We previously reported that messenger RNA expression of DENN (differentially expressed in normal and neoplastic cells) is considerably higher in cancer cell lines than in normal cells. In our present study, we established that certain cancer cell lines express conspicuously higher levels of the 2 DENN isoforms in contrast to the 2 pro-apoptotic IG20 isoforms. Antisense DENN oligodeoxynucleotide treatment of K36 cells in vitro induced extensive apoptosis, while antisense DENN silencing of K36 tumor-bearing mice caused significant tumor regression in vivo. Compared to wild-type murine embryonic fibroblasts, antisense treatment of NFB and TNFR1 KO cells resulted in markedly more pronounced cell death, whereas antisense-treated TNF␣ and TNFR2 knockouts exhibited less prominent apoptosis. Cell viability and apoptosis were authenticated by flow cytometry, membrane integrity, TUNEL, annexin V assays, histology and electron microscopy. Antisense abrogation of DENN expression culminated in upregulated expression of TNFR2, TRAIL and Fas, but downregulation of TNF␣, TNFR1 and cyclin D3.
Key words: DENN; MADD; IG20; splice variants; TNF␣; TNF␣ receptors; Fas; antisense; apoptosis; tumor regressionCellular homeostasis requires a delicate balance between cell proliferation and programmed cell death, or apoptosis. The latter constitutes a crucial mechanism that is tightly regulated by a network of molecular circuits governing cell death and survival. Proteins that either induce or inhibit apoptosis participate in signaling pathways that elicit or inhibit the activation of cell death proteases or caspases. The pathogenesis of cancer may involve dysregulated apoptosis, given that such defects are common in malignant cells. 1 Tumor necrosis factor alpha (TNF␣) is a multifunctional cytokine that belongs to a family of ligands with a related family of receptor molecules. Its multifarious activities range from cell proliferative, inflammatory and immune mechanisms, to destructive cellular events, such as apoptosis and necrosis. In order to orchestrate and achieve its cellular roles, TNF␣ triggers numerous signaling pathways and controls downstream molecules, such as transcription factors and G proteins. 2 For example, administration of anti-TNF␣ antibodies to young mice causes severe but transient retardation in growth and body weight. 3 Members of the TNF receptor (TNFR) family include the 2 TNFR isotypes, TNFR1 (55 kD) and TNFR2 (75 kD), which perform distinct roles in various tissues. TNFR proteins are involved in the regulation of cellular homeostatic phenomena including cellular development, differentiation, survival and apoptosis, as well as in pathophysiologic conditions. Death domain receptors represent a subgroup of TNFRs that are defined according to their functional similarities and the common death domain within their cytoplasmic tails. 4 Activated TNFRs associate with distinct adaptor proteins to modulate multiple signaling pathways. The most notable signaling processes involving TNFR family proteins are those culminat...