Journal: Oncogene

Article Title: The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis.

doi: 10.1038/onc.2008.146

Figure Lengend Snippet: Figure 4 Reduced level of CK2b stabilizes Wee1 kinase. H1299 cells were transfected with the indicated siRNAs for 72 h. Total cell lysates were analysed by western blot using the indicated antibodies. The densitometric analysis of Wee1 and phospho- CDK1 protein bands is expressed in percentage assigning a value of 100% to the Wee1 and phospho-CDK1 protein bands in lane 1.

Article Snippet: Proteins were detected by probing western blot membranes with the following antibodies: mouse monoclonal anti-CK2a, mouse monoclonal anti-CK2b (both from Calbiochem); rabbit polyclonal antiCK2a0 obtained by immunizing rabbits with specific peptide sequence of human CK2a0, SQPCADNAVLSSGTAAR; mouse monoclonal anti-CDC25A, rabbit polyclonal antiCDC25C, mouse monoclonal anti-Chk1, mouse monoclonal anti-CDK1, mouse monoclonal anti-Wee1, rabbit polyclonal anti-cyclin E, rabbit polyclonal anti-cyclin A, mouse monoclonal anti-PLK1 antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-phospho-CDK1 (Tyr15), rabbit monoclonal anti-PLK1 (both from Cell Signaling Technology); mouse monoclonal anti-b-actin (Sigma).

Techniques: Transfection, Western Blot

Journal: Oncogene

Article Title: The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis.

doi: 10.1038/onc.2008.146

Figure Lengend Snippet: Figure 8 Model of the proposed influence of CK2b on G2/M transition. Active cyclin B-CDK1 kinase promotes G2/M transition. The activity of CDK1 is negatively controlled by Wee1-dependent phosphorylation and positively regulated by CDC25A-dependent dephosphorylation. The stability of Wee1 is controlled by a series of phosphorylations, including phosphorylation by CK2, which creates the first phosphodegron (pSer121), responsible for Wee1 instability during interphase (Watanabe et al., 2004, 2005). At the onset of M phase, stabilized PLK1 binds to CDK-mediated phosphorylated Wee1. Subsequently, PLK1 phosphorylates Wee1 at Ser53 (that is, pSer53 creates the second phosphodegron) leading to ubiquitination and proteasome-dependent degradation of Wee1. The CDC25A phosphatase is also a target of CDK1 albeit the CDK1-dependent phosphorylation leads to CDC25A stabilization. Upon DNA damage, CDC25A is subjected to multiple phosphorylations from Chk1 and other protein kinases (‘PK’), including Chk2 and p38 MAPK, leading to destabilization of the phosphatase. Disruption of CK2b leads to stabilization of both Wee1 and CDC25A. At present, it is unclear exactly how CK2b regulates CDC25A stability but CK2b presumably enhances CDC25A phosphorylation via Chk1 and/or other protein kinases. Lack of CK2b, however, leads to partial inhibition of CDK1 and this is explained by the requirement of CK2b for PLK1–Wee1 interaction. Without CK2b the CK2- and PLK1-dependent Wee1 degradation is compromised, resulting in stabilization of Wee1 and increased phosphorylation of CDK1.

Article Snippet: Proteins were detected by probing western blot membranes with the following antibodies: mouse monoclonal anti-CK2a, mouse monoclonal anti-CK2b (both from Calbiochem); rabbit polyclonal antiCK2a0 obtained by immunizing rabbits with specific peptide sequence of human CK2a0, SQPCADNAVLSSGTAAR; mouse monoclonal anti-CDC25A, rabbit polyclonal antiCDC25C, mouse monoclonal anti-Chk1, mouse monoclonal anti-CDK1, mouse monoclonal anti-Wee1, rabbit polyclonal anti-cyclin E, rabbit polyclonal anti-cyclin A, mouse monoclonal anti-PLK1 antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-phospho-CDK1 (Tyr15), rabbit monoclonal anti-PLK1 (both from Cell Signaling Technology); mouse monoclonal anti-b-actin (Sigma).

Techniques: Activity Assay, Phospho-proteomics, De-Phosphorylation Assay, Ubiquitin Proteomics, Disruption, Inhibition

