Effect of acute HSP70 overexpression on activation of effector caspase-3 The initiator caspase-8 can directly activate the effector caspase-3 by proteolytic cleavage

Caspase-3 can also be activated by factors of the intrinsic apoptotic pathway. Due to their nuclear localisation sequence ICAD and CAD translocate into the nucleus (Neimanis et al. 2007) where CAD is released from its inhibitor ICAD by caspase-3 and CAD then induces DNA fragmentation. The flow cytometric analysis of caspase-3 is based on an increase in the percentage of cells positive for active caspase-3. To determine, whether the acute overexpression of HSP70 has an effect on GrB-induced caspase-3 activation, cells were treated with doxycycline for 24 hrs and apoptosis was induced by the addition of

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GrB. The activation of the effector caspase-3 was measured by intracellular flow cytometry with an antibody just detecting the active cleaved form of caspase-3 (figure 5.23). The experiments for the activation of caspase-3 were performed with the same cells as the experiments for the release of cytochrome c and therefore the induction of HSP70 was identical and is visualised in part A of figure 5.20 on page 89.

Figure 5.23: Acute HSP70 overexpression did not influence the activation of caspase-3 by GrB-induced apoptosisThe acute overexpression of HSP70 was induced in Ge-tet clones upon the addition of doxycyline (dox) for 24 hrs as determined by intracellular flow cytometry. Control cells (co) were left untreated. Apoptosis was induced using 1 ng/µl human GrB and AdV-β-gal (+AdV +GrB), or as a control just the AdV-β-gal was added (+AdV). Cells were harvested, fixed, and analysed by intracellular flow cytometry using an antibody specific for caspase-3. 5 independent experiments were performed with Ge-tra and Ge-tet-1 and 4 independent experiments with Ge-tet-2 cells. Error bars indicate SD.

No difference in the activation of caspase-3 after the acute overexpression of HSP70 was found in GrB-induced apoptosis. It must be noted, that the overall activation of caspase-3 was low in the Ge-tet-2 clone, in contrast to the Ge-tra and the Ge-tet-1 clone and that the SD were quiet high in the Ge-tra and the Ge-tet-1 clone.

The activation of caspase-3 was also determined after staurosporine-induced apoptosis.

Ge-tra and Ge-tet cells were treated with doxycycline for 24 hrs to induce HSP70 in both Ge-tet clones (part A of figure 5.24 on the next page). Apoptosis was induced by the addition of staurosporine for 20 hrs. The percentage of cells with activated caspase-3 was determined by intracellular flow cytometry (part B of figure 5.24 on the following page).

Strikingly, the acute overexpression of HSP70 reduced the percentage of cells with acti-vated caspase-3 in Ge-tet-1 cells significantly. Statistical analysis using the Wilcoxon-test determined a significant p-value of 0.01 for Ge-tet-1. The percentage of cells with

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Figure 5.24: Acute HSP70 overexpression significantly decreased the percentage of cells with active caspase-3 by staurosporine-induced apoptosisCells were treated with doxycycline and subsequently apoptosis was induced by the addition of staurosporine A The acute overexpression of HSP70 was induced in Ge-tet clones upon the addition of doxycyline (dox) for 24 hrs as determined by intracellular flow cytometry. Control cells (co) were left untreated. B Apoptosis was induced with 1 µM staurosporine (+Stau) for 20 hrs. The percentage of cells with activated caspase-3 was determined by intracellular flow cytometry with an antibody specific for the activated form of caspase-3. 14 independent experiments with Ge-tra, 10 independent experiments with Ge-tet-1 and 11 independent experiments with Ge-tet-2 cells contributed to the mean values shown. Error bars indicate SD. Statistical analysis using the Wilcoxon-test and anαof 0.05 was performed. An asterisk (*) indicates a significant p-value.

vated caspase-3 was unchanged in Ge-tet-2 cells after staurosporine-induced apoptosis (p

= 1.00). However, also in Ge-tra cells, the percentage of cells with active caspase-3 was significantly decreased upon addition of doxycycline (p = 0.01).

Caspase-3 is the key player in apoptosis, as it can be activated by the extrinsic and

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the intrinsic pathway and can induce DNA fragmentation through cleavage of ICAD, and therefore its activation was further analysed. GrB is just one component of the cytotoxic granules of CTLs and NK cells and we wanted to investigate whether apoptosis induced by cytotoxic cells would affect the percentage of cells with active caspase-3.

Apoptosis was induced by co-incubation of Ge cells with NK cells. To distinguish the activation of caspase-3 in tumour cells from the one in NK cells, tumour cells were stained with DiD beforehand. By evaluating the activation of caspase-3 only in DiD-positive cells (part C of figure 5.25), the percentage of tumour cells with active caspase-3 could be determined (part D of figure 5.25).

Figure 5.25: Flow cytometric analysis of the activation of caspase-3 in NK cell-mediated apoptosisTarget cells were stained with DiD before apoptosis was induced by NK cells. AEither target cells were incubated in the absence of NK cells (0:1), orBNK cells were added in an effector-target ratio of 5:1 (5:1). CA marker was set on all DiD-positive cells and was applied as a gate to the analysis of active caspase-3DThe percentage of DiD-positive cells with active caspase-3 is shown.

It was examined, whether NK cell-mediated activation of caspase-3 was influenced by the acute overexpression of HSP70. Human NK cells were generated from whole blood and stimulated for 4 days with IL-2. Ge-tra and Ge-tet-1 cells were treated with doxycycline for 24 hrs. The acute overexpression of HSP70 in Ge-tet-1 cells was confirmed (part A of figure 5.26 on the next page). Both Ge clones were stained with DiD to be able to distinguish them from unstained NK cells in flow cytometric analysis with an antibody specific for activated caspase-3 (part B of figure 5.26 on the following page). For measuring

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the activation of caspase-3, killer and target cells were co-incubated at a ratio of 5:1 for 4 hrs, or target cells were incubated in the absence of NK cells (0:1).

Figure 5.26: Acute HSP70 overexpression did not change the activation of caspase-3 by NK cell-induced apoptosisAcute induction of HSP70 in Ge-tet-1 cells and NK cell-induced apoptosis in these cells with flow cytometric measurement of activation of caspase-3. 4 independent experiments were performed with error bars indicating SD. A Confirmation of acute HSP70 overexpression in the Ge-tet-1 clone after addition of doxycycline (dox). Control (co) cells were left untreated. BHuman NK cells and DiD-stained target cells treated with doxycycline (dox) or as control (co) left untreated were incubated at a ratio of 5:1. As a control, target cells were incubated without killer cells (ratio of 0:1). After 4 hrs of (co-)incubation the activation of caspase-3 in target cells was analysed by intracellular flow cytometry by gating on DiD-stained cells using an antibody specific for the activated form of caspase-3.

The results of the activation of caspase-3 after NK cell-induced apoptosis showed for both clones, Ge-tra and Ge-tet-1, at both ratios, 0:1 and 5:1, that the addition of

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cycline not markedly altered the activation of caspase-3. A statistical analysis could not be done due to the small number of experiments performed.

Thus, no strong effects of the acute overexpression of HSP70 could be detected on activa-tion of caspase-3 by different means. There rather seemed to be a tendency implying that the effect of acute HSP70 overexpression seemed to be opposite in GrB and staurosporine-induced apoptosis in the Ge-tet-1 clone. This effect did not seem to be important for NK cell-mediated activation of caspase-3.

5.1.4.5 Effect of acute HSP70 overexpression on DNA fragmentation analysed by