[Effects of fluorouracil-induced Flt3 Ligand gene expression transcriptional regulated by Egr-1 promoter sequences].

AIM

To explore the regulatory effects of Egr-1 promoter sequences in transcriptional targeting by 5-fluorouracil(5-Fu) on the expression of hematopoietic growth factor genes.

METHODS

The human Flt3 Ligand(FL) cDNA and the enhanced green fluorescent protein (EGFP) cDNA were linked with IRES and then inserted into the expression vector pCIneo under control of the Egr-1 promoter(Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL. The transfected cells (HFCL/EF) were exposed to the clinically important anticancer agent 5-fluorouracil. The activity of EGFP in HFCL/EF cells was detected by FACS. The expression of FL in HFCL/EF postchemotherapy was confirmed by ELISA, Western blot and RT-PCR, repectively. The effect of FL in HFCL/EF cultural supernatants on the expansion of CD34(+); cells and CFU-GM was studied. The effect of N-acetylcysteine (a free radical scavenger) on FL production following the exposure to 5-Fu was examined.

RESULTS

The activity of EGFP and the amount of secreted FL in HFCL/EF cells exposed to 5-Fu increased compared to those in non-5-Fu group. The effects of FL in HFCL/EF cultural supernatants on the expansion of CD34(+); cells and CFU-GM were more significant than those of non-5-Fu group. N-acetylcysteine significantly decreased the concentration of FL produced by HFCL/EF treated with 5-FU.

CONCLUSION

The therapy of hematopoietic growth factor gene regulated by Egr-1 promoter can protect hematopoiesis from 5-Fu injury.