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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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Depletion of Human Api5 in p16INK4a-Deficient Squamous Cell Carcinoma Cells Results in Reduced Survival versus Normal Human Fibroblasts(A) RNAi-mediated depletion of transfected Api5. Various shRNA constructs were tested for their ability to deplete FLAG-Api5 transfected U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfected constructs strongly depleted Api5 expression after 3 d as determined by anti-FLAG immunoblot.(B) RNAi-mediated depletion of endogenous Api5 protein. JHU-029 cells were infected with lentiviral encoding scramble or API5-AB shRNAs and selected with puromycin for 3 d. Expression of endogenous Api5 was determined by immunoblotting with affinity-purified anti-Api5 polyclonal antibody (G3162).(C and D) Endogenous Api5 expression in JHU-029 cells is nuclear and excluded from the nucleolus. JHU-029 cells were stained with the G3162 polyclonal antibody after 4% paraformaldehyde fixation.(E) Api5 RNAi reduces survival of JHU-029 tumor cells as compared to normal human fibroblasts. JHU-029 cells, as well as normal human WI38 diploid fibroblasts, were infected with lentiviral empty vector or vectors encoding API5-AB shRNAs for 24 h and plated onto culture plates in 10% fetal calf serum–containing media. Cell survival was determined by MTT assay at the indicated days post–lentiviral infection. Api5 and control actin expression was determined from equally loaded protein from day 3 lysates.
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pgen-0020196-g008: Depletion of Human Api5 in p16INK4a-Deficient Squamous Cell Carcinoma Cells Results in Reduced Survival versus Normal Human Fibroblasts(A) RNAi-mediated depletion of transfected Api5. Various shRNA constructs were tested for their ability to deplete FLAG-Api5 transfected U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfected constructs strongly depleted Api5 expression after 3 d as determined by anti-FLAG immunoblot.(B) RNAi-mediated depletion of endogenous Api5 protein. JHU-029 cells were infected with lentiviral encoding scramble or API5-AB shRNAs and selected with puromycin for 3 d. Expression of endogenous Api5 was determined by immunoblotting with affinity-purified anti-Api5 polyclonal antibody (G3162).(C and D) Endogenous Api5 expression in JHU-029 cells is nuclear and excluded from the nucleolus. JHU-029 cells were stained with the G3162 polyclonal antibody after 4% paraformaldehyde fixation.(E) Api5 RNAi reduces survival of JHU-029 tumor cells as compared to normal human fibroblasts. JHU-029 cells, as well as normal human WI38 diploid fibroblasts, were infected with lentiviral empty vector or vectors encoding API5-AB shRNAs for 24 h and plated onto culture plates in 10% fetal calf serum–containing media. Cell survival was determined by MTT assay at the indicated days post–lentiviral infection. Api5 and control actin expression was determined from equally loaded protein from day 3 lysates.

Mentions: Tumor studies have shown that Api5 is preferentially expressed in squamous cell carcinoma versus adenocarcinoma in non–small cell lung cancer [38]. We hypothesized that endogenous Api5 might be an important regulator of survival in squamous cell carcinoma and tested this by reducing Api5 expression (Figure 8). shRNA constructs to Api5 were designed, tested for their ability to deplete transfected FLAG-tagged Api5 (Figure 8A), and then expressed from lentiviral vectors (LLP) to target the endogenous Api5 protein (51-kDa doublet) in human squamous cell carcinoma 029 cells (JHU-029) (Figure 8B). JHU-029 cells are deficient for p16INK4a [42] and endogenously express nuclear-localized Api5 (Figure 8C and 8D). Compared to control WI38 human diploid fibroblasts, endogenous Api5 is highly expressed and RNAi depletion of Api5 resulted in reduced survival with higher sensitivity in the tumor cells (Figure 8E). In keeping with the synthetic lethality between RBF1 and Aac11 depletion in SL2 cells, apoptosis was even more evident when Api5-depleted cells were maintained in low-serum (unpublished data). Taken together, the extensive pattern of genetic interactions between E2F1 and Api5, and the conservation of these interactions from flies to humans, underscores the significance of Api5 for E2F1-dependent apoptosis. Depletion of Api5 in E2F-deregulated tumor cells results in reduced survival, and this raises the possibility that Api5 may be a useful target for antineoplastic therapy.


