Characterization of G1 Phase Entry and Progression and the Cyclin- Dependent Kinases that Regulate These Processes.

Abstract

Transit through G1 phase of the cell cycle is controlled by the action of cyclin D-cdk4, cyclin D-cdk6 and cyclin E-cdk2 complexes. These enzymatic complexes act as serine/threonine kinases, specifically phosphorylating the substrates required for progression through G1 phase. Cdk4 and cdk6 are two structurally related proteins that were presumed to be functionally redundant due to their ability to phosphorylate and inhibit the activity of the members of the Retinoblastoma (Rb) protein family and also sequester the cdk inhibitors p21Cip1 and p27Kip1 as part of a regulatory mechanism to control cdk2 activity. This thesis project focused on comparing the two roles of cdk4 and cdk6 during continuous proliferation and the exit from G0 and applying our knowledge of cell cycle regulation to engineer a dual promoter vector system. The first project demonstrated that cdk4 and cdk6 are functionally distinct molecules. We used a tetracycline-repressible system in mink lung epithelial cells (Mv1Lu) to control the expression of different variants of cdk4 or cdk6 in order to directly compare the requirements for the catalytic and sequestration activity of these two kinases during continuous proliferation and upon exit from G0. We found that increased expression of a catalytically inactive/sequestrationonly cdk6 allele, but not the similar cdk4 allele, supported proliferation during TGF-β treatment and accelerated the exit from a quiescent state. Size exclusion chromatography identified a novel cyclin D-cdk6 dimer that is not detected with cdk4, demonstrating that cdk6 may have different requirements for assembly than cdk4. Collectively, our data indicates that cdk4 and cdk6 are not functionally redundant and may have distinct roles during cell cycle progression. In a separate set of experiments, I applied my knowledge of G1 phase regulation to develop a cell cycle-regulated, TGF-β-inducible vector that may have clinical implications. I was able to engineer a dual promoter construct that would allow for the expression of two genes simultaneously while the promoters retained individual cell cycle-regulated activity. TGF-β 7 signaling causes the direct transcriptional induction of target genes that fall into two classes: 1) those that were dependent only on Smad binding and have c-Myc independent activity, and 2) those that required both Smad binding and downregulation of c-Myc for activity. I capitalized on these two different types of promoters and constructed a bicistronic vector that was both TGF-β dependent as well as differentially regulated in breast cancer and normal cells. One use of this vector may be to target cell death to TGF-β-resistant, c-Myc overexpressing breast cancer cells while simultaneously preserving normal cells.