ROGDI is a novel gene which locates on human chromosome 16p13.3 and has unknown function. The length of ROGDI coding region is 864 bp which encodes 287 amino acids. According to our previous studies, overexpression of ROGDI increased proliferation in several human cell lines. CDK2 is a member of cyclin-dependent kinases (CDKs) which plays a key role in regulation of G1/S cell cycle transition and is activated during G1 and S phases. In this studies, we found that expression of CDK2 and cyclin A were up-regulated following overexpression of ROGDI in HEK293T. On the other hand, down-regulation of CDK2 and cyclin A was observed following silencing of ROGDI. In addition, the interaction between ROGDI and CDK2 was evidenced by using immunoprecipitation. Meanwhile, we observed co-localization of ROGDI and CDK2 in the nucleus. We also exploited a drug, olomoucine, known to selectively inhibit CDK2. The result demonstrated that ROGDI-enhanced cell proliferation was dependent on CDK2 activity. Flow cytometry profiles of depletion of ROGDI displayed G1/S phase arrest as well as up-reguated the expression of p53 and p21. Taken together, we suggest that ROGDI may be a positive regulator of cell cycle and its action is CDK2-depedent.

Oral cancer is now the fourth leading cause of male cancer mortality in Taiwan. Betel quid chewing is one of the main causes of oral cancer in Taiwan. CDK2AP1 is a growth suppressor gene that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12CDK2AP1 protein is reduced and/or lost in oral cancers. Mutations in microsatellite-like sequence of CDK2AP1 gene in microsatellite instability colorectal cancer are associated with down-regulated CDK2AP1 transcription. This mutation was due to down-regulation of one DNA repair protein, MLH1. In order to understand whether CDK2AP1 mRNA and protein expression levels associate with betel-chewing oral cancers, we firstly analyzed 44 oral cancer specimens (normal and tumor, in pairs) by quantitative reverse transcription polymerase chain reaction and Western blotting. Immunohistochemistry was used to examine p12CDK2AP1 protein expression in another 167 buccal mucosa squamous cell carcinoma tissues. We have demonstrated that the expression levels of CDK2AP1 mRNAs were slightly higher in normal oral tissues than those in tumor tissues (P>0.05). Similarly, p12CDK2AP1 and CDK2 protein expression levels were up-regulated in oral cancer tissues than in normal tissues by Western blot analysis (P<0.05). Among Ca9-22, CAL27, SAS and in betel-chewing oral cancer cells TW2.6 and human normal skin keratinolial cells (HaCaT) that we examined, p12CDK2AP1 and CDK2 proteins were detected to be highest expressed in Ca9-22 and TW2.6 cells, respectively, when compared to HaCaT cells. Immunocytochemistry indicated p12CDK2AP1 expressed in nucleus and cytoplasm in Ca9-22, CAL27, SAS and HaCaT cells, however predominant present in nucleus in TW2.6 cells. On the other hand, immunohistochemistry demonstrated that nuclear (P=0.157) and cytoplasmic p12CDK2AP1 (P=0.350) in 167 patients with buccal mucosa squamous cell carcinoma were slightly down-regulated. Reduction of nuclear p12CDK2AP1 was not significantly correlated with any clinicopathologic characteristics or prognosis. Direct sequencing indicated that lack of microsatellite-like instability of CDK2AP1 3¡¦-UTR in four oral cancer cell lines, HaCaT and six patients with down-regulated MLH1 protein. In conclusion, we demonstrated that: (1) p12CDK2AP1 was located in both the nucleus and cytoplasm in most oral cancer cell lines and HaCaT cells but predominate present in the nucleus in betel-chewing oral cancer cells, TW2.6; (2) Reduction of nuclear p12CDK2AP1 in buccal mucosa squamous cell carcinoma tissues were identified, however, not significantly correlated with any clinicopathologic characteristics, prognosis or betel chewing; (3) In six patients with down-regulated MLH1, lack of micorsatellite-like instability in the CDK2AP1 3¡¦-UTR region has been found.

No description available.

