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Molecular Cancer Research
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DNA Damage and Cellular Stress Responses

The Nuclear Factor-κB and p53 Pathways Function Independently in Primary Cells and Transformed Fibroblasts Responding to Genotoxic Damage

Dobrila Nesic, Raelene Grumont and Steve Gerondakis
Dobrila Nesic
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Raelene Grumont
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Steve Gerondakis
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DOI: 10.1158/1541-7786.MCR-07-2125 Published July 2008
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  • FIGURE 1.
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    FIGURE 1.

    An absence of c-Rel and RelA does not alter thymocyte sensitivity to p53-dependent apoptosis. A and B. wt and NF-κB–deficient thymocyte apoptosis following p53-dependent and p53-independent death stimuli. wt (closed square), c-rel−/− (open triangle), rela−/− (open circle), c-rel−/−rela−/− (open star), or p53−/− (open square) thymocytes were incubated in medium alone or treated with (A) p53-dependent apoptotic stimuli, γ-irradiation (500 Rad), and doxorubicin (500 ng/mL) or (B) p53-independent apoptotic stimuli dexamethasone (10 nmol/L) and TNFα (10 ng/mL). Cells were collected 12, 24, and 36 h later and stained with propidium iodide and/or Annexin V-FITC, and cell viability was determined by flow cytometry. Points, mean from separate experiments done on thymocytes of all genotypes isolated from multiple independently engrafted mice; bars, SD. C. tnfr1 expression is normal in Rel/NF-κB–deficient thymocytes. tnfr1 expression in wt, c-rel−/−, rela−/−, and c-rel−/−rela−/− thymocytes before or following TNF stimulation (10 ng/mL) for 24 h is represented as relative to levels observed in wt cells. tnfr1 levels in each RNA sample have been normalized relative to gapdh mRNA expression. Columns, mean of three independent experiments; bars, SD.

  • FIGURE 2.
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    FIGURE 2.

    NF-κB–deficient primary MEFs undergo normal cell cycle arrest in response to ionizing radiation. Short-term passage mouse primary MEFs (wt, c-rel−/−, rela−/−, c-rel−/−rela−/−, or p53−/−) were subjected to γ-irradiation (1,000 Rad). A. Viability of primary fibroblasts following γ-irradiation. Pooled adherent and nonadherent wt (closed square), c-rel−/− (open triangle), rela−/− (open circle), c-rel−/−rela−/− (open star), or p53−/− (open square) cells collected before and 12, 24, and 36 h after irradiation were stained with propidium iodide and viability was determined by flow cytometry. B. Cell cycle in primary fibroblasts following γ-irradiation. Viable primary fibroblasts from untreated (medium alone) and irradiated (1,000 Rad) cultures were fixed after 24 h and stained with propidium iodide, and the percentage of cells in G0-G1 (white columns), S (black columns), or G2-M (cross-hatched columns) phase was determined by flow cytometry. Columns, mean of MEFs isolated from three to five different embryos for each genotype derived from two independent experiments; bars, SD. C. puma expression induced in primary MEFs by genotoxic stress. puma expression in untreated, doxorubicin-treated, or γ-irradiated (6 h after treatment) wt or p53−/− primary MEFs was assessed by semiquantitative real-time reverse transcription-PCR. D. NF-κB activity in primary fibroblasts induced by genotoxic stimuli. Following the transfection of wt and p53−/− primary MEFs with the reporter plasmids pluc or κB-luc, cells were either untreated or exposed to γ-irradiation (500 Rad) or doxorubicin (500 ng/mL), after which time luciferase assays were done. Promoter activity is displayed as relative luciferase units. Columns, mean luciferase activity from three sets of experiments; bars, SD.

  • FIGURE 3.
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    FIGURE 3.

    Blocking NF-κB activation in wt and p53−/− MEFs and thymocytes does not alter survival or cell cycle responses to genotoxic stimuli. A. The expression of FLAG-tagged human IκBSR in wt and p53−/− MEFs infected with control (puro only) or FLAG-IκBSR (puro/IκBSR) retroviruses was monitored by Western blotting. B. Response of wt and p53−/− MEFs expressing the IκBSR to ionizing radiation, doxorubicin, and TNF. Control and IκBSR-expressing wt and p53−/− MEFs were treated with TNFα (10 ng/mL) and doxorubicin (200 ng/mL) or γ-irradiated (1,000 Rad), and viability was determined 24 h later. Columns, mean of three independent experiments; bars, SD. C. DNA synthesis in wt and p53−/− MEFs expressing IκBSR following genotoxic stress. wt and p53−/− MEFs infected with control or IκBSR-expressing virus that were untreated or subjected to γ-irradiation or doxorubicin treatment were pulsed with BrdUrd, fixed, and stained with FITC-anti-BrdUrd antibody and propidium iodide to detect cells in S phase (Y axis) or in the G0-G1 and G2-M phases (X axis), respectively. These data are representative of three independent experiments. D. Isolation of wt and p53−/− thymocytes expressing IκBSR. CD4+CD8+ thymocytes expressing high levels of GFP were isolated from mice engrafted with hemopoietic progenitors infected with MY-GFP/IκBSR virus. E. IκBSR inhibition of NF-κB activation in thymocytes. Electrophoretic mobility shift assay was done on nuclear extracts from untreated or PMA-activated (10 ng/mL) GFP+ wt and p53−/− thymocytes (Ly5.2+) isolated from Ly5.1+rag-1−/− mice engrafted with hemopoietic progenitors infected with MY-GFP (control) or MY-GFP/IκBSR virus. The C1 complex comprises p50NF-κB1 homodimers and C2 represents PMA-inducible NF-κB heterodimers comprising p50NF-κB1, RelA, and c-Rel. These data are representative of three independent experiments, each done using nuclear extracts isolated from thymocytes derived from different mice. F. PMA induced A1 expression in wt and p53−/− thymocytes. A1 expression in untreated or PMA-activated (2 h) wt and p53−/− thymocytes transduced with pMY-GFP (control virus) or pMY-GFP/IκBSR was assessed by semiquantitative real-time reverse transcription-PCR. G. Inhibiting NF-κB activation does not alter the survival of γ-irradiated wt or p53−/− thymocytes. wt (triangles) and p53−/− (circles) thymocytes carrying the MY-GFP control (open symbols) or MY-GFP/IκBSR (closed symbols) virus were irradiated (500 Rad), and survival was monitored at 12-h intervals over 36 h. Points, mean of three independent experiments each done in triplicate; bars, SD.

  • FIGURE 4.
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    FIGURE 4.

    NF-κB–deficient MEFs expressing E1A12S remain sensitive to p53-dependent apoptotic stimuli. wt, rela−/−, c-rel−/−rela−/−, nfkb1−/−rela−/−, and p53−/− MEFs infected with control (pHED) or E1A12S-expressing (pHEDE1A12S) retroviruses were enriched using puromycin selection. A. Morphology of MEFs infected with control and E1A12S-expressing retroviruses. B. Expression of E1A12S in virally transduced MEFs. Cell lysates isolated from wt cells infected with control virus (lane 1) or wt (lane 2), rela−/− (lane 3), nfkb1−/−rela−/− (lane 4), c-rel−/−rela−/− (lane 5), and p53−/− (lane 6) MEFs infected with pHEDE1A12S were subjected to Western blotting using antibodies for adenovirus 5 E1A and HSP70. These data are representative of multiple Westerns, including blots, in which cells of all genotypes were infected with control virus. C. Viability of MEFs expressing E1A12S following treatment with p53-dependent and p53-independent apoptotic stimuli. wt, rela−/−, c-rel−/−rela−/−, nfkb1−/−rela−/−, and p53−/− MEFs infected with control or E1A12S-expressing retroviruses were untreated, serum deprived (0.1% FCS), γ-irradiated (1,000 Rad), or stimulated with doxorubicin (200 ng/mL) or TNF (10 ng/mL). Twenty-four hours later, adherent and nonadherent cells were pooled and cellular viability was determined. Columns, mean of three independent experiments; bars, SD. D. S-phase profiles of MEFs expressing E1A12S in response to p53-dependent and p53-independent apoptotic stimuli. Cell cycle profiles of control and E1A12S-expressing MEFs described in C were measured by monitoring BrdUrd incorporation and DNA content. The data are representative of three independent experiments done in duplicate. E. Puma expression in E1A12S immortalized wt or p53−/− MEFs. Following γ-irradiation or doxorubicin treatment (6 h), puma expression was determined by semiquantitative real-time reverse transcription-PCR. F. NF-κB activity in E1A12S immortalized MEFs induced by genotoxic stimuli. Luciferase assays were done on low-passage-number E1A12S immortalized wt and p53−/− MEFs transfected with pluc or κB-luc that were untreated or exposed to γ-irradiation (500 Rad) or doxorubicin (500 ng/mL). Promoter activity is displayed as relative luciferase units. Columns, mean luciferase activity of three sets of experiments; bars, SD.

