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[H202] fiM

Fig. 6. Effect of H2O2 on AP-1 activity. Jurkat cells transfected with pAP-luc were stimulated with TPA (2 ng/ml) and varying doses of H2O2 (0 to 100 ¡iM) for 6 hr. Cells were harvested and luciferase activity in the lysate was measured. Data are represented as the percentage of luciferase activity found in TPA-stimulated, H202-untreated cells. Data represent the means of two individual experiments. [Reprinted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rusnak,./. Biol. Inorg. Chem. 4,632 (1999), copyright © 1999, Society of Biological Inorganic Chemistry.]

[H202] fiM

Fig. 6. Effect of H2O2 on AP-1 activity. Jurkat cells transfected with pAP-luc were stimulated with TPA (2 ng/ml) and varying doses of H2O2 (0 to 100 ¡iM) for 6 hr. Cells were harvested and luciferase activity in the lysate was measured. Data are represented as the percentage of luciferase activity found in TPA-stimulated, H202-untreated cells. Data represent the means of two individual experiments. [Reprinted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rusnak,./. Biol. Inorg. Chem. 4,632 (1999), copyright © 1999, Society of Biological Inorganic Chemistry.]

by H2O2 treatment. pAP-luc contains three tandem copies of the AP-1 promoter sequence upstream of the luciferase gene. After pAP-luc transfection, cells were incubated in low serum to prevent activation of AP-1 by serum. Jurkat cells were transfected with this plasmid and treated with TPA and various concentrations of H2O2 for 6 hr. This control experiment shows that AP-1 activity is stimulated by all H2O2 concentrations tested (Fig. 6).

Cell viability experiments should also be performed when treating cells with any potentially cytotoxic compound. An easy viability assay can be performed with trypan blue dye. The viability assay showed minimal cell death after cell exposure to H2O2 for 6 hr (data not shown).

The reporter plasmid assay herein described is an indirect, yet sensitive assay for calcineurin activity in vivo. Calcineurin is a redox-sensitive enzyme and therefore we propose that this assay can be used as a qualitative measure of H202-induced oxidative stress. The assay, however, does have limitations. The assay will not work in cell lines that fail to express either calcineurin or NF-AT. These problems can be circumvented by cotransfection with NF-AT and/or calcineurin expression plasmids. Electroporation has proved to be an easy and effective means of transfecting Jurkat cells with plasmid DNA, whereas other cell lines may require a different means of transfection (liposomes, retrovirus, CaCl2, etc.). This assay could be adapted to yield significant information about calcineurin activity in different human cell lines and provide a readout of hLC^-induced oxidative stress.

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