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First published online 3 April 2007
doi: 10.1242/jcs.003152

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Inflammatory ROS promote and cooperate with the Fanconi anemia mutation for hematopoietic senescence

¹ Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
² Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. TNF suppresses HSC/progenitor cell activity. (A) Effect of TNF on HSC frequency. Wild-type (WT) or Fancc–/– mice were injected intraperitoneally (i.p.) with mouse recombinant TNF in PBS at a dose of 0.1 mg/kg per day for two consecutive days. The mice were then sacrificed 24 hours later and BM mononuclear cells were prepared and stained with antibodies against lineage markers (Lin) and Sca-1-PE and c-Kit-PE-Cy7. Cells were then analyzed by flow cytometry to obtain fractions representing HSCs. The frequencies of LSK cells as a percentage of total BM mononuclear cells are indicated. (B) The effect of TNF on colony-forming activity of WT and Fancc–/– BM progenitor cells was evaluated using LSK cells isolated from control (PBS-injected) or TNF-injected mice. Data shown represent the mean ± s.d. of colony numbers from three independent experiments; *P<0.05. (C) TNF inhibited BM-progenitor proliferation. LSK cells isolated from TNF-injected mice were analyzed for series-plating efficiency of hematopoietic progenitor cells. Data represents the mean ± s.d. of three experiments. (D) TNF inhibited HSC renewal. 2x106 BM cells isolated from control (PBS-injected) or TNF-injected mice (CD45.2+) were transplanted together with 1x106 competitor cells from B6.BoyJ mice (CD45.1+) into lethally irradiated recipient (B6.BoyJ) mice and long-term engraftment was evaluated 16 weeks after transplantation. Shown are representative flow cytometric data of two independent experiments (n=6). Numbers in the corners indicate percent of events in that quadrant.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Analysis of differentiation, apoptosis and cell cycle of HSCs and progenitor cells from TNF-treated mice. (A) TNF did not affect the differentiation of long-term repopulating HSCs. Donor-derived (CD45.2+) leukocytes in peripheral blood of recipient mice transplanted with the indicated BM cells were stained with antibodies that recognize Gr-1 and Mac-1 or B220 and CD3 or Ter119. Data are expressed as mean ± s.d. of three independent experiments, each with three recipients (nine mice per group). (B) TNF induced apoptosis in BM LSK cells of mice. BM cells from PBS- or TNF-treated WT and Fancc–/– mice were stained with lineage maker antibodies together with Sca-1 and c-Kit antibodies, and then with annexin V. Percentages of apoptosis in the LSK population were analyzed by flow cytometry. Numbers in the quadrants indicate percent of cells labeled for 7-AADlowannexin V+ or 7-AADhighannexin V+. (C) BM LSK cells from TNF-injected Fancc–/– mice show increased G2-M arrest. BM cells from PBS- or TNF-treated WT and Fancc–/– mice were gated for LSK cells and stained with propidium iodide (PI) followed by analysis for cell cycle distribution. Shown are representative flow cytometric presentations of three independent experiments. Numbers in plots indicate percent of cells in G2-M phases.

View larger version (67K): [in this window] [in a new window]   Fig. 3. TNF induces premature senescence in HSC/progenitor cells. (A) WT or Fancc–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. The mice were then sacrificed 24 hours later and BM LSK cells were isolated and stained for SA--gal. The bar graph shows the percentages of the cells stained positive for SA--gal; cells were quantified by counting >100 cells in random fields on a slide for each of three independent experiments. The data represent the mean ± s.d. of three independent experiments; *P<.05. (B) Senescence HP1- staining. BM LSK cells isolated from PBS- or TNF-treated WT and Fancc–/– mice were stained for HP1-. DNA was then labeled with DAPI. (C) Frozen (SA--gal; magnification, 20x) or paraffin-embedded (HP1-; magnification, 40x) BM sections of PBS- or TNF-treated WT and Fancc–/– mice were subjected to SA--gal (left) or HP1- (right) staining. (D) Paraffin-embedded spleen sections of PBS- or TNF-treated WT and Fancc–/– mice, stained with antibody against the proliferation marker Ki-67 (magnification, 20x). (E) BM LSK cells of TNF-treated Fancc–/– mice showed strong immunostaining for p53 and p16Ink4a. The BM LSK cells isolated from PBS- or TNF-treated WT and Fancc–/– mice were stained with the antibodies against p53 and p16Ink4a and then counterstained with DAPI. The bar graph shows the percentages of the cells stained positive for p53 and p16Ink4a; cells were quantified by counting >100 cells in random fields on a slide for each of three independent experiments; *P<0.05.

