The Suicide Gene System Cytosine Deaminase::Uracylphosphoribosyltransferase/ 5-fluorocytosine Displays a Strong Cytotoxic Effect on Human Melanoma Cells.

Abstract

Introduction: Melanoma is considered the deadliest form of skin cancer. While monoclonal-antibodies and molecular targets marked milestones in melanoma therapy, more research is needed to overcome the advanced stages of this disease.

Objective: To explore the possible use of the yeast cytosine deaminase::uracil phosphoribosyltransferase fusion enzyme/5-fluorocytosine (CD::UPRT/5FC) suicide gene (SG) system for human melanoma.

Methods: In eight metastatic human melanoma cell lines, we determined: cytotoxicity, lipofection efficiencies, colony forming capacity and bystander effects due to soluble and/or particulate factors secreted to the conditioned media after treatments.

Results: CD::UPRT induced cell death in a prodrug (5FC) concentration-dependent manner and was able to eliminate the sub-population of surviving cells with clonogenic capacity. Compared with human interferon-β gene transfer or the herpes simplex virus thymidine kinase/ganciclovir system, at 100 μM 5FC, CD::UPRT was more efficient in inducing cell death. The strong cytotoxic response contrasted with the low lipofection efficiencies (<5%), indicating a potent bystander effect. We analyzed the contribution of soluble and particulate factors released by SG lipofected cells to the conditioned media (CM) finding that they were able to deliver CD::UPRT genetic information and/or recombinant enzyme to recipient cells. When exposed to 5FC, the cells that received either supernatant or 12000×g pellet fractions of CM, efficiently activated the prodrug because of the acquired CD::UPRT activity and caused cell death.

Conclusion: This suicide gene therapy approach, amplified by the release of free 5-fluorouracil and soluble and particulate factors containing CD::UPRT genetic information and/or enzyme, could have a great clinical potential for malignant melanoma.