Essential role of CK2α for the interaction and stability of replication fork factors during DNA synthesis and activation of the S-phase checkpoint.

The ataxia telangiectasia mutated and Rad3-related (ATR)-CHK1 pathway is the major signalling cascade activated in response to DNA replication stress. This pathway is associated with the core of the DNA replication machinery comprising CDC45, the replicative MCM2-7 hexamer, GINS (altogether forming the CMG complex), primase-polymerase (POLε, -α, and -δ) complex, and additional fork protection factors such as AND-1, CLASPIN (CLSPN), and TIMELESS/TIPIN. In this study, we report that functional protein kinase CK2α is critical for preserving replisome integrity and for mounting S-phase checkpoint signalling. We find that CDC45, CLSPN and MCM7 are novel CK2α interacting partners and these interactions are particularly important for maintenance of stable MCM7-CDC45, ATRIP-ATR-MCM7, and ATR-CLSPN protein complexes. Consistently, cells depleted of CK2α and treated with hydroxyurea display compromised replisome integrity, reduced chromatin binding of checkpoint mediator CLSPN, attenuated ATR-mediated S-phase checkpoint and delayed recovery of stalled forks. In further support of this, differential gene expression analysis by RNA-sequencing revealed that down-regulation of CK2α accompanies global shutdown of genes that are implicated in the S-phase checkpoint. These findings add to our understanding of the molecular mechanisms involved in DNA replication by showing that the protein kinase CK2α is essential for maintaining the stability of the replisome machinery and for optimizing ATR-CHK1 signalling activation upon replication stress.

Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1, which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells.

Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5' splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5'-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.

The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.

Protein kinase CK2 regulates redox homeostasis through NF-κB and Bcl-xL in cardiomyoblasts.

Oxygen consumption is particularly elevated in cardiac cells as they are equipped with a large number of mitochondria and high levels of respiratory chain components. Consequently, production of reactive oxygen species (ROS) is tightly controlled as an imbalance in redox reactions can lead to irreversible cellular damage. siRNA-mediated down-regulation of protein kinase CK2 has been implicated in the accumulation of ROS in cells. The present study was undertaken in order to investigate the role of CK2 in redox homeostasis in cardiomyoblasts. We found that inhibition or silencing of CK2 causes elevated levels of ROS, notably superoxide radical, and this is accompanied by suppression of NF-κB transcriptional activity and mitochondrial dysfunction. We show that CK2 regulates the expression of manganese superoxide dismutase, the enzyme catalyzing the dismutation of superoxide, in cancer cells but not in cardiomyoblasts. Furthermore, we report evidence that impaired expression of CK2 results in destabilization of the Bcl-2 mammalian homolog Bcl-xL, which is known to stabilize the mitochondrial membrane potential, through a mechanism involving disruption of the chaperone function of heat shock protein 90. Analysis of differential mRNA expression related to oxidative stress revealed that CK2 silencing caused a statistically significant deregulation of four genes associated with the oxidative damage, i.e., Fmo2, Ptgs1, Dhcr24, and Ptgs2. Overall, the results reported here are consistent with the notion that CK2 plays a role in conferring protection against oxidative stress by positively regulating pro-survival signaling molecules and the protein folding machinery in cardiomyoblasts.

Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics.

Common γ-chain family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, where IL stands for interleukin) are key regulators of the immune homeostasis that exhibit pleiotropic biological activities and even sometimes redundant roles as a result of the utilization of the same receptor subunit. However, they also exert distinct functions that make each of them to be indispensable. For instance, all family members can act as T-cell growth factors; however, we found that IL-15 but not IL-7 can replace IL-2 to promote and sustain the proliferation of Kit225T cells. In addition to the γ-chain, IL-2 and IL-15 share the ß-chain, which creates the paradox of how they can trigger diverse phenotypes despite signaling through the same receptors. To investigate this paradigm, we combined SILAC with enrichment of tyrosine-phosphorylated proteins and peptides followed by mass spectrometric analysis to quantitatively assess the signaling networks triggered downstream IL-2/IL-2R and IL-15/IL-15R. This study confirmed that the transduction pathways initiated by both cytokines are highly similar and revealed that the main signaling branches, JAK/STAT, RAS/MAPK and PI3K/AKT, were nearly equivalently activated in response to both ILs. Despite that, our study revealed that receptor internalization rates differ in IL-2- and IL-15-treated cells indicating a discrete modulation of cytokine signaling. All MS data have been deposited in the ProteomeXchange with identifier PXD001129 (http://proteomecentral.proteomexchange.org/dataset/PXD001129).

