Proton Radioprotection of Fanconi Anemia (Fanca-/-) Mouse Marrow Stromal Cell Lines by Mitochondrial Targeted GS-Nitroxide JP-4-039

Abstract

The increasing incidence of oral cavity squamous cell carcinomas in radiosensitive Fanconi Anemia (FA) patients suggested that protons might minimize normal tissue toxicity. We tested the radiation protector/mitigator, JP4-039, with Proton irradiated mesenchymal stem cell lines derived from Fanca-/- mouse marrow. Fanca-/- and control Fanca+/+ bone marrow stromal cell lines were irradiated to doses of 0 – 10 Gy via pencil beam scanning proton therapy system. Subgroups of cultures were treated with 100uM JP4-039 for 12 h prior to irradiation and after plating for clonogenic survival curves. Parallel cultures were irradiated using a JL Shepherd Model 68A cesium irradiator at 340 cGy per minute, subgroups treated with JP4-039 in a similar protocol. Cells were plated in 4 well limbro plates and incubated for 10 days in a CO2 tissue culture incubator. Colonies of greater than 50 cells were stained with crystal violet and counted. Data from triplicate experiments was analyzed using Linear Quadratic and Single Hit, Multiple Target model. Cultures irradiated to 5 or 10 Gy using proton or gamma irradiation were plated in T25 flasks and stained 10 days later for β-Galactosidase. Proton irradiation revealed radiosensitivity of Fanca-/- bone marrow stromal cell lines compared to Fanca+/+ cell lines (D0 = 2.11 ± 0.17, vs D0 = 3.28 ± 0.10 p = 0.0027, ñ = 1.1 ± 0.1, vs ñ = 1.0 ± 0.1, respectively). Fanca-/- cells were also radiosensitive to gamma irradiation, (D0 = 2.08 ± 0.17, vs D0 = 3.36 ± 0.05 p = 0.0017, ñ = 2.0 ± 0.5, ñ = 1.3 ± 0.3, respectively). JP4-039 provided significant radiation protection for both Fanca-/- and Fanca+/+ cell lines against both proton irradiation (D0 = 3.01 ± 0.19 p = 0.0125, ñ = 1.0 ± 0.1, vs D0 = 3.84 ± 0.11 p = 0.0196, ñ = 1.0 ± 0.1), and gamma irradiation ((D0 = 3.81 ± 0.72 p = 0.0423, ñ = 1.2 ± 0.2; D0 = 3.70 ± 0.06 p = 0.0133 p = 0.0133, ñ = 1.7 ± 0.7). Following proton irradiation, Fanca+/+ cells showed greater senescence compared to Fanca-/- cells: 28% vs 10% β-galactosidase positive (p < 0.05). In contrast, gamma irradiation of Fanca+/+ compared to Fanca-/- cells showed decreased senescence (7% compared to 17% (p < 0.05). Fanca-/- bone marrow stromal cell lines are radiosensitive to proton and gamma irradiation, but significantly protected by JP4-039. The data support intra-oral JP4-039 for Fanconi Anemia patients receiving proton radiotherapy for oral squamous cell carcinoma.