Acquired mutations in the BH3 domain of Bcl2 conferring resistance to ABT-199. (A) Scheme showing the generation of the resistant murine model. A detailed description is provided in “Methods.” (B) Cell lines were incubated with the BH3 mimetics for 48 hours; cell proliferation was measured using the MTS assay. Ly2Bcl2-6 and Ly2Bcl2-9 resistant clones did not show activation of the mitochondrial apoptotic pathway upon exposure to the BH3 mimetics, as shown by the annexin V-FITC measurement and the lack of caspases 3 and 9 cleavage. A representative example from 3 independent experiments is shown. (C) Treatment with 1 µM ABT-199 induced activation of the mitochondrial apoptotic pathway in sensitive but not in resistant cells. Expression analysis of Bcl2 family members by western blot (D) or quantitative real-time PCR (E). (F) Resistant cell lines cultured without ABT-199 (labeled as SA) remained resistant to the BH3 mimetics. (G-H) The protein expression profile in the SA cells was similar to those of the sensitive cell lines. (C-G) Representative data from 1 of 3 independent experiments performed in triplicate (mean ± SD, where indicated). (I) Sequence of Bcl2 identified 2 de novo missense mutations at the same codon (F101) located within the BH3 domain. These mutations were found in the ectopic Bcl2 mouse gene but not in the endogenous mouse Bcl2. Arrows indicate the nucleotide change.