Model systems for the study of the role of PADPRP in essential biological processes

Abstract

The nuclear enzyme poly(ADP-ribose) polymerase (PADPRP) is implicated in a number of cellular processes, including DNA repair, replication, and differentiation. We have been using several model systems to examine these potential roles of PADPRP. A human keratinocyte model system has been developed in which stable lines of epidermal cells contain an inducible construct harboring the antisense cDNA to PADPRP. When PADPRP antisense RNA is induced in culture, endogenous PADPRP mRNA, protein, and enzymatic activity is lowered, and the pattern of poly(ADP) ribosylation in response to alkylating agents is altered. 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 topical application of dexamethasone to the reconstituted epidermis, as determined by in situ hybridization. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the grafted transfected skin layers. Thus, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as these keratinocyte lines can be grafted to nude mice, whereupon they form a histologically and immunocytochemically normal human epidermis. Another system that we have been utilizing to examine the role of PADPRP in proliferation and differentiation is stable lines of mouse preadipocytes that contain an inducible antisense PADPRP RNA. Similar to the keratinocyte system, these cells can inducibly express antisense PADPRP RNA, and subsequently lower levels of endogenous PADPRP. In this system, the normal differentiation to adipocytes is blocked by the lowering of endogenous PADPRP, apparently resulting from inhibition of replication immediately preceding terminal differentiation. This inhibition in turn may stem from the requirement for the physical association between PADPRP and polymerase alpha during the S phase of the cell cycle. These systems will be useful tools to study the role of PADPRP in essential biological processes.