casein-kinase-ii and Liver-Neoplasms

Chromosome 1 open reading frame 109 (C1orf09) is a protein whose expression pattern and biological function in humans, particularly in malignant tumors, have not been explored. In this study, both bioinformatics and immunohistochemical staining revealed that C1orf109 was overexpressed in the cytoplasm of liver cancer cells, and the positive ratio of C1orf109 in liver cancer samples (42.5%, 37/87) was significantly higher than that in normal liver tissues (10%, 3/30, P = 0.0012). C1orf109 expression was correlated with an advanced TNM stage (P = 0.017) and vascular invasion (P = 0.023) and predicted the poor overall survival of patients with liver cancer (P = 0.001). C1orf109 facilitated tumor growth, colony formation, migration, and invasion by activating Wnt signaling by upregulating non-phosphorylated β-catenin and its downstream target genes such as CyclinD1, c-myc, and MMP7. Our results also suggest that C1orf109 interacts and co-localizes with casein kinase II (CK2) to activate Wnt signaling. Treatment with a CK2-specific inhibitor markedly counteracted the increased expression of CyclinD1, c-Myc, and MMP7, as well as the upregulation of tumor proliferation and invasion caused by C1orf109 overexpression. Taken together, our results indicate that C1orf109 accelerates liver cancer cell proliferation and invasion by strengthening the Wnt signaling pathway in a CK2-dependent manner.

Topics: beta Catenin; Casein Kinase II; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 7; Phosphoproteins; Wnt Signaling Pathway

To investigate mechanisms that TMEM2 activation inhibits hepatitis B virus (HBV) infection in hepatocarcinoma (HCC) cells, co-immunoprecipitation (Co-IP) and mass spectrometry were used in screening interacting proteins for TMEM2. Levels of casein kinase 2 subunit α3 (CSNK2A3) in HCC cells were found to be inhibited or overexpressed using siRNAs and pcDNA3.1-CSNK2A3, respectively. Effect of CSNK2A3 expression on cell proliferation was analyzed using MTS, while its effect on HBV infection was measured using ddPCR and IHC. Western blotting and JAK inhibitor ruxolitinib were also used to determine whether TMEM2-regulated CSNK2A3 expression and HBV infection were affected by JAK-STAT signaling. Co-IP and mass spectrometry results showed that CSNK2A3 interacts with TMEM2. Moreover, overexpression of CSNK2A3 significantly inhibited cell proliferation, while inhibition of CSNK2A3 promoted proliferation of HCC cells. In addition, overexpression of CSNK2A3 was observed to significantly enhance HBV infection, while siRNA knockdown of CSNK2A3 inhibited HBV infection. Notably, effect of CSNK2A3 overexpression on HBV infection was suppressed by TMEM2 overexpression. Further mechanistic analyses have revealed that TMEM2 could antagonize the effects of CSNK2A3 on cell proliferation and HBV infection via JAK-STAT pathway activation. In conclusion, TMEM2 has been determined to bind to CSNK2A3 to inhibit HBV infection via activation of the JAK-STAT pathway.

Topics: Brain; Carcinoma, Hepatocellular; Case-Control Studies; Casein Kinase II; Epilepsy, Temporal Lobe; Hepatitis B; Hepatitis B virus; Humans; Janus Kinase 1; Liver Neoplasms; Membrane Proteins; PPAR gamma; STAT Transcription Factors; Tumor Cells, Cultured

Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown.. The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot.. The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo.. Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Carcinogenesis; Carcinoma, Hepatocellular; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Disease Progression; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Liver Neoplasms; Male; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; Proteolysis; Tumor Suppressor Proteins; Ubiquitin; Vascular Endothelial Growth Factor A

