bromodeoxycytidine and Melanoma

Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.

Topics: Animals; Antiviral Agents; Bromodeoxycytidine; Cell Line, Tumor; Cell Separation; Cloning, Molecular; Deoxycytidine; Dependovirus; Enhancer Elements, Genetic; Extracellular Matrix Proteins; Female; Flow Cytometry; Ganciclovir; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Humans; Immunosuppressive Agents; Melanoma; Mice; Mice, Nude; Models, Genetic; Monophenol Monooxygenase; Neoplasm Proteins; Neoplasm Transplantation; Plasmids; Promoter Regions, Genetic; Proteins; Simplexvirus; Thymidine Kinase; Time Factors; Tissue Distribution

The halogenated pyrimidines 5-chloro-2'-deoxycytidine (CldCyd) and 5-bromo-2'-deoxycytidine (BrdCyd) can act as radiosensitizers and cytotoxic agents. It was hypothesized that tumor cells and normal cells might use different metabolic pathways to incorporate these halogenated deoxycytidines into DNA. This difference could potentially be exploited to produce selective radiosensitization and cytotoxicity of human tumor cells compared to normal human fibroblasts. This hypothesis was tested using two human melanoma cell lines and two normal fibroblast cell lines. Either CldCyd or BrdCyd alone caused both cytotoxicity and radiosensitization of tumor and normal cells. The addition of the cytidine deaminase inhibitor tetrahydrouridine (H4U) significantly protected the normal cells but had relatively little effect on the tumor cells. These data indicate that it may be possible to exploit differences between the pyrimidine metabolism of normal cells and melanoma cells to improve the therapeutic index of halogenated pyrimidines both as radiosensitizers and as cytotoxic agents.

Topics: Antineoplastic Agents; Bromodeoxycytidine; Cell Line; Deoxycytidine; Fibroblasts; Humans; In Vitro Techniques; Melanoma; Radiation-Sensitizing Agents; Tetrahydrouridine; Tumor Cells, Cultured; Uridine