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)

Depletion of Human Api5 in p16INK4a-Deficient Squamous Cell Carcinoma Cells Results in Reduced Survival versus Normal Human Fibroblasts(A) RNAi-mediated depletion of transfected Api5. Various shRNA constructs were tested for their ability to deplete FLAG-Api5 transfected U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfected constructs strongly depleted Api5 expression after 3 d as determined by anti-FLAG immunoblot.(B) RNAi-mediated depletion of endogenous Api5 protein. JHU-029 cells were infected with lentiviral encoding scramble or API5-AB shRNAs and selected with puromycin for 3 d. Expression of endogenous Api5 was determined by immunoblotting with affinity-purified anti-Api5 polyclonal antibody (G3162).(C and D) Endogenous Api5 expression in JHU-029 cells is nuclear and excluded from the nucleolus. JHU-029 cells were stained with the G3162 polyclonal antibody after 4% paraformaldehyde fixation.(E) Api5 RNAi reduces survival of JHU-029 tumor cells as compared to normal human fibroblasts. JHU-029 cells, as well as normal human WI38 diploid fibroblasts, were infected with lentiviral empty vector or vectors encoding API5-AB shRNAs for 24 h and plated onto culture plates in 10% fetal calf serum–containing media. Cell survival was determined by MTT assay at the indicated days post–lentiviral infection. Api5 and control actin expression was determined from equally loaded protein from day 3 lysates.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0020196-g008: Depletion of Human Api5 in p16INK4a-Deficient Squamous Cell Carcinoma Cells Results in Reduced Survival versus Normal Human Fibroblasts(A) RNAi-mediated depletion of transfected Api5. Various shRNA constructs were tested for their ability to deplete FLAG-Api5 transfected U2OS cells. The AB, CD, and EF (but not Scramble or AB-L) cotransfected constructs strongly depleted Api5 expression after 3 d as determined by anti-FLAG immunoblot.(B) RNAi-mediated depletion of endogenous Api5 protein. JHU-029 cells were infected with lentiviral encoding scramble or API5-AB shRNAs and selected with puromycin for 3 d. Expression of endogenous Api5 was determined by immunoblotting with affinity-purified anti-Api5 polyclonal antibody (G3162).(C and D) Endogenous Api5 expression in JHU-029 cells is nuclear and excluded from the nucleolus. JHU-029 cells were stained with the G3162 polyclonal antibody after 4% paraformaldehyde fixation.(E) Api5 RNAi reduces survival of JHU-029 tumor cells as compared to normal human fibroblasts. JHU-029 cells, as well as normal human WI38 diploid fibroblasts, were infected with lentiviral empty vector or vectors encoding API5-AB shRNAs for 24 h and plated onto culture plates in 10% fetal calf serum–containing media. Cell survival was determined by MTT assay at the indicated days post–lentiviral infection. Api5 and control actin expression was determined from equally loaded protein from day 3 lysates.
Mentions: Tumor studies have shown that Api5 is preferentially expressed in squamous cell carcinoma versus adenocarcinoma in non–small cell lung cancer [38]. We hypothesized that endogenous Api5 might be an important regulator of survival in squamous cell carcinoma and tested this by reducing Api5 expression (Figure 8). shRNA constructs to Api5 were designed, tested for their ability to deplete transfected FLAG-tagged Api5 (Figure 8A), and then expressed from lentiviral vectors (LLP) to target the endogenous Api5 protein (51-kDa doublet) in human squamous cell carcinoma 029 cells (JHU-029) (Figure 8B). JHU-029 cells are deficient for p16INK4a [42] and endogenously express nuclear-localized Api5 (Figure 8C and 8D). Compared to control WI38 human diploid fibroblasts, endogenous Api5 is highly expressed and RNAi depletion of Api5 resulted in reduced survival with higher sensitivity in the tumor cells (Figure 8E). In keeping with the synthetic lethality between RBF1 and Aac11 depletion in SL2 cells, apoptosis was even more evident when Api5-depleted cells were maintained in low-serum (unpublished data). Taken together, the extensive pattern of genetic interactions between E2F1 and Api5, and the conservation of these interactions from flies to humans, underscores the significance of Api5 for E2F1-dependent apoptosis. Depletion of Api5 in E2F-deregulated tumor cells results in reduced survival, and this raises the possibility that Api5 may be a useful target for antineoplastic therapy.

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