Lack of protection from tumorigenesis by loss of Puma in the absence of tissue depletion or by overexpression of Bcl-x in the T-lymphoid linage. (A) Cohorts of adult wild-type (n = 10) and puma^−/− (n = 11) mice received a single injection of 3-MC or vehicle (seven mice per genotype) i.m. in the gluteus. Local tumor growth was monitored over 255 d, and animals were sacrificed when tumors reached ≥1 cm in size. (B) Cohorts of lck-bcl-x tg (n = 9) mice were monitored for tumorigenesis triggered by fractionated IR, and were monitored for the development of thymic lymphomas over time. Triangles represent mice that developed tumors distinct from thymic lymphomas, and diamonds represent mice that were tumor-free at 730 d. (C) Wild-type, puma^−/−, and lck-bcl-x tg mice were exposed to a single dose of IR (1.75 Gy). Animals were sacrificed after 24 h, and thymic single-cell suspensions were counted and stained for cell surface markers, followed by flow cytometric analysis. Representative dot blots from three independent experiments and animals per genotype defining the changes in the percentages of CD4⁺8⁺ thymocytes upon IR are shown. (D) Quantification of BrdU incorporation and S-phase activity in lck-bcl-x tg transgenic mice, performed as described in Figure 1B. Statistically significant differences in BrdU uptake were observed between wild-type versus bcl-x tg mice on day 3 (P = 0.007).

Loss of Puma prevents compensatory proliferation of stem cells upon DNA damage. (A) To compare the rates of proliferation in different stem cell populations, the mice of the indicated genotpyes were exposed to IR, and, on days 3 and 7, were injected i.p. with a single dose of BrdU. Four hours later, mice were sacrificed, and the percentage of BrdU⁺ cells was assessed by flow cytometry in the individual stem cell subsets or total bone marrow. The relative increase in proliferation (BrdU⁺ cells) in relation to untreated controls was calculated by using the following equation: (IR-induced proliferation percent − spontaneous proliferation percent)/(100 − spontaneous proliferation percent). Bars represent fold induction of BrdU⁺ cycling cells as mean ± SEM of three to four animals per genotype and three independent experiments. (*) Significant differences in BrdU uptake were observed between wild-type and puma^−/− or p53^−/− MPP (P < 0.03) and LT-HSC (P < 0.01) subsets at days 3 or 7 post-IR. (B) Mice were fed BrdU in drinking water for 12 d, yielding between 70% and 90% of labeling efficiency in both genotypes alike. Then mice were exposed to 1.75 Gy IR at days 0 and 7. The percentage of BrdU⁺ stem cell subsets was assessed by flow cytometry. IR-induced loss of BrdU as an indirect measure of proliferation was calculated by subtracting the percentage of BrdU⁺ cells of mice pre-exposed to IR from the percentage of BrdU⁺ cells in control animals. Symbols represent mean ± SEM of three to four animals per genotype and time point. Spontaneous BrdU loss was not different between genotypes (P > 0.78). (*) BrdU loss was significantly different at all time points after IR in MPP (P < 0.014) and LSK (P < 0.026) cells at days 3 and 7, and in LT-HSCs (P < 0.014) at days 7 and 10. (C) Cell cycle analysis using Ki67 was performed in control mice or mice exposed to IR 7 d before. The percentage of cells in G0 is plotted. Bars represent mean ± SEM of three animals per genotype. (*) The percentage of stem/progenitor cells in G0 was significantly higher in puma^+/−, puma^−/−, and p53^−/− when compared with wild-type mice (P < 0.04).

