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 ...

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...

Transcription factor NF-kappa B comprises two proteins, p50 and p65, that have sequence similarity to the v-rel oncogene. In primary hematopoietic cell populations an alternatively spliced form of NF-kappa B p65 mRNA was observed that encoded a protein designated p65 delta. Expression of the p65 delta cDNA in Rat-1 fibroblasts resulted in focus formation, anchorage-independent growth in soft agar, and tumor formation in athymic nude mice, effects not obtained with expression of p65 or a p65 delta mutant that contains a disruption within the transcriptional activation domain. Thus, p65 delta, which associated weakly and interfered with DNA binding by p65, may sequester an essential limiting regulatory factor or factors required for NF-kappa B function.

The p50 and p65 subunits of NF-kappa B 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-kappa B 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-kappa B 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 I kappa B.