5482

The purpose of this study is to unravel the subcellular localization of the protein product of a recently discovered gene, epithelial-stromal interaction 1 (breast), EPSTI1. EPSTI1 is upregulated by epithelial-stromal interaction in invasive breast carcinomas as compared to normal breast tissue and is expressed primarily in the epithelial compartment (Genomics 79:703, 2002). We have examined the subcellular localization of the protein product, EPSTI1, in a human breast epithelial cancer cell line, MCF-7, transfected with pRev-Tet-Off and pRevTRE with a FLAG-EPSTI1 insert, in which expression of EPSTI1 is induced by the absence of doxycycline. Immunocytochemistry with a monoclonal anti-FLAG antibody suggests that EPSTI1 is a nucleocytoplasmic protein, and that shuttling between nucleus and cytoplasm is cell density dependent. Thus, whereas confluent cultures exhibit EPSTI1 expression primarily in the cytoplasm, subconfluent cultures show nuclear localization of the protein product. Western blot analysis with the FLAG-antibody on extracts from FLAG-EPSTI1-MCF-7 gives rise to a band of approximately 40 kDa which is not contradictory to a predicted molecular mass of 35,4 kDa. To identify the native EPSTI1 protein as well as to target EPSTI1 directly, we have raised a polyclonal rabbit antibody against the N-terminal epitope. Immunostaining of FLAG-EPSTI1-MCF-7 with the polyclonal antibody confirmed doxycycline-regulated expression and verified the density dependent subcellular localization of EPSTI1 in FLAG-EPSTI1-MCF-7. Western blot analysis of extracts of FLAG-EPSTI1-MCF-7 and human primary breast carcinomas showed immunoreactivity with a band of approximately 40 kDa. In addition, FLAG-EPSTI1-MCF-7 gave rise to a band of approximately 80 kDa. Moreover, immunohistochemistry on corresponding paraffin sections of invasive breast carcinoma with the EPSTI1 antibody further supported EPSTI1 expression by carcinoma cells rather than surrounding stromal cells. In conclusion, the protein product of EPSTI1 shuttles between the nucleus and cytoplasm in a cell density dependent manner. The polyclonal EPSTI1 antibody suggests a molecular weight of EPSTI1 of approximately 40 kDa. Furthermore, it confirms the existence of EPSTI1 in human breast carcinoma and the expression of EPSTI1 primarily by carcinoma cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]