Cdc2 Kinase Activity Research Articles

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

The High Mobility Group I/Y Protein Is Hypophosphorylated in Endoreduplicating Maize Endosperm Cells and Is Involved in Alleviating Histone H1-mediated Transcriptional Repression

During maize endosperm development, cells shift from a mitotic cycle to endoreduplication, driving the massive synthesis of storage proteins (zeins) and starch. In this developmental context, we studied changes in expression levels of histone H1 and high mobility group I/Y (HMG-I/Y), two chromatin architectural proteins that are known to affect gene transcription. Almost no change was found in the level of histone H1 during endosperm development, despite a dramatic increase in DNA content (endoreduplication); hence, the histone H1/DNA ratio decreased substantially. Concurrently with a reduction in the Cdc2 kinase activity at the shift to endoreduplication, significant changes were found in the level and mobility of the HMG-I/Y protein; the faster migrating forms were, at least partly, hypophosphorylated. Purified maize HMG-I/Y protein was found to be phophorylated in vitro by the Cdc2 kinase and bound efficiently to the gamma-zein promoter AT-rich tract (gammaZ-AT). Using an in vitro transcription assay, we demonstrated the capability of the maize HMG-I/Y protein to relieve the inhibitory effect exerted by histone H1 on templates containing the gammaZ-AT sequence. These data suggest that during maize endosperm development transcription and perhaps replication are controlled, at least partly, by the activity of the Cdc2 kinase and the interplay between histone H1 and HMG-I/Y proteins.

Identification of Novel Cdc7 Kinase Inhibitors as Anti-Cancer Agents that Target the Interaction with Dbf4 by the Fragment Complementation and Drug Repositioning Approach.

BackgroundCdc7-Dbf4 is a conserved serine/threonine kinase that plays an important role in initiation of DNA replication and DNA damage tolerance in eukaryotic cells. Cdc7 has been found overexpressed in human cancer cell lines and tumor tissues, and the knockdown of Cdc7 expression causes an p53-independent apoptosis, suggesting that Cdc7 is a target for cancer therapy. Only a handful Cdc7 kinase inhibitors have been reported. All Cdc7 kinase inhibitors, including PHA-767491, were identified and characterized as ATP-competitive inhibitors. Unfortunately, these ATP-competitive Cdc7 inhibitors have no good effect on clinical trial.MethodsHere, we have developed a novel drug-screening platform to interrupt the interaction between Cdc7 and Dbf4 based on Renilla reniformis luciferase (Rluc)-linked protein-fragment complementation assay (Rluc-PCA). Using drug repositioning approach, we found several promising Cdc7 inhibitors for cancer therapy from a FDA-approved drug library.FindingsOur data showed that dequalinium chloride and clofoctol we screened inhibit S phase progression, accumulation in G2/M phase, and Cdc7 kinase activity. In addition, in vivo mice animal study suggests that dequalinium chloride has a promising anti-tumor activity in oral cancer. Interestingly, we also found that dequalinium chloride and clofoctol sensitize the effect of platinum compounds and radiation due to synergistic effect. In conclusion, we identified non-ATP-competitive Cdc7 kinase inhibitors that not only blocks DNA synthesis at the beginning but also sensitizes cancer cells to DNA damage agents.InterpretationThe inhibitors will be a promising anti-cancer agent and enhance the therapeutic effect of chemotherapy and radiation for current cancer therapy.FundThis work was supported by grants from the Ministry of Science and Technology, Ministry of Health and Welfare, and National Health Research Institutes, Taiwan.

Notch1 activation enhances proliferation via activation of cdc2 and delays differentiation of myeloid progenitors

Accumulating evidence indicates that the Notch signaling pathway has crucial roles in the control of fate decision and differentiation in numerous cell types. However, the role of Notch signaling in regulating proliferation and differentiation of myeloid progenitor cells remains controversial. To elucidate this issue, we modulated Notch activity through transducing a constitutively activated form of Notch1 and/or a dominant-negative form of MAML1 (DNMAML1) into myeloid progenitor 32D cells and assessed their effects on cell proliferation and differentiation. We found that Notch1 activation enhances proliferation and delays granulocytic differentiation of 32D cells. The enhanced proliferation due to activated Notch1 signaling was associated with upregulation of c-Myc, followed by decreased expression of p21 and p27, and increased cdc2 kinase activity, through a mechanism that was not blocked by DNMAML1. Conversely, Notch1 activation significantly delayed granulocytic differentiation and maintained a part of myeloid progenitor cells in an immature stage, and this Notch1-mediated effect was dependent on MAML. The Notch1-induced effects on mye myeloid cell proliferation and differentiation were likely mediated by induction of c-Myc and repression of PU.1, respectively. Thus, Notch1 signaling plays an important part in modulating proliferation and differentiation in MAML-independent and -dependent manners and promoting expansion of myeloid progenitors.

