Massimo Cancemi

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.

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.

MePR: a novel human mesenchymal progenitor model with characteristics of pluripotency.

Human embryo stem cells or adult tissues are excellent models for discovery and characterization of differentiation processes. The aims of regenerative medicine are to define the molecular and physiological mechanisms that govern stem cells and differentiation. Human mesenchymal stem cells (hMSCs) are multipotent adult stem cells that are able to differentiate into a variety of cell types under controlled conditions both in vivo and in vitro, and they have the remarkable ability of self-renewal. hMSCs derived from amniotic fluid and characterized by the expression of Oct-4 and Nanog, typical markers of pluripotent cells, represent an excellent model for studies on stemness. Unfortunately, the limited amount of cells available from each donation and, above all, the limited number of replications do not allow for detailed studies. Here, we report on the immortalization and characterization of novel mesenchymal progenitor (MePR) cell lines from amniotic fluid-derived hMSCs, whose biological properties are similar to primary amniocytes. Our data indicate that MePR cells display the multipotency potential and differentiation rates of hMSCs, thus representing a useful model to study both mechanisms of differentiation and pharmacological approaches to induce selective differentiation. In particular, MePR-2B cells, which carry a bona fide normal karyotype, might be used in basic stem cell research, leading to the development of new approaches for stem cell therapy and tissue engineering.

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.

Transcriptional Control of Cell Cycle Progression by Estrogenic Hormones: Regulation of Human Cyclin D1 Gene Promoter Activity by Estrogen Receptor-a

Estrogens (Es) are mitogens for breast and uterine epithelial cells, where they exert also a tumor-promoting action that appears to be directly linked to their growth promoting effects (1-3). Although during recent years the general bases of estrogen receptor (ER) action, in particular the role of these nuclear proteins on regulation of gene transcription, have been elucidated (4), the mechanisms that underlie E control of cell proliferation still remain largely unclear, mainly due to our relatively poor knowledge of the effects of these steroids on the cell cycle regulatory pathways. In eukaryotic cells, these pathways have been found to consist of an orderly sequence of genetic and biochemical processes, controlled by extracellular mitogens as well as oncogenes, that are required for and allow completion of the different tasks leading to cell cycle progression and cell division. They include both growth regulatory or primary events, and growth regulated processes, consequent to the effects evoked in the cell by the primary events (5–6). The evident analogy of these pathways with the general mechanism of action of Es in target cells (7) raises the possibility that cell cycle regulatory (or ‘master’) genes and gene products could be target of the ER s and mediate their growth promoting actions. A better understanding of the molecular basis of the mitogenic activity of Es will be greatly fostered by the identification of such cell cycle regulatory genes and molecules, and by the subsequent elucidation of their functional interactions with the hormone and its receptors.