A Novel and Critical Role for Oct4 as a Regulator of the Maternal-Embryonic Transition

Compared to the emerging embryonic stem cell (ESC) gene network, little is known about the dynamic gene network that directs reprogramming in the early embryo. We hypothesized that Oct4, an ESC pluripotency regulator that is also highly expressed at the 1- to 2-cell stages in embryos, may be a critical regulator of the earliest gene network in the embryo.Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we show that Oct4 is required for development prior to the blastocyst stage. Specifically, Oct4 has a novel and critical role in regulating genes that encode transcriptional and post-transcriptional regulators as early as the 2-cell stage. Our data suggest that the key function of Oct4 may be to switch the developmental program from one that is predominantly regulated by post-transcriptional control to one that depends on the transcriptional network. Further, we propose to rank candidate genes quantitatively based on the inter-embryo variation in their differential expression in response to Oct4 knockdown. Of over 30 genes analyzed according to this proposed paradigm, Rest and Mta2, both of which have established pluripotency functions in ESCs, were found to be the most tightly regulated by Oct4 at the 2-cell stage.We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early embryo development is large and distinct from its established network in ESCs. Further, our experimental approach can be applied to dissect the gene regulatory network of Oct4 and other pluripotency regulators to deconstruct the dynamic developmental program in the early embryo

Monitoring the Antioxidant Mediated Chemosensitization and ARE-Signaling in Triple Negative Breast Cancer Therapy

<div><p>Chemotherapy often fails due to cellular detoxifying mechanisms, including phase-II enzymes. Activation of Nrf2-Keap1 pathway induces phase-II enzymes expression through ARE-signaling and prevents cancer development. Nrf2-overexpression in cancer cells results in chemo- and/or radioresistance. This necessitates understanding of Nrf2-regulation, and identification of Nrf2 activators/inhibitors sensitizing cancer cells to improve chemotherapy. N-terminal 435-amino acids of Nrf2 are crucial for Keap1 binding during ubiquitination. Identification of a minimum Nrf2-domain required for Keap1 binding without altering endogenous ARE-signaling would be a novel tool to study Nrf2-signaling. Current study developed firefly-luciferase reporter fusion with N-terminal Nrf2-domain of different lengths and examined its response to Nrf2-activators in cells. The results identified FLuc2 fusion with N-terminal 100-aa of Nrf2 is sufficient for measuring Nrf2-activation in cancer cells. We used MDA-MB231 cells expressing this particular construct for studying antioxidant induced Nrf2-activation and chemosensitization in triple-negative breast cancer therapy. While antioxidant EGCG showed chemosensitization of MDA-MB231 cells to cisplatin by activating Nrf2-ARE signaling, PTS, another antioxidant showed chemoprotection. Tumor xenograft study in mouse demonstrates that combinational treatment by cisplatin/EGCG resulted in tumor growth reduction, compared to cisplatin alone treatment. The results of this study highlight the importance of identifying selective combination of antioxidants/chemotherapeutic agents for customized treatment strategy.</p></div

Time (A) and concentration (B) dependent activation of Nrf2-100-FLuc2 fusions in response to Nrf2-activators in cells.

<p>Asterisk (*) denotes statistical significance (p<0.05) compared to DMSO control (A) and non-exposed cells control (B). Expression of antioxidant genes in response to Nrf2 activators in MDA-MB231 cells stably expressing Nrf2-100aa-FLuc2 fusion. C, Immunoblot analysis of Nrf2, NQO1 and GST expression normalized to GAPDH expression and to control sample. The images were acquired by optical CCD (IVIS) camera imaging. Error bars represent SEM (A, B) or standard deviations (C) of triplicate experiments.</p

