Shaonan Cao

Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation

Abstract Epperly, M., Jin, S., Nie, S., Cao, S., Zhang, X., Franicola, D., Wang, H., Fink, M. P. and Greenberger, J. S. Ethyl Pyruvate, a Potentially Effective Mitigator of Damage after Total-Body Irradiation. Radiat. Res. 168, 552–559 (2007). Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to improve survival and ameliorate organ damage in animal models of sepsis, ischemia/reperfusion injury and hemorrhagic shock. Incubating IL3-dependent mouse hematopoietic progenitor cell 32Dcl3 cells before or after irradiation with 10 mM EP increased resistance to radiation as assessed by clonogenic radiation survival curves, decreased release of mitochondrial cytochrome C into the cytoplasm, and decreased apoptosis. EP inhibited radiation-induced caspase 3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in 32Dcl3 cells in a concentration-dependent fashion. EP was given i.p. to C57BL/6NHsd mice irradiated with 9.75 Gy total-body irradiation (TBI). This treatment significantly improved survival. The survival benefit was apparent irrespective of whether treatment with EP was started 1 h before TBI and continued for 5 consecutive days after TBI or the compound was injected only 1 h before or only for 5 days after TBI. In all of the in vitro and in vivo experiments, ethyl lactate, an inactive analogue of EP, had no detectable radioprotective or mitigating effects. EP may be an effective radioprotector and mitigator of the hematopoietic syndrome induced by TBI.

Intraesophageal manganese superoxide dismutase-plasmid liposomes ameliorates novel total-body and thoracic radiation sensitivity of NOS1-/- mice.

Abstract The effect of deletion of the nitric oxide synthase 1 gene (NOS1−/−) on radiosensitivity was determined. In vitro, long-term cultures of bone marrow stromal cells derived from NOS1−/− were more radioresistant than cells from C57BL/6NHsd (wild-type), NOS2−/− or NOS3−/− mice. Mice from each strain received 20 Gy thoracic irradiation or 9.5 Gy total-body irradiation (TBI), and NOS1−/− mice were more sensitive to both. To determine the etiology of radiosensitivity, studies of histopathology, lower esophageal contractility, gastrointestinal transit, blood counts, electrolytes and inflammatory markers were performed; no significant differences between irradiated NOS1−/− and control mice were found. Video camera surveillance revealed the cause of death in NOS1−/− mice to be grand mal seizures; control mice died with fatigue and listlessness associated with low blood counts after TBI. NOS1−/− mice were not sensitive to brain-only irradiation. MnSOD-PL therapy delivered to the esophagus of wild-type and NOS1−/− mice resulted in equivalent biochemical levels in both; however, in NOS1−/− mice, MnSOD-PL significantly increased survival after both thoracic and total-body irradiation. The mechanism of radiosensitivity of NOS1−/− mice and its reversal by MnSOD-PL may be related to the developmental esophageal enteric neuronal innervation abnormalities described in these mice.

Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2–/–) Mice

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2–/– mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2–/– mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2–/– mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2+/+ stromal cells (Fancd2–/– D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2+/+ D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2–/– IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2+/+ (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2–/– marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2–/– stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2+/+ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2–/– IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2–/– stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.

Amelioration of Radiation-Induced Pulmonary Fibrosis by a Water-Soluble Bifunctional Sulfoxide Radiation Mitigator (MMS350)

A water-soluble ionizing radiation mitigator would have considerable advantages for the management of acute and chronic effects of ionizing radiation. We report that a novel oxetanyl sulfoxide (MMS350) is effective both as a protector and a mitigator of clonal mouse bone marrow stromal cell lines in vitro, and is an effective in vivo mitigator when administered 24 h after 9.5 Gy (LD100/30) total-body irradiation of C57BL/6NHsd mice, significantly improving survival (P = 0.0097). Furthermore, MMS350 (400 μM) added weekly to drinking water after 20 Gy thoracic irradiation significantly decreased: expression of pulmonary inflammatory and profibrotic gene transcripts and proteins; migration into the lungs of bone marrow origin luciferase+/GFP+ (luc+/GFP+) fibroblast progenitors (in both luc+ marrow chimeric and luc+ stromal cell line injected mouse models) and decreased radiation-induced pulmonary fibrosis (P < 0.0001). This nontoxic and orally administered small molecule may be an effective therapeutic in clinical radiotherapy and as a counter measure against the acute and chronic effects of ionizing radiation.

