CDK1 promotes the stemness of lung cancer cells through interacting with Sox2.

OBJECTIVES: The promoting roles of cyclin dependent kinase 1 (CDK1) have been revealed in various tumors, however, its effects in the progression of cancer stem cells are still confusing. This work aims to explore the roles of CDK1 in regulating the stemness of lung cancer cells. METHODS: Online dataset analysis was performed to evaluate the correlation between CDK1 exression and the survival of lung cancer patients. RT-qPCR, western blot, cell viability, sphere-formation analysis and ALDH activity detection were used to investigate the roles of CDK1 on lung cancer cell stemness, viability and chemotherapeutic sensitivity. Immunocoprecipitation (Co-IP) analysis and rescuing experiments were performed to reveal the underlying mechanisms contributing to CDK1-mediated effects on lung cancer cell stemness. RESULTS: CDK1 mRNA expression was negatively correlated with the overall survival of lung cancer patients and remarkably increased in tumor spheres formed by lung cancer cells compared to the parental cells. Additionally, CDK1 positively regulated the stemness of lung cancer cells. Mechanistically, CDK1 could interact with Sox2 protein, but not other stemness markers (Oct4, Nanog and CD133). Furthermore, CDK1 increased the phosphorylation, cytoplasm-nuclear translocation and transcriptional activity of Sox2 protein in lung cancer cells. Moreover, CDK1 positively regulated the stemness of lung cancer cells in a Sox2-dependent manner. Finally, we revealed that inhibition of CDK1 enhanced the chemotherapeutic sensitivity, which was also rescued by Sox2 overexpression. CONCLUSIONS: This work reveals a novel CDK1/Sox2 axis responsible for maintaining the stemness of lung cancer cells.

Flavonoids as CDK1 Inhibitors: Insights in Their Binding Orientations and Structure-Activity Relationship.

In the last years, the interactions of flavonoids with protein kinases (PKs) have been described by using crystallographic experiments. Interestingly, different orientations have been found for one flavonoid inside different PKs and different chemical substitutions lead to different orientations of the flavonoid scaffold inside one PK. Accordingly, orientation predictions of novel analogues could help to the design of flavonoids with high PK inhibitory activities. With this in mind, we studied the binding modes of 37 flavonoids (flavones and chalcones) inside the cyclin-dependent PK CDK1 using docking experiments. We found that the compounds under study adopted two different orientations into the active site of CDK1 (orientations I and II in the manuscript). In addition, quantitative structure-activity relationship (QSAR) models using CoMFA and CoMSIA methodologies were constructed to explain the trend of the CDK1 inhibitory activities for the studied flavonoids. Template-based and docking-based alignments were used. Models developed starting from docking-based alignment were applied for describing the whole dataset and compounds with orientation I. Adequate R2 and Q2 values were obtained by each method; interestingly, only hydrophobic and hydrogen bond donor fields describe the differential potency of the flavonoids as CDK1 inhibitors for both defined alignments and subsets. Our current application of docking and QSAR together reveals important elements to be drawn for the design of novel flavonoids with increased PK inhibitory activities.

Identification and structural-functional analysis of cyclin-dependent kinases of the cattle tick Rhipicephalus (Boophilus) microplus.

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of Rhipicephalus microplus, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a R. microplus transcriptome database in silico. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from R. microplus showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3-7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from R. microplus embryo cells were utilized in vitro inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation.

2D autocorrelation modelling of the inhibitory activity of cytokinin-derived cyclin-dependent kinase inhibitors.

The inhibitory activity towards p34(cdc2)/cyclin b kinase (CBK) enzyme of 30 cytokinin-derived compounds has been successfully modelled using 2D spatial autocorrelation vectors. Predictive linear and non-linear models were obtained by forward stepwise multi-linear regression analysis (MRA) and artificial neural network (ANN) approaches respectively. A variable selection routine that selected relevant non-linear information from the data set was employed prior to networks training. The best ANN with three input variables was able to explain about 87% data variance in comparison with 80% by the linear equation using the same number of descriptors. Similarly, the neural network had higher predictive power. The MRA model showed a linear dependence between the inhibitory activities and the spatial distributions of masses, electronegativities and van der Waals volumes on the inhibitors molecules. Meanwhile, ANN model evidenced the occurrence of non-linear relationships between the inhibitory activity and the mass distribution at different topological distance on the cytokinin-derived compounds. Furthermore, inhibitors were well distributed regarding its activity levels in a Kohonen self-organizing map (SOM) built using the input variables of the best neural network.

