Ntrol transfected cells; however, cells with reduced IGFBP3 expression were partially resistant to the cytotoxic effects of imatinib. Taken together, this data PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26080418 suggests that IGFBP3 sensitizes GIST882 cells to the anti-tumor effects of imatinib. In contrast to GIST882 cells, GIST-T1 cells have no endogenous IGFBP3 expression and there was no induction of IGFBP3 after imatinib treatment. Therefore, this IGFBP3negative cell line provided us a system to examine the effects of IGFBP3 on imatinib response using a gain-offunction approach. To test whether IGFBP3 expression altered GIST-T1 sensitivity to imatinib, cells infected with Ad-IGFBP3 or Ad-EV or mock infected were subsequently analyzed for changes in cell viability after imatinib. AdIGFBP3 infection alone was cytotoxic relative to Ad-EV at 25 moi (p = 0.0024) (Figure 5). Analyzing imatinib sensitivity after IGFBP3 overexpression with a two-way ANOVA (interaction: p = 0.0009), we observed that imatinib significantly reduced viability in cells infected with AdIGFBP3 or Ad-EV. Furthermore, IGFBP3 overexpression did not significantly alter imatinib sensitivity in GIST-T1 cells (Figure 5). Although IGFBP-3 is cytotoxic to GIST-T1 cells, our data suggests that IGFBP3 does not mediate GIST-T1 response to imatinib.Page 4 of(page number not for citation purposes)Molecular Cancer 2009, 8:http://www.molecular-cancer.com/content/8/1/AAd-EV Ad-IGFBP3 IGFBP3 -tubulin1 moi2 moi5 moi- + – + – + – + – + – + – – + – + – + – + – + – +3d5d3d5d3d5dBCFigure IGFBP3 overexpression on GIST-T1 cell viability Effect of3 Effect of IGFBP3 overexpression on GIST-T1 cell viability. (A) Whole cell ML390 web lysates isolated from GIST-T1 cells three or five days after mock infection or infection with indicated titers (moi) of adenovirus expressing IGFBP3 (Ad-IGFBP3) or empty vector (Ad-EV) were analyzed by immunoblotting for IGFBP3 expression. Viability of GIST-T1 cells mock infected or infected with indicated titers of Ad-EV or Ad-IGFBP3 was analyzed 3 days (B) or 5 days (C) post-infection with the MTS assay. Asterisks (*) denote significant differences in cell viability at the indicated doses of Ad-EV vs. Ad-IGFBP3.DiscussionIn this study, we examined the potential role of IGFBP3 as a mediator of the therapeutic effects of imatinib mesylate in GISTs. Our previous studies showed that IGFBP3 is upregulated after imatinib treatment in a responsive GIST cell line (GIST882), and we provide evidence that IGFBP3 does indeed partially mediate GIST882 cell response to imatinib in vitro. In contrast, IGFBP3 has no effect on imatinib sensitivity in the responsive GIST-T1 cell line, which has no detectable endogenous IGFBP3 levels before or after imatinib exposure. Further, our studies, using both gain-of-function and loss-of-function approaches, reveal that IGFBP3 is an important modulator of cell viability in GISTs, but the effect is cell-dependent. Similar to what has been reported for epithelial cancers [23,26-34], IGFBP3 also manifests dual functions on cell survival in GIST, a mesenchymal cancer. Up-regulation of IGFBP3 has been observed in response to a variety of anti-cancer agents [18-23], including celecoxib [24]. In addition, IGFBP3 potentiates the actionof paclitaxel [35] and sensitizes cancer cells to the cytotoxic effects of gefitinib [36] and other chemotherapeutic agents [37]. Because we observed IGFBP3 expression in GIST in response to imatinib [12], we hypothesized that IGFBP3 would mediate its anti-tumor effects. Afte.