Resveratrol reverses ET-1-evoked mitogenic effects in human coronary arterial cells by activating the kinase-G to inhibit ERK-enzymes.

In human coronary smooth muscle cells (HCSMC), treatment with the vascular mitogen; endothelin-1 (ET-1), induced cell proliferation and stimulated ERK-1/2 phosphorylation at active sites. Pretreatment with the MEK-ERK inhibitor (PD98059) appreciably reversed the mitogenic effects of ET-1. On the other hand, pretreatment with the polyphenolic stilbene resveratrol (RSVL, 1-100 microM) triggered more prominent inhibition of ET-1-evoked cell proliferation and ERK1/2 activation. Besides, RSVL also markedly (2-3 fold) and rapidly enhanced cGMP formation, but had no effect on cAMP levels. This RSVL-evoked upregulation of cGMP was insensitive to pretreatment with the soluble guanylyl cyclase (sGC)-inhibitor (ODQ, 10 microM), but was ablated with an inhibitor of pGC (PMA, 0.1 microM). Further, pretreatment with the specific cGMP-phosphodiesterase inhibitor, zaprinast (10 microM) appreciably augmented RSVL-evoked cGMP formation, ERK inhibition, and cytostatic response. Moreover, the RSVL-induced ERK-inhibitory effects were significantly reversed by the kinase-G inhibitor, KT-5823 (10 microM; 69%), but not by the kinase-A inhibitor (KT-5720). These results demonstrate a novel signaling pathway for RSVL that leads from activation of the pGC/kinase-G system to inhibition of ERK1/2 and their downstream nuclear targets. This pathway functions to counteract the atherogenic signaling induced by vascular mitogens.

Establishment of a gastric epithelial progenitor cell line from a transgenic mouse expressing the simian virus 40 large T antigen gene in the parietal cell lineage.

OBJECTIVE: In this study the gastric mucosa of transgenic mice expressing the simian virus 40 large T antigen gene in the parietal cell lineage is used to establish and characterize a new epithelial progenitor cell line. In these mice, proliferation and amplification of preparietal cells preclude their maturation into acid-secreting parietal cells leading to achlorohydria, hyperplasia, dysplasia and eventually gastric adenocarcinoma. MATERIALS AND METHODS: Enzymatically dispersed gastric epithelial cells were cultured, cloned and screened using immunohistochemical methods, for expression of a variety of biomarkers of differentiated pit, parietal, enteroendocrine and neck/zymogenic cells. RESULTS: A biomarker-deficient cell line whose ultrastructural features resembled those of mouse gastric epithelial progenitor cells was established. Treatment with either hydrocortisone or oestrogen significantly enhanced proliferation of these cells, whereas retinoic acid inhibited their growth. No change in differentiation was detected with any of these treatments; however, when these cells were injected subcutaneously into nude mice, they proliferated to form tumours and undergo partial differentiation towards parietal cell lineage. CONCLUSION: This mouse gastric epithelial progenitor cell line could be useful as an in vitro model to study growth properties, proliferation and differentiation of a subpopulation of gastric epithelial progenitor cells and also to study gastric carcinogenesis.

Nongenomic activation of the GC-A enzyme by resveratrol and estradiol downstream from membrane estrogen receptors in human coronary arterial cells.

BACKGROUND AND AIM: Resveratrol (RSVL), a polyphenolic phytoestrogen in grapes, confers multifaceted cardiovascular benefits. The cellular and molecular basis of RSVL actions has been largely undefined until now. METHODS AND RESULTS: In human coronary smooth muscle cells (HCSMCs), RSVL markedly (3.2-fold) enhanced cGMP formation (t(1/2): 6.3 min, EC(50): 1.8 microM) and stimulated kinase-G activity (4-fold). By contrast, RSVL had no effect on cAMP or PKA activity in these cells. The RSVL-enhanced cGMP/kinase-G activity was not abrogated by the nitric oxide synthase-inhibitor (L-NMMA, 10 microM), or the soluble guanylyl cyclase (sGC)-inhibitor (ODQ, 10 microM). In membrane preparations from HCSMCs, RSVL activated GC in the particulate-, but not in the soluble-membrane fraction. Similar effects were due to the specific particulate-GC-A agonist atrial natriuretic peptide (ANP, 0.1-1 microM). The combined effects of RSVL and ANP were competitive. By contrast, the selective GC-B agonist (BNP) showed no response on cGMP, whereas that for GC-C (guanylin) produced only slight increases in cGMP levels. Estradiol (E2) mimicked the effects of RSVL on cGMP, but showed a 46% lower maximal response. Combining E2 with RSVL showed a competitive, rather than an additive, response. Further, cGMP formation by RSVL or E2 was significantly attenuated by the pure estrogen receptor blocker, ICI-182,780 (10 microM). CONCLUSION: These findings are the first to link RSVL with pGC/kinase-G activation downstream from membrane ERs in the vasculature, thus substantiating its coronary protective effects, even in endothelium-disrupted coronary arteries.

