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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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Semithin resin sections (1 μm) stained with toluidine blue to show morphology of control HBL-100 cells (A), after 24 h treatment with 3 mM H2O2 (B), or 50 μM SSP (C). The H2O2-treated cells appeared swollen, the cell membranes disrupted and the nuclei appeared pyknotic, whereas the SSP-treated cells were shrunken with condensed nuclear material. At the ultrastructural level, the nuclear material appeared to have fragmented to give electron dense bodies (D). Magnification bars A–C=0.1 mm, D=5  μM.
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fig2: Semithin resin sections (1 μm) stained with toluidine blue to show morphology of control HBL-100 cells (A), after 24 h treatment with 3 mM H2O2 (B), or 50 μM SSP (C). The H2O2-treated cells appeared swollen, the cell membranes disrupted and the nuclei appeared pyknotic, whereas the SSP-treated cells were shrunken with condensed nuclear material. At the ultrastructural level, the nuclear material appeared to have fragmented to give electron dense bodies (D). Magnification bars A–C=0.1 mm, D=5  μM.

Mentions: Dose response assays (assessed by NR) for the HBL-100 and the T47D cell lines over a 24 h period. (A) Dose response of SSP-treated cells using the range from 10 pM to 50 μM. LD100 (no significant fraction of viable cells remaining)=50 μM. (B) Dose response of H2O2-treated cells using the range from 0.1 to 3 mM. The LD100 (no significant fraction of viable cells remaining)=3 mM.


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

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

Semithin resin sections (1 μm) stained with toluidine blue to show morphology of control HBL-100 cells (A), after 24 h treatment with 3 mM H2O2 (B), or 50 μM SSP (C). The H2O2-treated cells appeared swollen, the cell membranes disrupted and the nuclei appeared pyknotic, whereas the SSP-treated cells were shrunken with condensed nuclear material. At the ultrastructural level, the nuclear material appeared to have fragmented to give electron dense bodies (D). Magnification bars A–C=0.1 mm, D=5  μM.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Semithin resin sections (1 μm) stained with toluidine blue to show morphology of control HBL-100 cells (A), after 24 h treatment with 3 mM H2O2 (B), or 50 μM SSP (C). The H2O2-treated cells appeared swollen, the cell membranes disrupted and the nuclei appeared pyknotic, whereas the SSP-treated cells were shrunken with condensed nuclear material. At the ultrastructural level, the nuclear material appeared to have fragmented to give electron dense bodies (D). Magnification bars A–C=0.1 mm, D=5  μM.
Mentions: Dose response assays (assessed by NR) for the HBL-100 and the T47D cell lines over a 24 h period. (A) Dose response of SSP-treated cells using the range from 10 pM to 50 μM. LD100 (no significant fraction of viable cells remaining)=50 μM. (B) Dose response of H2O2-treated cells using the range from 0.1 to 3 mM. The LD100 (no significant fraction of viable cells remaining)=3 mM.

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