หน้าหลัก

The empty expression vector was established as the control. Removal of Dox in the medium induced the expression of FLAG.SCUBE2-FL, -ty97, and -D4 protein, as confirmed by anti-FLAG Western blot analysis (Fig. 1B). However, SCUBE2-FL or mutant protein was not induced in the presence of Dox in these stable MCF-7 cell lines or in the control MCF-7 Tet-Off vector clone (Fig. 1B). Overexpression of SCUBE2 or Its Mutant Proteins Suppresses Proliferation, but Does Not Increase Apoptosis, of MCF-7 Breast Cancer Cells Both in Vitro and in Vivo--To investigate the effects of SCUBE2 expression on breast cancer cell growth, the MCF-7 Tet-Off vector and SCUBE2-FL, ty97, and -D4 stable cells were cultured in the presence or absence of Dox for up to 6 days to suppress or induce the expression of ectopic FLAG. SCUBE2-FL, -ty97, or -D4 protein. Cell growth was then measured by MTT assay. Induction of ectopic SCUBE2-FL protein,doxycycline (( )Dox) or not (( )Dox). Growth of the MCF-7 Tet-Off vector or SCUBE2-ty97 or -D4 cells was measured as a function of time in the absence of doxycycline. Data points, mean tumor volumes (length width height 0.5236 (in mm3)); error bars, S.E. *, p 0.01 (n 10 or 7 for the MCF-7 Tet-Off vector tumors, n 10 for the SCUBE2-ty97 tumors, and n 7 for the SCUBE2-D4 tumors).JULY 29, 2011 ?VOLUME 286 ?NUMBERJOURNAL OF BIOLOGICAL CHEMISTRYMolecular Analysis of Breast Tumor Suppressor Gene SCUBEFIGURE 3. SCUBE2 antagonizes autocrine BMP-mediated signaling through its CUB domain. MCF-7 Tet-Off vector and SCUBE2-FL, -ty97, and -D4 stable cells were cultured in medium without doxycycline (( )Dox) for 5 days, and the induction of ectopic NH2-terminal FLAG-tagged SCUBE2-FL or deletion mutant protein (ty97 or D4) was determined by Western blot (WB) analysis with anti-FLAG antibody (bottom panel). Total protein lysates (20 g) from each cell line in duplicate were subjected to Western blot analysis with an antibody specific against the phosphorylated Smad1/5/8 (p-Smad1/5/8) or total Smad1 protein (top panel and second panel). Ponceau red staining of the PVDF membrane confirmed the relative loading of total proteins for each sample (third panel). Shown is a histographic representation of quantified data of the phosphorylated Smad1/5/8 and total Smad1 protein concentrations in respective groups (quantified by densitometric scanning and normalized by the total amount of protein loading). Data are means S.E. of two experiments. *, p 0.01 compare with ( )Dox.as well as SCUBE2-ty97 and -D4, suppressed the proliferation of these MCF-7 cell lines in the absence of Dox (Fig. 2A). The control MCF-7 Tet-Off vector clone and SCUBE-FL, -ty97, and -D4 cells did not differ in growth on culture with Dox to block the expression of ectopic SCUBE2 proteins (data not shown). Because overexpression of SCUBE2-ty97 or -D4 protein inhibited MCF-7 breast cancer cell growth in vitro, we next examined breast tumor growth in vivo in nude mice. MCF-7 Tet-Off vector or SCUBE2-ty97 or -D4 cells were injected into the mammary fat pads of nude mice that received estrogen pellets to promote the growth and development of breast tumors.