Rapamycin Inhibits Pre-B Acute Lymphoblastic Leukemia Cells By Downregulating DNA And RNA Polymerases

Objective:To explore the potential therapeutic efficiency of rapamycin in growth inhibition ofpre-B acute lymphoblastic leukemia cells and the underlying mechanism.Methods:(1) Use trypan blue, CCK-8kit, CFSE staining and PI staining by flowcytometry to analyse the effect of rapamycin on the697pre-B acute lymphoblasticleukemia cell’ growth, cell viability, cell proliferation and cell cycle.(2) Check cell death including apoptosis of different treated groups usingPI/Anexxin-Ⅴ staining, and construct GFP-LC3stably expression697cell line to checkautophagy and mTOR signaling of rapamycin treated cells by flow immunofluorescenceand western blotting.(3) Detect changes of cell cycle regulatory factors in response to rapamycintreatment using Cell Cycle RT2Profiler PCR Array.(4) Check whether there are rapamycin-induced changes in cellular DNA and RNAcontent using methyl-green pyronin staining by flow cytometry. Use quantitativereal-time PCR and western blotting to measure rapamycin-induced changes of DNA andRNA polymerases which determine DNA duplication and RNA translation.(5) Check whether DNA polymerases and RNA polymerases are colocalized withautophagosomes by flow immunofluorescence after the cells are treated withrapamycin.Results:(1) Rapamycin inhibited proliferation and induced cell cycle arrested at G0/G1ofpre-B ALL cells.(2) Rapamycin activated autophagy but not apoptosis in pre-B ALL cells.(3) Rapamycin downregulated cell cycle positive regulators. (4) Rapamycin downregulated DNA and RNA levels through downregulating DNAPol δ1and RNAPol Ⅱ in pre-BALL cells.(5) Rapamycin caused colocalization of DNA and RNA polymerases inautolysosomes in pre-B ALL cells.Conclusion:Rapamycin activates autophagy to inhibit the proliferation and growth of697cells,and to induce cell cycle arrest of pre-B ALL cells via targeting DNA polymerases δ1and RNA polymerase Ⅱ.