  • FIGURE 5.
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    FIGURE 5.

    An absence of NF-κB function does not alter cellular levels of p53 induced by apoptotic stimuli. A. p53 expression in doxorubicin-treated primary MEFs. Whole-cell lysates from primary wt and NF-κB–deficient (rela−/−, nfkb1−/−rela−/−, and c-rel−/−rela−/−) MEFs treated with doxorubicin (500 ng/mL) were subjected to Western blotting using anti-p53 and HSP70-specific antibodies. These data are representative of three independent experiments. B. p53 expression in E1A12S immortalized MEFs. Low-passage-number wt and NF-κB–deficient (rela−/−, nfkb1−/−rela−/−, and c-rel−/−rela−/−) MEFs immortalized with E1A12S were untreated, γ-irradiated (1,000 Rad), serum deprived (0.1% FCS), or treated with doxorubicin (200 and 500 ng/mL) or TNF (10 ng/mL) and, 24 h later, examined for p53 expression as described in A. The data are representative of three independent experiments. C. Doxorubicin induction of mdm2 in primary MEFs lacking NF-κB function. mdm2 expression in wt, c-rel−/−, rela−/−, and c-rel−/−rela−/− MEFs before or following doxorubicin treatment (500 ng/mL) for 24 h is represented as relative to levels observed in untreated wt cells. mdm2 levels in each RNA sample have been normalized relative to gapdh mRNA expression. Columns, mean of three independent experiments; bars, SD.

Tables

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  • Table 1.

    NF-κB–Deficient Primary MEFs Remain Viable after Doxorubicin Treatment

    Viabilitywtc-rel−/−rela−/−nfkb1−/−rela−/−c-rel−/−rela−/−p53−/−
    Medium78.7 ± 1.378.4 ± 1.676.2 ± 0.975.8 ± 0.878.4 ± 1.779 ± 0.7
    200 ng/mL doxorubicin74.6 ± 3.272.8 ± 071.9 ± 1.574 ± 1.674.5 ± 0.676.8 ± 1.7
    • NOTE: wt, NF-κB–deficient, and p53-deficient MEFs were exposed to 200 ng/mL doxorubicin for 24 h. Floating and adherent cells were combined and stained with FITC-conjugated Annexin V, and the percentage of viable cells was determined by fluorescence-activated cell sorting. The values represent the mean ± SD of duplicates from two independent experiments.

  • Table 2.

    NF-κB–Deficient Primary MEFs Undergo Normal Cell Cycle Arrest in Response to Doxorubicin Treatment

    BrdUrdwtc-rel−/−rela−/−nfkb1−/−rela−/−c-rel−/−rela−/−p53−/−
    Medium17.8 ± 413.9 ± 0.414.1 ± 0.99.2 ± 0.514.7 ± 0.835.5 ± 1.5
    200 ng/mL doxorubicin00.1 ± 000.2 ± 0.2011.8 ± 1.1
    • NOTE: wt, NF-κB–deficient, and p53-deficient MEFs were treated with 200 ng/mL doxorubicin for ∼20 h. Cells were then pulsed with BrdUrd for 5 h, fixed, incubated with FITC-conjugated anti-BrdUrd antibody, and analyzed by fluorescence-activated cell sorting. The percent of BrdUrd-positive MEFs represents the percentage of cells in the S phase of the cell cycle. The values represent the mean ± SD of duplicates from two independent experiments.

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Molecular Cancer Research: 6 (7)
July 2008
Volume 6, Issue 7
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The Nuclear Factor-κB and p53 Pathways Function Independently in Primary Cells and Transformed Fibroblasts Responding to Genotoxic Damage
Dobrila Nesic, Raelene Grumont and Steve Gerondakis
Mol Cancer Res July 1 2008 (6) (7) 1193-1203; DOI: 10.1158/1541-7786.MCR-07-2125

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The Nuclear Factor-κB and p53 Pathways Function Independently in Primary Cells and Transformed Fibroblasts Responding to Genotoxic Damage
Dobrila Nesic, Raelene Grumont and Steve Gerondakis
Mol Cancer Res July 1 2008 (6) (7) 1193-1203; DOI: 10.1158/1541-7786.MCR-07-2125
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