This project investigates cell cycle regulation in proliferating, quiescent, and differentiating lens cells through studies of proto- oncogenes, cyclins, and cyclin dependent kinases (Cdks). Our previous work has identified two members of the Cdk family that retain their kinase activity in differentiating lens fibers: Cdk1/Cyclin B and Cdk5/p35. A survey of six remaining members of the Cdk family in the fibers and epithelia of rat lenses, completed during the current year, has found no other Cdk activity in differentiating fiber cells, underscoring the potential importance of Cdk1 and Cdk5 in lens differentiation. Since Cdk5 has previously been considered a neuron-specific enzyme, we have focused on determining its possible role in the lens and other non-neuronal cells. Immunofluorescence studies have been carried out on paraffin sections of neonatal rat eyes to determine whether other ocular tissues express Cdk5. Results show strong expression in neural retina, with lower levels of expression in the lens, corneal epithelium, and possibly the trabecular meshwork. In the cornea, Cdk5 is especially high along the basal membrane of the basal cells, suggesting an association with the cortical cytoskeleton. To study the role of Cdk5 in lens differentiation, we have overexpressed wild type Cdk5 or dominant negative mutations of Cdk5 in differentiating lens explants in vitro using adenoviral vectors. Preliminary results from these experiments suggest that overexpression of Cdk5 in lens fibers accelerates the caspase-dependent cleavage of PARP, suggesting that Cdk5 may play a role in the apoptotic events of terminal differentiation. These studies have also provided evidence that Cdk5 is under stringent negative control in the lens. Additional studies to confirm these observations are in progress. The observation that Cdk5 is expressed in both corneal epithelium and lens, suggested that Cdk5 might have some general function in epithelial cells. To investigate this possibly we have used the well characterized Cos-1 monkey kidney epithelial cell line. We have constructed stably transfected Cos-1 cells carrying either Cdk5 or a dominant negative mutation of Cdk5 (Cdk5-T33). Both constructs were well expressed and the Cdk5 stable transfectants had elevated Cdk5 activity. RT-PCR has demonstrated that Cos-1 cells express both p35 and p39, both of which are known activators of Cdk5. Cells overexpressing Cdk5 grow more slowly and show higher levels of apoptosis than either untransfected cells or cells transfected with the dominant negative construct, supporting a role for Cdk5 activity in initiating apoptosis. Recently published studies indicate that Cdk5 may be a downstream effector of Rac, which would imply a role of Cdk5 in cytoskeletal organization and cell migration. Cell migration and adhesion studies performed with our stably transfected Cos-1/Cdk5 cells, using either a Boyden chamber assay or the ECIS technique, confirm this possibility. The association between apoptosis and Cdk5 activity suggested that Cdk5 might be activated by stresses that induce apoptosis, such as heat shock. We have explored this possibility using a glioma cell line, U373, which has been widely used for studies of heat shock. The results indicate that severe heat shock (46C, 30min) leads to transient activation of endogenous Cdk5 and cell death, while mild heat shock (42C, 30min) produces neither Cdk5 activation nor significant cell death. The mechanism of Cdk5 activation duringheat shock is under investigation. To study the possible role of Cdk5 in the corneal epithelium, we have generated transgenic mice that overexpress either Cdk5 or dominant negative Cdk5 in the corneal epithelium, using the aldehyde dehydrogenase III promoter (provided by Dr. Joram Piatigorsky) to target expression to this tissue. Histological examinationof corneas of these transgenic lines has revealed no gross morphological abnormalities, although a slight thickening of basement membranewas observed in some sections. Additional studies are being conducted to investigate this possibility.To test whether Cdk5 plays a role in cell migration in the corneal epithelium, we have established a collaboration with Dr. Mary Ann Stepp, George Washington University to study the rate of corneal wound healing in our Cdk5 and dominant negative Cdk5 transgenic lines. A second area of intense study is the regulation ofproto- oncogene expression andcell proliferation in lens epithelial cells. We have demonstrated that 12 (S) HETE, a lipoxy genase metabolite of arachidonic acid, is required by lens epithelial cells for c-fos transcription in response to growth factor stimulation. We have identified a rabbit lens epithelial cell line that is suitable for studies of the mechanism of this effect. Further studies with this cell line have shown that 12(S)HETE is required for activation of protein kinase C (PKC) and have shown that regulation of PKC by 12(S)HETE is sufficient to account for the regulation of c-fos transcription by 12(S)HETE. Moreover, our data indicate that PKC affects c-fos induction downstream of ERK activation. This novel pathway will be investigated using specific inhibitors to block individual enzymes in the EGF-activated signal transduction cascade and specific isoforms of PKC will be investigated to determine whether 12(S)HETE selectively regulates certain isoforms.