Low-dose radiation modulates human mesenchymal stem cell proliferation through regulating CDK and Rb

Abstract

Low-dose radiation (LDR) has been known to stimulate cell proliferation. The effect of LDR on human bone marrow mesenchymal stem cells (BMSCs), however, remains to be determined. The current study, therefore, aimed to investigate the effect of LDR on human BMSC proliferation and its mechanisms. To accomplish this, human BMSCs were isolated from ribs and cultured with or without exposition to LDR (75 mGy) for 24 h. Cell proliferation was assessed by MTT assay, the cytokines secreted by the BMSCs were quantified by ELISA, and the proteins associated with cell proliferation and cell cycle were evaluated by immunoblot analysis. BMSCs isolated from human ribs were capable of differentiating into osteoblasts and adipocytes. LDR stimulated human BMSC proliferation (0.580 ± 0.106 vs 0.419 ± 0.026 on day 4, P < 0.05; 0.794 ± 0.025 vs 0.689 ± 0.047 on day 7, P < 0.05) and increased S-phase proportion. LDR significantly enhanced the production of SCF, GM-CSF, and IL-11. Moreover, BMSCs modulated T-cell proliferation, and LDR further augmented the modulatory effect of BMSCs on T-cell proliferation. Cell cycle-associated proteins, such as Rb, CDK1, and CDC25B, appeared to mediate the stimulatory effect of LDR on BMSC proliferation. The findings of the current study indicate that physical stimulants, such as LDR, could be used for the large-scale expansion of human BMSCs, and thus may be used for MSC cellular therapy in clinic.

Keywords: CDK; Low-dose radiation; Rb; bone marrow mesenchymal stem cells.

Figure 1

BMSC culture and differentiation. Mouse BMSCs were isolated and cultured as described in the methods: A. Morphology of BMSCs at confluence; B. von Kossa staining following osteogenic differentiation; C. Oil red staining following adipogenic differentiation. Data presented was one representative of five separate experiments.

Figure 2

Effect of low-dose radiation on BMSC proliferation and cell cycle. BMSCs were exposed to low-dose radiation (LDR), and cell proliferation was measured by MTT as described in the methods. A. Cell proliferation assessed by MTT. Vertical axis: OD value; horizontal axis: time (days). Open circle: non-treated cells (control); slide square: low-dose radiation-treated cells; B. Cell cycle assessment by flow cytometry. Vertical axis: percentage; horizontal axis: cell cycles.

Figure 3

Effect of low-dose radiation on cytokine release. BMSCs were exposed to low-dose radiation for 24 h, and the medium was harvested for quantification of the cytokines by ELISA as described in the methods. A. Stem-cell factor (SFC); B. Granulocyte macrophage colony-stimulating factor (GM-CSF); C. Interleukin-6 (IL-6); D. Interleukin-11 (IL-11). Vertical axes: OD value; horizontal axes: time (day).

Figure 4

Modulation of lymphocyte proliferation by BMSCs exposed to low-dose radiation. BMSCs were treated with or without low-dose radiation followed by plating in 96-well plates at a density of 5 × 10⁴/well, 1 × 10⁵/well, and 2 × 10⁵/well and allowed to attach for 72 h. T lymphocytes were then plated in the 96-well plates and treated with Con A as described in the methods. The number of T lymphocytes was determined by MTT assay. Vertical axis: OD value; horizontal axes: BMSC cell density. Data presented are the average of five separate experiments.

Figure 5

Effect of low-dose radiation on the phosphorylation of Rb and CDK1, and protein levels of cyclin E, CDK2, and CDC25B. BMSCs were treated with or without low-dose radiation as described in the methods. Total cell lysates were subjected for immunoblotting as described in the methods. ß-actin or α-tubulin was used as an internal control. A. Phospho-Rb; B. α-cyclin E and α-CDK2; C. Phospho-CDK1, and α-cyclin B1; D. CDC25B. Data presented are representative of five separate experiments.