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Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo.

Morris EJ, Michaud WA, Ji JY, Moon NS, Rocco JW, Dyson NJ - PLoS Genet. (2006)

Bottom Line: Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1).The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions.Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Charlestown, Massachusetts, United States of America.

ABSTRACT
Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

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RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi(A–D) Transfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss.(E) Quantitative measurements of GFP in dE2F1 transfections demonstrated significant GFP loss from dE2F1 that could be rescued by either RBF1 or p35 cotransfection (*p < 0.05 by t-test). Transfection of dE2F1 induced (F) apoptotic chromatin condensation in DAPI-stained nuclei in GFP-positive cells, (G) caspase-3 activation, and (H) caspase-9 activation 48 h after transfection.(I) RBF1 or Aac11 RNAi significantly enhanced dE2F1-dependent apoptosis (p < 0.01 by t-test). Cell survival was determined by GFP assay 48 h after transfection.(J) Aac11 RNAi does not affect dE2F1 transcriptional activation of the Drosophila PCNA promoter.(K) Aac11 depletion does not alter cell cycle profiles in SL2 cells as determined by flow-cytometry analysis (div = days in vitro after RNAi).(L) Aac11 RNAi is synthetic lethal with RBF1 RNAi under conditions of low-serum stress. Cells were treated with dsRNA in serum-free media and split into media with and without serum, and cell survival was determined 3 d after RNAi.
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pgen-0020196-g006: RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi(A–D) Transfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss.(E) Quantitative measurements of GFP in dE2F1 transfections demonstrated significant GFP loss from dE2F1 that could be rescued by either RBF1 or p35 cotransfection (*p < 0.05 by t-test). Transfection of dE2F1 induced (F) apoptotic chromatin condensation in DAPI-stained nuclei in GFP-positive cells, (G) caspase-3 activation, and (H) caspase-9 activation 48 h after transfection.(I) RBF1 or Aac11 RNAi significantly enhanced dE2F1-dependent apoptosis (p < 0.01 by t-test). Cell survival was determined by GFP assay 48 h after transfection.(J) Aac11 RNAi does not affect dE2F1 transcriptional activation of the Drosophila PCNA promoter.(K) Aac11 depletion does not alter cell cycle profiles in SL2 cells as determined by flow-cytometry analysis (div = days in vitro after RNAi).(L) Aac11 RNAi is synthetic lethal with RBF1 RNAi under conditions of low-serum stress. Cells were treated with dsRNA in serum-free media and split into media with and without serum, and cell survival was determined 3 d after RNAi.

Mentions: In addition to apoptosis, wing gnarling and blistering can be induced by a variety of different mechanisms that include changes in cell fate, adhesion, and proliferation. To confirm that Aac11 affects dE2F1-dependent apoptosis, rather than simply causing a synergistic disruption in tissue development, we moved away from the context in which we had discovered the connection between Aac11 and dE2F1 and reconstructed this genetic interaction in cultured Drosophila cells (Figure 6). A dE2F1-expression construct, or lacZ as control protein, was introduced into SL2 cells together with a GFP-expression construct that allowed us to visualize the transfected cells. As expected from the proapoptotic activity of dE2F1, very few GFP-positive cells were found in dE2F1-transfected cultures compared to the lacZ control after 48 h (Figure 6A–6D). The level of GFP expression was measured by fluorimetry, and this enabled the extent of dE2F1-induced cell killing to be quantified (Figure 6E). The effects of dE2F1 were both time and dosage dependent and quantitatively similar to the changes seen when the proapoptotic Drosophila gene, hid, was expressed as a positive control (unpublished data). As expected, the effects of dE2F1 in this assay were inhibited by the coexpression of RBF1 (Figure 6E).


Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo.

