Francesco Bresciani

Estrogens and Progesterone Promote Persistent CCND1 Gene Activation during G1 by Inducing Transcriptional Derepression via c-Jun/c-Fos/Estrogen Receptor (Progesterone Receptor) Complex Assembly to a Distal Regulatory Element and Recruitment of Cyclin D1 to Its Own Gene Promoter

ABSTRACT Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G1-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor α complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G1-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G1-phase progression by different classes of NRs.

Estrogen induces c-fos expression specifically in the luminal and glandular epithelia of adult rat uterus

It was previously shown that injection of 17 beta-estradiol into adult ovariectomized rats induces a rapid and transient increase of c-fos gene transcription in the uterus. Immunohistochemical analysis now shows that estrogen activates c-fos specifically in the luminal and glandular epithelial cells of the endometrium, which are the only uterine cells responding to the hormone with DNA synthesis and cell proliferation, and not in estrogen receptor positive stromal and myometrial cells. This finding suggests that c-fos is involved in the mechanism of estrogen regulation of uterine epithelial cell proliferation and, furthermore, that the c-fos activation by estrogen is cell type dependent.

Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells

Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.

Molecular identification of ERα-positive breast cancer cells by the expression profile of an intrinsic set of estrogen regulated genes

Estrogens exert a key biological role in mammary gland epithelial cells and promote breast carcinogenesis and tumor progression. We recently identified a new large set of estrogen responsive genes from breast cancer (BC) cells by DNA microarray analysis of the gene expression profiles induced by 17β‐estradiol in ZR‐75.1 and MCF‐7 cells. The purpose of the present study was to test whether the expression pattern of hormone regulated genes from this set identifies estrogen receptor (ERα) positive, hormone responsive BC cells. To this aim, we carried out in silico metanalysis of ERα positive and ERα negative human BC cell line transcriptomes, focusing on two sets of 171 and 218 estrogen responsive genes, respectively. Results show that estrogen dependent gene activity in hormone responsive BC cells is significantly different from that of non‐responsive cells and, alone, allows to discriminate these two cellular phenotypes. Indeed, we have identified 61 genes whose expression profile specifically marks ERα positive BC cells, suggesting that this gene set may be exploited for phenotypic characterization of breast tumors. This possibility was tested with data obtained by gene expression profiling of BC surgical samples, where the ERα positive phenotypes were highlighted by the expression profile of a subset of 27 such hormone responsive genes and four additional BC marker genes, not including ERs. These results provide direct evidence that the expression pattern of a limited number of estrogen responsive genes can be exploited to assess the estrogen signaling status of BC cells both in vitro and ex‐vivo.

The antiestrogen ICI 182,780 inhibits proliferation of human breast cancer cells by interfering with multiple, sequential estrogen-regulated processes required for cell cycle completion.

Antiestrogens are widely used for breast cancer treatment, where they act primarily by inhibiting the mitogenic action of estrogens on tumor cells. The effects of the pure antiestrogen ICI 182,780 on estrogen-regulated cell cycle phase-specific events were investigated here in synchronously cycling human breast cancer (HBC) cells. In early G(1)-arrested MCF-7 or ZR-75.1 cells, 17beta-estradiol (E2) induces rapid activation of the cyclin/Cdk/pRb pathway, as demonstrated by D-type G(1) cyclins accumulation during the first few hours of hormonal stimulation, followed by sequential accumulation of E, A and B1 cyclins and progressive pRb phosphorylation, as cells progress through the cell cycle. When added to quiescent cells together with E2, ICI 182,780 prevents all of the above hormonal effects. Interestingly, in mid-G(1) cells (2-8 h into estrogen stimulation) the antiestrogen causes rapid reversal of hormone-induced D-type cyclins accumulation and pRb phosphorylation, and still fully inhibits G(1)-S transition rate, while in late-G(1) cells it does not prevent S phase entry but still inhibits significantly DNA synthesis rate, S-phase cyclins accumulation and pRb hyperphosphorylation. These results indicate that pure antiestrogens prevent multiple estrogen-induced cell cycle-regulatory events, each timed to allow efficient G(1) completion, G(1)-S transition, DNA synthesis and cell cycle completion.

Purification and properties of native oestradiol receptor.

Abstract A method is described for the purification to homogeneity of oestrogen receptor from calf uterus cytosol. This method consists of affinity chromatography on heparin-agarose followed by affinity chromatography on 17β-oestradiol-17-hemisuccinyl-ovalbumin-agarose, by a second adsorption on heparin-agarose and finally by gel filtration on Sephadex G-50. Elution from heparin-agarose was obtained using buffer containing heparin (4 mg/ml), and from the oestradiol-adsorbent using buffer containing NaSCN 0.5 M and oestradiol. The yield from 1 kg of uteri was about 1.2 mg of receptor protein with more than 50% recovery. A single protein band, co-migrating with the [2H]oestradiol, is seen in polyacrylamide gel electrophoresis. A single protein band, with an apparent mol. wt. of 70,000, is also seen on electrophoresis in sodium dodecyl sulfate gels. As computed from specific activity, there is one oestradiol binding site per 70,000 receptor subunit. The pure receptor on low salt sucrose gradient sediments at 8 S and on high salt or in chaotropic salt containing gradient sediments at about 4 S. The high tendency to form aggregates is still maintained by the pure receptor as judged by the elution pattern from Sephadex G-200 columns. Aggregation is avoided by small concentrations of heparin in the buffers.

Inhibition of sodium pump and increase of passive fluxes by x-rays in human erythrocytes.

X irradiation induces an increase of Na and a decrease of K content in erythrocytes. Results are reported from studies on the mechanisms that produce this effect. It was concluded that x radiation interferes with the biochemical mechanism of the active extrusion of Na through the cell membrane, but has only a slight effect on passive fluxes. (C.R.)

[30] Purification of estrogen receptors. I

Publisher Summary The word “receptor” was originally used by Ehrlich to indicate a cell component able to recognize and interact with a specific molecule of the cellular environment and included the concept that the informational content of the incoming molecule was expressed through the interaction with its cellular receptor. This full and original meaning of the word still holds among pharmacologists. Therefore, when speaking of estrogen receptors, one must preliminarily state that this term refers to cellular components. There is no definite information yet as to whether these proteins also fulfill the second part of Ehrlichs proposition—that is, whether the estrogenic ligand acts by inducing some modification of these proteins which, in turn, enables them to activate the cell machinery to produce the final estrogen effects. Thus, estrogen receptors are used here not in the strictest sense of the word but as synonymous with high affinity, cellular estrogen-binding proteins (EB-proteins).

Three protein kinases from calf uterus. subcellular distribution and physical properties

Abstract Three protein-kinases which phosphorylate preferentially either histones (kinase a ), protamine (kinase b ) or casein (kinase c ) were separated from calf uterus cytosol. Kinase a weights 120,000, sediments at 6 S and shows an isoelectric point (I.P.) of 5.0. Kinase b weights 65,000, sediments at 4.7 S and shows an I.P. of 5.5. Kinase c weights 200,000, sediments at 7.0 S and shows an I.P. of 6.0. Only kinase a binds, and is stimulated by, 3′,5′-cyclic AMP.

Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G1 phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.