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

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.

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.

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.