casein-kinase-ii and Colonic-Neoplasms

Topics: Apoptosis; Butyric Acid; Casein Kinase II; Cell Division; Colonic Neoplasms; Dietary Fiber; Humans; Protein Serine-Threonine Kinases

As highly expressed in several human cancers, Casein Kinase 2 (CK2) is involved in chemotherapy-induced resistance. As a new potent CK2 inhibitor, DN701 is used to overcome chemoresistance through its synergistic antitumor effect with 5-fluorouracil (5-FU). Translesion DNA synthesis (TLS) has drawn our attention because it is associated with the development of chemo-resistance and tumor recurrence. The in vitro biological properties of 5-FU-resistant colon cancer cells revealed that DN701 combined with 5-FU could overcome chemo-resistance via blocking CK2-mediated aryl hydrocarbon receptor (AhR) and TLS-induced DNA damage repair (DDR). Moreover, pharmacologic and genetic inhibitions of AhR potently reduced TLS-promoted genomic instability. The mechanistic studies showed that combined DN701 with 5-FU was investigated to inhibit CK2 expression level and AhR-TLS-REV1 pathway. Meanwhile, DN701 combined with 5-FU could reduce CK2-AhR-TLS genomic instability, thus leading to superior in vivo antitumor effect. The insights provide a rationale for combining DN701 with 5-FU as a therapeutic strategy for patients with colon cancer.

Topics: Casein Kinase II; Cell Line, Tumor; Colonic Neoplasms; DNA; Drug Resistance, Neoplasm; Fluorouracil; Humans; Receptors, Aryl Hydrocarbon

MicroRNA (miRNA) exerts an important part in colon cancer cell proliferation and apoptosis. Meanwhile, the dysregulation of some miRNAs is detected in colon cancer cells. However, it remains unclear about the underlying mechanism of their effects on tumor pathogenesis. The current work aimed to examine the miR-1184 effect on colon cancer cells. The differentially expressed miRNAs (DEMs), including miR-9-3p, miR-1184, miR-492, miR-92a-1-5p and miR-20a-3p, were obtained from the GSE115108 and GSE132619 data sets using the 'GEO2R' online tool. Based on the findings, miR-1184 was significantly down-regulated within colon cancer cells and tissues. Moreover, the experimental results of CCK8, flow cytometry, colony formation and Western blotting assays showed that, miR-1184 over-expression suppressed colon cancer cell proliferation through inhibiting Ki67 expression and promoted their apoptosis through up-regulating cleaved caspase-3 and down-regulating Bcl-2 expression. By contrast, miR-1184 inhibition exerted the opposite effects. A total of 110 target genes of miR-1184 were predicted using the TargetScan and miRTarBase databases, which were then used to construct the protein-protein interaction (PPI) network based on the DAVID and STRING websites and to perform GO and KEGG pathway enrichment analyses. The MCODE plug-in of cytoscape was utilized to verify that CSNK2A1 was the target gene and key gene in significant modules. MiR-1184 directly targets CSNK2A1 via using RNA immunoprecipitation assay and luciferase reporter gene assay. According to the results, CSNK2A1 over-expression reversed the functions of miR-1184 over-expression in suppressing colon cancer cell proliferation and enhancing their apoptosis. In conclusion, over-expression of miR-1184 inhibits colon cancer cell proliferation but promotes their apoptosis through down-regulating CSNK2A1 expression.

Topics: Apoptosis; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Databases, Genetic; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HCT116 Cells; HT29 Cells; Humans; Male; MicroRNAs

Protein kinase CK2 is active in cancer cells. Previously, we reported that increased CK2 activity could induce epithelial mesenchymal transition of cancer cells. CK2 also induced epithelial mesenchymal transition in colon cancer cell lines such as HT29 and SW620, and the transitioned cells (CK2α cells) became more proliferative than the controls. We assumed that CK2 could affect cancer cell growth by modulating their energy metabolism. Here, we examined the molecular effects of CK2 on the glucose metabolism of cancer cells. We found that CK2α cells consumed more glucose and produced more lactate than control cells did. An XF glycolysis stress test showed that aerobic glycolysis was augmented up to the cancer cell's maximal glycolytic capacity in CK2α cells. Molecular analysis revealed that pyruvate kinase M1 was downregulated and pyruvate kinase M2 was nuclear localized in CK2α cells. Consequently, the expression and activity of lactate dehydrogenase A (LDHA) were upregulated. Treatment with FX11-a specific LDHA inhibitor-or clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knockout of LDHA inhibited the CK2-driven proliferation of cancer cells. We conclude that CK2 augments the Warburg effect, resulting in increased proliferation of cancer cells.

