Abstract. Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SVinduced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 IxM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Overexpression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SVinduced NF-kappa B activation. To determine if NFkappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.

The NF-KB transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65A), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65A, and various mutants indicates that residues 222 to 231 are important for binding to KB DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65A with itself or with p50 was evident. However, p65A was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65A may be to negatively regulate NF-KB function through formation of nonfunctional heteromeric complexes.The NF-KB transcription factor complex is a pleiotropic activator that participates in the induction of numerous cellular and viral genes (4, 32). The active complex is composed of two subunits, designated p50 and p65 (3, 18). The genes encoding p50 (19, 29) and p65 (37, 45) have recently been cloned, and the amino termini of both proteins have been shown to have considerable homology to the rel oncogene product. The rel oncogene, originally identified in the avian retrovirus Rev T, causes lymphoid cell tumors in birds (41). It is now clear that the Rel family of proteins, of which the Drosophila maternal morphogen dorsal is a member (55), possess transcriptional regulatory properties (8,16,23,28,42,58), as best exemplified by the NF-KB transcription factor complex.NF-KB exists in both an active and an inactive state. The inactive complex is present in the cytosol as a complex bound to an inhibitor protein termed IKB...

Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, downregulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.The ends of eukaryotic chromosomes, called telomeres, consist of an array of tandem repeats of the hexanucleotide 5'-TTAGGG-3'. It is currently assumed that telomeres were evolved to protect the ends of chromosomes against exonucleases and ligases, to prevent the activation of DNA-damage checkpoints, and to counter the loss of terminal DNA segments that occurs when linear DNA is replicated (for a review, see refs.

The NF-KB transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes.Various cell adhesion molecules have NF-w.B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-cB. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumor- Cell-cell and cell-substratum adhesion plays an important role in the regulation of normal and neoplastic cell growth (1). These adhesion events are mediated by diverse cell adhesion molecules (CAMs) and integrins (1,2). Cell transformation is often associated with qualitative alteration in the integrin repertoire (3). The process of tumor progression is complex and requires malignant cells to modulate their adhesion properties at various points of tumor development (4). Inhibition of NF-KB function by antisense technology elicits a strong block in the adhesion of diverse cell types; if the p65 subunit is inhibited this effect can be observed in most cell types, but if the p50 subunit is inhibited the effect is dependent on the differentiative status of the cells (5). The cellular adhesion of differentiated HL-60 cells stimulated by phorbol 12-myristate 13-acetate is significantly altered, an effect associated with a marked reduction in CD llb integrin expression (6). These results suggested to us that antisense inhibition of p65 function could have profound effects on cellular adhesion.Utilizing diverse tumor-derived cell lines, we demonstrate a pronounced inhibition of adhesion and in vitro growth after treatment with p65 antisense oligonucleotides. Expression of dexamethasone (Dex)-inducible antisense RNA to p65 in a fibrosarcoma cell line inhibited tumorigenicity and caused regression of tumors in nude mice in a Dex-dependentThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.manner. Based on these results, we have attempted to establish the in vivo therapeutic efficacy of antisense p65 oligonucleotides utilizing nude mouse tumor models. MATERIALS AND METHODSAntisense Oligonucleotides. The antisense and sense phosphorothioate analogs of oligonucleotides to the 5' end of the different subunits of NF-KB, including the ATG initiation codon (18-to 24-mer), were synthesized using an automated synthesizer (model 394, Applied Biosystems) as described (5,6) following the procedure of Matsukura et al. (7).Cell Lines and Growth Assay. The K-BALB, B-16, SW-480, HT-29, and T-47D cell lines were obtained from the American Type Culture Collection; Rat-1 ras (8), HOS-MNNG (9), and Rat-1 p65A (10) have been described. In four independent experiments, cells (3 x 106) were trypsinized ...

Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. We tested this hypothesis by arresting human tumor cell lines at different stages of the cell cycle. Induction of quiescence by serum deprivation did not affect telomerase activity. Cells arrested at the G1/S phase of the cell cycle showed similar levels of telomerase to asynchronous cultures; progression through the S phase was associated with increased telomerase activity. The

Immune-mediated mechanisms have been implicated in the etiology of idiopathic bone marrow fibrosis (IMF). However, the mechanism remains poorly defined. Compared with healthy controls, IMF monocytes are overactivated, with increased production of TGF-β and IL-1. TGF-β is central to the progression of fibrosis in different organs. In the lung, fibrosis is associated with up-regulation of TGF-β-inducible genes. Because IL-1 and TGF-β have pro- and antiinflammatory properties and neither appears to regulate the high levels of each other in IMF, we studied the mechanism of this paradigm. We focused on the role of RelA, a subunit of the transcription factor, NF-κB that is associated with inflammatory responses. We transiently knocked out RelA from IMF monocytes with antisense oligonucleotides and showed that RelA is central to IL-1 and TGF-β production and to the adhesion of IMF monocytes. Because the NF-κB family comprises subunits other than RelA, we used aspirin and sodium salicylate to inhibit kinases that activate NF-κB and showed effects similar to those of the RelA knockout system. It is unlikely that RelA could be interacting directly with the TGF-β gene. Therefore, we determined its role in TGF-β production and showed that exogenous IL-1 could induce TGF-β and adherence of IMF monocytes despite the depletion of NF-κB. The results indicate that IL-1 is necessary for TGF-β production in IMF monocytes, but NF-κB activation is required for the production of endogenous IL-1. Initial adhesion activates NF-κB, which led to IL-1 production. Through autocrine means, IL-1 induces TGF-β production. In total, these reactions maintain overactivation of IMF monocytes.

The p5O and p65 subunits of NF-cB represent two members of a gene family that shares considerable homology to the rel oncogene. Proteins encoded by these genes form homo-and heterodimers which recognize a common DNA sequence motif. Recent data have suggested that homodimers of individual subunits of NF-KB can selectively activate gene expression in vitro. To explore this possibility in a more physiological manner, murine embryonic stem (ES) cells were treated with phosphorothio antisense oligonucleotides to either p50 or p65. Within 5 h after exposure to phosphorothio antisense p65 oligonucleotides, cells exhibited dramatic alterations in adhesion properties. Similar findings were obtained in a stable cell line that expressed a dexamethasone-inducible antisense mRNA to p65. Although antisense oligonucleotides raised against both p50 and p65 elicited a significant reduction in their respective mRNAs, only the cells treated with antisense p50 maintained a normal morphology. However, 6 days following removal of leukemia-inhibiting factor, a growth factor which suppresses embryonic stem cell differentiation, adhesion properties of cells treated with the antisense p50 oligonucleotides were markedly affected. The ability of the individual antisense oligonucleotides to elicit differential effects on cell adhesion, a property dependent upon the stage of differentiation, suggests that the p50 and p65 subunits of NF-cB regulate gene expression either as homodimers or as heterodimers with other rel family members. Furthermore, the finding that reduction in p65 expression alone had profound effects on cell adhesion properties indicates that p65 plays an important role in nonstimulated cells and cannot exist solely complexed with the cytosolic inhibitory protein IKB.The NF-KB transcription factor complex is a pleiotropic activator which participates in the induction of a wide variety of cellular and viral genes (2, 17). The active complex is composed of two subunits designated p50 and p65 (2, 7). The genes encoding p50 (8, 12) and p65 (21, 24) have been cloned, and the N termini of both proteins show considerable homology to the product of the oncogene rel.Although both subunits of NF-KB bind to a common decameric DNA motif as either homo-or heterodimers, it was recently demonstrated that the individual subunits exhibit distinct binding preferences (15). Furthermore, selected DNA motifs that bound only one of these subunits (p50 or p65) were significantly reduced in their ability to recognize NF-KB (i.e., p50/p65) and sometimes could not recognize it at all (15). These findings suggest the existence of rather distinct KB DNA motifs that have differential binding specificities for the Rel proteins, implicating a role for such motifs in selective regulation of gene expression. Consistent with this speculation, transfection studies demonstrate that the p65 subunit can function as a homodimeric transcriptional activator (3, 25). However, the physiological relevance of this observation remains unclear, as it is believed that in r...

Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.