Abstract 70: GS nitroxide (JP4-039) induces radiation resistance of conditional MnSOD tet/tet murine bone marrow stromal cells.
Authors: Kalash Ronny , Houghton Frank , Berhane Hebist , Wipf Peter , Shields Donna , Epperly Michael W. , Chaillet Richard , Cao Shaonan , Zhang Xichen , Greenberger Joel S. 
Published: August 2014

Background/Objectives: Manganese superoxide dismutase (MnSOD) is a mitochondrial targeted enzyme which dismutates irradiation induced superoxide. Transgenic MnSOD -/- mice die two to three days after birth, and embryo cell lines are radiosensitive in vitro (Epperly et al, Rad Res 154:365-374,2000). Transgene mediated increased expression of MnSOD increases cell line and tissue radioresistance in heterozygote MnSOD +/- mice (Epperly,et al, Rad Res 154:365-374,2000). To regulate MnSOD expression, we developed a mouse line in which expression of one or both endogenous MnSOD alleles is activated by doxycycline (DOX) administration. Homozygous (tet/tet) mice displayed very low levels of MnSOD in the absence of DOX. Both tet/tet and tet/+ cell lines increased MnSOD expression in the presence of DOX. We evaluated the effect of JP4-039 a Gramicidin S (GS) derived-nitroxide as a mitochondrial targeted radiation protector and mitigator on Mnsod tet/tet and MnSOD tet/+cell lines in vitro.

Materials/Methods: Bone marrow stromal cell lines were established from MnSOD tet/tet, tet/+ and +/+ mice and compared to embryonic fibroblast cell line from unconditional MnSOD -/- mice. Stress response and cytokine gene expression was measured using real time PCR, and clonagenic survival curves were generated relative to MnSOD biochemical activity. Cells were grown in DOX, JP4-039, or both, 24 hours prior to, or immediately following, irradiation.

Results: Incubation of MnSOD tet/tet cells in DOX increased MnSOD biological activity to control levels within 24 hrs. Control C57BL/6NHsd cells were radio-resistant (ñ = 7.90 ± 1.36) relative to MnSOD tet/tet in no DOX (ñ = 2.79 + 1.04, p = 0.0175). Radioresistance was restored in DOX treated MnSOD tet/tet cells (ñ = 8.69 ± 1.09 (p = 0.0060) .There was no significant DOX induced change in radiosensitivity of MnSOD +/+ or MnSOD -/- cells. A synergistic increase in cell survival followed addition of JP4-039 to DOX in MnSOD tet/tet and MnSODtet/+ cell lines.

RT-PCR showed an elevation in MnSOD expression, as well as an elevation in expression of stress response genes TGFb,NF-KB, NFE212 at 1 and 6 hours following irradiation in +/+ cells. In tet/+, tet/tet, and -/- cell lines decreased levels of MnSOD were seen at baseline and at both 1 and 6 hours after irradiation without the addition of DOX. DOX added to tet/+ and tet/tet cell lines increased MnSOD levels 1 and 6 hours following irradiation. The addition of DOX to both tet/+ and tet/tet cells caused an increase in TGFb and NF-KB post irradiation in comparison to the same cells without DOX post irradiation. A decrease in NFE212 was found in tet/+ and tet/tet cells post irradiation in comparison to the same cells without DOX post-irradiation.

Conclusions: MnSOD tet/tet cells should be valuable to study the cellular mechanisms involved in the response to ionizing irradiation.

Acknowledgements: This project supported by NIAID U191A168021.

Citation Format: Ronny Kalash, Frank Houghton, Hebist Berhane, Peter Wipf, Donna Shields, Michael W. Epperly, Richard Chaillet, Shaonan Cao, Xichen Zhang, Joel S. Greenberger. GS nitroxide (JP4-039) induces radiation resistance of conditional MnSOD tet/tet murine bone marrow stromal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 70. doi:10.1158/1538-7445.AM2013-70