Topics: Casein Kinase II; Cell Proliferation; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Glucose; Glycolysis; HEK293 Cells; HT29 Cells; Humans; Isoenzymes; Lactate Dehydrogenase 5; Lactic Acid; Naphthalenes; Pyruvate Kinase; Transcriptional Activation

Serine/threonine kinase B-Raf proto-oncogene (BRAF) mutant colon cancer has a poor prognosis and there is an absence of targeted treatments for this subtype. Here, we investigated the effects of inhibition of casein kinase 2 (CK2) on the inhibitory effects of BRAF and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibition in BRAF-mutant colon cancer cells.. Colon cancer cell lines with mutations in components of the mitogen-activated protein kinase (MAPK) and PI3K signalling pathway were used. Cell viability was determined after exposure to single agent and combinations of erlotinib (EGFR inhibitor), dabrafenib (MEK inhibitor), GDC0941 (PI3K inhibitor) and CX4945 (CK2 inhibitor). Western blots were used to examine MAPK and AKT serine/threonine kinase (AKT) pathway activation.. Addition of CX4945 to dabrafenib did not enhance the antiproliferative effects of single-agent dabrafenib. Use of GDC0941 alone was highly effective in controlling growth of both BRAF-mutant and wild-type cells and this effect was enhanced by CK2 inhibition.. Inhibition of the PI3K/AKT pathway is central to regulating growth of colon cancer cells and this can be enhanced by CK2 inhibition.

Topics: Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Drug Synergism; Erlotinib Hydrochloride; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Imidazoles; Indazoles; MAP Kinase Signaling System; Mutation; Naphthyridines; Oximes; Phenazines; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins B-raf; Sulfonamides

We previously showed that protein kinase CK2 downregulation mediates senescence through the reactive oxygen species (ROS)-p53-p21(Cip1/WAF1) pathway in various human cells. In the present study, we investigated whether the FoxO3a transcription factor is associated with ROS production during CK2 downregulation-induced senescence in human colon cancer HCT116 and breast cancer MCF-7 cells. FoxO3a overexpression suppressed ROS production and p53 stabilization induced by a CK2α knockdown. CK2α downregulation induced nuclear export of FoxO3a through stimulation of AKT-mediated phosphorylation of FoxO3a and decreased transcription of its target genes (Cu/ZnSOD, MnSOD, and catalase). In contrast, CK2α overexpression inhibited AKT-mediated FoxO3a phosphorylation. This resulted in nuclear accumulation of FoxO3a, and elevated expression of its target genes. Therefore, these data indicate for the first time that CK2 downregulation stimulates ROS generation by inhibiting FoxO3a during premature senescence in human colon and breast cancer cells.

Topics: Active Transport, Cell Nucleus; Breast; Breast Neoplasms; Casein Kinase II; Cellular Senescence; Colon; Colonic Neoplasms; Down-Regulation; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; MCF-7 Cells; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Up-Regulation

The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.

Topics: Actins; alpha Catenin; Amino Acid Sequence; Animals; Apoptosis; Blotting, Western; Cadherins; Casein Kinase I; Casein Kinase II; Cell Adhesion; Cell Membrane; Cell Movement; Cell Proliferation; Cells, Cultured; Colonic Neoplasms; Dogs; Drosophila melanogaster; Female; Fluorescent Antibody Technique; Humans; Immunoprecipitation; Madin Darby Canine Kidney Cells; Molecular Sequence Data; Ovary; Phosphorylation; Protein Structure, Tertiary; Sequence Homology, Amino Acid

Endothelin-converting enzyme-1c (ECE-1c) is a membrane metalloprotease involved in endothelin-1 synthesis, which has been shown in vitro to have a role in breast, ovary and prostate cancer cell invasion. N-terminal end of ECE-1c displays three putative phosphorylation sites for the protein kinase CK2. We studied whether CK2 phosphorylates N-terminal end of ECE-1c as well as whether this has a role in migration and invasion of colon cancer cells. CK2 phosphorylated the N-terminal end of ECE-1c and this was precluded upon inhibition of CK2. Inhibition also led to diminished protein levels of both endogen ECE-1 or GFP-fused N-terminal end of ECE-1c in 293T embryonic and DLD-1 colon cancer cells, which highlighted the importance of this motif on UPS-dependent ECE-1c degradation. Full-length ECE-1c mutants designed either to mimic or abrogate CK2-phosphorylation displayed increased or decreased migration/invasion of colon cancer cells, respectively. Moreover, ECE-1c overexpression or its silencing with a siRNA led to increased or diminished cell migration/invasion, respectively. Altogether, these data show that CK2-increased ECE-1c protein stability is related to augmented migration and invasion of colon cancer cells, shedding light on a novel mechanism by which CK2 may promote malignant progression of this disease.

