Study Of Ara-C Resistance In Yeasts, AML Cells And Mouse Model

Objective: In yeast, acute myeloid leukemia cells, non-immunodeficient mice model for the study, to look for Ara-C resistance-related genes, study the changes of deoxycytidine kinase, which is Ara-C key genes in metabolic pathways, and of drug-related anti-apoptotic pathways.Methods:(1) The mutagenesis system available with piggyBac(PB) requires that two plasmids be introduced into the strain of S. Pombe. One plasmid contains the transposase gene, The second plasmid introduced into S. pombe contains the donor sequence, which consists of kanMX6 flanked by the inverted terminal repeats, PB right and PB left;(2) YHL912 strain detection sensitivity to G418;(3) Introduce the plasmids containing the donor and expression cassettes separately into the strain of S. pombe using lithium acetate transformation;(4) Cell viability after exposure to Ara-C or ABT-737 were determined by MTT assay;(5) Cell apoptosis were determined by Annexin V/PI assay;(6) In AML mouse model, the combined treatment with Ara-C and ABT-737 impact on the survival of mice.Results:(1) PB transposon system can effectively inserted in the yeast genome;(2) G418 minimum inhibitory concentration to YHL912 yeast strains is 300ug/m L;(3) In Ara-C resistance AML cells, there are point mutations, deletion mutations in the DCK cDNA and reduced transcriptional levels. Moreover, with increased levels of drug-resistant, the proportion of mutations tended to increased;(4) ABT-737 in combination with Ara-C can effectively improve the survival time of mice shortened by cell internal resistance.Conclusions: PB transposon system can effectively inserted in the yeast genome, which makes it serves as an effective mutagenesis tool for genetic screens. The mutations and decreased expressions of DCK in AML resistant cells maybe an important reason for Ara-C chemotherapy resistance. The combined treatment with Ara-C and ABT-737 suppressed leukemia and prolonged the survival to an even greater extent than the group treated with Ara-C alone, which indicats that resistant leukemic cells retain or even upregulate their intrinsic anti-apoptotic mechanisms.