CoChat
Sign Up
Cited by 1,147

The DOI matches its Crossref record.

Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis

Brentnall, Matthew, Rodriguez-Menocal, Luis, De Guevara, Rebeka Ladron, Cepero, Enrique, Boise, Lawrence H·2013·BMC Cell Biology
DOI 10.1186/1471-2121-14-32PMC PMC3710246
Open this paper in CoChat — free account

Abstract

Background: Apoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochrome c release is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined. Results: Here we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochrome c , but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM. Conclusions: Taken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1186/1471-2121-14-32

Cites

Statistics

Figures

Figure 1 Bid is cleaved in a caspase-9-dependent fashion during IL-3 withdrawal and is necessary for ROS production. (A-B) FL5.12 Neo (±100 μM BocD-fmk), Bcl-xL, Casp9DN and CrmA cells were cultured in the presence or absence of IL-3 for 24 h. (A) Bid levels were determined by western blot and viability was determined. (B) Loss of membrane potential was determined by flow cytometry (filled histogram (+)IL-3, black line (-)IL-3, grey line (-)IL-3 +BocD). (C)In vitro translated Bid, BidD98A, BidD75A and BidD59A were subjected to cleavage by increasing levels of caspase-9 for 90 min. Bid cleavage was determined by western blot. (D) Bid and BidD59A were expressed in Bid-/- MEFs by retroviral transduction and Bid levels were determined by western blot (Bid expression from same blot). (E) Bid-/- pBabe, Bid and BidD59A MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. (light grey line (+)FBS, dark grey line (-)FBS, black line (-)FBS +BocD). Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control (red line). Data are representative of at least 3 independent experiments.
Figure 1 Bid is cleaved in a caspase-9-dependent fashion during IL-3 withdrawal and is necessary for ROS production. (A-B) FL5.12 Neo (±100 μM BocD-fmk), Bcl-xL, Casp9DN and CrmA cells were cultured in the presence or absence of IL-3 for 24 h. (A) Bid levels were determined by western blot and viability was determined. (B) Loss of membrane potential was determined by flow cytometry (filled histogram (+)IL-3, black line (-)IL-3, grey line (-)IL-3 +BocD). (C)In vitro translated Bid, BidD98A, BidD75A and BidD59A were subjected to cleavage by increasing levels of caspase-9 for 90 min. Bid cleavage was determined by western blot. (D) Bid and BidD59A were expressed in Bid-/- MEFs by retroviral transduction and Bid levels were determined by western blot (Bid expression from same blot). (E) Bid-/- pBabe, Bid and BidD59A MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. (light grey line (+)FBS, dark grey line (-)FBS, black line (-)FBS +BocD). Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control (red line). Data are representative of at least 3 independent experiments.CC BY · PubMed Central
Figure 2 Caspase-7 and-3 have distinct effects on ROS production during apoptosis. (A) Expression levels of effector caspases in Casp KO MEFs were determined by western blot. (B) WT, Casp7-/-, Casp3-/- and Casp3-/-7-/- MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control. Data are representative of at least 3 independent experiments.
Figure 2 Caspase-7 and-3 have distinct effects on ROS production during apoptosis. (A) Expression levels of effector caspases in Casp KO MEFs were determined by western blot. (B) WT, Casp7-/-, Casp3-/- and Casp3-/-7-/- MEFs were withdrawn from serum for 12 hr. ROS production was determined by flow cytometry. Treatment with 50 μg/ml antimycin A for 30 min was used as a positive control. Data are representative of at least 3 independent experiments.CC BY · PubMed Central
