Hemmingsson O1, Still M1, Kao G2, Tuck S2 and Naredi P1. Background: Cisplatin is a widely used platinum-based drug against solid tumors in humans. However, the cytotoxic effect is limited by aquired resistance. Cross-resistance is reported between cisplatin and other metal salts, including arsenite and antimonite. Reduced accumulation of cisplatin and arsenite is found in resistant cells, suggesting a shared transport mechanism. In bacteria, the ars-pump is responsible for efflux of arsenite and antimonite. The catalytic subunit of the Ars-pump is an ATPase termed ArsA. ASNA-1 is the eukaryotic homologue of ArsA. Reduced ASNA1 expression in human cells results in increased sensitivity to cisplatin and arsenite. ZK637.5, the C. elegans counterpart of human ASNA1, encodes a predicted 342 residue protein with an ATP binding site and 53% homology to human ASNA1. The purpose with this study is to establish C. elegans as a model for toxicity trials and to study the effect of depleted ASNA-1 expression on nematode sensitivity to cisplatin. Methods: Double stranded C. elegans
asna-1 RNA is injected into the gonads of adult C. elegans worms and their progeny is studied. In addition, two
asna-1 deletion mutants,
sv42 and
ok938 are used. A NheI/KpnI cassette containing the full length C. elegans
asna-1 cDNA is used when placing the gene in front of the P
elt-2, P
unc-119 and P
daf-28 promoters. Worms are transferred to metal containing plates and grown at 20C for 24 hours. The number of living and dead worms is determined. A binary logistic regression model is used for statistical analysis. Results: The ASNA-1 null phenotype in RNAi treated L1 worms includes a statistically significant increase in sensitivity to cisplatin, arsenite and antimonite but not to zinc chloride or cadmium chloride. Cisplatin IC50 for ASNA-1 null phenotype L1 worms is 197g/ml compared to 360g/ml in wild type L1s (p<0.001). Adult wild type worms are resistant to cisplatin at the maximal concentration of 500g/ml cisplatin in agar. Adult
asna-1 deletion mutants
sv42 and
ok938 are sensitive to cisplatin, displaying IC50 242g/ml and 248g/ml respectively. The sensitivity to cisplatin at 300g/ml in
sv42 is completely rescued by expression of C. elegans
asna-1 (p<0.001). Three tissue specific promotors for
asna-1 (intestinal, neuronal and
daf-28) were tested and all rescued the phenotype. Expression of human ASNA1 in the
sv42 background improved the survival from 56% to 86% at 300g/ml cisplatin (p<0.05). We conclude that depletion of ASNA1 results in a phenotype of increased sensitivity to cisplatin, arsenite and antimonite. This phenotype is rescued by expression of C. elegans ASNA-1 or human ASNA1. These findings are consistent with our earlier observations that cells selected for resistance to cisplatin exhibit cross resistance to arsenite and antimonite. We propose that ASNA-1 can be a target to increase cisplatin sensitivity and that C. elegans can be a suitable model to identify proteins involved in cisplatin toxicity.