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Introduction Akt1, Akt2 and Akt3 kinases are downstream components of phosphoinositol 3-kinase derived signals from receptor tyrosine kinases, which influence cell growth, proliferation and survival. Akt2 overexpression and amplification have been described in breast, ovarian and pancreatic cancers. The present study was designed to investigate the prognostic significance of activated Akt in primary breast cancer and its association with other tumour biomarkers.

The effects of the tumor promoter phorbol 12-tetradecanoate 13-acetate (TPA) on the epidermal growth factor (EGF) receptor levels were investigated in hormonedependent (MCF-7, T-47-D, and ZR-75-1) and hormoneindependent (MDA-MB-231, HBL-100, and BT-20) human mammary carcinoma cell lines. In the absence of TPA, hormone-independent cell lines contained high concentrations of low-affinity EGF receptors (apparent Kd = 8 x 1010 M), whereas hormone-dependent cell lines exhibited low concentrations of high-affinity receptors (apparent Kd = 1 x 10-10 M). TPA causes a change of the receptor from a high-to the low-affinity state in hormone-dependent cell lines (MCF-7, T-47-D, and ZR-75-1), as well as in the hormone-independent HBL-100, whereas the affinity remained unchanged in MDA-MB-231 and BT-20 cells. In addition, progesterone receptor levels are decreased after TPA treatment in the hormonedependent cell lines MCF-7, T-47-D, and ZR-75-1, whereas the estrogen receptor levels remained unchanged. Tumor promoters such as TPA or teleocidin inhibited the proliferation of these cell lines at concentrations above 10 ,uM with the exception of the T-47-D cells. The most sensitive cell line towards growth inhibition by tumor promoter was the hormone-dependent MCF-7 cell line. Evaluation of different TPA analogs indicated a positive correlation between the growth-inhibitory effects and their ability to stimulate the subcellular redistribution of protein kinase C activity in MCF-7 cells. These data suggest a protein kinase C-mediated down-regulation of the progesterone receptor concentration and of the EGF receptor affinity, which is supposed to mediate the mitogenic response. Furthermore, these results support the hypothesis that the tumorderived growth factors induced by estradiol act via the EGF receptor in hormone-dependent mammary carcinoma cells.

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Heregulins (HRG) are known as soluble secreted growth factors that, on binding and activating ErbB3 and ErbB4 cell surface receptors, are involved in cell proliferation, metastasis, survival, and differentiation in normal and malignant tissues. Previous studies have shown that some HRG1 splice variants are translocated to the nucleus. By investigating the subcellular localization of HRGA 1-241 , nuclear translocation and accumulation in nuclear dot-like structures was shown in breast cancer cells. This subcellular distribution pattern depends on the presence of at least one of two nuclear localization sequences and on two domains on the HRG construct that were found to be necessary for nuclear dot formation. Focusing on the nuclear function of HRG, a mammary gland cDNA library was screened with the mature form of HRGA in a yeast two-hybrid system, and coimmunoprecipitation of endogenous HRG was done. The data reveal positive interactions of HRGA 1-241 with nuclear factors implicated in different biological functions, including transcriptional control as exemplified by interaction with the transcriptional repressor histone deacetylase 2. In addition, HRGA 1-241 showed transcriptional repression activity in a reporter gene assay. Furthermore, a potential of HRG proteins to form homodimers was reported and the HRG sequence responsible for dimerization was identified. These observations strongly support the notion that HRG1 splice variants have multifunctional properties, including previously unknown regulatory functions within the nucleus that are different from the activation of ErbB receptor signaling.