Introduction of exogenous DNA into mammalian cells represents a powerful approach for manipulating signal transduction. The available techniques, however, are limited by low transduction efficiency and low cell viability after transduction. Here we report a highly efficient molecular delivery technique, named nanotube spearing, based on the penetration of nickel-embedded nanotubes into cell membranes by magnetic field driving. DNA plasmids containing the enhanced green fluorescent protein (EGFP) sequence were immobilized onto the nanotubes, and subsequently speared into targeted cells. We have achieved an unprecedented high transduction efficiency in Bal17 B-lymphoma, ex vivo B cells and primary neurons with high viability after transduction. This technique may provide a powerful tool for highly efficient gene transfer into a variety of cells, especially the hard-to-transfect cells.

The bioenergetic response of B lymphocytes is subject to rapid changes following antigen encounter in order to provide ATP and anabolic precursors necessary to support growth. However, the pathways involved in glucose acquisition and metabolism are unknown. We find that B lymphocytes rapidly increase glucose uptake and glycolysis following B-cell antigen receptor (BCR) crosslinking. Inhibition of glycolysis blocks BCR-mediated growth. Prior to S-phase entry, glucose metabolism shifts from primarily glycolytic to include the pentose phosphate pathway. BCR-induced glucose utilization is dependent upon phosphatidylinositol 3-kinase (PI-3K) activity as evidenced by inhibition of glucose uptake and glycolysis with LY294002 treatment of normal B cells and impaired glucose utilization in B cells deficient in the PI-3K regulatory subunit p85␣. Activation of Akt is sufficient to increase glucose utilization in B cells. We find that glucose utilization is inhibited by coengagement of the BCR and Fc␥RIIB, suggesting that limiting glucose metabolism may represent an important mechanism underlying Fc␥RIIB-mediated growth arrest. Taken together, these findings demonstrate that both growth-promoting BCR signaling and growth-inhibitory Fc␥RIIB signaling modulate glucose energy metabolism. Manipulation of these pathways may prove to be useful in the treatment of lymphoproliferative disorders, wherein clonal expansion of B lymphocytes plays a role. IntroductionIn response to antigen challenge, resting B lymphocytes exit the G 0 phase of the cell cycle and undergo a period of growth before committing to genome replication. 1,2 Growth corresponds to an accumulation of cell mass that is accompanied by increased size and is linked to increased de novo macromolecular synthesis. [3][4][5] That mammalian cell growth may be necessary for genome replication underscores its importance in adaptive immunity in that the clonal expansion of antigen-specific B lymphocytes is a prerequisite for humoral immune responses. Most investigations in B cells have focused on the role of genes whose function are important for B-cell antigen receptor (BCR)-induced protein synthesis and increased cell size. 4,5 It is recognized, however, that antigen receptor-triggered macromolecular synthesis and gene expression places enormous bioenergetic demands on lymphocytes. 5,6 Therefore, one of the fundamental aspects of B-cell responses to antigen challenge that may be critical in vivo is the provision of metabolic substrates to provide ATP and anabolic precursors for cellular growth.Early studies in lectin-stimulated thymocytes highlighted the importance of glucose uptake and catabolism in providing energy and carbon for macromolecular synthesis. 7,8 Further, proliferating thymocytes meet their ATP demand mainly by glycolytic catabolism when sufficient glucose is available. 9 It is widely viewed that glucose metabolism is regulated by homeostatic mechanisms wherein mammalian cells respond to a decreased ATP/ADP ratio by adjusting nutrient uptake and catabolism t...

The dynamic processes of cell migration and invasion are largely coordinated by Rho family GTPases. The scaffolding protein IQGAP1 binds to Cdc42, increasing the amount of active Cdc42 both in vitro and in cells. Here we show that overexpression of IQGAP1 in mammalian cells enhances cell migration in a Cdc42-and Rac1-dependent manner. Importantly, cell motility was significantly decreased both by knock down of endogenous IQGAP1 using small interfering RNA and by transfection of a dominant negative IQGAP1 construct, IQGAP1⌬GRD. Cell invasion was similarly altered by manipulating intracellular IQGAP1 concentrations. Moreover, invasion mediated by constitutively active Cdc42 was attenuated by IQGAP1⌬GRD. Thus, IQGAP1 has a fundamental role in cell motility and invasion.Cell migration is a multistep process that is essential for normal development, angiogenesis, wound repair, and metastasis (1). Specifically, it involves protrusion of the plasma membrane at the leading edge, configuration of new sites of adhesion to the extracellular matrix at the front, the release of old adhesions in the back of the cell, and finally, contraction of actomyosin-based cytoskeletal filaments in the cell body (2). Coordination of the actin cytoskeleton, adhesion molecules, and microtubules is required for cell movement; this function is largely orchestrated by the Rho family of GTPases. For example, Cdc42 and Rac, which regulate the production of filopodia and lamellipodia, respectively (3, 4), are important for mediating new protrusions and adhesions at the cell periphery during migration.Cdc42 and Rac1 participate in cell function by interacting with a diverse array of proteins (5). One of these, IQGAP1, regulates cytoskeletal function by integrating multiple targets, including Cdc42 and Rac1 (6, 7), actin (8, 9), calmodulin (7, 10), and CLIP-170 (11). We previously documented that IQGAP1 inhibits the intrinsic GTPase activity of Cdc42 (10), thereby significantly increasing levels of active Cdc42 in cells (12). In addition, a dominant negative IQGAP1 construct, IQGAP1⌬GRD, 1 substantially reduced active Cdc42, preventing the formation of filopodia (12). Moreover, in vivo analysis revealed that IQGAP1 induced superficial ectodermal lesions in Xenopus embryos, indicating that IQGAP1 is likely to affect cytoskeletal architecture and cell adhesion (13).We therefore hypothesized that IQGAP1 may play a role in mediating cell motility and invasion. Here we demonstrate that IQGAP1 overexpression significantly increased cell migration in several different cell types. IQGAP1 also increased cell speed on both glass and plastic substrata. Dominant negative Cdc42 and Rac1, but not RhoA, inhibited the IQGAP1-mediated increase in motility. Moreover, cell migration was significantly slowed by both IQGAP1⌬GRD and knock down of IQGAP1 by both transient and stable expression of small interfering RNA (siRNA) for IQGAP1. Stable overexpression of IQGAP1 also led to a significant increase in cell invasive capacity. These data imply that IQGAP1 is an impor...

The scaffold protein IQGAP1 integrates signaling pathways and participates in diverse cellular activities. IQGAP1 is overexpressed in a number of human solid neoplasms, but its functional role in tumorigenesis has not been previously evaluated. Here we report that IQGAP1 contributes to neoplastic transformation of human breast epithelial cells. The amount of IQGAP1 in breast carcinoma is greater than that in normal tissue, with highly metastatic breast epithelial cells expressing the highest levels. Overexpression of IQGAP1 enhances proliferation of MCF-7 breast epithelial cells. Reduction of endogenous IQGAP1 by RNA interference impairs both serum-dependent and anchorage-independent growth of MCF-7 cells. Consistent with these in vitro observations, immortalized MCF-7 cells overexpressing IQGAP1 form invasive tumors in immunocompromised mice, whereas tumors derived from MCF-7 cells with stable knockdown of IQGAP1 are smaller and less invasive. In vitro analysis with selected IQGAP1 mutant constructs and a chemical inhibitor suggests that actin, Cdc42/Rac1, and the mitogen-activated protein kinase pathway contribute to the mechanism by which IQGAP1 increases cell invasion. Collectively, our data reveal that IQGAP1 enhances mammary tumorigenesis, suggesting that it may be a target for therapeutic intervention.Tumor progression that culminates in clinically relevant metastatic lesions is the end point of a complex sequence of interrelated cellular events. After the initial transforming event, tumor cell proliferation, invasion, and migration, as well as vascularization of the tumor mass, occur. A thorough understanding of the molecular mechanisms that regulate tumor progression will provide the biological foundation for improving the efficacy of current therapeutic interventions (1-3).IQGAP1 is a 189-kDa scaffolding protein that contains multiple protein-interacting domains (for reviews see Refs. 4 -7). These include a calponin homology domain, a polyprolinebinding domain, four calmodulin-binding motifs, and a Ras-GAP-related domain. The motifs present in IQGAP1 are involved in the interaction of IQGAP1 with specific proteins, such as actin, calmodulin, members of the Rho GTPase family (i.e. Rac1 and Cdc42), Rap1, E-cadherin, ␤-catenin, members of the mitogen-activated protein kinase (MAPK) 4 pathway, and adenomatous polyposis coli (7,8). By interacting with these proteins, IQGAP1 regulates multiple fundamental cellular activities including cytoskeletal organization, cell-cell adhesion, cell migration, transcription, and signal transduction. For example, binding of IQGAP1 to ␤-catenin both disrupts the E-cadherin-catenin complex, inhibiting epithelial cell-cell adhesion (9), and increases ␤-catenin-mediated transcriptional activation (10). IQGAP1 increases active Cdc42 in mammalian cells, resulting in formation of actin filopodia and microspikes (11), and promotion of cell migration and invasion (12). These morphological and functional changes are not observed with a mutant IQGAP1 construct with impeded Cdc42-me...