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W2009348177 abstract "E2F-1 is capable of promoting both cell cycle progression and apoptosis. The latter is important for suppressing untoward expansion of proliferating cells. In this study, we investigated its underlying mechanisms. E2F-1-induced apoptosis was accompanied by caspase-9 activation and inhibited by a specific inhibitor of caspase-9 in K562 sublines overexpressing E2F-1. E2F-1 enhanced the expression of Apaf-1 without the cytosolic accumulation of cytochrome c. Apaf-1-deficient melanoma cell lines were resistant to E2F-1, indicating that Apaf-1 is an essential element of E2F-1-mediated apoptosis. Finally, we isolated the promoter region of the Apaf-1 gene and found a putative binding site for E2F. A chromatin immunoprecipitation assay revealed that E2F-1 bound to Apaf-1 promoter upon E2F-1 overexpression, suggesting that Apaf-1 is under transcriptional regulation of E2F-1. These data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade. E2F-1 is capable of promoting both cell cycle progression and apoptosis. The latter is important for suppressing untoward expansion of proliferating cells. In this study, we investigated its underlying mechanisms. E2F-1-induced apoptosis was accompanied by caspase-9 activation and inhibited by a specific inhibitor of caspase-9 in K562 sublines overexpressing E2F-1. E2F-1 enhanced the expression of Apaf-1 without the cytosolic accumulation of cytochrome c. Apaf-1-deficient melanoma cell lines were resistant to E2F-1, indicating that Apaf-1 is an essential element of E2F-1-mediated apoptosis. Finally, we isolated the promoter region of the Apaf-1 gene and found a putative binding site for E2F. A chromatin immunoprecipitation assay revealed that E2F-1 bound to Apaf-1 promoter upon E2F-1 overexpression, suggesting that Apaf-1 is under transcriptional regulation of E2F-1. These data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade. isopropyl-β-d-thiogalactoside green fluorescent protein enzyme-linked immunosorbent assay caspase-recruitment domain enhanced green fluorescent protein 1,4-piperazinediethanesulfonic acid E2F is a family of transcription factors that control G1/S transition of eukaryotic cells by regulating the expression of various growth-related genes (1Harbor J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar). Target genes of the E2F family include those encoding enzymes for DNA synthesis (DNA polymerase α, thymidine kinase, thymidylate synthase, dihydrofolate reductase, and ribonucleotide reductase), regulators of DNA replication (HsOrc1, Cdc6, MCM5, MCM6, and proliferating cell nuclear antigen), and components of the cell cycle machinery (Cdc2, Cdk2, cyclins A, D1, D2, and E, E2F-1, E2F-2, pRB, p107, and the Myc and Myb families) (2Kel A.E. Kel-Margoulis O.V. Farnham P.J. Bartley S.M. Wingender E. Zhang M.Q. J. Mol. Biol. 2001; 309: 99-120Crossref PubMed Scopus (156) Google Scholar). Among E2F family members, E2F-1 is unique in its ability to induce apoptosis, which may play a role in the cellular homeostasis of multicellular organisms (3Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 4Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (808) Google Scholar, 5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar).In response to mitogenic stimuli, E2F-1 is induced in quiescent cells and promotes both cell cycle progression and apoptosis (6Du W. Xie J.E. Dyson N. EMBO J. 1996; 15: 3684-3692Crossref PubMed Scopus (105) Google Scholar). The apoptosis-inducing ability of E2F-1 is important for suppressing untoward expansion of proliferating cells and, thus, provides an internal defense mechanism against tumor development. The importance of E2F-1-induced apoptosis under physiological conditions is clearly demonstrated by spontaneous development of multiple tumors in mice lacking E2F-1 (7Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 8Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). In addition, it has been reported that deregulation of E2F-1 activity contributes to enhanced proliferation and resistance to cytotoxic drugs in human melanoma cells (9Halaban R. Cheng E. Smicun Y. Germino J. J. Exp. Med. 2000; 191: 1005-1015Crossref PubMed Scopus (69) Google Scholar). E2F-1 is also involved in the accelerated apoptosis of hematopoietic progenitor cells, which is considered the major mechanism of bone marrow failure in myelodysplastic syndrome (10Mundle S.D. Mativi B.Y. Cartlidge J.D. Dangerfield B. Broady- Robinson L., Li, B. Shetty V. Venugopal P. Gregory S.A. Preisler H.D. Raza A. Br. J. Haematol. 