Xichen Zhang

Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase Transgene

Manganese superoxide dismutase plasmid liposomes (MnSOD-PL) confer organ-specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new minicircle construct was tested. Minicircle MnSOD was purified and then cotransfected into 32D cl 3 murine interleukin-3-dependent hematopoietic progenitor cells along with another plasmid carrying the neo gene. Cells were selected in G418 (50 microg/ml) and cloned by limiting dilution. Biochemical analysis of minicircle MnSOD-transfected cells showed an MnSOD biochemical activity level of 5.8 +/- 0.5 U/mg compared with 2.7 +/- 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D-mc-MnSOD cells were as radioresistant as full-length MnSOD-PL transgene-expressing 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 +/- 0.2 and n = 4.6 +/- 0.2, respectively, compared with 1.5 +/- 0.5 for 32D cl 3 cells; p = 0.007). C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DsRed-PL control, full-length MnSOD-PL, or blank-PL and then were irradiated 24 hr later with 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL showed increased survival compared with control mice or mice treated with mc-DsRed-PL (p = 0.0003 and 0.039, respectively), and comparable to full-length MnSOD-PL. Intravenous, systemic administration of mc-MnSOD-PL protected mice from total body irradiation (9.75 Gy). Therefore, minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

Bone Marrow Origin of Cells with Capacity for Homing and Differentiation to Esophageal Squamous Epithelium

Abstract Epperly, M. W., Guo, H., Shen, H., Niu, Y., Zhang, X., Jefferson, M., Sikora, C. A. and Greenberger, J. S. Bone Marrow Origin of Cells with Capacity for Homing and Differentiation to Esophageal Squamous Epithelium. Radiat. Res. 162, 233–240 (2004). Our goal was to determine whether esophageal progenitor cells could be isolated from adult mouse esophagus or bone marrow and shown to home to and proliferate in the irradiated esophagus of recipient mice. Esophageal progenitor cells were isolated from adult male C3H/HeNsd or C57BL/6J green fluorescent protein (GFP+) mice by a serial in vitro preplate technique or the technique of side population cell sorting. When injected intravenously (i.v.), these cells homed to the 30-Gy-irradiated esophagus of GFP− female recipient mice and formed donor-origin esophageal foci. GFP+ whole murine bone marrow cells injected i.v. also formed donor-origin esophageal squamous cell foci and protected recipient GFP− mice from upper-body irradiation in a cell dose-dependent manner. Marrow chimeric GFP− mice reconstituted with GFP+ cells showed migration of GFP+ marrow cells to the esophagus after 30 Gy irradiation. Purified esophageal progenitor cells isolated from first-generation preplate cell recipients engrafted after i.v. injection to the esophagus of second-generation-irradiated recipient mice. These data establish that esophageal progenitor cells can home to the irradiated esophagus and show limited differentiation capacity to squamous epithelium.

A Phase I Study of Concurrent Chemotherapy (Paclitaxel and Carboplatin) and Thoracic Radiotherapy with Swallowed Manganese Superoxide Dismutase Plasmid Liposome Protection in Patients with Locally Advanced Stage III Non-Small-Cell Lung Cancer

Manganese superoxide dismutase (MnSOD) is a genetically engineered therapeutic DNA/liposome containing the human MnSOD transgene. Preclinical studies in mouse models have demonstrated that the expression of the human MnSOD transgene confers protection of normal tissues from ionizing irradiation damage. This is a phase I study of MnSOD plasmid liposome (PL) in combination with standard chemoradiation in surgically unresectable stage III non-small-cell lung cancer. Chemotherapy (carboplatin and paclitaxel) was given weekly (for 7 weeks), concurrently with radiation. MnSOD PL was swallowed twice a week (total 14 doses), at three dose levels: 0.3, 3, and 30 mg. Dose escalation followed a standard phase I design. Esophagoscopy was done at baseline, day 4, and 6 weeks after radiation with biopsies of the squamous lining cells. DNA was extracted and analyzed by PCR for the detection of the MnSOD transgene DNA. Ten patients with AJCC stage IIIA (three) and IIIB (seven) completed the course of therapy. Five had squamous histology, two adenocarcinoma, one large cell, and two not specified. Patients were treated in three cohorts at three dose levels of MnSOD PL: 0.3 (three patients), 3 (three patients), and 30 mg (four patients). The median dose of radiation was 77.7 Gy (range 63-79.10 Gy). Overall response rate for the standard chemoradiation regimen was 70% (n = 10). There were no dose-limiting toxicities reported in all three dosing tiers. It is concluded that the oral administration of MnSOD PL is feasible and safe. The phase II recommended dose is 30 mg.

