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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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Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transgenic StockVarious alleles were analyzed for modification of the dE2f1-dependent phenotype in trans. The wild-type wing phenotype is depicted in Act88F-Gal4,UAS-EGFP/+ (w1118) recombinant stock as control (A). The Act88F-Gal4,UAS-dE2f1/+ (w1118) recombinant stock phenotype (B) is strongly phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) completely suppressed the dE2f1 phenotype. The dE2f1-dependent phenotype was also suppressed by various apoptotic regulators including coexpression of the caspase inhibitor baculovirus p35 (F) or dIAP1 (G) or a heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog.(I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-Gal4,UAS-dE2f1 recombinant chromosome was crossed at 25 °C to various transgenic and mutant alleles and phenotypes analyzed in transheterozygous progeny. Modification of the dE2F1 phenotype was compared relative to control chromosome w1118. See Materials and Methods for mutant alleles used in this study.
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pgen-0020196-g003: Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transgenic StockVarious alleles were analyzed for modification of the dE2f1-dependent phenotype in trans. The wild-type wing phenotype is depicted in Act88F-Gal4,UAS-EGFP/+ (w1118) recombinant stock as control (A). The Act88F-Gal4,UAS-dE2f1/+ (w1118) recombinant stock phenotype (B) is strongly phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) completely suppressed the dE2f1 phenotype. The dE2f1-dependent phenotype was also suppressed by various apoptotic regulators including coexpression of the caspase inhibitor baculovirus p35 (F) or dIAP1 (G) or a heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog.(I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-Gal4,UAS-dE2f1 recombinant chromosome was crossed at 25 °C to various transgenic and mutant alleles and phenotypes analyzed in transheterozygous progeny. Modification of the dE2F1 phenotype was compared relative to control chromosome w1118. See Materials and Methods for mutant alleles used in this study.

Mentions: As a further test of the processes involved in this dE2F1-induced phenotype, we crossed Act88F-Gal4,UAS-dE2f1 recombinant stocks with various UAS and mutant alleles (Figure 3) and examined their genetic interactions. As expected, the wing phenotype was completely suppressed by the coexpression of the Drosophila pRB ortholog, Rbf (Figure 3D), or by the coexpression of a dominant-negative form of the dE2F1 heterodimerization partner, dDp (Figure 3E). These proteins also blocked dE2F1-induced apoptosis, as measured by the loss of EGFP-expressing cells (Figure 2K). Conversely, expression of additional dE2f1 or the coexpression of functional dDp strongly enhanced the wing defects (Figure 3I). Taken together, with the suppression observed by dominant-negative dDp, these data strongly suggest that the wing phenotype is dependent on dE2F1-induced transcription. Control UAS transgenes, such as UAS-EGFP and UAS-beta-galactosidase (lacZ), had no effect on the Act88F-Gal4,UAS-dE2f1 phenotype (Figure 3I).


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)

Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transgenic StockVarious alleles were analyzed for modification of the dE2f1-dependent phenotype in trans. The wild-type wing phenotype is depicted in Act88F-Gal4,UAS-EGFP/+ (w1118) recombinant stock as control (A). The Act88F-Gal4,UAS-dE2f1/+ (w1118) recombinant stock phenotype (B) is strongly phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) completely suppressed the dE2f1 phenotype. The dE2f1-dependent phenotype was also suppressed by various apoptotic regulators including coexpression of the caspase inhibitor baculovirus p35 (F) or dIAP1 (G) or a heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog.(I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-Gal4,UAS-dE2f1 recombinant chromosome was crossed at 25 °C to various transgenic and mutant alleles and phenotypes analyzed in transheterozygous progeny. Modification of the dE2F1 phenotype was compared relative to control chromosome w1118. See Materials and Methods for mutant alleles used in this study.
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pgen-0020196-g003: Genetic Characterization of a Recombinant Act88F-Gal4,UAS-dE2f1 Transgenic StockVarious alleles were analyzed for modification of the dE2f1-dependent phenotype in trans. The wild-type wing phenotype is depicted in Act88F-Gal4,UAS-EGFP/+ (w1118) recombinant stock as control (A). The Act88F-Gal4,UAS-dE2f1/+ (w1118) recombinant stock phenotype (B) is strongly phenocopied by caspase expression (C). Coexpression of Rbf (D) or dDpDN (E) completely suppressed the dE2f1 phenotype. The dE2f1-dependent phenotype was also suppressed by various apoptotic regulators including coexpression of the caspase inhibitor baculovirus p35 (F) or dIAP1 (G) or a heterozygous dominant allele of dArk (H), the Drosophila APAF1 homolog.(I) Summary of the genetic interactions with Act88F-Gal4,UAS-dE2f1. The Act88F-Gal4,UAS-dE2f1 recombinant chromosome was crossed at 25 °C to various transgenic and mutant alleles and phenotypes analyzed in transheterozygous progeny. Modification of the dE2F1 phenotype was compared relative to control chromosome w1118. See Materials and Methods for mutant alleles used in this study.
Mentions: As a further test of the processes involved in this dE2F1-induced phenotype, we crossed Act88F-Gal4,UAS-dE2f1 recombinant stocks with various UAS and mutant alleles (Figure 3) and examined their genetic interactions. As expected, the wing phenotype was completely suppressed by the coexpression of the Drosophila pRB ortholog, Rbf (Figure 3D), or by the coexpression of a dominant-negative form of the dE2F1 heterodimerization partner, dDp (Figure 3E). These proteins also blocked dE2F1-induced apoptosis, as measured by the loss of EGFP-expressing cells (Figure 2K). Conversely, expression of additional dE2f1 or the coexpression of functional dDp strongly enhanced the wing defects (Figure 3I). Taken together, with the suppression observed by dominant-negative dDp, these data strongly suggest that the wing phenotype is dependent on dE2F1-induced transcription. Control UAS transgenes, such as UAS-EGFP and UAS-beta-galactosidase (lacZ), had no effect on the Act88F-Gal4,UAS-dE2f1 phenotype (Figure 3I).

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