Novel mechanisms involved in the resistance to cytotoxic agents have been recently described both in fission yeast and in mammalian cells. The work described in this thesis was focused on novel determinants of multi-drug resistance in mammalian cells and their possible mechanism of action.
Human Pohl, a highly conserved subunit of the regulatory particle of the 26 S proteasome complex, has been shown to confer moderate resistance to chemotherapeutic drugs and ultraviolet light in mammalian cells by a P-gp-independent mechanism. The mechanism by which HA-Pohl causes drug resistance was investigated. Using constitutive and inducible expression of HA-tagged Pohl in mammalian cells, it was found that most overexpressed Pohl is not associated with the 26S proteasome complex. The cells expressing HA-Pohl exhibited elevated protein levels of the two AP-1 components, c-Jun and c-Fos, associated with an increased stabilisation of c-Jun possibly caused by an interaction between HA-Pohl and c-Jun. Furthermore, HA-Pohl overexpression led to an increase in AP-1 transcriptional and DNA binding activities. As c-Jun and c-Fos are both degraded by the ubiquitin/proteasome pathway, it is proposed that Pohl is able to negatively modulate the ubiquitin-dependent proteolysis of transcription factors with consequent alteration of cellular drug susceptibility.
PWPl is member of the WD-40 repeat protein family and is the closest known human relative of the fission yeast pwpl+ multi-drug resistance gene. It is shown here that overexpression of human Pwpl in mammalian cells leads to multi-drug sensitivity possibly via positive modulation of the AP-1 DNA binding and transcriptional activity. Preliminary data obtained using an RNA anti-sense strategy, together with data obtained elsewhere, suggest that human Pwpl might have a role in the regulation of cell growth
