title: E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation.
abstract: BACKGROUND
  PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells.
  
  
  METHODS
  PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit.
  
  
  RESULTS
  Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway.
  
  
  CONCLUSIONS
  Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling pathways provided here will further enhance insights on the mechanisms of E2F-1-induced cancer cell apoptosis as a strategy for cancer therapy.
authors.affiliation: Department of Surgery, University of Louisville, Louisville, KY 40292, USA. h0hao001@louisville.edu <h0hao001@louisville.edu>
authors.forename: Hongying
authors.initials: H
authors.lastname: Hao
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Dong
authors.forename: Maria T
authors.initials: MT
authors.lastname: Bowling
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: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D001665:Binding Sites; D053453:Caspase 9 / Q000502:physiology; D045744:Cell Line, Tumor; D050687:E2F1 Transcription Factor / Q000502:physiology*; D006801:Humans; D008545:Melanoma / Q000473:pathology*; D008928:Mitochondria / Q000378:metabolism; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D015533:Transcriptional Activation; D015854:Up-Regulation; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D011518:Proto-Oncogene Proteins; D051028:bcl-2-Associated X Protein; D053453:Caspase 9