3 MATERIALS AND METHODS
4.1 Death receptor agonist-induced apoptosis
4.1.1 HeLa model for death receptor-mediated apoptosis
The cervix carcinoma derived HeLa cell line is known as a good model for apoptosis induced by death receptor agonists. According to literature 41, TNFα was used to induce apoptosis in sensitized HeLa cells in most of experiments. Since TNFα induces both an apoptotic and anti-apoptotic branch of signal transduction, sensitization by inhibition of either transcription or translation is necessary to shift balance towards apoptosis. This study revealed that 100µM of the translation inhibitor cycloheximide (CHX) was suitable (data not shown). Because this work contributes to inhibition of apoptosis, all following experiments were performed with that combination.
DEVD cleavage activity [pmol/(min⋅mg)] Cytotoxicity [%]
Figure 4.1 | Induction of apoptosis in HeLa cells
(A) Concentration-dependent induction of apoptosis by TNFα in sensitized HeLa cells. Cells were treated with 100µM CHX 30 minutes prior addition of TNFα as indicated. After 18h, cytotoxicity was determined. (B) Time course of cytotoxicity (■) and caspase-3-like activity (□) after treatment of HeLa cells with 100ng/ml TNFα in the presents of 100µM CHX. Data represents mean ± SEM.
Apoptosis was induced in a concentration-dependent manner (Figure 4.1A) and 100 ng/ml TNFα is required to induce a maximal apoptosis rate in sensitized cells. Next, the kinetics of apoptosis and caspase-3-like activity were evaluated. For measurement of caspase activity and determination of cytotoxicity 3 and 18 hours
4 RESULTS
were suitable (Figure 4.1B), respectively. In addition, cytotoxicity seemed to be a consequence of caspase activity.
4.1.2 Apoptosis after caspase arrest
Investigations in the hepatoma carcinoma cell line HepG2 revealed that caspase inhibition with the broadband inhibitor zVAD-fmk did not protect cells 37. Therefore, investigations whether HeLa cells also could undergo apoptosis-like cell death under caspase arrest was one object in this study.
4.1.2.1 Caspase inhibition with zVAD-fmk
To test the hypothesis that HeLa cells could undergo cell death without caspase activity, experiments were performed to determine whether cytotoxicity directly was accompanied by caspase-3-like activity.
C 0 25 50 75
0.01 0.1 1 10 100 0 25 50 75 100
zVAD-fmk [µM]
DEVD cleavage activity [pmol/(min⋅mg)] Cytotoxicity [%]
Figure 4.2 | Effect of zVAD-fmk on caspase activity and cytotoxicity
Indicated amounts of zVAD-fmk were added together with 100µM CHX 30 minutes prior addition of 100ng TNFα/ml. Caspase-3-ike activity (□) and cytotoxicity (■) were measured 3 and 18 hours after induction, respectively. Data represent mean ± SEM.
As shown in Figure 4.2, inhibition of caspase-3-like activity by the caspase broadband inhibitor zVAD-fmk did not result in inhibition of cytotoxicity. Only high inhibitor concentrations conferred protection to cells. Because low concentrations of zVAD-fmk already inhibited caspase activity efficiently, the observed protection might be due to unspecific effects at high concentration of the inhibitor. These observations indicate that a second pathway of cell death independent of caspase activity exists in HeLa cells.
4 RESULTS
Furthermore, it can be assumed that this second pathway is a common phenomenon for death receptor induced apoptosis in HeLa cells since the death receptor agonists TRAIL and αCD95 had the same effect. The effective concentration (EC50) values of inhibition of cytotoxicity and the corresponding inhibitory concentration (IC50) values of the caspase inhibition are compiled in Table 4.1. For protection of cells, more than 10times higher concentrations were necessary as compared with inhibition of caspase activity. Moreover, at 1µM zVAD-fmk, caspase activity was abolished completely while cytotoxicity was only reduced to some extent. For this reason, this concentration of zVAD-fmk was used for caspase arrest in the following experiments.
IC50 EC50
(Inhibition of caspases) (Inhibition of cytotoxicity)
[µM zVAD-fmk] ratio
TNFα 0.14 ± 0.03 4.3 ± 0.9 1:31
TRAIL 0.17 ± 0.03 2.3 ± 0.4 1:13
αCD95 0.16 ± 0.04 1.9 ± 0.8 1:12
Table 4.1 | Comparison of IC50 and EC50 values of TNFα, TRAIL, and αCD95
Comparison of the IC50 values of caspase inhibition and the EC50 values of inhibition of cytotoxicity in sensitized HeLa cells. TNF, TRAIL, and αCD95 were applied 30 minutes after indicated zVAD-fmk concentrations and 100µM CHX in the concentrations 100ng/ml, 10ng/ml, and 50ng/ml, respectively.
Data are given as mean ± std. error of at least three experiments.
4.1.2.2 Caspase inhibition with zDEVD-fmk
In addition, the inhibitor of caspase-3-like activity zDEVD-fmk was used together with TNFα to compare the result with that of zVAD-fmk experiments. Analyses with zDEVD-fmk revealed that the ratio between inhibition of caspases and inhibition of cytotoxicity is more prominent, about 1:200 (data not shown).
4 RESULTS
4.1.2.3 Protective effects of serine protease inhibitors
Because HeLa cells are regarded as a good model for caspase-dependent apoptosis, it is astonishing to detect caspase-independent cell death. To define this newly discovered way to death in HeLa cells, we tested other protease inhibitors next.
4.1.2.3.1 Serine protease inhibitors: protection after caspase arrest According to the situation revealed in HepG2 37, serine protease inhibitors were employed for the characterization of this death style. Figure 4.3 depicts the results for these experiments. Whereas the unspecific serine protease inhibitors TLCK (Trypsin-like proteases) and AEBSF (broad range of serine proteases) had no inhibitory effect on the caspase-mediated cell death, they have proved to inhibit cell death after caspase-arrest significantly. Light microscopic analysis confirmed these results (data not shown). Thus, we could describe this cell death as TLCK/AEBSF-sensitive.
Control TLCK AEBSF
0 25 50 75
100 Vehicle
zVAD-fmk
*** ***
Cytotoxicity [%]
Figure 4.3 | Serine protease inhibitors: inhibition of cell death after caspase arrest
Effects of serine protease inhibitors on cell death in HeLa cells treated with saline, TLCK (75µM), or AEBSF (300µM), the vehicle (DMSO), or zVAD-fmk (1µM), and CHX (100µM). TNFα (100ng/ml) was added after 30 minutes and cytotoxicity was determined by MTS reduction after 18 hours. Data represents mean ± SEM. Two-way ANOVA, Bonferroni post-test vehicle vs. zVAD-fmk.
4 RESULTS