Molecular models of cyclin-dependent kinase 1 complexed with inhibitors.

Roscovitine and flavopiridol have been shown to potently inhibit cyclin-dependent kinase 1 and 2 (CDK1 and 2). The structures of CDK2 complexed with roscovitine and deschoroflavopiridol have been reported, however no crystallographic structure is available for complexes of CDK1 with inhibitors. The present work describes two molecular models for the binary complexes CDK1:roscovitine and CDK1:flavopiridol. These structural models indicate that both inhibitors strongly bind to the ATP-binding pocket of CDK1 and structural comparison of the CDK complexes correlates the structures with differences in inhibition of these CDKs by flavopiridol and roscovitine. This article explains the structural basis for the observed differences in activity of these inhibitors.

Sodium salicylate induces the expression of the immunophilin FKBP51 and biglycan genes and inhibits p34cdc2 mRNA both in vitro and in vivo.

One of the mechanisms proposed to explain the anti-inflammatory activity of sodium salicylate (NaSal) is based, at least in part, on its ability to inhibit nuclear factor-kappaB activation and inhibition of nuclear factor-kappaB-dependent gene expression. On the other hand, little is known about the ability of NaSal to activate gene expression. By differential display reverse transcription polymerase chain reaction, we identified several genes that are modulated upon treatment of mouse fibroblasts with NaSal. From the various cDNA fragments recovered from autoradiograms, we found that NaSal can increase the levels of mRNA for biglycan, the mouse homologue of the human eIF-3 p47 unit, and immunophilin FKBP51. NaSal-induced expression of these genes was time- and dose-dependent. Moreover, FKBP51 gene expression was augmented in vivo, in mice treated orally or intraperitoneally with NaSal. We also found that treating cells with NaSal can inhibit the expression of the p34(cdc2) kinase. The impact this inhibition on cell cycle was evaluated by measuring the content of DNA during the cell cycle. Treatment of cells with NaSal led to a G(2)/M arrest. By investigating the signaling events that regulate the expression of these genes and their biological activities, we can contribute to the understanding of the mechanism of NaSal.

The crk3 gene of Leishmania mexicana encodes a stage-regulated cdc2-related histone H1 kinase that associates with p12.

A cdc2-related protein kinase gene, crk3, has been isolated from the parasitic protozoan Leishmania mexicana. Data presented here suggests that crk3 is a good candidate to be the leishmanial cdc2 homologue but that the parasite protein has some characteristics which distinguish it from mammalian cdc2. crk3 is predicted to encode a 35.6-kDa protein with 54% sequence identity with the human cyclin-dependent kinase cdc2 and 78% identity with the Trypanosoma brucei CRK3. The trypanosomatid CRK3 proteins have an unusual, poorly conserved 19-amino acid N-terminal extension not present in human cdc2. crk3 is single copy, and there is 5-fold higher mRNA in the replicative promastigote life-cycle stage than in the non-dividing metacyclic form or mammalian amastigote form. A leishmanial suc-binding cdc2-related kinase (SBCRK) histone H1 kinase, has previously been described which binds the yeast protein, p13(suc1), and that has stage-regulated activity (Mottram J. C., Kinnaird, J., Shiels, B. R., Tait, A., and Barry, J. D. (1993) J. Biol. Chem. 268, 21044-21051). CRK3 from cell extracts of the three life-cycle stages was found to bind p13(suc1) and the leishmanial homologue p12(cks1). CRK3 fused with six histidines at the C terminus was expressed in L. mexicana and shown to have SBCRK histone H1 kinase activity. Depletion of histidine-tagged CRK3 from L. mexicana cell extracts, by Ni-nitrilotriacetic acid agarose selection, reduced histone H1 kinase activity binding to p13(suc1). These data imply that crk3 encodes the kinase subunit of SBCRK. SBCRK and histidine-tagged CRK3 activities were inhibited by the purine analogue olomoucine with an IC50 of 28 and 42 microM, respectively, 5-6-fold higher than human p34(cdc2)/cyclinB.

Phosphorylation of connexin43 and the regulation of neonatal rat cardiac myocyte gap junctions.

The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of 32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of 32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of 32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived from in vitro phosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243-382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.