Overexpression of wild-type p53 gene renders MCF-7 breast cancer cells more sensitive to the antiproliferative effect of progesterone.

We have recently shown that growth inhibition of breast cancer cells by progesterone is due to the induction of cell differentiation, but not apoptosis. Because the tumor suppressor protein p53 plays a central role in normal cell growth and in tumor suppression, we have examined the effect of progesterone on the levels of this protein in MCF-7 cells. We show here that the antiproliferative effect of progesterone is accompanied with down-regulation of endogenous p53 protein. To study the effect of progesterone on cell growth in the presence of normal levels of p53 protein, we used transient transfection to overexpress p53 protein. MCF-7 cells were transfected with a p53 expressing vector that contains p53 human cDNA under the control of a cytomegalovirus promoter. Cell growth, cell viability, and apoptosis were analyzed in the transfected cells after six days of exposure to 100 nM progesterone. We show here that progesterone significantly enhances growth inhibition and apoptosis in MCF-7 cells overexpressing p53, but not in cells transfected with the control vector. These data suggest that re-establishing p53 function in MCF-7 breast cancer cells renders them more sensitive to the growth inhibitory effect of progesterone.

Growth inhibition of MCF-7 human breast cancer cells by progesterone is associated with cell differentiation and phosphorylation of Akt protein.

Progesterone inhibits the proliferation of normal breast epithelial cells as well as breast cancer cells. The molecular mechanisms of this inhibition are not fully understood. The purpose of this study was to investigate the capacity of progesterone to induce apoptosis and to alter the activity of a key regulator of cell growth and differentiation, the Akt protein. We show here that (i) growth inhibition of breast cancer cells by progesterone is due to the induction of cell differentiation and not to apoptosis; (ii) progesterone activates the PI3-kinase/Akt pathway as shown by the increase in the phosphorylation of Akt protein; (iii) inhibiting PI3-kinase/Akt pathway with LY294002 causes stimulation of apoptosis; and (v) progesterone enhances LY294002 induced-growth inhibition and apoptosis. These results suggest that progesterone may protect breast cancer cells from apoptosis by altering PI3-kinase activity and that MCF-7 cells become more sensitive to progesterone and die by apoptosis upon inhibition of the PI3-kinase/Akt pathway.

MDM2 overexpression generates a skin phenotype in both wild type and p53 null mice.

The MDM2 proto-oncogene is overexpressed in human tumours and regulates the activities of the tumour suppressors p53 and pRB. We created mice that overexpress MDM2 under the control of the CMV promoter. These mice did not display an increased tumour incidence, but rather a specific skin phenotype, characterized by desquamation and hyperkeratosis. Transgenic MDM2 was found to be overexpressed in the epidermis, a tissue that normally expresses high levels of MDM2. The phenotype appeared during the first week after birth and then lessened with age, closely following the level of expression of the transgene. MDM2 overexpression was associated with an increase in proliferation in the basal layer, thickening of the epidermis, altered expression of the differentiation markers cytokeratin CK14, CK10 and CK1, and a decrease in the size and the number of granules that contain products of differentiation. Transgenic mice on a p53 null background displayed similar although not identical changes, showing that the effects of MDM2 are to a certain degree p53 independent. The skin is a major site of MDM2 expression in mice, raising the possibility that MDM2 overexpression perturbs the normal pattern of MDM2 expression and inhibits differentiation of the epidermis.

Regulation of gene expression in T-47D human breast cancer cells by progestins and antiprogestins.

The molecular mechanisms by which progestins and antiprogestins inhibit human breast cancer cell growth are essentially unknown. The mechanisms by which they mediate growth inhibition in human breast cancer cells and the expression of the putative autocrine/paracrine growth factors, epidermal growth factor and transforming growth factors alpha and beta-1 were studied under conditions in which progestin and antiprogestin inhibit the growth of T-47D human breast cancer cells in culture. Under the same conditions, the expression of genes such as c-myc, c-jun and c-fos, which are known to have important roles in growth and differentiation, has been measured. The results indicate that progestins and antiprogestins differentially regulate expression of these genes. The data are consistent with the conclusion that the mechanism of growth inhibition of these two agents differs, although an initial interaction with the progesterone receptor is a necessary first step in initiating the as yet ill-defined cascade of events leading to growth inhibition.

Enhanced c-jun activity alters responsiveness to medroxyprogesterone acetate in Ishikawa human endometrial carcinoma cells.

