ICAM-2 and/or actin in transfected neuroblastoma cells. Proteins in lysates from pIRESneo and pIRES.ICAM2 transfectants were immunoprecipitated with a monoclonal antibody to ICAM-2, and an aliquot of precipitated proteins was analyzed by immunoblot with a polyclonal antiserum to this protein. The blot in the lower panel of Fig. 1D shows the expected difference in level of ICAM-2 precipitated from ICAM-2 compared to control transfectants. A second aliquot of precipitated proteins was separated by electrophoresis and stained with SyproRuby to detect co- precipitated proteins. SyproRuby-stained gels showed a distinct band of ,42 kDa, unique to the SK-N-ASpIRES.ICAM2 cells. This band (#1, in upper panel of Fig. 1D) was confirmed to be .99% identical to human actin by mass spectrometry, indicating that in SK-N-ASpIRES.ICAM2 cells, actin associated with ICAM-2. Co-immunoprecipitations were then done with antibod- ies to a-actinin, actin, or ezrin, and precipitated products identified by immunoblot. The immunoprecipitation/immunoblot per- formed with a monoclonal antibody to a-actinin or control immunoglobulin/polyclonal antiserum (Fig. 1E) showed that control precipitations were appropriately negative and that equal amounts of a-actinin were precipitated from SK-N-ASpIRESneo and SK-N-ASpIRES.ICAM2 transfectants. Subsequent immuno- blots to detect proteins that co-precipitated with a-actinin revealed that both actin and ICAM-2 associated with a-actinin in lysates from SK-N-ASpIRES.ICAM2 cells, but little or no actin or ICAM-2 co-precipitated with a-actinin in lysates from SK-N- ASpIRESneo cells (Fig. 1F). Data in Fig. 1F also suggested that little actin associated with a-actinin in the absence of ICAM-2 expression. The specificity of the observed association of ICAM-2, a-actinin, and actin was verified by immunoprecipitating ICAM-2 and immunoblotting for ICAM-2, a-actinin, or actin (Supporting Information, Fig. S1). Ezrin was not detected to co-precipitate with ICAM-2 or with actin in either cell line, nor was actin or ICAM-2 detected to co-precipitate with ezrin (data not shown). These co-immunoprecipitation data showed that ICAM-2 associated with a-actinin and actin in neuroblastoma cell lysates. We note that the data are qualitative and do not necessarily reflect the ratio in which these proteins associate. ICAM-2 is a heavily glycosylated protein that exists in multiple forms that migrate as a diffuse band having an area6density signal approximately 300%– 400% greater than that for the same amount of de-glycosylated ICAM-2. Further, a-actinin induces actin self-association [32], resulting in variable numbers of actin molecules associating with a single a-actinin molecule. Nevertheless, the data indicate that ICAM-2, a-actinin, and actin associate with each other in neuroblastoma cell lysates. ICAM-2-mediated redistribution of actin fibers is disrupted by competitive peptides to inhibit ICAM-2/a- actinin or a-actinin/actin binding, and is associated with decreased tumor cell motility The observed ICAM-2/a-actinin/actin association in cell lysates suggested that ICAM-2 expression in neuroblastoma cells might affect the function of the actin cytoskeleton. Alternatively, in other types of transfected cells, ICAM-2 has been reported to stimulate immune responses [22] and affect AKT phosphorylation and chemosensitivity [25]. Therefore, we evaluated the effect of ICAM-2 expression by SK-N-AS cells on AKT phosphorylation and sensitivity to vincristine, doxorubicin, or etoposide, and on actin fiber distribution and cell motility, using in vitro assays in which immune stimulation was not a potential factor. Effect of ICAM-2 expression on AKT phosphorylation, drug sensitivity, and growth characteristics. In contrast to ICAM-2 Limits Metastasis published reports, we did not detect ICAM-2-mediated increases in AKT phosphorylation as determined with a Ser473-specific antibody on immunoblots, or in sensitivity to etoposide or doxorubicin (data not shown). Neither did we detect ICAM-2 mediated-effects on cell cycle distribution, cell volume, or cell doubling time (data not shown). ICAM-2 expression did affect the morphology of cells as adherent monolayers. Adherent cells expressing ICAM-2 appeared larger and less spheroid in shape than cells expressing little or no ICAM-2 (evident in Fig. 2A). Effect of ICAM-2 expression on actin fiber distribution and cell motility. FITC-phalloidan staining showed that, ICAM-2 redistributed intracellular actin fibers from a transverse to a juxtamembrane localization (Fig. 2A). This redistribution was observed irrespective of cell density (data not shown). We reasoned that if redistribution depended on the interaction of ICAM-2 with a- actinin and actin, peptides designed to competitively inhibit these interactions would be predicted to reverse the observed actin fiber redistribution pattern in SK-N-ASpIRES.ICAM2 transfectants. Consistent with this hypothesis, fibers reverted to a transverse distribution in SK-N-ASpIRES.ICAM2 cells exposed to each competitive peptide (Fig. 2B), but remained co-localized with cell membranes in cells exposed to scrambled control peptides. Similar results were also observed with NB-1691 neuroblastoma cells transfected with pIRESneo or pIRES.ICAM2 (data not shown). Co-incident with observed ICAM-2-mediated redistribution of actin fibers was an inhibition of tumor cell motility (Figs. 2C and D), as assessed with two types of in vitro assays: scratch (wound) assays and modified Boyden chamber invasion assays. In scratch assays, cell monolayers were overlaid with Matrigel immediately after scratching, and the original scratch width was recorded in pixels. Photomicrographs taken at 0 hours and 72 hours (Fig. 2C) showed initial scratch widths of ,1125 pixels and ,1050 pixels for the pIRESneo and pIRES.ICAM2 transfectants, respectively. At 72 hours pIRESneo transfectants had completed closed the scratch, while the SK-N-ASpIRES.ICAM2 cells remained separated by a width of ,450 pixels. In the second type of motility assay, a modified Boyden chamber invasion assay, significantly fewer SK-N- ASpIRES.ICAM2 cells migrated through a layer of Matrigel to the distal side of porous membranes (P,0.0003, Fig. 2D), compared to SK-N-ASpIRESneo cells. ICAM-2 expression also inhibited the motility of NB-1691 cells transfected with pIRESneo and pIRES.ICAM2 in this invasion assay (P,0.001, data not shown). Inhibition of tumor cell motility by ICAM-2 has not been reported previously. Most notably, in vitro results of phalloidan staining and cell motility assays demonstrated a function for ICAM-2 indepen- dent of its known immune stimulatory effect. ICAM-2-mediated redistribution of actin fibers and inhibition of tumor cell motility depended on an intact intracellular domain To then corroborate observations with competitive peptides indicating that ICAM-2/a-actinin binding affected actin fiber distribution, we transfected SK-N-AS cells to express a truncated form of ICAM-2. ICAM-2 and ICAM-2DCD (ICAM-2 deleted cytoplasmic domain) transfectants expressed similar levels of their respective forms of ICAM-2 (Fig. 3A) and both forms of this protein localized to cell membranes (Figs. 1C and S2), but ICAM2DCD lacked the 26-amino acid intracellular domain to which a-actinin binds. If ICAM-2/a-actinin binding were essential to the observed ICAM-2-mediated redistribution of actin fibers, expression of ICAM2DCD would have no effect on actin fiber distribution and, similar to SK-N-ASpIRESneo cells, actin fibers in SK-N-ASpIRES.ICAM2DCD cells would be in a transverse configuration. Immunoprecipitation/immunoblot experiments us- PLoS ONE | www.plosone.org 3 November 2008 | Volume 3 | Issue 11 | e3629