Selective killing of glioma cell lines using an astrocyte-specific expression of the herpes simplex virus-thymidine kinase gene (original) (raw)
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Cancer Gene Therapy, 2000
Herpes simplex virus thymidine kinase (HSV-tk) gene transfer and ganciclovir (GCV) administration have been suggested for the treatment of malignant gliomas. To understand tissue responses and possible ways to improve the treatment effect, we studied tumor growth, tissue reactions, and survival time after HSV-tk/GCV treatment in a syngeneic BT4C rat glioma model by mixing various ratios of stably transfected HSV-tk-expressing BT4C-tk glioma cells with wild-type BT4C glioma cells (percentage of BT4C-tk cells: 0%, 1%, 10%, 30%, 50%, and 100%), followed by injection into BDIX rat brains (n ϭ 79). With the exception of some animals with end-stage tumors, very little astroglia or microglia reactivity was detected in the wild-type tumors as analyzed by immunocytochemistry using glial fibrillary acid protein (GFAP)-, vimentin-, human histocompatibility leukocyte antigen-DR-, OX-42-, and CD68-specific monoclonal antibodies. After 14 days of GCV treatment, tumors induced with Ն10% BT4C-tk cells showed a significant reduction in tumor size (P Ͻ .05) and prolonged survival time (P Ͻ .01). Astrogliosis, as indicated by a strong GFAP and vimentin immunoreactivity, was seen in the tumor scar area. GFAP and vimentin reactivity was already present after the GCV treatment in tumors induced with 1% BT4C-tk cells. Much less human histocompatibility leukocyte antigen-DR-positive microglia was seen in the treated animals, indicating low microglia reactivity and immunoactivation against the tumor. However, GCV-treated tumors were positive for apoptosis, indicating that apoptosis is an important mechanism for cell death in the BT4C-tk glioma model. Our results suggest that Ն10% transfection efficiency is required for a successful reduction in BT4C glioma tumor size with HSV-tk/GCV treatment in vivo. Tissue reactions after 14 days of GCV treatment are characterized by astrogliosis and apoptosis, whereas microglia response and immunoactivation of the brain cells appear to play a minor role. Stimulation of the microglia response by gene transfer or other means might improve the efficacy of the HSV-tk/GCV treatment in vivo. Cancer Gene Therapy (2000) 7, 413-421
Cancer Gene Therapy, 2000
Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: Ͻ2 g/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: Ͼ75 g/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.
Cancer Gene Therapy, 2005
To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkB expressing both HSV-TK and a mutant form of the NF-kB inhibitor IkBa (IkBaM) was developed. TOIkB was constructed by recombining the IkBaM gene into the U L 41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkBaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kB was already translocated to the nucleus without receptor-dependent signaling by TNF-a. Following infection with TOIkB, nuclear translocation of NF-kB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIkB and 80-85% for TOZ.1. TOIkB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIkB injection and GCV administration. Survival of TOIkB þ GCV group was significantly longer (Po.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkB only, PBS or PBS þ GCV). These results suggest that IkBaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.
Cancer Gene Therapy, 2000
Transfer of the herpes simplex virus thymidine kinase (HSVtk) gene, followed by administration of ganciclovir (GCV), generates the "bystander effect," in which HSVtk-negative wild-type cells are killed by GCV, as are HSVtk-expressing cells. Our previous study demonstrated that intracranial 9L gliomas could be efficiently treated due to this bystander effect by injecting the 9L glioma cells transduced with the HSVtk gene in the vicinity of the preimplanted wild-type 9L glioma and then administering GCV. For a possible clinical application of the bystander effect-mediated cell killing, we tested HSVtk gene-transduced allogeneic C6 glioma cells (C6tk) instead of syngeneic 9L glioma cells transduced with the HSVtk gene. Fisher rats were implanted intracranially with wild-type 9L glioma cells, subsequently injected with C6tk cells at the same brain coordinate, and thereafter treated with GCV or saline. When the rats were treated with GCV, a significant retardation of tumor growth was observed by serial magnetic resonance imaging, although this growth retardation was less prominent than that observed with 9L glioma cells transduced with the HSVtk gene; consequently, survival was prolonged (P Ͻ .01). Tumors that received C6tk cells contained almost no HSVtk-positive cells after treatment with GCV. Rejection of allogeneic tumor cells, although possibly incomplete in the brain, can also contribute to the safety of this therapeutic strategy. Cancer Gene Therapy (2000) 7, 947-953
Proceedings of the National Academy of Sciences, 1995
Genetically engineered viruses and viral genes inserted into retroviral vectors are increasingly being considered for experimental therapy of brain tumors. A primary target of these viruses and vectors is human gliomas, the most frequently occurring primary human brain tumor. To investigate the potential of genetically engineered herpes simplex viruses (HSVs) in the therapy of these tumors, we compared the attributes of two viruses, a recombinant from which the gamma 1(34.5) gene had been deleted (R3616) and a recombinant in which the gamma 1(34.5) gene had been interrupted by a stop codon (R4009). Previous studies have shown that these recombinants were completely devoid of the ability to multiply in the central nervous system of rodents. To pursue these studies, we developed a scid mouse glioma model. Tumor cell response (survival) for 10(3), 10(4), and 10(5) implanted MT539MG glioma cells was 38, 23, and 15 days, respectively. The results were as follows: (i) both R3616 and R4009...
