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Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

McKeague AL, Wilson DJ, Nelson J - Br. J. Cancer (2003)

Bottom Line: In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells.Although complete apoptosis of both cell lines was induced by 50 microM SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h).Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

View Article: PubMed Central - PubMed

Affiliation: School of Biology and Biochemistry, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.

ABSTRACT
The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H(2)O(2)-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5-48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 microM SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

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Related in: MedlinePlus

Comparative analysis of the cytotoxicity assays (MTT and NR) vs the differential dye uptake assay (Hoechst/PI) in the T47D cell line. Graphs (A) 50 μM SSP, (B) 50 nM SSP and (C) 3 mM H2O2 represent the results of the MTT and NR assays over the 0.5 to 48 h time course. Graphs (D) 50 μM SSP, (E) 50 nM SSP (note the scale on the ordinate axis in this figure) and (F) 3 mM H2O2 represent the results of the percentage of apoptotic and necrotic cell death detected by the Hoechst/PI assay over the 0.5–48 h time course. The values of 100% cell death represent when the entire population of cells analysed were apoptotic (graph D) or necrotic (graph F).
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fig5: Comparative analysis of the cytotoxicity assays (MTT and NR) vs the differential dye uptake assay (Hoechst/PI) in the T47D cell line. Graphs (A) 50 μM SSP, (B) 50 nM SSP and (C) 3 mM H2O2 represent the results of the MTT and NR assays over the 0.5 to 48 h time course. Graphs (D) 50 μM SSP, (E) 50 nM SSP (note the scale on the ordinate axis in this figure) and (F) 3 mM H2O2 represent the results of the percentage of apoptotic and necrotic cell death detected by the Hoechst/PI assay over the 0.5–48 h time course. The values of 100% cell death represent when the entire population of cells analysed were apoptotic (graph D) or necrotic (graph F).

Mentions: Comparison between all assays and treatments using the T47D cell line showed that the 50 μM SSP treatment resulted in a marked increase in cell death (Figure 5AFigure 5


Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

McKeague AL, Wilson DJ, Nelson J - Br. J. Cancer (2003)

Comparative analysis of the cytotoxicity assays (MTT and NR) vs the differential dye uptake assay (Hoechst/PI) in the T47D cell line. Graphs (A) 50 μM SSP, (B) 50 nM SSP and (C) 3 mM H2O2 represent the results of the MTT and NR assays over the 0.5 to 48 h time course. Graphs (D) 50 μM SSP, (E) 50 nM SSP (note the scale on the ordinate axis in this figure) and (F) 3 mM H2O2 represent the results of the percentage of apoptotic and necrotic cell death detected by the Hoechst/PI assay over the 0.5–48 h time course. The values of 100% cell death represent when the entire population of cells analysed were apoptotic (graph D) or necrotic (graph F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376787&req=5

fig5: Comparative analysis of the cytotoxicity assays (MTT and NR) vs the differential dye uptake assay (Hoechst/PI) in the T47D cell line. Graphs (A) 50 μM SSP, (B) 50 nM SSP and (C) 3 mM H2O2 represent the results of the MTT and NR assays over the 0.5 to 48 h time course. Graphs (D) 50 μM SSP, (E) 50 nM SSP (note the scale on the ordinate axis in this figure) and (F) 3 mM H2O2 represent the results of the percentage of apoptotic and necrotic cell death detected by the Hoechst/PI assay over the 0.5–48 h time course. The values of 100% cell death represent when the entire population of cells analysed were apoptotic (graph D) or necrotic (graph F).
Mentions: Comparison between all assays and treatments using the T47D cell line showed that the 50 μM SSP treatment resulted in a marked increase in cell death (Figure 5AFigure 5

Bottom Line: In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells.Although complete apoptosis of both cell lines was induced by 50 microM SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h).Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

View Article: PubMed Central - PubMed

Affiliation: School of Biology and Biochemistry, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.

ABSTRACT
The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H(2)O(2)-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5-48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 microM SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

Show MeSH
Related in: MedlinePlus