No abstract

␤-Catenin is a mediator of the Wnt-signaling pathway. In many cancers, ␤-catenin is stabilized and accumulates in the nucleus where it associates with lymphoidenhancing factor 1/ T-cell transcription factors to activate genes involved in cell transformation. Previously, we showed that adenomatous polyposis coli (APC) protein can regulate ␤-catenin localization by nuclear export. In this study, we used in vitro transport assays to test whether cellular ␤-catenin can exit the nucleus independent of APC and the CRM1 export receptor. In digitonin-permeabilized SW480 (APC mut/mut ) tumor cells, nuclear ␤-catenin decreased >60% in export reactions in the absence of exogenous factors. Under similar conditions, nuclear c-ABL was only exported after the addition of cytosolic extract, and the export was blocked by the CRM1-specific inhibitor, leptomycin B. The nuclear export of ␤-catenin was not blocked by leptomycin B treatment, revealing a CRM1-and APC-independent pathway. The export of ␤-catenin was sensitive to lower temperatures and the removal of ATP, indicating an active process. Ectopically expressed yellow fluorescent protein-␤-catenin also displayed CRM1-independent export. Conversely, the overexpression of the CRM1 transporter moderately stimulated export of nuclear ␤-catenin, confirming that ␤-catenin exits the nucleus by at least two distinct pathways. The shuttling ability of tumor cell ␤-catenin has implications for its regulation and its role in transferring signals between the nucleus and plasma membrane.

We have synthesized a series of bis(9-aminoacridine-4-carboxamides) linked via the 9-position with neutral flexible alkyl chains, charged flexible polyamine chains, and a semirigid charged piperazine-containing chain. The carboxamide side chains comprise N,N-dimethylaminoethyl and ethylmorpholino groups. The compounds are designed to bisintercalate into DNA by a threading mode, in which the side chains are intended to form hydrogen-bonding contacts with the O6/N7 atoms of guanine in the major groove, and the linkers are intended to lie in the minor groove. By this means, we anticipate that they will dissociate slowly from DNA, and be cytotoxic as a consequence of template inhibition of transcription. The dimers remove and reverse the supercoiling of closed circular DNA with helix unwinding angles ranging from 26 degrees to 46 degrees, confirming bifunctional intercalation in all cases, and the DNA complexes of representative members dissociate many orders of magnitude more slowly than simple aminoacridines. Cytotoxicity for human leukemic CCRF-CEM cells was determined, the most active agents having IC(50) values of 35-50 nM in a range extending over 20-fold, with neither the dimethylaminoethyl nor the ethylmorpholino series being intrinsically more toxic. In common with established transcription inhibitors, the morpholino series, with one exception, have no effect on cell cycle distribution in randomly dividing CCRF-CEM populations. By contrast, the dimethylaminoethyl series, with two exceptions, cause G2/M arrest in the manner of topoisomerase poisons, consistent with possible involvement of topoisomerases in their mode of action. Thus, the cellular response to these bisintercalating threading agents is complex and appears to be determined by both their side chain and linker structures. There are no simple relationships between structure, cytotoxicity, and cell cycle arrest, and the origins of this complexity are unclear given that the compounds bind to DNA by a common mechanism.