Dysregulated in vitro hematopoiesis, radiosensitivity, proliferation, and osteoblastogenesis with marrow from SAMP6 mice.

The senescence accelerated-prone mouse variant 6 (SAMP6) shows normal growth followed by rapid aging, development of osteopenia, and shortened lifespan, compared with control R1 mice. Because oxidative stress is a fundamental mechanism of tissue aging, we tested whether cellular parameters that are associated with oxidative stress are impaired with marrow from SAMP6 mice. We compared in vitro hematopoiesis, irradiation sensitivity, proliferative potential, and osteoblastogenesis with marrow cells from SAMP6 and R1 mice. Marrow cells from SAMP6 mice showed shortened in vitro hematopoiesis; their stromal cells showed greater radiation sensitivity and decreased proliferation. Consistent with those properties, there was constitutive upregulation of transforming growth factor-β(1), an inhibitor of hematopoiesis, and of cell cycle inhibitory genes, p16(INK4A) and p19(ARF). Paradoxically, there was constitutive expression of osteoblast genes in stromal cells from SAMP6 mice, but in vitro matrix mineralization was impaired. These studies and data included in other reports indicate that impaired proliferation of osteoblast progenitors in SAMP6 marrow may be a major factor contributing to accelerated loss of bone mass. In sum, marrow from SAMP6 mice had diminished capacity for long-term hematopoiesis, increased radiosensitivity, and reduced proliferative capacity.

Abstract 1655: Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Introduction: Transgenic FVB/n mice with the HPV E7 oncogene under the control of the CK14 promoter were bred with Fancd2+/- (129/Sv background) mice to obtain K14E7 Fancd2-/- mice Park, et al. Cancer Research, 70(23): 9959, 2010), in which delivery of 4-nitroquinoline N-oxide in drinking water produces oral and esophageal adenomas. The hematopoietic system, of these mice was studied, using long term bone marrow cultures (LTBMCs) from Fancd2-/- (SV129), K14E7 transgenic (FVB/n), and K14E7 Fancd2-/- mice. Methods: LTBMCs were established by flushing bone marrow into T-25 flasks from which bone marrow stromal cell lines and IL3-dependent hematopoietic cell lines were developed. The cell lines were analyzed using irradiation survival curves, Western analysis for protein expression. K14E7 Fancd2-/- IL3 dependent clonal cell lines of the IL3-dependent cell lines were established by flow cytometry. Clonal lines were expanded and tested for tumorigenicity by injecting 1 × 106 cells of each of four clones into flanks of K14E7 Fancd2+/+ mice. Results: K14E7 Fancd2-/- LTBMCs were similar to those from Sv/129 Fancd2-/-: 1) hematopoiesis was shortened when compared to K14E7 Fancd2+/+ or Fancd2+/+ LTBMCs, respectively. Stromal cell line irradiation survival curves with K14E7 Fancd2+/+ and Fancd2-/- cell lines showed (Do = 1.48 ± 0.05 and 1.52 ± 0.15 Gy) they were radiosensitive compared to those from K14E7 Fancd2+/+ or Sv/129 Fancd2+/+ cells (Do = 2.33 ± 0.11 and 2.23 ± 0.01 Gy, p = 0.0043 and 0.0368, respectively). Fancd2-/- stromal cells were more radiosensitive than Fancd2+/+ cells (decreased shoulder on the survival curve of n = 1.5 ± 0.5 compared to 4.7 ± 0.3 (p = 0.0126). K14E7Fancd2+/+ cells had an n of 1.9 ± 0.3 compared to 3.5 ± 0.1 (p = 0.0309) for Sv/129 cells. In contrast, hematopoietic IL-3 dependent K14E7Fancd2+/+ and Sv/129 Fancd2+/+ cell lines were similar (Do = 2.15 ± 0.4 and 1.92 ± 0.06, p = 0.6469, respectively). Confirming prior data, (Berhane et. al, Rad Res 182: 35, 2014) Sv/129 Fancd2-/- IL-3 dependent cell lines were radioresistant compared to Fancd2+/+ cell lines (Do = 2.12 ± 0.12 and 1.92 ± 0.06, respectively, p = 0.0236). In contrast, K14E7 Fancd2-/- cell lines were radiosensitive compared to K14E7 Fancd2+/+ cell lines (1.44 ± 0.13 and 2.15 ± 0.28, respectively, p = 0.0498). Hematopoietic and marrow stromal cell lines from K14E7Fancd2-/- marrow expressed cytokeratin 14 and E7 oncogene by Western analysis. K14E7 Fancd2-/- (but no other genotype derived) IL-3 dependent cell lines transformed in vitro to dense tumor cells lines. Cloned sublines were injected into K14E7 Fancd2+/- mice producing plasmacytomas. Conclusions: Expression of the E7 oncogene in the K14/E7 Fancd2-/- mouse marrow derived IL-3 dependent cell lines leads to radiosensitivity and tumorgenicity. Supported by NIAID/NIH U19-A 1068021 and the Fanconi Anemia Research Foundation. Citation Format: Joel S. Greenberger, Lora Rigatti, Aranee Sivanathan, Shaonan Cao, Xichen Zhang, Donna Shields, Darcy Franicola, Michael Epperly. Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1655.