The spacer region of pRb2/p130 was reported to be able to inhibit the kinase activity of Cdk2. The region responsible for the inhibitory effect was further narrowed down to a 39-amino-acid sequence, which was named as Spa310. In this dissertation, the anti-cancer functions and mechanisms of Spa310 were studied. The synthesized Spa310 peptide was able to inhibit the kinase activities of Cdk2/Cyclin E/A complexes. In vitro kinase assays showed the inhibition occurred in a dose-dependent manner. The half maximal inhibition concentration of the Spa310 in the kinase assay was 1.67mM. In addition, it has been shown that Spa310 peptide is able to inhibit the kinase activities of both Cdk2/Cyclin E and Cdk2/Cyclin A. Intra-cellular distribution study using fluorescein-labeled Spa310 peptide showed that Spa310 was able to localize to the nuclei of A549 cancer cells. Some data indicated the endoplasmic reticulum might play a role in transporting Spa310 peptide from cytoplasm to the nucleus. At high concentration, the treatment of Spa310 peptide was able to arrest cells at the G0/G1 phase of the cell cycle and reduce the growth of xenografted tumors in nude mice. Further studies indicated Spa310 peptide is not a specific inhibitor for Cdk2/Cyclin E/A. It is also able to inhibit the kinase activities of Cdk1/Cyclin B, Cdk4/Cyclin D and Cdk9/Cyclin T/K. Result of a binding assay using GST-Spa310 and in vitro transcribed/translated Cdk2 did not support a direct binding between Spa310 and Cdk2. Additionally, GST-Spa310 was unable to bind to the in vitro transcribed/translated Cyclin E. At first, co-immunoprecipitation experiments indicated a weak binding between Spa310 peptide and Cdk2. However, later this weak binding was proven to be unspecific and only occurred when the concentration of Spa310 peptide was high. Thus, the hypothesized mechanism of the inhibitory effect of Spa310 was not supported. After noticing three classic Cdk phosphorylation sites present in Spa310, it was proven that Spa310 is a substrate for Cdk1, 2, 4 and 9. Results of kinase assays supported the inhibitory effect of Spa310 on the different Cyclin-dependent kinases was resulted from a substrate-competitive mechanism. Although the data generated from this study does not support Spa310 is a potent peptide inhibitor for the Cdks, knowledge gained from and the approach used in this research can be applied to design and develop more potent and specific Cdk2 peptide inhibitors, which have their potentials to work as powerful anti-cancer reagents. / Biology

The radiosensitivity of tumour cells depends mainly on their capacity to maintain genomic integrity. This requires efficient repair of radiation-induced DNA double strand breaks, a process governed by the cell cycle. Based on their functions in cell cycle regulation and DNA damage repair, we hypothesised that targeting of CDK2 and CDK9 modifies cancer cell response to radiotherapy. Therefore, we evaluated the significance of CDK2 and CDK9 for the cellular radiation response in a panel of human head and neck squamous cell carcinoma (HNSCC) cell lines. In order to achieve our goal, we performed a series of experiments to measure several key parameters such as clonogenic radiation survival, cell cycling, DNA damage repair and apoptosis. We found that loss of CDK2 radiosensitises mouse embryonic fibroblasts (MEFs) as well as HNSCC two dimensional (2D) cell cultures. However, under more physiological three dimensional (3D) growth conditions in laminin-rich extracellular matrix, targeting of CDK2 failed to modulate the radiosensitivity of HNSCC cells. Moreover, CDK2 attenuated the repair of radiogenic double strand breaks (DSBs) in MEFs as well as SAS and FaDu HNSCC cells indicating a possible role of CDK2 in DNA damage repair. However, we found that CDK2 is dispensable for cell cycle and checkpoint regulation in response to irradiation in SAS and FaDu cells. Taken together, our results suggest that targeting of CDK2 may not provide a therapeutic benefit to overcome HNSCC cell resistance to radiotherapy. We also showed that depletion of CDK9 clearly enhances the radiosensitivity of HNSCC cultures. In addition, the ectopic expression of CDK9 has a radioprotective effect. These findings suggest a potential role of CDK9 in the radiation response of HNSCC cells. Moreover, our study indicates a possible role of CDK9 in the DNA damage repair response and cell cycling of HNSCC cells. Conclusively, on the basis of these data, targeting of CDK9 in addition to conventional radiotherapy might be a viable strategy to overcome cancer cell resistance.

Protein-protein interactions (PPIs) are key drug targets and recent breakthroughs in this area are providing insight into the types of molecules needed to selectively and potently inhibit a target traditionally seen as untractable. The rules that have been used to design classic substratecompetitive drugs (for example Lipinski's rule of five) may not apply in this new field in the same way. Here I present work performed in three systems that are well-validated drug targets for oncogenesis: the CDK2/cyclin A complex, the PLK1 Polobox domain and MDM2. In each case the site of the protein-protein interaction is defined and understood and the rationale for pharmaceutical intervention is clear. I use these as a model system to evaluate the characteristics of drugs that target protein-protein interaction sites and present work on the development of inhibitors as potential leads for subsequent drug development. In Chapter 1 I introduce the problems, challenges and rewards of PPI drug development; in Chapter 2 I present co-crystal structures of MDM2 with isoindolinone inhibitors; in Chapter 3 I detail attempts to co-crystallise the Plk1 Polobox with inhibitors and screen potential inhibitors; in Chapter 4 I present the results of screening to identify inhibitors of Cyclin A recruitment; and in Chapter 5 I discuss other strategies for inhibition of the CDK2/cyclin A complex, including results with a covalent inhibitor. Through these projects I have been able to demonstrate the wide applicability of the PPI inhibition approach, identify key features of drugs able to inhibit PPIs and contribute to drug design in each system.

