Optimization of radiation controlled gene expression by adenoviral vectors in vitro.

Institut für Experimentelle Onkologie & Therapieforschung, München, Germany.
Cancer Gene Therapy (Impact Factor: 2.95). 08/2005; 12(7):640-6. DOI: 10.1038/sj.cgt.7700829
Source: PubMed

ABSTRACT The radiation-inducible EGR-1-promoter has been used in different gene therapy approaches in order to enhance and locally restrict therapeutic efficacy. The aim of this study was to reduce nonspecific gene expression in the absence of irradiation (IR) in an adenoviral vector. Rat rhabdomyosarcoma R1H tumor cells were infected with adenoviral vectors expressing either EGFP or HSV-TK under control of the murine EGR-1 promoter/enhancer. Cells were irradiated at 0-6 Gy. Gene expression was determined by FACS-analysis (EGFP), or crystal violet staining (HSV-TK). The bovine growth hormone polyadenylation signal (BGH pA) was used as insulating sequence and was introduced upstream or upstream and downstream of the expression cassette. Infected R1H cells displayed IR dose-dependent EGFP expression. Cells treated with IR, AdEGR.TK and ganciclovir displayed a survival of 17.3% (6 Gy). However, significant gene expression was observed in the absence of IR with EGR.TK and EGR.EGFP constructs. Introduction of BGHpA upstream or upstream and downstream of expression cassette resulted in decreased nonspecific cytotoxicity by a factor of 1.6-2.3 with minor influence on the induced level of cytotoxicity. Introduction of insulating sequences in adenoviral vectors might allow tighter temporospatial control of gene expression by the radiation-inducible EGR-1 promoter.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Significant evidence has accumulated indicating that certain genes are induced by ionising radiation. An implication of this observation is that their promoter regions include radiation-responsive sequences. These sequences have been isolated in the promoter of several genes including Erg-1, p21/WAF-1, GADD45alpha and t-PA. The mechanism by which radiation induces gene expression remains unclear but involves putative binding sites for selected transcription factors and/or p53. Consensus CC(A/T)6GG sequences have been localized in the Erg-1 promoter and are referred to as serum response elements or CArG elements. The tandem combination of CArG elements has been shown to improve gene expression levels, with a 9-copy motif conferring maximum inducibility. The response of these genes to ionising radiation appears to follow a sigmoid relationship with time and dose. Therapeutic induction of suicide genes and significant cytotoxicity can be achieved at clinically relevant x-rays doses both in vitro and in vivo but was found to be cell-type dependent. Radiation-inducible gene therapy can be potentially enhanced by exploiting hypoxia through the inclusion of hypoxia-response element motifs in the expression cassette, the use of the anaerobic bacteria or the use of neutron irradiation. These results are encouraging and provide significant evidence that gene therapy targeted to the radiation field is a reasonably attractive therapeutic option and could help overcome hypoxic radioresistant tumors.
    Cancer biology & therapy 06/2007; 6(7):1005-12. · 3.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tumor microenvironment is composed of different cell types including immune cells. Far from acting to eradicate cancer cells, these bone marrow-derived components could be involved in carcinogenesis and/or tumor invasion and metastasis. Here, we describe an alternative approach to treat solid tumors based on the genetic modification of hematopoietic stem and progenitor cells with lentiviral vectors. To achieve transgene expression in derivative tumor infiltrating leukocytes and to try to decrease systemic toxicity, we used the stress inducible human HSP70B promoter. Functionality of the promoter was characterized in vitro using hyperthermia. Antitumor efficacy was assessed by ex vivo genetic modification of lineage-negative cells with lentiviral vectors encoding the dominant-negative mutant of the human transforming growth factor-β receptor II (TβRIIDN) driven by the HSP70B promoter, and reinfusion of cells into recipient mice. Subsequently, syngeneic GL261 glioma cells were subcutaneously injected into bone marrow-transplanted mice. As a result, a massive antitumor response was observed in mice harboring TβRIIDN under the HSP70B promoter, without the need of any external source of stress. In summary, this study shows that stem cell-based gene therapy in combination with spatial and temporal control of transgene expression in derivative tumor-infiltrating cells represents an alternative strategy for the development of novel antitumor therapies.
    Cancer gene therapy 03/2012; 19(5):352-7. · 3.13 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating reactive oxygen intermediates, which have been reported to activate the early growth response-1 (Egr-1) promoter through specific cis-acting sequences, termed CArG elements. The aim of this study was to construct an adenoviral vector containing CArG elements cloned upstream of the cDNA for human wt-p53, and to observe the effect of this vector on human non-small cell lung cancer (NSCLC) xenografts in athymic nude mice when combined with cisplatin treatment. The adenoviral vector AdEgr-p53 was generated by inserting CArG elements upstream of human wt-p53 cDNA. Two human NSCLC cell lines of varying p53 gene status, A549 (containing wild-type p53) and H358 (containing an internal homozygous deletion of the p53 gene) were used for in vitro and in vivo experiments. Wt-p53 production in cultured tumor cells and xenografts treated with the combination of AdEgr-p53 and cisplatin were detected by enzyme-linked immunosorbent assays. The antitumor responses in nude mice with the A549 or H358 xenografts following treatment with AdEgr-p53 and cisplatin were observed. We found that p53 was produced in tumor cells and xenografts treated with a combination of AdEgr-p53 and cisplatin. Furthermore, the Egr-1 promoter is induced by cisplatin, and this induction is mediated in part through the CArG elements. There was an enhanced antitumor response without an increase in toxicity following treatment with AdEgr-p53 and cisplatin, compared with either agent alone. Cisplatin-inducible p53 gene therapy may provide a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents. This is a novel treatment for human NSCLC.
    Cancer Science 11/2005; 96(10):706-12. · 3.48 Impact Factor

Full-text (2 Sources)

Available from
Jun 10, 2014