Liposome-mediated functional expression of multiple drug resistance gene in human bone marrow CD34⁺ cells

Abstract

The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34⁺ cells was observed. Human normal bone marrow CD34⁺ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34⁺ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34⁺ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34⁺ cells was obviously higher than that in non-transfected CD34⁺ cells. The amount of P-gp in non-transfected CD34⁺ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.01). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34⁺ cells.

Keywords

gene transfection / hematopoietic progenitor cell / multiple drug resistance gene / P-glycoprotein

References

[1] WardM, RichardsonC, PioliP, et al. . Transfer and expression of the human multiple drug resistance gene in human CD34⁺ cells. Blood, 1994, 84(5): 1408-1408

[2] Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, 1989.

[3] CaoW J, ZouP. The study on isolation and expansion of bone marrow CD34⁺ cellsin vitro. Inspection & Chinics (Chinese), 2002, 2(3): 14-14

[4] ChaudharyP M, RoninsonI B. Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell, 1991, 6685-85

[5] RomanoG, MichellP, PacilioC, et al. . Latest developments in gene transfer technology: achievements, perspectives, and controversies over therapeutic applications. Stem Cells, 2000, 18(1): 19-19

[6] PetrizJ, SanchezJ, BertranJ. Comparative effect of verapamil, cyclosporin A and SDZ PSC 833 on rhodamine 123 transport and cell cycle in vinblastine-resistant Chinese hamster ovary cells overexpressing P-glycoprotein. Anticancer Drugs, 1997, 8(9): 869-869