FIGURE 9.

NF-κB inhibitors block PKCϵ-induced cyclin D1 up-regulation. A, NF-κB inhibitors block up-regulation of cyclin D1 by PMA in IEC-18 cells. Cells were treated with PMA (P) or vehicle (C) for 8 h in the presence of 100 μm PDTC (i), CAPE (ii), or vehicle (DMSO), and levels of cyclin D1 and β-actin were determined by Western blot analysis. Data are representative of at least three independent experiments. In each panel, data are from the same Western blot, with dashed lines indicating where lanes have been rearranged for clarity. PDTC and CAPE showed some mild toxicity under the conditions used, with protein recovery relative to control as follows: 100 μm PDTC, 93 ± 6%; 10 μm CAPE, 91 ± 3%; 100 μm CAPE, 80 ± 7%. These values were not affected by PMA treatment. B, NF-κB inhibitors block up-regulation of cyclin D1 by PMA in DLD1 colon cancer cells; as in A except that DLD1 cells were used, and PMA treatment was for 4 h. CAPE showed some mild toxicity under the conditions used, with protein recoveries for samples treated with PDTC and CAPE relative to control as follows: 100 μm PDTC, 98 ± 3%; 10 μm CAPE, 91 ± 3%; 100 μm CAPE, 77 ± 7%. These values were not affected by PMA treatment. C, NF-κB inhibitors block PKC-induced up-regulation of cyclin D1 mRNA. IEC-18 (i) or DLD1 (ii) cells were treated with PMA or vehicle (EtOH) in the presence of 100 μm PDTC, 100 μm CAPE, or vehicle (DMSO). After 8 h (i) or 4 h (ii) of PMA treatment, cells were harvested and analyzed by quantitative RT-PCR. Cyclin D1 mRNA expression was normalized to 18 S rRNA levels and is shown relative to that in control (vehicle only-treated) cells. Data are averages ± S.E. (error bars) of at least four independent experiments except for data for CAPE-treated DLD1 cells, which are from two experiments. *, statistically different from vehicle (EtOH)-treated control cells (p < 0.05). #, statistically different from corresponding treatment of cells not exposed to PDTC or CAPE (p < 0.05). D, NF-κB inhibitors block PMA-induced increase in cyclin D1 promoter activity. DLD1 cells were transfected with the −963CD1Luc reporter and treated with PMA or vehicle (EtOH) for 4 h in the presence of 100 μm PDTC, 100 μm CAPE, or vehicle. Promoter activity was measured by the Dual-Luciferase assay and is normalized to the activity in control cells treated with vehicle only. Data are averages ± S.E. of four independent experiments. *, statistically different from vehicle (EtOH)-treated cells (p < 0.05). #, statistically different from corresponding treatment of cells not exposed to PDTC or CAPE (p < 0.05). E, up-regulation of cyclin D1 transcription by PMA does not require canonical activation of NF-κB. IEC-18 (i) or DLD1 (ii) cells, transfected with the NF-κB reporter along with pRL-TK and dominant active IκBα-expressing vector (DA IκB) or empty vector (Con), were treated with PMA or vehicle (EtOH) for 8 h (i) or 5 h (ii). Firefly and Renilla luciferase activities were determined using the Dual-Luciferase assay. Differences in transfection efficiency were controlled for by normalizing against pRL-TK activity in vehicle-treated cells. These data are expressed relative to empty vector-transfected, vehicle-treated cells and are the averages ± S.E. of at least three independent experiments. *, statistically different from corresponding vehicle (EtOH)-treated control cells (p < 0.05).