Sumi Dinda

Regulation of tumor suppressor proteins, p53 and retinoblastoma, by estrogen and antiestrogens in breast cancer cells

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 β-estradiol (E2) 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 E2 had no effect on either of the above parameters. The antiestrogen, ICI 164,384 (ICI, 1 μM), 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 differential 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 E2 (1 nM) increased cell proliferation, p53 expression and phosphorylation of pRB. The antiestrogens ICI and OHT blocked these E2 effects, 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 E2-induced proliferation of T47D cells.

Estrogen-like effects of thyroid hormone on the regulation of tumor suppressor proteins, p53 and retinoblastoma, in breast cancer cells.

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 (E2) 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 effects of E2 and triiodothyronine (T3) on these phenomena. Cells were grown in a medium containing charcoal-treated serum to deplete the levels of endogenous steroids. Upon confluency, the cells were treated with T3 (10−12 to 10−7 M) for 24 h and the presence of p53 and pRb was detected by Western analysis. E2 treatment of cells caused a 2–3-fold increase in the level of p53. Presence of T3 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 E2 and T3. Supplementation of growth medium with T3 (1 μM) caused an increase in the rate of proliferation of T47D cells and induced hyperphosphorylation of pRb within 4 h; this effect was maintained for up to 12 h. When ICI 164 384 (ICI) (1 μM), an ER antagonist, was combined with E2 (1 nM) or T3 (1 μM), effects 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 fluorescence microscopy, which revealed an ICI-sensitive increase in the abundance of p53 in hormone-treated cells. Steroid binding analysis revealed lack of competition by T3 for the [3H]E2 binding. These results indicate that T3 regulates T47D cell cycle progression and proliferation raising the p53 level and causing hyperphosphorylation of pRb by a common mechanism involving ER and T3 receptor (T3R)-mediated pathways.

Hormonal regulation of the p53 tumor suppressor protein in T47D human breast carcinoma cell line.

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 17β-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 17β-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 down-regulation 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.

Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells

Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 β estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells.

Hormonal regulation of tumor suppressor proteins in breast cancer cells.

This laboratory is studying hormonal regulation of tumor suppressor proteins, p53 and retinoblastoma (pRB). Estrogen receptor and progesterone receptor positive human breast cancer cell lines, T47D and MCF-7, were utilized for determining influence of hormonal and antihormonal agents on the level of expression of p53, state of phosphorylation of pRB, and rate of cell proliferation. The expression of p53 in T47D cells grown for 4-5 days in culture medium containing charcoal-treated (stripped) fetal bovine serum declined gradually to 10% of the level seen in control (whole serum, non charcoal-treated) groups. Supplementation of culture medium containing stripped serum with 0.1-1 nM estradiol (E(2)) restored p53 to its level seen in the control within 6-24 h. Under above conditions, treatment of cells with R5020 or RU486 reduced (15-30%) the level of p53. Incubation of cells in E(2)-containing growth medium caused cell proliferation and hyperphosphorylation of pRB; the latter effect was seen maximally between 24-72 h. The E(2)-induced hyperphosphorylation of pRB and increase in the level of p53 were sensitive to the presence of ICI and 4-hydroxy tamoxifen (OHT). T47D and MCF-7 cells were also transiently transfected with a P1CAT reporter plasmid containing c-Myc responsive element and the levels of chloramphenicol acetyltransferase (CAT) activity were observed in response to various treatments. E(2) and OHT caused P1CAT induction as seen by increased CAT activity: E(2) caused an endogenous increase in the expression of an ICI-sensitive c-Myc form. These data suggest that estrogen upregulates p53 expression while progesterone downregulates this process. Further, E(2) regulates p53 level and pRB activity in a coordinated manner.

EFFECTS OF ANTIPROGESTINS ON THE RATE OF PROLIFERATION OF BREAST CANCER CELLS

We have examined the influence of progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31710 and Org 31806) on the rate of proliferation of wild type T47D cells cultured in whole fetal bovine serum (FBS) or in single charcoal stripped fetal bovine serum (SSFBS). All of the progesterone antagonists RU486, ZK98299 and two novel antiprogestins Org 31710 and Org 31806 inhibited cell proliferation when cells were cultured in FBS. In contrast, all of the antiprogestins with the exception of ZK98299 enhanced cell growth when cells were cultured in SSFBS. This stimulatory effect of RU486 was observed only at a high concentration of the ligand (1 μM). The effect of R5020, however, was concentration independent. The number of cells in the presence of RU486 was ~ 600% followed by R5020 ~ 400% above control values after a 28 day culturing period. In contrast, when the cells were grown in the presence of medium containing non-stripped whole serum, RU486 inhibited the extent of cell proliferation by 45%. Estradiol (E2) stimulated the rate of proliferation in cells cultured in SSFBS. Similar to when cells were cultured in whole serum, the antiprogestins inhibited cell growth in E2-supplemented SSFBS. Detection of the growth enhancement effects of progesterone receptor (PR) ligands such as RU486 and R5020 on the cells grown in charcoal-stripped medium appear to require the removal of E2 by charcoal stripping of the serum.

