Virinder K. Moudgil

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.

Hormone specific regulation of natural killer cells by cortisol Direct inactivation of the cytotoxic function of cloned human NK cells without an effect on cellular proliferation

Corticosteroids have previously been reported to partially inhibit the natural cytotoxic activity of peripheral blood lymphocytes. However, since only a few percent of peripheral lymphocytes are natural killer (NK) cells, it has not been possible to determine whether corticosteroids directly inhibit NK cells or mediate this effect via other cell types. This report documents direct functional inactivation, but unimpeded proliferation, of cloned human NK cells by subphysiologic levels of cortisol. In contrast, high concentrations of testosterone, progesterone or estradiol had no significant effect on proliferation or cytotoxic activity of the cloned NK cells. The kinetics of inhibition of NK function by cortisol are consistent with a transcription‐dependent mechanism.

Phosphorylation of steroid hormone receptors.

IV. Steroid receptors as substrates for protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 A. Progesterone receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 B. Estrogen receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 C. Glucocorticoid receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 D. Androgen receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 E. Vitamin D-3 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Activation of glucocorticoid receptor by ATP.

Summary Glucocorticoid-receptor complex from rat liver cytosol acquired the ability to bind to isolated nuclei when incubated with 5–10 mM ATP at 4°C. This nuclear uptake resulting from the ‘activation’ of cytosolic glucocorticoid-receptor complex was comparable to the activation achieved by warming the steroid-receptor complex at 23°C. ATP was more effective in causing this transformation when compared with ADP, whereas AMP failed to show any such effect. Both ATP and temperature-caused activation of glucocorticoid receptor was blocked by the presence of 10 mM molybdate. The presence of ATP did not alter the steroid binding properties or the sedimentation characteristics of glucocorticoid receptor under the above conditions. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor at mild assay conditions.

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.

Steroid receptors in health and disease

The influence of steroid hormones on various complex physiological and developmental processes is well known. Their role in the fetal brain development, attainment of puberty, sexual differentiation, regulation of reproductive function, normal and tumor cell proliferation, and maintenance of mineral balance has long been recognized. During the past twenty years, progress in research on the effects and mode of action of steroid hormones has resulted in the development of new approaches to contraception as well as diagnosis and treatment of endocrine disorders and cancers. It is now known that proliferation of almost one-third of reported breast and uterine cancers is influenced by circulating hormones: a high percentage of these cancers respond favorably to endocrine manipulations.

Evidence for separate binding sites for progesterone and RU486 in the chick oviduct

Binding characteristics of synthetic steroid, mifepristone (RU38486 - also referred to as RU486), were examined in cytosol prepared from the chick oviduct and the calf uterus, and were compared with those of progesterone and synthetic progestin R5020. Unlike [3H]progesterone binding, the [3H]RU486 binding in the oviduct cytosol did not saturate at 50 nM ligand concentration. The [3H]progesterone binding could not be eliminated in the presence of excess RU486, and [3H]RU486 binding was seen to be indisplaceable upon pretreatment of the chick oviduct cytosol with a 1000-fold excess progesterone. It is apparent that the chick oviduct cytosol is endowed with two separate sets of sites which interact with progesterone and RU486 independently. Furthermore, [3H]RU486 binding in the chick oviduct cytosol remained intact when incubated for 60 min at 37 degrees C; it exhibited a single ionic form upon elution from DEAE-Sephacel and the [3H]RU486-associated radioactivity sedimented in the 4 S region both in salt-free and 0.3 M KCl-containing 5-20% sucrose gradients. In the calf uterus cytosol, both steroids exhibited comparable binding profiles. Our results provide evidence that chick oviduct possesses distinct binding sites that accept either progesterone or RU486, but not both, as is the case in the calf uterus.

Activation of rat liver glucocorticoid receptor bound to the antiglucocorticoid Ru38486

The kinetics of steroid binding to rat liver glucocorticoid receptor (GR) and receptor denaturation were dependent upon the nature of the molecule occupying GR. Both the agonist [triamcinolone acetonide (TA)] and the antagonist (Ru38486) however competed for the same saturable binding site. Despite opposing physiological action, both steroid analogues permitted receptor activation as evident by binding to DNA-cellulose and 9S to 4S shift on sucrose gradient sedimentation. It therefore seems necessary to reevaluate a current notion that antagonist action of RU38486 in rat liver is a result of impaired receptor activation.