Article Figures & Data
Figures
- Figure 1.
Cisplatin represses NF-κB–dependent transcription. A, TNF activates, whereas cisplatin and daunorubicin repress NF-κB reporter plasmid activity. U-2 OS cells were transfected with 2 μg of 3× κB concanavalin A luciferase NF-κB reporter plasmid or a control lacking κB sites as indicated. Cells were either unstimulated or stimulated for 8 hours with 10 ng/mL TNF, 4 μg/mL cisplatin, or 1 μmol/L daunorubicin, 36 hours after transfection. Fold activation or repression relative to levels seen in untreated controls. Normalized such that no change in luciferase activity has a value of 0. B, cisplatin represses NF-κB activity in HeLa cells. HeLa 57A cells, which contain a chromosomally integrated 3× κB reporter plasmid, were treated as in (A). C-D, cisplatin inhibits expression of Bcl-xL but not XIAP mRNA. RNA was prepared from U-2 OS cells treated with either 4 μg/mL cisplatin or 15 μmol/L etoposide for the indicated times (C), 1 μmol/L daunorubicin, or 40 J/m2 UV-C for 0 or 6 hours (D, right) or the U-2 OS cell derivative NARF2, where ARF was induced by 1 mmol/L IPTG for 0 or 24 hours (D, left). Semiquantitative PCR analysis was done using 5 ng total RNA, with primers specific to human XIAP, Bcl-xL, or a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control.
- Figure 2.
Cisplatin and ARF do not induce NF-κB DNA binding or RelA nuclear translocation. A, EMSA analysis using nuclear protein extracts from U-2 OS cells treated with either 4 μg/mL cisplatin (left) or 15 μmol/L etoposide (right) for the indicated times. A lane using 1 μmol/L daunorubicin-treated cells (3 hours) is also shown. EMSA analysis was done using a 32P-labeled consensus Ig/HIV κB probe. B, immunofluorescence analysis of RelA nuclear localization following cisplatin treatment. U-2 OS were plated onto coverslips and fixed after 4 μg/mL cisplatin (4 hours) or 40 J/m2 UV-C (4 hours) treatment. Cells were stained with rabbit anti-RelA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or 4′,6-diamidino-2-phenylindole (DAPI) to reveal DNA. In addition, NARF2 cells were treated with 1 mmol/L IPTG for 0 or 24 hours to induce ARF and similarly stained for RelA. Cells were analyzed, and images were acquired using a DeltaVision microscope. C, cisplatin treatment represses TNF-induced NF-κB activity. U-2 OS cells were transfected as in Fig. 1A, except that some transfected cells were also treated with TNF (8 hours) and/or cisplatin (6 hours). D, cisplatin does not inhibit TNF-induced NF-κB DNA binding. EMSA analysis was done as in (A), except that cells were treated with 10 ng/mL TNF for 30 minutes or cisplatin for 4 hours either alone or in combination. Where indicated, supershift analysis was done with a specific anti-RelA antibody.
- Figure 3.
Cisplatin does not affect RelA binding to the Bcl-xL promoter but does induce HDAC1 recruitment. Control or cisplatin-treated U-2 OS cells were harvested, and chromatin immunoprecipitation analysis was done with the indicated RelA, HDAC1, acetylated H3 (Lys9 and Lys14) or Gal4 control antibodies using primers to the Bcl-xL or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters.
- Figure 4.
Cisplatin repression of NF-κB requires the ATR/Chk1 pathway. A, cisplatin represses the RelA transactivation domain in a Thr505-dependent manner. U-2 OS cells were transfected with 2 μg of Gal4 luciferase reporter plasmid together with expression plasmids encoding either the Gal4 RelA (TAD) or Gal4 RelA (TAD T505A) expression plasmids as indicated. Thirty-two hours after transfection, cells were either left untreated or stimulated for (16 hours) with 4 μg/mL cisplatin. Relative luciferase activity per microgram of protein. The RelA transactivation domain (TAD) used in this study encodes amino acids 428 to 551 of human RelA. B, cisplatin-mediated repression of NF-κB activity requires ATR. U-2 OS cells were transfected as in Fig. 1A and treated with either 4 μg/mL cisplatin or 40 J/m2 UV-C for 0 or 8 hours. Cells also included 1 μg of kinase-dead ATR expression plasmid or a control plasmid as indicated. C, siRNAs targeting ATR and Chk1 abolish cisplatin-induced repression of Bcl-xL. PCR analysis of Bcl-xL expression was done following treatment of U-2 OS cells with the indicated siRNAs. Analysis of ATR, Chk1, and ATM expression, as well as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control, is also included. D, cisplatin induces phosphorylation of Chk1. Whole-cell lysates were prepared from U-2 OS cells treated with 4 μg/mL cisplatin for the indicated times. Western blot analysis was done using the indicated Chk1 or phosphospecific Chk1 antibodies.
- Figure 5.
Cisplatin induces Thr505 phosphorylation of RelA. A, cisplatin induces Thr505 phosphorylation. Whole-cell lysates were prepared from NARF2 cells either treated with IPTG to induce ARF for 24 hours or nuclear protein extracts were prepared from U-2 OS cells stimulated with 4 μg/mL cisplatin (4 hours), 1 μmol/L daunorubicin (4 hours), or 40 J/m2 UV-C (4 hours) or left untreated. Phosphorylated RelA was then immunopreciptated by incubating 100 μg of lysate with purified rabbit phospho-RelA T505A antibody. The immunoprecipitate was resolved by SDS-PAGE before Western blotting with an anti-RelA antibody (Santa Cruz Biotechnology). B, experiment was done as in (A), except that whole-cell lysates from U-2 OS cells were used in combination with the goat phospho-RelA T505A antibody and anti-RelA antibody (Santa Cruz Biotechnology). Both antibodies were used to either immunoprecipitate or Western blot RelA as indicated. C, cisplatin induced Thr505 phosphorylation is prevented by the Chk1 inhibitor Gö6976. Experiment was done as in (A), except that indicated cells were incubated with 1 μmol/L Gö6976 for 30 minutes before the addition of cisplatin and the preparation of whole-cell lysates. The same result is also seen with the goat phospho-RelA T505 antibody (data not shown).
Volume 66, Issue 2