Acetoxymethyl Ester of Tetrabromobenzimidazole-Peptoid Conjugate for Inhibition of Protein Kinase CK2 in Living Cells.

CK2 is a ubiquitous serine/threonine protein kinase, which has the potential to catalyze the generation of a large proportion of the human phosphoproteome. Due to its role in numerous cellular functions and general anti-apoptotic activity, CK2 is an important target of research with therapeutic potential. This emphasizes the need for cell-permeable highly potent and selective inhibitors and photoluminescence probes of CK2 for investigating the protein phosphorylation networks in living cells. Previously, we had developed bisubstrate inhibitors for CK2 (CK2-targeted ARCs) that showed remarkable affinity (KD < 1 nM) and selectivity, but lacked proteolytic stability and plasma membrane permeability. In this report, the structures of CK2-targeted ARCs were modified for the application in live cells. Based on structure-activity studies, proteolytically stable achiral oligoanionic peptoid conjugates of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) were constructed. Affinity of the conjugates toward CK2 reached subnanomolar range. Acetoxymethyl (AM) prodrug strategy was applied for loading TBBz-peptoid conjugates into living cells. The uptake of inhibitors was visualized by live cell imaging and the reduction of the phosphorylation levels of two CK2-related phosphosites, Cdc37 pSer13 and NFκB pSer529, was demonstrated by Western blot analysis.

Identification of a novel potent, selective and cell permeable inhibitor of protein kinase CK2 from the NIH/NCI Diversity Set Library.

The anti-apoptotic protein kinase CK2 increasingly becomes an attractive target in cancer research with great therapeutic potential. Here, we have performed an in vitro screening of the Diversity Set III of the DTP program from the NCI/NIH, comprising 1600 compounds. We have identified 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] dibenzo(b,d) furan-2,7-diol (referred to as D11) to be a potent and selective inhibitor of protein kinase CK2. The D11 compound was tested against 354 eukaryotic protein kinases. By setting the threshold for inhibition to <2% remaining kinase activity, only DYRK1B, IRAK1 and PIM3 were inhibited to an extent as the tetrameric CK2 holoenzyme and its catalytic subunits α and α'. The IC50 values for the CK2α and CK2α' were on average 1-2 nM in comparison to the DYRK1B, IRAK1 and PIM3 kinases, which ranged from 18 to 49 nM. Cell permeability and efficacy of D11 were tested with cells in culture. In MIA PaCa-2 cells (human pancreatic carcinoma cell line), the phosphorylation of the CK2 biomarker CDC37 at S13 was almost completely inhibited in the presence of D11. This was observed both under normoxia and hypoxia. In the case of the human non-small cell lung carcinoma cell line, H1299, increasing amounts of D11 led to an inhibition of S380/T382/383 phosphorylation in PTEN, another biomarker for CK2 activity.

Evidence for aggregation of protein kinase CK2 in the cell: a novel strategy for studying CK2 holoenzyme interaction by BRET(2).

