PMC

Analysis of MRP expression and CKII activity in primary foreskin keratinocyte NFK cells and the HPV16-immortalized 16MT cell line. (A) For immunofluorescence assays, cells were fixed and immunostained with MAbs to MRP-8, MRP-14, and MRP-8/14. The MRPs are shown in green, and the cell nuclei are red. (B) CKII was immunoprecipitated, and its kinase activity was measured in an in vitro phosphorylation assay. The error bars indicate standard errors (n = 3).

Complex formation of MRP-8 and MRP-14. Equal concentrations, i.e., 1 μg each of MRP-8 (a) and MRP-14 (b), were mixed in the presence of 0.5 mM CaCl₂ (c) and incubated at room temperature for 30 min. To determine the dependence of MRP-8/14 complex formation on calcium ions, the MRP-8 and MRP-14 mixture was incubated with various concentrations of calcium (d). Proteins were resolved by SDS-PAGE, and MRP-8, MRP-14, and their complex formation were analyzed by Western blot assay using mouse MAbs 8-5 C2, S36.48, and 27 E10, which recognize MRP-8, MRP-14, and MRP-8/14, respectively. (a and b) Reducing; (c and d) nonreducing.

Inhibition of CKII activity in HeLa cells by exogenous MRP-8/14. (A) HeLa cells were incubated with various concentrations of MRP-8/14 proteins for 24 h at 37°C and extracted in RIPA buffer. In parallel experiments cells were treated with an increasing concentration of apigenin, a CKII inhibitor, as a positive control. (B) HeLa cells were incubated with 2.5 μg of MRP-8, MRP-14, and MRP-8/14 proteins per ml and 10 μM apigenin for 24 h at 37°C and then extracted in RIPA buffer. CKII was immunoprecipitated, and kinase activity was measured in triplicate samples. Controls were untreated cells and DMSO-treated cells, since DMSO was used as an organic solvent for apigenin. Similar results were obtained in three independent experiments. The error bars indicate standard errors (n = 3).

Inhibition of HPV16 E7 phosphorylation by exogenous MRP-8/14. (A) HPV16 E7-GST phosphorylation was examined in an in vitro phosphorylation assay with CKII and [γ-³²P]ATP. Bovine casein was used as a positive control. One hundred nanograms each of MRP-8, MRP-14, and MRP-8/14 proteins was added into the reaction mixture for 30 min. The phosphorylated E7 and casein bands were visualized by exposing the gel to X-ray films. Control, E7 phosphorylation in absence of MRP proteins; w/o CKII, without CKII. (B) HPV16 GST-E7 phosphorylation was examined in an in vitro phosphorylation assay with nonradioactive ATP, CKII, and E7-GST in the presence of an increasing concentration of MRP-8/14 protein. (a) HPV16 E7-GST protein was detected by mouse MAb. (b) The phosphorylated E7 protein was detected by Western blot assay using rabbit polyclonal antiphosphoserine antibodies. (c) Intensity of E7 phosphorylation was measured by determining the mean density of pixels of phosphorylated E7 bands. Control, E7 phosphorylation in the absence of MRP-8/14.

MRP-8/14-mediated inhibition of HPV16 E7 during the cell cycle. (A) Inhibition of E7 phosphorylation by endogenous MRP-8/14 protein. HSC-3 cells were transfected with HPV16 E7, and 12 h later cells were growth arrested in 0.2% serum for the next 38 h. To induce MRP-8/14 expression at 24 h poststarvation, cells were treated with 10 ng of dexamethasone/ml. At 38 h posttransfection, cell growth was activated by adding 15% serum in the presence of 10 ng of dexamethasone/ml and 1 μCi of [³H]thymidine/ml. At 2-h intervals after growth activation, the cell cycle (a), MRP-8/14 (b), CKII activity (c), total E7 expression (d), and E7 phosphorylation (e) were analyzed. *, MRP-8/14 expression in E7-transfected HSC-3 cells before MRP-8/14 induction; **, CKII activity in E7-transfected HSC-3 cells before MRP-8/14 induction. (B) Inhibition of E7 phosphorylation by exogenous MRP-8/14 protein. Experiments were performed as described above, but instead of induction of endogenous MRP-8/14 with dexamethasone, 3 μg of exogenous MRP-8/14/ml was added at 24 h posttransfection. At 2-h intervals after growth activation, the cell cycle (a), CKII activity (b), E7 expression (c), and E7 phosphorylation (d) were analyzed in the same samples. *, CKII activity in E7-transfected HSC-3 cells before adding MRP-8/14.

