Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

KUL'TIVIROVANIE V BESSYVOROTOChNOY SREDE USILIVAET TsISPLATIN-INDUTsIROVANNUYu PROGRAMMIRUEMUYu GIBEL' OPUKhOLEVYKh KLETOK NEZAVISIMO OT AUTOFAGII КУЛЬТИВИРОВАНИЕ В БЕССЫВОРОТОЧНОЙ СРЕДЕ УСИЛИВАЕТ ЦИСПЛАТИН-ИНДУЦИРОВАННУЮ ПРОГРАММИРУЕМУЮ ГИБЕЛЬ ОПУХОЛЕВЫХ КЛЕТОК НЕЗАВИСИМО ОТ АУТОФАГИИ