Differential signaling mechanisms involving TRAIL-DR4 and DR5 selective variants

Cruz, Marisa Costa da

http://hdl.handle.net/10400.1/4088

Use this identifier to reference this record.

Name:	Description:	Size:	Format:
Tese_TRAIL receptors.pdf		4.44 MB	Adobe PDF	Download

Send Feedback

Authors

Cruz, Marisa Costa da

Advisor(s)

Quax, Wim. J.

Reis, Carlos R.

Power, Deborah

Abstract(s)

TRAIL is a type II transmembrane protein, which can be cleaved from the cell surface to form a soluble ligand. TRAIL can bind to several receptors leading to their activation and the induction of apoptosis in several cancer cells. The protein shows selectivity towards tumor cells while leaving normal cells unharmed and it is considered a potential anti-cancer therapeutic and is currently being evaluated in the clinic. Binding to the death receptors DR4 and DR5 triggers apoptosis in several cancer cell types. TRAIL can also bind to three other receptors which act as devoys of functional cell death. These include DcR1, DcR2 and the secreted OPG receptor. Computational protein design methods have been previously used to generate new improved variants of TRAIL with death receptor selectivity and enhanced agonistic properties, which were further characterized in the present study. This study aims to understand why death receptor selective TRAIL variants with enhanced therapeutic potential for cancer show differential activities in different cell types. Several cell lines respond to only one of the two death receptors implicated in the activation of the so-called apoptosis extrinsic pathway. Using these death receptor TRAIL variants, we characterized the functionality of these receptors (apoptosis) in cancer cells and found differences in internalization mechanisms by both death. In addition, we present several combinations responsible for the re-sensitization towards DR4 and DR5-mediated apoptosis in several cancer cells. By using labelled rhTRAIL wild-type and mutants (DR4 and DR5 selective variants) using different fluophores we accessed the endocytosis profile of specific death receptors in different cell lines, and the binding characteristics of death receptor TRAIL variants was also studied in the context of the cell. Confocal microscopy and flow cytometry techniques as well as combination therapies allowed us to conclude that TRAIL-DR5 mainly follows a dynamin dependent endocytosis route, whereas DR4 follows a dynamin independent route. The apoptotic potential of TRAIL does not seem to be affected by the inhibition of endocytosis, as shown by the impact of cholesterol depletion in several cancer cell lines. Finally, the importance of COX-2 shows that this protein is an important player in the apoptotic pathway mediated by TRAIL, and that ROS production is essential for the downstream signaling cascade mediated via both death receptors.