Figure 6: TDP1/TOP1 ratio predicts the cellular response of SCLC to topotecan. (A) Whole cell extracts (40 μg) from the indicated SCLC cell lines were separated by 10 % SDS-PAGE and immunblotted using antibodies against TDP1, TOP1 and actin. A quantitation of relative TDP1 (B) and TOP1 (C) protein level is shown. Data are the average of three independent experiments ± STD normalised to HCC33 for TDP1 and H510 for TOP1. (D) The indicated SCLC cell lines were seeded in triplicate at 10,000 cells per well in a 96-well plate in 100 μl media containing the indicated concentration of topotecan (TPT) and incubated at 37 °C for 4 days. Viability was assessed by the CellTiter-Blue viability assay. Fluorescence was determined using a GloMax Multi Detection System (Promega, Southamption, UK) at excitation and emission wavelengths of λex 560 nm and λem 590 nm, respectively. For each cell line, percentage viability (%) was calculated as [(Fluorescence readtreatment/Fluorescence readuntreated)*100%] and the graphical representation shows the results obtained from three independent experiments ± STD. (E) Graphical representations depicting Pearson’s correlation coefficient (r) between SCLC viability at 120 nM TPT treatment and TDP1/TOP1 levels. The solid line represents the correlation coefficient for all 10 SCLC cell lines whereas the dotted line represents the correlation coefficient for the eight SCLC cell lines HCC33, N417, H2171, H209, H510, H82, H146 and H345, excluding H69 and H187 (triangles), which exhibit high levels of TDP1 and an unexpected hypersensitivity to topotecan.