KNOCKDOWN OF CASEIN KINASE 1e INHIBITS CELL PROLIFERATION AND INVASION OF COLORECTAL CANCER CELLS VIA INHIBITION OF THE Wnt/ß-CATENIN SIGNALING.

Deregulation of casein kinase 1 epsilon (CK1ε) is involved in the development of multiple pathological disorders such as cancer, however the function and molecular mechanism of CK1εin cancer are still unclear. In the present study, we aimed to investigate the role of CK1ε in human colorectal cancer (CRC). The expression of CK1ε was examined by immunohistochemical assay using a tissue microarray procedure. A loss-of-function experiment was performed to observe the effects of lentivirus-mediated CK1ε shRNA (Lv-shCK1ε) on cell proliferation and invasive potential by MTT and Transwell assays in CRC cell line (SW480). As a result, we found that the expression of CK1ε protein was significantly increased in CRC tissues compared with that in adjacent non-cancerous tissues (ANCT) (68.9% vs 42.2%, P=0.017) and was correlated with the Duke’s staging and depth of invasion in CRC patients (P=0.012; P=0.015). Knockdown of CK1ε reduced cell proliferation and invasion of CRC cells followed by the downregulation of wnt3α, ß-catenin, PCNA and MMP-9. In conclusion, our findings show that high expression of CK1ε is positively associated with the Duke’s staging and depth of invasion in CRC patients, and knockdown of CK1ε suppresses the growth and invasion of CRC cells through inhibition of the wnt/ß-catenin signaling, suggesting that CK1ε may serve as a promising therapeutic target for the treatment of CRC.

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae

BACKGROUND: The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to familyRosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties. RESULTS: Among observed plant species, the highest amount of total flavonoids has been found in leaves ofHelianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU). CONCLUSIONS: This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.

A novel mechanism controlling resetting speed of the circadian clock to environmental stimuli.

Many aspects of mammalian physiology are driven through the coordinated action of internal circadian clocks. Clock speed (period) and phase (temporal alignment) are fundamental to an organism's ability to synchronize with its environment. In humans, lifestyles that disturb these clocks, such as shift work, increase the incidence of diseases such as cancer and diabetes. Casein kinases 1δ and ε are closely related clock components implicated in period determination. However, CK1δ is so dominant in this regard that it remains unclear what function CK1ε normally serves. Here, we reveal that CK1ε dictates how rapidly the clock is reset by environmental stimuli. Genetic disruption of CK1ε in mice enhances phase resetting of behavioral rhythms to acute light pulses and shifts in light cycle. This impact of CK1ε targeting is recapitulated in isolated brain suprachiasmatic nucleus and peripheral (lung) clocks during NMDA- or temperature-induced phase shift in association with altered PERIOD (PER) protein dynamics. Importantly, accelerated re-entrainment of the circadian system in vivo and in vitro can be achieved in wild-type animals through pharmacological inhibition of CK1ε. These studies therefore reveal a role for CK1ε in stabilizing the circadian clock against phase shift and highlight it as a novel target for minimizing physiological disturbance in shift workers.

Casein kinase I epsilon somatic mutations found in breast cancer cause overgrowth in Drosophila.

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.

[cAMP/PKA signaling underlies age-related memory impairment].

Physiological aging of the brain is inevitable, regardless of the occurrence of pathological diseases such as Alzheimer disease or cerebral vascular disorders. AMI (age-related memory impairment) is an important phenotype of brain aging. In contrast to organismal aging, the molecular mechanisms underlying AMI are poorly understood and hindered by the lack of specific mutants for AMI. We used the fruit fly Drosophila as a novel model for genetic analyses of AMI since it has a short lifespan and is suitable for quantitative analysis of learning and memory. The molecular mechanisms underlying learning and memory in Drosophila are similar to those in mammals. In a screen for AMI mutants, we found that heterozygous mutations of DC0 gene, which encodes the major catalytic subunit of PKA (cAMP-dependent kinase), delayed AMI onset by more than 2-fold without affecting lifespan and memory at young age. The first identification of AMI mutant provides provocative insights into the role of cAMP/PKA signaling and the genetic relationship between organismal aging and brain aging.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo.

BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. AIMS: We analysed the expression levels of CK1 delta and epsilon (CK1delta/in) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. METHODS: CK1delta/in expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. RESULTS: We found that CK1delta/in are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1delta/in by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. CONCLUSIONS: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.

Novel insights from genetic and molecular characterization of the human clock.

