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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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Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by Tet treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell death.(E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-dosing every other day.(F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells.(H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells.(I) Api5 expression blocks death induced by E2F1 (+T) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT.
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pgen-0020196-g007: Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by Tet treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell death.(E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-dosing every other day.(F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells.(H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells.(I) Api5 expression blocks death induced by E2F1 (+T) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT.

Mentions: An underlying premise to this work is the idea that our understanding of E2F-dependent apoptosis is incomplete. Since much of the current information about E2F is derived from studies in mammalian cells, it is important to know whether novel functional interactions discovered in a genetic screen in Drosophila are also relevant to studies of the mammalian factor. To test the evolutionary conservation of the genetic interaction between dE2F1 and Aac11, we examined the effects of their homologs in human cells. Lines of Saos-2 cells, a human osteosarcoma cell line that is both Rb and p53 deficient, were generated containing a tetracycline (Tet)-responsive transgene controlling human E2F1 expression. A cDNA for the human API5 gene was cloned and used to generate paired cell lines, with or without exogenous Api5. Using these lines, we examined the effects of elevated E2F1 (Figure 7).


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)

Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by Tet treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell death.(E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-dosing every other day.(F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells.(H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells.(I) Api5 expression blocks death induced by E2F1 (+T) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0020196-g007: Human Api5 Specifically Abrogates E2F1-Dependent Apoptosis without Generally Affecting E2F1-Dependent Transcription(A–D) API5 stably expressing Saos-2 cells were generated with a Tet-inducible E2F1 transgene in the background. Following E2F1 induction by Tet treatment, the parental cells undergo rapid widespread apoptosis; however, the Api5-expressing cells are highly resistant to E2F1-induced cell death.(E) Api5-expressing cells survive and proliferate even following high and sustained levels of E2F1 expression. Cells were grown for 6 d after Tet re-dosing every other day.(F and G) Api5 reduces the levels of E2F1-mediated caspase-3 and PARP cleavage in both stable and Tet-inducible Api5 Saos-2 cells.(H) Api5 expression does not inhibit the E2F1-mediated induction of target genes CycE and p14ARF in Saos-2 cells.(I) Api5 expression blocks death induced by E2F1 (+T) but not by treatment with the DNA-damaging agent camptothecin (CPT) as compared to DMSO vehicle control (Veh). Saos-2 cell survival was assayed at 48 h by MTT.
Mentions: An underlying premise to this work is the idea that our understanding of E2F-dependent apoptosis is incomplete. Since much of the current information about E2F is derived from studies in mammalian cells, it is important to know whether novel functional interactions discovered in a genetic screen in Drosophila are also relevant to studies of the mammalian factor. To test the evolutionary conservation of the genetic interaction between dE2F1 and Aac11, we examined the effects of their homologs in human cells. Lines of Saos-2 cells, a human osteosarcoma cell line that is both Rb and p53 deficient, were generated containing a tetracycline (Tet)-responsive transgene controlling human E2F1 expression. A cDNA for the human API5 gene was cloned and used to generate paired cell lines, with or without exogenous Api5. Using these lines, we examined the effects of elevated E2F1 (Figure 7).

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