Review Therapeutic Evaluation of microRNAs by Molecular Imaging

licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2013.07.26; Accepted: 2013.09.22; Published: 2013.12.06 MicroRNAs (miRNAs) function as regulatory molecules of gene expression with multifaceted activities that exhibit direct or indirect oncogenic properties, which promote cell proliferation, differentiation, and the development of different types of cancers. Because of their extensive functional involvement in many cellular processes, under both normal and pathological conditions such as various cancers, this class of molecules holds particular interest for cancer research. MiRNAs possess the ability to act as tumor suppressors or oncogenes by regulating the expression of different apoptotic proteins, kinases, oncogenes, and other molecular mechanisms that can cause the onset of tumor development. In contrast to current cancer medicines, miRNA-based therapies function by subtle repression of gene expression on a large number of oncogenic factors, and therefore are anticipated to be highly efficacious. Given their unique mechanism of action, miRNAs are likely to yield a new class of targeted therapeutics for a variety of cancers. More than thousand miRNAs have been identified to date, and their molecular mechanisms and functions ar

Time dependent activation of Nrf2-100-FLuc2 fusions in response to varying anticancer drug (A, RRx-001 and B, cisplatin) concentration in the presence of antioxidant (EGCG) in MDA-MB231 cells, and Nrf2 target genes (GST and NQO1) expression in MDA-MB231-Nrf2-100-FLuc2 cells in response to anticancer drug (RRx-001) in the presence of antioxidant (EGCG).

<p>Asterisk (*) denotes statistical significance (p<0.05) of a signal compared to that from the untreated cells (A) and from cells treated with 50 μM EGCG alone (B). C, Immunoblot of cell lysates treated with drug and/or antioxidant for 24 hours (Nrf2, GST and NQO1 expression was normalized to GAPDH expression). Error bars represent standard deviations of triplicate experiments.</p

Effect of anticancer drugs in response to antioxidant-Nrf2-activators in MDA-MB231 cells.

<p>Apoptotic effect of anticancer drug RRx-001 (2.5 μM) in response to antioxidants EGCG (0–50 μM) (A) and PTS (0–10 μM) (B). C, Effect of GSH pathway inhibitor sulfasalazine on apoptotic rates of cells treated with anticancer drug RRx-001 (2.5 μM) with or without EGCG (0–50 μM). Error bars represent standard deviations of triplicate experiments.</p

Schematic illustration of Nrf2 pathway in cells and Nrf2-FLuc2 construct, the measure of Nrf2 nuclear translocation in cells in response to its activators.

<p>Schematic illustration of Nrf2 pathway in cells and Nrf2-FLuc2 construct, the measure of Nrf2 nuclear translocation in cells in response to its activators.</p

A, Rate of MDA-MB231 tumor xenografts growth in mice treated by vehicle control, cisplatin, EGCG and cisplatin/EGCG combination.

<p>Asterisk (*) denotes statistical significance of data obtained from mice treated with EGCG (Day 14, P = o.046), cisplatin (Day 14, p = 0.036) and combinational treatment (Day 6, p = 0.048; Day 11, p = 0.014; Day 12, p = 0.019 and Day 14, p = 0.016) compared to vehicle control treated mice. Error bars represent SEM of multiple tumors. <b>B, <i>Ex-vivo</i> analysis of MDA-MB231 tumor xenografts</b>. H&E staining, top row, TUNEL staining for apoptotic cells, bottom row. Arrows represent nuclear TUNEL staining.</p

Construction and evaluation of Nrf2-Luciferase (Nrf2-FLuc2) truncation constructs in cells.

<p>A, Schematic illustration of Nrf2-FLuc2 truncation constructs. B, Expression of Nrf2-FLuc2 fusions in HeLa cells, immunoblot and its signal quantification normalized to GAPDH expression signal C, D, E, Activation of Nrf2-FLuc2 fusions in response to Nrf2-activators in cells (Nrf2-100-FLuc2, Nrf2-250-FLuc2, Nrf2-350-FLuc2). The activators used are DMSO, 5 μM PTS, 50 μM EGCG, 20 μM RES, 1 μM THA, 10 μM MMS, 10 μM DEM and 10 μM sodium arsenite. Asterisk (*) represents statistical significance (p<0.05) of signals compared to the ones obtained from DMSO treated cells. Error bars represent standard deviations (B) or SEM (C, D, E) of triplicate experiments.</p