RESUMEN

Antisense inhibition of the RelA subunit of NF-kappa B transcription factor (but not the NFKB1 subunit) causes pronounced inhibition of tumor cell growth in vitro and in vivo. Inhibition of either subunit, however, results in inhibition of the heterodimeric NF-kappa B complex in antisense-treated cells. Either of the subunits of NF-kappa B can form homo- or heterodimers with other members of the Rel oncogene family. In an effort to decipher the role of homo- vs heterodimeric NNF-kappa B in regulating tumor cell growth, we have used a decoy approach to trap these complexes in vivo. Using double-stranded phosphorothioates as a direct in vivo competitor for homo- vs heterodimeric NF-kappa B, we demonstrate that decoys more specific to RelA inhibit growth tumor cell growth in vitro. We demonstrate that RelA, either as a homodimer or a heterodimer with some other members of the Rel family and not the classical NF-kappa B (RelA/NFKB1), is involved in the differential growth control of tumor cells. Our results indicate that such transcription factor decoys can be a non-antisense tool to study the function of DNA-binding transcription factors.

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RESUMEN

NFKB: is a pleiotropic transcription factor that participates in the induction of various cellular and viral genes (for a review, see refs. 1 and 2). The principal form of this active complex is composed of two polypeptides, p65 (recently renamed rel A) and p50 (recently renamed NFKB1) (3, 4). Both of these sub-units belong to the rel famtly of transcription factors with homology in the amino terminus (5, 6). Since the original identification of these factors, several others have been identified in this family of transcription factors: P49, c-rel, p100, rel B, dorsal, Bcl-3, and p105 (1). The NF-kheterodimer is associated in the cytoplasm with the IkB subunit, which sequesters this factor in an inactive state (7). On activation by various stimuli, the IkB subunit separates from the active NFheterodimer, and the active complex is translocated to the nucleus where it binds to its nuclear DNA target sequence (Fig. 1). The principal cause for disassociation of IkB from NFis phosphorylation (8), but it may involve proteolysis of IkB (9) Several autoregulatory loops have been proposed for the involvement of NF-kB in the regulation of the inhibitor IkB. Furthermore, NF-kB has been shown to regulate the transcription of IkB and NFKB1 (10-12). Fig. 1. NF-K: B transcription factor. This figure depicts the basis for the function of the NF-K: B transcription factor. The principal form of the NF-K: B transcription complex is as a heterodimer composed of the two subunits: rel A and NF-K: B 1. The inactive complex is sequestered in the cytoplasm associated with the inhibitor protein, IK: B. On activation, the IK: B protein separates from the active complex through a mechanism involving the phosphorylation and proteolysis Of IK: B. The active NF-K: B complex is then translocated to the nucleus where it binds DNA in a sequence-specific manner and interacts with the transcriptional machinery to modulate the rate of transcription of target genes.

RESUMEN

Antisense inhibition of the RelA subunit but not the NFKB1 subunit of NK-kappa B transcription factor results in a block of cellular adhesion and inhibition of tumor cell growth in vitro and in vivo. Studies aimed at dissecting the molecular mechanism of antisense relA action led to our identification of a kappa B-like motif present in aV integrin promoter. The alpha V/kappa B motif is closely related to RelA/c-Rel-binding sequences, such as 65-2 and TF-1. However, unlike these two kappa Blike motifs, the alpha V/kappa B motif detected a nuclear Sp1 activity distinct from kappa B activity, which was subsequently confirmed to be derived from Sp1. In comparison to the conventional GC box-containing Sp1 motif, the alpha V/kappa B motif also binds in vitro to c-Rel and RelA but not to NFKB1. Antisense inhibition of RelA inhibited the alpha V/kappa B activity. Direct in vivo competition of alpha V/kappa B-binding activity by a decoy approach also resulted in inhibition of alpha V/kappa B activity in intact cells. A variant of the alpha V/kappa B motif was found to retain the dual ability to detect Sp1 and the NF-kappa B complex in the nuclear and cytoplasmic extracts. Such dual interacting ability of a DNA motif offers yet another way of gene regulation in vivo and hence can affect cellular growth. Our results identify alpha V integrin as one of the molecular targets for relA/NF-kappa B and may explain growth inhibition by antisense relA.

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RESUMEN

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.

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