Article Figures & Data
Figures
- Fig. 1.
53BP2 levels increase following UV irradiation-induced DNA damage. (A) Western blots of lysates (30 μg of total protein per lane) prepared from GM38 cells (left panels) and HT1080 cells (right panels) 24 h after UV irradiation with 0, 5, or 10 J/m2. The fold increase of 53BP2 protein levels compared to no-UV-irradiation levels are indicated below, normalized for tubulin. 53BP2 was detected with monoclonal antibody DX547; p53 was detected with monoclonal antibody 1801. (B) Northern blot of total RNA (15 μg/lane) prepared from HT1080 cells 24 h after UV irradiation at the indicated doses, probed for 53BP2, stripped, and reprobed for actin. The fold increase of 53BP2 mRNA is indicated below, normalized for actin.
- Fig. 2.
53BP2 expression in cell lines containing wild-type or mutant p53. (A) The top panels show Western blots of lysates (30 μg of total protein per lane) prepared from human 041 and 087 LFS skin fibroblasts heterozygous for wild-type p53 (+/−) (lanes 2 and 4, respectively) or homozygous for p53 mutations (−/−) (lanes 3 and 5, respectively) and from normal primary human skin fibroblasts (GM38) homozygous for wild-type p53 (+/+) (lane 1). The fold differences, compared to GM38 cells, of 53BP2 protein normalized for tubulin are indicated below. 53BP2 protein was detected with monoclonal antibody DX547. The bottom panels show Northern blots of total RNA (15 μg/lane) prepared from cells as indicated above, probed for 53BP2, stripped, and reprobed for actin. The fold differences, compared to GM38 cells, of 53BP2 mRNA normalized for actin are indicated below. (B) Western blots of lysates (30 μg of total protein per lane) prepared from mouse fibroblasts homozygous for wild-type p53 (3T3, p21-null, and XPA+/−) or from p53 knockout mouse fibroblasts (p53-null). The fold differences, compared to 3T3 cells, of 53BP2 protein normalized for tubulin are indicated below. 53BP2 detected with rabbit antisera Rab-1.
- Fig. 3.
Expression of wild-type p53 in the absence of cellular damage decreases 53BP2 levels. (A) Western blot of lysates (30 μg of total protein per lane) prepared from 041TR cells at the indicated times after tetracycline withdrawal. The decreases in 53BP2 protein, as a fraction of baseline levels determined at time zero, are indicated below, normalized for tubulin. 53BP2 was detected with monoclonal antibody DX547; p53 was detected with monoclonal antibody 1801. (B) Northern blot of total RNA (15 μg/lane) prepared from the 041TR cells above at the indicated time points, probed for 53BP2, stripped, and reprobed for GAPDH. The decreases in 53BP2 mRNA, as a fraction of baseline levels determined at time zero, are indicated below, normalized for GAPDH.
- Fig. 4.
p53-independent mechanisms participate in the UV irradiation induction of 53BP2. (A) Western blot of lysates (15 μg of total protein per lane) prepared from 041−/− cells 24 h after UV irradiation with 0, 5, or 10 J/m2. The fold increase of 53BP2 protein compared to no-UV-irradiation lysates are indicated below, normalized for tubulin. (B) Western blot of equivalent amounts of lysates prepared from 041TR cells maintained in 2 μg of tetracycline per ml at the indicated times after UV irradiation at 20 J/m2. The fold increases of 53BP2 protein compared to the levels at time zero are indicated below, normalized for tubulin. (C) The upper panels show Western blots of equivalent amounts of lysates prepared from 041TR cells at the indicated times after tetracycline withdrawal. At time zero, cells were UV irradiated with 20 J/m2. The fold increases of 53BP2 protein compared to levels at time zero are indicated below, normalized for tubulin. 53BP2 protein was detected with monoclonal antibody DX547; p53 was detected with monoclonal antibody 1801. The lower panels show Northern blots of total RNA (15 μg/lane) prepared from the 041TR cells above at the indicated time points, probed for 53BP2, stripped, and reprobed for GAPDH. The fold increases of 53BP2 mRNA compared to the levels at time zero are indicated below, normalized for GAPDH.
- Fig. 5.
Expression of 53BP2 sensitizes cells to UV irradiation-induced apoptosis. (A) Percentage of 293/53BP2 and 293/LZ cells with apoptotic nuclear morphology 48 h after UV irradiation (shaded bars) or no UV irradiation (open bars). Cells induced to express 53BP2 or β-galactosidase (+) were treated with 2.5 μM ponasterone A for 24 h prior to UV irradiation. Mock-induced cells (−) were treated with an equivalent volume of control carrier (ethanol). Shown are the means and standard deviations from triplicate plates. (B) Western blot using anti-53BP2 monoclonal antibody DX547 of equivalent amounts of lysates prepared from 293/53BP2 cells induced for 24 h with 2.5 μM ponasterone A (+) or ethanol (−).
- Fig. 6.
Expression of 53BP2 decreases clonogenic survival in response to UV irradiation. (A) The left graph shows the surviving fraction of HT/53BP2 cells after induction of 53BP2 expression with 5 μM ponasterone A (■) or ethanol control carrier (⋄) for 48 h prior to UV irradiation at the indicated doses. Shown are the means and standard deviations from triplicate plates. The right graph shows the surviving fraction of HT/LZ cells after the induction of LacZ expression with 5 μM ponasterone A (■) or ethanol control carrier (⋄) for 48 h prior to UV irradiation at the indicated doses. Colonies were fixed, stained, and counted after 12 days. (B) Western blot using anti-53BP2 monoclonal antibody DX547 of equivalent amounts of lysates prepared from HT/53BP2 cells induced for 48 h with 5 μM ponasterone A (+) or ethanol (−).
- Fig. 7.
Attenuation of 53BP2 induction enhances clonogenic survival following UV irradiation. (A) Surviving fraction of HT1080 cells after introduction of AS-oligo (■) or control-oligo (⋄). Oligonucleotide-treated cells were incubated for 5 h, followed by counting, replating, and UV irradiation at the indicated doses. Shown is a representative experiment with the means and standard deviations from triplicate plates. (B) Western blot using anti-53BP2 monoclonal antibody DX547 of equivalent amounts of lysates (50 μg of total protein per lane) prepared from HT1080 cells after the introduction of AS-oligo or control-oligo. Oligonucleotide treated cells were incubated and UV irradiated in parallel with the clonogenic assay. Lysates were prepared 24 h after UV irradiation. Anti-hsc70 monoclonal antibody was used to confirm equal loading.
- Fig. 8.
Model of 53BP2 pathways. In undamaged cells, p53 mediates the suppression of 53BP2 levels. UV damage can relieve this suppression and increase 53BP2 levels. Through unknown mechanisms, 53BP2 can inhibit cell growth and promote damage-induced apoptosis after UV irradiation, as well as enhance p53 transactivation and cell cycle arrest. Lines with arrowheads denote positive regulation; the slashed line denotes negative regulation. “p53*” denotes the DNA damage-activated state.