Morris EJ, Michaud WA, Ji JY, Moon NS, Rocco JW, Dyson NJ - PLoS Genet. (2006)

RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi(A–D) Transfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss.(E) Quantitative measurements of GFP in dE2F1 transfections demonstrated significant GFP loss from dE2F1 that could be rescued by either RBF1 or p35 cotransfection (*p < 0.05 by t-test). Transfection of dE2F1 induced (F) apoptotic chromatin condensation in DAPI-stained nuclei in GFP-positive cells, (G) caspase-3 activation, and (H) caspase-9 activation 48 h after transfection.(I) RBF1 or Aac11 RNAi significantly enhanced dE2F1-dependent apoptosis (p < 0.01 by t-test). Cell survival was determined by GFP assay 48 h after transfection.(J) Aac11 RNAi does not affect dE2F1 transcriptional activation of the Drosophila PCNA promoter.(K) Aac11 depletion does not alter cell cycle profiles in SL2 cells as determined by flow-cytometry analysis (div = days in vitro after RNAi).(L) Aac11 RNAi is synthetic lethal with RBF1 RNAi under conditions of low-serum stress. Cells were treated with dsRNA in serum-free media and split into media with and without serum, and cell survival was determined 3 d after RNAi.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1636698&req=5

pgen-0020196-g006: RNAi of Aac11 Enhances dE2F1-Induced Apoptosis and Is Synthetic-Lethal with RBF1 RNAi(A–D) Transfection of dE2F1 in Drosophila SL2 cells induces cell death as determined by co-transfected GFP loss.(E) Quantitative measurements of GFP in dE2F1 transfections demonstrated significant GFP loss from dE2F1 that could be rescued by either RBF1 or p35 cotransfection (*p < 0.05 by t-test). Transfection of dE2F1 induced (F) apoptotic chromatin condensation in DAPI-stained nuclei in GFP-positive cells, (G) caspase-3 activation, and (H) caspase-9 activation 48 h after transfection.(I) RBF1 or Aac11 RNAi significantly enhanced dE2F1-dependent apoptosis (p < 0.01 by t-test). Cell survival was determined by GFP assay 48 h after transfection.(J) Aac11 RNAi does not affect dE2F1 transcriptional activation of the Drosophila PCNA promoter.(K) Aac11 depletion does not alter cell cycle profiles in SL2 cells as determined by flow-cytometry analysis (div = days in vitro after RNAi).(L) Aac11 RNAi is synthetic lethal with RBF1 RNAi under conditions of low-serum stress. Cells were treated with dsRNA in serum-free media and split into media with and without serum, and cell survival was determined 3 d after RNAi.
Mentions: In addition to apoptosis, wing gnarling and blistering can be induced by a variety of different mechanisms that include changes in cell fate, adhesion, and proliferation. To confirm that Aac11 affects dE2F1-dependent apoptosis, rather than simply causing a synergistic disruption in tissue development, we moved away from the context in which we had discovered the connection between Aac11 and dE2F1 and reconstructed this genetic interaction in cultured Drosophila cells (Figure 6). A dE2F1-expression construct, or lacZ as control protein, was introduced into SL2 cells together with a GFP-expression construct that allowed us to visualize the transfected cells. As expected from the proapoptotic activity of dE2F1, very few GFP-positive cells were found in dE2F1-transfected cultures compared to the lacZ control after 48 h (Figure 6A–6D). The level of GFP expression was measured by fluorimetry, and this enabled the extent of dE2F1-induced cell killing to be quantified (Figure 6E). The effects of dE2F1 were both time and dosage dependent and quantitatively similar to the changes seen when the proapoptotic Drosophila gene, hid, was expressed as a positive control (unpublished data). As expected, the effects of dE2F1 in this assay were inhibited by the coexpression of RBF1 (Figure 6E).

Bottom Line: Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1).The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions.Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

View Article: PubMed Central - PubMed

Affiliation: Massachusetts General Hospital Cancer Center, Laboratory of Molecular Oncology, Charlestown, Massachusetts, United States of America.

ABSTRACT
Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

Show MeSH
Related in: MedlinePlus