Hepatocellular carcinoma (HCC) pathogenesis involves the interplay of multiple signalling pathways. Notch and Hedgehog (Hh) are two major developmental pathways that act in concert to regulate adult cell repair. CK2α -serine-threonine kinase-down-regulation enhanced apoptotic activity and was proven beneficial for HCC patients. Quercetin is a bioactive flavonoid and has been shown to protect against HCC through its antioxidant activity. This study was carried out to elucidate the antineoplastic effect of quercetin through regulating both Notch and Hh pathways, apoptosis, cell proliferation and CK2α activity.. Hepatocellular carcinoma was induced in male Sprague Dawley rats by thioacetamide. Quercetin was administered in both protective and curative doses. Parameters of liver function and oxidative stress were assessed. CK2α, Notch and Hh pathways were evaluated using RT-PCR and ELISA. Apoptosis was investigated by detecting caspase-3, caspase-8 and p53. Proliferative and cell cycle markers as cyclin D1 and Ki-67 were detected immunohistochemically.. Quercetin inhibited CK2α and downregulated mRNA and protein expression of Notch1 and Gli2. Quercetin also suppressed caspase-3 expression but not caspase-8. Quercetin elevated p53 expression whereas proliferative and cell cycle markers cyclin D1 and Ki-67 were downregulated. Markers of hepatic cellular integrity such as AST, ALT, ALP, GGT, albumin and bilirubin were significantly ameliorated. This was confirmed by histological examination. Quercetin also alleviated oxidative stress as shown by SOD, GSH, MDA and NO levels.. We can conclude that in addition to its antioxidant power, quercetin blocked Notch, Hedgehog, regulated the apoptotic and proliferative pathways and inhibited CK2α in HCC.

Topics: Animals; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Casein Kinase II; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Liver Neoplasms; Male; Oxidative Stress; Quercetin; Rats; Rats, Sprague-Dawley; Receptors, Notch; Signal Transduction

Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying insulin-like growth factor-I (IGF-I) bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mechanistic target of rapamycin (mTOR) and casein kinase (CSNK)-2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2β, and mTOR as a prerequisite for protein-protein interaction. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2β and a nuclear co-localization between CSNK-2β and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2β as well as mTOR and CSNK-2β but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2β interactions were in the perinuclear region and mTOR and CSNK-2β interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2β co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2β (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia and rapamycin treatment increased IGFBP-1 phosphorylation at Ser98/Ser101/Ser119/Ser174 but most considerably (106-fold) at Ser169. We report interactions between CSNK-2β and IGFBP-1 as well as mTOR and CSNK-2β, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2.

Topics: Carcinoma, Hepatocellular; Casein Kinase II; Fluorescent Antibody Technique; Hep G2 Cells; Humans; Insulin-Like Growth Factor Binding Protein 1; Liver Neoplasms; Phosphorylation; Signal Transduction; TOR Serine-Threonine Kinases

Protein kinase CK2 alpha (CK2α), one isoform of the catalytic subunit of serine/threonine kinase CK2, has been indicated to participate in tumorigenesis of various malignancies. We conducted this study to investigate the biological significances of CK2α expression in hepatocellular carcinoma (HCC) development. Real-time quantitative polymerase and western blotting analyses revealed that CK2α expression was significantly increased at mRNA and protein levels in HCC tissues. Immunohistochemical analyses indicated that amplified expression of CK2α was highly correlated with poor prognosis. And functional analyses (cell proliferation and colony formation assays, cell migration and invasion assays, cell cycle and apoptosis assays) found that CK2α promoted cell proliferation, colony formation, migration and invasion, as well as inhibited apoptosis in hepatoma cell lines in vitro. CK2α-silenced resulted in significant apoptosis in cells that was demonstrated been associated with downregulation of expression of Bcl-2, p-AKT (ser473) and upregulation of expression of total P53, p-P53, Bax, caspase3 and cleaved-caspase3 in HCC cells. In addition, experiments with a mouse model revealed that the stimulative effect of CK2α on tumorigenesis in nude mice. Our results suggest that CK2α might play an oncogenic role in HCC, and therefore it could serve as a biomarker for prognostic and therapeutic applications in HCC.

Topics: Aged; Animals; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoma, Hepatocellular; Casein Kinase II; Cell Movement; Cell Proliferation; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Prognosis; Proportional Hazards Models; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Transfection