Biological rhythms govern the ebb and flow of life on planet Earth. Animals have an internal timekeeping mechanism that precisely regulates 24-hour rhythms of body function and behavior and synchronizes them to the day/night cycle. Circadian pacemakers trigger behavioral and physiological processes that dictate our daily rhythms. Despite the importance of the circadian clock to all aspects of our physiology and behavior, the opportunity to probe the human circadian clock only recently became possible with the recognition of Mendelian circadian variants in people (familial advanced sleep phase syndrome, FASPS). We have now cloned several genes and identified mutations causing FASPS. Study of these genes and the proteins they encode and engineering of the human mutations into mouse models are allowing study of this fascinating phenotype and yielding novel insights into circadian regulation in humans. Ultimately, such work will allow us to understand the similarities and differences between the human clock and those of model organisms. In addition, recent studies have also linked disruption of the circadian clock with numerous ailments, including cancer, cardiovascular diseases, asthma, and learning disorders. Thus, studying the molecular mechanism of human circadian rhythmicity will have an enormous impact on our understanding of human health and disease. It should also lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag, various clock-related sleep and psychiatric disorders, and other human diseases.

CKIepsilon/discs overgrown promotes both Wnt-Fz/beta-catenin and Fz/PCP signaling in Drosophila.

The related Wnt-Frizzled(Fz)/beta-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I epsilon (CKIepsilon) may act as a molecular "switch," promoting Wnt/beta-catenin while inhibiting Fz/PCP signaling. Here, we demonstrate in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIepsilon acts positively for signaling in both pathways, rather than as a switch. Our data suggest that the kinase activity of CKIepsilon is required for peak levels of Wnt/beta-catenin signaling. In contrast, CKIepsilon is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, we have identified the primary kinase target residue of CKIepsilon on Dsh. Thus, our data suggest that CKIepsilon modulates Wnt/beta-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms.

Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila.

Members of the casein kinase I (CKI) family have been implicated in regulating canonical Wnt/Wingless (Wg) signaling by phosphorylating multiple pathway components. Overexpression of CKI in vertebrate embryos activates Wg signaling, and one target is thought to be the cytoplasmic effector Dishevelled (Dsh), which is an in vitro target of CKI phosphorylation. Phosphorylation of Dsh by CKI has also been suggested to switch its activity from noncanonical to canonical Wingless signaling. However, in vivo loss-of-function experiments have failed to identify a clear role for CKI in positive regulation of Wg signaling. By examining hypomorphic mutations of the Drosophila CKIepsilon homolog discs overgrown (dco)/double-time, we now show that it is an essential component of the noncanonical/planar cell polarity pathway. Genetic interactions indicate that dco acts positively in planar polarity signaling, demonstrating that it does not act as a switch between canonical and noncanonical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Furthermore, in these mutants, Dishevelled fails to adopt its characteristic asymmetric subcellular localisation at the distal end of pupal wing cells, and the site of hair outgrowth is disrupted. Finally, we also find that dco function in polarity is partially redundant with CKIalpha.

Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.

The circadian clock and tumor suppression by mammalian period genes.

Period (Per) genes are key circadian rhythm regulators in mammals. Expression of mouse Per (mPer) genes has a diurnal pattern in the suprachiasmatic nucleus and in peripheral tissues. Genetic ablation mPER1 and mPER2 function results in a complete loss of circadian rhythm control based on wheel-running activity in mice. In addition, these animals also display apparent premature aging and a significant increase in neoplastic and hyperplastic phenotypes. When challenged by gamma radiation, mPer2-deficient mice respond by rapid hair graying, are deficient in p53-mediated apoptosis in thymocytes, and have robust tumor occurrences. Studies have demonstrated that the circadian clock function is very important for cell cycle, DNA damage response, and tumor suppression in vivo. The temporal expression of genes involved in cell cycle regulation and tumor suppression, such as c-Myc, Cyclin D1, Cyclin A, Mdm-2, and Gadd45alpha, is deregulated in mPer2 mutant mice. Genetic studies have demonstrated that many key regulators of cell cycle and growth control are also important circadian clock regulators, confirming the critical role of circadian function in organismal homeostasis.

Pharmacological modulation of circadian rhythms: a new drug target in psychotherapeutics.

Daily variation in an organism's physiology and behaviour is regulated by the synchrony that is achieved between the internal timing mechanisms - the circadian rhythms of the biological clock - and the prevailing environmental cues. Proper synchrony constitutes an adaptive response; improper or lost synchrony may well yield maladaptation and, in the case of humans, a psychiatric disorder. On a basic level, the circadian system is comprised of three parts: a central oscillator, its various neuronal inputs and its outputs. For all three of these parts, the dissemination of new information is moving at an unprecedented pace, and the number of molecular targets for the opportunistic pharmacologist is growing in step. Monoamines, neuropeptides, kinases - sorting through all these, much less developing one into a drug discovery programme, may be the biggest challenge. However, the potential benefits in targeting a basic flaw in a fundamental biological system may be enormous.