Combined gene therapy with suicide gene and interleukin-12 is more efficient than therapy with one gene alone in a murine model of hepatocellular carcinoma

doi:10.1016/S0168-8278(00)80073-2

Journal of Hepatology

Volume 32, Issue 2, February 2000, Pages 279-286

https://doi.org/10.1016/S0168-8278(00)80073-2 Get rights and content

Abstract

Background/Aims: Gene therapy has emerged as a new form of treatment for unresectable hepatocellular carcinoma (HCC). We evaluate here the effect of IL-12 and the suicide gene thymidine kinase as single agents and in combination to treat experimental liver cancer.

Methods: Recombinant adenoviruses expressing mouse interleukin-12 (AdCMVIL-12) or thymidine kinase of herpes simplex virus (AdCMVtk) or lacZ reporter gene (AdCMVlacZ) were constructed. The efficacy of the treatment was evaluated in a murine HCC model based on subcutaneous implantation of liver tumor cells (BNL).

Results: Transduction of BNL cells after in vitro infection with AdCMVlacZ was very low at multiplicity of infection (moi) of 100, whereas 10–15% of cells were transduced when using moi 1000. Similarly, production of IL-12 was detectable only in BNL cells infected with AdCMVIL-12 at moi 1000. In vitro infection of BNL cells with AdCMVIL-12 at moi 100 did not abrogate tumorigenicity, whereas moi 1000 resulted in inhibition of tumor growth in all mice as well as in abrogation of tumor formation in 3 out of 8 animals. In vivo studies showed that intratumor injection of AdCMVIL-12 induced a dose-dependent effect on tumor regression. However, none of the animals exhibited complete tumor elimination with this treatment. We observed that suppression of tumor growth was more intense in animals treated with the combination of AdCMVIL-12 plus AdCMVtk than in animals which received AdCMVtk or AdCMVIL-12 alone. The combined treatment resulted in a significant increase in animal survival, and 25% of treated animals were free of tumor for over 100 days without recurrence of the disease.

Conclusions: Combination of AdCMVIL-12 and AdCMVtk is more efficient than either of the two vectors alone for the treatment of the murine model of HCC used in this study.

Section snippets

Animals

Female BALB/c mice aged 6–8 weeks were obtained from Charles Rivers Laboratories (Barcelona, Spain). During the experimental period animals were housed in standard conditions, and all animal procedures were performed according to approved protocols and in accordance with recommendations for proper care and use of laboratory animals.

Cell lines and cell culture

Murine BNL 1MEA.7R.1 (BNL) methylcholanthrene transformed liver cell line and 293 were obtained from American Type Culture Collection (Rockville, MD, USA). The cells

Gene transfer efficiency in BNL cells with Ad vectors

As shown in Fig. 1, BNL cells are relatively resistant to infection by AdCMVlacZ. At moi 100, only a few cells were infected, and an increase of moi to 1000 raised the number of transduced cells up to 10–15%. Intratumoral injection of AdCMVlacZ 5×10⁹ resulted in transduction of cells surrounding the site of injection. More distant parts of the tumor were not transduced.

IL-12 production after infection of BNL cells with AdCMVIL-12

Supernatants collected from non-infected BNL cells and cells infected in vitro with AdCMVlacZ at moi 100 and 1000 did not show

Discussion

Immunogene therapy represents remarkable progress in the field of cancer gene therapy. Many cytokines have been studied so far, including IL-2, IL-4, IL-7, IL-10, IL-12, IFN-γ, TNF-α, and GM-CSF 6., 7., 8., 9.. Among these cytokines, IL-12 is of particular interest because of its critical role in the stimulation of cellmediated immunity (10). In addition, IL-12 has antiangiogenic effect by induction of chemokines such as IP-10 and Mig 14., 18.. IL-12 has been shown to be very effective in

Acknowledgements

This work was supported by SAF 98–0146 from CICYT. M.D. is supported by a fellowship from the Fundación Empresa Universidad de Navarra, R.B. by Beca del Programa de Formación de Investigadores del Departamento de Educacion, Universidades e Investigacion del Gobiermo Vasco, and G.M. by an Ines Bemberg Grant. We thank Pilar Alzuguren for her excellent technical assistance.

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Combined gene therapy with suicide gene and interleukin-12 is more efficient than therapy with one gene alone in a murine model of hepatocellular carcinoma

Abstract

Section snippets

Animals

Cell lines and cell culture

Gene transfer efficiency in BNL cells with Ad vectors

IL-12 production after infection of BNL cells with AdCMVIL-12

Discussion

Acknowledgements

Immunol Today

Adv Immunol

Immunol Today

Immunol Today

Immunol Today

Immunol Today

Hepatology

Hepatocellular carcinoma

J Hepatol

Treatment of hepatocellular carcinoma: too many options?

J Clin Oncol

Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells

Nat Med

IL-12: a key cytokine in immune regulation

Immunol Today

Cloning of cDNA for natural killer cell stimulatory factor, a heterdimeric cytokine with multiple biologic effect on T and natural killer cells

J Immunol

IL-12 acts directly on CD4+ T cells to enhance priming for IFN-γ production and diminishes IL-4 inhibition of such priming

Proc Natl Acad Sci USA

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocytes activity and induces production of IFN-γ in vivo

Int Immunol

Interleukin-12 inhibits angiogenesis in vivo

J Natl Cancer Inst

Antitumor and antimetastatic activity of interleukin-12 against murine tumors

J Exp Med

Recombinant interleukin-12 administration induces tumor regression in association with interferon-γ production

J Immunol

Administration of recombinant interleukin-12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes

Cancer Res

Cytokine and chemokine expression in tumors of mice receiving systemic therapy IL-12

J Immunol

Effect of interleukin 12 on tumor induction by 3-methylcolantrene

Proc Natl Acad Sci USA

Cancer trial of interleukin-12 halted

Science

After initial setback, IL-12 regaining popularity

J Natl Cancer Inst

Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12

Hum Gene Ther

IL-12 acts directly on CD4⁺ T cells to enhance priming for IFN-γ production and diminishes IL-4 inhibition of such priming