Quantification of transforming capacity and cooperation of defined genetic alterations in myeloid malignancies.
OBJECTIVE
Mutations found in myeloid malignancies are qualitatively classified as conferring proliferative and survival advantages or impairing cellular differentiation. However, no suitable experimental model to quantify transforming potential of individual mutations and functional cooperation between defined genetic/epigenetic alterations has been established so far.


METHODS
Based on cytokine-independent proliferation as a marker for cellular transformation, we used limiting dilution and clonal expansion of retrovirally transduced cells in the presence or absence of cytokines to quantify the transformation potential of constitutively active receptor mutants and short hairpin RNAs (shRNA) targeting transcription factors by RNA interference. Interleukin-3-dependent 32D cells were transduced with betaGMR-I374N, c-KitV558D, or c-MplS368C, and cloning efficiencies were normalized to viral integration numbers as determined by quantitative polymerase chain reaction.


RESULTS
In this assay, c-KitV558D and c-MplS368C were about 25-fold more effective than betaGMR-I374N. To study cooperation of defined genetic/epigenetic aberrations, receptor mutants were coexpressed with shRNAs targeting PU.1 and p53. In p53-hypomorphic, but not in 32D wild-type cells, RNA interference against PU.1 significantly enhances transformation efficacy by c-KitV558D, but not by c-MplS368C, as compared to control shRNA. These data demonstrate nonredundant, receptor-specific and p53-dependent responses to reduced PU.1 expression in 32D cells.


CONCLUSIONS
This cell culture model represents a useful tool to quantify hematopoietic cell transformation by defined genetic and epigenetic alterations.
Dogan
Yildirim
Y
Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany.
Ganser
Arnold
A
Scherr
Michaela
M
Eder
Matthias
M
D001483:Base Sequence; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D019337:Hematologic Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D016133:Polymerase Chain Reaction
D017931:DNA Primers