Protein kinase CK2 is a ubiquitous pro-survival kinase whose substrate targets are involved in various cellular processes. Crystal structure analysis confirmed constitutive activity of the kinase, yet CK2 activity regulation in the cell is still obscure. In-vitro studies suggest autoinhibitory aggregation of the hetero-tetrameric CK2 holoenzyme as a basis for CK2 regulation. In this study, we applied bioluminescent resonance energy transfer (BRET) technology to investigate CK2 holoenzyme aggregation in living cells. We designed a BRET(2) pair consisting of the fusion proteins CK2α-Rluc8 and CK2α-GFP(2). This BRET(2) sensor reported specific interaction of CK2 holoenzyme complexes. Furthermore, the BRET(2) sensor was applied to study modulators of CK2 aggregation. We found that CK2 aggregation is not static and can be influenced by the CK2-binding protein alpha subunit of the heterotrimeric G-protein that stimulates adenylyl cyclase (Gαs) and the polycationic compound polylysine. Gαs, but not the CK2 substrate ß-arrestin2, decreased the BRET(2) signal by up to 50%. Likewise polylysine, but not the CK2 inhibitor DRB, decreased the signal in a dose-dependent manner up to 50%. For the first time, we present direct experimental evidence for CK2 holoenzyme aggregates in the cell. Our data suggest that CK2 activity may be controlled by holoenzyme aggregation, to our knowledge a novel mechanism for protein kinase regulation. Moreover, the BRET(2) sensor used in our study is a novel tool for studying CK2 regulation by aggregation and pharmacological screening for novel allosteric CK2 effectors.

SHAPES Marketplace: Transparency, Trust and Fair Competition in the Healthy Ageing Market.

Access to digital health and care solutions and services that promote healthy ageing, independent living, and ageing in place is limited due to significant market barriers and challenges. The SHAPES project addresses the challenge of ageing populations by developing a sociotechnical ecosystem comprising a variety of health and care digital solutions, tools and services to enable and facilitate active, independent, and healthy ageing at home. Within the SHAPES project, the SHAPES Marketplace serves as a one-stop-shop for digital solutions and services designed for the Silver Economy that target the smart and healthy ageing and independent living markets. Delivering a dynamic catalogue of health and care digital solutions and services, the Marketplace promotes a transparent expansion of a trusted market offer on digital solutions and services for healthy ageing and independent living on a pan-European scale, thereby preventing vendor lock-in and enhancing the agile and fair competitiveness of the health and care industry, particularly in Europe. This paper introduces the SHAPES Marketplace and considers its function as a market driver to raise awareness on the benefits and impact of health and care digital solutions and services, as well as to shape the healthy ageing market, upholding the Systems-Market for Assistive and Related Technologies (SMART) Thinking Matrix to stimulate transparency, trust and fair competition.

Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage.

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. RESULTS: In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. CONCLUSIONS: Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

NFκB signaling is important for growth of antiestrogen resistant breast cancer cells.

Resistance to endocrine therapy is a major clinical challenge in current treatment of estrogen receptor-positive breast cancer. The molecular mechanisms underlying resistance are yet not fully clarified. In this study, we investigated whether NFκB signaling is causally involved in antiestrogen resistant cell growth and a potential target for re-sensitizing resistant cells to endocrine therapy. We used an MCF-7-derived cell model for antiestrogen resistant breast cancer to investigate dependence on NFκB signaling for antiestrogen resistant cell growth. We found that targeting NFκB preferentially inhibited resistant cell growth. Antiestrogen resistant cells expressed increased p50 and RelB, and displayed increased phosphorylation of p65 at Ser529 and Ser536. Moreover, transcriptional activity of NFκB after stimulation with tumor necrosis factor α was enhanced in antiestrogen resistant cell lines compared to the parental cell line. Inhibition of NFκB signaling sensitized tamoxifen resistant cells to the growth inhibitory effects of tamoxifen but was not sufficient to fully restore sensitivity of fulvestrant resistant cells to fulvestrant. In support of this, depletion of p65 with siRNA in tamoxifen resistant cells increased sensitivity to tamoxifen treatment. Our data provide evidence that NFκB signaling is enhanced in antiestrogen resistant breast cancer cells and plays an important role for antiestrogen resistant cell growth and for sensitivity to tamoxifen treatment in resistant cells. Our results imply that targeting NFκB might serve as a potential novel treatment strategy for breast cancer patients with resistance toward antiestrogen.

Implementation of a pan-European ecosystem and an interoperable platform for Smart and Healthy Ageing in Europe: An Innovation Action research protocol.

