Quantification of transforming capacity and cooperation of defined genetic alterations in myeloid malignancies
Received 2 July 2009; received in revised form 2 September 2009; accepted 14 October 2009. published online 19 October 2009.
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.
Materials and 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 βGMR-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 βGMR-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.
Conclusion
This cell culture model represents a useful tool to quantify hematopoietic cell transformation by defined genetic and epigenetic alterations.
Hannover Medical School, Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover, Germany
Offprint requests to: Matthias Eder, M.D., Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany
∗ Drs. Scherr and Eder contributed equally to this work.
ABSTRACT