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.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the most active metabolite of vitamin D, inhibits the proliferation of a variety of cell types including adenocarcinoma of the prostate. The primary mechanism for the antiproliferative effects of 1,25-(OH)2D3 in prostate cancer cells is inhibition of G1 to S phase cell cycle progression. While 1,25-(OH)2D3-mediated growth inhibition requires the vitamin D receptor (VDR), a ligand activated transcription factor, expression of functional VDR is not sufficient. To define target genes that might participate in the antiproliferative actions of 1,25-(OH)2D3, we developed a derivative of the human prostate cancer cell line, LNCaP, which retains transcriptionally active VDRs but unlike parental LNCaP cells, is not growth inhibited by 1,25-(OH)2D3. Gene expression profiling of these resistant cells (termed VitD.R) compared to control LNCaP cells revealed two novel 1,25-(OH)2D3-inducible genes, GADD45G and MIG6. The expression of GADD45G and MIG6 genes was induced by 1,25-(OH)2D3 in LNCaP but not in the resistant VitD.R or in ALVA31 cells, human prostate cancer cells that exhibit natural resistance to growth inhibition by 1,25-(OH)2D3 despite expression of functional VDR. Ectopic expression of GADD45G but not MIG6 in either LNCaP or ALVA31 cells resulted in accumulation of cells in G1 and inhibition of proliferation equal to or greater than that caused by 1,25-(OH)2D3 treatment. While GADD45G is induced by androgens in prostate cancer cells, up-regulation of GADD45G by 1,25-(OH)2D3 was not dependent on androgen receptor signaling further refuting a requirement for androgens/androgen receptor in vitamin D-mediated growth inhibition in prostate cancer cells. These data introduce two novel 1,25-(OH)2D3-regulated genes and establish GADD45G as a growth inhibitory protein in prostate cancer. Further, defects in vitamin D-mediated induction of GADD45G may underlie vitamin D resistance in prostate cancer cells. We previously demonstrated that the antiproliferative actions of 1,25-(OH)2D3 in prostate cancer cells are associated with decreased CDK2 activity and increased stability of the cyclin dependent kinase inhibitor (CKI) p27KIP1. We defined a novel mechanism that may underlie these antiproliferative effects, 1,25-(OH)2D3 -mediated cytoplasmic relocalization of CDK2, which would provide a unifying mechanism for the observed effects of 1,25-(OH)2D3 on CDK2 and p27. In the present study, we investigated the role of CDK2 cytoplasmic relocalization in the antiproliferative effects of 1,25-(OH)2D3. CDK2 was found to be necessary for prostate cancer cell proliferation. In contrast, while p27KIP1 is induced by 1,25-(OH)2D3, this CKI was completely dispensable for 1,25-(OH)2D3-mediated growth inhibition. Reduction of CDK2 activity by 1,25-(OH)2D3 was associated with decreased T160 phosphorylation, a residue whose phosphorylation in the nucleus is essential for CDK2 activity. Since cyclin E is important for nuclear translocation of CDK2, we investigated cyclin E effects on 1,25D-mediated growth inhibition. Ectopic expression of cyclin E blocked 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2 and all antiproliferative effects of 1,25-(OH)2D3, yet endogenous levels of cyclin E or binding to CDK2 were not affected by 1,25-(OH)2D3. Similarly, knockdown of the CDK2 substrate retinoblastoma (Rb), which causes cyclin E up-regulation, resulted in resistance to 1,25-(OH)2D3 mediated growth inhibition. VitD.R cells did not exhibit 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2. Importantly, targeting CDK2 to the nucleus of LNCaP cells blocked G1 accumulation and growth inhibition by 1,25-(OH)2D3. These data establish central roles for CDK2 nuclear-cytoplasmic trafficking and uncoupling of VDR in the regulation of antiproliferative target genes in the mechanisms of 1,25-(OH)2D3-mediated growth inhibition in prostate cancer cells. Since 1,25-(OH)2D3 continues to be evaluated for its chemotherapeutic and chemopreventative potential, elucidating the mechanism of 1,25-(OH)2D3 antiproliferative effects is critical in the determination of 1,25-(OH)2D3 responsiveness and the design of individualized treatment strategies.