Frontiers in Neurology
IntroductionThe prognosis for glioblastoma multiforme (GBM), a malignant brain tumor, is poor despite recent advancements in treatments. Suicide gene therapy is a therapeutic strategy for cancer that requires a gene to encode a prodrug-activating enzyme which is then transduced into a vector, such as mesenchymal stem cells (MSCs). The vector is then injected into the tumor tissue and exerts its antitumor effects.Case presentationA 37-year-old man presented to our department with two evident foci of glioblastoma multiforme at the left frontal and left parietal lobes. The patient received an injection of bone marrow-derived MSCs delivering the herpes simplex virus thymidine kinase (HSV-tk) gene to the frontal focus of the tumor, followed by ganciclovir administration as a prodrug for 14 days. For follow-up, the patient was periodically assessed using magnetic resonance imaging (MRI). The growth and recurrence patterns of the foci were assessed. After the injection on 09 February 2019,...
Treatment of rat gliosarcoma brain tumors by HSV-based multigene therapy combined with radiosurgery
Molecular Therapy, 2003
Our laboratory has employed replication-defective herpes simplex virus type 1 gene transfer vectors for treatment of animal models of human malignant glioblastoma. The base vectors were defective for the immediate early (IE) genes ICP4, ICP27, and ICP22 but expressed the IE gene ICP0, which can arrest tumor cell division, and an IE thymidine kinase (␣-tk) gene construct that mediates suicide gene therapy (SGT) in the presence of ganciclovir (GCV). Previously, we reported that SGT using ICP0/␣-tk vectors in nude mouse models of glioblastoma was improved by coexpression of the gap-junction-forming protein connexin43 (Cx43) or human tumor necrosis factor ␣ (TNF␣). We also showed that further gains in therapeutic outcome could be achieved by combining TNF␣-enhanced SGT with gamma-knife radiosurgery (GKR). To expand these observations, we have first repeated these studies in immunocompetent rats with brain tumors derived from implanted 9L gliosarcoma cells and second compared the most efficient vector from this study with a new recombinant vector, NUREL-C2, which expressed both TNF␣ and Cx43 along with ICP0 and ␣-tk. Results from the first part indicated that our ICP0/␣-tk/TNF␣ vector in combination with GKR provides an effective therapy although this treatment was not statistically better than GKR combined with the ICP0/␣-tk/Cx43 vector. Our observations in the second part suggested that NUREL-C2 may be more effective than the ICP0/␣-tk/TNF␣ vector in combination treatments with GCV (P ؍ 0.08) or GCV plus GKR (P ؍ 0.10). GKR significantly enhanced the efficacy of NUREL-C2/GCV treatment (P ؍ 0.02) as well as other virus/GCV treatments (P < 0.05). Conversely, the efficacy of GKR was significantly improved by both the ICP0/␣-tk/TNF␣ vector and NUREL-C2 in combination with GCV (P ؍ 0.02 and P < 0.01, respectively). Together these results indicate that NUREL-C2 may be an attractive candidate for Phase I gene-therapy safety studies in patients with recurrent malignant glioblastoma.