and negatively impact overall survival rates [17,18]. The expression of versican G3 domain does not only appear to enhance breast cancer cell proliferation in vitro and in the mammary gland, but also promotes tumor cell migration in vitro and systemic metastasis in syngenetic orthotopic models in vivo [19,20]. Increased expression of EGFR occurs frequently in human breast cancer and is associated with a poor prognosis [21]. Anti- apoptotic and drug resistant effects have been implicated in EGFR signaling. Some molecules in the pathway, however, may promote cell cycle arrest and enhanced sensitivity to chemotherapeutic drugs [22]. Direct targeting of EGFR is a promising therapeutic strategy for breast cancers with abnormalities in this pathway and may be beneficial in breast cancer patients who cannot tolerate surgery or traditional chemotherapy, or in advanced recalcitrant cases with poor prognoses [23,24]. There is a desire to improve our understanding of the cellular mechanisms involved in versican G3 mediated tumor growth and invasiveness. Understanding EGFR signaling that influences cell sensitivity to apoptosis as well as effects that are elicited by chemotherapy may help guide our understanding towards identifying other potential target molecules in the pathway from an immunotherapeutic perspective. To investigate the effects of versican G3 domain on breast cancer cell apoptosis, we exogenously expressed versican G3 in mouse mammary tumor cell lines 66c14, 4T07, 4T1 [25], and human breast cancer cell lines MT1, MDA-MB-231, MCF-7, MDA-MB-468. We evaluated the effect of apoptotic agent C2-ceramide as well as chemother- apeutic drugs such as Doxorubicin, Epirubicin, and Docetaxil on cell activity and EGFR downstream signaling. Materials and Methods Materials and cell cultures The monoclonal antibodies against ERK2, pERK, CDK2, and Caspase-3 were obtained from Santa Cruz Biotechnology. The polyclonal antibodies against SAPK/JNK and pSAPK/JNK were obtained from Cell Signaling. EGF, selective EGFR inhibitor AG 1478, selective MEK inhibitor PD 98059, selective SAPK/JNK inhibitor SP 600125, hydroxyurea, and the monoclonal antibody against b-actin used in the study were obtained from Sigma. Glycogen synthase kinase-3ß serine-9 phosphorylation (GSK-3b, S9P), and polyclonal antibodies against versican V1 were obtained from Abcam. Horseradish peroxidase-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. Immunoblotting was performed using the ECL Western blot detection kit. Cell Proliferation Reagent WST-1 was obtained from Roche Applied Science. Mouse mammary tumor cell lines 67NR, 66c14, 4T07, 4T1 [26], and human breast cancer cell line MDA-MB-231 were cultured in DMEM media[27], and human breast cancer cell line MT-1 [28], MCF-7 [29], MDA-MB-468 were cultured in RPMI- 1640 media [30], which were supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) and maintained at 37uC in a humidified atmosphere of 5% CO2. In selected experiments, cell suspensions were cultured with EGF (20 ng/ml), EGFR inhibitor AG 1478 (2.0 mM), selective MEK inhibitor PD 98059 (50 mM), and selective SAPK/JNK inhibitor SP 600125 (100 nM). The pcDNA1 - G3 construct and pcDNA1 - G3 fragment lacking the EGF-like motifs (G3DEGF) construct were generated by us [31,32,33]. Mouse mammary tumor cell lines 66c14, 4T07, 4T1 and human breast cancer cell line MT-1, MDA-MB-231, MCF-7, and MDA-MB-468 cells were transfected with pcDNA1- vecor and G3 constructs. The 66c14 cells were transiently Versican G3 Modulates Breast Cancer Cell Apoptosis transfected with G3 construct, G3DEGF construct, or the control vector. A leading sequence that has been shown to be efficient in product secretion was engineered to both construct by us previously [32,34,35]. Cell viability assays G3 and vector-transfected 66c14 cells (26105) were cultured in 10% FBS/DMEM medium in culture dishes and maintained at 37uC for 12 hours. After cell attachment, we changed the medium to serum free DMEM medium or 10% FBS/DMEM medium which contained different concentrations of chemotherapeutic compounds. Cells were harvested daily and cell number was analyzed by Coulter Counter. Cell survival assays were also performed with colorimetric proliferation assays (Cell Proliferation Reagent WST-1). Versican G3 and control vector transfected breast cancer cells (16104 cells/well) were inoculated and cultured in 10% FBS/DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, we changed the medium into serum free DMEM medium or 10% FBS/DMEM medium containing different concentrations of chemotherapeutic agents, and then cultured cells with 10 ml WST-1 reagent for 4 hours. The absorbance of the samples against a background blank control was measured by a microplate reader. Western blot analysis Protein samples were subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) on separating gel containing 7–10% acrylamide. Separated proteins were trans- blotted onto a nitrocellulose membrane in 16Tris/glycine buffer containing 20% methanol at 60 V for 2 h in a cold room. The membrane was blocked in TBST (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% non-fat dry milk powder (TBSTM) for 1 hour at room temperature, and then incubated with primary antibodies at 4uC overnight. The membranes were washed with TBST (3630 minutes) and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies in TBSTM for 1 hour. After washing as described above, the bound antibodies were visualized with an ECL detection kit as described previously [36,37]. Cell cycle analysis The expression of cell cycle-related proteins was analyzed by immunoblotting probed with appropriate antibodies as described above. G3- and vector-transfected 66c14 cell lines were cultured in 10% FBS/DMEM media at 37uC, 5% CO2 with or without EGFR inhibitor AG 1478 (0.2, 2.0, and 5.0 mM), and selective MEK inhibitor PD 98059 (20, 50, and 100 mM). The cells were washed and resuspended in cold PBS and incubated in ice-cold 70% ethanol for 3 hours. The cells were then centrifuged at 1,500 rpm for 10 minutes and resuspended in propidium iodide (PI) master mix (40 mg/ml PI and 100 mg/ml RNase in PBS) at a density of 56105/ml and incubated at 37uC for 30 minutes before analysis by flow cytometry. Annexin V assays An Annexin V-FITC apoptosis detection kit (Biovision Inc, Mountain View, CA, USA) was used to detect apoptotic activity. Cells (16106) were collected and resuspended in binding buffer, and Annexin V-FITC and propidium iodide were added to each sample and incubated in the dark for 5 minutes. Annexin V-FITC binding was determined by flow cytometry (Ex = 488 nm; Em = 530 nm) using FITC signal detector (FL1) and propidium staining by the phycoerythrin emission signal detector (FL2). PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e26396