Antiapoptotic cytokine IL-3 + SCF + FLT3L influence on proliferation of gamma-irradiated AC133+/CD34+ progenitor cells.
Recovery from radiation-induced bone marrow aplasia depends on appropriate cytokine support. The aim of our work was to find a cytokine combination allowing in vitro gamma-irradiated (2.5 Gy) CD34+/AC133+ haematopoietic stem cells to evade radiation-induced apoptosis and to enhance damage reparation, which should enable proliferation and ex vivo expansion of cells. Cells were isolated using separation in a Cobe separator followed by immunomagnetic selection by antibody against the AC133 antigen. Thus isolated cells were 80% AC133+/CD34+ and 10% of them expressed the CD33+ antigen. Ten thousand of AC133+ cells formed 1146 CFU-GM and 304 BFU-E. We proved a high expansion efficiency of cytokine combination SCF + IL-3 + FLT3L in comparison with the combination SCF + IL-3 + IL-11 in both, non-irradiated cells and cells irradiated with a dose of 2.5 Gy. The D0 value for AC133+ cells was determined by the clonogeneity test. The D0 value for CFU-GM was estimated to be 1.08 Gy and for BFU-E 0.95 Gy. The results of DNA analysis showed that the majority of isolated AC133+ cells were in G0/G1 phase of the cell cycle. We proved that the dose of 2.5 Gy induced massive apoptosis (80%) of these cells without progression through the cell cycle, which indicates interphase cell death. Under the influence of cytokine combination (SCF + IL-3 + FLT3L), the surviving 20% of cells entered the cell cycle and, similarly to non-irradiated control cells, on 7th day 35% of cells were in S phase.
Vávrová
J
J
Institute of Radiobiology and Immunology, Purkynĕ Military Medical Academy, Hradec Králové, Czech Republic. vavrova@pmfhk.cz
Vokurková
D
D
Mareková
M
M
Bláha
M
M
Jebavý
L
L
Filip
S
S
D017209:Apoptosis; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D005720:Gamma Rays; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D016130:Immunophenotyping; D007377:Interleukin-3 / Q000494:pharmacology*; D008565:Membrane Proteins / Q000494:pharmacology*; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology*
D007377:Interleukin-3; D008565:Membrane Proteins; D011837:Radiation-Protective Agents; D011994:Recombinant Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein