title: Effects of selective checkpoint kinase 1 inhibition on cytarabine cytotoxicity in acute myelogenous leukemia cells in vitro.
abstract: OBJECTIVE
  Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro.
  
  
  METHODS
  AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays.
  
  
  RESULTS
  Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors.
  
  
  CONCLUSIONS
  These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted.
authors.affiliation: Department of Oncology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Schenk
authors.forename: Brian D
authors.initials: BD
authors.lastname: Koh
authors.forename: Karen S
authors.initials: KS
authors.lastname: Flatten
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Peterson
authors.forename: David
authors.initials: D
authors.lastname: Parry
authors.forename: Allan D
authors.initials: AD
authors.lastname: Hess
authors.forename: B Douglas
authors.initials: BD
authors.lastname: Smith
authors.forename: Judith E
authors.initials: JE
authors.lastname: Karp
authors.forename: Larry M
authors.initials: LM
authors.lastname: Karnitz
authors.forename: Scott H
authors.initials: SH
authors.lastname: Kaufmann
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage; D017209:Apoptosis / Q000187:drug effects; D064007:Ataxia Telangiectasia Mutated Proteins; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D003561:Cytarabine / Q000008:administration & dosage*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011720:Pyrazoles / Q000008:administration & dosage; D011743:Pyrimidines / Q000008:administration & dosage
chemical_list: D018797:Cell Cycle Proteins; C559815:MK-8776; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D003561:Cytarabine; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases