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.

Experimental targeted radioiodide therapy following transfection of the sodium iodide symporter gene: effect on clonogenicity in both two-and three-dimensional models.

To evaluate the potential of the expression of the sodium/iodide symporter (NIS) as a means of targeting radioiodine to tumor cells, we have employed plasmid-mediated transfection of the NIS gene into a range of mammalian cell hosts. We observed perchlorate-inhibitable iodide uptake up to 41-fold over control in all NIS-transfected cells. We assessed the effect of NIS expression followed by exposure to 131I- on the clonogenic survival of UVW glioma cells. After exposure of two-dimensional monolayer cultures of UVW-NIS cells to 131I- at a radioactive concentration of 4 MBq/mL, clonogenic survival was reduced to 21%. Similar treatment of UVW-NIS cells in three-dimensional spheroid cultures resulted in a reduction of clonogenic survival to 2.5%. This increase in sensitivity to 131I- exposure is likely to be due to a radiological bystander effect. These results are very encouraging for the development of a novel cytotoxic gene-therapy strategy in which a radiological bystander effect plays a significant role in tumor cell sterilization.

Applications of gene transfer to targeted radiotherapy.

For targeted radionuclide therapy to succeed as a single modality treatment, schemes must be devised which will enable the deposition in malignant cells of sterilising doses of radiation. Until such methods have been perfected, it is necessary to combine targeted radiotherapy in a rational manner with conventional anti-cancer treatments. Several means of delivery of therapeutic radionuclides are being evaluated but none of these yet appears to be as powerful as the simplest and most effective example, viz: sodium [131I]iodide treatment of disseminated thyroid carcinoma. The radiopharmaceutical [131I]meta-iodobenzylguanidine ([131I]MIBG) is an effective single agent for the treatment of neuroblastoma. However, uptake of the drug in malignant sites is heterogeneous, suggesting that this therapy alone is unlikely to cure disease. A growing body of experimental evidence indicates exciting possibilities for the integration of gene transfer with radionuclide targeting. This review covers aspects of the combination of gene manipulation and targeted radiotherapy, emphasising the potential of gene transfer to facilitate tumour targeting with low molecular weight radiopharmaceuticals.

No-carrier-added iodine-131-MIBG: evaluation of a therapeutic preparation.

UNLABELLED: Iodine-131-metaiodobenzylguanidine ([131I]MIBG) is a radiopharmaceutical for imaging as well as targeted radiotherapy of neuroblastoma. It is predicted that the use of no-carrier-added [131I]MIBG, rather than the conventional low specific activity preparation, will result in an enhanced therapeutic ratio because of different transport processes in neuroblastoma compared with most normal tissues. METHODS: The main aims of the study were: (1) to determine whether [131I]MIBG of substantially greater specific activity is transported into tumor cells by the same process as the existing compound; (2) to evaluate the effect of nonradiolabeled MIBG on the cytotoxicity of no-carrier-added [131I]MIBG; and (3) to compare the biodistribution of both preparations of the radiochemical in neuroblastoma xenografts. RESULTS: Active uptake of no-carrier-added [131I]MIBG was temperature-, sodium- and oxygen-dependent; ouabain- and desmethylimipramine-inhibitable; and could be blocked competitively by monoamine inhibitors of the noradrenaline transport mechanism. The rank order of specific uptake capacity in a panel of neuroblastoma cell lines was the same for both low and high specific activity drug. Neuroblastoma spheroid regrowth was 85% inhibited by no-carrier-added [131I]MIBG at 2 MBq.ml-1. Inhibitory potency was reduced in a dose-dependent manner by nonradiolabeled MIBG. The accumulation of no-carrier-added [131I]MIBG was significantly greater in tumor, adrenal, heart and skin of tumor-bearing mice than that of the conventional therapy preparation of [131I]MIBG. CONCLUSION: These data indicate that there may be clinical advantages in the use of no-carrier-added [131I]MIBG rather than conventional [131I]MIBG.

The effect of cisplatin pretreatment on the accumulation of MIBG by neuroblastoma cells in vitro.

[131I]meta-iodobenzylguanidine ([131I]MIBG) provides a means of selectively delivering radiation to neuroblastoma cells and is a promising addition to the range of agents used to treat neuroblastoma. As MIBG is now being incorporated into multimodal approaches to therapy, important questions arise about the appropriate scheduling and sequencing of the various agents employed. As the ability of neuroblastoma cells to actively accumulate MIBG is crucial to the success of this therapy, the effect of chemotherapeutic agents on this uptake capacity needs to be investigated. We report here our initial findings on the effect of cisplatin pretreatment on the neuroblastoma cell line SK-N-BE (2c). After treating these cells with therapeutically relevant concentrations of cisplatin (2 microM and 20 microM), a stimulation in uptake of [131I]MIBG was observed. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that this effect was due to increased expression of the noradrenaline transporter. These results suggest that appropriate scheduling of cisplatin and [131I]MIBG may lead to an increase in tumour uptake of this radiopharmaceutical with consequent increases in radiation dose to the tumour.

Noradrenaline transporter gene transfer for radiation cell kill by 131I meta-iodobenzylguanidine.

Meta-iodobenzylguanidine conjugated to 131I-iodine is an effective agent for the targeted radiotherapy of tumors of neural crest origin which express the noradrenaline transporter (NAT). The therapeutic application of 131I MIBG is presently limited to the treatment of phaeochromocytoma, neuroblastoma, carcinoid and medullary thyroid carcinoma. To determine the feasibility of MIBG targeting for a wider range of tumor types, we employed plasmid-mediated transfer of the NAT gene into a human glioblastoma cell line (UVW) which does not express the NAT gene. This resulted in a 15-fold increase in uptake of MIBG by the host cells. A dose-dependent toxicity of 131I MIBG to the transfectants was demonstrated using three methods: (1) survival of clonogens derived from monolayer culture; (2) survival of clonogens derived from disaggregated multicellular spheroids; and (3) spheroid growth delay. 131I MIBG was twice as toxic to cells in spheroids compared with those in monolayers, consistent with a greater effect of radiation cross-fire (radiological bystander effect) from 131I beta-radiation in the three-dimensional tumor spheroids. The highest concentration of 131I MIBG tested (1 MBq/ml) was nontoxic to UVW control cells or spheroids transfected with the NAT gene in reverse orientation. These findings are encouraging for the development of NAT gene transfer-mediated 131I MIBG therapy.

Toxicity to neuroblastoma cells and spheroids of benzylguanidine conjugated to radionuclides with short-range emissions.

Radiolabelled meta-iodobenzylguanidine (MIBG) is selectively taken up by tumours of neuroendocrine origin, where its cellular localization is believed to be cytoplasmic. The radiopharmaceutical [131I]MIBG is now widely used in the treatment of neuroblastoma, but other radioconjugates of benzylguanidine have been little studied. We have investigated the cytotoxic efficacy of beta, alpha and Auger electron-emitting radioconjugates in treating neuroblastoma cells grown in monolayer or spheroid culture. Using a no-carrier-added synthesis route, we produced 123I-, 125I-, 131I- and 211At-labelled benzylguanidines and compared their in vitro toxicity to the neuroblastoma cell line SK-N-BE(2c) grown in monolayer and spheroid culture. The Auger electron-emitting conjugates ([123I]MIBG and [125I]MIBG) and the alpha-emitting conjugate ([211At]MABG) were highly toxic to monolayers and small spheroids, whereas the beta-emitting conjugate [131I]MIBG was relatively ineffective. The Auger emitters were more effective than expected if the cellular localization of MIBG is cytoplasmic. As dosimetrically predicted however, [211At]MABG was found to be extremely potent in terms of both concentration of radioactivity and number of atoms ml(-1) administered. In contrast, the Auger electron emitters were ineffective in the treatment of larger spheroids, while the beta emitter showed greater efficacy. These findings suggest that short-range emitters would be well suited to the treatment of circulating tumour cells or small clumps, whereas beta emitters would be superior in the treatment of subclinical metastases or macroscopic tumours. These experimental results provide support for a clinical strategy of combinations ('cocktails') of radioconjugates in targeted radiotherapy.

Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality.

BACKGROUND: Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart. METHODS: Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect on the regulation of cell-cell communication. RESULTS: The 25 normal subjects and 23 of the 30 children with heart disease had no amino acid substitutions in connexin43. All six children with syndromes that included complex heart malformations had substitutions of one or more phosphorylatable serine or threonine residues. Four of these children had two independent mutations, suggesting an autosomal recessive disorder. Five of these children had substitutions of proline for serine at position 364. A seventh child, with a different heart condition, also had a point mutation in connexin43. Transfected cells expressing the Ser364Pro mutant connexin43 sequence showed abnormalities in the regulation of cell-cell communication, as compared with cells expressing normal connexin43. CONCLUSIONS: Mutations in the connexin43 gap-junction gene, which lead to abnormally regulated cell-cell communication, are associated with visceroatrial heterotaxia.

A gene therapy/targeted radiotherapy strategy for radiation cell kill by.

BACKGROUND: 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. METHODS: NAT transfected UVW cells, grown as monolayers and spheroids, were treated with various doses of [131I]MIBG and assessed for cell kill by clonogenic survival and measurement of spheroid volume over time (growth delay). Spheroids were left intact for different time periods to assess the effect of radiation crossfire on cell death. RESULTS AND CONCLUSIONS: Total clonogen sterilisation was observed when the cells were grown as three-dimensional spheroids and treated with 7 MBq/ml [131I]MIBG. The added benefit of radiation crossfire was demonstrated by the improvement in cell kill achieved by prolongation of the maintenance of [131I]MIBG treated spheroids in their three-dimensional form, before disaggregation and clonogenic assay. When left intact for 48 h after treatment, spheroid cure was achieved by exposure to 6 MBq/ml [131I]MIBG. These results demonstrate that the efficiency of cell kill by [131I]MIBG targeted therapy is strongly dependent on beta-particle crossfire irradiation. This gene therapy/targeted radiotherapy strategy has potential for [131I]MIBG mediated cell kill in tumours other than those derived from the neural crest.