Journal: Molecular Cancer Therapeutics

Article Title: Mechanism and Efficacy of Sub–50-nm Tenfibgen Nanocapsules for Cancer Cell–Directed Delivery of Anti-CK2 RNAi to Primary and Metastatic Squamous Cell Carcinoma

doi: 10.1158/1535-7163.mct-14-0166

Figure Lengend Snippet: Figure 1. s50-TBG nanoencapsulated RNAi targets both a and a0 subunits of CK2 in HNCC lines in vitro. A, sequence and chemical modifications of the OGN. DNA residues are uppercase and italic indicating phosphorothioate linkages; RNA residues are lowercase with italics indicating 20-O-methyl–modified RNA residues. The single-base mismatch between the OGN used and the CK2 a0 subunit mRNA is shown in gray. The 30 overhanging bases of the siRNA and the 30 propyl modifications of the single-stranded OGN are indicated by TT and Pr, respectively. B, immunoblot showing the effect on CK2a and a0 subunits 72 hours after addition of 15 mmol/L s50-TBG nanocapsules containing RNAi-CK2 with 6 RNA residues (RNAi-CK2), siCK2, RNAi-CK2 with 12 RNA residues (RNAi-CK2-12R), sugar (s50 control), phosphorothioate antisense (AS-CK2), or siRNA targeting red fluorescent protein (siRFP). CK2 purified from rat liver was used as a positive control. Lactate dehydrogenase (LDH) was used as a loading control. C, growth inhibition of human HNSCC lines was determined by [3H]-thymidine incorporation 48 hours after treatment with 20 mmol/L s50-TBG-RNAi-CK2 versus s50-TBG-sugar. Mean SE is shown. , P < 0.002. D, mRNA levels of CK2a and CK2a0 were determined by SYBR green qRT-PCR 24 hours after treatment with 20 mmol/L s50-TBG-RNAi-CK2 (black bars) or the CK2 sense-RNAi control (white bars). Levels were normalized using GAPDH transcript and are expressed relative to 20 mmol/L s50-TBG-sugar–treated cells. Mean SE is shown. , P < 0.05.

Article Snippet: Western analysiswas performed onwhole cell, nuclear, and cytoplasmic lysates as described (20) using the following antibodies: CK2a, CK2a0 (Bethyl Laboratories; A300-197A, A300-199A), caveolin-1, SP-3 (Santa Cruz Biotechnology; sc-894, sc-644), Ago2 (Abnova; 00027161), LDH (Cell Signaling; 2012), Keratin-14 (Covance; PRB155P), and FITC (Biodesign; K9006G). qRT-PCR analysis For SYBR green qRT-PCR, cDNAwas transcribed from 0.5 mg of total RNA using Superscript III (Invitrogen).

Techniques: In Vitro, Sequencing, Western Blot, Control, Positive Control, Inhibition, SYBR Green Assay, Quantitative RT-PCR

Journal: Molecular Cancer Therapeutics

Article Title: Mechanism and Efficacy of Sub–50-nm Tenfibgen Nanocapsules for Cancer Cell–Directed Delivery of Anti-CK2 RNAi to Primary and Metastatic Squamous Cell Carcinoma

doi: 10.1158/1535-7163.mct-14-0166

Figure Lengend Snippet: Figure 4. Biodistribution and acute efficacy of s50-TBG nanocapsules in FaDu tumor xenograft mice. A, biodistribution was determined in vivo 2 hours after i.v. administration of s50-TBG nanocapsules containing I127-siRNA to nude mice bearing FaDu tumors (n ¼ 3) by neutron activation analysis of the tissues. Levels of endogenous tissue iodine were measured in FaDu tumor mice treated with s50-TBG-sugar (n ¼ 2). The results are expressed as % ID per gram of tissue. B, acute effects in primary tumor after i.v. administration of 25 mg/kg s50-TBG-RNAi-CK2 in FaDu xenograft mice. Cryosections were labeled with anti-CK2 (green) or anti–NF-kB p65 p-Ser 529 (red). Nuclei (blue) were counterstained with bisbenzamide. C, immunoblot showing the inhibition of CK2 expression in FaDu xenograft metastatic spleens 3 days after i.v. administration of 25 mg/kg s50-TBG-RNAi-CK2. Densitometric analysis of the immunoblots is depicted. Nuclear CK2a (white) protein is normalized to Sp-3; cytosolic CK2a (black) and keratin-14 (K-14, red) proteins are normalized to LDH. End-point qRT-PCR analysis crossing the predicted cleavage site targeted in the human CK2a (dark green) and CK2a0 (light green) transcripts in the tumor-burdened FaDu spleens is also graphed. Transcript values were normalized to GAPDH, and the endogenous naïve mouse spleen baseline transcript values for CK2a0 were subtracted. The data shown are mean SE from 5 animals per group. , P values are given in Results. D, confocal micrographs confirming the colocalization of K-14 and FaDu tumor and the efficacy of s50-TBG-RNAi-CK2 in reducing spleen metastases. Single signals are K-14 (blue) and GFP (red). DNA was counterstained with Sytox Green. Merged signals are GFP and Sytox Green (yellow), and K-14 and Sytox Green (cyan).

