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Cited by 105Liver physiology and pathologyTelomeres, Telomerase, and SenescenceGenetics, Aging, and Longevity in Model Organisms

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The senotherapeutic drug ABT-737 disrupts aberrant p21 expression to restore liver regeneration in adult mice

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Ritschka, Birgit, Knauer-Meyer, Tania, Gonçalves, Daniel Sampaio, Mas, Alba, Plassat, Jean-Luc, Durik, Matej, Jacobs, Hugues, Pedone, Elisa, Di Vicino, Umberto, Cosma, Maria Pia, Keyes, William M.·2020·Genes & Development
DOI 10.1101/gad.332643.119PMC PMC7111259

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Abstract

Young mammals possess a limited regenerative capacity in some tissues, which is lost upon maturation. We investigated whether cellular senescence might play a role in such loss during liver regeneration. We found that following partial hepatectomy, the senescence-associated genes p21, p16 Ink4a , and p19 Arf become dynamically expressed in different cell types when regenerative capacity decreases, but without a full senescent response. However, we show that treatment with a senescence-inhibiting drug improves regeneration, by disrupting aberrantly prolonged p21 expression. This work suggests that senescence may initially develop from heterogeneous cellular responses, and that senotherapeutic drugs might be useful in promoting organ regeneration.

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Figure 1. p21 exhibits prolonged expression in adult livers with impaired regeneration. (A) Liver regeneration of young (2- to 3-mo-old) and adult (6- to 8-mo-old) mice was calculated as liver/body weight ratio at different days after PH (n = 4–13). (B) H&E staining of young and adult liver sections before (day 0) and at different time points after PH. All images are representative of at least five biological replicates. Scale bars, 20 µm. (C–E) qPCR analysis for the senescence markers p21 (C), p16Ink4a (D), and p19Arf (E) of young and adult livers at different days after PH normalized to young livers at day 0 (n = 5–27). (F) Immunohistochemistry for p21 in young and adult liver sections at different days after PH and quantification of p21-positive hepatocytes/field of view (n = 3). All images are representative of at least three biological replicates. Scale bars, 20 µm. (G,H) p21 expression in whole liver lysates at 0 d and 1 d (G) and 3 d (H) after PH measured by Western blot. Tubulin was used as a loading control (n = 2–3). (I) RNA in situ hybridization (ISH) staining and quantification for CDKN2A in young and adult liver sections before PH (day 0). Scale bars, 20 µm. Boxed area shows higher magnification of positive staining. Scale bar, 10 µm. All images are representative of at least five biological replicates. Quantification shows number of cells/field of view that have three or more dots/cell. (PC) Parenchymal cells; (NPC) nonparenchymal cells (n = 5–6). (J) RNA co-ISH staining for CDKN2A (blue) and ADGRE1 (red) in young and adult liver sections before PH (day 0). Scale bars, 20 µm. Boxed area shows higher magnification of positive staining. Scale bar, 10 µm. All images are representative of at least three biological replicates. Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001.
Figure 1. p21 exhibits prolonged expression in adult livers with impaired regeneration. (A) Liver regeneration of young (2- to 3-mo-old) and adult (6- to 8-mo-old) mice was calculated as liver/body weight ratio at different days after PH (n = 4–13). (B) H&E staining of young and adult liver sections before (day 0) and at different time points after PH. All images are representative of at least five biological replicates. Scale bars, 20 µm. (C–E) qPCR analysis for the senescence markers p21 (C), p16Ink4a (D), and p19Arf (E) of young and adult livers at different days after PH normalized to young livers at day 0 (n = 5–27). (F) Immunohistochemistry for p21 in young and adult liver sections at different days after PH and quantification of p21-positive hepatocytes/field of view (n = 3). All images are representative of at least three biological replicates. Scale bars, 20 µm. (G,H) p21 expression in whole liver lysates at 0 d and 1 d (G) and 3 d (H) after PH measured by Western blot. Tubulin was used as a loading control (n = 2–3). (I) RNA in situ hybridization (ISH) staining and quantification for CDKN2A in young and adult liver sections before PH (day 0). Scale bars, 20 µm. Boxed area shows higher magnification of positive staining. Scale bar, 10 µm. All images are representative of at least five biological replicates. Quantification shows number of cells/field of view that have three or more dots/cell. (PC) Parenchymal cells; (NPC) nonparenchymal cells (n = 5–6). (J) RNA co-ISH staining for CDKN2A (blue) and ADGRE1 (red) in young and adult liver sections before PH (day 0). Scale bars, 20 µm. Boxed area shows higher magnification of positive staining. Scale bar, 10 µm. All images are representative of at least three biological replicates. Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001.CC BY-NC · PubMed Central
Figure 2. Adult p21-deficient mice have partially improved regenerative capacity. (A) Liver/body weight ratios of 2- to 3-mo-old and 6- to 8-mo-old WT, p21−/−, and p19−/− mice at 3 and 7 d after PH (n = 4–12). (B) H&E staining of 6- to 8-mo-old p21−/− livers before and at different days after PH. All images are representative of at least four biological replicates. Scale bars, 20 µm. (C) Oil Red O staining of 6- to 8-mo-old WT and p21−/− livers 3 d after PH. All images are representative of at least four biological replicates. Scale bars, 50 µm. (D,E) qPCR analysis for (D) p16Ink4a and (E) p19Arf of 2- to 3-mo-old WT and p21−/− and 6- to 8-mo-old p21−/− livers at different days after PH normalized to 2- to 3-mo-old WT day 0. (n = 4–27). (F) Liver/body weight ratios of 6- to 8-mo-old WT and p16−/− mice 7 d after PH (n = 4–5). Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001.