2000; 109: 376-381Crossref PubMed Scopus (12) Google Scholar). To clarify the molecular basis of these diseases, it is essential to understand the precise mechanisms of E2F-1-induced apoptosis.There are a few reports dealing with the mechanisms of E2F-1-mediated apoptosis. First, Bates et al. (11Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (810) Google Scholar) reported that E2F-1 induces transcriptional activation of ARF, which stabilizes p53 by sequestering MDM2, a ubiquitin ligase for p53. The stabilization of p53 results in facilitation of p53-dependent apoptosis. Obviously, this is not the sole mechanism of E2F-1-induced apoptosis, because E2F-1 can trigger cell death in p53-deficient tumors, including most leukemia cell lines (12Fueyo J. Gomez-Manzano C. Yung W.K.A. Liu T.J. Alemany R. McDonnel T.J. Shi X. Rao J.S. Levin V.A. Kyritsis A.P. Nat. Med. 1998; 4: 685-690Crossref PubMed Scopus (153) Google Scholar). p53 is also shown to be dispensable for E2F-1-mediated cell death by genetic approaches (13Phillips A.C. Bates S. Ryan K.M. Helin K. Vousden K.H. Genes Dev. 1997; 11: 1853-1863Crossref PubMed Scopus (242) Google Scholar). Second, the lack of NF-κB activation has been described as a mechanism of increased sensitivity of E2F-1-overexpressing cells to apoptotic stimuli through the death receptor pathways (14Phillips A.C. Ernst M.K. Rice N.R. Vousden K.H. Mol. Cell. 1999; 4: 771-781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). However, this mechanism is not applicable to all cell types, because E2F-1 can induce apoptosis in a death receptor-independent manner in certain settings. Most recently, three groups demonstrated the involvement of the p53 homologue p73 in E2F-1-mediated apoptosis (15Lissy N.A. Davis P.K. Irvin M. Kaelin W.G. Dowdy S.F. Nature. 2000; 407: 642-645Crossref PubMed Scopus (288) Google Scholar, 16Stiewe T. Putzer B.M. Nat. Genet. 2001; 26: 464-469Crossref Scopus (307) Google Scholar, 17Irvin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (533) Google Scholar). Although p73 is known to cause apoptotic cell death, its underlying mechanism is still unclear (18Sheikh M.S. Fornace Jr., A.J. J. Cell. Physiol. 2000; 182: 171-181Crossref PubMed Scopus (169) Google Scholar). In view of the fact that p73 is also a transcription factor, it is possible that other direct mediators act downstream of p73 in E2F-induced apoptosis.To explore the mechanisms of E2F-1-induced apoptosis, we established K562 sublines that can overexpress E2F-1 conditionally. Using this system, we found that E2F-1 was capable of activating caspase-9 to initiate the caspase cascade without mitochondrial damage. We further demonstrated that the activation of caspase-9 was caused by up-regulation of Apaf-1, which is a direct transcriptional target of E2F-1.DISCUSSIONIn this study, we investigated the mechanisms of E2F-1-induced apoptosis using K562 sublines that can overexpress E2F-1 conditionally. When E2F-1 was overexpressed in these cells, apoptosis was readily induced after 72 h, following a transient increase in the S phase at 12 h. This confirms the notion that E2F-1 promotes cell cycle progression and apoptosis simultaneously to suppress untoward expansion of proliferating cells (3Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 4Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (808) Google Scholar, 5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar, 6Du W. Xie J.E. Dyson N. EMBO J. 1996; 15: 3684-3692Crossref PubMed Scopus (105) Google Scholar, 12Fueyo J. Gomez-Manzano C. Yung W.K.A. Liu T.J. Alemany R. McDonnel T.J. Shi X. Rao J.S. Levin V.A. Kyritsis A.P. Nat. Med. 1998; 4: 685-690Crossref PubMed Scopus (153) Google Scholar).We found that E2F-1 was capable of activating caspase-9 to initiate a caspase cascade in the absence of mitochondrial damage. The activation of caspase-9 is usually triggered by the release of cytochromec from damaged mitochondria in response to certain apoptotic stimuli such as anticancer drugs, ultraviolet radiation, and serum deprivation (40Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar). Subsequently, cytochrome c binds to Apaf-1 and, in association with adenine nucleotides, facilitates a conformational change of Apaf-1 to expose its CARD domain (35Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar, 36Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). Apaf-1 oligomerizes through the exposed CARD domain and recruits procaspase-9 by a homophilic interaction involving CARDs, which results in catalytic autoactivation of caspase-9. However, E2F-1 activates caspase-9 through a different pathway; it bypasses the translocation of cytochrome c from mitochondria to cytosol and directly induces autoactivation of caspase-9 via the increase in intracellular Apaf-1 levels. The involvement of other direct activators of caspase-9, including a CARD-containing adapter Nod-1/CARD4 (29Inohara N. Koseki T. del Peso L., Hu, Y. Yee C. Chen S. Carrio R. Merino J. Liu D., Ni, J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 33Bertin J. Nir W.-J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H.W. