title: [Effect of Fluorouracil-inducible GM-CSF gene therapy regulated by Egr-1 promoter on chemotherapeutic hematopoietic damage of tumor-bearing mice].
abstract: OBJECTIVE
  In order to explore the regulating 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 GM-CSF 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 Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin(TM). The transfected cell clones (HFCL/EG) have been selected by the addition of G418. The cells are exposed to the clinically important anticancer agent 5-fluorouracil. The activity of EGFP in HFCL/EG cells was detected by flow cytometry. The post-chemotherapeutical expression of GM-CSF in HFCL/EG was confirmed with ELISA and Western blot and RT-PCR respectively. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production post-exposure to 5-Fu was examined. The HFCL/EG cells were transplanted intravenously into B16 melanoma in C. B-17 combined immunodeficient (SCID) mice. 5-Fu was given i.p. at Day 3. The white blood cell number in peripheral blood, the expression of EGFP and GM-CSF and in human stromal cell engrafted in recipient mice were detected by flow cytometry and RT-PCR respectively. Tumor volume in tumor-bearing mice was calculated.
  
  
  RESULTS
  The results indicated that the activity of EGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to 5-Fu increased as compared to non-5-Fu group with flow cytometry, RT-PCR and ELISA respectively. N-acetylcysteine significantly decreased the concentration of GM-CSF in HFCL/EG cells treated with 5-FU. In contrast to two control groups, HFCL/EG (Egr-1 regulatory element-derived expression of GM-CSF gene therapy) resulted in a proportionally obvious increase in the number of white blood cell after chemotherapy and no significant difference was found for tumor inhibition in recipient mice.
  
  
  CONCLUSIONS
  These in vitro data provide an experimental basis for use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from 5-Fu-injury effects.
authors: First Affiliated Hospital of General PLA Hospital, Beijing 100037, China. dunan05@yahoo.com.cn
authors: Nan
authors: N
authors: Du
authors: Xue-Tao
authors: XT
authors: Pei
authors: Wen-Hua
authors: WH
authors: Xiao
authors: Jun-Zhong
authors: JZ
authors: Sun
authors: Yan
authors: Y
authors: Fu
authors: Hui
authors: H
authors: Zhao
authors: Xi-Liang
authors: XL
authors: Wang
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects; D002460:Cell Line; D051766:Early Growth Response Protein 1 / Q000235:genetics*; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology*; D015870:Gene Expression; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006410:Hematopoiesis; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005472:Fluorouracil