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

O ciclo celular é controlado pela atividade das quinases dependentes de ciclinas (Ciclin-dependent kinases, CDKs). As CDKs são inativas como monômeros, e a sua ativação necessita da ligação às ciclinas, uma família diversa de proteínas cujos os níveis oscilam durante o ciclo celular, e fosforilação pela CAK (CDK-activating kinase) sobre um resíduo de treonina específico. As CDKs são capazes de fosforilar muitas proteínas que estão envolvidas nos eventos do ciclo celular, incluindo histonas e proteínas supressoras de tumores como pRb. Além da função de regulação positiva das ciclinas e CAK, muitas proteínas inibidoras de CDKs (CDK inhibitors, CKIs) têm sido descobertas, tais como p16, p21 e p28. Visto que, a desregulação das ciclinas e/ou alteração ou ausência de CKIs têm sido associadas com muitos cânceres, há um forte interesse em inibidores químicos de CDKs que possam ter uma função importante na descoberta de novas famílias de agentes anti-tumores. Vistoque, ATP é o autêntico co-fator da CDK2 este pode ser considerado como um \"pseudo-composto líder\" para a descoberta de inibidores de CDK2. Entretanto, há duas preocupações maiores a serem consideradas: composto contendo adenina são ligantes comuns para muitas enzimas nas células, desta forma, qualquer composto altamente carregado como ATP não será absorvido pelas células. Nós descrevemos aqui as estruturas determinadas por difração de raios-X da CDK2 em complexo com dois inibidores diferentes, descloro-flavopiridol (DFP) e Roscovitine. A estrutura do complexo binário CDK2-DFP foi resolvida por substituição molecular e refinada até um Rfactor=20,3% e a estrutura da CDK-2Roscovitine foi refinada até um Rfactor=18%. O descloro-flavopiridol é uma flavona com uma nova estrutura,comparável àquelas de flavonas polihidroxiladas. Estudos prévios mostraram que flavopiridol, um flavonóide, pode inibir cânceres de mama e de pulmão. O Roscovitine é um derivado de adenina e um potente inibidor de CDK2. A comparação das estruturas tridimensionais de CDK2-DFP e CDK2-Roscovitine com a de CDK2-ATP mostraram que o bolsão hidrofóbico de ligação de adenina tem a habilidade surpreendente de acomodar estruturas moleculares diferentes daquelas da ATP / Cell cycle progression is tightly controlled by the activity of ciclin-dependent kinases (CDKs). CDKs are inactive as monomers, and activation requires binding to cyclins, a diverse family of proteins whose levels oscillate during cell cycle, and phosphorilation by CDK-activating kinase (CAK) on a specific threonine residue. CDKs are able to phosphorylate many proteins that are in volvedin cell cycle events, including histones and tumor suppressor proteins like the retinoblastoma gene product pRb. In addition to the positive regulatory role of cyclins and CAK, many negative regulatory proteins (CDK Inhibitors, CIGs) have been discovered, such as p16, p21, and p28. Since deregulation of cyclins and/or alteration or absence ofCKIs have been associated with many cancers, there is strong interest in chemical inhibitors of CDKs that could play an important role in the discovery of new family of antitumor agents. Since ATP is the authentic cofactor of CDK2 it can be considered as a \"pseudo-lead compound\" for discovery of CDK2 inhibitors. However there are two major concerns: adenine containing compounds are common ligants for many enzymes in cells, thus, any adenine derivatives may inhibit many enzymes in the cells: second, any highly charged compounds such as ATP will prevent them from uptake by cells. We report here the x-ray structures of CDK2 in complex with two different inhibitors, deschloro-flavopiridol(DFP) and Roscovitine. The structure of the binary complex CDK2-DFP was solved by molecular replacement and refined to Rfactor = 20.3% and the structure ofCDK2-Roscovitine was refined to Rfactor = 18.0 %. The deschloro-flavopiridol(DFP) is a flavone with a novel structure, compared to that of polyhydroxylated flavones. Previous studies have shown that flavopiridol, a flavonoid, can inhibit growth of breast and lung carcinoma cell lines. The Roscovitine is an adenine derivative and a potent CDK2 inhibitor. The two inhibitors are competitive inhibitors for ATP binding to CDK2 and bind to the ATP binding pocket ofCDK2. The comparison of the three-dimensional structures of CDK2-DFP and CDK2-Roscovitine with the CDK2-ATP shows that the hydrophobic adenine-binding pocket has a surprising ability to accommodate molecular structures that are different from ATP.