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) and an inhibitor of apoptosis-binding factor Smac/DIABLO (30Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2887) Google Scholar, 34Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar), is to be determined because E2F-1 also modulates the expression of these molecules. Up-regulation of Apaf-1 is also observed during apoptosis caused by the adenoviral oncogene E1A, but it occurs as a secondary event of cytoplasmic accumulation of cytochrome c and therefore is distinct from that of E2F-1 (41Fearnhead H.O. Rodriguez J. Govek E.-E. Guo W. Kobayashi R. Hannon G. Lazebnik Y.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13664-13669Crossref PubMed Scopus (159) Google Scholar). Although the mechanism by which caspase-9 is activated by the increase in Apaf-1 is at present unclear, a high concentration of Apaf-1 may increase the probability of physical interaction between procaspase-9 molecules even in the absence of mitochondrial damage, resulting in oligomerization and subsequent autoactivation of procaspase-9. This hypothesis is substantiated by studies in which forced expression of Apaf-1 solely promoted apoptosis in human myeloid leukemia cell lines (42Perkins C. Kim C.N. Fang G. Bhalla K.N. Cancer Res. 1998; 58: 4561-4566PubMed Google Scholar, 43Perkins C. Fang G. Kim C.N. Bhalla K.N. Cancer Res. 2000; 60: 1645-1653PubMed Google Scholar).Finally, we provide evidence that Apaf-1 is a direct transcriptional target of E2F-1 by analyzing the 5′-untranslated regions of the Apaf-1 gene. This finding is consistent with the results of a recent effort to identify E2F-responsive genes by global analysis of gene expression using high density oligonucleotide arrays; Apaf-1 is one of the newly identified E2F-1-inducible genes (44Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar). Interestingly, E2F-2 and E2F-3 did not up-regulate Apaf-1 in this analysis, which is consistent with the fact that E2F-1 has the strongest ability in promoting apoptosis among E2F family members (5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar). However, there are some reports suggesting that other E2F proteins, especially E2F-2 and E2F-4, are also capable of inducing apoptosis (45Chang Y.-C. Nakajima H. Illenye S. Lee Y.-S. Honjo N. Makiyama T. Fujiwara I. Mizuta N. Sawai K. Saida K. Mitsui Y. Heintz N.H. Magae J. Oncogene. 2000; 19: 4713-4720Crossref PubMed Scopus (14) Google Scholar, 46Dirks P.B. Rutka J.T. Hubbard S.L. Mondal S. Hamel P.A. Oncogene. 1998; 17: 867-876Crossref PubMed Scopus (46) Google Scholar). The mechanisms underlying apoptosis induced by E2F-2 and E2F-4 may be different from that of E2F-1, because E2F-2 and E2F-4 cannot transactivate Apaf-1 (44Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, 47Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H.-M. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar). Because E2F-4 was not able to induce apoptosis in our system (data not shown), it is likely that E2F-4-mediated apoptosis involves p53-dependent pathways.The structure of the Apaf-1 promoter is characteristic; it lacks either a typical TATA motif or initiator elements and is devoid of canonical binding sites for known transcription factors except E2F, Sp-1, and p53. Instead, the Apaf-1 promoter shows an extremely high G and C content (>60%) with an observed/expected CpG ratio of greater than 0.8, which fulfills the requirement of a CpG island (21Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Crossref PubMed Scopus (2631) Google Scholar). These features suggest that methylation of the CpG island serves as the major mechanism of transcriptional regulation of Apaf-1. Indeed, Apaf-1 expression is defective in approximately half of melanoma cell lines, which is reversed by 5-aza-2′-deoxycytidine, an inhibitor of methylation (39Soengas M.S. Capodieci P. Polsky D. Mora J. Esteller M. Opitz-Araya X. McCombie R. Herman J.G. Gerald W.L. Lazebnik Y.A. Cordon-Cardo C. Lowe S.W. Nature. 2001; 409: 207-211Crossref PubMed Scopus (857) Google Scholar). This implies that the Apaf-1 gene is silenced by methylation of the CpG island, and the modulation of the methylation status alleviates the repression to induce Apaf-1 transcription. In this study, we demonstrated the binding of E2F-1 to the Apaf-1 promoter in E2F-1-overexpressing cells using the chromatin immunoprecipitation assay. This result indicates that E2F-1 participates in the derepression of the Apaf-1 gene probably by affecting promoter methylation. The mechanisms by which E2F-1 modulates the methylation status of the Apaf-1 promoter are currently under investigation in our laboratory.While this manuscript was being prepared, an almost identical observation was published by Moroni et al. (48Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Mullar H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar). Moreover, Fortin et al. (49Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar) reported that p53 up-regulates Apaf-1 as a direct transcriptional target during neuronal cell death. Notably, the former study clearly demonstrated that cytoplasmic translocation of cytochrome c is accompanied by E2F-1-induced apoptosis, which contradicts our present finding. This