Limits...
p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.

Hilpert M, Legrand C, Bluteau D, Balayn N, Betems A, Bluteau O, Villeval JL, Louache F, Gonin P, Debili N, Plo I, Vainchenker W, Gilles L, Raslova H - Stem Cell Reports (2014)

Bottom Line: We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition.Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion.This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U1009, Equipe labellisée Ligue Nationale contre le Cancer, 114 rue Edouard Vaillant, 94805 Villejuif, France; University Paris Sud, 114, rue Edouard Vaillant, 94805 Villejuif, France; Gustave Roussy, IFR54, 114, rue Edouard Vaillant, 94805 Villejuif, France; University Paris Diderot, 5 rue Thomas-Mann, 75205 Paris, France.

Show MeSH

Related in: MedlinePlus

Hematopoietic Stress in p19INK4d−/− Mice Leads to a Defect in Survival due to Increased HSC Exhaustion and Apoptosis(A) Survival rate of mice injected weekly with 5-FU. One of two independent experiments with similar results is presented. For each experiment, five mice were injected.(B–D) Cell cycle analysis by Ki-67/Hoechst costaining four days after 5-FU injection. Immunophenotype (B) and cell cycle distribution in LSK (C) and SLAM (D) populations are shown (n = 4).(E–G) Immunophenotype of DCF staining (E) and DCF mean fluorescence in LSK (F) and SLAM (G) populations before and after oxidative stress induction by menadione (n = 4).(H and I) Annexin-V staining before and after oxidative stress induction by menadione in LSK (H) and SLAM (I) populations (n = 4).(J and K) P-H2AX staining in LSK cells before and after oxidative stress induction with menadione. Representative pictures (J) are shown. Nuclei are stained with DAPI (blue) and DNA-DSBs with an anti-P-H2AX antibody (red). (K) The number of P-H2AX foci per 100 LSK cells.(L) Survival rate of sublethally irradiated mice reconstituted with 2 × 105 Lin− cells and injected with 5-FU 1 month after reconstitution. One of two independent experiments with similar results is presented. For each experiment, six mice were injected.(M and N) The distribution of cells in the cell cycle was determined by Ki-67/Hoechst costaining 2 days after 5-FU injection. Immunophenotype and frequencies of cell cycle phases in LSK (M) and SLAM (N) populations in 2-month-old transplanted lethally irradiated mice are shown (n = 6).(O and P) Annexin-V staining two days after 5-FU treatment in LSK (O) and SLAM (P) populations (n = 4).Mice that were 8 to 10 weeks old were used. KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4264042&req=5

fig3: Hematopoietic Stress in p19INK4d−/− Mice Leads to a Defect in Survival due to Increased HSC Exhaustion and Apoptosis(A) Survival rate of mice injected weekly with 5-FU. One of two independent experiments with similar results is presented. For each experiment, five mice were injected.(B–D) Cell cycle analysis by Ki-67/Hoechst costaining four days after 5-FU injection. Immunophenotype (B) and cell cycle distribution in LSK (C) and SLAM (D) populations are shown (n = 4).(E–G) Immunophenotype of DCF staining (E) and DCF mean fluorescence in LSK (F) and SLAM (G) populations before and after oxidative stress induction by menadione (n = 4).(H and I) Annexin-V staining before and after oxidative stress induction by menadione in LSK (H) and SLAM (I) populations (n = 4).(J and K) P-H2AX staining in LSK cells before and after oxidative stress induction with menadione. Representative pictures (J) are shown. Nuclei are stained with DAPI (blue) and DNA-DSBs with an anti-P-H2AX antibody (red). (K) The number of P-H2AX foci per 100 LSK cells.(L) Survival rate of sublethally irradiated mice reconstituted with 2 × 105 Lin− cells and injected with 5-FU 1 month after reconstitution. One of two independent experiments with similar results is presented. For each experiment, six mice were injected.(M and N) The distribution of cells in the cell cycle was determined by Ki-67/Hoechst costaining 2 days after 5-FU injection. Immunophenotype and frequencies of cell cycle phases in LSK (M) and SLAM (N) populations in 2-month-old transplanted lethally irradiated mice are shown (n = 6).(O and P) Annexin-V staining two days after 5-FU treatment in LSK (O) and SLAM (P) populations (n = 4).Mice that were 8 to 10 weeks old were used. KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S2.

Mentions: p19INK4d−/− mice exhibited a markedly increased mortality after weekly injections of 5-FU (Figure 3A). To confirm that the increased mortality is related to a more rapid exhaustion of p19INK4d−/− HSCs as a consequence of the enhanced G0/G1 transition that is followed by apoptosis, we analyzed the cell cycle of WT and p19INK4d−/− HSC/Ps 4 days after 5-FU injection (Figure 3B). As expected, we observed a substantial decrease in quiescent p19INK4d−/− LSK (2.6-fold) and SLAM (11.6-fold) cell numbers, with a concomitant increase in G1 phase cells, whereas no differences in the number of cells in S/G2-M phases were observed (Figures 3C and 3D).


p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.