Figure 3 Caspase-7 and-3 have distinct functions during apoptosis. (A-B) WT, Casp7-/-, Casp3-/- and Casp3-/-7-/- MEFs were withdrawn from serum for 4 days. (A) At the indicated time points, cell death was determined by Annexin V-FITC staining. (B) At the indicated time points, percent detachment was determined by separating non-adherent from adherent cells and counting with a hemocytometer. Data are presented as the mean ± SEM of at least 3 independent experiments. (C) Casp7−/− MEFs were grown on glass coverslips for 24 hr and then were washed with PBS and the medium was replaced with full medium or serum free medium for 48 hr. After, cells were fixed, stained for actin and DNA and visualized by fluorescent microscopy.
Figure 3 Caspase-7 and-3 have distinct functions during apoptosis. (A-B) WT, Casp7-/-, Casp3-/- and Casp3-/-7-/- MEFs were withdrawn from serum for 4 days. (A) At the indicated time points, cell death was determined by Annexin V-FITC staining. (B) At the indicated time points, percent detachment was determined by separating non-adherent from adherent cells and counting with a hemocytometer. Data are presented as the mean ± SEM of at least 3 independent experiments. (C) Casp7−/− MEFs were grown on glass coverslips for 24 hr and then were washed with PBS and the medium was replaced with full medium or serum free medium for 48 hr. After, cells were fixed, stained for actin and DNA and visualized by fluorescent microscopy.CC BY · PubMed Central
Figure 4 Reconstitution of caspase-deficient MEFs with caspase-3 or caspase-7 rescues the WT phenotype. (A) Caspase-deficient MEFs were reconstituted with the appropriate caspase by retroviral transduction and caspase expression was determined by western blot. (B) WT, Casp3-/- pBabe and Casp3-/- C3 MEFs were withdrawn from serum for 4 days. At the indicated time points, cell death was determined by Annexin V-FITC staining. Data are presented as the mean ± SEM of at least 3 independent experiments. (C) WT, Casp7-/- pBabe and Casp7-/- C7 MEFs were withdrawn from serum for 4 days. At the indicated time points, percent detachment was determined by separating non-adherent from adherent cells and counting with a hemocytometer. Data are presented as the mean ± SEM of at least 3 independent experiments.
Figure 4 Reconstitution of caspase-deficient MEFs with caspase-3 or caspase-7 rescues the WT phenotype. (A) Caspase-deficient MEFs were reconstituted with the appropriate caspase by retroviral transduction and caspase expression was determined by western blot. (B) WT, Casp3-/- pBabe and Casp3-/- C3 MEFs were withdrawn from serum for 4 days. At the indicated time points, cell death was determined by Annexin V-FITC staining. Data are presented as the mean ± SEM of at least 3 independent experiments. (C) WT, Casp7-/- pBabe and Casp7-/- C7 MEFs were withdrawn from serum for 4 days. At the indicated time points, percent detachment was determined by separating non-adherent from adherent cells and counting with a hemocytometer. Data are presented as the mean ± SEM of at least 3 independent experiments.CC BY · PubMed Central
Figure 5 Model of caspase activation downstream of cytochrome c release during apoptosis. Cell death signals induce MOMP, which leads to cytochrome c release and the activation of caspase-9. Caspase-9 can cleave and activate Bid, caspase-7 and caspase-3. tBid can remodel the mitochondria and make conditions favorable for ROS production, which is enhanced by caspase-7 and inhibited by caspase-3.
Figure 5 Model of caspase activation downstream of cytochrome c release during apoptosis. Cell death signals induce MOMP, which leads to cytochrome c release and the activation of caspase-9. Caspase-9 can cleave and activate Bid, caspase-7 and caspase-3. tBid can remodel the mitochondria and make conditions favorable for ROS production, which is enhanced by caspase-7 and inhibited by caspase-3.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

