p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice - PubMed (original) (raw)

p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice

Irene M Ward et al. Mol Cell Biol. 2003 Apr.

Abstract

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.

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Figures

FIG. 1.

FIG. 1.

Targeted disruption of the mouse 53BP1 gene. (A) Map of the genomic locus surrounding the targeted exon, the targeting vector containing the PGK-neo cassette, and the targeted locus. A 5′-flanking probe used for screening ES cell clones and mice is indicated. (B) Southern blot analysis of _Eco_RI-digested genomic DNA. (C) Multiplex PCR genotype analysis with a primer pair for the neo gene (resulting in a 455-bp product) and a 5′ external exon (resulting in a 270-bp product). (D) Western blot of cell extracts from mouse testes with an antibody specific for the N terminus of 53BP1. (E) Immunofluorescence analysis of irradiated (1 Gy) 53BP1+/+ and 53BP1−/− MEFs with polyclonal antibodies raised against the N terminus of 53BP1.

FIG. 2.

FIG. 2.

53BP1 deficiency results in growth retardation, cell cycle defect, radiosensitivity, and chromosomal instability. (A) Body weights of female 53BP1 wild-type, heterozygous, and knockout mice at 5 months of age. (B) G2/M arrest of 53BP1+/+ and 53BP1−/− embryonic cells in response to 0.5 or 5 Gy of IR. Cells were stained with anti-P-Histone 3 antibody 1 h after IR and analyzed by fluorescence-activated cell sorting. (C) G2 accumulation of 53BP1+/+ and 53BP1−/− MEFs several hours after IR. Cells were pulse-labeled for 1 h with BrdU before exposure to 6 Gy of IR. The cell cycle profile of BrdU-positive cells was analyzed by staining with propidium iodide. Consistent data were obtained in three independent experiments. (D) IR-induced intra-S-phase checkpoint in 53BP1+/+ and 53BP1−/− MEFs. DNA synthesis was assessed by [3H]thymidine incorporation 30 min after exposure to 20 Gy of IR. (E) Sensitivity of 10 pairs of female 53BP1−/− and 53BP1+/+ littermates to 8 Gy of whole body IR. Similar results were observed with 10 male pairs. (F) Chromosomal instability in 53BP1−/− MEFs. 100 metaphase spreads from genetically matched passage three 53BP1+/+ and 53BP1−/− MEFs were analyzed. Consistent data were obtained from three different experiments.

FIG. 3.

FIG. 3.

53BP1−/− mice are tumor prone. (A) CD4 and CD8 cell surface expression of cells from two different thymic lymphomas was assessed by flow cytometry. (B) Massive thymic lymphoma (T) in a 4-month-old 53BP1−/− mouse. (C) Hematoxylin-and-eosin-stained sections from the thymus, spleen, and kidney of a 53BP1−/− animal with thymic lymphoma. Monomorphic lymphoblastic tumor cells are dominant in all three tissues. The arrows indicate mitotic figures. G, glomerulus; PT, proximal tubulus. (D) Overall survival of 53BP1+/+ (n = 54), 53BP1+/− (n = 97), and 53BP1−/− (n = 101) mice over a period of 10 months.

FIG. 4.

FIG. 4.

Irradiated 53BP−/− MEFs show impaired Chk2 activation after low doses of IR. (A) γ-H2AX and NBS1 focus formation 6 h after exposure to 6 Gy of IR is unaffected in 53BP1−/−MEFs compared to 53BP1 wild-type MEFs, as assessed by immunofluorescence staining. (B) Coimmunoprecipitation of 53BP1 and Chk2 in untreated or irradiated 293T cells. (C) Activated Chk2 phosphorylated at Thr 68 (Chk2T68) forms foci in 53BP1+/+ MEFs 1 h after exposure to 1 Gy of IR. No foci are detectable in 53BP1−/− MEFs at this low dose of IR. γ-H2AX staining is shown as a control. (D) Chk2T68P foci form in both 53BP1+/+ and 53BP1−/− cells in response to 20 Gy of IR. (E) Chk2 mobility shift is reduced in 53BP1−/− MEFs in response to low-dose radiation. Cell lysates from 53BP1+/+ and 53BP1−/− MEFs were prepared 1 h after IR and immunoblotted with anti-Chk2 antibody. (F) For better comparison of the Chk2 mobility shift, lysates from irradiated 53BP1+/+ and 53BP1−/− cells were run side by side. (G) The guinea pig anti-Chk2T68P antibody specifically recognizes Chk2.

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