Article Snippet: Western analysiswas performed onwhole cell, nuclear, and cytoplasmic lysates as described (20) using the following antibodies: CK2a, CK2a0 (Bethyl Laboratories; A300-197A, A300-199A), caveolin-1, SP-3 (Santa Cruz Biotechnology; sc-894, sc-644), Ago2 (Abnova; 00027161), LDH (Cell Signaling; 2012), Keratin-14 (Covance; PRB155P), and FITC (Biodesign; K9006G). qRT-PCR analysis For SYBR green qRT-PCR, cDNAwas transcribed from 0.5 mg of total RNA using Superscript III (Invitrogen).

Techniques: In Vivo, Activation Assay, Labeling, Western Blot, Inhibition, Expressing, Quantitative RT-PCR

Journal: Molecular and cellular biochemistry

Article Title: Systemic administration of antisense oligonucleotides simultaneously targeting CK2α and α' subunits reduces orthotopic xenograft prostate tumors in mice.

doi: 10.1007/s11010-011-0943-x

Figure Lengend Snippet: Fig. 1 a Downregulation of CK2a in prostate tumors causes increased expression of CK2a0. Whole cell lysates from orthotopic xenograft prostate tumors treated via tail vein injection with 200 ll of 2 mg/ml AS-CK2a phosphodiester OGN in PBS or 200 ll of PBS were subjected to immunoblot analysis using antibodies directed against CK2a/a0 and actin. Signal density was quantitated using Image J and normalized to actin. Average expression in AS-CK2a treated tumors for CK2a and a0 was 0.38 ± 0.10 and 0.88 ± 0.02 compared to 0.58 and 0.24 for PBS treated tumor. b A bispecific antisense sequence reduces cell viability better than a or a0-specific sequences alone. PTO (7.5 lM) were directly administered to PC3- LN4 cells grown on tenascin and fibronectin. Cell proliferation was assessed by means of 3H-Thy incorporation over a 24 h period. Both sense-CK2a and AS-GAPDH were included as controls. bs-AS-CK2 #1 and #2 represent different bispecific OGN sequences designed to simultaneously target CK2a and a0. Data are presented as the mean ± SEM

Article Snippet: Antibodies used were Actin (sc-1616) and LDH-A (sc-27230) from Santa Cruz Biotechnology; CK2a (A300197A) and CK2a0 (A300-199A) from Bethyl Laboratories; Casein Kinase II a/a0 from BD Transduction Laboratories (611611); CK2b (218712) from Calbiochem; Lamin B1 (33-2000) from Invitrogen; NF-jB p65 (610868) from BD Transduction Laboratories; Akt (9272) and Phospho-Akt Ser473 (9271) from Cell Signaling.

Techniques: Expressing, Injection, Western Blot, Sequencing

Journal: Molecular and cellular biochemistry

Article Title: Systemic administration of antisense oligonucleotides simultaneously targeting CK2α and α' subunits reduces orthotopic xenograft prostate tumors in mice.

doi: 10.1007/s11010-011-0943-x

Figure Lengend Snippet: Fig. 2 a bs-AS-CK2 downregulates both CK2a and a0 proteins in cultured PC3-LN4 prostate cancer cells. PC3-LN4 cells were transfected with 80 or 160 nM bs-AS-CK2 or 160 nM AS-RFP (red fluorescent protein) PTO using DOTAP reagent. Cells were collected at 48 and 72 h post-transfection and analyzed by immunoblot for CK2a and a0 and actin expression. b bs-AS-CK2 decreases DNA synthesis in cultured prostate cells. Cultured PC3-LN4 cells grown on tenascin and fibronectin and BPH-1 cells grown on laminin cells were transfected using Dharmafect reagent with 50 or 100 nM bs-AS-CK2 or AS-RFP PTO. After 48 h, 3H-Thy was added, and cells were harvested for counting at 72 h. *Significant difference from Dharmafect (P \ 0.005). ^ Significant difference from AS-RFP (P \ 0.05). c bs-AS-CK2 induces apoptosis in cultured PC3-LN4 prostate cancer cells. PC3-LN4 cells were transfected with 160 or 320 nM bs-AS-CK2 or 160 nM AS-GAPDH PTO using Dharmafect reagent. After 48 h, the cells were plated onto cover slips. Cells were fixed at 72 h post-transfection, and analyzed using a TUNEL staining kit

Article Snippet: Antibodies used were Actin (sc-1616) and LDH-A (sc-27230) from Santa Cruz Biotechnology; CK2a (A300197A) and CK2a0 (A300-199A) from Bethyl Laboratories; Casein Kinase II a/a0 from BD Transduction Laboratories (611611); CK2b (218712) from Calbiochem; Lamin B1 (33-2000) from Invitrogen; NF-jB p65 (610868) from BD Transduction Laboratories; Akt (9272) and Phospho-Akt Ser473 (9271) from Cell Signaling.

Techniques: Cell Culture, Transfection, Western Blot, Expressing, DNA Synthesis, TUNEL Assay, Staining