Histone deacetylase 2 (HDAC2) is aberrantly regulated and plays a pivotal role in the development of hepatocellular carcinoma (HCC) through regulation of cell-cycle components at the transcriptional level, but the underlying mechanism leading to oncogenic HDAC2 remains unknown. In this study, we show that expression of CK2α (casein kinase II α subunit) was up-regulated in a large cohort of human HCC patients, and that high expression of CK2α was significantly associated with poor prognosis of HCC patients in terms of five-year overall survival. It was also found that CK2α over-expression positively correlated with HDAC2 over-expression in a subset of HCCs. We observed that treatment with epidermal growth factor (EGF) elicited an increase in CK2α expression and Akt phosphorylation, causing induction of HDAC2 expression in liver cancer cells. It was also observed that ectopic expression of dominant-negative CK2α blocked EGF-induced HDAC2 expression, and that ectopic CK2α expression attenuated the suppressive effect of Akt knockdown on HDAC2 expression in liver cancer cells. Targeted disruption of CK2α influenced the cell cycle, causing a significant increase in the number of liver cancer cells remaining in G₂/M phase, and suppressed growth via repression of Cdc25c and cyclin B in liver cancer cells. Taken together, our findings suggest the oncogenic potential of CK2α in liver tumorigenesis. Furthermore, a regulatory mechanism for HDAC2 expression is proposed whereby EGF induces transcriptional activation of HDAC2 by CK2α/Akt activation in liver cancer cells. Therefore, this makes CK2α a promising target in cancer therapy.

Topics: Carcinogenesis; Carcinoma, Hepatocellular; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cohort Studies; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Humans; Liver; Liver Neoplasms; Mutant Proteins; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Recombinant Proteins; RNA Interference; Signal Transduction; Survival Analysis

Histone deacetylase (HDAC) inhibitors exhibit a unique ability to degrade topoisomerase (topo)IIα in hepatocellular carcinoma (HCC) cells, which contrasts with the effect of topoII-targeted drugs on topoIIβ degradation. This selective degradation might foster novel strategies for HCC treatment in light of the correlation of topoIIα overexpression with the aggressive tumor phenotype and chemoresistance. Here we report a novel pathway by which HDAC inhibitors mediate topoIIα proteolysis in HCC cells. Our data indicate that HDAC inhibitors transcriptionally activated casein kinase (CK)2α expression through increased association of acetylated histone H3 with the CK2α gene promoter. In turn, CK2 facilitated the binding of topoIIα to COP9 signalosome subunit (Csn)5 by way of topoIIα phosphorylation. Furthermore, we identified Fbw7, a Csn5-interacting F-box protein, as the E3 ligase that targeted topoIIα for degradation. Moreover, knockdown of CK2α, Csn5, or Fbw7 reversed HDAC inhibitor-induced topoIIα degradation. Mutational analysis indicates that the (1361) SPKLSNKE(1368) motif plays a crucial role in regulating topoIIα protein stability. This motif contains the consensus recognition sites for CK2 (SXXE), glycogen synthase kinase (GSK)3β (SXXXS), and Fbw7 (SPXXS). This study also reports the novel finding that topoIIα may be a target of GSK3β phosphorylation. Evidence suggests that CK2 serves as a priming kinase, through phosphorylation at Ser1365, for GSK3β-mediated phosphorylation at Ser1361. This double phosphorylation facilitated the recruitment of Fbw7 to the phospho-degron (1361) pSPKLpS(1365) of topoIIα, leading to its ubiquitin-dependent degradation.. This study shows a novel pathway by which HDAC inhibitors facilitate the selective degradation of topoIIα, which underlies the complexity of the functional role of HDAC in regulating tumorigenesis and aggressive phenotype in HCC cells.

Topics: Animals; Antigens, Neoplasm; Carcinoma, Hepatocellular; Casein Kinase II; Cell Cycle Proteins; Cell Line, Tumor; COP9 Signalosome Complex; DNA Topoisomerases, Type II; DNA-Binding Proteins; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Female; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Mice; Mice, Nude; Peptide Hydrolases; Phenylbutyrates; Phosphorylation; Ubiquitin-Protein Ligases

