Abstract
Normal haematopoietic stem cells are particularly sensitive to radiation-induced apoptosis. Overexpression of the multidrug resistance 1 (MDR1) gene product, P-glycoprotein (P-gp), leads to suppression of apoptosis and is radioprotective in the human lymphoblastoid cell line TK6. Therefore, gene therapy with MDR1 might protect bone marrow cells during tumour-radiotherapy. The aim of this study was to test if human CD34(+) blood stem cells can be protected from the effect of radiation by MDR1 gene transduction. MDR1 cDNA was cloned into the lentiviral SIN-vector pHR’SINcPPT-SEW to replace the eGFP cDNA. CD34(+) cells isolated from four individual donors were exposed to lentiviral supernatants with an MOI of 10 of either HR’SIN-MDR1 or HR’SINcPPT-SEW as a control. After lentiviral transduction, 0.8×105 cells of transduced and non-transduced CD34(+) control cells, respectively, were irradiated with 0–8 Gy and held in liquid culture under differentiation conditions. Ten days after irradiation, the amount of MDR1 expressing cells was determined by the Rhodamine-(Rh−)123 efflux assay and the MDR1-expression rate was monitored by real-time PCR. Additionally, the differentiation status was tested with FACS-analyses for CD11b (myeloid and natural killer cells), CD15 (neutrophils, eosinophils, monocytes), CD33 (myeloid progenitor cells, monocytes), and CD34 (hematopoietic precursors) expression. To test the potential of MDR1 to protect differentiated cells, unirradiated cells were irradiated after 10 days in liquid culture. Apoptotic cells were detected 48 hours later by staining with Annexin V. The transduction efficiency for the MDR1-virus was 3–18%, for the GFP-virus 12–60%. The proportion of RH-123-negative (=MDR1-positive) cells of all four donors increased with escalating radiation doses (e.g. 18–54% from 0–8 Gy). Irradiation of the differentiated cells after ten days of liquid culture also led to an increase of RH-123-negative cells with escalating radiation doses (e.g. 12.5–17% from 0–8 Gy). We found a correlation between radiation dose and differentiation status: independent on transduction the amount of CD11b-cells increased at 2–4 Gy (e.g from 23–45%) and decreased to 9% with 8 Gy; a similar course was also seen for CD15 expression. Our results clearly indicate the radioprotective effect of human blood stem cells by lentiviral MDR1-overexpression. Thus, enhancing repopulation by surviving stem cells may increase the irradiation tolerance of hematopoietic cells and thus contribute to widening the therapeutic range in radiotherapy.
An isoform of Dicer protects mammalian stem cells against multiple RNA viruses