As life expectancy continues to increase in most EU Member States, smart technologies can help enable older people to continue living at home, despite the challenges accompanying the ageing process. The Innovation Action (IA) SHAPES 'Smart and Healthy Ageing through People Engaging in Supportive Systems' funded by the EU under the Horizon 2020 Research and Innovation Programme (grant agreement number 857159) attends to these topics to support active and healthy ageing and the wellbeing of older adults. This protocol article outlines the SHAPES project's objectives and aims, methods, structure, and expected outcomes. SHAPES seeks to build, pilot, and deploy a large-scale, EU-standardised interoperable, and scalable open platform. The platform will facilitate the integration of a broad range of technological, organisational, clinical, educational, and social solutions. SHAPES emphasises that the home is much more than a house-space; it entails a sense of belonging, a place and a purpose in the community. SHAPES creates an ecosystem - a network of relevant users and stakeholders - who will work together to scale-up smart solutions. Furthermore, SHAPES will create a marketplace seeking to connect demand and supply across the home, health and care services. Finally, SHAPES will produce a set of recommendations to support key stakeholders seeking to integrate smart technologies in their care systems to mediate care delivery. Throughout, SHAPES adopts a multidisciplinary research approach to establish an empirical basis to guide the development of the platform. This includes long-term ethnographic research and a large-scale pan-European campaign to pilot the platform and its digital solutions within the context of seven distinct pilot themes. The project will thereby address the challenges of ageing societies in Europe and facilitate the integration of community-based health and social care. SHAPES will thus be a key driver for the transformation of healthcare and social care services across Europe.

Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts.

Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na(+) dependent transporter TauT and taurine release are increased and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA(2) activity, exposure to exogenous reactive oxygen species as well as inhibition of protein tyrosine phosphotases (PTP) potentiate the swelling-induced taurine loss. Inhibition of PI3K and PTEN reduces and potentiates swelling-induced taurine release, respectively. Inhibition of CK2 has no effect on PLA(2) activity and ROS production by NADPH oxidase, whereas it lifts the effect of PTEN and PTP inhibition. It is suggested that CK2 regulates the taurine release downstream to known swelling-induced signal transducers including PLA(2), NADPH oxidase and PI3K.

Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity.

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Phytochemicals in cancer and their effect on the PI3K/AKT-mediated cellular signalling.

Protein kinases belong to the largest family of enzymes controlling every aspect of cellular activity including gene expression, cell division, differentiation and metabolism. They are part of major intracellular signalling pathways. Hence, it is not surprising that they are involved in the development of major diseases such as cardiovascular disorders, diabetes, dementia and, most importantly, cancer when they undergo mutations, modifications and unbalanced expression. This review will explore the possibility to draw a connection between the application of natural phytochemicals and the treatment of cancer. We have chosen to focus on the PI3K/AKT cellular signalling pathway which has been shown to be a major target by natural compounds in cell cultures and animal models.

Shaping the Future of Digitally Enabled Health and Care.

People generally need more support as they grow older to maintain healthy and active lifestyles. Older people living with chronic conditions are particularly dependent on healthcare services. Yet, in an increasingly digital society, there is a danger that efforts to drive innovations in eHealth will neglect the needs of those who depend on healthcare the most-our ageing population. The SHAPES (Smart and Healthy Ageing through People Engaging in Supportive Systems) Innovation Action aims to create an open European digital platform that facilitates the provision of meaningful, holistic support to older people living independently. A pan-European pilot campaign will evaluate a catalogue of digital solutions hosted on the platform that have been specifically adapted for older people. 'Medicines control and optimisation' is one of seven themes being explored in the campaign and will investigate the impact of digital solutions that aim to optimise medicines use by way of fostering effective self-management, while facilitating timely intervention by clinicians based on remote monitoring and individualised risk assessments powered by artificial intelligence. If successful, the SHAPES Innovation Action will lead to a greater sense of self-sufficiency and empowerment in people living with chronic conditions as they grow older.

Reciprocal activating interaction between natural killer cells and dendritic cells.

