Antisense abrogation of DENN expression induces apoptosis of leukemia cells in vitro, causes tumor regression in vivo and alters the transcription of genes involved in apoptosis and the cell cycle

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 NFkappaB and TNFR1 KO cells resulted in markedly more pronounced cell death, whereas antisense-treated TNFalpha 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 TNFalpha, TNFR1 and cyclin D3. Conversely, DENN overexpression stimulated cell proliferation and led to upregulated TRPM2 and cyclin B1, but diminished expression of Fas, TNFR2, TRAIL and Egr-1. The participation of TNFalpha, TNFR1, TNFR2 and Fas in the inhibition of DENN expression was also demonstrated. These data support the anti-apoptotic and cell survival role of DENN, especially in malignant cells, and its interaction with specific genes and proteins involved in the apoptotic and cell cycle pathways. (C) 2003 Wiley-Liss, Inc.