3 MATERIALS AND METHODS
5.4 Breakdown of mitochondrial membrane potential and apoptosis Early after discovery of XIAP, its protective capability was proven for all conceivable
inducers of apoptosis. It was shown that XIAP could block apoptosis induced by the redistribution of cytochrome c but not its release from mitochondria 81. For cytochrome c release, BH-only proteins forming a pore within the outer mitochondrial membrane 1. In addition, the breakdown of membrane potential was described as an element that contributes to apoptosis induction 158-161. However, the point that mitochondrial depolarization is accompanied by cytochrome c release is contradictory discussed in literature 160.
To investigate whether depolarization of the mitochondria is an early event in execution of cell death in the used model, experiments using the specifically staining of polarized mitochondria, was performed. The experiments revealed that the potential of most cells was intact after three hours despite of about three thirds of cells displayed apoptotic morphology in their nuclei. A decrease of stain was observed after six hours. Because of the high apoptosis rate at three hours, the decrease was probably due to the advanced death of cells. Therefore, general breakdown of mitochondrial potential was rather a consequence than a cause of apoptosis. In literature, several reports supporting the obtained result. For HeLa cells, Gao et al.
showed that cytochrome c release was faster than swelling of mitochondria in UV and TNFα treated cells and this was not caspase-dependent 175. However, Waterhouse et al. and others described a reduction of membrane potential-dependent staining of mitochondria, which could partially be prevented by caspase inhibition 160,162. In addition, they could demonstrate that cytochrome c was even able to maintain mitochondrial potential and ATP generation when released into cytosol. Strikingly, single cells could restore the membrane potential of mitochondria after caspase arrest whereas cytochrome c release was not reversible. Supposing that the reduction of mitochondrial membrane potential was slower than cytochrome c release and dependent on caspase activity, XIAP was probably able to bridge the time for regeneration of cytochrome c in mitochondria. Therefore, XIAP could rescue cells even with released cytochrome c.
In conclusion, HeLa cells showed destroyed nuclei while membrane potential of mitochondria was hardly affected after three hours. These cells with condensed nuclei cannot be rescued and therefore mitochondrial membrane potential is not involved for
5 DISCUSSION
induction of apoptosis in the used model. This does not mean that mitochondria play no role because release of some cytochrome c might be sufficient to induce the intrinsic pathway without destroying membrane potential 175.
6 SUMMARY
6 SUMMARY
Cell death independent of caspase activity and sensitive to serine protease inhibitors has been described for many different cellular systems. However, this type of cell death has not been demonstrated for the cervix carcinoma derived HeLa cell line so far.
In the presented study, the ability of HeLa cells to undergo cell death after caspase arrest was investigated. For this purpose, concentration-dependent caspase inhibitor studies were performed together with analyses of serine protease inhibitors in cell death lacking caspase activity. As a result, induction of apoptosis with TNFα revealed a serine protease inhibitor-sensitive cell death after caspase arrest. Since over-expression of XIAP protects HeLa cells, different XIAP mutants were employed to investigate this serine protease inhibitor-sensitive cell death. Expression of a mutant abolished to bind and inhibit caspases completely failed to protect HeLa cells from TNFα induced apoptosis. This result demonstrates that the caspase binding sites of XIAP are decisive for the inhibitory abilities. To distinguish between caspase-dependent and other functions of XIAP, a mutant lacking the C-terminal RING domain was assayed. This mutant significantly protected cells but protection was accompanied by a dramatic decrease in potency. Since the E3 ubiquitin protein ligase is localized within the RING domain, a XIAP with abolished ligase activity was assayed to characterize this ineffectiveness. This defect caused an ineffectiveness of protection comparable to the complete removal of the RING domain. Finally, experiments combining the caspase inhibitor experiments with the expression of XIAP mutants did not revealed any caspase-independent effects of the protection by XIAP.
These results show that the caspase-binding abilities of XIAP are essential for protection of HeLa cells from caspase independent and dependent cell death.
To investigate whether caspase-8 was able to induce this serine protease inhibitor-sensitive cell death after caspase arrest, experiments were performed, which short-circuited the receptor pathway with an active caspase-8. Over-expression of caspase-8 caused a cell death that was independent of caspase-3-like activity and hardly preventable by XIAP. However, it was sensitive towards serine protease inhibitors after caspase arrest indicating that caspase-8 is able to induce this described cell death in HeLa cells.
7 ZUSAMMENFASSUNG
7 ZUSAMMENFASSUNG
Apoptose, unabhängig von Caspaseaktivität, wurde für verschiedeneZelltypen bereits gezeigt – nicht aber für die von einem Cervixcarcinom stammende HeLa Zelllinie.
Deshalb wurde in der vorliegenden Arbeit untersucht, ob HeLa Zellen, nachdem Caspasen inhibiert wurden, immer noch zu einem apoptotischen Zelltod fähig sind.
Aus diesem Grund wurden vergleichende Studien zur Caspaseaktivität und der entsprechenden Apoptoserate durchgeführt. Diese Studien zeigten, dass HeLa Zellen auch nach einer Inhibition der Caspasen weiterhin in der Lage sind apoptotisch zu sterben. Diese Art des Zelltodes wurde weiter untersucht und dabei konnte gezeigt werden, dass unspezifische Serinproteaseinhibitoren in der Lage sind, nach Inhibition der Caspasen diesen durch TNFα ausgelösten Zelltod zu hemmen.
HeLa Zellen können durch Überexpression von XIAP geschützt werden. Deshalb boten sich verschiedene XIAP Mutanten zur genaueren Untersuchung dieses Zelltodes an. XIAP, das nicht mehr in der Lage war Caspasen zu binden, war auch nicht mehr in der Lage, Zellen zu schützen. Dieses Ergebnis streicht die essentielle Bedeutung der Caspasebindungstellen von XIAP für die protektive Wirkung heraus.
Um Caspase unabhängige XIAP Funktionen von abhängigen zu unterscheiden, wurde ein XIAP ohne RING-Domäne verwendet. Diese Mutante konnte zwar noch HeLa Zellen vor TNFα induziertem Zelltod schützen aber nur mit deutlich geringerer Wirksamkeit. Die Ergebnisse eines XIAPs mit unterbundener Ubiquitinligaseaktivität waren vergleichbar zu denen des XIAPs ohne RING-Domäne. Diese Untersuchungen zusammen mit Untersuchungen, die die XIAP Mutanten mit der Inhibition von Caspasen durch synthetische Caspaseinhibitoren verbanden, zeigt, dass XIAP seine Caspasebindestellen für den Schutz von Zellen benötigt, sogar für den Zelltod ohne Caspaseaktivität.
Um zu untersuchen, ob Caspase-8 in der Lage war, diesen Zelltod zu induzieren, wurde mittels aktiver Caspase-8 der rezeptorvermittelte Apoptoseinduktionsweg kurzgeschlossen. Die Überexpression von Caspase-8 führte zu apoptotischem Zelltod, der unabhängig von Caspase-3-Aktivität war und außerdem nur bedingt von XIAP gehemmt werden konnte. Da dieser Zelltod unter Bedingungen der Caspasehemmung von Serineproteaseinhibitoren gehemmt werden konnte, wird klar, dass Caspase-8 in der Lage ist, in HeLa Zellen diese Art des
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