title: Novel leukemic cell lines resistant to clofarabine by mechanisms of decreased active metabolite and increased antiapoptosis.
abstract: Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA-resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20-fold and 80-fold more CAFdA-resistant than HL-60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL-60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL-60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4-h incubation with 10 μM CAFdA was 20 pmol/10(7) cells in HL/CAFdA20 and 3 pmol/10(7) cells in HL/CAFdA80 compared with 63 pmol/10(7) cells in HL-60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA-induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL-60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.
authors: Division of Hematology and Oncology, University of Fukui, Eiheiji, Japan.
authors: Hiroko
authors: H
authors: Shigemi
authors: Takahiro
authors: T
authors: Yamauchi
authors: Yukie
authors: Y
authors: Tanaka
authors: Takanori
authors: T
authors: Ueda
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000378:metabolism*; D001087:Arabinonucleosides / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor / Q000166:cytology / Q000378:metabolism; D000077866:Clofarabine; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000227:Adenine Nucleotides; D000970:Antineoplastic Agents; D001087:Arabinonucleosides; D000077866:Clofarabine