No references could be resolved yet.

Research works printed but not linked: 25

  1. Danial NN, Korsmeyer SJ: Cell death: critical control points. Cell. 2004, 116 (2): 205-219. 10.1016/S0092-8674(04)00046-7. · DOI · Paper page
  2. Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S: Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ. 2012, 19 (1): 107-120. 10.1038/cdd.2011.96. · DOI · Paper page
  3. Wei MC, Lindsten T, Mootha VK, Weiler S, Gross A, Ashiya M, Thompson CB, Korsmeyer SJ: tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes Dev. 2000, 14 (16): 2060-2071. · DOI
  4. Eskes R, Desagher S, Antonsson B, Martinou JC: Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol. 2000, 20 (3): 929-935. 10.1128/MCB.20.3.929-935.2000. · DOI
  5. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ: Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 2001, 292 (5517): 727-730. 10.1126/science.1059108. · DOI · Paper page
  6. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997, 91 (4): 479-489. 10.1016/S0092-8674(00)80434-1. · DOI · Paper page
  7. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES: Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell. 1998, 1 (7): 949-957. 10.1016/S1097-2765(00)80095-7. · DOI
  8. Woo M, Hakem R, Soengas MS, Duncan GS, Shahinian A, Kagi D, Hakem A, McCurrach M, Khoo W, Kaufman SA: Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev. 1998, 12 (6): 806-819. 10.1101/gad.12.6.806. · DOI
  9. Shi Y: Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell. 2002, 9 (3): 459-470. 10.1016/S1097-2765(02)00482-3. · DOI
  10. Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, Mannella CA, Korsmeyer SJ: A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Dev Cell. 2002, 2 (1): 55-67. 10.1016/S1534-5807(01)00116-2. · DOI · Paper page
  11. Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M, Kroemer G: Mitochondrial control of nuclear apoptosis. J Exp Med. 1996, 183 (4): 1533-1544. 10.1084/jem.183.4.1533. · DOI
  12. Martin SJ, Green DR: Protease activation during apoptosis: death by a thousand cuts?. Cell. 1995, 82 (3): 349-352. 10.1016/0092-8674(95)90422-0. · DOI
  13. Cepero E, King AM, Coffey LM, Perez RG, Boise LH: Caspase-9 and effector caspases have sequential and distinct effects on mitochondria. Oncogene. 2005, 24 (42): 6354-6366. · DOI
  14. Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES: Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999, 144 (2): 281-292. 10.1083/jcb.144.2.281. · DOI
  15. Stennicke HR, Jurgensmeier JM, Shin H, Deveraux Q, Wolf BB, Yang X, Zhou Q, Ellerby HM, Ellerby LM, Bredesen D: Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem. 1998, 273 (42): 27084-27090. 10.1074/jbc.273.42.27084. · DOI
  16. Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP: A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997, 272 (29): 17907-17911. 10.1074/jbc.272.29.17907. · DOI
  17. Lakhani SA, Masud A, Kuida K, Porter GA, Booth CJ, Mehal WZ, Inayat I, Flavell RA: Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science. 2006, 311 (5762): 847-851. 10.1126/science.1115035. · DOI
  18. Walsh JG, Cullen SP, Sheridan C, Luthi AU, Gerner C, Martin SJ: Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc Natl Acad Sci USA. 2008, 105 (35): 12815-12819. 10.1073/pnas.0707715105. · DOI
  19. Gregory CD, Pound JD: Microenvironmental influences of apoptosis in vivo and in vitro. Apoptosis. 2010, 15 (9): 1029-1049. 10.1007/s10495-010-0485-9. · DOI
  20. Johnson BW, Cepero E, Boise LH: Bcl-xL inhibits cytochrome c release but not mitochondrial depolarization during the activation of multiple death pathways by tumor necrosis factor-alpha. J Biol Chem. 2000, 275 (40): 31546-31553. · DOI
  21. Saraste M: Oxidative phosphorylation at the fin de siecle. Science. 1999, 283 (5407): 1488-1493. 10.1126/science.283.5407.1488. · DOI
  22. Cai J, Jones DP: Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem. 1998, 273 (19): 11401-11404. 10.1074/jbc.273.19.11401. · DOI · Paper page
  23. Wen LP, Fahrni JA, Troie S, Guan JL, Orth K, Rosen GD: Cleavage of focal adhesion kinase by caspases during apoptosis. J Biol Chem. 1997, 272 (41): 26056-26061. 10.1074/jbc.272.41.26056. · DOI
  24. Johnson BW, Boise LH: Bcl-2 and caspase inhibition cooperate to inhibit tumor necrosis factor-alpha-induced cell death in a Bcl-2 cleavage-independent fashion. J Biol Chem. 1999, 274 (26): 18552-18558. 10.1074/jbc.274.26.18552. · DOI
  25. Swift S, Lorens J, Achacoso P, Nolan GP: Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems. Curr Protoc Immunol. 2001, Chapter 10: Unit 10 17C-

Cited by

Sorted by citations

See all 1,147

Metadata

Authors
Brentnall, M., Rodriguez-Menocal, L., De Guevara, R. L., Cepero, E., & Boise, L. H.
Venue
BMC Cell Biology
Publisher
Springer Science and Business Media LLC
Published
2013-07-09
Volume
14
Issue
1
ISSN
1471-2121
DOI
10.1186/1471-2121-14-32
PMC
PMC3710246
Cited by
1,147
References
0
Language
en

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. https://app.cochat.ai/papers/10.1186/1471-2121-14-32
The paper
Brentnall, M., Rodriguez-Menocal, L., De Guevara, R. L., Cepero, E., & Boise, L. H. (2013). Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biology. https://doi.org/10.1186/1471-2121-14-32