Establishment of a Screening System for Chemicals that Upregulate a Melanoma Antigen, Melan-A/MART-1

Abstract

Immunotherapy is well-practiced as one of the main adjuvant therapies for melanoma patients. Until now, many immunotherapeutic investigations have focused on improving the effector side of the antitumor response, but only a few studies have been concerned with preventing the loss of tumor-associated antigen (TAA) expression. Loss of TAA should be an important problem for the recognition of tumor cells by cytotoxic T lymphocytes. If any agents that augment the expression of melanoma antigens were found, they could improve the efficacy of immunotherapy by increasing the antigens. To detect effective chemicals, we made a fluorescent cellular reporter system for screening promising candidate chemicals. In this system, the fusion gene of the Melan-A/MART-1 promoter sequence followed by the green fluorescent protein (GFP) coding region was stably transfected into MUX human melanoma cells which are known to express little or no Melan-A/MART-1. Melan-A/MART-1 is a well-known melanoma antigen recognized by autologous cytotoxic T cells, and is a glycoprotein associated with the melanosome, the organelle in which melanin synthesis proceeds. By using this screening system, daunorubicin, doxorubicin and cytochalasin D, which enhanced the green fluorescent, were selected and then were confirmed to actually increase the expression of Melan-A/MART-1 mRNA and protein in human melanoma cells of MU89, MM96L⁺ and SK-MEL-28, but also in low-antigen presenting cells such as MM96L^-, MUX, and A375. In conclusion, we have successfully established a well-functioning screening system, which will allow us to find candidate chemicals that up-regulate or maintain the melanoma antigen expression.