Under normal culturing conditions, the T47D human breast cancer cell line expresses progesterone receptor constitutively and is responsive to estrogen. Because the tumor suppressor protein p53 plays a central role in determining genetic stability and cell proliferation, we have examined the effects of 17␤-estradiol, the synthetic progestin R5020, and the antiprogestin RU486 on the levels of this protein in T47D cells. Western blot analysis of cellular extracts, performed with a monoclonal antibody capable of quantitatively supershifting a specific p53-p53 response element complex in a gel mobility shift assay, detected a single immunoreactive band representing p53. When cells were grown for 4 -5 days in culture medium containing charcoal-treated fetal calf serum, p53 levels declined to 10% of the level seen in the control (no charcoal treatment) group. Supplementation of culture medium containing charcoal-treated calf serum with 0.1-1 nM 17␤-estradiol restored p53 to its normal levels. A 4-day treatment of cells with R5020 or RU486 lowered the p53 levels in cells grown in normal culturing conditions to 15 and 30% of control levels, respectively. R5020 and RU486 treatments also caused down-regulation and/or hyperphosphorylation of the progesterone receptor, which correlated with the downregulation of p53. These observations indicate that in T47D cells, p53 is up-regulated by estradiol while R5020 down-regulates this protein. Since estradiol is known to promote cell proliferation, the induction of p53 observed in this study leads us to propose that estradiol stimulates p53 to regulate proliferation of T47D cells in culture.The development, growth, and differentiation of human breast is under the influence of a number of hormones including the sex steroids, estradiol (E 2 ) 1 and progesterone. In cases where the breast tissue transforms into a tumorous entity, it often continues to respond to circulating levels of these hormones provided it expresses receptors for the corresponding hormone. While hormone-insensitive malignancies do not appear to express functional receptors, hormone-sensitive cancers may overexpress progesterone receptor (PR) and estradiol receptor (McGuire, 1978). Treatment of breast cancers with hormones or antihormones presumes the presence of functional receptors, which via activation or inactivation of receptors mediate regression of cancerous tissues. Although breast cancers may initially respond to endocrine therapy, the tissue can transform into a hormone-insensitive entity. The mechanisms underlying the evolution of hormone-sensitive tumors to hormone-insensitive states are not known (Horwitz, 1994). It is, therefore, crucial to determine whether treatment with hormones and/or antihormones might affect a shift toward a progressively more malignant state of breast cancer. The biological activity of the tumor suppressor protein p53 is associated with suppression of cell growth. It is now widely recognized that p53 may be the most frequently mutated protein in human cancers (Oren, 1992). Beca...

We have utilized the estrogen receptor (ER)-positive human breast carcinoma cell line, T47D, to determine the role of ER in regulating cell proliferation, the level of expression of p53 and the state of phosphorylation of retinoblastoma protein (pRB) by 17 b-estradiol (E 2 ) and antiestrogens. T47D cells cultured for 7 days proliferated rapidly expressing maximal levels of p53 in medium containing 5% fetal bovine (whole) serum. Exogenously added E 2 had no e ect on either of the above parameters. The antiestrogen, ICI 164,384 (ICI, 1 mM), decreased cell number and p53 level to nearly 20% of the control. Comparatively, a treatment of the cells with 100 nM 4OH-tamoxifen (OHT) decreased cell number to 40% of the control without a concomitant decrease in the p53 levels suggesting a di erential ability of these antiestrogens to regulate p53 levels in cells cultured in whole serum. When cells were cultured in medium containing serum depleted of endogenous steroids (charcoal stripped serum), cell number and p53 levels declined. Treatment with exogenous E 2 (1 nM) increased cell proliferation, p53 expression and phosphorylation of pRB. The antiestrogens ICI and OHT blocked these E 2 e ects, demonstrating a direct antagonism of ER by ICI and OHT. These results indicate an ER-mediated mechanism for coordinate expression of p53 and hyperphosphorylation of pRB during E 2 -induced proliferation of T47D cells.

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Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK) and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser 63 phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser 63 phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.