Hepatocellular carcinoma (HCC), a highly aggressive form of solid tumor, has been increasing in South East Asia. The lack of effective therapy necessitates the introduction of novel chemopreventive strategies to counter the substantial morbidity and mortality associated with the disease. Recently, we reported that dimethoxy flavone (DMF), a methylated flavone derived from chrysin, significantly suppressed the development of preneoplastic lesions induced by N-nitrosodiethylamine (DEN) in rats, although the mechanism of action was not known. In the present study, we have investigated the effects of DMF administration on gene expression changes related to the inflammation-mediated NF-kB pathway, Wnt pathway and apoptotic mediators in DEN-induced preneoplastic nodules. There was a significant increase in inflammatory markers like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and a decrease in apoptotic mediators like p53, caspase-3 and bax in DEN-treated rats when compared to the control group. Activation of NF-kB was noticed by an elevated expression of nuclear protein expression of NF-kB and cytoplasmic phospho-IkBαSer(32/36) in the same animals. Likewise, upregulation of canonical Wnt pathway was noticed by elevated expression of nuclear protein levels of phospho-β-cateninThr(393) and cytoplasmic casein kinase-2 (CK2), Dvl2 and cyclin D1 levels, along with a simultaneous decrease in expression of phospho-GSK3β(Ser9). Dietary DMF (100mg/kg) administration inhibited liver nodule incidence and multiplicity by 82% and 78%, respectively. DMF also reversed the activation of NF-kB and Wnt pathway as shown by the decrease in protein expression of several proteins. Results of the present investigation provide evidence that attenuation of Wnt pathway and suppression of inflammatory response mediated by NF-kB could be implicated, in part, in the chemopreventive effects of methylated flavone. Therefore, the present findings hold great promise for the utilization of DMF as an effective chemotherapeutic agent in treating early stages of liver cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Hepatocellular; Casein Kinase II; Caspase 3; Cyclin D1; Cyclooxygenase 2; Diethylnitrosamine; Disease Models, Animal; Flavones; Flavonoids; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Male; NF-kappa B; Nitric Oxide Synthase Type II; Precancerous Conditions; Rats; Rats, Wistar; Signal Transduction; Tumor Suppressor Protein p53; Up-Regulation; Wnt Proteins

The multi-kinase-inhibitor Sorafenib has been shown to prolong survival of patients suffering from hepatocellular carcinoma (HCC). We investigated effects of the serine/threonine kinase inhibitor 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) on experimental HCC growth, and identified mechanisms and target kinases of DMAT. Our results show that DMAT application in vivo reduced tumor growth in a xenotransplant model by interference with tumor cell proliferation. Biochemical parameters and histology following DMAT administration revealed no alterations in liver tissue. Similar to Sorafenib, DMAT interfered with NFκB activation and Wnt-signaling. Of the kinases inhibited by DMAT at almost equimolar IC50, CK2 and PIM-3 were found to be over-expressed or more active in hepatoma cells and human HCC tissue. Knockdown of PIM-3 or CK2 by shRNA revealed that both kinases are important for hepatoma cell proliferation and survival. In conclusion, DMAT reduces HCC growth by interference with NFκB- and Wnt-signaling. PIM-3 and CK2 seem to be important target kinases. Inhibition of these kinases by application of inhibitors, e.g., DMAT, might represent a promising therapeutic approach in future HCC therapy.

Topics: Animals; Antineoplastic Agents; Benzenesulfonates; Benzimidazoles; Carcinoma, Hepatocellular; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Gene Knockdown Techniques; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; NF-kappaB-Inducing Kinase; Niacinamide; Phenylurea Compounds; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Pyridines; Signal Transduction; Sorafenib; Wnt Proteins; Xenograft Model Antitumor Assays

SEPT2 plays an important role in cell division through its effect on cytoskeletons. It is a GTP-binding protein and can also form filament with SEPT6 and SEPT7. Knockdown of SEPT2, 6, and 7 causes stress fibers to disintegrate and then cells lose polarity and divide abnormally. Increasing evidence has shown that septins are related to the regulation of cell proliferation. In this study, the expression of SEPT2 was first identified to be up-regulated in human hepatoma carcinoma cells (HCC). In addition, SEPT2 was found to be phosphorylated on Ser218 by casein kinase 2 (CK2), which was also overexpressed in HCC. By overexpressing SEPT2 and its S218A mutant in SMMC7721 and L02 cell lines, we confirmed that the phosphorylation of SEPT2 on Ser218 by CK2 was crucial to the proliferation of HCC. These results suggest that SEPT2 might be a promising target for liver cancer therapy.

Topics: Base Sequence; Blotting, Northern; Carcinoma, Hepatocellular; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; DNA Primers; Humans; Liver Neoplasms; Mutagenesis, Site-Directed; Phosphoric Monoester Hydrolases; Phosphorylation; Polymerase Chain Reaction; Serine