The involvement of altered c-jun activity in medroxyprogesterone acetate (MPA)-induced growth responses in human endometrial carcinoma cells is examined in this paper. Under conditions of MPA-induced growth inhibition, c-jun mRNA and protein levels are decreased in Ishikawa cells. This decrease is accompanied by an overall decrease in endogenous AP-1 activity in these cells. Only a transient decrease in c-jun mRNA level without any effect on endogenous AP-1 activity is seen in HEC-50 human endometrial carcinoma cells after MPA treatment. Increased expression and activity of c-jun was achieved in Ishikawa cells by transient transfection of Rous sarcoma virus (RSV)-c-jun alone or RSV-c-jun plus AP-1 binding sites (5x-4-beta-phorobol 12-myristate 13-acetate response element-thymidine kinase-chloramphenicol acetyltransferase), respectively. These treatments were accompanied by an increase in cell numbers due to MPA treatment in Ishikawa cells. In contrast, MPA treatment of mock-transfected, RSV-jun-B-transfected, or 5x-4 beta-phorbol 12-myristate 13-acetate response element-thymidine kinase-chloramphenicol acetyltransferase alone transfected Ishikawa cells resulted in the expected decrease in cell numbers. The data presented in this paper are consistent with the hypothesis that altered c-jun activity in a target cell can alter proliferative responsiveness to MPA and suggest that such a mechanism may be associated with resistance to hormonal manipulative therapies used in the treatment of both human breast and endometrial cancer.

Regulation of c-jun and jun-B by progestins in T-47D human breast cancer cells.

To investigate further the molecular mechanisms of progestin regulation of human breast cancer cell growth, we studied the effect of progestins on expression of the protooncogene c-jun and other members of the jun family, jun-B and jun-D, in T-47D human breast cancer cells. The progestin medroxyprogesterone acetate (MPA) increased c-jun mRNA levels in a time- and dose-dependent fashion. Maximal effects were seen after 3 h of treatment with 10-100 nM MPA. Under these conditions, the c-jun mRNA was increased 5.4-fold above the control level. Although the c-jun mRNA level was increased by cycloheximide alone, a further 2.4-fold increase was seen when the cells were treated with MPA in the presence of cycloheximide. The p39 c-jun protein was also increased 3.8-fold by this treatment. Maximum levels of p39 c-jun protein were achieved 9 h after treatment, and this level was maintained for at least 24 h. Dexamethasone and dihydrotestosterone did not increase the p39 c-jun protein level under these conditions. However, MPA treatment of T-47D cells resulted in a 55% decrease in overall AP-1 activity, as measured by transient transfection of an AP-1-regulated chloramphenicol acetyltransferase reporter gene. These effects were all reversible by cotreatment with a 10-fold higher concentration of the antiprogestin RU 486. MPA decreased jun-B mRNA levels 50% 1 h after treatment in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural changes in guinea pig endometrial cells during the estrous cycle.

In the guinea pig, the estrous cycle is characterized by a constant measurable level of plasma progesterone with two peaks: the first one associated with the peak of plasma estradiol-17 beta occurring at proestrus and the second, during diestrus, more pronounced at the time at which the level of estradiol-17 beta is undetectable. The progesterone receptor content is the highest on day 1 and the lowest on day 10 of the estrous cycle, which lasts 16.3 +/- 1.5 days (n = 37; mean +/- SD). There is a positive correlation between the plasma level of estradiol-17 beta and the progesterone receptors detected immunocytochemically in both endometrial epithelial and stromal cells. The general morphology of the endometrium during proestrus and estrus is consistent with an estrogenic stimulation, i.e., a smooth and regular surface of the endometrium and the presence of numerous microvilli on the cell surface. However, a moderate secretory activity also occurs in proestrus and estrus. During postestrus, the glandular cells display an increase in characteristic secretory features which parallels the rise of progesterone in the plasma.

Mechanisms of growth inhibition by antiestrogens and progestins in human breast and endometrial cancer cells.

Marked changes in both growth factor and proto-oncogene expression occur due to treatment of hormonally-responsive human cancers with progestins and antiestrogens. In human endometrial cancer cell lines the antiproliferative effects of progestins and antiestrogens in a particular cell line appear to be associated with similar effects on growth factor and/or proto-oncogene expression. This suggests that although these compounds initially interact with different steroid hormone receptors, the molecular mechanisms of their growth inhibition may be essentially similar. In the case of human breast cancer cell lines, however, the effects of progestins and antiestrogens on gene regulation are often different, suggesting that the molecular mechanisms of progestin and antiestrogen growth inhibition may be essentially dissimilar.

Characterization of estrogen receptor variant mRNAs from human breast cancers.

