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Maria Cekanova MS, RNDr, PhD.
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R Fernando, JS Foster,
A Bible,
A Ström,
RG Pestell,
M Rao,
Arnold Saxton,
SJ Baek,
K Yamaguchi,
R Donnell,
Maria Cekanova,
J Wimalasena
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ABSTRACT: Recent investigations suggest that functions of the proapoptotic BCL2 family members, including BAD, are not limited to regulation of apoptosis. Here we demonstrate that BAD inhibits G(1) to S phase transition in MCF7 breast cancer cells independent of apoptosis. BAD overexpression inhibited G(1) transit and cell growth as well as cyclin D1 expression. Inhibition of cyclin D1 expression was mediated through inhibition of transcription activated by AP1. Chromatin immunoprecipitation assays indicated that BAD is localized at the 12-O-tetradecanoylphorbol-13-acetate-response element (TRE) and cAMP-response element (CRE) in the cyclin D1 promoter. This was shown to reflect direct binding interactions of BAD with c-Jun, and this interaction inhibited the activity of AP1 complexes at TRE. BAD did not interact with phosphorylated forms of c-Jun. Our data suggest that inhibitory TRE/CRE-c-Jun-BAD complexes are present at the cyclin D1 promoter in quiescent cells. Estrogen stimulation displaced BAD from TRE/CRE elements in MCF7 cells, whereas BAD overexpression inhibited estrogen-induced cyclin D1 synthesis and cell proliferation. Inhibition of endogenous BAD in MCF7 cells markedly increased the proliferative fraction and DNA synthesis, activated Cdks, and increased cyclin D1 protein levels. This action of BAD required serine residues Ser(75) and Ser(99). Both phosphorylated and unphosphorylated forms of BAD localized to the nuclei of human breast epithelial cells. Thus, we demonstrate a novel role for BAD in cell cycle regulation dependent upon its phosphorylation state and independent of the BAD/BCL2 interaction and apoptosis.
Maria Cekanova MS, RNDr, PhD.
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ABSTRACT: Estrogens play an important role in the regulation of placental function, and 17-beta-estradiol (E2) production rises eighty fold during human pregnancy. Although term placenta has been found to specifically bind estrogens, cellular localization of estrogen receptor alpha (ER-alpha) in trophoblast remains unclear. We used western blot analysis and immunohistochemistry with h-151 and ID5 monoclonal antibodies to determine the expression and cellular localization of ER-alpha protein in human placentae and cultured trophoblast cells. Western blot analysis revealed a ~65 kDa ER-alpha band in MCF-7 breast carcinoma cells (positive control). A similar band was detected in five normal term placentae exhibiting strong expression of Thy-1 differentiation protein in the villous core. However, five other term placentae, which exhibited low or no Thy-1 expression (abnormal placentae), exhibited virtually no ER-alpha expression. In normal placentae, nuclear ER-alpha expression was confined to villous cytotrophoblast cells (CT), but syncytiotrophoblast (ST) and extravillous trophoblast cells were unstained. In abnormal placentae no CT expressing ER-alpha were detected. Normal and abnormal placentae also showed ER-alpha expression in villous vascular pericytes and amniotic (but not villous) fibroblasts; no staining was detected in amniotic epithelial cells or decidual cells. All cultured trophoblast cells derived from the same normal and abnormal placentae showed distinct ER-alpha expression in western blots, and the ER-alpha expression was confined to the differentiating CT, but not to the mature ST. Trophoblast cells from six additional placentae were cultured in normal medium with phenol red (a weak estrogen) as above (PhR+), or plated in phenol red-free medium (PhR-) without or with mid-pregnancy levels of E2 (20 nM). Culture in PhR- medium without E2 caused retardation of syncytium formation and PhR-medium with E2 caused acceleration of syncytium formation compared to cultures in PhR+ medium. These data indicate that the considerable increase in estrogen production during pregnancy may play a role, via the ER-alpha, in the stimulation of CT differentiation and promote function in normal placentae. This mechanism, however, may not operate in abnormal placentae, which show a lack of ER-alpha expression.
Maria Cekanova MS, RNDr, PhD.
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ABSTRACT: Cyclin E, a G(1) cyclin serving to activate cyclin-dependent kinase 2, is the only cyclin gene for which alternative splicing leading to structurally different proteins has been described. Different cyclin E proteins are present in tumor tissues but absent from normal (steady) tissues. Cyclin E contributes to the regulation of cell proliferation and ongoing differentiation and aging. Because trophoblast has invasive properties and differentiates into syncytium and placental aging may develop at term, we examined cyclin E protein variants in human placenta. Placental samples were collected from 27 deliveries between 33 and 41 wk and were compared with ovarian cancer (positive control). Both placental and tumor tissues showed seven cyclin E low molecular weight (LMW) bands migrating between 50 and 36 kDa. Placental expression of cyclin E showed certain variability among cases. Lowest cyclin E expression was detected in normal placentas (strong expression of Thy-1 differentiation protein in villous core and low dilatation of villous blood sinusoids). Abnormal placentas (significant depletion of Thy-1 and more or less pronounced dilatation of sinusoids) showed significant increase either of all (early stages of placental aging) or only certain cyclin E proteins (advanced aging). Our studies indicate that a similar spectrum of cyclin E protein variants is expressed in the placental and tumor tissues. Low cyclin E expression in normal placentas suggests a steady state. Overexpression of all cyclin E proteins may indicate an activation of cellular proliferation and differentiation to compensate for developing placental insufficiency. However, an enhanced expression of some cyclin E LMW proteins only might reflect an association of cyclin E isoforms with placental aging or an inefficient placental adaptation.
Maria Cekanova MS, RNDr, PhD.