contradiction may stem from the difference in the p53 status of host cells used in each study; p53 expression is defective in our cell line, whereas Moroni et al. (48Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Mullar H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar) used primary human fibroblasts and U2OS cells, both of which possess normal p53. Although our finding may be unique to p53-deficient cells, it instead makes two important points clear. First, E2F-1 can transactivate the Apaf-1 gene in the absence of p53. This is clinically important because most cancer cells lost the function of p53. Second, it is suggested that the E2F-1/Apaf-1 pathway (growth-associated intrinsic route) works in parallel with the p53/Bax/cytochrome c pathway (DNA damage-triggered extrinsic route) in promoting apoptotic cell death.In conclusion, these data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels induced by E2F-1 results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade. Because both E2F-1 and Apaf-1 are frequently deregulated in various pathologic conditions, this finding may contribute to a better understanding of the pathophysiology of many diseases. E2F is a family of transcription factors that control G1/S transition of eukaryotic cells by regulating the expression of various growth-related genes (1Harbor J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar). Target genes of the E2F family include those encoding enzymes for DNA synthesis (DNA polymerase α, thymidine kinase, thymidylate synthase, dihydrofolate reductase, and ribonucleotide reductase), regulators of DNA replication (HsOrc1, Cdc6, MCM5, MCM6, and proliferating cell nuclear antigen), and components of the cell cycle machinery (Cdc2, Cdk2, cyclins A, D1, D2, and E, E2F-1, E2F-2, pRB, p107, and the Myc and Myb families) (2Kel A.E. Kel-Margoulis O.V. Farnham P.J. Bartley S.M. Wingender E. Zhang M.Q. J. Mol. Biol. 2001; 309: 99-120Crossref PubMed Scopus (156) Google Scholar). Among E2F family members, E2F-1 is unique in its ability to induce apoptosis, which may play a role in the cellular homeostasis of multicellular organisms (3Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 4Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (808) Google Scholar, 5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar). In response to mitogenic stimuli, E2F-1 is induced in quiescent cells and promotes both cell cycle progression and apoptosis (6Du W. Xie J.E. Dyson N. EMBO J. 1996; 15: 3684-3692Crossref PubMed Scopus (105) Google Scholar). The apoptosis-inducing ability of E2F-1 is important for suppressing untoward expansion of proliferating cells and, thus, provides an internal defense mechanism against tumor development. The importance of E2F-1-induced apoptosis under physiological conditions is clearly demonstrated by spontaneous development of multiple tumors in mice lacking E2F-1 (7Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 8Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). In addition, it has been reported that deregulation of E2F-1 activity contributes to enhanced proliferation and resistance to cytotoxic drugs in human melanoma cells (9Halaban R. Cheng E. Smicun Y. Germino J. J. Exp. Med. 2000; 191: 1005-1015Crossref PubMed Scopus (69) Google Scholar). E2F-1 is also involved in the accelerated apoptosis of hematopoietic progenitor cells, which is considered the major mechanism of bone marrow failure in myelodysplastic syndrome (10Mundle S.D. Mativi B.Y. Cartlidge J.D. Dangerfield B. Broady- Robinson L., Li, B. Shetty V. Venugopal P. Gregory S.A. Preisler H.D. Raza A. Br. J. Haematol. 2000; 109: 376-381Crossref PubMed Scopus (12) Google Scholar). To clarify the molecular basis of these diseases, it is essential to understand the precise mechanisms of E2F-1-induced apoptosis. There are a few reports dealing with the mechanisms of E2F-1-mediated apoptosis. First, Bates et al. (11Bates S. Phillips A.C. Clark P.A. Stott F. Peters G. Ludwig R.L. Vousden K.H. Nature. 1998; 395: 124-125Crossref PubMed Scopus (810) Google Scholar) reported that E2F-1 induces transcriptional activation of ARF, which stabilizes p53 by sequestering MDM2, a ubiquitin ligase for p53. The stabilization of p53 results in facilitation of p53-dependent apoptosis. Obviously, this is not the sole mechanism of E2F-1-induced apoptosis, because E2F-1 can trigger cell death in p53-deficient tumors, including most leukemia cell lines (12Fueyo J. Gomez-Manzano C. Yung W.K.A. Liu T.J. Alemany R. McDonnel T.J. Shi X. Rao J.S. Levin V.A. Kyritsis A.P. Nat. Med. 1998; 4: 685-690Crossref PubMed Scopus (153) Google Scholar). p53 is also shown to be dispensable for E2F-1-mediated cell death by genetic approaches (13Phillips A.C. Bates S. Ryan K.M. Helin K. Vousden K.H. Genes Dev. 1997; 11: 1853-1863Crossref PubMed Scopus (242) Google Scholar). Second, the lack of NF-κB activation has been described as a mechanism of increased sensitivity of E2F-1-overexpressing cells to apoptotic stimuli through the death receptor pathways (14Phillips A.C. Ernst M.K. Rice N.R. Vousden K.H. Mol. Cell. 1999; 4: 771-781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). However, this mechanism is not applicable to all cell types, because E2F-1 can induce apoptosis in a death receptor-independent manner in certain settings. Most recently, three groups demonstrated the involvement of the p53 homologue p73 in E2F-1-mediated apoptosis (15Lissy N.A. Davis P.K. Irvin M. Kaelin W.G. Dowdy S.F. Nature. 