title: Induction of marked apoptosis in mammalian cancer cell lines by antisense DNA treatment to abolish expression of DENN (differentially expressed in normal and neoplastic cells).
abstract: We previously reported the isolation of the novel human DENN gene, which is differentially expressed in normal and neoplastic cells. DENN is identical to MADD (mitogen-activated protein kinase-activating death domain), which interacts with tumor necrosis factor receptor 1 through their death domains. DENN is also homologous to Rab3 GEP, a rat Rab3 GDP/GTP exchange protein. Real-time reverse transcription-polymerase chain reaction analysis showed that DENN expression in cancer cell lines was 26-50 times that in normal cells. The Jurkat human leukemia, PLC/PRF/5 human hepatoma, and NS-1 mouse myeloma cell lines as well as the MRC-5 human fetal lung and Vero monkey kidney cell lines were treated successfully with four separate DENN-targeted antisense oligodeoxynucleotides (ODNs) to abrogate DENN expression. Quantitative assessment of cell viability and apoptosis by flow cytometry via fluorescein diacetate and propidium iodide membrane-integrity tests, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling, and annexin V assays showed that antisense silencing of DENN resulted in markedly more pronounced cell death in cancer cells compared with nonmalignant cells. Antisense-treated cell lines exhibited extensive loss of DNA content, forming distinct sub-G(1) peaks, while cell proliferation diminished significantly. Ultrastructural features of programmed cell death in cells subjected to antisense ODNs were authenticated by electron microscopy. In contrast, transfection of cell lines with a plasmid construct to achieve DENN overexpression augmented cellular proliferation and could reverse the apoptotic effect of antisense and staurosporine treatment. Our findings suggest that DENN is intimately involved in anti-apoptotic and cell-survival processes.
authors.affiliation: Human Genome Laboratory, Department of Microbiology, Faculty of Medicine, National University of Singapore, Kent Ridge, Singapore.
authors.forename: K M
authors.initials: KM
authors.lastname: Lim
authors.forename: Vincent T K
authors.initials: VT
authors.lastname: Chow
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002522:Chlorocebus aethiops; D016373:DNA, Antisense / Q000494:pharmacology*; D053418:Death Domain Receptor Signaling Adaptor Proteins; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014709:Vero Cells
chemical_list: D017304:Annexin A5; D016373:DNA, Antisense; D053418:Death Domain Receptor Signaling Adaptor Proteins; D004791:Enzyme Inhibitors; D020662:Guanine Nucleotide Exchange Factors; C105852:MADD protein, human; C491371:Madd protein, mouse; C491370:Madd protein, rat; D019311:Staurosporine