Abstract 3343: Identifying kinases and phosphatases that regulate STAT3 activity

Abstract The STAT3 transcription factor is hyperactivated in many cancers where it induces cancer progression by driving drug resistance, metastasis and pro-cancer inflammation. We therefore sought new strategies to target the activation of STAT3. Many kinases and signalling pathways can cause STAT3 activation, and it is not well understood how this is regulated in cancer cells. In some rare solid and hematological cancers, such as NK-/T-cell large granular lymphocytic leukemia and inflammatory hepatocellular adenomas, STAT3 has also been found to be mutated, resulting in a constitutively active STAT3 protein. In testing mock transfected vs. STAT3(Y640F)-transfected cells, as a means to introduce activated STAT3 in already transformed cells, in a screen against 525 anticancer agents, we identified that activated STAT3 protects the cells from cytotoxic effects of several classes of anticancer compounds. Therefore, we performed RNAi screens to identify kinases and phosphatases that regulate STAT3 activity and therefore may act as drug-sensitizing targets. Using STAT3 transcription-driven luciferase reporter cell lines expressing active wild type (WT) or mutant (Y640F) STAT3, we identified several candidate genes (CDC7, CDK8, CSK, CSNK2A1, PI4KII, PTPRH, DDR2) whose downregulation led to differential STAT3 transcriptional activity. Strikingly, none of the hits caused highly selective effect on either mutant or wild-type STAT3 expressing cells, suggesting that the phosphoregulation of hyperactive mutant and IL6 induced wild-type STAT3 are mechanistically similar. Using small molecule inhibitors targeting CDK8, CSNK2A1, DDR2 and CDC7, we confirmed that STAT3 transcriptional activity was inhibited in a dose and time dependent fashion without severely affecting cell viability. Small molecules targeting of CSNK2A1, DDR2 and CDC7 kinase activity caused a slow inhibition of STAT3 transcriptional activity where the strongest effects were only seen after 72 h. CDK8 kinase inhibition, on the other hand, reduced STAT3 activity effectively already after 4 h. This suggests that CDK8 regulates STAT3 activity more directly than CSNK2A1, DDR2 and CDC7. Inhibition of CDK8 and CSNK2A1 resulted in decrease of STAT3 phosphorylation in the STAT3(Y640F)-transfected cells, and knockdown of CSNK2A1 inhibited the nuclear localization of STAT3. In conclusion, we have found that hyperactive STAT3(Y640F) can protect cancer cells from several drug classes, mimicking the case when hyperactivated wild type STAT3 is acting as malignancy promoting protein in cancers. In a functional genetics screen, we found six kinases and a phosphatase that regulate transcriptional activity of STAT3. Using small molecule inhibitors, we could confirm that targeting the hit kinases regulate STAT3 transcriptional activity. Together, our data suggest that there are multiple kinases that may be targeted to counteract STAT3 mediated drug resistance in cancers. Citation Format: Elina Parri, Heikki Kuusanmäki, Arjan van Adrichem, Meri Kaustio, Laura Turunen, Krister Wennerberg. Identifying kinases and phosphatases that regulate STAT3 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3343.

DNA Replication Dynamics and Cellular Responses to ATP Competitive CDC7 Kinase Inhibitors.

