Effect of acute HSP70 overexpression on release of cytochrome c from mi- mi-tochondria

The second hallmark of the intrinsic apoptotic pathway, which was investigated, was the release of cytochrome c from mitochondria. Cytochrome c is a heme protein belonging to the electron transport chain and is loosely associated with the inner mitochondrial

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Figure 5.18: Acute HSP70 overexpression did not change the mitochondrial membrane potential∆Ψafter staurosporine-induced apoptosis Acute induction of HSP70 in Ge-tet clones and staurosporine-induced change of ∆Ψ. AConfirmation of acute HSP70 overexpression in the Ge-tet clones after addition of doxycycline (dox). Control (co) cells were left untreatedB1µM staurosporine (+Stau) was added for 20 hrs for induction of apoptosis. As a control cells were left untreated. Cells were harvested, stained with JC-1 and fixed for flow cytometric measurement. Shown is the percentage of cells with reduced ∆Ψ. Depicted are the mean values of 4 independent experiments with error bars indicating SD.

membrane. In the flow cytometric analysis the release of cytochrome c from mitochondria was evaluated as an increase of cells with a lower content of mitochondrial cytochrome c upon induction of apoptosis by AdV and GrB (Stahnke et al. 2004). This increase in the percentage of cells with a reduced mitochondrial cytochrome c content was determined as shown in figure 5.19 on the next page.

It was tested, whether the acute overexpression of HSP70 had an effect on the release of cytochrome c in GrB-induced apoptosis. Acute HSP70 overexpression was induced in Ge-tet clones after the addition of doxycycline for 24 hrs (part A of figure 5.20 on page 89).

Apoptosis was induced by the addition of AdV-β-gal and GrB for 24 hrs. The release of

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Figure 5.19: Flow cytometric analysis of the release of cytochrome c from mitochondriaApoptosis was induced by the addition of AdV and 1 ng/µl human GrB (+AdV +GrB), or as control, just the AdV (+AdV) was added for 24 hrs. Cells were fixed and stained with a cytochrome c-specific antibody and subsequently analysed by flow cytometry. For the evaluation of the data, the percentage of cells with a lower content of mitochondrial cytochrome c (under the marker) were summarised.

mitochondrial cytochrome c is presented as percentage of cells with reduced mitochondrial cytochrome c content and was determined with a cytochrome c-specific antibody (part B of figure 5.20 on the next page).

There seemed to be a tendency that in GrB-induced apoptosis in the Ge-tet-1 clone the acute overexpression of HSP70 further increased the proportion of cells with reduced mitochondrial cytochrome c. Statistical analysis using the Wilcoxon-test determined a p-value of 0.06 for the Ge-tet-1 clones in GrB induced apoptosis between cells acutely overexpressing HSP70 and cells with normal HSP70 expression. This effect was not seen in the other inducible clone Ge-tet-2 (p = 0.35). Notably, in the Ge-tet-2 clone, the overall amount of cells with reduced mitochondrial cytochrome c content did increase less upon addition of GrB-induced apoptosis compared to both other clones.

Next, it was determined, whether the same or a contrary tendency would be seen upon staurosporine-induced apoptosis. Doxycycline was again added to Ge-tra and Ge-tet clones for the acute overexpression of HSP70 in the inducible Ge-tet clones (part A of figure 5.21 on page 90). Apoptosis was elicited upon the addition of staurosporine for 20 hrs. The percentage of cells with a reduced amount of mitochondrial cytochrome c was analysed by flow cytometry (part B of figure 5.21 on page 90).

The acute overexpression of HSP70 in the Ge-tet-1 and Ge-tet-2 clones showed no protection against staurosporine-induced release of cytochrome c as determined by the percentage of cells possessing a reduced mitochondrial cytochrome c content. Statistical analysis using the Wilcoxon-test did not show a significant decrease for the Ge-tet-1 clone

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Figure 5.20: Acute HSP70 overexpression does not significantly increase the release of cytochrome c from mitochondria after GrB-induced apoptosisAcute induction of HSP70 in Ge-tet clones and GrB-induced apoptosis in these cells with flow cytometric measurement of mitochondrial cytochrome c release afterwards. AConfirmation of acute HSP70 overexpression in the Ge-tet clones. Cells were either treated with doxycycline (dox) for 24 hrs or left untreated (co). BAdV-β-gal and 1 ng/µl human GrB were added for induction of apoptosis. Cells were harvested, fixed, and stained for intracellular flow cytometric analysis with an anti-cytochrome c antibody and a FITC-conjugated secondary one. Depicted is the amount of cells, which shows a lower mitochondrial (mitoch.) cytochrome c (cyt c) content upon induction of apoptosis by GrB and AdV in comparison to living cells. Shown are the mean values of 6 independent experiments of Ge-tra and Ge-tet-2 and 7 independent experiments of Ge-tet-1 with error bars indicating SD.

(p = 0.60). The control clone Ge-tra (p = 1.00) and the other inducible clone Ge-tet-2 (p

= 0.60) showed no difference in the percentage of cells with reduced cytochrome c content upon the addition of doxycycline. Thus, the acute overexpression of HSP70 in the Ge-tet-1 clone showed no significant protection of cells against staurosporine-induced mitochondrial cytochrome c release but a tendency to increase GrB-induced mitochondrial cytochrome

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Figure 5.21: Acute HSP70 overexpression does not protect Ge-tet-1 cells from the release of cytochrome c from mitochondria after staurosporine-induced apoptosisAcute induction of HSP70 in Ge-tet clones and staurosporine-induced apoptosis in these cells with flow cytometric measurement of cytochrome c release afterwards. A Confirmation of acute HSP70 overexpression in the Ge-tet clones. Cells were either treated with doxycycline (dox) for 24 hrs or left untreated (co). B1µM staurosporine (+Stau) was added for 20 hrs for induction of apoptosis. Cells were harvested, fixed, and stained for intracellular flow cytometric analysis with an anti-cytochrome c antibody and a FITC-conjugated secondary one. Depicted is the amount of cells, which shows a lower mitochondrial cytochrome c (cyt c) content upon induction of apoptosis by staurosporine in comparison to living cells. Shown are the mean values of 7 independent experiments of Ge-tra and 6 independent experiments of Ge-tet-1 and 2 with error bars indicating SD.

c release.

5.1.4.3 Effect of acute HSP70 overexpression on activation of initiator caspase-8