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

Abstract

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...

MADD/DENN splice variant of the IG20 gene is necessary and sufficient for cancer cell survival

“…These observations indicated that IG20-SVs could differentially regulate cell proliferation and death. Recent studies (Lim and Chow, 2002;Lim et al, 2004) in which all four endogenous IG20-SVs were knocked down using oligodeoxynucleotides (ODNs) revealed increased susceptibility of cancer cells to apoptosis; an intriguing finding given our previous Selective knockdown of IG20-SVs in cancer cell survival N Mulherkar et al observations. Therefore, we set out to determine the specific IG20-SV responsible for cancer cell survival.…”

“…Knockdown of all endogenous IG20-SVs, using antisense oligonucleotides, resulted in spontaneous apoptosis of cancer cells in vitro and in vivo (Lim and Chow, 2002;Lim et al, 2004). Therefore, contrasting effects of IG20-SVs, noted earlier, can only be clarified by knocking down individual IG20-SVs and determining the consequent effects.…”

“…The IG20 gene plays an important role in cancer cell proliferation, apoptosis and survival Lee, 1996, 1998;Schievella et al, 1997;Brinkman et al, 1999;Murakami-Mori et al, 1999;Telliez et al, 2000;AlZoubi et al, 2001;Lim and Chow, 2002;Efimova et al, 2003Efimova et al, , 2004Lim et al, 2004;Ramaswamy et al, 2004). Additionally, it plays an important role in neurotransmission (Zhang et al, 1998;Tanaka et al, 2001;Yamaguchi et al, 2002), neurodegeneration (Del Villar and Miller, 2004) and guanine nucleotide exchange (Wada et al, 1997;Brown and Howe, 1998;Iwasaki and Toyonaga, 2000;Levivier et al, 2001).…”

MCP-1 Antibody Treatment Enhances Damage and Impedes Repair of the Alveolar Epithelium in Influenza Pneumonitis

“…Total RNA was isolated from frozen lung tissues using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany), and reverse-transcribed into cDNA with the MMLV Reverse Transcription system (Promega, Madison, WI) using random primers (32). Aliquots of cDNA (1 ml each) were amplified by PCR using SP-C, T1-a, HGF, and b-actin primers ( Table 1).…”

The U95 Protein of Human Herpesvirus 6B Interacts with Human GRIM-19: Silencing of U95 Expression Reduces Viral Load and Abrogates Loss of Mitochondrial Membrane Potential

“…At 0, 1, 4, and 7 h postinfection, both infected and uninfected cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 1 h. The fixed samples were then postfixed with 2% osmium tetroxide at room temperature for 1 h, dehydrated in a graded alcohol series, and embedded in Epon 812. Ultrathin sections (20 nm thick) were cut with a microtome, stained with uranyl acetate and lead citrate for 20 and 10 min, respectively, and observed using a Philips model 208S electron microscope (26).…”