PI3Kinase/AKT pathway [25,26] and promotes cell migration [27], suggesting that this adhesion molecule has multiple, apparently unrelated functions. Neither the expression nor the function of ICAM-2 in neuroblastoma cells has been investigated previously. Neuroblastoma is a pediatric tumor thought to arise from cells of the neural crest. Patients diagnosed with neuroblastoma frequently achieve complete remissions, but up to 80% of high- risk patients develop metastatic disease that does not respond to therapy [28]. As is true for most types of solid tumors, the primary cause of death is the development of metastatic disease; but no current therapies specifically target the metastatic process, as a potential approach for limiting the occurrence or the progression of disseminated tumors. The integral role of cell motility (migration and invasion) in the metastatic process and data in the literature suggesting the potential involvement of CAMs in regulating cell motility provided the rationale for the studies reported here. The long-range goal of the work is to identify molecular interactions that regulate the metastatic potential of neuroblastoma, and to determine if these molecular interactions represent potential therapeutic targets. Results ICAM-2 associates with a-actinin and actin to establish a membrane-to-actin linkage In tumor cells, proteins of the cytoplasmic actin linker families contribute to tumorigenesis and metastasis. RT-PCR analysis of a ICAM-2 Limits Metastasis panel of neuroblastoma cell lines (data not shown) showed that neuroblastoma cells express two of these linker proteins: ezrin and a-actinin. Ezrin and a-actinin bind to the intracellular domain of the intercellular adhesion molecule-2 (ICAM-2) [29,30] and to actin. Therefore, we postulated that membrane-bound ICAM-2 binds to cytoplasmic ezrin or a-actinin which, in turn, binds to actin to regulate the motility—and hence the metastatic potential—of neuroblastoma cells. We first demonstrated using immunoblotting, that 6 of 6 neuroblastoma cells express ICAM-2 (Fig. 1A). ICAM-2 protein had the expected apparent mass of 55–60 kDa [31] in all six neuroblastoma cell lines (as well as other types of solid tumor cell lines [data not shown]), and the level of ICAM-2 expression varied with SJNB-1A.NB-1691, SK-N-SH and NB-1643.SK-N-AS and IMR-32. We then transfected a cell line with low endogenous ICAM-2 expression (SK-N-AS) with control pIRESneo or with the ICAM- 2-encoding plasmid, and compared the phenotypes of the stable congenic transfected cell lines. Immunoblots of whole cell lysates of SK-N-ASpIRESneo and SK-N-ASpIRES.ICAM2 cells confirmed that the transfectants expressed similar levels of actin, a-actinin, and ezrin, but differed in levels of ICAM-2 expression (Fig. 1B). As reported for normal endothelial cells [19], immunoperoxidase staining of paraffin-embedded cells showed that ICAM-2 in transfected SK-N-ASpIRES.ICAM2 cells localized to cell mem- branes (Fig. 1C). Using these transfectants, we conducted co-immunoprecipita- tion assays to determine whether a-actinin or ezrin associated with Figure 1. In neuroblastoma cells, membrane-bound ICAM-2 associates with a-actinin and actin. A) Immunoblot of whole cell lysates (35 mg protein/lane) of six neuroblastoma cell lines, comparing endogenous levels of expression of ICAM-2. B) Immunoblots of SK-N-AS cells transfected with control plasmid (pIRESneo) or with plasmid encoding ICAM-2 (pIRES.ICAM2) show that transfectants expressed similar levels of a- actinin, ezrin, and actin, but SK-N-ASpIRES.ICAM2 cells expressed a higher level of ICAM-2 than SK-N-ASpIRESneo cells (25 mg protein/lane). C) ICAM-2 expressed in SK-N-ASpIRES.ICAM2 cells localized to cell membranes (examples indicated by red arrows). D) Co-immunoprecipitation co-precipitation/ mass spectroscopic analysis (actin) showed that actin co-precipitated with ICAM-2 in ICAM-2 transfectants. Band #1 was .99% identical to human actin. E) Immunoprecipitation/immunoblot showing equal amounts of a-actinin precipitated from lysates of SK-N-ASpIRESneo and SK-N- ASpIRES.ICAM2 cells. F) Immunoprecipitation/immunoblots showing that ICAM-2 and actin co-precipitated with a-actinin in lysates from SK-N- ASpIRES.ICAM2 cells, but little if any ICAM-2 and actin co-precipitated with a-actinin in SK-N-ASpIRESneo cells. doi:10.1371/journal.pone.0003629.g001 PLoS ONE | www.plosone.org 2 November 2008 | Volume 3 | Issue 11 | e3629