Abstract 70: GS nitroxide (JP4-039) induces radiation resistance of conditional MnSOD tet/tet murine bone marrow stromal cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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 (n = 7.90 ± 1.36) relative to MnSOD tet/tet in no DOX (n = 2.79 + 1.04, p = 0.0175). Radioresistance was restored in DOX treated MnSOD tet/tet cells (n = 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

Abstract 4355: Use of MnSOD tet on transgenic mice as a model for evaluating irradiation protection and mitigation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme responsible for the dismutation or reduction of superoxide produced by cellular respiration. Absence of MnSOD is lethal two to three days after birth. Following irradiation there is an increase in superoxide production which combined with nitric oxide can result in the formation of the highly reactive radical peroxynitrite. Increased expression of MnSOD in a cell increases survival by the reduction of superoxide produced following irradiation. Decreased MnSOD production has resulted in decreased survival following irradiation. To better understand the role of MnSOD in cellular respiration as well as protecting the cell from irradiation damage, we have developed a MnSOD tet on mouse which has no MnSOD activity. In the absence of doxycycline the homozygous MnSOD tet on mouse does not survive past 2 or 3 days after birth. If doxycycline (2 µg/ml) is added to the water of heterozygous mothers, the MnSOD tet on mice develop normally and are fertile as long as they remain on the doxycycline treated water. Long term bone marrow cultures (LTBMCs) were established from homozygous MnSOD tet on mice, heterozygous tet on mice and control C57BL/6J mice. The cultures from the control mice had more hematopoietic cell activity than the MnSOD tet on cultures. To determine when the MnSOD gene is turned on after addition of doxocycline, bone marrow stromal cell lines were developed from the LTBMCs were incubated in 1 µg doxocycline/ml media for time points up to 24 hr, RNA was extracted and RT-PCR for the MnSOD transgene was performed. By 12 hours there was an increase in MnSOD RNA expression in the MnSOD tet on cells. Removal of the doxycycline resulted in a decrease in MnSOD RNA by 1 hr in the MnSOD tet on cells as well as the heteroxyzous cells. This mouse model will be useful for testing new compounds for radiation protection and mitigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4355. doi:1538-7445.AM2012-4355