The cDNAs for variant estrogen receptor (ER) mRNAs previously identified in human breast cancer biopsy samples have been cloned and characterized. Some of these cDNAs are unique to a tumor sample (e.g. clones 24 and 5), while others are present in multiple breast tumor samples (e.g. clone 4). The 5' ends of the variant cDNAs are essentially identical to sequences present in exons 1, 2, and 3 of the normal ER mRNA. However, at points which mark either the exon 2/intron or exon 3/intron boundaries, the variant cDNA sequences diverge and are unrelated to the normal ER mRNA. The unique sequences of clones 24 and 5 are unknown, and the unique sequence of clone 4 is related to the long interspersed repetitive LINE-1 sequences. The variant mRNAs contain open reading frames which could encode proteins containing known functional domains of the normal ER but missing others. In particular, the hormone binding domain of the normal ER is always missing. Furthermore, some of the variant transcripts may encode other unique proteins. In transient expression assays the proteins encoded by the variant ER mRNAs are unable to activate transcription of an estrogen-responsive reporter gene; neither are they able to modulate the ability of normal ER proteins to activate transcription.

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin.

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.

Effects of 17 beta-estradiol and growth factors on c-fos gene expression in endometrial epithelial cells in primary culture.

Primary culture of guinea-pig endometrial cells was made quiescent by serum depletion. When added to quiescent cells, 17 beta-estradiol (E2) alone affected neither c-fos and c-myc gene expression, nor DNA synthesis and cell proliferation. Insulin or epidermal growth factor (EGF) only induced DNA synthesis. An association of both growth factors allowed significant cell proliferation without inducing c-fos or c-myc expression. A response including c-fos induction (maximal expression at 75 min), but not c-myc expression, DNA synthesis and a marked cell proliferation was only obtained when 17 beta-estradiol was associated with insulin plus EGF. In this case, cycloheximide raised the c-fos gene expression. These data suggest that in endometrial epithelial cells, E2 is mitogenic only when it acts in association with EGF plus insulin and the c-fos gene expression may not be correlated with cell proliferation.

Uterine epithelial cells in primary culture: a model system to study cell proliferation.

Guinea-pig uterine glandular epithelial cells were grown in primary culture. During the 4-day initial culture period, a 6.7 fold increase in DNA synthesis and a doubling time of approximately 30 hours were observed. Then the cells were submitted to serum depletion (60 hours) and the quiescent cells were stimulated with 15% fetal calf serum (FCS). The control cells were submitted to 1% heated and dextran-coated charcoal stripped FCS. In stimulated cells, the DNA synthesis increased and peaked between the 12th and 24th hour. In these cells, c-fos mRNAs increased rapidly, within 30 min., peaked at 75 min. (ratio to the control = 2.5), and returned to basal level within 90 min. These results prove that uterine epithelial cells in primary culture are able to respond to unspecific mitogen by both rapid expression of c-fos gene and DNA synthesis, suggesting that this cell culture system will be useful in studying the growth regulation in endometrium.

Regulation of c-fos expression in primary culture of guinea pig glandular epithelial cells stimulated by growth factors and estradiol.

The c-fos expression was investigated in primary culture of guinea pig glandular epithelial cells. These cells were made quiescent by serum deprivation and stimulated with fetal calf serum (FCS, 15%), 17 beta-estradiol (E2 10(-8) mol/l) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) and insulin (I, 10 micrograms/ml). Low levels of c-fos mRNA were detectable in quiescent cells and were not increased in cells stimulated with either E2, EGF, I, or EGF plus I. On the contrary, the c-fos mRNA were early and transiently increased by FCS or E2 plus EGF plus I (4.5 and 9.5 fold induction, respectively). This effect was independent of de novo protein synthesis since it was not abolished in the presence of cycloheximide. It appears that E2 acts in a multiple step process including the stimulation by EGF plus insulin.

Specific effect of oestrone sulphate on protein synthesis and secretion by cultured epithelial cells from guinea-pig endometrium.

Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated. Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium. For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells. Only experiments in which the control dishes displayed more than 80% of anticytokeratin-immunostained cells were further processed. After this period oestradiol-17 beta (20 nmol/l; control), oestradiol-17 beta (20 nmol/l) plus progesterone (0.5 mumol/l), oestrone sulphate (1 mumol/l) or oestrone sulphate (1 mumol/l) plus progesterone (0.5 mumol/l) were added to the medium for 48 h. An immunocytochemical progesterone receptor assay showed that oestradiol-17 beta increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17 beta induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells. In these culture conditions and after 16 h of incubation, oestradiol-17 beta induced a 1.7-fold increase in [3H]thymidine incorporation into DNA, and [35S]methionine incorporation into cellular proteins was linearly increased up to 8 h. Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of [35S]methionine. The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)