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Text: HEYL Localization Is Altered in Cancer Tissue-We have previously shown that in prostate cancer biopsies HEY1 expression is predominantly cytoplasmic rather than nuclear, and we hypothesized that this cellular localization provides a growth advantage in cancer cells, circumventing the repressive effects of nuclear HEY1 (25,26). To understand better the mechanism(s) of repression of AR by HEY proteins, we examined the subcellular distribution of HEYL protein by confocal microscopy with a monoclonal antibody. In normal and benign transformed prostate epithelial lines, RWPE-1 and BPH-1, respectively, endogenous HEYL was entirely nuclear (Fig. 7A). In our inducible LNCaP:HEYL cell line treated with doxycycline, the exogenously expressed HEYL was also nuclear, demonstrating that the transfected HEYL behaves similarly to endogenous protein, and furthermore, we observed a co-localization of HEYL and AR in the nucleus of these cells in the presence of androgen (Fig. 7A, bottom panel). In addition, we verified the specificity of the HEYL antibody by immunofluorescence and Western blotting of LNCaP:HEYL cells treated with or without doxycycline (Fig. 7A). To examine expression patterns of endogenous HEYL in human prostate tissue, we used prostate needle biopsies from patients with prostate cancer and stained them for HEYL. As shown in Fig. 7B, endogenous HEYL was expressed in the epithelial cell compartment of the prostate and showed nuclear localization in benign cells (Fig. 7B, left panels) with little expression detected in the cytoplasm. However, in all stages of cancer examined, HEYL nuclear intensity decreased, in most cases correlating with increasing cytoplasmic expression (Fig. 7, B and C). The expression score for each patient is plotted in Fig. 7C. This shows that nuclear expression decreased in all stages of the disease studied but remained high in adjacent benign tissue. Furthermore, expression of HEYL in the cytoplasm remained low in adjacent tissue but at the same level or higher in prostate cancer. Collectively, these **data** suggest that HEYL is excluded from the nuclei of cancer cells but not benign cells, indicating that nuclear exclusion of HEYL occurs in the early stages of prostate cancer formation or progression.<|eot_id|><|start_header_id|>assistant<|end_header_id|>

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