Expression of MRPs in HSC-3 cells by confocal microscopy (A) and Western blot analysis (B and C). (A) Cells were grown on chamber slides and treated with dexamethasone for 4 days. For confocal microscopy analysis, cells were fixed and immunostained for MRPs with appropriate mouse MAbs (in green). The cell nuclei were stained with propidium iodide (PI; red). Yellow in the merged panels shows nuclear localization of MRPs. (B) For Western blot assays, approximately 10⁶ HSC-3 cells were treated with 10 ng of dexamethasone/ml for 4 days and MRPs were detected using mouse MAbs. (a and b) Reducing; (c) nonreducing. (C) To analyze induction of MRP-8/14 expression in HSC-3 cells, approximately 3 × 10⁶ cells were treated with 10 ng of dexamethasone/ml for 4 days. MRP-8/14 protein expression was examined in a Western blot assay under nonreducing conditions at 1, 2, 3, and 4 days postinduction.

Inhibition of HPV16 E7 phosphorylation in HSC-3 cells by endogenous MRP-8/14 protein complex. A total of 3 × 10⁶ HSC-3 cells were transfected with HPV16 E7 and 8 h later were treated with 10 ng of dexamethasone/ml, 10 ng of calcium ionophore/ml, and 10 μM apigenin. (A and B) MRP-8/14 (A) and HPV16 E7 (B) were detected using a Western blot assay with appropriate mouse mAbs. (C) At 48 h posttransfection, HPV16 E7 was immunoprecipitated and E7 phosphorylation was examined in a Western blot assay with rabbit antiphosphoserine antibodies. (D) Intensity of E7 phosphorylation was measured by determining the mean density of pixels of phosphorylated E7 bands. (E) CKII activity was examined in an in vitro phosphorylation assay. The error bars indicate standard errors (n = 3). MRP-8/14 and E7 expression, E7 phosphorylation, and CKII activity were analyzed in the same samples. (F) Colocalization of MRP-8/14, E7, and CKII in HSC-3 cells. Cells were fixed and immunostained for CKII (red) and MRP-8/14 (blue). HPV16 E7 protein was detected by GFP (green). In the merge panel, white shows colocalization of MRP-8/14, CKII, and E7.

Expression of MRPs and assessment of CKII activity in normal and tumor cell lines. MRP expression was examined in the HPV-negative primary NFK, OCO, and NCK and immortalized C33A cell lines, and in the following HPV-positive immortalized cell lines: 16MT (derived by HPV16-mediated transformation of NFK cells), HOK-16, HeLa, and SiHa cells. (A) For Northern blot assays, cellular mRNA was hybridized with probes to MRP-8 and MRP-14. A probe for GAPDH was used as a housekeeping gene control. (B and E) For Western blot assays, MRPs were separated under reducing (for MRP-8 and MRP-14) and nonreducing (for MRP-8/14 complex) conditions and immunoblotted with appropriate mouse MAbs to MRP-8, MRP-14, and MRP-8/14. (C and F) To measure CKII activity, CKII was immunoprecipitated and its kinase activity was measured in an in vitro phosphorylation assay. The error bars indicate standard errors (n = 3). (D) For the immunofluorescence assay, NCK and C33A cells grown on chamber slides were fixed and immunostained with MAbs to MRP-8, MRP-14, and MRP-8/14. The MRPs are shown in green, and the cell nuclei are red.

Antiproliferative and anti-E7 phosphorylation activity of the MRP-8/14 protein complex. (A) Antigrowth activity of MRP-8/14 in HPV-positive and HPV-negative cervical cell lines. Cells were grown in the presence of the MRP-8/14 protein complex at various concentrations for 14 days. The number of live cells was quantified with trypan blue staining at day 14. Similar results were obtained in three independent experiments. The error bars indicate standard errors; n = 3. Control, live cells without treatment. (B) Inhibition of HPV-16 E7 phosphorylation by MRP-8/14 in HPV16-infected SiHa and Caski cell lines. Approximately 5 × 10⁶ cells were grown in the presence of 1, 2, and 3 μg of exogenous MRP-8/14 per ml. (a) At 5 days posttreatment, cells were extracted and E7 was immunoprecipitated with mouse MAb and E7 was detected by Western blotting. (b) E7 phosphorylation in the same samples was examined using rabbit antiphosphoserine antibodies. (c) Intensity of E7 phosphorylation was measured by the mean density of pixels in the E7 phosphorylated bands. (C) Dephosphorylation of immunoprecipitated E7 by λ phosphatase treatment. Control (all panels), untreated cells.