Radiation Research

Published by: Radiation Research Society

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Radiation Research 166(2):352-359. 2006
doi: 10.1667/RR3603.1

Live Attenuated Salmonella Carrying Platelet Factor 4 cDNAs as Radioprotectors

Bin Liua, Lihua Zhaoa, Xiaofei Yua, Zhibo Hanb, Shihong Lua, Renchi Yanga, and Zhong Chao Han1a

aState Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College,

bNational Research Center for Cell Products, Tianjin, China

11Address for correspondence: Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, People's Republic of China;

Abstract

Liu, B., Zhao, L., Yu, X., Han, Z., Lu, S., Yang, R. and Han, Z. C. Live Attenuated Salmonella Carrying Platelet Factor 4 cDNAs as Radioprotectors. Radiat. Res. 166, 352–359 (2006).

To determine whether live attenuated Salmonella carrying platelet factor 4 cDNAs can protect mice from radiation damage, the attenuated Salmonella SL3261 was used as oral vector for targeted gene delivery. The recovery of mice receiving sublethal total-body irradiation (TBI) was investigated after the oral administration of attenuated Salmonella carrying cDNA for platelet factor 4 (PF4) or truncated PF4. This oral gene therapy protected mice from radiation damage after TBI. The number of bone marrow cells and high proliferative potential colony-forming cells (HPP-CFCs) increased significantly at day 7. Similarly, the administration of PF4 or PF417–70 protein also improved the survival of mice after TBI. Both PF4 gene therapy and protein administration accelerated hematopoietic recovery in vivo in mice after irradiation. In vitro, PF4 also promoted survival and proliferation of 5-fluorouracil-resistant hematopoietic stem/progenitor cells after irradiation. These data demonstrate a novel biological function of PF4 as a protector against radiation injury and suggest that attenuated Salmonella could be used in vivo as a PF4 DNA delivery vector in the management of radiation injury.

Received: September 1, 2005; Accepted: April 17, 2006



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FIG. 1. Effects of PF4 (panel A) or PF417–70 (panel B) on the survival of mice irradiated at 7 Gy. PF4 or PF417–70 at 1 μg/ml was injected subcutaneously into mice 20 h before and 4 h after TBI at 7 Gy. Nine mice were in the PF4-treated groups and 30 mice were in the PF417–70-treated groups; the experiments were repeated three times. Both PF4 and PF417–70 significantly prolonged the survival of mice (P < 0.05).

FIG. 2. In vivo effects of PF4 or PF417–70 on the expansion of bone marrow HPP-CFCs and CFU-GM in mice at day 14 after TBI at 7 Gy. Each group consisted of six mice, and the experiments were repeated three times. All mice received two subcutaneous injections of PF4 or PF417–70 (50 μg/kg) or PBS of similar volume at 20 h before and 4 h after TBI (7 Gy). Three control mice and one mouse treated with PF4 died at day 14 after TBI. The living mice were killed and their femurs were collected. The numbers of HPP-CFCs and CFU-GM obtained from one whole femur were measured. There was a statistically significant increase in the numbers of HPP-CFCs and CFU-GM in the femurs of PF4-treated (panel A) or PF417–70-treated (panel B) mice compared to those of control mice (*P < 0.05).

FIG. 3. Panel A: After oral administration of attenuated Salmonella SL3261 harboring the eukaryotic expression vector, the serum of mice was collected. Protein expression of PF4 and PF417–70 was detected by Western blotting. No PF4 protein expression could be detected in the serum from mice treated with SL3261/eGFP. Panel B: GFP expression was detected in liver (a), kidney (b), spleen (c), intestine (d), peripheral blood cells (e), and bone marrow cells (f) of the SL3261/eGFP treated mice based on confocal microscope examinations.

FIG. 4. Effects of attenuated Salmonella typhimurium carrying PF4 or PF417–70 on the survival of mice and the proliferation of HSPCs after TBI at 7 Gy. Panel A: The orally administered attenuated Salmonella typhimurium carrying PF4 or PF417–70 cDNA sequence significantly prolonged the survival time of mice after TBI (P < 0.05). PF417–70 was as effective as PF4 in protecting mice from death, and there was no significant difference between the two. Increased numbers of bone marrow CFU-GM (panel B) and HPP-CFCs (panel C) were observed at day 7 and day 14 after irradiation in the groups of mice receiving SL3261/PF4 or SL3261/PF417–70 (*P < 0.05). There was no significant difference between the two treatments (P > 0.05). Fifteen mice per group were used for survival analysis, and the experiments were repeated three times.

FIG. 5. Panel A: In vitro effect of PF4 on the survival of 5-FU-resistant murine bone marrow cells. 5-FU-resistant cells (2 × 106/ml) were incubated with various concentrations of PF4 at 37°C for 24 h immediately after irradiation with 7 Gy. The survival of the cells was measured using the MTT method. Data represent the means ± standard deviations of determinations obtained from triplicate experiments. There was a statistically significant difference in cell survival (P < 0.01) between the cells treated with and without PF4 at different PF4 concentrations. Panel B: Time course of PF4 effects on in vitro survival of 5-FU-resistant murine cells. 5-FU-resistant cells (2 × 106/ml) were subjected to 7 Gy irradiation and were then incubated with or without PF4 (500 ng/ml) at 37°C for different times. The survival of cells was measured using the MTT method. Data represent the means ± standard deviations of determinations obtained from triplicate experiments. There was a statistically significant difference (P < 0.05) in cell survival between control cells and PF4-treated cells for each PF4 concentration. Panel C: Effect of PF4 on the production of MNCs from long-term bone marrow culture. Stromal layers obtained from long-term bone marrow culture were irradiated at 15 Gy and were then seeded with irradiated (7 Gy) bone marrow cells (2 × 106/ml). The cells were treated three times with PF4 (500 ng/ml) or PBS at 4 h before, immediately after and 24 h after irradiation. Increased production of MNCs was observed in the PF4 group compared with the PBS group. Panel D: Effect of PF4 on the production of CFCs from the suspension and adherent layers of long-term bone marrow culture after irradiation. Stromal layers obtained from long-term bone marrow culture were irradiated with 15 Gy and were then seeded with bone marrow cells irradiated with 7 Gy. The seeded bone marrow cells were then incubated with PF4 (500 ng/ml) or PBS 4 h before, immediately after, and 24 h after irradiation. Increased CFC production from non-adherent cells was observed in the PF4 group compared with the PBS group. There was a statistically significant difference (P < 0.01) for the cumulative CFCs produced in week 3 to week 5 cultures between PF4-treated cells and control cells.

 
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