Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Status of B23 in the cytoplasm and NM fractions of prostate cancer cells in response to CK2 specific inhibitor TBB mediated induction of apoptosis. A, TUNEL staining of ALVA-41 and PC-3 cells treated with varying doses of TBB (from 50 to 150 µM). Induction of apoptosis is shown. Cells were counterstained with Hoechst 33342 to identify nuclear component. B, cytoplasmic and NM associated B23 in ALVA-41 and PC-3 cells treated with TBB at varying concentrations as under A. The relative change in the protein bands is calculated compared to the respective control and normalized to the β-actin in the sample (e.g., lanes 2 and 3 from left in Fig. 1B had a higher amount of protein to ensure visualization of B23 signal in the nuclear matrix). C, effect of TBB and apigenin on CK2 activity at the concentrations of the inhibitors shown. D, measurement of the message expression for CK2α in ALVA-41 and PC-3 cells under the same conditions as for C. E, measurement of B23 message in ALVA-41 and PC-3 cells under the same conditions as for C.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: TUNEL Assay, Staining, Control, Activity Assay, Expressing

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Status of B23 in the cytoplasm and NM fractions of prostate cancer cells in response to siRNA-mediated downregulation of CK2. A, cell viability was determined by WST-1 assay in ALVA-41 and PC-3 cells transfected with varying concentrations of CK2••′ siRNA for the periods of time shown. B, ALVA-41 and PC-3 cells were transfected with varying concentrations of CK2αα′ siRNA as under A. Cyclophilin B (Dharmacon, Catalog: D-001136-01-05) was employed as a control. All other details were as described under Experimental Procedures. TUNEL staining shows the induction of apoptosis in cells treated with CK2 siRNA. Cells were counterstained with Hoechst 33342 to identify nuclei. C, immunoblot analysis of cytoplasmic and NM associated B23 and CK2••′ was carried out in ALVA-41 and PC-3 cells transfected with varying concentrations of CK2••′ siRNA. Lane a, untreated control; lane b, 5 nM CK2••′ siRNA; lane c, 10 nM CK2••′ siRNA; lane d, 50 nM CK2αα′ siRNA; lane e, untreated control; lane f, DharmaFECT control, lane g, 10 nM CK2αα′ siRNA; lane h, 100 nM CK2αα′ siRNA. The relative change in the protein bands is calculated compared to the respective control.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: WST-1 Assay, Transfection, Control, TUNEL Assay, Staining, Western Blot

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Effect of apoptosis inducing conditions on NM-associated B23. A, prostate cancer cells (ALVA-41) were treated with doses of etoposide, or CK2 inhibitors (TBB or Apigenin) at doses that are sub-optimal for induction of apoptosis. The results show a minimal effect on cell death under these conditions. When the sub-optimal doses of the same inhibitors are combined it results in induction of apoptosis, as shown in the chart. Illustrated in A are the data employing etoposide (an apoptosis-inducing agent) with and without CK2 inhibitors TBB or apigenin. Similar effects have previously been reported using TRAIL (an apoptosis-inducing agent) with and without TBB at sub-optimal doses (27) (the latter conditions were employed in the experiment shown under C. B, NM-associated B23 immunoreactive protein in cells treated with TBB, apigenin and etoposide, as shown. Lane a, control; lane b, 20 µM etoposide (sub-optimal for induction of apoptosis); lane c, TBB at 40 µM (sub-optimal for induction of apoptosis); lane d, etoposide plus TBB (combined effect to produce apoptotic activity); lane e, apigenin at 20 µM (insufficient for induction of apoptosis); lane f, apigenin plus etoposide (combined effect to produce apoptotic activity). Densitometric values are calculated relative to the level in control cells. C, NM-associated B23 immunoreactive protein in cells treated with TBB, apigenin and TRAIL as shown. Lane a, control; lane b, TRAIL 2 ng/ml (sub-optimal dose which is insufficient for induction of apoptosis); lane c, TBB at 40 µM (sub-optimal dose for induction of apoptosis); lane d, TBB plus TRAIL (combined effect evokes apoptotic activity); lane e, apigenin at 20 µM (insufficient dose for induction of apoptosis); lane f, TRAIL plus apigenin (conditions to induce apoptotic activity). Relative densitometric values are based on level in the control cells. D, combined effect of TBB and TRAIL (both at sub-optimal doses) in causing induction of apoptosis is illustrated by cleavage of lamin A in treated cells, as shown. Lane a, control; lane b, 60 µM TBB (insufficient for induction of apoptosis) showing no cleavage of Lamin A; lane c, 2 ng/ml of TRAIL (insufficient for apoptosis induction) as indicated by a minimal cleavage of Lamin A; lane d, 60 µM TBB plus 2 ng/ml of TRAIL resulting in potent induction of apoptosis as indicated by extensive cleavage of Lamin A. These results accord with the corresponding effect on NM-associated B23 as shown under C. In all cases, equal amount of protein was loaded in the gels, and each experiment was confirmed at least three times.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Control, Activity Assay