View larger version (46K): [in this window] [in a new window]   Fig. 4. TNF-induced senescence is mediated by oxidative stress in Fancc-deficient HSCs and progenitor cells. (A) The ROS scavenger NAC mitigated TNF-induced senescence in HSCs and progenitor cells. WT and Fancc–/– mice were injected i.p. with TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse per day) was administered 30 minutes before and after each TNF injection. The isolated BM LSK cells (top and middle groups of panels) or paraffin-embedded BM sections (bottom group of panels; magnification, 40x) were stained with the antibody against the senescence marker HP1-. The LSK cell slides were also counterstained with DAPI. (B) BM LSK cells from WT and Fancc–/– mice were cultured in the absence or presence of H2O2 (100 µM) for 45 minutes, washed and further cultured in fresh medium for 24 hours. Cells were then stained with the antibody against the senescence marker HP1-. Shown is quantification of HP1--positive cells by scoring >100 cells in random fields on a slide for each of three independent experiments; *P<0.05. (C) BM LSK cells from WT and Fancc–/– mice were cultured in the absence or presence of H2O2 (100 µM) for 45 minutes, washed and further cultured in fresh medium for 24 hours. 1000 cells were mixed with 1x106 competitor cells were transplanted into lethally irradiated recipient mice and long-term engraftment was evaluated 16 weeks after transplantation. Data are expressed as mean ± s.d. of two independent experiments, each with six recipients (12 mice per group). P<0.05 between untreated and treated Fancc–/– samples. (D) ROS production. BM cells from PBS- or TNF-treated WT and Fancc–/– mice were labeled with CM-H2DCFDA followed by flow cytometry. (E) ROS production was mediated by TNF. WT, Fancc–/– mice or their littermates lacking the Tnfr1 gene were injected with PBS or TNF at 0.1 mg/kg per day for 2 consecutive days. 24 hours after TNF injection, BM cells from individual animals were isolated and labeled with CM-H2DCFDA followed by flow cytometry. Data represent the mean ± s.d. of two independent experiments, each with six mice (total 12 mice per group). (F) WT, Fancc–/– mice or their littermates lacking the Tnfr1 gene were injected with TNF (0.1 mg/kg per day) for 2 consecutive days. TNF-treated mice were injected with 20 µg of a TNF-neutralizing antibody 30 minutes after each TNF injection. At 24 hours after TNF injection, frozen spleen sections were prepared and subjected to SA--gal staining.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Role of TNF and ROS in hematopoietic suppression. (A) The ROS scavenger NAC or deletion of Tnfr1 gene rescued progenitor growth. WT, Fancc–/– mice or their littermates lacking Tnfr1 were injected with PBS or TNF (0.1 mg/kg per day) for 2 consecutive days. Mice were injected with NAC (1 mg/mouse per day) 30 minutes before and after TNF injection. Twenty-four hours later, BM cells were isolated and subjected to clonogenic assay. Data shown represent the mean ± s.d. of the total number of colonies from three independent experiments; *P<0.05. (B) Anti-oxidant NAC or deletion of the Tnfr1 gene restored HSC self-renewal ability. 2x106 BM mononuclear cells isolated from the mice described in A were transplanted together with 1x106 competitor cells from B6.BoyJ mice (CD45.1+) into lethally irradiated recipient mice; long-term engraftment was evaluated 16 weeks after transplantation. Data represent the mean ± s.d. of three independent experiments with three recipients per group for each experiment. *P<0.05.

View larger version (44K): [in this window] [in a new window]   Fig. 6. TNF-induced senescence of HSCs and progenitor cells is associated with oxidative DNA damage and genomic instability. (A) WT or Fancc–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse per day) was administered 30 minutes before and after each TNF injection. The mice were then sacrificed 24 hours later and BM cells from individual mice were analyzed for DNA strand breaks in a the comet assay. Larger tails represents higher levels of DNA damage. For each treatment, at least 100 cells were scored from random sampling. Data are expressed as the mean ± s.d. of two independent experiments, each with three mice (six mice per group). (B) WT or Fancc–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse/day) was administered 30 minutes before and after each TNF injection. The mice were then sacrificed 24 hours later and BM LSK cells were isolated and stained for the oxidative DNA damage marker 8-oxo-deoxyguanosin (8-oxodG). The bar graph shows the percentages of the cells stained positive for 8-oxodG and quantified by counting >100 cells in random fields on a slide for each of two experiments with total 6 mice. (C) Examples of metaphase chromosomes prepared from TNF-treated WT and Fancc–/– BM cells. Arrows indicate aberrant chromosomes. (D) WT or Fancc–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse/day) was administered 30 minutes before and after each TNF injection. Mice were sacrificed 24 hours later and BM LSK cells were then isolated and stained for p53Ser20 and H2AX. Bar graphs show the percentages of the cells stained positive for p53Ser20 and H2AX. Cells were quantified by counting >100 cells in random fields on a slide for each of two experiments with total six mice. *P<0.05. (E) Kinetics of DNA repair of oxidative DNA damage and DNA strand breaks. BM cells from WT and Fancc–/– mice were treated with or without H2O2 (100 µM) for the indicated time periods, and protein extracts were prepared and analyzed by immunoblotting with antibody against phosphorylated p53Ser20 (p53Ser20) and anti-H2AX and anti-actin antibodies. Extracts were also prepared from cells 2 hours (2-) after H2O2 withdrawal after the cells had been treated with H2O2 for 4 hours.

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