Mitochondrial Targeting of a Catalase Transgene Product by Plasmid Liposomes Increases Radioresistance In Vitro and In Vivo

Abstract Epperly, M. W., Melendez, J. A., Zhang, X., Nie, S, Pearce, L., Peterson, J., Franicola, D., Dixon, T., Greenberger, B. A., Komanduri, P., Wang, H. and Greenberger, J. S. Mitochondrial Targeting of a Catalase Transgene Product by Plasmid Liposomes Increases Radioresistance In Vitro and In Vivo. Radiat. Res. 171, 588-595 (2009). To determine whether increased mitochondrially localized catalase was radioprotective, a human catalase transgene was cloned into a small pSVZeo plasmid and localized to the mitochondria of 32D cl 3 cells by adding the mitochondrial localization sequence of MnSOD (mt-catalase). The cell lines 32D-Cat and 32D-mt-Cat had increased catalase biochemical activity as confirmed by Western blot analysis compared to the 32D cl 3 parent cells. The MnSOD-overexpressing 32D cl 3 cell line, 2C6, had decreased baseline catalase activity that was increased in 2C6-Cat and 2C6-mt-Cat subclonal cell lines. 32D-mt-Cat cells were more radioresistant than 32D-Cat cells, but both were radioresistant relative to 32D cl 3 cells. 2C6-mt-Cat cells but not 2C6-Cat cells were radioresistant compared to 2C6 cells. Intratracheal injection of the mt-catalase-plasmid liposome complex (mt-Cat-PL) but not the catalase-plasmid liposome complex (Cat-PL) increased the resistance of C57BL/6NHsd female mice to 20 Gy thoracic irradiation compared to MnSOD-plasmid liposomes. Thus mitochondrially targeted overexpression of the catalase transgene is radioprotective in vitro and in vivo.

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.

The autophagy-inducing drug carbamazepine is a radiation protector and mitigator

Abstract Purpose: To determine whether Carbamazepine (CBZ) is a radiation protector and/or mitigator. Materials and methods: Murine hematopoietic progenitor 32D cl 3 cells were incubated in 1, 10, or 100 μM CBZ 1 h before or immediately after 0–8 Gy irradiation and assayed for clonogenic survival. Autophagy was assayed by immunoblot for microtubule-associated protein light chain 3 (LC3). In vivo radioprotection and mitigation were determined with C57BL/6NTac mice. Results: CBZ treatment at 1, 10 or 100 μM for 1 h prior to irradiation increased radioresistance (the dose for 37% survival or D0) from control 1.5 ± 0.1 Gy to 2.1 ± 0.2 Gy (P = 0.012), 2.3 ± 0.1 Gy (P = 0.010), and 3.6 ± 0.7 Gy (P = 0.003), respectively; after irradiation increased the extrapolation number (ñ) from 1.5 ± 0.3 to 10.1 ± 4.2 (P = 0.011), 5.5 ± 1.7 (P = 0.019), and 3.6 ± 0.8 (P = 0.014), respectively, and increased autophagy. CBZ treated mice 10 min or 24 h before or 10 min or 12 h after 9.25 Gy total body irradiation (TBI) showed increased survival (P = 0.012, 0.011, 0.0002, and 0.017, respectively). Conclusion: CBZ may be a useful radiation protector and mitigator.

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.

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines.

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG–/– (PD326) and FancD2–/– (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2–/– cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG–/– cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2–/– cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2–/– cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.

Overexpression of the MnSOD Transgene Product Protects Cryopreserved Bone Marrow Hematopoietic Progenitor Cells from Ionizing Radiation

Abstract Epperly, M. W., Epperly, L. D., Niu, Y., Wang, H., Zhang, X., Franicola, D. and Greenberger, J. S. Overexpression of the MnSOD Transgene Product Protects Cryopreserved Bone Marrow Hematopoietic Progenitor Cells from Ionizing Radiation. Radiat Res. 168, 560–566 (2007). We determined whether manganese superoxide dismutase (MnSOD)-plasmid liposome (PL) transfection of C57BL/ 6NHsd mouse bone marrow protected cells irradiated at room temperature (24°C) or in the cryopreserved state. MnSOD-overexpressing hematopoietic progenitor 2C6 cells were radioresistant compared to the parent 32D cl 3 cells when irradiated frozen or at 24°C. Fresh whole marrow from mice injected intravenously with MnSOD-PL prior to explant as well as explanted marrow single cell suspensions transfected in vitro were irradiated at 24°C or −80°C. In vivo or in vitro transfection of marrow with MnSOD-PL produced significant radiation protection of irradiated marrow progenitor cells compared to controls at 24°C or −80°C. (in vivo transfection D0 2.19 ± 0.21 at 24°C, D0 2.10 ± 0.07 at −80°C compared to control D0 1.56 ± 0.06 or 1.66 ± 0.04, P = 0.047 and 0.017 respectively; in vitro transfection D0 2.35 ± 0.11 at 24°C, D0 3.42 ± 0.13 at −80°C compared to D0 1.81 ± 0.01 or 2.53 ± 0.05, P = 0.0087 and 0.0026, respectively). Thus the MnSOD transgene product protects frozen marrow cells as well as marrow cells irradiated at 24°C.

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.