title: Reduced drug incorporation into DNA and antiapoptosis as the crucial mechanisms of resistance in a novel nelarabine-resistant cell line.
abstract: BACKGROUND
  Nine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis.
  
  
  METHODS
  In the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting.
  
  
  RESULTS
  CEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells.
  
  
  CONCLUSIONS
  An ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.
authors: Department of Hematology and Oncology, Faculty of Medical Sciences, University of Fukui, 23-3, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. tyamauch@u-fukui.ac.jp.
authors: Takahiro
authors: T
authors: Yamauchi
authors: Kanako
authors: K
authors: Uzui
authors: Rie
authors: R
authors: Nishi
authors: Hiroko
authors: H
authors: Shigemi
authors: Takanori
authors: T
authors: Ueda
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001087:Arabinonucleosides / Q000378:metabolism* / Q000494:pharmacology; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006801:Humans; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; D001087:Arabinonucleosides; C028771:9-arabinofuranosylguanine; C104457:nelarabine; D004247:DNA