Recent studies have shown that the inhibition of casein kinase 2 (CK2) sensitizes many cancer cells to Fas ligand- and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, it has not been demonstrated directly whether CK2 inhibition can also enhance the cytotoxicity of natural killer (NK) cells, which actually use the death ligands to kill cancer cells in vivo. To address whether NK cell-mediated cancer cell death is affected by the inhibition of CK2, we first checked whether the death ligand-induced apoptosis of hepatocellular carcinoma cells (HCCs) and HeLa were affected by CK2 inhibition. We then investigated the effect of CK2 inhibition on NK cytotoxicity against HCCs and HeLa cells and its mechanistic features. Inhibition of CK2 by emodin increased the apoptotic cell death of HepG2, Hep3B and HeLa when the cancer cell lines were treated with a soluble form of recombinant TRAIL or an agonistic antibody of Fas. This phenomenon appeared to be correlated with the expression level of death receptors on the cancer cell surface. More interestingly, the inhibition of CK2 also greatly increased the NK cell-mediated cancer cell killing. The NK cytotoxicity against the cancer cells increased about twofold when the target cells were pretreated with a specific CK2 inhibitor, emodin or 4,5,6,7-tetrabromobenzotriazole. Furthermore, the increase of the NK cytotoxicity against cancer cells by CK2 inhibition was granule-independent and mediated possibly by the death ligands on the NK cell surface. This suggests that CK2 inhibitors could be used to enhance the cytotoxicity of NK cells and consequently increase host tumour immunity.

Topics: Apoptosis; Carcinoma, Hepatocellular; Casein Kinase II; Cytoplasmic Granules; Cytotoxicity, Immunologic; Emodin; Fas Ligand Protein; Humans; Killer Cells, Natural; Liver Neoplasms; Protein Kinase Inhibitors; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

HIF-1 (hypoxia-inducible factor-1) is the main transcription factor involved in the adaptation of cells to hypoxia. In addition to regulation of HIF-1alpha protein level, HIF-1 activity is also enhanced by several pathways involving asparagine hydroxylation and phosphorylation. Here, we investigated the relationship between casein kinase 2 (CK2), p53 and HIF-1. An increase in p53 protein level and transcriptional activity was observed when CK2 was inhibited by different inhibitors under normoxia and hypoxia. This increase was in parallel with a decrease in HIF-1 activity without changes in HIF-1alpha protein level, indicating a regulation of its transcriptional activity. Similar results were obtained using CK2alpha siRNA. Ectopic overexpression of p53 also led to an inhibition of HIF-1 activity. Conversely, CK2 inhibition had no effect in p53-null cells indicating that the inhibitory effect of CK2 inhibitors requires the presence of p53. p53 activity was not required because overexpression of a p53 mutated in its DNA-binding domain exerted the same effect as wild-type p53 and because the effect of CK2 inhibitors was still observed when p53 activity was inhibited by pifithrin-alpha. Since CK2 activity is increased in hypoxic conditions, this process provides one more mechanism to ensure enhanced HIF-1 activity under such conditions.

Topics: Carcinoma, Hepatocellular; Casein Kinase II; Cell Hypoxia; Cells, Cultured; Enzyme Inhibitors; HeLa Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Transcription, Genetic; Tumor Suppressor Protein p53

Post-translational modification of MTF-1 (metal-response-element-binding transcription factor-1) was suggested to play a role in its metalloregulatory functions. In the present study, pulse labelling and two-dimensional electrophoresis-Western blotting were used to demonstrate that, although MTF-1 is highly modified in vivo, its phosphorylation level does not rapidly change in response to metals, nor does its overall modification pattern. Recombinant MTF-1 was found to serve as an in vitro substrate for casein kinase II, c-Jun N-terminal kinase and protein kinase C, but inhibition of these kinases in vivo did not significantly change the modification pattern of MTF-1. Northern blotting revealed that inhibitors of casein kinase II and c-Jun N-terminal kinase severely attenuate the metal-induced transcription of the native chromatin-packaged metallothionein-I and zinc transporter-1 genes, whereas protein kinase C inhibitors exerted gene- and cell-type-specific effects on the metal regulation and basal expression of these two genes. A chromatin immunoprecipitation assay was used to demonstrate that none of these inhibitors prevent the metal-dependent recruitment of MTF-1 to the MT-I promoter. In brief, results of the present study suggest that protein kinases may not alter the phosphorylation state of MTF-1 during the rapid-response phase to metals, nor do they regulate the metal-dependent formation of a stable MTF-1-chromatin complex. Instead, protein kinases may exert their interdependent effects on metal-induced gene expression by acting on cofactors that interact with MTF-1.

