ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X L and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 'druggable' targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top 'hits' from the screen were found to result from off-target gene silencing. Interestingly, the three offtarget siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3 0 untranslated region (3 0 UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.

To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against f3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2

Formation of branched glucan, glucan-glucan cross links, and glucan-chitin cross links most likely involves the action of fungal wall glucanases and transglycosylases. We developed an HPLC assay using radiolabeled substrates in order to study the kinetics of interaction of donor and acceptor molecules with a glucosyltransferase present in the cell walls of both Saccharomyces cerevisiae and Candida albicans. Purified transferase first forms an activated intermediate from a donor p-1,3 glucan, releasing free disaccharide. The activated intermediate is transferred, in the presence of an appropriate acceptor /3-1,3 glucan, yielding a linear glucan containing a p-1,6 linkage at the transfer site [Yu, L., Goldman, R., Sullivan, P., Walker, G. & Fesik, S. W. (1993) J. Biomol. NMR 3, 429-4411, An apparent K,, of 0.41 mM for the acceptor site was determined using laminaritetraose as the acceptor. An apparent K, of 31 mM for the donor site was determined using increasing concentrations of laminaripentaose, and monitoring formation of laminaribiose. The enzyme functioned as a glucanase at low concentrations of acceptor molecules, with excess H,O competing for reaction at the activated donor site, thus resulting in hydrolysis.However, as the concentration of acceptor increased, the reaction shifted from hydrolysis to glucosyltransfer. The reaction appeared specific for p-1,3 glucan as acceptor, in as much as no transfer was detected when either hexa-N-acetyl-chitohexaose or maltooligosaccharides were used as acceptors. The roles of such an enzymic activity in cell wall metabolism is discussed in terms of repair, cross linking and incorporation of newly synthesized chains of p-1,3 glucan into the previously existing cell wall structure.Keywords. Glucosyltransferase ; fungal wall ; BGL2 protein ; wall assembly ; Saccharomyces.Synthesis and assembly of the fungal cell wall is a complex process about which we know much less compared to the analogous events occurring in bacteria. We do know that linear polymers of chitin, p-1,4 N-acetylglucosamine, and glucan, p-1,3 glucose, are synthesized by microsomal fractions prepared from fungal cells when iincubated in the presence of their appropriate UDP-sugars [l, 21. The process appears to be vectorial in that UDP-sugar presenf in the cytoplasm is polymerized, with concurrent translocation through the membrane of the growing polymeric chain. However, little if anything is known about the following aspects of p-1,3 polymer synthesis: (a) initiation of chain synthesis, (b) regulation of chain initiation, (c) translocation through the membrane, (d) mechanism and regulation of termination of chain synthesis, and (e) localization of chain synthesis during different phases of wall expansion. Furthermore, glucan found in the mature cell wall consists of highly branched molecules containing both p-1,3 and p-1,6 linkages.

The coupling of apoptosis (programmed cell death) to the cell division cycle is essential for homeostasis and genomic integrity. Here, we report the crystal structure of survivin, an inhibitor of apoptosis, which has been implicated in both control of cell death and regulation of cell division. In addition to a conserved N-terminal Zn finger baculovirus IAP repeat, survivin forms a dimer through a symmetric interaction with an intermolecularly bound Zn atom located along the molecular dyad axis. The interaction of the dimer-related C-terminal alpha helices forms an extended surface of approximately 70 A in length. Mutagenesis analysis revealed that survivin dimerization and an extended negatively charged surface surrounding Asp-71 are required to counteract apoptosis and preserve ploidy. These findings may provide a structural basis for a dual role of survivin in inhibition of apoptosis and regulation of cell division.