Abstract Topotecan (TPT) is an agent which targets topoisomerase I (Topo I) and is used in the treatment of platinum-resistant and/or recurrent ovarian cancer. We have previously reported that topotecan inhibits hypoxia inducible factor 1α (HIF1α) stabilization, leading to decreased HIF-1α protein and depression of vascular endothelial growth factor (VEGF) levels in ovarian cancer cell lines. Furthermore, we have shown a Topo I: HIF-1α mRNA association using RNA-immunoprecipitation. The aim of this study was to further elucidate the mechanism by which topotecan knocks down HIF-1 alpha and the nature of its dependence on Topoisomerase I. CAOV3 (high Topo I expressing) ovarian cancer cells were used to carry out Topo I knockdown studies using Topo I siRNA in both normoxic and hypoxic (0.5% O2) conditions. Topo I and HIF-1α transcript levels were determined by quantitative PCR and protein expression was evaluated via western blot analysis. Topo I and HIF-1α protein levels were normalized to B-Actin controls. RT-PCR analysis did not show a significant decrease in HIF-1α transcript levels with Topo I siRNA treatment compared to untreated and negative control siRNA treated cells. An 80% decrease in Topo I protein expression was obtained using 30nM Topo I siRNA for both normoxic and hypoxic conditions. An ∼2.30 fold increase in HIF-1α protein expression was seen in Topo I knockdown cells compared to negative control siRNA treated cells after 800nM topotecan treatment under hypoxic conditions. 800nM TPT yielded an 80% decrease in Topo I protein expression with negative control siRNA treated cells under both normoxic and hypoxic conditions. However, 800nM TPT showed complete inhibition of Topo I protein stabilization in Topo I siRNA treated cells. Our results suggest that Topo I is required for Topotecan to inhibit HIF-1α protein stabilization. Furthermore, they also suggest that Topo I may be modulating its own transcript levels. Further studies are being carried out to verify these findings and will be reported at the AACR conference. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3511.