Loss of Puma delays IR-induced lymphomagenesis. Wild-type, puma^−/−, and p53^−/− mice 4–6 wk of age were exposed to 1.75 Gy of whole-body IR, and were sacrificed at the indicated time points. (A) The number of CD4⁺8⁺ thymocytes was quantified by cell counting and cell surface staining, followed by flow cytometric analysis. Each data point represents the mean ± SEM of three to six animals per genotype and time point. Statistically significant differences in cell number were observed between wild-type versus puma^−/− mice on days 1 and 3 after the first IR and day 1 after the second IR (P < 0.025); between wild-type versus bim^−/− mice on day 1 (P = 0.005); and between wild-type and p53^−/− mice on days 1, 3, and 7 after the first IR and day 1 after the second IR (P < 0.014). Untreated puma^−/− mice also showed increased cellularity compared with the other genotypes (P < 0.001). (B) Mice of the indicated genotypes were exposed to 1.75 Gy of IR at the indicated time points and were injected i.p. with BrdU 4 h before sacrifice. The percentage of cycling CD4⁺8⁺ thymocytes was defined by combined staining of cell surface markers, followed by intracellular staining of BrdU, followed by flow cytometric analysis. Data points represent the mean ± SEM of three to six animals per genotype and time point. Statistically significant differences in BrdU uptake were observed between wild-type versus puma^−/− mice on day 3 (P = 0.003), wild-type versus bim^−/− on day 3 (P < 0.015), and wild-type versus p53^−/− at day 3 after the first IR and day 1 after the second IR (P < 0.02). Untreated controls were not different across genotypes. Cohorts of wild-type (n = 16), puma^+/− (n = 12), puma^−/− (n = 20), bim^−/− (n = 9), and p53^−/− (n = 9) mice were subjected to fractionated IR (four times at1.75 Gy) at weekly intervals, starting at 4 wk of age, and were monitored for the development of tumors up to 730 d. (C) Kaplan-Meier analysis of thymic lymphoma-free survival of mice of the indicated genotypes. (D) Tumor-free survival of cohorts of mice shown in C. Mean survival in days ± SE: 201 ± 9 for wild type versus 570 ± 52 for puma^+/− versus 558 ± 38 for puma^−/− (Fig. 2A,B); P < 0.0001. Circles represent mice still alive, triangles represent mice that developed tumors distinct from thymic lymphomas, and diamonds represent mice that were tumor-free at the end of the observation period; i.e., 730 d.

Loss of Puma prevents stem cell apoptosis upon IR. (A) Cellularity of both femora and tibiae was assessed in mice of the indicated genotypes after exposure to a single dose of IR. (B–D) Cell counting and staining with cell surface marker-specific antibodies were used to identify and enumerate the different stem cell populations. Bars represent mean ± SEM of three to six animals per genotype and time point from three independent experiments. (*) LSK cell numbers were significantly different between wild-type or bim^−/− versus puma^−/− (P < 0.0002) or p53^−/− mice (P < 0.0014) at all time points analyzed after IR; MPP numbers were different between wild-type or bim^−/− versus puma^−/− (P < 0.04) or p53^−/− mice (P < 0.035) at days 1, 3, and 7 after IR; LT-HSC numbers were different between wild-type or bim^−/− versus puma^−/− (P < 0.038) or p53^−/− mice (P < 0.003) at day 7 after IR. (§) In untreated mice, LT-HSC numbers were different between bim^−/− and all other genotypes (P < 0.05).

Replication stress-associated DNA damage in wild-type HSCs after IR and restoration of thymic lymphoma development in puma^−/− mice by concomitant loss of p53. (A) Quantification of DNA damage foci in LSK cells and LT-HSCs derived from mice of the indicated genotypes. Six weeks after the fourth cycle of IR, stem cells were sorted from the bone marrow onto poly-L-lysine-coated coverslips and stained for γH2AX. (B) Quantification of the percentage of LT-HSCs containing a given number of γH2AX foci. (Insert) Immunofluorescence detection of γH2AX foci in LT-HSCs and LSK cells derived from wild-type, puma^−/−, and p53^−/− mice 6 wk after the last dose of irradiation. (Green) γH2AX; (blue) DAPI. Images were acquired with a Leica confocal scanning microscope, and the numbers of foci per cell were quantified by using the CellProfiler software, measuring an average of 100 cells per sample from four to six mice per genotype. (*) IR wild type versus puma^−/− or p53^−/− (P < 0.001). LT-HSCs from untreated p53^−/− mice show reduced foci number (P < 0.05). (B) Assessment of foci number per cell, in relation to the total number of cells analyzed. Cohorts of p53^+/− and p53^−/− mice proficient or deficient for puma were monitored for spontaneous tumorigenesis and tumor formation triggered by fractionated IR. (C,D) Kaplan-Meier analysis of tumor-free survival of untreated p53^+/− (n = 15), p53^−/− (n = 15), p53^+/−puma^−/− (n = 16), and p53^−/−puma^−/− (n = 15) mice (C), or thymic lymphoma-free survival of irradiated p53^+/− (n = 10), p53^−/− (n = 9), p53^+/−puma^−/− (n = 9), and p53^−/−puma^−/− (n = 5) mice (D). Mean survival in days ± SE: 176 ± 8 for p53^+/− versus 307 ± 37 for p53^+/−puma^−/−; P < 0.0001.