1,25-(OH)2 vitamin D3 inhibits cell proliferation of a variety of cancers including prostate. In the human prostate cancer cell line LNCaP, 1,25-(OH)2 vitamin D3-mediated growth inhibition is attributed to cell cycle G1 accumulation which correlates with a robust decrease of cyclin-dependent kinase 2 (CDK2) activity and pronounced relocalization of CDK2 into the cytoplasm. Nuclear targeting CDK2 blocks the 1,25-(OH)2 vitamin D3-mediated growth inhibition and cell cycle G1 accumulation. Further, the nuclear targeted CDK2 blocks 1,25-(OH)2 vitamin D3-mediated inhibition of CDK2 activity and nuclear exclusion in LNCaP cells. Therefore, CDK2 cytoplasmic relocalization is the key mechanism for 1,25-(OH)2 vitamin D3 effects. Since cyclin E is important for CDK2 nuclear localization and activation, 1,25-(OH)2 vitamin D3 may exert its effects through regulation of cyclin E. Cyclin E but not a cyclin E mutant deficient in CDK2 binding reverses 1,25-(OH)2 vitamin D3-mediated antiproliferation which suggests the involvement of cyclin E as a mechanism. However, the studies showed no effects of 1,25-(OH)2 vitamin D3 on cyclin E levels, intracellular localization or binding to CDK2. In order to develop a model for studying 1,25-(OH)2 vitamin D3-mediated antiproliferative effects, LNCaP vitD.R cell line, a vitamin D resistant LNCaP derivative, was generated by continuously culturing of LNCaP cells in medium supplemented with 10 nM 1,25-(OH)2 vitamin D3 for over 9 months. The initial characterization of this cell line showed complete resistance to 1,25-(OH)2 vitamin D3-mediated effects. Analysis of vitamin D regulation of VDR target gene expression revealed that vitamin D resistance in LNCaP vitD.R cells was not due to deregulation of VDR signaling. HDAC inhibitor Trichostatin A (TSA) did not confer sensitivity of LNCaP vitD.R cells to vitamin D treatment suggested the resistance to 1,25(OH)2 vitamin D3 effect of LNCaP vitD.R cells is not due to histone deacetylase remodeling of the chromatin structure which leads to inhibition of gene transcription. While the partial sensitization of LNCaP vitD.R cells to 1,25(OH)2 vitamin D3 effect by demethylation reagent 5-Aza-2¡¯-deoxycytidine treatment suggested a set of genes involved in 1,25(OH)2 vitamin D3-mediated antiproliferative effects is silenced via hypermethylation in LNCaP vitD.R cells. These results suggested LNCaP vitD. R cell line is a useful tool and further studies to elucidate the genes involved in this effect will help uncover the mechanisms of 1,25(OH)2 vitamin D3-mediated antiproliferative effects.

During the early development of Xenopus laevis embryos, cells divide without checkpoints. At the midblastula transition (MBT), the cell cycle is remodeled as the division time lengthens and checkpoints are acquired. Initiation of the MBT depends upon the degradation of maternally supplied cyclin E, which is the regulatory partner of the cyclin dependent kinase, Cdk2. To study the program that drives cyclin E degradation and cell cycle remodeling at the MBT, embryos were treated with two cell cycle inhibitors, GST-D34Xic1 and XChk1. Injection of embryos with GST-D34Xic1, a stoichiometric inhibitor of cyclin E/Cdk2, delays degradation of cyclin E and onset of the MBT. GST-D34Xic1 lowers Wee1 level, a kinase that maintains Cdks in an inactivate state. Eventual degradation of cyclin E is preceded by degradation of GST-D34Xic1. The mathematical modelers, Andrea Ciliberto and John Tyson, incorporated the data into a kinetic model and set of ordinary differential equations. The model accurately described the experimental data and made additional predictions, which were tested experimentally. Additionally, embryos were injected with mRNA encoding XChk1, a kinase that activates Wee1 and inhibits Cdc25, the phosphatase opposing Wee1. Like GST-D34Xic1, XChk1 inhibits cyclin E/Cdk2 and delays the degradation of cyclin E. In contrast to GST-D34Xic1, XChk1 elevates the level of Wee1 at a time when sibling controls begin the MBT, despite cell cycle arrest. Since XChk1 inhibits both Cdk1 and Cdk2, and GST-D34Xic1 inhibits only Cdk2, we propose Cdk1 destabilizes Wee1, whereas Cdk2 elevates Wee1 level. Prior to the MBT, when cyclin E/Cdk2 is active, Wee1 is maintained. After cyclin E/Cdk2 is destroyed at the MBT, Wee1 is degraded. / Master of Science