Anne Livingstone

Expression in UVW glioma cells of the noradrenaline transporter gene, driven by the telomerase RNA promoter, induces active uptake of [131I]MIBG and clonogenic cell kill.

One of the most effective ways to kill cancer cells is by treatment of tumours with radiation. However, the administered dose of radiation to the tumour is limited by normal tissue toxicity. Strategies which decrease normal tissue exposure relative to tumour dose are urgently sought. One such promising scheme involves gene transfer, leading to the introduction of transporters specific for pharmaceuticals which can be labelled with radionuclides. We have previously demonstrated in vitro, that transfer of the noradrenaline transporter (NAT) gene, under viral promoter control, induces in host cells the active accumulation of the radiopharmaceutical [131I]meta-iodobenzylguanidine ([131I]MIBG) which results in kill of clonogens. We now report 17-fold enhancement of [131I]MIBG uptake by UVW glioma cells transfected with the NAT gene whose expression is driven by the human telomerase RNA (hTR) promoter (70% the uptake achieved by the strong viral promoter). Multicellular spheroids composed of hTR–NAT-transfected UVW cells exhibited dose-dependent susceptibility to treatment with [131I]MIBG. This was demonstrated by decreased survival of clonogens and complete sterilization of clonogens derived from spheroids and also failure of spheroids to regrow after administration of 7 MBq/ml [131I]MIBG. These data suggest hTR regulated expression of NAT may be an effective gene therapy strategy.

Transfectant mosaic spheroids: a new model for evaluation of tumour cell killing in targeted radiotherapy and experimental gene therapy

We describe an in vitro tumour model for targeted radiotherapy and gene therapy that incorporates cell population heterogeneity.

A gene therapy/targeted radiotherapy strategy for radiation cell kill by [131I]meta‐iodobenzylguanidine

Although [131I]meta‐iodobenzylguanidine (MIBG) is currently one of the best agents available for targeted radiotherapy, its use is confined to a few neural crest derived tumours which accumulate the radiopharmaceutical via the noradrenaline transporter (NAT). To determine whether this drug could be used for the treatment of non‐NAT expressing tumours following genetic manipulation, we previously showed that plasmid mediated transfection of NAT into a non‐NAT expressing glioblastoma cell line, UVW, endowed the host cells with the capacity to actively accumulate [131I]MIBG. We now present data defining the conditions required for complete sterilisation of NAT transfected cells cultured as multicellular spheroids and treated with [131I]MIBG.

A gene therapy approach to enhance the targeted radiotherapy of neuroblastoma.

BACKGROUND The aims of this study were to determine whether the introduction and expression of the noradrenaline transporter (NAT) gene into NAT-negative neuroblastoma cell lines would make them amenable to targeted radiotherapy using [(131)I]MIBG. PROCEDURE Neuroblastoma cell lines were transfected with a eukaryotic expression vector containing the bovine noradrenaline transporter cDNA under the expression of the CMV promoter. Stable transfectants were created by selection in geneticin (G418) and were characterised for their MIBG uptake ability and susceptibility to [(131)I]MIBG therapy. RESULTS The cell line SK-N-MC, which normally shows no ability to take up MIBG, was successfully transfected with bNAT. SK-N-MC.bNAT transfectants exhibited uptake and release kinetics similar to those of the natural NAT-expressing cell line SK-N-BE(2c). Levels of [(131)I]MIBG uptake were 33% of those of the highest naturally NAT-expressing cell line SK-N-BE(2c). Growth delay assays using multicellular spheroids indicated that this degree of [(131)I]MIBG uptake was sufficient to inhibit growth at radioactive concentrations of 4 Mbq/ml. CONCLUSIONS These results demonstrate the feasibility of combining gene therapy with targeted radiotherapy to enhance uptake, and hence radiation dose, to neuroblastoma tumours using [(131)I]MIBG. With the appropriate delivery vehicle and tumour-specific control of expression, the introduction of noradrenaline transporter molecules may be a viable means of enhancing the response of neuroblastoma tumours to [(131)I]MIBG therapy.