Topics: Animals; Cadmium; Carcinoma, Hepatocellular; Casein Kinase II; Cation Transport Proteins; Cell Line; Cell Line, Tumor; DNA-Binding Proteins; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Mice; Phosphates; Phosphorylation; Promoter Regions, Genetic; Protein Kinase C; Signal Transduction; Species Specificity; Substrate Specificity; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Zinc

Nuclear factor kappaB (NF-kappaB) is an antiapoptotic factor involved in development, regeneration, and neoplastic progression of the liver. Previously, we have shown that stabilization of inhibitor kappaB (IkappaB)-alpha protein following treatment of hepatocytes with transforming growth factor (TGF)-beta1 promoted NF-kappaB repression, which then permitted induction of AP-1/SMAD-mediated liver cell death. Because basal IkappaB-alpha protein turnover is regulated by protein kinase CK2, here we have elucidated the regulation of CK2 kinase activity and its role in control of NF-kappaB levels following treatment with TGF-beta1. We show that both messenger RNA (mRNA) and protein levels of the CK2alpha catalytic subunit are down-regulated following TGF-beta1 stimulation in murine hepatocyte cells. The ensuing inhibition of CK2 kinase activity promotes stabilization of IkappaB protein, which is followed by the shutoff of constitutive NF-kappaB activity and induction of apoptosis. Ectopic expression of CK2alpha inhibits TGF-beta1-induced apoptosis through sustained activation of NF-kappaB. Conversely, expression of a kinase-dead mutant of CK2alpha potentiates TGF-beta1 cell killing. Importantly, we show that hepatocellular carcinomas (HCCs) derived from TGF-beta1 transgenic mice and human HCC cell lines display enhanced CK2 IkappaB kinase activity that contributes in part to an elevated NF-kappaB activity in vivo. In conclusion, inhibition of CK2 expression levels by TGF-beta1 is crucial for the induction of apoptosis of hepatocytes. Circumvention of this process by up-regulation of CK2 activity in transformed cells may contribute to the promotion of TGF-beta1-induced liver carcinogenesis.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Casein Kinase II; Cells, Cultured; Down-Regulation; Hepatocytes; Humans; I-kappa B Proteins; Liver Neoplasms; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Serine-Threonine Kinases; Transforming Growth Factor beta; Transforming Growth Factor beta1

Protein kinase CK2 and phosphorylated ERK1/2 accumulated in nucleus after serum stimulation of quiescent HepG2 cells. Nonetheless, phospho-ERK1/2 accumulated mainly in the nuclease-extracted fraction (NE) whereas the increases in nuclear CK2 (either CK2alpha or CK2beta) occurred initially in the nuclease-resistant fraction (NR). Transient decreases in CK2 were observed in cytoplasm and NE in the first 3h but thereafter they either reverted (cytoplasm) or increased above the control (NE). CK2 levels in both NE and NR were high in cells arrested at G1/S. Maximal nuclear accumulation of CK2 was blocked by cycloheximide but little affected by PD98059, SB203580 or apigenin, all of which affected nuclear phopho-ERK1/2. Thus, nuclear accumulation of CK2 during G1 phase is independent of ERK1/2 pathway. Although this process may initially relay on intracellular redistribution of the preexisting enzyme, active protein synthesis is required to attain maximal nuclear CK2 levels.

Topics: Active Transport, Cell Nucleus; Carcinoma, Hepatocellular; Casein Kinase II; Cell Cycle; Cell Nucleus; Cycloheximide; G1 Phase; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; Tumor Cells, Cultured

A previously isolated endocytic trafficking mutant (TRF1) isolated from HuH-7 cells is defective in the distribution of subpopulations of cell-surface receptors for asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin, and mannose-terminating glycoproteins. The pleiotropic phenotype of TRF1 also includes an increased sensitivity to Pseudomonas toxin and deficient assembly and function of gap junctions. HuH-7xTRF1 hybrids exhibited a normal subcellular distribution of ASGR, consistent with the TRF1 mutation being recessive. A cDNA expression library derived from HuH-7 mRNA was transfected into TRF1 cells, which were subsequently selected for resistance to Pseudomonas toxin. Sequence analysis of a recovered cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2alpha". Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2alpha expression. Consistent with this finding, the hybrids HuH-7xHuH-7 and HuH-7xTRF1 expressed equivalent amounts of total CK2alpha. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2alpha and CK2alpha' and the novel CK2alpha" isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2alpha and CK2alpha', the mutant did not express CK2alpha". Based on the genomic DNA sequence, RNA transcripts encoding CK2alpha" apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2alpha or the newly cloned CK2alpha" restored the parental HuH-7 phenotype, including Pseudomonas toxin resistance, cell-surface ASGR binding activity, phosphorylation, and the assembly of gap junctions. This study suggests that HuH-7 cells express at least three CK2alpha isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.

