line: MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved.In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c.Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Studies, Division of Genomic Medicine, Medical School, University of Sheffield, Sheffield S10 2RX, UK.

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Staurosporine induced apoptosis in T47D and MCF-7 human breast cancer cells. (A) Cells were treated with 1 μM STS for the times indicated and apoptosis was determined by DNA fragmentation (ISNT) on fixed and permeabilised cells. Data are presented as per cent DNA fragmentation: error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated with 1 μM STS for the times indicated, lysates were analysed by ELISA according to the manufacturer's instructions. Fold induction in apoptosis is expressed as amount of cytoplasm DNA-histone complexes in treated cells compared to untreated controls. Error bars represent the mean±s.e.m. of three independent experiments where * indicates statistically significant increase in apoptosis compared to untreated control (0 h), (ANOVA: F=5.44, P<0.05, d,f 2,9. Tukey multiple comparison test, P<0.05). (C) Apoptotic nuclear morphology. (i) T47D cells (ii) MCF-7 cells. Cells were treated with 1 μM STS for 16 h, then washed in PBS, fixed, permeabilised and stained with 1 μg ml−1 DAPI. Cells were then analysed by fluorescent microscopy.
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fig1: Staurosporine induced apoptosis in T47D and MCF-7 human breast cancer cells. (A) Cells were treated with 1 μM STS for the times indicated and apoptosis was determined by DNA fragmentation (ISNT) on fixed and permeabilised cells. Data are presented as per cent DNA fragmentation: error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated with 1 μM STS for the times indicated, lysates were analysed by ELISA according to the manufacturer's instructions. Fold induction in apoptosis is expressed as amount of cytoplasm DNA-histone complexes in treated cells compared to untreated controls. Error bars represent the mean±s.e.m. of three independent experiments where * indicates statistically significant increase in apoptosis compared to untreated control (0 h), (ANOVA: F=5.44, P<0.05, d,f 2,9. Tukey multiple comparison test, P<0.05). (C) Apoptotic nuclear morphology. (i) T47D cells (ii) MCF-7 cells. Cells were treated with 1 μM STS for 16 h, then washed in PBS, fixed, permeabilised and stained with 1 μg ml−1 DAPI. Cells were then analysed by fluorescent microscopy.

Mentions: Studies of apoptosis, as determined by DNA fragmentation (ISNT), demonstrated that T47D and MCF-7 cells, when exposed to STS (0.2–1 μM), exhibited a concentration-dependent increase in apoptosis, with maximal effects for both cells being at 1 μM STS (data not shown). Apoptosis was also a time-dependent process. When exposed to 1 μM STS, a significant increase in cells displaying DNA fragmentation was evident at 14 h in T47D cells, yet apoptotic changes were detected as early as 4 h in MCF-7 cells (Figure 1AFigure 1


Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells.

Mooney LM, Al-Sakkaf KA, Brown BL, Dobson PR - Br. J. Cancer (2002)

Staurosporine induced apoptosis in T47D and MCF-7 human breast cancer cells. (A) Cells were treated with 1 μM STS for the times indicated and apoptosis was determined by DNA fragmentation (ISNT) on fixed and permeabilised cells. Data are presented as per cent DNA fragmentation: error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated with 1 μM STS for the times indicated, lysates were analysed by ELISA according to the manufacturer's instructions. Fold induction in apoptosis is expressed as amount of cytoplasm DNA-histone complexes in treated cells compared to untreated controls. Error bars represent the mean±s.e.m. of three independent experiments where * indicates statistically significant increase in apoptosis compared to untreated control (0 h), (ANOVA: F=5.44, P<0.05, d,f 2,9. Tukey multiple comparison test, P<0.05). (C) Apoptotic nuclear morphology. (i) T47D cells (ii) MCF-7 cells. Cells were treated with 1 μM STS for 16 h, then washed in PBS, fixed, permeabilised and stained with 1 μg ml−1 DAPI. Cells were then analysed by fluorescent microscopy.
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fig1: Staurosporine induced apoptosis in T47D and MCF-7 human breast cancer cells. (A) Cells were treated with 1 μM STS for the times indicated and apoptosis was determined by DNA fragmentation (ISNT) on fixed and permeabilised cells. Data are presented as per cent DNA fragmentation: error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated with 1 μM STS for the times indicated, lysates were analysed by ELISA according to the manufacturer's instructions. Fold induction in apoptosis is expressed as amount of cytoplasm DNA-histone complexes in treated cells compared to untreated controls. Error bars represent the mean±s.e.m. of three independent experiments where * indicates statistically significant increase in apoptosis compared to untreated control (0 h), (ANOVA: F=5.44, P<0.05, d,f 2,9. Tukey multiple comparison test, P<0.05). (C) Apoptotic nuclear morphology. (i) T47D cells (ii) MCF-7 cells. Cells were treated with 1 μM STS for 16 h, then washed in PBS, fixed, permeabilised and stained with 1 μg ml−1 DAPI. Cells were then analysed by fluorescent microscopy.
Mentions: Studies of apoptosis, as determined by DNA fragmentation (ISNT), demonstrated that T47D and MCF-7 cells, when exposed to STS (0.2–1 μM), exhibited a concentration-dependent increase in apoptosis, with maximal effects for both cells being at 1 μM STS (data not shown). Apoptosis was also a time-dependent process. When exposed to 1 μM STS, a significant increase in cells displaying DNA fragmentation was evident at 14 h in T47D cells, yet apoptotic changes were detected as early as 4 h in MCF-7 cells (Figure 1AFigure 1

Bottom Line: MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved.In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c.Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Studies, Division of Genomic Medicine, Medical School, University of Sheffield, Sheffield S10 2RX, UK.

Show MeSH
Related in: MedlinePlus