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


CONCLUSIONS
The therapy of hematopoietic growth factor gene regulated by Egr-1 promoter can protect hematopoiesis from 5-Fu injury.
First Hospital Attaching General Hosptal of PLA, Beijing, China. dunan05@yahoo.com.cn
Nan
N
Du
Xue-tao
XT
Pei
Jin-ming
JM
Zhou
Jun-zhong
JZ
Sun
Yi-xin
YX
Hao
D000111:Acetylcysteine / Q000494:pharmacology; D018952:Antigens, CD34 / Q000097:blood; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D051766:Early Growth Response Protein 1 / Q000235:genetics*; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D005472:Fluorouracil / Q000494:pharmacology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018952:Antigens, CD34; D000964:Antimetabolites, Antineoplastic; D017077:Culture Media, Conditioned; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D016166:Free Radical Scavengers; D008565:Membrane Proteins; C084881:flt3 ligand protein; D049452:Green Fluorescent Proteins; D005472:Fluorouracil; D000111:Acetylcysteine