Abstract
BackgroundHSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed.MethodsStable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL.ResultsDexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade.ConclusionThe overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment.
Highlights
Herpes Simplex virus thymidine kinase gene (HSV-tk)/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect
Dexamethasone reduces the gap junction intercellular communication (GJIC) of glioma cells In iontophoresis experiments, Lucifer Yellow dye diffused to 10.3 ± 10.6 C6 cells (n = 33) per injected cell in control conditions, and to 7.5 ± 6.5 cells (n = 13) in the presence of 10-6M of dexamethasone (NS tendency, Student's t-test, mean ± SD, Figure 2A)
A similar inhibition of GJIC occurred in LN18 and U87 cells treated with 1 μM dexamethasone for 24 hours (Figure 2C)
Summary
HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Herpes Simplex virus thymidine kinase gene (HSV-tk) suicide gene therapy has lately been used in a variety of cancer models to sensitize replicating cells to the antiviral drug ganciclovir (GCV) [1,2,3]. The bystander effect relies on the transfer of phosphorylated ganciclovir molecules between cells via their gap junctions [6,7], other mechanisms have been described in some models [8,9,10,11] Given their resistance to conventional treatments and their confinement to the brain, malignant gliomas have undergone a variety of human trials of HSV-tk gene therapy. Given the widespread use of corticosteroids in the symptomatic treatment of malignant gliomas patients, we have assessed the effect of dexamethasone on the bystander effect in this type of cancer
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