Cliff Hurd

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.

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.

Protein kinase D complexes with C-Jun N-terminal kinase via activation loop phosphorylation and phosphorylates the C-Jun N-terminus.

Protein kinase D (PKD), a downstream effector of protein kinase C (PKC), is implicated in suppression of the c-Jun N-terminal kinase (JNK) signaling pathway, however, its mechanism of action is unclear. Transphosphorylation of the PKD activation loop at serines 744/748 by a PKC mediated signal transduction pathway enhances its catalytic activity. Here we show that PKD activation loop phosphorylation at serines 744/748 via PKC, or mutation of these serines to glutamic acid (PKD-S744/748E) also results in complex formation with JNK, indicating that suppression of JNK signaling by PKD involves a direct interaction with JNK. Because catalytically active PKD associates with JNK we determined whether it could phosphorylate the c-Jun N-terminus as a potential mechanism by which it suppresses c-Jun Ser 63 phosphorylation when it complexes with JNK. Purified human PKD and either wild-type PKD from phorbol 12, 13-dibutyrate (PDB)-stimulated cells or unstimulated constitutively active PKD (PKD-S744/748E), phosphorylated the c-Jun N-terminus between amino acids 1–89 at sites distinct from those phosphorylated by JNK. These results demonstrate, for the first time, phosphorylation dependent association of PKD with another signaling molecule and reveal a potential mechanism by which PKD could modulate the ability of JNK to phosphorylate c-Jun by phosphorylating alternative sites in the c-Jun N-terminus when it is complexed with JNK.

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.

Induced Overexpression of Protein Kinase D1 Stimulates Mitogenic Signaling in Human Pancreatic Carcinoma PANC-1 Cells

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 Ser63 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 Ser63 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. 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. J. Cell. Physiol. 223: 309–316, 2010.

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.

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.

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.

Uncoupling of protein kinase D from suppression of EGF-dependent c-Jun phosphorylation in cancer cells.

Protein kinase D (PKD) has been established as a negative modulator of the c-Jun N-terminal kinase (JNK) signaling pathway. We previously demonstrated that induced expression of constitutively active PKD (PKD-S744/748E) that mimics phosphorylation by PKC is sufficient to attenuate epidermal growth factor (EGF) stimulated c-Jun Ser 63 phosphorylation, a natural substrate of JNK, in HEK 293 cells. Because the JNK pathway has been implicated in sustaining both lung and pancreatic cancerous phenotypes, we have utilized stable inducible expression of PKD-S744/748E in clones of A549 non-small cell lung cancer (NSCLC) and Panc1, pancreatic cancer cells to determine its effects on JNK signaling in the context of the cancerous phenotype. In contrast to HEK 293 cells, induced expression of PKD-S744/748E in either A549 NSCLC or Panc1 cells failed to attenuate EGF dependent phosphorylation of c-Jun, indicating that EGF stimulated JNK phosphorylation of c-Jun is uncoupled from PKD suppression in these cancer cells.

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.