2000; 407: 642-645Crossref PubMed Scopus (288) Google Scholar, 16Stiewe T. Putzer B.M. Nat. Genet. 2001; 26: 464-469Crossref Scopus (307) Google Scholar, 17Irvin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (533) Google Scholar). Although p73 is known to cause apoptotic cell death, its underlying mechanism is still unclear (18Sheikh M.S. Fornace Jr., A.J. J. Cell. Physiol. 2000; 182: 171-181Crossref PubMed Scopus (169) Google Scholar). In view of the fact that p73 is also a transcription factor, it is possible that other direct mediators act downstream of p73 in E2F-induced apoptosis. To explore the mechanisms of E2F-1-induced apoptosis, we established K562 sublines that can overexpress E2F-1 conditionally. Using this system, we found that E2F-1 was capable of activating caspase-9 to initiate the caspase cascade without mitochondrial damage. We further demonstrated that the activation of caspase-9 was caused by up-regulation of Apaf-1, which is a direct transcriptional target of E2F-1. DISCUSSIONIn this study, we investigated the mechanisms of E2F-1-induced apoptosis using K562 sublines that can overexpress E2F-1 conditionally. When E2F-1 was overexpressed in these cells, apoptosis was readily induced after 72 h, following a transient increase in the S phase at 12 h. This confirms the notion that E2F-1 promotes cell cycle progression and apoptosis simultaneously to suppress untoward expansion of proliferating cells (3Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 4Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (808) Google Scholar, 5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar, 6Du W. Xie J.E. Dyson N. EMBO J. 1996; 15: 3684-3692Crossref PubMed Scopus (105) Google Scholar, 12Fueyo J. Gomez-Manzano C. Yung W.K.A. Liu T.J. Alemany R. McDonnel T.J. Shi X. Rao J.S. Levin V.A. Kyritsis A.P. Nat. Med. 1998; 4: 685-690Crossref PubMed Scopus (153) Google Scholar).We found that E2F-1 was capable of activating caspase-9 to initiate a caspase cascade in the absence of mitochondrial damage. The activation of caspase-9 is usually triggered by the release of cytochromec from damaged mitochondria in response to certain apoptotic stimuli such as anticancer drugs, ultraviolet radiation, and serum deprivation (40Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar). Subsequently, cytochrome c binds to Apaf-1 and, in association with adenine nucleotides, facilitates a conformational change of Apaf-1 to expose its CARD domain (35Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar, 36Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). Apaf-1 oligomerizes through the exposed CARD domain and recruits procaspase-9 by a homophilic interaction involving CARDs, which results in catalytic autoactivation of caspase-9. However, E2F-1 activates caspase-9 through a different pathway; it bypasses the translocation of cytochrome c from mitochondria to cytosol and directly induces autoactivation of caspase-9 via the increase in intracellular Apaf-1 levels. The involvement of other direct activators of caspase-9, including a CARD-containing adapter Nod-1/CARD4 (29Inohara N. Koseki T. del Peso L., Hu, Y. Yee C. Chen S. Carrio R. Merino J. Liu D., Ni, J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 33Bertin J. Nir W.-J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H.W. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) and an inhibitor of apoptosis-binding factor Smac/DIABLO (30Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2887) Google Scholar, 34Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar), is to be determined because E2F-1 also modulates the expression of these molecules. Up-regulation of Apaf-1 is also observed during apoptosis caused by the adenoviral oncogene E1A, but it occurs as a secondary event of cytoplasmic accumulation of cytochrome c and therefore is distinct from that of E2F-1 (41Fearnhead H.O. Rodriguez J. Govek E.-E. Guo W. Kobayashi R. Hannon G. Lazebnik Y.