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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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Summary of the Gal4-Driven Phenotypic Comparison Screen(A) Gal4 driver lines were crossed to various UAS alleles at 25 °C in order to identify novel dominant phenotypes. Phenotype modification was compared relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable “escaper” flies from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P).(B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested.
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pgen-0020196-g001: Summary of the Gal4-Driven Phenotypic Comparison Screen(A) Gal4 driver lines were crossed to various UAS alleles at 25 °C in order to identify novel dominant phenotypes. Phenotype modification was compared relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable “escaper” flies from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P).(B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested.

Mentions: We sought visible phenotypes that were caused by E2F-induced apoptosis and were suitable for genetic screening. We used the Gal4-UAS system to express the Drosophila E2f gene (dE2f1) in a tissue-specific manner [29] and screened a collection of 50 Gal4 drivers that provided a broad assortment of developmentally regulated patterns. We compared the effects of expressing dE2f1 with the effects of expressing known regulators of cell cycle progression such as cyclin E (CycE), dacapo (dap), and human p21, or apoptosis regulators such as reaper (rpr) or baculovirus caspase inhibitor, p35 (Figure 1). Each of these transgenes caused lethality when combined with specific subsets of drivers, and in some cases gene expression resulted in visible phenotypes. Interestingly, in this general survey we noted that the consequences of expressing dE2f1 closely paralleled the effects of expressing the Drosophila proapoptic gene, rpr, but showed far less similarity with the effects of expressing the cell cycle regulator CycE. Because we sought to study dE2F1-dependent apoptosis, we selected the combinations of transgenes in which dE2f1 expression gave a visible phenotype that was phenocopied by the expression of rpr, but not by CycE, and we used these to generate stable stocks bearing dE2F1-dependent phenotypes.


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)

Summary of the Gal4-Driven Phenotypic Comparison Screen(A) Gal4 driver lines were crossed to various UAS alleles at 25 °C in order to identify novel dominant phenotypes. Phenotype modification was compared relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable “escaper” flies from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P).(B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0020196-g001: Summary of the Gal4-Driven Phenotypic Comparison Screen(A) Gal4 driver lines were crossed to various UAS alleles at 25 °C in order to identify novel dominant phenotypes. Phenotype modification was compared relative to control chromosome w1118. Crosses lethal to progeny are indicated in black, while viable crosses are indicated in white. Viable “escaper” flies from lethal crosses are indicated (E). Phenotypes generated in viable or escaper progeny are also indicated for each cross (P).(B) Expression of rpr and dE2f1 resulted in significant lethality in the majority of Gal4 lines tested.
Mentions: We sought visible phenotypes that were caused by E2F-induced apoptosis and were suitable for genetic screening. We used the Gal4-UAS system to express the Drosophila E2f gene (dE2f1) in a tissue-specific manner [29] and screened a collection of 50 Gal4 drivers that provided a broad assortment of developmentally regulated patterns. We compared the effects of expressing dE2f1 with the effects of expressing known regulators of cell cycle progression such as cyclin E (CycE), dacapo (dap), and human p21, or apoptosis regulators such as reaper (rpr) or baculovirus caspase inhibitor, p35 (Figure 1). Each of these transgenes caused lethality when combined with specific subsets of drivers, and in some cases gene expression resulted in visible phenotypes. Interestingly, in this general survey we noted that the consequences of expressing dE2f1 closely paralleled the effects of expressing the Drosophila proapoptic gene, rpr, but showed far less similarity with the effects of expressing the cell cycle regulator CycE. Because we sought to study dE2F1-dependent apoptosis, we selected the combinations of transgenes in which dE2f1 expression gave a visible phenotype that was phenocopied by the expression of rpr, but not by CycE, and we used these to generate stable stocks bearing dE2F1-dependent phenotypes.

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