Assessment In Vitro of a Novel Therapeutic Strategy for Glioma, Combining Herpes Simplex Virus HSV1716-mediated Oncolysis with Gene Transfer and Targeted Radiotherapy

Genetically engineered herpes simplex virus ICP34.5 null mutants replicate only in dividing cells and have shown potential for the treatment of malignant disease, including glioma. Phase I trials have demonstrated the safety of these viruses in various clinical settings but it is envisaged that for full efficacy they will be used in combination with other therapeutic modalities. To enhance virus-induced tumour cytotoxicity, we have engineered an ICP34.5 null mutant (HSV1716) of HSV1 which expresses the noradrenaline transporter gene (NAT). This virus is designated HSV1716/NAT. We have shown previously that introduction of the NAT gene into a range of tumour cells, via plasmid-mediated transfection, conferred the capacity for active uptake of the radiopharmaceutical [131I]MIBG and resulted in dose-dependent toxicity. In this study, combination therapy utilising HSV1716/NAT and [131I]MIBG was assessed in vitro by the MTT assay. We demonstrate that the NAT gene, introduced by HSV1716/NAT into cultured glioma cells, was expressed 1 h after viral infection, enabling active uptake of [131I]MIBG. The combination of viral oncolysis and induced radiopharmaceutical uptake resulted in significantly enhanced cytotoxicity compared to either agent alone and the response was dose- and time-dependent. These studies show that the combination of oncolytic HSV therapy with targeted radiotherapy has the potential for effective tumour cell kill and warrants further investigation as a treatment for malignant glioma.

Combining a targeted radiotherapy and gene therapy approach for adenocarcinoma of prostate

A targeted radiotherapy/gene therapy approach for prostate cancer, using the radiopharmaceutical [131I]meta-iodobenzylguanidine ([131I]MIBG), would restrict the effects of radiotherapy to malignant cells, thereby increasing efficacy and decreasing morbidity of radiotherapy. Prostate cancer cells were transfected with a transgene encoding the noradrenaline transporter (NAT) under the control of tumour-specific telomerase promoters, enabling them to actively take up [131I]MIBG. This led to tumour-specific cell kill. This strategy has the advantage of generating a radiological bystander effect, leading to the destruction of neighbouring tumour cells that have escaped transfection. This targeted approach could be a promising tumour-specific treatment option for prostate cancer.

N-myc amplification and its relationship to experimental therapy

N-myc amplification correlates with poor prognosis in neuroblastoma patients. Although the reason for this is unclear, it is possible that amplified N-myc confers resistance to certain agents used in the therapy of the disease. The acquisition of resistance to cytotoxic drugs in human tumour cells is multifactorial. One mechanism involved in the development of drug resistance is an increased efficiency of DNA repair. This could reduce the effectiveness of both cisplatin and etoposide (VP-16). Previous studies on human neuroblastoma cells have shown a relationship between N-myc copy number and cisplatin sensitivity [1]. We now report the response to VP-16 treatment of five human neuroblastoma cell lines with a range of N-myc gene copy numbers. After exposure of cells to drug for 24 hours, survival curves were constructed from clonogenic assay data and the iso-effective dose (the dose required to produce 1 log cell kill) was derived. The relationship between N-myc copy number or expression and response to VP-16 was assessed. A significant correlation was established between VP-16 resistance and copy number (r = 0.82; P < 0.05). However, no association was found between N-myc expression and isoeffective dose of VP-16. These results indicate that N-myc amplification may be responsible for treatment failure in those patients receiving cisplatin or VP-16.

Radiation studies on multicellular tumour spheroids derived from human neuroblastoma: Absence of sparing effect of dose fractionation

In vitro experiments were carried out to compare the effects of single-dose and split-dose irradiation on a cell line (NB1-G) derived from human neuroblastoma and grown as multicellular tumour spheroids (MTS). The radiation response was evaluated in terms of regrowth delay; estimates of in situ cell survival were made by back-extrapolation of regrowth curves. These studies showed no significant difference in the effectiveness of single as compared to split dose irradiation i.e. no sparing effect of fractionation. If MTS constitute a realistic model for micrometastases in vivo, these results provide a radiobiological rationale for hyperfractionated treatment regimes in the adjuvant radiotherapy of neuroblastoma.