Figure 2. Adult p21-deficient mice have partially improved regenerative capacity. (A) Liver/body weight ratios of 2- to 3-mo-old and 6- to 8-mo-old WT, p21−/−, and p19−/− mice at 3 and 7 d after PH (n = 4–12). (B) H&E staining of 6- to 8-mo-old p21−/− livers before and at different days after PH. All images are representative of at least four biological replicates. Scale bars, 20 µm. (C) Oil Red O staining of 6- to 8-mo-old WT and p21−/− livers 3 d after PH. All images are representative of at least four biological replicates. Scale bars, 50 µm. (D,E) qPCR analysis for (D) p16Ink4a and (E) p19Arf of 2- to 3-mo-old WT and p21−/− and 6- to 8-mo-old p21−/− livers at different days after PH normalized to 2- to 3-mo-old WT day 0. (n = 4–27). (F) Liver/body weight ratios of 6- to 8-mo-old WT and p16−/− mice 7 d after PH (n = 4–5). Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001.CC BY-NC · PubMed Central
Figure 3. ABT-737 improves regenerative capacity in adult livers. (A) Schematic representation of the experimental protocol. Intraperitoneal injection of DMSO or ABT-737 twice over 2 d was followed 1 d later by PH and mice were analyzed 1, 3, and 7 d after PH. (B) Liver/body weight ratios of 6- to 8-mo-old DMSO- or ABT-737-treated mice at different time points after PH (n = 5–11). (C) Representative macroscopic images of livers from DMSO- or ABT-737-treated mice 3 and 7 d after PH. Scale bars, 5 mm. (D) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) measured 7 d after PH (n = 4–5). (E) H&E staining of DMSO- and ABT-737-treated livers before and at different days after PH. All images are representative of at least five biological replicates. Scale bars, 20 µm. (F) Oil Red O staining of 6- to 8-mo-old DMSO- or ABT737-treated mice 3 d after PH. All images are representative of at least four biological replicates. Scale bars, 50 µm. Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01.
Figure 3. ABT-737 improves regenerative capacity in adult livers. (A) Schematic representation of the experimental protocol. Intraperitoneal injection of DMSO or ABT-737 twice over 2 d was followed 1 d later by PH and mice were analyzed 1, 3, and 7 d after PH. (B) Liver/body weight ratios of 6- to 8-mo-old DMSO- or ABT-737-treated mice at different time points after PH (n = 5–11). (C) Representative macroscopic images of livers from DMSO- or ABT-737-treated mice 3 and 7 d after PH. Scale bars, 5 mm. (D) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) measured 7 d after PH (n = 4–5). (E) H&E staining of DMSO- and ABT-737-treated livers before and at different days after PH. All images are representative of at least five biological replicates. Scale bars, 20 µm. (F) Oil Red O staining of 6- to 8-mo-old DMSO- or ABT737-treated mice 3 d after PH. All images are representative of at least four biological replicates. Scale bars, 50 µm. Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01.CC BY-NC · PubMed Central
Figure 4. ABT-737 decreases expression of p21 and SASP factors. (A) qPCR analysis for p21 in DMSO- and ABT-737-treated livers at different days after PH normalized to DMSO day 0 (n = 5–21). (B) Immunohistochemistry for p21 in DMSO- and ABT-737-treated livers at different time points after PH. All images are representative of at least four biological replicates. Scale bars, 20 µm. (C,D) p21 expression following treatment with DMSO or ABT-737 in whole liver lysates 1 d (C) and 3 d (D) after PH measured by Western blot. Tubulin was used as a loading control (n = 3). (E) qPCR analysis for p16Ink4a in DMSO- and ABT-737-treated livers at different days after PH normalized to DMSO day 0 (n = 5–21). (F) RNA ISH staining and quantification for CDKN2A in DMSO and ABT737 liver sections before PH (day 0). All images are representative of at least three biological replicates. Scale bars, 20 µm. Quantification shows number of cells/field of view that have three or more dots/cell. (n = 3) (PC) Parenchymal cells; (NPC) nonparenchymal cells. (G) qPCR analysis for p19Arf in DMSO- and ABT-737-treated livers at different days after PH normalized to DMSO day 0 (n = 5–21). (H) Venn diagram showing the overlap in 6- to 8-mo-old liver lysates ≥1.5-fold up-regulated proteins (compared with 2- to 3-mo-old liver lysates) and in ABT737-treated liver lysates ≤0.6-fold down-regulated proteins (compared with DMSO-treated liver lysates) (n = 2). (I) List of 19 overlapping proteins of Venn diagram in H. (J) qPCR analysis for p21 and selected SASP factors in DMSO- or ABT737-treated dermal fibroblasts 4 d after continuous treatment (n = 3). Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01 (***) P ≤ 0.001.
Figure 4. ABT-737 decreases expression of p21 and SASP factors. (A) qPCR analysis for p21 in DMSO- and ABT-737-treated livers at different days after PH normalized to DMSO day 0 (n = 5–21). (B) Immunohistochemistry for p21 in DMSO- and ABT-737-treated livers at different time points after PH. All images are representative of at least four biological replicates. Scale bars, 20 µm. (C,D) p21 expression following treatment with DMSO or ABT-737 in whole liver lysates 1 d (C) and 3 d (D) after PH measured by Western blot. Tubulin was used as a loading control (n = 3). (E) qPCR analysis for p16Ink4a in DMSO- and ABT-737-treated livers at different days after PH normalized to DMSO day 0 (n = 5–21). (F) RNA ISH staining and quantification for CDKN2A in DMSO and ABT737 liver sections before PH (day 0). All images are representative of at least three biological replicates. Scale bars, 20 µm. Quantification shows number of cells/field of view that have three or more dots/cell. (n = 3) (PC) Parenchymal cells; (NPC) nonparenchymal cells. (G) qPCR analysis for p19Arf in DMSO- and ABT-737-treated livers at different days after PH normalized to DMSO day 0 (n = 5–21). (H) Venn diagram showing the overlap in 6- to 8-mo-old liver lysates ≥1.5-fold up-regulated proteins (compared with 2- to 3-mo-old liver lysates) and in ABT737-treated liver lysates ≤0.6-fold down-regulated proteins (compared with DMSO-treated liver lysates) (n = 2). (I) List of 19 overlapping proteins of Venn diagram in H. (J) qPCR analysis for p21 and selected SASP factors in DMSO- or ABT737-treated dermal fibroblasts 4 d after continuous treatment (n = 3). Error bars, mean ± SEM, unpaired two-tailed Student's t-test. (*) P ≤ 0.05; (**) P ≤ 0.01 (***) P ≤ 0.001.CC BY-NC · PubMed Central

