RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Abstract

Nuclear factor (NF)-kappaB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-kappaB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65(-/-) murine fibroblasts immortalize at considerably faster rates than RelA/p65(+/+) cells. The ability of RelA/p65(-/-) fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65(-/-) fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-kappaB. Altogether, our findings present a fresh perspective on the role of NF-kappaB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.

Figure 1

p65^−/− MEFs immortalize at a faster rate than wild type cells. (A,B) 3T3 analysis of p65^+/+ and p65^−/− MEFs. Graph indicates the passage numbers at which C57BL/6 (A; n=17 pairs) or BALB/c (B; n=3 pairs) MEFs entered senescence and became immortalized. Arrowheads indicate passage numbers at which MEFs immortalize. (C) Immortalization of p65^+/+ MEFs expressing pBabepuro vector (V) or pBabeIκBα-SR (SR) by a 3T3 protocol (n=3). (D) Immortalization of p65^−/− MEFs expressing pBabepuro vector (V) or pBabep65 (p65) (n=3). (E,F) p50^+/+ or p50^−/− (E) and Bcl-3^+/+ or Bcl-3^−/− (F) MEFs were prepared and passaged by a 3T3 protocol. MEF, mouse embryonic fibroblast.

Figure 2

p65 functions to regulate genomic stability. (A,B) Chromosomes were counted in (A) immortalized and (B) primary p65^+/+ and p65^−/− MEFs from metaphase-arrested cells. (C,D) SKY of immortalized p65^+/+ and p65^−/− cells at P42. (E,F) LacZ genomic stability assays (n=3) from primary p65^+/+ and p65^−/− MEFs (P6, P9, P12, P15, P<0.02). (G) Quantitative RT–PCR of incorporated LacZ genes. (H) Early passage p65+/+ and p65−/− MEFs were stably infected with pBabepuro–LacZ. The LacZ gene was amplified from DNA isolated from MEFs at indicated passages using high-fidelity PCR and then cloned into pBluescript SK+. Data show the percentage of white bacterial colonies grown on X-GAL LB agar plates. LB, Luria-Bertani; MEF, mouse embryonic fibroblast; RT–PCR, real-time PCR; SKY, spectrum karyotyping.

Figure 3

p65^−/− MEFs and tissues show defects in DNA repair. (A) P4–P6 MEFs were subjected to 8 Gy of IR. Whole-cell extracts were prepared at indicated time points and western blots were probed for γ-H2AX. (B) P4–P6 MEFs were treated with IR and recovery from DNA damage was analysed by using γ-H2AX immunofluorescence. (C) Basal levels of DNA damage in primary MEFs was analysed by γ-H2AX immunoflourescence. (D) Quantification of DNA-damage-positive nuclei in proliferating (P4) and senescent (P11) cells. Nuclei containing five or more brightly stained γ-H2AX foci were considered as positive. (E,F) Frozen sections from TNF-α;p65^+/+ or TNF-α;p65^−/− livers and colons were stained for γ-H2AX. For each analysis, data are representative of three independent experiments. For quantitative analysis, data were derived from a minimum of 100 nuclei (D). Inserts represent γ-H2AX foci at × 1,000 magnification (C,E,F). Cells or sections were counterstained with Hoechst to visualize nuclei (C,E,F). IR, γ-irradiation; MEF, mouse embryonic fibroblast.

Figure 4

Early immortalization of p65^−/− MEFs is dependent on continuous DNA damage. (A) Oxidative DNA damage in p65^+/+ and p65^−/− cells cultured under 20% O₂ or 3% O₂ was analysed by FLARE comet assay. Tail length and percentage of tail DNA contents were calculated using Cometscore software (TriTek Corporation). DNA damage index was derived from three independent experiments, with at least 50 nuclei counted per experiment. (B) p65^+/+ and p65^−/− cells were cultured by a 3T3 method after IR-induced senescence under 3% O₂. Data represent experiments from six pairs of primary MEFs. (C) 3T3 analysis (n=3) of p65^+/+ or p65^−/− MEFs after cells were induced to senesce with IR and switched from 3% to 20% O₂. Arrowhead indicates the passage at which cells were irradiated. IR, γ-irradiation; MEF, mouse embryonic fibroblast.

Figure 5

p65 is required to maintain cellular senescence in HFs. (A) HFs were sequentially infected with retroviruses expressing human TERT and pBabepuro (HF-TERT/V) or pBabe-IκBα-SR (HF-TERT/SR) in a total of eight independent lines (supplementary Fig S8D online). Cells were treated with 400 μM H₂O₂ for 10 days to induce cellular senescence and subsequently immortalized by using intermittent H₂O₂ treatment. Cell numbers were determined on a weekly basis for 50 weeks. (B) Karyotyping results of immortalized HF from line HF-TERT/SR1-1. Chromosomes 1 and 9 are shown to indicate the unbalanced translocation event, whereas chromosome 8 is shown as a reference. Arrowheads indicate break points. (C) Western blot of γ-H2AX in proliferating (P35) HF-TERT/V and HF-TERT/SR cells following IR similar to that described in Fig 3A. (D) Expression levels of γ-H2AX at indicated time points in (C) were quantified by NIH image software from three independent experiments. HF, human fibroblast; IR, γ-irradiation; NIH, National Institutes of Health; SR, super repressor; TERT, telomerase reverse transcriptase.