Mitigation of Irradiation Induced Potentially Lethal Damage (PLD) in Hematopoietic Cells by Mitochondrial Localized GS-Nitroxide, JP4-039

Abstract PLD is an ionizing irradiation pathway believed to apply to quiescent hematopoietic cells in the marrow microenvironment. To determine if the mitochondrial targeted JP4-039 which has been shown to mitigate sublethal irradiation damage (SLD) in non-quiescent clonagenic cells protected cells from PLD, 32D cl 3 hematopoietic cells were irradiated in three sets of cultures to doses ranging from 0 to 8 Gy. To one group of cells, to measure SLD mitigation effects, JP4-039 (10 μM) was added immediately, then cells plated in methycellulose, and incubated at 37°C for seven days at which time colonies of greater than 50 cells counted. The second set of irradiated cells was grown in flasks in 10 ml of liquid media. The third set was tested for PLD effects and cells were centrifuged and maintained in pellets in 1 ml of media (PLD conditions preventing cell growth or division) and these latter two sets were kept at 37°C. After 24, 48 or 72 hrs irradiation, subsets of each were removed, viability and cell counts were determined and JP4-039 was then added to subgroups of cultured cells at each time point, cells re-suspended in methycellulose, and incubated at 37°C for seven days at which time colonies of greater than 50 cells counted. Viability of cells in non-irradiated pellets decreased from 24 to 72 hours from 60.3 ± 2.7% to 37.3 ± 2.3% (p = 0.0030), while after 8 Gy the cell viability was 57. 3 ± 0.3% at 24 hr and 25.7 ± 6.1% at 72 hr (p = 0.0065). There was no significant decrease in percent viability at 24 hrs of non-irradiated or 8 Gy irradiated cells (60.3 ± 2.7% and 57.3 ± 0.3%, respectively) or at 72 hr (37.3 ± 2.3% and 25.7 ± 6.1%, respectively). In contrast, non-irradiated cells re-suspended in flasks grew and thus viability increased from 77.0 ± 4.0% at 24 hr to 96.7 ± 0.3% at 72 hr (p = 0.0080). Cell viability after 8 Gy of surviving irradiated cells grown in flasks was not significantly changed from 64.0 ± 10.6% at 24 hr to 49.3 + 3.2% at 72 hr. There was no significant change in cell viability in flasks at 24 hrs of non-irradiated or 8 Gy irradiated cells (77 ± 4.0% and 64.0 ± 10.6%, respectively). Cell viability was significantly decreased at 72 hr in non-irradiated cell flasks of 96.7 ± 0.3% compared to 8 Gy irradiated flasks with a viability of 49.3 ± 3.2% (p = 0.0001). Cells maintained in pellets showed there was no cell growth between 24 to 72 hours while those maintained in flasks did significantly grow for non-irradiated cells from 6.3 ± 0.2 × 105 cells at 24 hr to 85.3 ± 6.8 × 105 cells at 72 hr (p = 0.0003). For 8 Gy irradiated cells grown in flasks, there was no significant change in the number of cells between 24 and 72 hr (4.9 ± 0.9 × 105 and 4.9 ± 1.2 × 105, respectively). Thus, PLD conditions were established in pellets. Irradiation survival curves showed a JP4-039 mediated PLD radiation resistance when cells were incubated in JP4-039 at 24 hr or 72 hr after irradiation by increase in the Do. At 24 hr, the Do for pelleted cells incubated in JP4-039 was 1.92 ± 0.01 Gy compared to pelleted irradiated control 32D cl 3 cells, 1.36 ± 0.02 Gy (p = 0.0014). At 24 hrs, cells grown in flasks and incubated in JP4-039 had a Do of 1.70 ± 0.02 Gy compared to 1.40 ± 0.05 Gy for irradiated control 32D cl 3 cells (p = 0.0261). By 72 hrs after irradiation, the Do for pelleted cells was increased to 1.97 ± 0.06 Gy by adding JP4-039 compared to the pelleted irradiated control 32D cl 3 cells with a Do of 1.51 ± 0.04 Gy (p = 0.0218). In cells grown in flasks for 72 hr before the addition of JP4-039 the Do was 2.01 ± 0.02 Gy compared to irradiated controls which had a Do of 1.62 ± 0.07 Gy (p = 0.0346). Therefore, JP4-039 displays a clear potential to mitigate irradiation induced PLD damage. These data correlate with significant mitigation of the hematopoietic syndrome by JP4-039 in total body irradiated C57BL/6NHsd mice.