Topics: Aspartic Acid Endopeptidases; Blotting, Western; Casein Kinase II; Cell Line, Tumor; Chromatin Immunoprecipitation; Colonic Neoplasms; Endothelin-Converting Enzymes; Humans; Metalloendopeptidases; Microscopy, Confocal; Neoplasm Invasiveness; Protein Stability; RNA, Small Interfering; Transfection

Cellular senescence is an important tumor suppression process in vivo. Tamoxifen is a well-known anti-breast cancer drug; however, its molecular function is poorly understood. Here, we examined whether tamoxifen promotes senescence in breast cancer and colon cancer cells for the first time.. Human breast cancer MCF-7, T47D, and MDA-MB-435 and colorectal cancer HCT116 cells were treated with tamoxifen. Cellular senescence was measured by SA-β-gal staining and based on the protein expression of p53 and p21(Cip1/WAF1). The production of reactive oxygen species (ROS) was determined by staining with CM-H2DCFDA and dihydroethidium (DHE). CK2 activity was assessed with a specific peptide substrate.. Tamoxifen promoted senescence phenotype and ROS generation in MCF-7 and HCT116 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), and the NADPH oxidase inhibitor, apocynin, almost completely abolished this event. Tamoxifen inhibited the catalytic activity of CK2. Overexpression of CK2α antagonized senescence mediated by tamoxifen, indicating that tamoxifen induced senescence via a CK2-dependent pathway. A well-known CK2 inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), also stimulated ROS production and senescence in MCF-7 cells. Finally, experiments using T47D (wild-type p53) and MDA-MB-435 (mutant p53) cell lines suggested that tamoxifen induces p53-independent ROS production as well as p53-dependent senescence in breast cancer cells.. These results demonstrate that tamoxifen promotes senescence through a ROS-p53-p21(Cip1/WAF1) dependent pathway by inhibiting CK2 activity in breast cancer and colon cancer cells.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cellular Senescence; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Female; HCT116 Cells; Humans; Reactive Oxygen Species; Tamoxifen; Tumor Suppressor Protein p53

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC50 of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent Ki value of 7.67 μM. Coumestrol at 50μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21(Cip1/WAF1) pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22(phox) siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.

Topics: Breast Neoplasms; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Colonic Neoplasms; Coumestrol; Down-Regulation; Female; Glycine max; HCT116 Cells; Humans; Kinetics; Male; Plant Extracts; Reactive Oxygen Species

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.

Topics: Casein Kinase II; Cell Line, Tumor; Colonic Neoplasms; Dual Specificity Phosphatase 1; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Models, Biological; Phosphorylation; RNA Stability; RNA, Small Interfering; Transforming Growth Factor beta1; Tristetraprolin; Vascular Endothelial Growth Factor A

We have shown that protein kinase CKII (CKII) inhibition induces senescence through the p53-dependent pathway in HCT116 cells. Here we examined the molecular mechanism through which CKII inhibition activates p53 in HCT116 cells. CKII inhibition by treatment with CKII inhibitor or CKIIalpha small-interfering RNA (siRNA) increased intracellular hydrogen peroxide and superoxide anion levels. These effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine. Additionally, NADPH oxidase (NOX) inhibitor apocynin and p22(phox) siRNA significantly reduced p53 expression and suppressed the appearance of senescence markers. CKII inhibition did not affect mitochondrial superoxide generation. These data demonstrate that CKII inhibition induces superoxide anion generation via NOX activation, and subsequent superoxide-dependent activation of p53 acts as a mediator of senescence in HCT116 cells after down-regulation of CKII.

