The Use of C. Elegans to Identify Mutations That Confer Drug Resistance, Using Hemiasterlin as an Example
Cardenas, Eduardo Cardenas
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Hemiasterlin is a natural product isolated from the several species of marine sponges. Hemiasterlin inhibits microtubule polymerization resulting in cell cycle arrest and apoptosis. Cells over-expressing efflux pumps associated with multidrug resistance are not resistant to the toxic effects of hemiasterlin. Because of these characteristics, hemiasterlin or hemiasterlin derivatives have been involved in several clinical trials. The Roth lab set out to discover novel modes of hemiasterlin resistance. To that effect, a mutagenic screen to identify hemiasterlin resistant mutant C. elegans was performed. As a result of this screen, seven independent true-breeding hemiasterlin-resistant worms were identified. Studies in this lab have identified hemiasterlin resistance in these worms is due to a mitochondrial dysfunction that results in elevated levels of reactive oxygen species. Interestingly this mitochondrial dysfunction also results in a multi-drug resistant phenotype. This dissertation describes the identification of the previously unmapped hemiasterlin resistant worm har-2. There is no known homolog or function of HAR-2 outside of other nematode species. HAR-2 has been predicted to be an E3 ligase. Through genetic, biochemical and pharmacological analysis, it is determined that har-2 is not within the same class of previous hemiasterlin-resistant mutants. har-2 does not exhibit a mitochondrial dysfunction leading to elevated reactive oxygen species and a multi-drug resistant phenotype. Interestingly it is hypersensitive to the microtubule polymerizer Paclitaxel. In order to identify possible interacting partners in the hopes of gaining insight into HAR-2 function, a yeast-two-hybrid screen of a C. elegans cDNA library was performed using HAR-2 cDNA as the bait. The yeast-two-hybrid experiment identified Rps-1 most frequently as an interactor with HAR-2. Rps-1 is the homolog to the human S3A small ribosomal protein. Published reports show that increased expression of S3A is correlated with drug resistance and that S3A interacts with the anti-apoptotic factor Bcl-21. The yeast-two-hybrid result identifying S3A as an interactor of HAR-2 suggest a possible mechanism of hemiasterlin resistance in which HAR-2 is involved in the modulation of the interaction of S3A with Bcl-2.