title: E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK.
abstract: E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.
authors.affiliation: Department of Surgery, University of Louisville School of Medicine, and J. Graham Brown Cancer Center, Louisville, KY, USA.
authors.forename: Hongying
authors.initials: H
authors.lastname: Hao
authors.forename: Canming
authors.initials: C
authors.lastname: Chen
authors.forename: Xiao-Mei
authors.initials: XM
authors.lastname: Rao
authors.forename: Jorge G
authors.initials: JG
authors.lastname: Gomez-Gutierrez
authors.forename: H Sam
authors.initials: HS
authors.lastname: Zhou
authors.forename: Kelly M
authors.initials: KM
authors.lastname: McMasters
mesh_terms: D020413:3' Untranslated Regions; D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D045744:Cell Line, Tumor; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D051605:Kv Channel-Interacting Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D051017:Apoptosis Regulatory Proteins; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C484586:HRK protein, human; C496749:KCNIP3 protein, human; D051605:Kv Channel-Interacting Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins