Expression of the Bcl-3 proto-oncogene suppresses p53 activation

Abstract

While Bcl-3 expression in cancer was originally thought to be limited to B-cell lymphomas with a 14;19 chromosomal translocation, more recent evidence indicates that expression of this presumptive oncoprotein is significantly more widespread in cancer. However, an oncogenic role for Bcl-3 has not been clearly identified. Experiments presented here indicate that Bcl-3 is inducible by DNA damage and is required for the induction of Hdm2 gene expression and the suppression of persistent p53 activity. Furthermore, constitutive expression of Bcl-3 suppresses DNA damage-induced p53 activation and inhibits p53-induced apoptosis through a mechanism that is at least partly dependent on the up-regulation of Hdm2. The results provide insight into a mechanism whereby altered expression of Bcl-3 leads to tumorigenic potential. Since Bcl-3 is required for germinal center formation, these results suggest functional similarities with the unrelated Bcl-6 oncoprotein in suppressing potential p53-dependent cell cycle arrest and apoptosis in response to somatic hypermutation and class switch recombination.

Figure 1.

Overexpression of Bcl-3 inhibits DNA damage-induced apoptosis. (A) Expression of Bcl-3 in MCF-7, MCF-7B, Karpas 299, and HT-1080 cells. Western blots of extracts from indicated cell lines were probed with antibodies against Bcl-3 and β-tubulin. (B) MCF-7B cells are protected against DNA damage-induced apoptosis. MCF-7 and MCF-7B cells were left untreated or were treated with 40 J/m² UV-C or 10 μg/mL cisplatin as indicated. Eighteen hours after treatment, apoptosis was measured by flow cytometric analysis of Annexin-V staining. (C) UV-induced apoptosis in MCF-7 cells is p53 dependent. MCF-7 cells were treated with DMSO, 10 μM pifithrin-α, 40 J/m² UV-C plus DMSO, or 40 J/m² UV-C plus 10 μM pifithrin-α as indicated. Eighteen hours after treatment, apoptosis was measured by flow cytometric analysis of Annexin-V staining.

Figure 2.

Bcl-3 overexpression inhibits DNA damage-induced p53 activity. (A) UV-induced p53 protein levels are reduced in MCF-7B cells. MCF-7 and MCF-7B cells were treated with 40 J/m² UV-C for the indicated times, and Western analysis was performed on whole-cell extracts using antibodies against Bcl-3, p53, and β-tubulin. (B) Overexpression of Bcl-3 does not affect p53 mRNA levels. MCF-7 and MCF-7B cells were treated with 40 J/m² UV-C for the indicated times, and relative expression of p53 was measured by quantitative real-time PCR. (Lane 1) Expression levels were normalized to expression of glucuronidase-β, and the values represent the fold increase or decrease relative to untreated MCF-7 cells. (C) Transient expression of Bcl-3 leads to decreased p53 protein levels following UV treatment. MCF-7 cells were transfected with either empty vector or 2 or 4 μg of pCMV-Flag-Bcl-3. Two days after transfection, the cells were left untreated or were treated with 50 J/m² UV-C for 4 h, and Western analysis was performed with antibodies against the Flag epitope, p53, or β-tubulin. (D) Transient expression of Bcl-3 inhibits p53 transcriptional activity. MCF-7 cells were transfected with 50 ng of pg-13-luciferase and 5 ng of renilla luciferase plus 100 ng of pCMV-Flag-Bcl-3 and pCMV-Flag-p53 where indicated. Firefly luciferase activity was measured and normalized to renilla luciferase. (Lane 1) Values represent fold increase over basal activity. (E) DNA damage-induced expression of p53 target genes is lost in MCF-7B cells. MCF-7 and MCF-7B cells were treated with 40 J/m² UV-C for the indicated times, and relative expression of p21, Noxa, and Puma was measured by quantitative real-time PCR. (Lane 1) Expression levels were normalized to expression of glucuronidase-β, and the values represent the fold increase or decrease relative to untreated MCF-7 cells.

Figure 3.

Loss of Bcl-3 leads to increased levels of p53 following DNA damage and sensitivity to DNA damage-induced apoptosis. (A) Knockdown of Bcl-3 results in an increase of UV-induced p53 protein. HT-1080 cells were transfected with a control siRNA or an siRNA targeting Bcl-3. Forty-eight hours after transfection, cells were treated with 40 J/m² UV-C for the indicated time points. Western analysis was performed on whole-cell extracts using antibodies against Bcl-3, p53, and β-tubulin. (B) Bcl-3-null MEFs have increased p53 levels following UV treatment. Wild-type and Bcl-3-deficient MEFs were treated with 40 J/m² UV-C for the indicated time points. Western analysis was performed on whole-cell extracts using antibodies against p53 and β-tubulin. (C) Bcl-3-null MEFs are sensitized to p53-dependent UV-induced apoptosis. Bcl-3^+/+ and Bcl-3^-/- MEFs were treated with DMSO, 10 μM pifithrin-α, 40 J/m² UV-C plus DMSO, or 40 J/m² UV-C plus 10 μM pifithrin-α as indicated. Eighteen hours after treatment, apoptosis was measured by flow cytometric analysis of Annexin-V staining.

