title: Biochemical modulation of aracytidine (Ara-C) effects by GTI-2040, a ribonucleotide reductase inhibitor, in K562 human leukemia cells.
abstract: GTI-2040 is a potent antisense to the M2 subunit of the ribonucleotide reductase (RNR), an enzyme involved in the de novo synthesis of nucleoside triphosphates. We hypothesized that combination of GTI-2040 with the cytarabine (Ara-C) could result in an enhanced cytotoxic effect with perturbed intracellular deoxynucleotide/nucleotide (dNTP/NTP) pools including Ara-C triphosphate (Ara-CTP). This study aims to provide a direct experimental support of this hypothesis by monitoring the biochemical modulation effects, intracellular levels of Ara-CTP, dNTPs/NTPs following the combination treatment of Ara-C, and GTI-2040 in K562 human leukemia cells. GTI-2040 was introduced into cells via electroporation. A hybridization-ligation ELISA was used to quantify intracellular GTI-2040 concentrations. Real-time PCR and Western blot methods were used to measure the RNR M2 mRNA and protein levels, respectively. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay was used to measure the cytotoxicity following various drug treatments. A non-radioactive HPLC-UV method was used for measuring the intracellular Ara-CTP, while a LC-MS/MS method was used to quantify intracellular dNTP/NTP pools. GTI-2040 was found to downregulate M2 mRNA and protein levels in a dose-dependent manner and showed significant decrease in dNTP but not NTP pool. When combining GTI-2040 with Ara-C, a synergistic cytotoxicity was observed with no further change in dNTP/NTP pools. Importantly, pretreatment of K562 cells with GTI-2040 was found to increase Ara-CTP level for the first time, and this effect may be due to inhibition of RNR by GTI-2040. This finding provides a laboratory justification for the current phase I/II evaluation of GTI-2040 in combination with Ara-C in patients with acute myeloid leukemia.
authors.affiliation: College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Josephine
authors.initials: J
authors.lastname: Aimiuwu
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Xie
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Wei
authors.forename: Shujun
authors.initials: S
authors.lastname: Liu
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klisovic
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Chan
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000032:analysis / Q000494:pharmacology*; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003561:Cytarabine / Q000032:analysis / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007202:Indicators and Reagents; D020014:K562 Cells; D013058:Mass Spectrometry; D009711:Nucleotides / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000964:Antimetabolites, Antineoplastic; D007202:Indicators and Reagents; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D003561:Cytarabine; C464617:GTI2040; D012264:Ribonucleotide Reductases