Effects of Estrogen Metabolite 2-Methoxyestradiol on Tumor Suppressor Protein p53 and Proliferation of Breast Cancer Cells

An endogenous 17β-estradiol (E2) metabolite, 2-methoxyestradiol (2-ME2), has been reported to exhibit estrogen receptor (ER)-independent anti-angiogenic and anti-tumor effects. Several mechanisms have been proposed for 2-ME2 actions, but there is a lack of evidence for a common pathway for all of the cell-types sensitive to this metabolite. We have examined potential alterations in p53 in response to 2-ME2, E2 and the microtubule disruptor taxol in T47D breast cancer cells. Cells were cultured for six days in medium depleted of endogenous steroids or effectors. Semi-confluent cells were treated with 2-ME2 (1 nM – 10 µM), 10 nM E2 and/or 1 µM taxol and subjected to SDS-PAGE and Western blot analysis, quantitative analysis, or laser-scanning confocal microscopy. Western blot analysis revealed a concentration-dependent biphasic trend in p53 levels. Addition of 10 nM – 1 µM 2-ME2 induced significant up-regulation in p53, and this response gradually diminished to levels comparable to the control upon treatment with higher concentrations (2.5 – 10 µM). The observed upregulation of p53 induced by 2-ME2 is inhibited by concurrent treatment with 1 µM taxol. Cell quantitation revealed a significant decrease (50 – 90%) in cell number upon treatment with 1 – 10 µM 2-ME2 with minimal effect at lower concentrations. No additional effect on cell proliferation was observed when taxol was combined with 10 nM or 1 µM 2-ME2. In a concentration dependent manner, treatment with 2-ME2 for 24 h differentially influenced cellular localization of p53. These results may aid in further understanding the relationship between steroid receptors, tumor suppressor proteins, and effects of hormone metabolites on breast cancer cells.

The effects of turmeric (curcumin) on tumor suppressor protein (p53) and estrogen receptor (ERα) in breast cancer 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.

Isolation and comparative analysis of potential stem/progenitor cells from different regions of human umbilical cord.

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 Whartons 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.

Inhibition of proliferation of T47D human breast cancer cells: Alterations in progesterone receptor and p53 tumor suppressor protein

We have investigated the influence of three structurally different but functionally related compounds [1, 10 ortho-phenanthroline (phenanthroline), Rifampicin and aurin tricarboxylic acid (ATA)] on the rate and the extent of proliferation of progesterone-responsive T47D human breast cancer cells. These compounds have previously been used in this laboratory and have been shown to modulate properties of nucleic acid binding proteins. Because p53 and the progesterone receptor (PR) are both DNA binding proteins that appear to regulate proliferation of breast cells, alterations in T47D cell p53 and PR levels were examined to determine their relevance in cell proliferation. T47D cells were grown in the absence of phenol red and in the presence of 5% fetal calf serum with or without charcoal stripping in the presence of the inhibitors. The rate of proliferation of cells grown in Rifampicin containing medium exhibited nearly 70% inhibition. Phenanthroline, a known metal chelator, was an effect ive inhibitor of proliferation at 3 mM reducing the cell number by more than 75%. ATA (0.24–2.4 µg/ml) inhibited the growth of the cells by nearly 50%. Analysis of the mechanism of action of these compounds revealed that treatment with these compounds caused specific changes in the molecular composition of T47D cell PR. Whereas ATA caused increased stability of PR isoforms, Rifampicin induced a upshift in the mobility of PR in SDS gels – a phenomenon associated with hyperphosphorylation of steroid receptors (SRs). Phenanthroline treatment (> 2 mM) caused a complete down-regulation of PR and the tumor suppressor protein, p53. The downregulation of p53 paralleled the changes in the molecular composition of PR. We propose that the inhibition of T47D cell proliferation by phenanthroline, Rifampicin and ATA results from a number of cellular changes that include regulation of p53 and PR. (Mol Cell Biochem 175: 81–89, 1997)