Figure 2
Figure 2. DNA damage and gene expression changes are reverted in stimulated, telomere dysfunctional HSCs that enter the cell cycle. (A-C) To analyze gene expression and DNA damage in quiescent and cycling HSCs under homeostatic conditions, HSCs were freshly isolated from 12-month-old, nonstimulated G3mTerc−/− mice and mTerc+/+ mice: quantitative polymerase chain reaction analysis of differentially expressed genes in G0-HSCs (gray bars) and G1/S/G2/M HSCs (black bars) (A). Note that gene expression differences between HSCs of G3mTerc−/− mice and mTerc+/+ mice were more pronounced in quiescent HSCs. Three genes (Dusp2, Fyb, and Clasp1), which were not differentially regulated in gene array analysis of HSCs from G3mTerc−/− compared with mTerc+/+ mice, were chosen as negative control (A, right). Values are shown as mean ± standard error of the mean (SEM). *P < .05. (B-C) The histograms show the percentage of cell nuclei staining positive for the indicated numbers of γH2AX foci in quiescent HSCs (B) and cycling HSCs (C) (100 nuclei were counted per cell type and group) of G3mTerc−/− and mTerc+/+ mice. (D-E) Twelve-month-old G3mTerc−/− mice were treated with interferon-α to stimulate cell cycle activity (supplemental Figure 1A-B). Interferon-α (10 000 U per mouse) or PBS was injected into 12-month-old G3mTerc−/− mice intraperitoneally. Freshly isolated bone marrow cells were analyzed 16 hours after stimulation. (D) Messenger RNA expression of Puma in cycling HSCs (black bars) compared with quiescent HSCs (gray bars). Data are shown as mean ± SEM (n = 3 for each group). (E) Rate of apoptosis (annexin V–positive cells) in quiescent HSCs and activated HSCs of G3mTerc−/− mice treated with either PBS or interferon-α. Data are shown as mean ± SEM (n = 3 mice per group). (F) This histogram shows the relative expression of p16 in quiescent HSCs from 12-month-old G3mTerc−/− and age-matched mTerc+/+ mice treated with PBS or interferon-α. Data are shown as mean ± SEM (n = 3 mice per group). (G) The histogram shows the percentage of cell nuclei staining of γH2AX foci for quiescent HSCs from G3mTerc−/− mice treated with either PBS (gray bar) or interferon-α (black bar) (100 nuclei were counted per group). (H-I) The pie charts depict the composite of clones generated from freshly isolated single HSCs from 12-month-old G3mTerc−/− (332) and age-matched mTerc+/+ (310) mice. Cell numbers were counted on day 6 (H) and day 12 (I) after plating. HSCs were cultured individually in stem cell medium (Stem Cell Technology) with stem cell factor (30 ng/mL) and thrombopoietin (20 ng/mL).

DNA damage and gene expression changes are reverted in stimulated, telomere dysfunctional HSCs that enter the cell cycle. (A-C) To analyze gene expression and DNA damage in quiescent and cycling HSCs under homeostatic conditions, HSCs were freshly isolated from 12-month-old, nonstimulated G3mTerc−/− mice and mTerc+/+ mice: quantitative polymerase chain reaction analysis of differentially expressed genes in G0-HSCs (gray bars) and G1/S/G2/M HSCs (black bars) (A). Note that gene expression differences between HSCs of G3mTerc−/− mice and mTerc+/+ mice were more pronounced in quiescent HSCs. Three genes (Dusp2, Fyb, and Clasp1), which were not differentially regulated in gene array analysis of HSCs from G3mTerc−/− compared with mTerc+/+ mice, were chosen as negative control (A, right). Values are shown as mean ± standard error of the mean (SEM). *P < .05. (B-C) The histograms show the percentage of cell nuclei staining positive for the indicated numbers of γH2AX foci in quiescent HSCs (B) and cycling HSCs (C) (100 nuclei were counted per cell type and group) of G3mTerc−/− and mTerc+/+ mice. (D-E) Twelve-month-old G3mTerc−/− mice were treated with interferon-α to stimulate cell cycle activity (supplemental Figure 1A-B). Interferon-α (10 000 U per mouse) or PBS was injected into 12-month-old G3mTerc−/− mice intraperitoneally. Freshly isolated bone marrow cells were analyzed 16 hours after stimulation. (D) Messenger RNA expression of Puma in cycling HSCs (black bars) compared with quiescent HSCs (gray bars). Data are shown as mean ± SEM (n = 3 for each group). (E) Rate of apoptosis (annexin V–positive cells) in quiescent HSCs and activated HSCs of G3mTerc−/− mice treated with either PBS or interferon-α. Data are shown as mean ± SEM (n = 3 mice per group). (F) This histogram shows the relative expression of p16 in quiescent HSCs from 12-month-old G3mTerc−/− and age-matched mTerc+/+ mice treated with PBS or interferon-α. Data are shown as mean ± SEM (n = 3 mice per group). (G) The histogram shows the percentage of cell nuclei staining of γH2AX foci for quiescent HSCs from G3mTerc−/− mice treated with either PBS (gray bar) or interferon-α (black bar) (100 nuclei were counted per group). (H-I) The pie charts depict the composite of clones generated from freshly isolated single HSCs from 12-month-old G3mTerc−/− (332) and age-matched mTerc+/+ (310) mice. Cell numbers were counted on day 6 (H) and day 12 (I) after plating. HSCs were cultured individually in stem cell medium (Stem Cell Technology) with stem cell factor (30 ng/mL) and thrombopoietin (20 ng/mL).

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