Transcriptional control of Flt3 ligand targeted by fluorouracil-induced Egr-1 promoter in hematopoietic damage

Nan Du,Jinming Zhou,Xiaosong Li,Yixin Hao,Yan Fu,Xuetao Pei,Hui Zhao

doi:10.1186/1423-0127-16-85

Nan Du, Jinming Zhou + Show 5 more

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https://doi.org/10.1186/1423-0127-16-85

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Abstract

BackgroundIonizing radiation (IR) activate the early growth response-1 (Egr-1) promoter by production of radical oxygen intermediates (ROIs). Egr-EF, an expression vector pCIneo containing Egr-1 promoter cloned upstream of the cDNA for Flt3 ligand, was used to treat hematopoietic damage. 5-fluorouracil, a commonly used chemotherapeutic agent, cause tumor cell death by producing DNA damage and generating ROIs. We therefore hypothesized that clinically employed chemotherapeutic agents that increase ROIs could also be employed to activate Egr-EF in a chemoinducible gene therapy strategy. The goal of this study was to explore the effect of Flt3 Ligand gene transcription regulated by fluorouracil-induced Egr-1 promoter on hematopoietic recovery.MethodsHuman Flt3 Ligand (FL) cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and inserted into the expression vector pCI-neo under control of the Egr-1 promoter (Egr-EF). The vector was transfected into the HFCL human bone marrow stromal cell line, and these cells were exposed to 5-FU, a chemotherapeutic drug. Expression of FL by HFCL/EF cells after 5-FU treatment was determined with ELISA, western blot and RT-PCR assays. In addition, the effect of FL from HFCL/EF cell culture supernatants on growth of CD34+ cells from cord blood was also studied. HFCL/EF cells were injected into CB-17 combined immunodeficient (SCID) mice with B16 melanoma. 5-FU was given three days after injection of the HFCL/EF cells. In the recipient mice, white blood cell levels in peripheral blood and expression of EGFP and FL in human stromal cells were measured. Tumor volumes in tumor-bearing mice were also measured.Results5-FU treatment increased EGFP levels and secreted FL levels in HFCL/EF cells. Supernatants from HFCL/EF cell cultures treated with 5-FU increased CD34+ cell growth significantly. HFCL/EF exhibited an increase in the number of white blood cells after chemotherapy.ConclusionThe data presented here support the use of transcriptional control mediated by chemoinducible gene therapy to reduce hematopoietic injury associated with 5-FU.

Highlights

Ionizing radiation (IR) activate the early growth response-1 (Egr-1) promoter by production of radical oxygen intermediates (ROIs)
Based on our previous study on Egr-1 promoter regulated Flt3 ligand (FL) or GM-CSF expression induced by ionizing radiation (IR) or chemotherapy [9,29], we report here that 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent that stimulates ROIs generation, induces the production of Flt3 Ligand (FL) in human bone stromal cells transfected with Egr-EF containing CArG elements cloned upstream of the cDNA for human recombinant FL. 5-FU was used to recover hematopoiesis from chemotherapy- induced marrow failures and CFUGM in culture and as xenografts in tumor-bearing SCID mice
Effect of 5-FU treatment on enhanced green fluorescent protein (EGFP) expression in HFCL/EF EGFP expression in cultured HFCL/EF cells was higher in cells treated with 5-FU over a range of doses (0 - 400 mmol/L, Figure 1.A) than in those not treated with 5-FU

Summary

Introduction

Ionizing radiation (IR) activate the early growth response-1 (Egr-1) promoter by production of radical oxygen intermediates (ROIs). One approach to control gene expression is through the use of tissue-selective promoters to activate transcription of transgenes [3] Another more interesting approach is the eukaryotic inducible expression system [4,5]. Cells exposed to IR generate reactive oxygen intermediates (ROIs) that activate radio-inducible CArG [CC (A/ T)6GG] DNA elements of the early growth response gene (Egr-1) [7]. Therapeutic genes can be inserted downstream of Egr-1 and their expression can be induced by IR This approach is known as radiationgene therapy [8]. This strategy has been used with cDNA encoding human recombinant Flt Ligand (FL) that has been ligated into a eukaryotic expression vector. The combination of FL with chemotherapeutic agents that damage DNA, such as 5-FU and adriamycin, has resulted in synergistic anticancer effects and reduced hematopoietic cytotoxicity in experimental models [11,12]

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