Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer

J Worthington, T Robson, S Scott, D Hirst

    Research output: Contribution to journalArticle

    27 Citations (Scopus)

    Abstract

    Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase ( iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine ( PE). CMVi-NOS transfection also reduced response to PE, but by only 52%. A single injection of 25 mu g of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO center dot, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO center dot. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.
    LanguageEnglish
    Pages1417-1423
    JournalGene Therapy
    Volume12
    Issue number19
    DOIs
    Publication statusPublished - Oct 2005

    Fingerprint

    Nitric Oxide Synthase Type II
    Nitric Oxide Synthase
    Genetic Therapy
    Transfection
    Phenylephrine
    Neoplasms
    Transgenes
    Lipids
    Neoplasm Genes
    Vasoconstrictor Agents
    Nitrites
    Tumor Burden
    Sarcoma
    Proteins
    Radiotherapy
    Endothelial Cells
    Arteries
    Radiation
    Apoptosis
    Gene Expression

    Cite this

    Worthington, J ; Robson, T ; Scott, S ; Hirst, D. / Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer. In: Gene Therapy. 2005 ; Vol. 12, No. 19. pp. 1417-1423.
    @article{6004692b11a44cd4a49c2678f524ad94,
    title = "Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer",
    abstract = "Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase ( iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83{\%} inhibition of response to the vasoconstrictor phenylephrine ( PE). CMVi-NOS transfection also reduced response to PE, but by only 52{\%}. A single injection of 25 mu g of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO center dot, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO center dot. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.",
    author = "J Worthington and T Robson and S Scott and D Hirst",
    year = "2005",
    month = "10",
    doi = "10.1038/sj.gt.3302552",
    language = "English",
    volume = "12",
    pages = "1417--1423",
    journal = "Gene Therapy",
    issn = "0969-7128",
    number = "19",

    }

    Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer. / Worthington, J; Robson, T; Scott, S; Hirst, D.

    In: Gene Therapy, Vol. 12, No. 19, 10.2005, p. 1417-1423.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer

    AU - Worthington, J

    AU - Robson, T

    AU - Scott, S

    AU - Hirst, D

    PY - 2005/10

    Y1 - 2005/10

    N2 - Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase ( iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine ( PE). CMVi-NOS transfection also reduced response to PE, but by only 52%. A single injection of 25 mu g of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO center dot, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO center dot. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.

    AB - Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase ( iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine ( PE). CMVi-NOS transfection also reduced response to PE, but by only 52%. A single injection of 25 mu g of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO center dot, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO center dot. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.

    U2 - 10.1038/sj.gt.3302552

    DO - 10.1038/sj.gt.3302552

    M3 - Article

    VL - 12

    SP - 1417

    EP - 1423

    JO - Gene Therapy

    T2 - Gene Therapy

    JF - Gene Therapy

    SN - 0969-7128

    IS - 19

    ER -