title: Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells.
abstract: OBJECTIVE
  This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph(+)) cells.
  
  
  METHODS
  shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining.
  
  
  RESULTS
  Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0-10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells.
  
  
  CONCLUSIONS
  These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment.
authors.affiliation: Section of Experimental Haematology, Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, University of Glasgow, Glasgow, UK.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Myssina
authors.forename: G Vignir
authors.initials: GV
authors.lastname: Helgason
authors.forename: Alan
authors.initials: A
authors.lastname: Serrels
authors.forename: Heather G
authors.initials: HG
authors.lastname: Jørgensen
authors.forename: Ravi
authors.initials: R
authors.lastname: Bhatia
authors.forename: Hardik
authors.initials: H
authors.lastname: Modi
authors.forename: Janet W
authors.initials: JW
authors.lastname: Baird
authors.forename: Joanne C
authors.initials: JC
authors.lastname: Mountford
authors.forename: Ashley
authors.initials: A
authors.lastname: Hamilton
authors.forename: Mirle
authors.initials: M
authors.lastname: Schemionek
authors.forename: Steffen
authors.initials: S
authors.lastname: Koschmieder
authors.forename: Valerie G
authors.initials: VG
authors.lastname: Brunton
authors.forename: Tessa L
authors.initials: TL
authors.lastname: Holyoake
mesh_terms: D017304:Annexin A5 / Q000096:biosynthesis / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D053148:Caspase 3 / Q000096:biosynthesis / Q000235:genetics; D000069439:Dasatinib; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus; D010677:Philadelphia Chromosome; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013844:Thiazoles / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D017304:Annexin A5; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D012313:RNA; D016044:Fusion Proteins, bcr-abl; C505407:CASP3 protein, human; D053148:Caspase 3; D000069439:Dasatinib