8-Methoxypsoralen and ultra-violet A (PUVA) therapies are used in the treatment of psoriasis and cutaneous T cell lymphoma (CTCL). Recently, a new phototherapy for CTCL, photopheresis, has been developed which may serve as a prototype of a new form of photopharmacology. The efficacy of PUVA in these diseases has been attributed to the formation of 8-MOP photoadducts in cellular DNA. The nature and quantity of these adducts in mammalian systems has not been determined, nor has their repair been fully characterized. AlphaDNA, a 92 base pair fragment widely distributed throughout the human genome, will be used to elucidate the effects of specific base sequences on photoadduct formation. Three psoralens (8-methoxypsoralen, 4'-aminomethyl-4,5',8-trimethylpsoralen, and 4',5-dimethylangelicin) will be used in comparative studies to discern the effects of base sequences on monoadduct and crosslink formation. The overall objective of the proposed studies is to determine the nature, quantity and location of the UVA-induced psoralen photoadducts in AlphaDNA. Specifically the following aspects will be examined. 1. the quantitation of psoralen photoadducts in AlphaDNA by HPLC analysis of enzymatically hydrolyzed AlphaDNA samples. AlphaDNA from PUVA treated lymphoid cells will also be analyzed in the same way. 2. the determination of base sequences in which photoadducts have formed, as well as the identity of adducts in specific DNA sites using footprinting techniques in combination with psoralen-DNA monoclonal antibodies. 3. cytofluorometric determination of the effects of PUVA on cultured lymphoid cells using both propidium iodide staining and specific psoralen-DNA monoclonal antibodies. The development of the techniques described in this proposal will eventually lead to the detailed characterization of the repair of psoralen photoadducts. Knowledge of the sites of unrepaired photoadducts will lead to a better understanding of phototherapies based on psoralen plus UVA and culminate in the rational design of new, potentially more effective, psoralens.