The CDC7 kinase, by phosphorylating the MCM DNA helicase, is a key switch for DNA replication initiation. ATP competitive CDC7 inhibitors are being developed as potential anticancer agents; however how human cells respond to the selective pharmacological inhibition of this kinase is controversial and not understood. Here we have characterized the mode of action of the two widely used CDC7 inhibitors, PHA-767491 and XL-413, which have become important tool compounds to explore the kinase's cellular functions. We have used a chemical genetics approach to further characterize pharmacological CDC7 inhibition and CRISPR/CAS9 technology to assess the requirement for kinase activity for cell proliferation. We show that, in human breast cells, CDC7 is essential and that CDC7 kinase activity is formally required for proliferation. However, full and sustained inhibition of the kinase, which is required to block the cell-cycle progression with ATP competitor compounds, is problematic to achieve. We establish that MCM2 phosphorylation is highly sensitive to CDC7 inhibition and, as a biomarker, it lacks in dynamic range since it is easily lost at concentrations of inhibitors that only mildly affect DNA synthesis. Furthermore, we find that the cellular effects of selective CDC7 inhibitors can be altered by the concomitant inhibition of cell-cycle and transcriptional CDKs. This work shows that DNA replication and cell proliferation can occur with reduced CDC7 activity for at least 5 days and that the bulk of DNA synthesis is not tightly coupled to MCM2 phosphorylation and provides guidance for the development of next generation CDC7 inhibitors.

The Cdc2/Cdk1 inhibitor, purvalanol A, enhances the cytotoxic effects of taxol through Op18/stathmin in non-small cell lung cancer cells invitro.

PurvalanolA is a highly selective inhibitor of Cdc2[also known as cyclin-dependent kinase1(CDK1)]. Taxol is an anti-tumor chemotherapeutic drug which is widely used clinically. In this study, the CDK1 inhibitor, purvalanolA was applied to explore the relevance of Cdc2 signaling and taxol sensitivity through analyses, such as cellular proliferation and apoptosis assays, ELISA, western blot analysis and immunoprecipitation. We demonstrated that purvalanolA effectively enhanced the taxol-induced apoptosis of NCI-H1299 cells, as well as its inhibitory effects on cellular proliferation and colony formation. In combination, purvalanolA and taxol mainly decreased the expression of oncoprotein18(Op18)/stathmin and phosphorylation at Ser16 andSer38, while purvalanolA alone inhibited the phosphorylation of Op18/stathmin at all 4serine sites. Co-treatment with purvalanolA and taxol weakened the expression of Bcl-2 and activated the extrinsic cell death pathway through the activation of caspase-3 and caspase-8. Further experiments indicated that Cdc2 kinase activities, including the expression of Cdc2 and the level of phospho-Cdc2(Thr161) were significantly higher in taxol-resistant NCI-H1299 cells compared with the relatively sensitive CNE1 cells before and following treatment with taxol. These findings suggest that Cdc2 is positively associatd with the development of taxol resistance. The Cdc2 inhibitor, purvalanolA, enhanced the cytotoxic effects of taxol through Op18/stathmin. Our findings may prove to be useful in clinical practice, as they may provide a treatment strategy with which to to reduce the doses of taxol applied clinically, thus alleviating the side-effects.

Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin.

HER2/neu positive breast tumors predict a high mortality and comprise 25%–30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2-overexpressing breast cancer cell lines (i.e., SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e., MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has a minimal effect on the growth of non-malignant breast epithelial MCF10A cells. FKA induces G2M arrest in cell cycle progression of HER2-overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and downregulation of expression of Myt1 and Wee1 leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl2, BclX/L, XIAP, and survivin. FKA also downregulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to an enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through downregulation of Myt1, Wee1, Skp2, survivin, and XIAP. Our results suggest FKA as a promising and novel apoptosis inducer and G2 blocking agent that, in combination with Herceptin, enhances for the treatment of HER2-overexpressing breast cancer.

