title: Cellular uptake and intracellular levels of the bcl-2 antisense g3139 in cultured cells and treated patients with acute myeloid leukemia.
abstract: OBJECTIVE
  Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated. This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation.
  
  
  METHODS
  Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISA-based assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells.
  
  
  RESULTS
  The assay was fully validated and showed a limit of quantification of 50 pmol/L. When exposed to 0.33 to 10 mumol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10- to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139.
  
  
  CONCLUSIONS
  This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.
authors.affiliation: Division of Pharmaceutics, College of Medicine and Public Health, Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Guowei
authors.initials: G
authors.lastname: Dai
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Chan
authors.forename: Shujun
authors.initials: S
authors.lastname: Liu
authors.forename: Dale
authors.initials: D
authors.lastname: Hoyt
authors.forename: Susan
authors.initials: S
authors.lastname: Whitman
authors.forename: Marko
authors.initials: M
authors.lastname: Klisovic
authors.forename: Tiansheng
authors.initials: T
authors.lastname: Shen
authors.forename: Michael A
authors.initials: MA
authors.lastname: Caligiuri
authors.forename: John
authors.initials: J
authors.lastname: Byrd
authors.forename: Michael
authors.initials: M
authors.lastname: Grever
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
mesh_terms: D000208:Acute Disease; D001483:Base Sequence; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D020014:K562 Cells; D007951:Leukemia, Myeloid / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013873:Thionucleotides / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*; D013997:Time Factors
chemical_list: D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D013873:Thionucleotides; C408162:oblimersen