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13664-13669Crossref PubMed Scopus (159) Google Scholar). Although the mechanism by which caspase-9 is activated by the increase in Apaf-1 is at present unclear, a high concentration of Apaf-1 may increase the probability of physical interaction between procaspase-9 molecules even in the absence of mitochondrial damage, resulting in oligomerization and subsequent autoactivation of procaspase-9. This hypothesis is substantiated by studies in which forced expression of Apaf-1 solely promoted apoptosis in human myeloid leukemia cell lines (42Perkins C. Kim C.N. Fang G. Bhalla K.N. Cancer Res. 1998; 58: 4561-4566PubMed Google Scholar, 43Perkins C. Fang G. Kim C.N. Bhalla K.N. Cancer Res. 2000; 60: 1645-1653PubMed Google Scholar).Finally, we provide evidence that Apaf-1 is a direct transcriptional target of E2F-1 by analyzing the 5′-untranslated regions of the Apaf-1 gene. This finding is consistent with the results of a recent effort to identify E2F-responsive genes by global analysis of gene expression using high density oligonucleotide arrays; Apaf-1 is one of the newly identified E2F-1-inducible genes (44Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar). Interestingly, E2F-2 and E2F-3 did not up-regulate Apaf-1 in this analysis, which is consistent with the fact that E2F-1 has the strongest ability in promoting apoptosis among E2F family members (5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar). However, there are some reports suggesting that other E2F proteins, especially E2F-2 and E2F-4, are also capable of inducing apoptosis (45Chang Y.-C. Nakajima H. Illenye S. Lee Y.-S. Honjo N. Makiyama T. Fujiwara I. Mizuta N. Sawai K. Saida K. Mitsui Y. Heintz N.H. Magae J. Oncogene. 2000; 19: 4713-4720Crossref PubMed Scopus (14) Google Scholar, 46Dirks P.B. Rutka J.T. Hubbard S.L. Mondal S. Hamel P.A. Oncogene. 1998; 17: 867-876Crossref PubMed Scopus (46) Google Scholar). The mechanisms underlying apoptosis induced by E2F-2 and E2F-4 may be different from that of E2F-1, because E2F-2 and E2F-4 cannot transactivate Apaf-1 (44Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, 47Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H.-M. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar). Because E2F-4 was not able to induce apoptosis in our system (data not shown), it is likely that E2F-4-mediated apoptosis involves p53-dependent pathways.The structure of the Apaf-1 promoter is characteristic; it lacks either a typical TATA motif or initiator elements and is devoid of canonical binding sites for known transcription factors except E2F, Sp-1, and p53. Instead, the Apaf-1 promoter shows an extremely high G and C content (>60%) with an observed/expected CpG ratio of greater than 0.8, which fulfills the requirement of a CpG island (21Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Crossref PubMed Scopus (2631) Google Scholar). These features suggest that methylation of the CpG island serves as the major mechanism of transcriptional regulation of Apaf-1. Indeed, Apaf-1 expression is defective in approximately half of melanoma cell lines, which is reversed by 5-aza-2′-deoxycytidine, an inhibitor of methylation (39Soengas M.S. Capodieci P. Polsky D. Mora J. Esteller M. Opitz-Araya X. McCombie R. Herman J.G. Gerald W.L. Lazebnik Y.A. Cordon-Cardo C. Lowe S.W. Nature. 2001; 409: 207-211Crossref PubMed Scopus (857) Google Scholar). This implies that the Apaf-1 gene is silenced by methylation of the CpG island, and the modulation of the methylation status alleviates the repression to induce Apaf-1 transcription. In this study, we demonstrated the binding of E2F-1 to the Apaf-1 promoter in E2F-1-overexpressing cells using the chromatin immunoprecipitation assay. This result indicates that E2F-1 participates in the derepression of the Apaf-1 gene probably by affecting promoter methylation. The mechanisms by which E2F-1 modulates the methylation status of the Apaf-1 promoter are currently under investigation in our laboratory.While this manuscript was being prepared, an almost identical observation was published by Moroni et al. (48Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Mullar H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar). Moreover, Fortin et al. (49Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar) reported that p53 up-regulates Apaf-1 as a direct transcriptional target during neuronal cell death. Notably, the former study clearly demonstrated that cytoplasmic translocation of cytochrome c is accompanied by E2F-1-induced apoptosis, which contradicts our present finding. This contradiction may stem from the difference in the p53 status of host cells used in each study; p53 expression is defective in our cell line, whereas Moroni et al. (48Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Mullar H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar) used primary human fibroblasts and U2OS cells, both of which possess normal p53. Although our finding may be unique to p53-deficient cells, it instead makes two important points clear. First, E2F-1 can transactivate the Apaf-1 gene in the absence of p53. This is clinically important because most cancer cells lost the function of p53. Second, it is suggested that the E2F-1/Apaf-1 pathway (growth-associated intrinsic route) works in parallel with the p53/Bax/cytochrome c pathway (DNA damage-triggered extrinsic route) in promoting apoptotic cell death.In conclusion, these