Abstract A180: Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity

Abstract Introduction> Cell cycle kinases have been recognized as potential anticancer targets, because loss of regulated cell cycle progression would lead to uncontrolled cell growth and development of cancer. Cdc7 kinase is involved in regulation of the cell cycle and plays important roles in DNA replication. Notably, inhibition of Cdc7 in cancer causes lethal S phase or M phase progression, whereas normal cells can survive, most likely through induction of cell cycle arrest at the DNA replication checkpoints. Therefore Cdc7 kinase has emerged as a promising therapeutic target for the treatment of cancers. Here we present a novel series of furanone derivatives with potent inhibitory effects on Cdc7 kinase. <Methods> To identify hit compounds that are insensitive or resistant to high ATP concentration, a high-throughput screening (HTS) was performed in the presence of 100 microM ATP which corresponds to 36-times higher than the Km value of ATP for Cdc7 kinase activity. To characterize inhibitors, ATP dependency and pre-incubation effects were examined. To determine the efficacy of Cdc7 inhibitors, phosphorylation of MCM2 protein in cells were analyzed by western blotting. Selected compounds were subjected to a kinase selectivity panel assay. <Results> Only a single hit compound was identified from the HTS, reflecting extremely low hit rate compared to that of other kinase targets in our past HTS. Subsequent hit to lead studies led to the identification of a novel furanone compound as a potential lead compound for further optimization study. Interestingly, these furanone derivatives showed slow-binding inhibition against Cdc7, and exhibited strong inhibitory activity for Cdc7 even in the presence of 1 mM ATP that was added after the pre-incubation of the kinase and compound. Notably this pre-incubation effect was not observed with other kinases tested. Detailed results using cancer cells will also be presented in this poster. Citation Format: Takayuki Irie, Tokiko Asami, Ayako Sawa, Chika Taniyama, Yoko Funakoshi, Yuko Uno, Hisao Masai, Masaaki Sawa. Discovery of furanone derivatives as novel Cdc7 inhibitors with anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A180.

Two Distinct Cdc2 Pools Regulate Cell Cycle Progression and the DNA Damage Response in the Fission Yeast S.pombe

The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.

WEE1, histone and tumor

WEE1 is an important factor for histone transcription, chromosome condensation and regulation of cell cycle progression. WEE1 kinase can phosphorylate Cdc2 and down-regulate Cdc2 kinase activity. It can regulate G2 to M phase transition and cell mitosis. It plays a key role in chromosome condensation delay and histone synthesis, suggesting the important functions of WEE1 in the occurrence and development in cancer. At present, a multiple WEE1 inhibitors have been discovered. A great progress has been made in combination of WEE1 inhibitors with DNA damage treatment (chemotherapy or radiotherapy), which makes WEE1 an important target in cancer treatment.

Fission Yeast Cdk7 Controls Gene Expression through both Its CAK and C-Terminal Domain Kinase Activities

Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

Silibinin Combination with Arsenic Strongly Inhibits Survival and Invasiveness of Human Prostate Carcinoma Cells

Effects of silibinin, a naturally occurring flavanone, on prostate carcinoma (PCa) cells in presence of arsenic are not known. Arsenic is clinically approved for leukemia treatment; however, studies are not enough to support its role in the management of solid tumors. In the present study, we observed that silibinin (100 µM) modulated the oxidative status of human PCa DU145 cells exposed to arsenic (0.5 or 5 µM) and inhibited cell growth and survival by primarily inducing autophagy and apoptosis. The silibinin–arsenic combination also inhibited the growth, survival, and clonogenic potential of 22Rv1 PCa cells. Silibinin with 0.5 or 5 µM arsenic induced G1 or G2/M phase arrest, respectively, and decreased the protein levels of CDK2, -4, and -6 and cyclin D1, D3, and E and increased CDK inhibitors p21 and p27. Arsenic alone increased cyclin B1 level and Cdc2 kinase activity which were reduced in silibinin combination. Cell motility and invasiveness along with expression of MMP-2 and vimentin were suppressed. Together, these in vitro findings suggest that in presence of arsenic, silibinin strongly inhibits tumorigenic and metastatic potential of PCa cells.

Characterization of a Drosophila Ortholog of the Cdc7 Kinase: A ROLE FOR Cdc7 IN ENDOREPLICATION INDEPENDENT OF CHIFFON

Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.

Maturation-associated Dbf4 expression is essential for mouse zygotic DNA replication.

Cdc7 is an S-phase-promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one-cell embryos at zygotic S-phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one-cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi-mediated knockdown of maternal Dbf4 expression prevents the maturation-associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one-cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3' untranslated region (3'UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.

