Expression and activity of cell cycle-regulatory proteins in normal and transformed corneal endothelial cells

Corneal endothelial cells have a limited capacity for proliferation. Upon t ransformation with the SV40 large T antigen, however, these cells undergo d ivision and grow rapidly. In order to gain insight into the control mechani sms that determine this proliferative switch, we investigated the expressio n level and activity of various known cell cycle-regulatory proteins in the se cells. Primary human and rabbit corneal endothelial cells were transduce d in vitro with a replication-defective adenovirus containing SV40 large T antigen, and subsequently the expression and activity of cell cycle-regulat ory proteins was analyzed. Cells transduced with large T antigen exhibited strongly increased activity of cyclin-dependent kinases. This increase corr elated with the elevated expression of various cyclin-dependent kinase subu nits, such as cyclin A, and to a lesser extent, cyclin D, cdk2, and cdk4. F urthermore, the expression of two cyclin-dependent kinase inhibitors, p21(W AF1) and p27(KIP1), which was high in primary human cells (but not in prima ry rabbit cells), was strongly reduced in large T-antigen transduced cells. Thus, the remarkably low proliferative activity of normal human corneal en dothelial cells appears to be regulated at two levels: the expression of ce rtain cell cycle-regulatory proteins that are essential for cell cycle prog ression is extremely low (cyclin A) or somewhat low (cdk2 and cdt4); but th e amount of p21 and p27, inhibitors of cell cycle progression, is very high . As a consequence, the enzymatic activity of cyclin-dependent kinase is be low detectable levels. However, the growth-inhibitory status of these compo nents is clearly reversible: upon transduction with large T antigen, the ex pression of cyclin A, cyclin D, cdk2, and cdk4 is induced, whereas the expr ession of p21 and p27 is inhibited, and the cells proliferate. Thus, our st udy provides insight into the molecular basis of the attenuated proliferati on of corneal endothelial cells and suggests potential targets that could b e manipulated for the purpose of therapeutic interventions aimed at renewed cell growth. (C) 1999 Academic Press.