Figure 4.

Overexpression of Bcl-3 leads to an increase in Hdm2 expression. (A) Both transient and stable overexpression of Bcl-3 lead to higher basal levels of Hdm2 protein. Whole-cell extracts were prepared from MCF-7 and MCF-7B cells as well as MCF-7 cells transfected with 8 μg of either empty vector or pCMV-Flag-Bcl-3, and Western analysis was performed using antibodies against Bcl-3, Hdm2, and β-tubulin. (B) MCF-7B cells have higher basal levels of Hdm2 RNA. Real-time quantitative PCR was performed on cDNA prepared from MCF-7 and MCF-7B cells using primers specific for Bcl-3 and Hdm2. (Lane 1) Expression levels were normalized to expression of glucuronidase-β, and values represent fold difference relative to MCF-7 for each gene tested. (C) Transient overexpression of Bcl-3 leads to an increase in Hdm2 levels in U-2OS cells. U-2OS cells were transfected with 0-200 ng of pCMV-Flag-Bcl-3 in 50-ng increments. Total DNA content was brought to 200 ng with pCMV-Flag vector. Forty-eight hours after transfection, Western analysis was performed on whole-cell extracts using antibodies against Bcl-3, Hdm2, and β-tubulin. (D) Bcl-3 is present at the Hdm2 promoter at higher levels in MCF-7B cells. MCF-7 and MCF-7B cells were treated with 40 J/m² UV-C for either 0 or 4 h, and ChIP was performed using antibodies specific for p53 or Bcl-3, or no antibody. Real-time quantitative PCR was performed on precipitated DNA using primers specific for the p2 promoter region of the Hdm2 gene and for the promoter region of the β-actin gene. Values are normalized against the input DNA and are represented as the percentage of input for each given sample. Each value represents the mean of three independent measurements of the precipitated DNA, and the error bars represent one standard deviation. The experiment was repeated three times with identical results.

Figure 5.

Loss of Bcl-3 leads to a decrease in basal and DNA damage-inducible Hdm2 expression. (A) Knockdown of Bcl-3 in human cancer cells leads to loss of Hdm2 expression. MCF-7 and Karpas 299 cells were transfected with either control siRNAs or siRNAs specific for Bcl-3. Forty-eight hours after transfection, Western analysis was performed on whole-cell extracts using antibodies specific for Bcl-3, Hdm2, and β-tubulin. (B) DNA damage fails to induce Mdm2 RNA in Bcl-3-deficient fibroblasts. Wild-type and Bcl-3-null MEFs were treated with either 40 J/m² UV-C or 10 μg/mL cisplatin for the indicated times, and Mdm2 gene expression was measured by quantitative real-time PCR. (Lane 1) Expression levels were normalized to expression of GAPDH, and the values represent the fold increase or decrease relative to untreated wild-type MEFs. (C) DNA damage fails to induce Mdm2 protein in Bcl-3-deficient fibroblasts. Wild-type and Bcl-3-null MEFs were treated with 40 J/m² UV-C for the indicated times, and Western blots were performed on whole-cell extracts using antibodies specific for Mdm2 and β-tubulin. (D) Knockdown of Bcl-3 impairs the ability of DNA damage to induce Hdm2 in HT-1080 cells. HT-1080 cells were transfected with either control siRNAs or siRNAs specific for Bcl-3. Forty-eight hours after transfection, cells were treated with 40 J/m² UV-C for the indicated times, and Western analysis was performed on whole-cell extracts using antibodies specific for Bcl-3, Hdm2, and β-tubulin.

Figure 6.

Disruption of the p53-Hdm2 interaction rescues the effects of Bcl-3 overexpression. (A) Disruption of the p53-Hdm2 interaction restores the ability of UV to induce p53 in MCF-7B cells. MCF-7 and MCF-7B cells were pretreated for 30 min with either DMSO or 2 μM RITA and then treated with 40 J/m² UV-C for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using antibodies specific for p53 and β-tubulin. (B) Knockdown of Hdm2 restores the ability of UV to induce p53 in MCF-7B cells. MCF-7B cells were transfected with either a control siRNA or an siRNA specific for Hdm2. Forty-eight hours after transfection, cells were treated with 40 J/m² UV-C for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using antibodies specific for p53, Hdm2, and β-tubulin. (C) Disruption of the p53-Hdm2 interaction restores the ability of UV to induce apoptosis in MCF-7B cells. MCF-7 and MCF-7B cells were left untreated, or were treated with 40 J/m² UV-C or with 40 J/m² UV-C plus 2 μM RITA. Eighteen hours after treatment, apoptosis was measured by flow cytometric analysis of Annexin-V staining.