Topics: Acetophenones; Acetylcysteine; Aging; Antioxidants; Casein Kinase II; Cellular Senescence; Colonic Neoplasms; Down-Regulation; Genes, p53; HCT116 Cells; Humans; Hydrogen Peroxide; Mitochondria; NADPH Oxidases; Reactive Oxygen Species; RNA, Small Interfering; Superoxides

We have previously shown that the down-regulation of protein kinase CKII activity is tightly associated with cellular senescence of human fibroblast IMR-90 cells. Here, we examined the roles of p53 and p21(Cip1/WAF1) in senescence development induced by CKII inhibition using wild-type, isogenic p53-/- and isogenic p21-/- HCT116 human colon cancer cell lines. A senescent marker appeared after staining for senescence-associated beta-galactosidase activity in wild-type HCT116 cells treated with CKII inhibitor or CKIIalpha siRNA, but this response was almost abolished in p53- or p21(Cip1/WAF1)-null cells. Increased cellular levels of p53 and p21(Cip1/WAF1) protein occurred with the inhibition of CKII. CKII inhibition upregulated p53 and p21(Cip1/WAF1) expression at post-transcriptional level and transcription level, respectively. RB phosphorylation significantly decreased in cells treated with CKII inhibitor. Taken together, this study shows that the activation of the p53-p21(Cip1/WAF1) pathway acts as a major mediator of cellular senescence induced by CKII inhibition.

Topics: Casein Kinase II; Cellular Senescence; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Phosphorylation; Retinoblastoma Protein; Tumor Suppressor Protein p53; Up-Regulation

A series of macrocyclic derivatives has been designed and synthesized based on the X-ray co-crystal structures of pyrazolo[1,5-a] [1,3,5]triazines with corn CK2 (cCK2) protein. Bioassays demonstrated that these macrocyclic pyrazolo[1,5-a] [1,3,5]triazine compounds are potent CK2 inhibitors with K(i) around 1.0 nM and strongly inhibit cancer cell growth with IC(50) as low as approximately 100 nM.

Topics: Animals; Antineoplastic Agents; Casein Kinase II; Cell Line, Tumor; Colonic Neoplasms; Crystallography, X-Ray; Drug Design; Humans; Male; Molecular Conformation; Prostatic Neoplasms; Protein Kinase Inhibitors; Structure-Activity Relationship; Triazines

Protein kinase casein kinase II (CK2) is increased in response to diverse growth stimuli, as well as being elevated in many human cancers examined. We have demonstrated that CK2 is a key survival factor that protects human colon carcinoma cells from TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. We determined that inhibition of CK2 phosphorylation events by DRB (5,6-dichlorobenzimidazole) resulted in dramatic sensitization of tumor cells to TRAIL-induced apoptosis, in the absence of effects in normal cells. Sensitization was caspase dependent, and independent of regulation via NF-kappaB. Further, inhibition of phosphorylation by CK2 did not modify the expression level of antiapoptotic proteins. Analysis of TRAIL-induced death-inducing signaling complex (DISC) formation demonstrated enhanced formation of the DISC, enhanced cleavage of caspase-8 and cleavage of Bid in the presence of DRB, thereby facilitating the release of proapoptotic factors from the mitochondria with subsequent downregulation of the expression of XIAP and c-IAP1. Further, silencing of CK2alpha in HT29 cells following transfection of CK2alpha shRNA abrogated CK2 kinase activity while simultaneously increasing TRAIL sensitivity. These findings demonstrate that CK2 plays a critical antiapoptotic role by conferring resistance to TRAIL at the level of the DISC.

Topics: Adenocarcinoma; Apigenin; Apoptosis; Apoptosis Regulatory Proteins; Casein Kinase II; Cell Death; Cell Line, Tumor; Colonic Neoplasms; Death Domain Receptor Signaling Adaptor Proteins; Enzyme Inhibitors; Genetic Vectors; Humans; Indoles; Kinetics; Maleimides; Membrane Glycoproteins; Receptors, Tumor Necrosis Factor; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Necrosis Factor-alpha

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine involved in the expression of many genes integral to the inflammatory response. In addition, it activates both apoptotic and survival pathways, the latter being mediated through the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Protein kinase CK2, a serine-threonine kinase that is universally upregulated in human malignancies, may be involved at multiple levels in this process. However, its role in mediating a survival response within colon cancer cells remains incompletely understood. Here we report that inhibition of CK2 in HCT-116 and HT-29 cells with the use of two specific CK2 inhibitors, 5,6-dichloro-ribifuranosylbenzimidazole (DRB) and apigenin, effected a synergistic reduction in cell survival when used in conjunction with TNF-alpha. Furthermore, there was a demonstrable synergistic reduction in colony formation in soft agar with the use of the same combinations. Western blot analysis showed that poly-ADP ribose polymerase and procaspase-3 cleavage complemented the fluorescence-activated cell sorter analysis findings of significantly increased subdiploid DNA-containing cell populations using these conditions. Remarkably, these events occurred in the absence of any reduction in the expression of the Bcl-2 family members Bcl-2, Mcl-1, and Bcl-xL or any change in the proapoptotic molecules Bad or Bax. One-hybrid NF-kappaB promoter assays utilizing a Gal4-p65 transactivation domain construct revealed that the TNF-induced transactivation was inhibited by both DRB and apigenin. This was associated with a concomitant reduction in the expression of a recognized anti-apoptotic NF-kappaB target, manganese superoxide dismutase, demonstrated by Q-PCR. Our findings indicate a potentially novel strategy for the treatment of colon cancer, one that targets CK2 simultaneous with TNF-alpha administration.