Hilpert M, Legrand C, Bluteau D, Balayn N, Betems A, Bluteau O, Villeval JL, Louache F, Gonin P, Debili N, Plo I, Vainchenker W, Gilles L, Raslova H - Stem Cell Reports (2014)

Hematopoietic Stress in p19INK4d−/− Mice Leads to a Defect in Survival due to Increased HSC Exhaustion and Apoptosis(A) Survival rate of mice injected weekly with 5-FU. One of two independent experiments with similar results is presented. For each experiment, five mice were injected.(B–D) Cell cycle analysis by Ki-67/Hoechst costaining four days after 5-FU injection. Immunophenotype (B) and cell cycle distribution in LSK (C) and SLAM (D) populations are shown (n = 4).(E–G) Immunophenotype of DCF staining (E) and DCF mean fluorescence in LSK (F) and SLAM (G) populations before and after oxidative stress induction by menadione (n = 4).(H and I) Annexin-V staining before and after oxidative stress induction by menadione in LSK (H) and SLAM (I) populations (n = 4).(J and K) P-H2AX staining in LSK cells before and after oxidative stress induction with menadione. Representative pictures (J) are shown. Nuclei are stained with DAPI (blue) and DNA-DSBs with an anti-P-H2AX antibody (red). (K) The number of P-H2AX foci per 100 LSK cells.(L) Survival rate of sublethally irradiated mice reconstituted with 2 × 105 Lin− cells and injected with 5-FU 1 month after reconstitution. One of two independent experiments with similar results is presented. For each experiment, six mice were injected.(M and N) The distribution of cells in the cell cycle was determined by Ki-67/Hoechst costaining 2 days after 5-FU injection. Immunophenotype and frequencies of cell cycle phases in LSK (M) and SLAM (N) populations in 2-month-old transplanted lethally irradiated mice are shown (n = 6).(O and P) Annexin-V staining two days after 5-FU treatment in LSK (O) and SLAM (P) populations (n = 4).Mice that were 8 to 10 weeks old were used. KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264042&req=5

fig3: Hematopoietic Stress in p19INK4d−/− Mice Leads to a Defect in Survival due to Increased HSC Exhaustion and Apoptosis(A) Survival rate of mice injected weekly with 5-FU. One of two independent experiments with similar results is presented. For each experiment, five mice were injected.(B–D) Cell cycle analysis by Ki-67/Hoechst costaining four days after 5-FU injection. Immunophenotype (B) and cell cycle distribution in LSK (C) and SLAM (D) populations are shown (n = 4).(E–G) Immunophenotype of DCF staining (E) and DCF mean fluorescence in LSK (F) and SLAM (G) populations before and after oxidative stress induction by menadione (n = 4).(H and I) Annexin-V staining before and after oxidative stress induction by menadione in LSK (H) and SLAM (I) populations (n = 4).(J and K) P-H2AX staining in LSK cells before and after oxidative stress induction with menadione. Representative pictures (J) are shown. Nuclei are stained with DAPI (blue) and DNA-DSBs with an anti-P-H2AX antibody (red). (K) The number of P-H2AX foci per 100 LSK cells.(L) Survival rate of sublethally irradiated mice reconstituted with 2 × 105 Lin− cells and injected with 5-FU 1 month after reconstitution. One of two independent experiments with similar results is presented. For each experiment, six mice were injected.(M and N) The distribution of cells in the cell cycle was determined by Ki-67/Hoechst costaining 2 days after 5-FU injection. Immunophenotype and frequencies of cell cycle phases in LSK (M) and SLAM (N) populations in 2-month-old transplanted lethally irradiated mice are shown (n = 6).(O and P) Annexin-V staining two days after 5-FU treatment in LSK (O) and SLAM (P) populations (n = 4).Mice that were 8 to 10 weeks old were used. KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S2.
Mentions: p19INK4d−/− mice exhibited a markedly increased mortality after weekly injections of 5-FU (Figure 3A). To confirm that the increased mortality is related to a more rapid exhaustion of p19INK4d−/− HSCs as a consequence of the enhanced G0/G1 transition that is followed by apoptosis, we analyzed the cell cycle of WT and p19INK4d−/− HSC/Ps 4 days after 5-FU injection (Figure 3B). As expected, we observed a substantial decrease in quiescent p19INK4d−/− LSK (2.6-fold) and SLAM (11.6-fold) cell numbers, with a concomitant increase in G1 phase cells, whereas no differences in the number of cells in S/G2-M phases were observed (Figures 3C and 3D).

Bottom Line: We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition.Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion.This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U1009, Equipe labellisée Ligue Nationale contre le Cancer, 114 rue Edouard Vaillant, 94805 Villejuif, France; University Paris Sud, 114, rue Edouard Vaillant, 94805 Villejuif, France; Gustave Roussy, IFR54, 114, rue Edouard Vaillant, 94805 Villejuif, France; University Paris Diderot, 5 rue Thomas-Mann, 75205 Paris, France.

Show MeSH
Related in: MedlinePlus