[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.