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Effect of overexpression of CK2α on the status of NM-associated B23 in prostate cancer cells treated with apoptotic inducers. ALVA-41 and PC-3 cells were treated with etoposide or TRAIL at apoptosis inducing concentrations in the absence or presence of forced overexpression of the catalytic subunit CK2α as described under Experimental Procedures. A, treatment with TRAIL at 10 ng/ml for ALVA-41 and 20 ng/ml for PC-3 cells (for 24 h) was carried out to induce apoptotic condition in cells. pcDNA6-CK2α expression vector (2.0 µg/ml) was employed to achieve overexpression of CK2α in ALVA-41 and PC-3 cells, as indicated. NM-associated B23 is shown for each experimental condition; there was no change in cytoplasmic B23 immunoreactive B23 under these conditions (not shown). Lane a, control cells treated with pcDNA6; lane b, pcDNA6 plus TRAIL; lane c, pcDNA6-CK2α; and lane d, pcDNA6-CK2α plus TRAIL. B, treatment of ALVA-41 and PC-3 cells with 50 µM etoposide (apoptosis-inducing level). Overexpression of CK2α was achieved as described under A. NM-associated immunoreactive B23 is shown. Lane a, pcDNA6 control; lane b, pcDNA6 plus 50 µM etoposide for 48 h; lane c, pcDNA6-CK2α; and lane d, pcDNA6-CK2α plus etoposide. C, a representative experiment showing confirmation of CK2α overexpression in PC-3 cells by transient transfection with pcDNA6-CK2α is illustrated by analysis of the message and protein levels.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Transfection

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Colocalization of CK2α and B23 in the nucleus. A, double immunofluorescence staining for anti-CK2α and anti-B23 was carried out in ALVA-41 cells treated with varying doses of TBB, as shown. Merged image of the immunofluorescence stains for anti-CK2α and anti-B23 shows co-localization of the two proteins in the nucleus. Downregulation of CK2 by TBB treatment (producing apoptotic condition) shows co-ordinate loss of B23 and CK2α in the nucleus. B, double immunofluorescence staining for anti-CK2α and anti-B23 was carried out in PC-3 cells treated with varying doses of TBB. All details are the same as for A. Results confirm the co-localization of CK2α and B23 in the nucleus and its reduction on induction of apoptotic condition induced by TBB mediated inhibition of CK2 as observed for ALVA-41 cells under A.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Double Immunofluorescence Staining, Immunofluorescence, Inhibition

Journal:

Article Title: Protein B23/Nucleophosmin/Numatrin nuclear dynamics in Relation to Protein Kinase CK2 and Apoptotic Activity in Prostate Cells †

doi: 10.1021/bi9021928

Figure Lengend Snippet: Effect of altered androgenic status on CK2α and B23 levels in rat ventral prostate tissue. Lysate, cytoplasm and nuclear matrix fractions were isolated from prostatic tissue of rats subjected to various treatments as shown. Each fraction was analyzed by Western blotting for the presence of immunoreactive CK2α, B23 and lamin A. A, level of B23 in total lysates from prostatic tissues isolated from rats with altered androgenic status. Lane a, control rats; lanes b to d, rats orchiectomized for 1, 2, and 3 days, respectively. B, detection of protein CK2α, B23 and lamin A in cytoplasm and NM fractions isolated from prostate tissue of normal rats, or rats subjected to altered androgenic status. Lane a, control rats; lane b, 3-day orchiectomized rats; lane c, 6-day orchiectomized rats; lane d, 6-day orchiectomized rats given 5α-DHT for 4 days; lane e, same as lane d except that 5α-DHT was administered for 7 days. Breakdown of lamin A (to 28 kDa fragment) in the NM fraction indicates apoptotic condition. All other details are as described under Experimental Procedures.

Article Snippet: Primer sets were from SuperArray as follows: CK2α #PPH01514A; B23 #PPH19534A; and Actin #PPH00073A.

Techniques: Isolation, Western Blot, Control