Limits...
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.

Show MeSH

Related in: MedlinePlus

Staurosporine induced apoptosis activation of the caspase cascade in both MCF-7 and T47D cells. (A) Cells were pre-incubated with 100 μM z-VAD-fmk (Z-VAD) for 1 h prior to the addition of 1 μM STS for 16 h. Cells were fixed, permeabilised and DNA fragmentation detected by ISNT. Error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated for different time periods with 1 μM STS. Cell lysates were prepared and incubated with Ac-DEVD-pNA (caspase-3/7). The reaction products were measured at 405 nm after a 4 h incubation. Error bars represent the mean±s.e.m. of four independent experiments where * indicates statistically significant increase in DEVDase activity compared to untreated control (0 h), (ANOVA: F=3.38, P<0.05, d,f 3,12. Tukey multipe comparison test, P<0.05). (C) Full length caspase-6 protein is cleaved in MCF-7 but not in T47D cells after treatment. Cells were treated with 1 μM STS for indicated times, lysates prepared and 50 μg protein subjected to SDS–PAGE followed by Western transfer and probed with caspase-6 antibody. C indicates untreated cells. The results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2376174&req=5

fig3: Staurosporine induced apoptosis activation of the caspase cascade in both MCF-7 and T47D cells. (A) Cells were pre-incubated with 100 μM z-VAD-fmk (Z-VAD) for 1 h prior to the addition of 1 μM STS for 16 h. Cells were fixed, permeabilised and DNA fragmentation detected by ISNT. Error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated for different time periods with 1 μM STS. Cell lysates were prepared and incubated with Ac-DEVD-pNA (caspase-3/7). The reaction products were measured at 405 nm after a 4 h incubation. Error bars represent the mean±s.e.m. of four independent experiments where * indicates statistically significant increase in DEVDase activity compared to untreated control (0 h), (ANOVA: F=3.38, P<0.05, d,f 3,12. Tukey multipe comparison test, P<0.05). (C) Full length caspase-6 protein is cleaved in MCF-7 but not in T47D cells after treatment. Cells were treated with 1 μM STS for indicated times, lysates prepared and 50 μg protein subjected to SDS–PAGE followed by Western transfer and probed with caspase-6 antibody. C indicates untreated cells. The results are representative of three independent experiments.

Mentions: DNA fragmentation is mostly mediated by caspase-3 cleavage of the inhibitory protein DFF45 resulting in release of the endonuclease DFF40 which is then free to cleave DNA (Enari et al, 1998; Wolf et al, 1999). It has been shown that MCF-7 cells lack caspase-3 protein. Caspase-3 expression was also absent in the MCF-7 cells used in this study, however we detected caspase-3 expression in T47D cells (data not shown). Therefore in contrast recent studies where lack of DNA fragmentation in response to TNFα and STS was reported (Janicke et al, 1998b), these results (Figure 3AFigure 3


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 activation of the caspase cascade in both MCF-7 and T47D cells. (A) Cells were pre-incubated with 100 μM z-VAD-fmk (Z-VAD) for 1 h prior to the addition of 1 μM STS for 16 h. Cells were fixed, permeabilised and DNA fragmentation detected by ISNT. Error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated for different time periods with 1 μM STS. Cell lysates were prepared and incubated with Ac-DEVD-pNA (caspase-3/7). The reaction products were measured at 405 nm after a 4 h incubation. Error bars represent the mean±s.e.m. of four independent experiments where * indicates statistically significant increase in DEVDase activity compared to untreated control (0 h), (ANOVA: F=3.38, P<0.05, d,f 3,12. Tukey multipe comparison test, P<0.05). (C) Full length caspase-6 protein is cleaved in MCF-7 but not in T47D cells after treatment. Cells were treated with 1 μM STS for indicated times, lysates prepared and 50 μg protein subjected to SDS–PAGE followed by Western transfer and probed with caspase-6 antibody. C indicates untreated cells. The results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376174&req=5

fig3: Staurosporine induced apoptosis activation of the caspase cascade in both MCF-7 and T47D cells. (A) Cells were pre-incubated with 100 μM z-VAD-fmk (Z-VAD) for 1 h prior to the addition of 1 μM STS for 16 h. Cells were fixed, permeabilised and DNA fragmentation detected by ISNT. Error bars represent the mean±s.e.m. of three independent experiments. (B) Cells were treated for different time periods with 1 μM STS. Cell lysates were prepared and incubated with Ac-DEVD-pNA (caspase-3/7). The reaction products were measured at 405 nm after a 4 h incubation. Error bars represent the mean±s.e.m. of four independent experiments where * indicates statistically significant increase in DEVDase activity compared to untreated control (0 h), (ANOVA: F=3.38, P<0.05, d,f 3,12. Tukey multipe comparison test, P<0.05). (C) Full length caspase-6 protein is cleaved in MCF-7 but not in T47D cells after treatment. Cells were treated with 1 μM STS for indicated times, lysates prepared and 50 μg protein subjected to SDS–PAGE followed by Western transfer and probed with caspase-6 antibody. C indicates untreated cells. The results are representative of three independent experiments.
Mentions: DNA fragmentation is mostly mediated by caspase-3 cleavage of the inhibitory protein DFF45 resulting in release of the endonuclease DFF40 which is then free to cleave DNA (Enari et al, 1998; Wolf et al, 1999). It has been shown that MCF-7 cells lack caspase-3 protein. Caspase-3 expression was also absent in the MCF-7 cells used in this study, however we detected caspase-3 expression in T47D cells (data not shown). Therefore in contrast recent studies where lack of DNA fragmentation in response to TNFα and STS was reported (Janicke et al, 1998b), these results (Figure 3AFigure 3

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