ENGINEERED HUMAN SKIN MODEL USING POLY(ADP-RIBOSE) POLYMERASE ANTISENSE EXPRESSION SHOWS A REDUCED RESPONSE TO DNA-DAMAGE

Poly(ADP-ribose) polymerase (PADPRP) modifies nuclear proteins in resp onse to DNA-damaging agents. The principal organ subject to exposure t o many of these agents is the skin. To understand the role of PADPRP i n the maintenance of the epidermis, a model system has been developed in which we have selectively lowered the levels of this enzyme by the use of induced expression of antisense RNA. Human keratinocyte lines w ere stably transfected with the cDNA for human PADPRP in the antisense orientation under an inducible promoter. Induction of this antisense RNA in cultured cells selectively lowers the levels of PADPRP mRNA, pr otein, and enzyme activity. Induction of antisense RNA also led to a r eduction in the levels of PADPRP in individual cell nuclei, as well as the loss of the ability of cells to synthesize and modify proteins by poly(ADP-ribose) polymer in response to DNA damage. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice, they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topic al application of dexamethasone to the reconstituted epidermis. In add ition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the gra fted transfected skin layers. Accordingly, a model system has been dev eloped in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as well. This system will be a useful tool to study the rol e of PADPRP and DNA repair in general in essential biologic processes in the epidermis.