The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes.

Histone H3K4 methylation is a feature of meiotic recombination hotspots shared by many organisms including plants and mammals. Meiotic recombination is initiated by programmed double-strand break (DSB) formation that in budding yeast takes place in gene promoters and is promoted by histone H3K4 di/trimethylation. This histone modification is recognized by Spp1, a PHD finger containing protein that belongs to the conserved histone H3K4 methyltransferase Set1 complex. During meiosis, Spp1 binds H3K4me3 and interacts with a DSB protein, Mer2, to promote DSB formation close to gene promoters. How Set1 complex- and Mer2- related functions of Spp1 are connected is not clear. Here, combining genome-wide localization analyses, biochemical approaches and the use of separation of function mutants, we show that Spp1 is present within two distinct complexes in meiotic cells, the Set1 and the Mer2 complexes. Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation. Finally, we provide evidence that Spp1 preserves normal H3K4me3 levels independently of the Set1 complex. We propose a model where Spp1 works in three ways to promote recombination initiation: first by depositing histone H3K4 methylation (Set1 complex), next by "reading" and protecting histone H3K4 methylation, and finally by making the link with the chromosome axis (Mer2-Spp1 complex). This work deciphers the precise roles of Spp1 in meiotic recombination and opens perspectives to study its functions in other organisms where H3K4me3 is also present at recombination hotspots.

Indole Derivative Interacts with Estrogen Receptor Beta and Inhibits Human Ovarian Cancer Cell Growth.

Ovarian cancer remains the leading cause of mortality among gynecological tumors. Estrogen receptor beta (ERß) expression has been suggested to act as a tumor suppressor in epithelial ovarian cancer by reducing both tumor growth and metastasis. ERß expression abnormalities represent a critical step in the development and progression of ovarian cancer: for these reasons, its re-expression by genetic engineering, as well as the use of targeted ERß therapies, still constitute an important therapeutic approach. 3-{[2-chloro-1-(4-chlorobenzyl)-5-methoxy-6-methyl-1H-indol-3-yl]methylene}-5-hydroxy-6-methyl-1,3-dihydro-2H-indol-2-one, referred to here as compound 3, has been shown to have cytostatic as well cytotoxic effects on various hormone-dependent cancer cell lines. However, the mechanism of its anti-carcinogenic activity is not well understood. Here, we offer a possible explanation of such an effect in the human ovarian cancer cell line IGROV1. Chromatin binding protein assay and liquid chromatography mass spectrometry were exploited to localize and quantify compound 3 in cells. Molecular docking was used to prove compound 3 binding to ERß. Mass spectrometry-based approaches were used to analyze histone post-translational modifications. Finally, gene expression analyses revealed a set of genes regulated by the ERß/3 complex, namely CCND1, MYC, CDKN2A, and ESR2, providing possible molecular mechanisms that underline the observed antiproliferative effects.

Identification of a new bisindolinone arresting IGROV1 cells proliferation.

In an initial screening, a series of novel Knoevenagel adducts were submitted to the National Cancer Institute for evaluation of antitumor activity in human cell lines. In particular, compound 5f showed remarkable selectivity against IGROV1, an ovarian cancer cell line, without affecting healthy human fibroblast cells. Analyses of cytotoxicity, cell proliferation, cell migration, epigenetic changes, gene expression, and DNA damage were performed to obtain detailed information about its antitumor properties. Our results show that 5f causes proliferation arrest, decrease in motility, histone hyperacetylation, downregulation of cyclin D1 and α5 subunit of integrin ß1 gene transcription. In addition, 5f treatment reduces transcript and protein levels of cyclin D1, which increases sensitivity to ionizing radiation and results in DNA damage comparable to cyclin D1 gene silencing.

Chitosan nanoparticles for lipophilic anticancer drug delivery: Development, characterization and in vitro studies on HT29 cancer cells.

The aim of this study was to develop chitosan-based nanoparticles that could encapsulate lipophilic molecules and deliver them to cancer cells. Nanoparticles were prepared with different molar ratios of chitosan, hyaluronic acid and sulphobutyl-ether-ß-cyclodextrin and with or without curcumin. The nanosystems were characterized in terms of their size, zeta potential, morphology, encapsulation efficiency and stability in different media. Intestinal epithelial and colorectal cancer cells were treated with unloaded nanoparticles in order to study their effect on cellular membrane organization and ROS production. Finally, in vitro assays on both cellular lines were performed in order to evaluate the ability of nanoparticles to promote curcumin internalization and to study their effect on cell proliferation and cell cycle. Results show that nanoparticles were positively charged and their size increased with the increasing amounts of the anionic excipient. Nanoparticles showed good encapsulation efficiency and stability in water. Unloaded nanoparticles led to a change in lipid organization in the cellular membrane of both cell lines, without inducing ROS generation. Confocal microscopy, cell proliferation and cell cycle studies allowed the selection of the best formulation to limit curcumin cytotoxicity in normal intestinal epithelial cells and to reduce cancer cell proliferation. The latter was the result of the increase of expression for genes involved in apoptosis.