Radiation Resistance of C57Bl/6 Mouse Bone Marrow Stromal Cell Lines Induced By Apoptosis Inhibitor JP4-039 or Necroptosis Inhibitor Necrostatin-1, but Not By Simultaneous Administration of Both Mitigators

Introduction: Necroptosis is a TNFa activated, caspase-independent cell death pathway that involves active disintegration of mitochondrial, lysosomal and plasma membranes (Vandenabeele, et al, Nat Rev Mol Cell Biol., 2010 Oct; 11(10):700-14). We measured clonogenic radiation survival of C57/Bl6 mouse bone marrow stromal cell lines in the presence of a mitochondrial targeted inhibitor of apoptosis (JP4-039), an inhibitor of necroptosis (Necrostatin-1) or both drugs.

Materials and Methods: C57BL/6 mouse long term bone marrow cultures (LTBMCs) were established from adult female C57Bl/6 mice. Bone marrow stromal cell lines were derived from the adherent layers of LTBMCs. Radiosensitivity of C57Bl/6 bone marrow stromal cell lines and fresh bone marrow CFU-GEMM (semisolid medium with added hemopoietic growth factors) was measured in clonogenic radiation survival curves. Stromal Cells were irradiated to doses of 0 to 8 Gy, plated in T-75 tissue culture flasks, incubated for 8 days at 37^oC, stained with crystal violet and colonies of greater than 50 cells counted at day 7. CFU-GEMMs from fresh bone marrow were scored at day 14. Both cultured cell populations were tested with addition of radiation mitigators: JP4-039 (10µM), Necrostatin-1 (40µM), or both drugs added to culture medium 30 minutes post irradiation. C57BL/6 mouse LTBMCs generated hemopoietic cells for 20 weeks. Marrow stromal cell lines were derived from 20 week old hemopoieticly-inactive culture adherent layer cells. Statistical analysis was performed using Student's t-test, with a two-tail p-value < 0.05 considered significant.

Results: Radiation survival curves showed that C57Bl/6 fresh bone marrow CFU-GEMM and stromal cell lines were comparable to those for other mouse strains (CFU-GEMM Do = 1.67 ± 0.02, stromal cells Do=1.43 ± 0.14). Both fresh marrow CFU-GEMM and stromal cells (ň = 5.26 ± 0.72) were radioresistant when grown in presence of JP4-039 (ň = 8.85 ± 1.23, p = 0.012) or necrostatin-1 (n=7.97 ± 1.12, p = 0.024). Addition of both JP4-039 and Necrostatin-1 (ň = 5.79 ± 1.44) at the same time after irradiation (30 min) did not improve clonogenic survival of either fresh marrow CFU-GEMM or stromal cell lines.

Conclusions: Both freshly explanted CFU-GEMM marrow hematopoietic progenitor cells and LTBMC derived marrow stromal cell lines display radioresistance when treated with each of two radiation mitigators that target different cell death pathways. While each drug delivered independently induced radioresistance, combined delivery of JP4-039 and Necrostatin-1 at 30 min after irradiation did not. Thus, the timing of delivery of drugs that target different cell death pathways is likely critical in designing effective protocols for radiation mitigation.