Somatostatin Analog Inhibits the Growth of Insulinoma Cells by p27-Mediated G1 Cell Cycle Arrest

Although the somatostatin analog octreotide (OCT) has been used for uncontrollable insulinoma, the mechanism involved is still unknown. The aim of this study was to elucidate the therapeutic effect of OCT for insulinoma. Mouse insulinoma cell line MIN6 cells were cultured with OCT to clarify its antiproliferative effects, the expression of somatostatin receptor subtypes, cell cycle, p27 expression, and cdc2 kinase activity. The changes of the messenger RNA expression profiles were examined by microarray analysis. Intraperitoneal OCT treatment was given to insulinoma model IT6 mice for 4 weeks. MIN6 cells expressed somatostatin receptor 2A, 3, and 5 under the OCT treatment. Octreotide showed a dose-dependent antiproliferative effect on MIN6 cells but not on the other cell lines. p27 expression and cdc2 kinase activity in MIN6 cells became prominent with OCT treatment. At the messenger RNA level, several molecules in the mitogen-activated protein kinase signaling pathway were downregulated. The sizes of the individual tumors tended to be smaller in the OCT-treated group. p27 expression was seen in the tumor tissue, but no apoptotic marker was detected. Octreotide acted through a cytostatic mechanism and could be an effective therapy for insulinoma.

Steroid-induced oocyte maturation in Indian shad Tenualosa ilisha (Hamilton, 1822) is dependent on phosphatidylinositol 3 kinase but not MAP kinase activation

Fully grown fish and amphibian oocytes exposed to a maturation-inducing steroid (MIS) activates multiple signal transduction pathways, leading to formation and activation of maturation-promoting factor (MPF) and induction of germinal vesicle breakdown (GVBD). The present study was to investigate if phosphatidylinositol 3 kinase (PI3 kinase) and mitogen-activated protein kinase (MAP kinase) activation are required for naturally occurring MIS, 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced cdc2 activation and oocyte maturation (OM) in Tenualosa ilisha. We observed that17,20β-P-induced OM was significantly inhibited by PI3 kinase inhibitors Wortmannin and LY29400. 17,20β-P was shown to activate PI3 kinase maximally at 90min and cdc2 kinase at 16h of treatment. Relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in 17,20β-P-induced OM was examined. MAP kinase was rapidly phosphorylated and activated (60–120min) after MIS treatment and this response preceded the activation of cdc2 kinase by several hours. A selective inhibitor of MAP kinase (MEK), PD98059, sufficiently blocked the phosphorylation and activation of MAP kinase. Inhibition of MAP kinase activity using PD98059 however, had no effect on MIS-induced cdc2 kinase activation and GVBD. These results demonstrate that activation of the PI3 kinase is required for 17,20β-P-induced cdc2 kinase activation and OM in T. ilisha. MAP kinase although was activated in response to 17,20β-P and PI3 kinase activation, it is not necessary for cdc2 activation and OM in this species.

Synthesis and structure–activity relationship of trisubstituted thiazoles as Cdc7 kinase inhibitors

The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7 inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited DNA synthesis and cell viability in vitro.

EBV-LMP1-targeted DNAzyme induces DNA damage and causes cell cycle arrest in LMP1-positive nasopharyngeal carcinoma cells

This study aimed to determine the molecular mechanisms underlying the effect of the LMP1-targeted DNAzyme 1 (DZ1) on cell cycle progression in nasopharyngeal carcinoma (NPC) cells. We showed that the active DZ1 inhibited the expression of latent membrane protein 1 (LMP1) and induced a G1 phase arrest. In addition, this cell cycle deregulation was shown to be accompanied by upregulation of the DNA damage marker γ-H2AX, downregulation of the DNA damage response factor p-p53-Ser15 and cell proliferation inhibition. To investigate what affected the cell cycle progression, we examined the expression of two checkpoint-related cyclins and cyclin-dependent kinases (CDKs). We found a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DZ1 treatment. Moreover, we observed inhibition of CDK4 activity and decreased cyclin D1 expression in the complexes immunoprecipitated with CDK4 antibody. We also found a reduction in cdc2 phosphorylation at Thr161 which partially stands for the cdc2 kinase activity in DZ1-treated CNE1-LMP1 cells, although the downregulation of LMP1 expression had no effect on the cyclin B1 and cdc2 expression. Further, we analyzed changes in cdc2 kinase activity induced by DZ1 and found that the downregulation of the LMP1 expression resulted in a 5-fold reduction in cdc2 kinase activity in CNE1-LMP1. The data suggest that the downregulation of the LMP1 expression by DZ1 was able to induce DNA damage, which then further inhibited the cell proliferation and resulted in malfunction of cell cycle checkpoints that led to G1 phase arrest and the decrease in number of cells in G2/M phase.

Cdc7 inhibition as a novel approach for pancreas cancer therapy.

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.