data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels induced by E2F-1 results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade. Because both E2F-1 and Apaf-1 are frequently deregulated in various pathologic conditions, this finding may contribute to a better understanding of the pathophysiology of many diseases. In this study, we investigated the mechanisms of E2F-1-induced apoptosis using K562 sublines that can overexpress E2F-1 conditionally. When E2F-1 was overexpressed in these cells, apoptosis was readily induced after 72 h, following a transient increase in the S phase at 12 h. This confirms the notion that E2F-1 promotes cell cycle progression and apoptosis simultaneously to suppress untoward expansion of proliferating cells (3Qin X.Q. Livingston D.M. Kaelin Jr., W.G. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 4Wu X. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3602-3606Crossref PubMed Scopus (808) Google Scholar, 5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar, 6Du W. Xie J.E. Dyson N. EMBO J. 1996; 15: 3684-3692Crossref PubMed Scopus (105) Google Scholar, 12Fueyo J. Gomez-Manzano C. Yung W.K.A. Liu T.J. Alemany R. McDonnel T.J. Shi X. Rao J.S. Levin V.A. Kyritsis A.P. Nat. Med. 1998; 4: 685-690Crossref PubMed Scopus (153) Google Scholar). We found that E2F-1 was capable of activating caspase-9 to initiate a caspase cascade in the absence of mitochondrial damage. The activation of caspase-9 is usually triggered by the release of cytochromec from damaged mitochondria in response to certain apoptotic stimuli such as anticancer drugs, ultraviolet radiation, and serum deprivation (40Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar). Subsequently, cytochrome c binds to Apaf-1 and, in association with adenine nucleotides, facilitates a conformational change of Apaf-1 to expose its CARD domain (35Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar, 36Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (961) Google Scholar). Apaf-1 oligomerizes through the exposed CARD domain and recruits procaspase-9 by a homophilic interaction involving CARDs, which results in catalytic autoactivation of caspase-9. However, E2F-1 activates caspase-9 through a different pathway; it bypasses the translocation of cytochrome c from mitochondria to cytosol and directly induces autoactivation of caspase-9 via the increase in intracellular Apaf-1 levels. The involvement of other direct activators of caspase-9, including a CARD-containing adapter Nod-1/CARD4 (29Inohara N. Koseki T. del Peso L., Hu, Y. Yee C. Chen S. Carrio R. Merino J. Liu D., Ni, J. Nunez G. J. Biol. Chem. 1999; 274: 14560-14567Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar, 33Bertin J. Nir W.-J. Fischer C.M. Tayber O.V. Errada P.R. Grant J.R. Keilty J.J. Gosselin M.L. Robison K.E. Wong G.H.W. Glucksmann M.A. DiStefano P.S. J. Biol. Chem. 1999; 274: 12955-12958Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar) and an inhibitor of apoptosis-binding factor Smac/DIABLO (30Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2887) Google Scholar, 34Verhagen A.M. Ekert P.G. Pakusch M. Silke J. Connolly L.M. Reid G.E. Moritz R.L. Simpson R.J. Vaux D.L. Cell. 2000; 102: 43-53Abstract Full Text Full Text PDF PubMed Scopus (1959) Google Scholar), is to be determined because E2F-1 also modulates the expression of these molecules. Up-regulation of Apaf-1 is also observed during apoptosis caused by the adenoviral oncogene E1A, but it occurs as a secondary event of cytoplasmic accumulation of cytochrome c and therefore is distinct from that of E2F-1 (41Fearnhead H.O. Rodriguez J. Govek E.-E. Guo W. Kobayashi R. Hannon G. Lazebnik Y.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13664-13669Crossref PubMed Scopus (159) Google Scholar). Although the mechanism by which caspase-9 is activated by the increase in Apaf-1 is at present unclear, a high concentration of Apaf-1 may increase the probability of physical interaction between procaspase-9 molecules even in the absence of mitochondrial damage, resulting in oligomerization and subsequent autoactivation of procaspase-9. This hypothesis is substantiated by studies in which forced expression of Apaf-1 solely promoted apoptosis in human myeloid leukemia cell lines (42Perkins C. Kim C.N. Fang G. Bhalla K.N. Cancer Res. 1998; 58: 4561-4566PubMed Google Scholar, 43Perkins C. Fang G. Kim C.N. Bhalla K.N. Cancer Res. 2000; 60: 1645-1653PubMed Google Scholar). Finally, we provide evidence that Apaf-1 is a direct transcriptional target of E2F-1 by analyzing the 5′-untranslated regions of the Apaf-1 gene. This finding is consistent with the results of a recent effort to identify E2F-responsive genes by global analysis of gene expression using high density oligonucleotide arrays; Apaf-1 is one of the newly identified E2F-1-inducible genes (44Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar). Interestingly, E2F-2 and E2F-3 did not up-regulate Apaf-1 in this analysis, which is consistent with the fact that E2F-1 has the strongest ability in promoting apoptosis among E2F family members (5DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (601) Google Scholar). However, there are some reports suggesting that other E2F proteins, especially E2F-2 and E2F-4, are also capable of inducing apoptosis (45Chang Y.