CDK8/19 Inhibition Attenuates G1 Arrest Induced by BCR-ABL Antagonists and Accelerates Death of Chronic Myelogenous Leukemia Cells
The aim of the project is investigating the mechanism of synergistic induction of cell death by CDK8/19 and Bcr-Abl inhibitors. Human BCR-ABL-positive chronic myelogenous cell lines K562 (Russian Collection of Cell Cultures, Saint-Petersburg, Russia) were propagated in RPMI-1640 (PanEco, Moscow, Russia) supplemented with 10percent fetal bovine serum (HyClone, GE Healthcare Life Sciences, Chicago, IL), 2 mM L-glutamine, 100 U/ml penicillin and 100 mcg/ml streptomycin (PanEco) at 37C, 5percent CO2 in humidified atmosphere. The K562 cells (2?106 in 10 mL medium) were treated with the vehicle (0.02percent DMSO), 1 mcM IM, 1 mcM SenB or their combination (two replicates per each treatment) for 8 h. Total RNA was extracted with TRI reagent. Four mcg RNA was used to isolate poly(A)-enriched RNA with NEBNext Poly(A) mRNA Magnetic Isolation Module (NE Biolabs, Ipswich, MA) that was immediately used to prepare RNA sequencing libraries with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NE Biolabs). Actinomycin D was used for first strand cDNA synthesis; then cDNA libraries ligated with the Illumina suitable adaptor sequences were generated and amplified with Q5 DNA polymerase (NE Biolabs). After purification from dimers by size selection in the agarose gel the libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA).