Estrogen and IGF-I are potent mitogens for most breast cancer cell lines, and although their signaling pathways contrast, there is considerable interaction between them. Recent evidence indicating that IGF-I can alter estrogen receptor (ER) action led us to investigate whether an inhibitor of IGF-I action. IGF-binding protein-1 (IGFBP-1), could affect transcriptional activation of ER. First, we confirmed that tamoxifen (TAM) could inhibit IGF-I-mediated proliferation of MCF-7 cells. Although TAM can increase IGFBP-3 expression in MCF-7 cells, and this binding protein has been shown to be able to inhibit IGF action, TAM had no effect on IGF-I-stimulated tyrosine phosphorylation of IGF-I receptor or the downstream signaling molecule, insulin receptor substrate-1. Therefore, to confirm that IGF-I was affecting transcriptional activation of ER, we utilized a gene reporter assay using a single consensus estrogen response element (ERE-tk-luc) upstream of luciferase. As expected, estradiol (E2; 1nM) increased transcriptional activation three- to fivefold from the ERE in three ER-positive breast cancer cell lines (MCF-7, ZR-75 and T47D). A 2.5-to 4-fold increase was also seen with IGF-I (5 nM). TAM (1 microM) effectively blocked activation by E2 and IGF-I, indicating disruption of ER-mediated transcription. As expected, human recombinant IGFBP-1 (80 nM) completely inhibited IGF-I-mediated activation of ER, however, IGFBP-1 also caused a significant decrease in E2-mediated activation. We also noticed that IGF-I increased the activity of all plasmids that we cotransfected including TATA-luc, SV40-luc and pGL Basic. This effect was post-transcriptional, as it was not affected by actinomycin D (2 micrograms/ml), while we were able to completely inhibit E2-mediated transcriptional activation of ERE-tk-luc. Unlike the complete inhibition of ER-mediated transcriptional activation by actinomycin D, IGF-I-mediated transactivation was reduced by only 50%, indicating that the activation by IGF-I represented both transcriptional and post-transcriptional effects. This study confirmed that IGF-I can cause transcriptional activation of endogenous ER in human breast cancel cells, and inhibition of ER action by IGFBP-1 suggests that IGF-1 signaling may be necessary for maximal ER activation.

The estrogen receptor (ER) is a ligand-dependent transcription factor that acts in a cell- and promoter-specific manner. Evidence suggests that the activity of the ER can be regulated by a number of other stimuli (e.g. growth factors) and that the effects of the ER are modulated by nuclear factors termed coregulators. While the interplay among these factors may in part explain the pleiotropic effects elicited by the ER, there are several other less well described mechanisms of control, such as interactions with the nuclear matrix. Here we report that the nuclear matrix protein/scaffold attachment factor HET/SAF-B is an ER-interacting protein. ER and HET/SAF-B interact in in vitro binding assays, with HET binding to both the ER DNA-binding domain and the hinge region. Coimmunoprecipitation experiments reveal that HET/SAF-B and ER associate in cell lines in the presence or absence of estradiol, but binding is increased by the antiestrogen tamoxifen. HET/SAF-B enhances tamoxifen antagonism of estrogen-induced ER-mediated transactivation, but at high concentrations can inhibit both estrogen and tamoxifen-induced ER activity. HET/SAF-B-mediated repression of ER activity is dependent upon interaction with the ER-DBD. While the existence of high-affinity binding sites for the ER in the nuclear matrix has been known for some time, we now provide evidence of a specific nuclear matrix protein binding to the ER. Furthermore, our data showing that HET/SAF-B binds to ER particularly strongly in the presence of tamoxifen suggests that it may be important for the antagonist effect of tamoxifen.

The detection of IGF-IR signaling in animal models has important implications for determining the role of this receptor in normal physiology and tumor growth. While many reports have correlated changes in plasma IGF-I levels in vivo with biological responses, few have shown that altered IGF-I levels can directly affect signaling within normal or tumor tissue. Here, we present new data that shows how the intravenous (IV) injection of IGF-I can be used to directly examine IGF signaling at the tissue level. Tail-vein IV injection of IGF-I into mice resulted in a rapid and dose-dependent activation of the IGF-I receptor and downstream phosphorylation of Akt and ERK1/2 in liver, kidney, and mammary gland. Similarly, IV IGF-I rapidly stimulated signaling in HT-29 colorectal and in MCF-7 breast cancer xenografts. This study shows how IV IGF injection can be used to examine the signaling mechanisms used by IGF-IR, in both normal mammary tissue and during tumor growth, and may provide a model for the characterization of IGF inhibitors.

#4060 Introduction: Over the last few years, much attention has been paid to the development and utilization of three-dimensional (3D) cell culture to replace classical two-dimensional (2D) monolayer cell culture systems grown as a flat layer on plastic. Not completely surprisingly, there are tremendous differences between cells in 2D and 3D culture. However, currently available 3D cell culture media are expensive for regular use by the majority of research laboratories and thus large scale use of 3D cell culture system is presently cost prohibited. In our search for a reliable and economically viable replacement for the expensive commercial 3D media, we hypothesized that avian egg white could be a potential alternative.
 Methods: Egg white-based chamber slide: Separated egg white from the chicken eggs and coated each wells of an eight well culture chamber with 80μl of egg white by careful heating at 600C on a heating block. After 30-60 minutes, the egg white becomes a semi-solid that adheres to the bottom of the well. The unstuck egg white is washed out by adding 500µl of growth medium and removing it slowly using a Pasteur pipette.
 Cell culture and analysis: To each well 2x103 immortalized human breast epithelial cells (MCF10A), were added in 0.5ml of appropriate culture medium. We have analyzed the growth curve of the acini, lumen formation, apoptosis and cell proliferation at different days post-culture. Apico/basal polarization of the acini was analyzed using appropriate antibodies. We have also cultured IGFR1-transformed MCF10A cells, different established human cell lines (MCF7, HEK293, HeLa, LNCaP, and Saos-2), and MMTV-PyMT transformed mouse mammary epithelial cells in their appropriate growth media. For comparative studies, we used a well-established reconstituted basement membrane matrix preparation (BM).
 Results: Our analysis shows that this simple avian egg white based system supports growth of cells in 3D, with significantly decreased cost. Specifically, the growth of MCF10A in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apico/basal polarization comparable to what has been described using established 3D culture media. There was no significant difference in MCF10A proliferation and acini size between egg white and BM. We have also observed similar morphology for different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells between egg white and BM.
 Conclusion: Our data convincingly argue that egg white can be used as a suitable alternative model for 3D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3D cell culture on a regular basis, and thus result in a dramatic increase of utilization of the 3D cell culture in research. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4060.

No abstract