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 exami
ne 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 PADP
RP antisense RNA is induced in culture, endogenous PADPRP mRNA, protei
n, and enzymatic activity is lowered, and the pattern of poly(ADP) rib
osylation in response to alkylating agents is altered. When keratinocy
te clones containing the antisense construct or empty vector alone wer
e grafted onto nude mice, they formed histologically normal human skin
. The PADPRP antisense construct was also inducible in vivo by the top
ical application of dexamethasone to the reconstituted epidermis, as d
etermined by in situ hybridization. In addition, poly(ADP-ribose) poly
mer could be induced and detected in vivo following the topical applic
ation of a DNA alkylating agent to the grafted transfected skin layers
. Thus, a model system has been developed in which the levels of PADPR
P can be selectively manipulated in human keratinocytes in cell cultur
e, and potentially in reconstituted epidermis as these keratinocyte li
nes can be grafted to nude mice, whereupon they form a histologically
and immunocytochemically normal human epidermis. Another system that w
e have been utilizing to examine the role of PADPRP in proliferation a
nd 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 subseque
ntly lower levels of endogenous PADPRP. In this system, the normal dif
ferentiation to adipocytes is blocked by the lowering of endogenous PA
DPRP, apparently resulting from inhibition of replication immediately
preceding terminal differentiation. This inhibition in turn may stem f
rom the requirement for the physical association between PADPRP and po
lymerase alpha during the S phase of the cell cycle. These systems wil
l be useful tools to study the role of PADPRP in essential biological
processes.