We analyzed the interaction between human peripheral blood natural killer (NK) cells and monocyte-derived immature dendritic cells (DC). Fresh NK cells were activated, as indicated by the induced expression of the CD69 antigen, and their cytolytic activity was strongly augmented by contact with lipopolysaccharide (LPS)-treated mature DC, or with immature DC in the presence of the maturation stimuli LPS, Mycobacterium tuberculosis or interferon (IFN)-alpha. Reciprocally, fresh NK cells cultured with immature DC in the presence of the maturation stimuli strongly enhanced DC maturation and interleukin (IL)-12 production. IL-2--activated NK cells directly induced maturation of DC and enhanced their ability to stimulate allogeneic naive CD4(+) T cells. The effects of NK cells were cell contact dependent, although the secretion of IFN-gamma and TNF also contributed to DC maturation. Within peripheral blood lymphocytes the reciprocal activating interaction with DC was restricted to NK cells, because the other lymphocyte subsets were neither induced to express CD69, nor induced to mature in contact with DC. These data demonstrated for the first time a bidirectional cross talk between NK cells and DC, in which NK cells activated by IL-2 or by mature DC induce DC maturation.

Enhancing chemosensitivity to gemcitabine via RNA interference targeting the catalytic subunits of protein kinase CK2 in human pancreatic cancer cells.

BACKGROUND: Pancreatic cancer is a complex genetic disorder that is characterized by rapid progression, invasiveness, resistance to treatment and high molecular heterogeneity. Various agents have been used in clinical trials showing only modest improvements with respect to gemcitabine-based chemotherapy, which continues to be the standard first-line treatment for this disease. However, owing to the overwhelming molecular alterations that have been reported in pancreatic cancer, there is increasing focus on targeting molecular pathways and networks, rather than individual genes or gene-products with a combination of novel chemotherapeutic agents. METHODS: Cells were transfected with small interfering RNAs (siRNAs) targeting the individual CK2 subunits. The CK2 protein expression levels were determined and the effect of its down-regulation on chemosensitization of pancreatic cancer cells was investigated. RESULTS: The present study examined the impact on cell death following depletion of the individual protein kinase CK2 catalytic subunits alone or in combination with gemcitabine and the molecular mechanisms by which this effect is achieved. Depletion of the CK2alpha or -alpha' subunits in combination with gemcitabine resulted in marked apoptotic and necrotic cell death in PANC-1 cells. We show that the mechanism of cell death is associated with deregulation of distinct survival signaling pathways. Cellular depletion of CK2alpha leads to phosphorylation and activation of MKK4/JNK while down-regulation of CK2alpha' exerts major effects on the PI3K/AKT pathway. CONCLUSIONS: Results reported here show that the two catalytic subunits of CK2 contribute differently to enhance gemcitabine-induced cell death, the reduced level of CK2alpha' being the most effective and that simultaneous reduction in the expression of CK2 and other survival factors might be an effective therapeutic strategy for enhancing the sensitivity of human pancreatic cancer towards chemotherapeutic agents.

Role of Protein Kinase CK2 in Aberrant Lipid Metabolism in Cancer.

Uncontrolled proliferation is a feature defining cancer and it is linked to the ability of cancer cells to effectively adapt their metabolic needs in response to a harsh tumor environment. Metabolic reprogramming is considered a hallmark of cancer and includes increased glucose uptake and processing, and increased glutamine utilization, but also the deregulation of lipid and cholesterol-associated signal transduction, as highlighted in recent years. In the first part of the review, we will i) provide an overview of the major types of lipids found in eukaryotic cells and their importance as mediators of intracellular signaling pathways ii) analyze the main metabolic changes occurring in cancer development and the role of oncogenic signaling in supporting aberrant lipid metabolism and iii) discuss combination strategies as powerful new approaches to cancer treatment. The second part of the review will address the emerging role of CK2, a conserved serine/threonine protein kinase, in lipid homeostasis with an emphasis regarding its function in lipogenesis and adipogenesis. Evidence will be provided that CK2 regulates these processes at multiple levels. This suggests that its pharmacological inhibition combined with dietary restrictions and/or inhibitors of metabolic targets could represent an effective way to undermine the dependency of cancer cells on lipids to interfere with tumor progression.