Topics: Antineoplastic Agents; Apigenin; Apoptosis; Casein Kinase II; Cell Division; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Dichlororibofuranosylbenzimidazole; Diploidy; Drug Synergism; Enzyme Inhibitors; Flavonoids; HCT116 Cells; Humans; I-kappa B Kinase; Mitogen-Activated Protein Kinases; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Superoxide Dismutase; Transcriptional Activation; Tumor Necrosis Factor-alpha

Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; Casein Kinase II; CASP8 and FADD-Like Apoptosis Regulating Protein; Colonic Neoplasms; Drug Synergism; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; NF-kappa B; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

p53 undergoes phosphorylation on several residues in response to cellular stresses that include UV and ionizing radiation, however the influence of spindle damage on this parameter is relatively unclear. Consequently, the effect of nocodazole on serine 392 phosphorylation was examined in two epithelial cell lines. We show that this process is dependent upon the stepwise activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase casein kinase 2 (CK2). Furthermore, this activation correlated with the biochemical regulation of the maturation-promoting factor (MPF, cdc2/cyclin B), as both DRB and antisense depletion of CK2, as well as SB203580 were associated with an inhibition of its activation in response to nocodazole. Strikingly, when the cell cycle characteristics of nocodazole treated cells were examined, we observed that depletion or inhibition of the catalytic subunit of CK2, in the presence of microtubule inhibitors, resulted in a compromise of the G2 arrest (spindle checkpoint). Furthermore, CK2-depleted, nocodazole treated cells demonstrated a dramatic reduction in the apoptotic cell fraction, confirming that these cells had been endowed with oncogenic properties. These changes were observed in both HeLa cells and HCT116 cells. We also show that this effect is dependent on the presence of functional wild-type p53, as this phenomenon is not apparent in HCT116 p53(-/-) cells. Collectively, our results indicate two novel roles for CK2 in the spindle checkpoint arrest, in concert with p53. Firstly, to maintain increased cyclinB/cdc2 kinase activity, as a component of G2 arrest, and secondly, a role in p53-mediated apoptosis. These findings may have implications for an improved understanding of abnormalities of the spindle checkpoint in human cancers, which is a prerequisite for defining future therapies.

Topics: Apoptosis; Casein Kinase II; Cell Line; Colonic Neoplasms; Cyclin B; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; G2 Phase; Genes, cdc; Genes, p53; HeLa Cells; Humans; Imidazoles; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Nocodazole; Oligodeoxyribonucleotides, Antisense; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Pyridines; Recombinant Fusion Proteins; Spindle Apparatus; Stress, Physiological; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells.

Topics: Alanine; Casein Kinase II; Colonic Neoplasms; Enzyme Inhibitors; Flavonoids; GTP Phosphohydrolases; Humans; Immunoblotting; Indoles; Kinetics; Maleimides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Phosphoproteins; Phosphorylation; Precipitin Tests; Protein Kinase C; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RGS Proteins; Serine; Signal Transduction; Transfection; Tumor Cells, Cultured

Butyrate, a dietary fiber derivative, is a well-known differentiating agent in cultured cell lines. In addition, its antineoplastic activity toward colon-rectum cancers has been documented both in vivo and in vitro. Despite the large amount of information on the potential clinical efficacy of butyrate, its mechanism of action at the molecular level has only been partially investigated. Here, we show that serine/threonine protein kinase CKII is a target of butyrate activity. In the human adenocarcinoma cell line, HT29, treated with 2 mM sodium butyrate, CKII activity decreases 50% at 24 and 48 hours after drug addition. The enzyme down-regulation is not due to changes in protein amount since the levels of the different CKII subunits remain constant during butyrate treatment. The data reported provide the first evidence that CKII down-regulation is involved in the signal transduction pathway started by butyrate.

Topics: Adenocarcinoma; Amino Acid Sequence; Butyrates; Butyric Acid; Casein Kinase II; Cell Differentiation; Colonic Neoplasms; Down-Regulation; Enzyme Inhibitors; Humans; Oligopeptides; Protein Serine-Threonine Kinases; Signal Transduction; Substrate Specificity; Tumor Cells, Cultured