1012. Immortalization of Murine Hematopoietic Stem and Progenitor Cells by AML1-ETO and HoxB4
Authors: Michaela Scherr, Gernot Beutel, Karin Battmer, Letizia Venturini, Daniel Schaefer, Arnold Ganser, Matthias Eder
Journal: Molecular Therapy (2006)

The analysis of molecular events sufficient and/or necessary to transform hematopoietic stem and progenitor cells is hampered by the lack of suitable models which allow the stepwise analysis oftransforming events. In an effort to generate such models we isolated lineage negative (lin-) murine bone marrow cells and expressed either AML1-ETO or HoxB4 along with EGFP as a quantitative reporter by retroviral gene transfer. Retrovirally transduced cells were initially cultured in the presence of SCF+IL-3+IL-6 in serum free medium, and EGFP+ cells were sorted 3 days after transduction to start long- term cultures with a ratio of about 50% EGFP + cells. Interestingly, long-term cultures for AML1-ETO remain strictly SCF-dependent wheras HOXB4 cultures only require IL-3 for cell survival and proliferation. Beside cytokine stimulation, cell survival requires expression of AML1-ETO and HoxB4, respectively, as demonstrated by RNA interference targeting EGFP which is encoded along with the respective transgene on bicistronic transcripts. Although AML1- ETO and HoxB4 cultures display similar proliferation kinetics and blast like morphology (with some degree of granulocytic maturation in HoxB4 cultures), the immunophenotype of immortalized cells differ with more than 90% of AML1-ETO cells being c-Kit and Sca-1 positive whereas HoxB4 cells are c-Kit and Sca-1 negative. Interestingly, AML1-ETO cells form only blast like colonies with very low plating efficiency (<10-4), whereas HoxB4 cultures give rise to large colonies of all lineages with mature cells at a plating efficiency of more than 10%. Molecular genetic analysis reveals poly- or oligoclonal proliferation at least for the six months for both cultures, whereas at later time points, the HoxB4 cells were found to turn to a mono- or biclonal pattern. Both cell lines can be transduced for a second time by retro- or lentiviral gene transfer with high efficacy. We generated shRNAs targeting both the human and murine transcription factor PU.1, and cloned lentiviral plasmids encoding anti-PU.1 shRNA (small hairpin RNA) along with red fluorescence protein (RFP). High-titer VSV.G pseudotyped lentiviruses were generated and used to transduce K562 and 32D cells. Anti-PU.1 shRNAs strongly reduce both PU-1 mRNA and protein levels as determined by real-time RT-PCR and immunoblotting, respectively, as well as surface expression of CD11b, a known transcriptional target of PU.1. Lentiviral transduction of HoxB4 immortalized cells results in more than 95% RFP+ cells, accompanied by a reduction in colony numbers in PU.1 shRNA expressing cells. Interestingly, almost no mature granulocytes are found in PU.1 shRNA expressing cells as compared to non-specific shRNA controls. This data demonstrates that murine lin- bone marrow cells can be immortalized by different molecular mechanisms leading to cell lines with different phenotypes. These cells are useful to further analyze cooperative molecular events required for malignant transformation.