-C. Nakajima H. Illenye S. Lee Y.-S. Honjo N. Makiyama T. Fujiwara I. Mizuta N. Sawai K. Saida K. Mitsui Y. Heintz N.H. Magae J. Oncogene. 2000; 19: 4713-4720Crossref PubMed Scopus (14) Google Scholar, 46Dirks P.B. Rutka J.T. Hubbard S.L. Mondal S. Hamel P.A. Oncogene. 1998; 17: 867-876Crossref PubMed Scopus (46) Google Scholar). The mechanisms underlying apoptosis induced by E2F-2 and E2F-4 may be different from that of E2F-1, because E2F-2 and E2F-4 cannot transactivate Apaf-1 (44Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (627) Google Scholar, 47Weinmann A.S. Yan P.S. Oberley M.J. Huang T.H.-M. Farnham P.J. Genes Dev. 2002; 16: 235-244Crossref PubMed Scopus (391) Google Scholar). Because E2F-4 was not able to induce apoptosis in our system (data not shown), it is likely that E2F-4-mediated apoptosis involves p53-dependent pathways. The structure of the Apaf-1 promoter is characteristic; it lacks either a typical TATA motif or initiator elements and is devoid of canonical binding sites for known transcription factors except E2F, Sp-1, and p53. Instead, the Apaf-1 promoter shows an extremely high G and C content (>60%) with an observed/expected CpG ratio of greater than 0.8, which fulfills the requirement of a CpG island (21Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Crossref PubMed Scopus (2631) Google Scholar). These features suggest that methylation of the CpG island serves as the major mechanism of transcriptional regulation of Apaf-1. Indeed, Apaf-1 expression is defective in approximately half of melanoma cell lines, which is reversed by 5-aza-2′-deoxycytidine, an inhibitor of methylation (39Soengas M.S. Capodieci P. Polsky D. Mora J. Esteller M. Opitz-Araya X. McCombie R. Herman J.G. Gerald W.L. Lazebnik Y.A. Cordon-Cardo C. Lowe S.W. Nature. 2001; 409: 207-211Crossref PubMed Scopus (857) Google Scholar). This implies that the Apaf-1 gene is silenced by methylation of the CpG island, and the modulation of the methylation status alleviates the repression to induce Apaf-1 transcription. In this study, we demonstrated the binding of E2F-1 to the Apaf-1 promoter in E2F-1-overexpressing cells using the chromatin immunoprecipitation assay. This result indicates that E2F-1 participates in the derepression of the Apaf-1 gene probably by affecting promoter methylation. The mechanisms by which E2F-1 modulates the methylation status of the Apaf-1 promoter are currently under investigation in our laboratory. While this manuscript was being prepared, an almost identical observation was published by Moroni et al. (48Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Mullar H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar). Moreover, Fortin et al. (49Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar) reported that p53 up-regulates Apaf-1 as a direct transcriptional target during neuronal cell death. Notably, the former study clearly demonstrated that cytoplasmic translocation of cytochrome c is accompanied by E2F-1-induced apoptosis, which contradicts our present finding. This contradiction may stem from the difference in the p53 status of host cells used in each study; p53 expression is defective in our cell line, whereas Moroni et al. (48Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Mullar H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar) used primary human fibroblasts and U2OS cells, both of which possess normal p53. Although our finding may be unique to p53-deficient cells, it instead makes two important points clear. First, E2F-1 can transactivate the Apaf-1 gene in the absence of p53. This is clinically important because most cancer cells lost the function of p53. Second, it is suggested that the E2F-1/Apaf-1 pathway (growth-associated intrinsic route) works in parallel with the p53/Bax/cytochrome c pathway (DNA damage-triggered extrinsic route) in promoting apoptotic cell death. In conclusion, these data demonstrate a novel mechanism of apoptosis in which an increase in Apaf-1 levels induced by E2F-1 results in direct activation of caspase-9 without mitochondrial damage, leading to the initiation of a caspase cascade. Because both E2F-1 and Apaf-1 are frequently deregulated in various pathologic conditions, this finding may contribute to a better understanding of the pathophysiology of many diseases. We thank Drs. Tadashi Mayumi, Kiyoshi Kawakami, and Keiko Ikeda (Jichi Medical School) for helpful discussions. We are grateful to Yukiko Fukuda-Kamiyoshihara for excellent technical assistance." @default.
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W2009348177 title "Apaf-1 Is a Mediator of E2F-1-induced Apoptosis" @default.
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