Topics: Amino Acid Sequence; Bacterial Toxins; Carcinoma, Hepatocellular; Casein Kinase II; Cloning, Molecular; DNA, Complementary; Gap Junctions; Genetic Complementation Test; Humans; Hybrid Cells; Liver Neoplasms; Membrane Proteins; Molecular Sequence Data; Protein Serine-Threonine Kinases; Protein Transport; Pseudomonas; Sequence Homology, Amino Acid; Tumor Cells, Cultured

Hepatitis delta virus (HDV) contains two virus-specific delta antigens (HDAgs), large and small forms, which are identical in sequence except that the large one contains 19 extra amino acids at the C terminus. HDAgs are nuclear phosphoproteins with distinct biological functions; the small form activates HDV RNA replication, whereas the large form suppresses this process but is required for viral particle assembly. In this study, we have characterized the phosphorylative property of HDAg in a human hepatoma cell line (HuH-7) and examined the role of phosphorylation in HDAg function. As demonstrated by in vivo labeling and kinase inhibitor experiments, the phosphorylation levels of both HDAgs were diminished by the inhibitor of casein kinase II (CKII). Nevertheless, phosphorylation of only the small form could be markedly reduced by the protein kinase C (PKC) inhibitor, suggesting different phosphorylation properties between the two HDAgs. When these two kinase inhibitors were added separately to the transient-expression system, HDV RNA replication was profoundly suppressed. In contrast, the inhibitors did not affect the assembly of empty HDAg particle from HDAgs and hepatitis B virus surface antigen. To further examine the role of phosphorylation in HDAg function, two conservative CKII recognition sites at Ser-2 and Ser-123 of both HDAgs and one potential PKC recognition site at Ser-210 of the large HDAg were altered to alanine by site-directed mutagenesis. Transfection experiments indicated that mutation at Ser-2, but not Ser-123, significantly impaired the activity of the small HDAg in assisting HDV RNA replication. This property is in accordance with our observation that Ser-2, not Ser-123, was the predominant CKII phosphorylation site in the small HDAg. Our studies also excluded the possibility that the phosphorylation of Ser-2, Ser-123, or Ser-210, had roles in the trans-suppression activity of the large HDAg, in the assembly of empty virus-like HDAg particle, and in the nuclear transport of HDAgs. In conclusion, our results indicate that both CKII and PKC positively modulate HDV RNA replication but not the assembly of empty HDAg particle. The role of CKII in HDV replication may at least in part be accounted for by the phosphorylation of Ser-2 in the small HDAg. The effect of PKC on HDV RNA replication is, however, not to mediate the phosphorylation of the conservative Ser-210 in the large HDAg but rather to act on as-yet-unidentified Ser or Thr resid

Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Carcinoma, Hepatocellular; Casein Kinase II; Cell Line; Genes, Viral; Genetic Variation; Genotype; Hepatitis Antigens; Hepatitis B Antigens; Hepatitis B virus; Hepatitis delta Antigens; Hepatitis Delta Virus; Humans; Liver Neoplasms; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Proteins; Oligodeoxyribonucleotides; Phosphoproteins; Point Mutation; Protein Kinase C; Protein Serine-Threonine Kinases; Recombinant Proteins; RNA, Viral; Sequence Homology, Amino Acid; Transfection; Tumor Cells, Cultured; Virus Replication

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M(r) 150,000 (peak I kinase) and M(r) 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC50 = 2.5 and 16.5 micrograms/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC50 = 50 microM and 100 microM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-32P]ATP lowered the incorporation of 32P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-32P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases.

Topics: Amino Acid Sequence; Carcinoma, Hepatocellular; Casein Kinase II; Casein Kinases; Enzyme Inhibitors; Guanosine Triphosphate; Heparin; Humans; Insulin-Like Growth Factor Binding Protein 1; Isoquinolines; Liver Neoplasms; Molecular Sequence Data; Molecular Weight; Peptide Mapping; Phosphorylation; Phosphoserine; Protein Kinases; Protein Serine-Threonine Kinases; Tumor Cells, Cultured