br Acknowledgement br Introduction Estrogens play an

Acknowledgement
Introduction Estrogens play an important role in the development and progression of breast cancer (McGuire et al., 1976; Clemons and Goss, 2001). 17β-Estradiol (E2), the most biologically active estrogen in breast tissue, functions via its nuclear receptors, estrogen receptor (ER) α and β (Gronemeyer and Laudet, 1995). Binding of E2 to ER triggers a cascade of events leading to the modulation of gene expression reflected by changes in synthesis of specific mRNAs and proteins and by pleiotropic effects on cell proliferation (Pietras and Szego, 1999). The sensitivity to E2 is strongly influenced by culture conditions; in particular, the mitogenic effect of E2 on breast cancer A 61603 hydrobromide requires a sufficient concentration of serum and can also be modulated by serum (Soto and Sonnenschein, 1985). Addition of E2 to media containing more than 2.5% charcoal-dextran (CD)-stripped human female serum significantly increases the proliferation rate of the cloned human breast tumor cell line C7MCF7-173, which behaves as an estrogen-dependent tumor in nude mice, but has no effect on cell proliferation in a serum-free medium. The highest cell growth stimulation of human breast cancer cells was achieved when E2 was added in the presence of 5% CD-stripped fetal calf serum; removing serum from the culture medium or reducing its concentration resulted in a lower stimulation of cell proliferation (Ruedl et al., 1990). In view of these results, most studies on E2 effects on cultured cells have been performed in the presence of serum. However, interestingly, E2 can stimulate the induction of its response gene, progesterone receptor (PR) gene, in low serum conditions (Cho et al., 1994), but further study to show the direct transcriptional/translational activity of E2 alone has not been done yet. Therefore, in this study, the stimulation of transcription by E2 in ER-positive human MCF-7 breast cancer cells cultured in the absence of serum was investigated using proteomics. The transcript levels of E2-stimulated proteins were evaluated by real-time PCR and compared with those in cells cultured in the presence of serum.
Materials and methods
Results Proteins differentially expressed in the MCF-7 cells after E2 treatment in serum-free condition were identified (Fig. 1). Of ∼1000 proteins evaluated in this study, 19 proteins were up-regulated and 7 proteins down-regulated; these are listed in Tables 2 and 3, respectively. The spot densities of these proteins in E2-treated MCF-7 cells differed from untreated cells by at least 2-fold. Using real-time PCR, the transcript levels of 13 of the 19 up-regulated proteins were evaluated in MCF-7 cells cultured without serum (Fig. 2A). Four of the transcripts were significantly up-regulated: heat shock protein-90α (Hsp90α; U1), protein disulfide isomerase (PDI) related protein (U4), unactive 23-kDa PR, p23 (U17) and XTP3-transactivated protein A (U18). Four others, of which the proteins were shown to be up-regulated in proteomics, were significantly down-regulated: 2-oxoglutarate dehydrogenase (U3), phosphatase 2A (PP2A; U9), cytoskeleton associated protein 1 (CKAP1; U11) and cytosolic phospholipase A2 (cPLA2; U15). The transcript levels of 5 of the 7 down-regulated proteins were also evaluated (Fig. 2B). The transcript level of aminoacylase 1 (D3) was significantly down-regulated, but those of chaperonin containing t-complex protein (TCP) subunit 2 (TCP-1β; D2) and stathmin 1 (D6) were significantly up-regulated by E2. The cellular action of E2 can be confirmed by measuring the transcript expression of PR; this is a classical example of ER-mediated gene expression (Horwitz and McGuire, 1978; Nardulli et al., 1988). As shown in Fig. 2C, E2 can stimulate the transcript expression of PR by 12-fold in serum-free condition. The expression levels of 2 up-regulated proteins (Hsp90α and Hsp90β) and 2 down-regulated proteins (Hsp60 and TCP-1β) revealed by proteomics were evaluated using specific antibodies (Fig. 3). The actin-normalized protein expression levels of Hsp90α, Hsp90β and TCP-1β were increased, but that of Hsp60 was slightly decreased by E2 in MCF-7 cells cultured without serum.