title: The MEK inhibitor, PD98059, reduces survival but does not block acute myeloid leukemia blast maturation in vitro.
abstract: The appearance of blasts in acute myeloid leukemia (AML) reflects a shift from cellular processes inducing maturation and cell death to those favouring survival and accumulation. We have monitored changes in the growth factor signalling molecule MAPKinase, in the cytoprotective protein Bcl-2 and in the cell death protein Bax, during maturation of proliferating and non-proliferating AML blasts in vitro. Eighteen AML samples were cultured for 7 d in serum-free medium with or without a supplement of recombinant cytokines comprising c-kit ligand, IL3 and GMCSF. Maturation of AML blasts, as assessed by morphology on Romanowsky-stained slides of 7/18 samples and by changes in surface CD markers on all 18 leukemias, occurred in both the absence and presence of cytokines. Cell numbers decreased to a mean of 71% after 7 d of cytokine-free culture, but increased to 210% in cytokine-supplemented cultures. The proportion of CD15-positive cells, assessed by flow cytometry, increased over 7 d in 17/18 samples, from a mean of 22% to 68% in cytokine-free cultures and to 72% in cytokine-supplemented cultures (p = < 0.0001 for both). By immunofluorescence/flow cytometry, there was no significant change in Bcl-2 over 7 d of culture, while Bax increased, particularly in cytokine-free cultures (2.2-fold), which led to a significant decrease in the Bcl-2/Bax ratio. Immunoblotting demonstrated that ERK was briefly phosphorylated after seeding AML blasts into culture. PD98059, an inhibitor of MAPKinase kinase (MEK) which activates MAPKinase, inhibited this transient ERK phosphorylation but was unable to block maturation as measured by acquisition of CD15 in samples from 12 patients with low starting numbers of CD15-positive cells. PD98059, however, reduced cell numbers in 7-d liquid culture and, in cytokine-supplemented cultures, this was associated with a 1.3-fold increase in Bcl-2 (p = 0.012) and a 1.4-fold increase in Bax (p = 0.02). Overall, these data demonstrate that most leukemic populations can partially differentiate in vitro without the need for cytokines or inducers. The MAPKinase pathway is not required for this maturation, but it does maintain cell viability in the absence or presence of cytokines. A rise in Bcl-2 may not protect AML blasts in the face of elevated Bax.
authors.affiliation: Haematology Department, University Hospital of Wales and University of Wales College of Medicine, Cardiff, UK. BainesP@CF.Ac.UK
authors.forename: P
authors.initials: P
authors.lastname: Baines
authors.forename: J
authors.initials: J
authors.lastname: Fisher
authors.forename: L
authors.initials: L
authors.lastname: Truran
authors.forename: E
authors.initials: E
authors.lastname: Davies
authors.forename: M
authors.initials: M
authors.lastname: Hallett
authors.forename: T
authors.initials: T
authors.lastname: Hoy
authors.forename: A K
authors.initials: AK
authors.lastname: Burnett
mesh_terms: D000208:Acute Disease; D000328:Adult; D000368:Aged; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D005260:Female; D005419:Flavonoids / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D007951:Leukemia, Myeloid / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D004791:Enzyme Inhibitors; D005419:Flavonoids; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one