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5-fluorouracil enhances Egr1 promoter upregulate expression of granulocytemacrophage colonystimulating factor in human bone marrow stromal cells
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Abstract:
Abstract Objective: To explore 5fluorouracilinduced regulating effect of Egr1 promoter on expression of granulocytemacrophage colonystimulating factor (GM-CSF) in human bone marrow stromal cells. Methods:The human GMCSF cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and then inserted into the expression vector pCIneo under control of the Egr1 promoter(Egr-EG). The vector was then transferred into human bone marrow stromal cell line HFCL by lipofection. The transfected cell clones (HFCL/EG) were selected by the addition of G418. The cells were exposed to the anticancer agent 5-fluorouracil (5-FU). The activity of EGFP in HFCL/EG cells were detected by FACS. The amounts of GMCSF in HFCL/EG postchemotherapy were determined with ELISA. The effects of GMCSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood were also studied. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production was examined following exposure to 5FU. Results: We successfully constructed vector EgrEG with Egr-1 promoter, and its transfectant HFCL/EG was obtained. The results indicated that the activity of EGFP and the amounts of secreted GMCSF in HFCL/EG cells exposed to 5-FU were increased compared to non5-FU group. The content of GM-CSF in HFCL/EG cultural supernatants was significantly higher than that in the non5-FU group (P<0.01). N-acetylcysteine significantly decreased the content of GM-CSF produced by HFCL/EG treated with 5-FU (P<0.01). Conclusion: 5-FU can enhance Egr-1 upregulate GM-CSF expression in human bone marrow stromal cells, and thus contribute to the recovery of hematopoietic function after chemotherapy.
Keywords:
early growth response1(Egr-1) ; 5-fluorouracil ; granulocytemacrophage colonystimulating factor ; bone marrow stromal cell ; radical oxygen intermediate
Project Supported:
Surpported by the National Natural Science Foundation of China(N0.39900040); the New Major Technology Program of the First Hospital Affiliated to PLA General Hospital (No.ZD200502)
