T47D cells represent an estrogen-responsive human ductal carcinoma cell line which expresses detectable levels of estrogen receptor (ER). We have previously shown that estradiol (E 2 ) treatment of T47D cells causes an increase in the level of p53 and a concomitant phosphorylation of retinoblastoma protein (pRb). In the present study, we have analysed the expression of p53 and phosphorylation state of pRb and compared the eects of E 2 and triiodothyronine (T 3 ) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon con¯uency, the cells were treated with T 3 (10 712 to 10 77 M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E 2 treatment of cells caused a 2 ± 3-fold increase in the level of p53. Presence of T 3 in the medium caused a gradual increase in the level of p53 in a concentration-dependent manner. Under the above conditions, pRb was phosphorylated (detected as an upshift during SDS ± PAGE) in the presence of E 2 and T 3 . Supplementation of growth medium with T 3 (1 mM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this eect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 mM), an ER antagonist, was combined with E 2 (1 nM) or T 3 (1 mM), eects of hormones on cell proliferation and hyperphosphorylation of pRb were blocked. Western analysis of p53 was supplemented with its cytolocalization by immuno-labeling using laser scanning confocal uorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T 3 for the [ 3 H]E 2 binding. These results indicate that T 3 regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T 3 receptor (T 3 R)-mediated pathways.

Human umbilical cord (hUC) blood and tissue are non-invasive sources of potential stem/progenitor cells with similar cell surface properties as bone marrow stromal cells (BMSCs). While they are limited in cord blood, they may be more abundant in hUC. However, the hUC is an anatomically complex organ and the potential of cells in various sites of the hUC has not been fully explored. We dissected the hUC into its discrete sites and isolated hUC cells from the cord placenta junction (CPJ), cord tissue (CT), and Wharton's jelly (WJ). Isolated cells displayed fibroblastoid morphology, and expressed CD29, CD44, CD73, CD90, and CD105, and showed evidence of differentiation into multiple lineages in vitro. They also expressed low levels of pluripotency genes, OCT4, NANOG, SOX2 and KLF4. Passaging markedly affected cell proliferation with concomitant decreases in the expression of pluripotency and other markers, and an increase in chondrogenic markers. Microarray analysis further revealed the differences in the gene expression of CPJ-, CT- and WJ-hUC cells. Five coding and five lncRNA genes were differentially expressed in low vs. high passage hUC cells. Only MAEL was expressed at high levels in both low and high passage CPJ-hUC cells. They displayed a greater proliferation limit and a higher degree of multi-lineage differentiation in vitro and warrant further investigation to determine their full differentiation capacity, and therapeutic and regenerative medicine potential.

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.

Curcumin (CUR) is a compound that has antibacterial, antiviral, anti-inflammatory, and anticancer properties. In this study, we have analyzed the effects of CUR on the expression of ERα and p53 in the presence of hormones and anti-hormones in breast cancer cells. Cells were cultured in a medium containing charcoal-stripped fetal bovine serum to deplete any endogenous steroids and treated with CUR at varying concentrations or in combination with hormones and anti-hormones. Protein analysis revealed a relative decrease in the levels of p53 and ERα upon treatment with 5–60 µM CUR. In cell proliferation studies, CUR alone caused a 10-fold decrease compared with the treatment with estrogen, which suggests its antiproliferative effects. Delineating the role of CUR in the regulation of p53, ERα, and their mechanisms of action may be important in understanding the influence of CUR on tumor suppressors and hormone receptors in breast cancer.