Figure 2.
Figure 2. Overexpression of HOXB6 in bone marrow progenitors results in enhanced self-renewal and immortalization in vitro. (A) Results of 2 independent serial replating assays performed in duplicate demonstrated increased myeloid CFC frequency with successive replating for HOXB6 and HOXB6WG vectors, whereas CFC frequency rapidly declined in bone marrow transduced with HOXB6NA or MIG control. Results shown are means ± SD. P < .005 for comparison of HOXB6 or HOXB6WG with MIG in secondary and tertiary cultures. (B) HOXB6- and HOXB6WG-transduced marrow frequently produced dense colonies with little peripheral migration (left). Wright-Giemsa–stained cytospins of well-isolated dense colonies revealed these cells had blastlike morphology and thus the colonies were subclassified as CFU-blasts (right). CFC and cytospin images were obtained using an Axiovert 25 inverted microscope equipped with an AccuPlan 4 ×/0.10 objective lens (Carl Zeiss, Thornwood, NY) or a BH2 upright microscope equipped with a D Plan 100 ×/1.25 objective lens (Olympus America, Melville, NY). Images were captured with a Nikon Coolpix 4500 digital camera (Nikon USA, Torrence, CA) and enhanced using Nikon Photoimpression 4.0 software (Nikon USA). (C) Percentage of CFC composed of CFU-blasts increased with successive replatings in HOXB6- and HOXB6WG-transduced marrow, whereas HOXB6NA and MIG cultures had few or no CFU-blasts. P less than .005 for frequency of CFU-B in HOXB6 and HOXB6WG marrow versus MIG or HOXB6NA for all platings. Results shown are means ± SD for 2 independent experiments performed in duplicate. (D) Cell proliferation assays demonstrate exponential growth of HOXB6- and HOXB6WG-transduced bone marrow, whereas MIG-transduced cell cultures ceased to grow after approximately 4 weeks. The apparent intermediate growth of the HOXB6NA marrow was due to outgrowth of mast cells. (E) Western blot of lysates from HOXB6-immortalized cell lines showing robust expression of HOXB6. A HOXA9-immortalized cell line is used as a negative control. (F) Southern blot analysis of DNA isolated from immortalized cell lines using a fragment of EGFP cDNA as a probe. Digestion of genomic DNA with BamHI or XhoI alone cuts the provirus once liberating a unique-sized fragment for each proviral integration. One proviral integration was detected for all lines tested except HOXB6WG line 4, which may be oligoclonal or represent a single clone with multiple viral integrations. NS indicates nonspecific band. (G) FACS analysis of HOXB6-immortalized cell lines. In the top left panel, EGFP expression is shown on the x axis and FL-2 fluorescence in the unstained specimen is shown on the y axis. In the remaining 3 FACS histograms, Mac-1, B220, and c-kit staining are shown on the x axis (dark lines). The unstained cell line is overlaid as a control (light lines). Cell lines also had high Gr-1, intermediate F4/80, and negative CD8 expression (data not shown).

Overexpression of HOXB6 in bone marrow progenitors results in enhanced self-renewal and immortalization in vitro. (A) Results of 2 independent serial replating assays performed in duplicate demonstrated increased myeloid CFC frequency with successive replating for HOXB6 and HOXB6WG vectors, whereas CFC frequency rapidly declined in bone marrow transduced with HOXB6NA or MIG control. Results shown are means ± SD. P < .005 for comparison of HOXB6 or HOXB6WG with MIG in secondary and tertiary cultures. (B) HOXB6- and HOXB6WG-transduced marrow frequently produced dense colonies with little peripheral migration (left). Wright-Giemsa–stained cytospins of well-isolated dense colonies revealed these cells had blastlike morphology and thus the colonies were subclassified as CFU-blasts (right). CFC and cytospin images were obtained using an Axiovert 25 inverted microscope equipped with an AccuPlan 4 ×/0.10 objective lens (Carl Zeiss, Thornwood, NY) or a BH2 upright microscope equipped with a D Plan 100 ×/1.25 objective lens (Olympus America, Melville, NY). Images were captured with a Nikon Coolpix 4500 digital camera (Nikon USA, Torrence, CA) and enhanced using Nikon Photoimpression 4.0 software (Nikon USA). (C) Percentage of CFC composed of CFU-blasts increased with successive replatings in HOXB6- and HOXB6WG-transduced marrow, whereas HOXB6NA and MIG cultures had few or no CFU-blasts. P less than .005 for frequency of CFU-B in HOXB6 and HOXB6WG marrow versus MIG or HOXB6NA for all platings. Results shown are means ± SD for 2 independent experiments performed in duplicate. (D) Cell proliferation assays demonstrate exponential growth of HOXB6- and HOXB6WG-transduced bone marrow, whereas MIG-transduced cell cultures ceased to grow after approximately 4 weeks. The apparent intermediate growth of the HOXB6NA marrow was due to outgrowth of mast cells. (E) Western blot of lysates from HOXB6-immortalized cell lines showing robust expression of HOXB6. A HOXA9-immortalized cell line is used as a negative control. (F) Southern blot analysis of DNA isolated from immortalized cell lines using a fragment of EGFP cDNA as a probe. Digestion of genomic DNA with BamHI or XhoI alone cuts the provirus once liberating a unique-sized fragment for each proviral integration. One proviral integration was detected for all lines tested except HOXB6WG line 4, which may be oligoclonal or represent a single clone with multiple viral integrations. NS indicates nonspecific band. (G) FACS analysis of HOXB6-immortalized cell lines. In the top left panel, EGFP expression is shown on the x axis and FL-2 fluorescence in the unstained specimen is shown on the y axis. In the remaining 3 FACS histograms, Mac-1, B220, and c-kit staining are shown on the x axis (dark lines). The unstained cell line is overlaid as a control (light lines). Cell lines also had high Gr-1, intermediate F4/80, and negative CD8 expression (data not shown).

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