No abstract

Although the cancer cell cytoskeleton is a clinically validated target, few new strategies have emerged for selectively targeting cell division by modulating the cytoskeletal structure, particularly ways that could avoid the cardiotoxic and neurotoxic effects of current agents such as taxanes. We address this gap by describing a novel class of small-molecule agonists of the mammalian Diaphanous (mDia)-related formins, which act downstream of Rho GTPases to assemble actin filaments, and their organization with microfilaments to establish and maintain cell polarity during migration and asymmetric division. GTP-bound Rho activates mDia family members by disrupting the interaction between the DID and DAD autoregulatory domains, which releases the FH2 domain to modulate actin and microtubule dynamics. In screening for DID-DAD disruptors that activate mDia, we identified two molecules called intramimics (IMM-01 and -02) that were sufficient to trigger actin assembly and microtubule stabilization, serum response factor-mediated gene expression, cell-cycle arrest, and apoptosis. In vivo analysis of IMM-01 and -02 established their ability to slow tumor growth in a mouse xenograft model of colon cancer. Taken together, our work establishes the use of intramimics and mDia-related formins as a new general strategy for therapeutic targeting of the cytoskeletal remodeling machinery of cancer cells. Cancer Res; 73(22); 6793-803. Ó2013 AACR.

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Monoclonal anti-NGF antibodies that specifically inhibit the biological activity of mouse beta-NGF were used to study the structural determinants involved in the interaction of NGF with its receptors gp75LNGFR and Trk. None of the three antibodies--N60, M15, and 27/21--showed any reactivity toward denatured NGF. Three experimental methods--radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots--detected no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlapping antigenic site, but 27/21 recognizes a different epitope. Only 27/21, and not N60 or M15, immunoprecipitated beta-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/21 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purified Fab fragments of N60 and M15 gave similar results to the intact antibodies. The other subunits present in the 7S complex of NGF, i.e. the alpha and gamma subunits, competitively inhibited binding of antibodies to beta-NGF. Only the gamma subunit inhibited phosphorylation of Trk and biological activity of beta-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF receptor. The region encompassing the N-terminus, the C-terminus, and the loop on the surface of beta-NGF containing residues 60-80 is proposed as important for binding to the Trk receptor.

We tested the mitogenic response to staphylococcal enterotoxin (SE) type A and SE type B in spleen cells from five strains of mice and found consistent and significant differences among the strains. We chose to study the mitogenic responses of two of these strains, C58BL/6J and BALB/cJ, in greater detail. We investigated the effects of specific monoclonal antibodies to cell surface determinants on SE-induced mitogenesis. Monoclonal antibodies against Ia (class II major histocompatibility complex) determinants blocked SE-induced mitogenesis. Both I-A and I-E molecules can participate in the stimulation, and in BALB/cJ mice which express both types of class II molecules both must be blocked to prevent mitogenesis. Mitogenesis was not inhibited by monoclonal antibodies specific for class I major histocompatibility complex antigens or monoclonal antibodies specific for Mac-1, Lyt-1, or Lyt-2 cell surface proteins. Monoclonal antibodies specific for the T-cell surface antigens L3T4 and T3 also substantially inhibited SE-induced mitogenesis. This implicates participation of the T-cell antigen receptor complex in stimulation induced by the SEs. Elimination of L3T4+ helper-inducer T cells abolished the mitogenic response of spleen cells to SE. Reconstitution of L3T4-depleted spleen cells with L3T4+ T cells showed that the level of the mitogenic response was directly proportional to the number of L3T4+ cells added. Elimination of Lyt-2+ cells resulted in a 50% decrease in the response to SEs. These results indicate that L3T4+ T cells are required for the mitogenic response to SE, but both L3T4+ and Lyt 2+ T cells participate in SE-induced mitogenesis. Our results suggest that both Ia and the T-cell antigenic receptor complex are involved in SE-induced mitogenesis.