Evaluating BRCA2 Variant Sensitivity to PARP Inhibitors

DLD1 BRCA2 (−/−) cells harboring 20 variants of BRCA2 cDNA and empty vector, untransduced DLD1 BRCA2 (−/ −) cells, and DLD1 parental cells were seeded in 96-well plates at a density of 2.0 × 10³ cells/well with 100 µl of medium/well, and each drug was added at various concentrations: olaparib (50 nM–5 µM, Selleckchem), niraparib (10 nM–1 µM, Selleckchem), rucaparib (50 nM–5 µM, LC Laboratories), and CBDCA (50 nM–5 µM, Selleckchem). DMSO (Nacalai Tesque) was added to a final concentration of 0.01% (volume/volume) to wells without drugs. Ten microliters of PrestoBlue cell viability reagent (Thermo Fisher Scientific) was added to each well 144 h after exposure to these drugs, and fluorescence intensity was measured with a 2030 ARVO X3 microplate reader and PerkinElmer 2030 Software v4.0 (PerkinElmer) (excitation; 530 nm, emission; 590 nm)^{55 (link)}. Wells without cells were assessed as the negative controls, and survival data were graphically analyzed as a sigmoid curve by GraphPad Prism software v8.02 for Mac (GraphPad Software, Inc.).