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  1. 10.1016/j.cell.2008.06.049 · DOI · Paper page

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Metadata

Authors
Ritschka, B., Knauer-Meyer, T., Gonçalves, D. S., Mas, A., Plassat, J.-L., Durik, M., Jacobs, H., Pedone, E., Di Vicino, U., Cosma, M. P., & Keyes, W. M.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • Barcelona Institute of Science and Technology (ES)
  • Institució Catalana de Recerca i Estudis Avançats (ES)
ORCID
Venue
Genes & Development
Publisher
Cold Spring Harbor Laboratory
Published
2020-03-05
Volume
34
Issue
7-8
Pages
489-494
ISSN
  • 0890-9369 (ISSN-L)
  • 1549-5477
DOI
10.1101/gad.332643.119
PMC
PMC7111259
OpenAlex
W3009846583
Cited by
105
References
34
Citations by year
2026: 10 · 2025: 17 · 2024: 18 · 2023: 20 · 2022: 14 · 2021: 17 · 2020: 9
Type
posted content
Language
en
Open access
Licence
cc-by-nc
Locations
  • Genes & Development (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
  • HAL (submittedVersion, cc-by-nc)
  • HighWire Press Open Archive (submittedVersion)
  • Repositori digital de la UPF (Universitat Pompeu Fabra) (submittedVersion, cc-by-nc)
Funders
  • Spanish Ministry for Economy and Competitiveness (SAF2013-49082-P)
  • La Fondation Recherche Medicale (AJE20160635985)
  • Fondation ARC (PJA20181208104)
  • IDEX Attractivité-University of Strasbourg (IDEX2017)
  • La Fondation Schlumberger pour l'Education et la Recherche (FSER 19-Year 2018)
  • ANR (ANR-19-CE13-0023-03, ANR-10-LABX-0030-INRT)
  • Agence Nationale de la Recherche (ANR-10-IDEX-0002-02)
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
Keywords
cellular senescence, ABT-737, p21, liver regeneration, partial hepatectomy, p16INK4a, p19ARF, senescence-associated gene expression
MeSH terms
Animals, Biphenyl Compounds, Cells, Cultured, Female, Gene Expression Regulation, Liver, Male, Mice, Inbred C57BL, Nitrophenols, Piperazines, Regeneration, Sulfonamides, Cellular Senescence, Cyclin-Dependent Kinase Inhibitor p16, Models, Animal, Cyclin-Dependent Kinase Inhibitor p21, Mice

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