Intronic elements in the Na+/I- symporter gene (NIS) interact with retinoic acid receptors and mediate initiation of transcription

Activity of the sodium/iodide symporter (NIS) in lactating breast is essential for iodide (I–) accumulation in milk. Significant NIS upregulation was also reported in breast cancer, indicating a potential use of radioiodide treatment. All-trans-retinoic acid (tRA) is a potent ligand that enhances NIS expression in a subset of breast cancer cell lines and in experimental breast cancer models. Indirect tRA stimulation of NIS in breast cancer cells is very well documented; however, direct upregulation by tRA-activated nuclear receptors has not been identified yet. Aiming to uncover cis-acting elements directly regulating NIS expression, we screened evolutionary-conserved non-coding genomic sequences for responsiveness to tRA in MCF-7. Here, we report that a potent enhancer in the first intron of NIS mediates direct regulation by tRA-stimulated nuclear receptors. In vitro as well as in vivo DNA–protein interaction assays revealed direct association between retinoic acid receptor-α (RARα) and retinoid-X-receptor (RXR) with this enhancer. Moreover, using chromatin immunoprecipitation (ChIP) we uncovered early events of NIS transcription in response to tRA, which require the interaction of several novel intronic tRA responsive elements. These findings indicate a complex interplay between nuclear receptors, RNA Pol-II and multiple intronic RAREs in NIS gene, and they establish a novel mechanistic model for tRA-induced gene transcription

The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

Background Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential. © 2010 Mumcuoglu et al

Coiled-coil domain containing protein 124 is a novel centrosome and midbody protein that interacts with the Ras-guanine nucleotide exchange factor 1B and is involved in cytokinesis.

Cytokinetic abscission is the cellular process leading to physical separation of two postmitotic sister cells by severing the intercellular bridge. The most noticeable structural component of the intercellular bridge is a transient organelle termed as midbody, localized at a central region marking the site of abscission. Despite its major role in completion of cytokinesis, our understanding of spatiotemporal regulation of midbody assembly is limited. Here, we report the first characterization of coiled-coil domain-containing protein-124 (Ccdc124), a eukaryotic protein conserved from fungi-to-man, which we identified as a novel centrosomal and midbody protein. Knockdown of Ccdc124 in human HeLa cells leads to accumulation of enlarged and multinucleated cells; however, centrosome maturation was not affected. We found that Ccdc124 interacts with the Ras-guanine nucleotide exchange factor 1B (RasGEF1B), establishing a functional link between cytokinesis and activation of localized Rap2 signaling at the midbody. Our data indicate that Ccdc124 is a novel factor operating both for proper progression of late cytokinetic stages in eukaryotes, and for establishment of Rap2 signaling dependent cellular functions proximal to the abscission site

Active endogenous Rap2 relocates to midzone at anaphase, and to midbody during cytokinetic abscission.

<p>(A) HeLa cells were arrested at G2/M phase by sequential double thymidine and nocodazole treatments as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069289#pone-0069289-g002" target="_blank">Figure 2</a>, and they were classified according to phases of mitosis, and cytokinesis. Samples of cells were then stained with anti-Rap2 antibody, and with DAPI to visualize DNA. At anaphase Rap2 was detected at the midzone with staining characteristics reminiscent of endosomes, and at telophase/cytokinesis Rap2 was observed as puncta at the middle of the intercellular bridge, a position typically occupied by midbody associated factors. (<b>B</b>) Following synchronization of cells as above, 80 mins. after nocodazole was washed-off samples were taken with four consecutive intervals of 10 minutes (I, II, III, and IV), the last one (IV) corresponding to ∼120 minutes after the drug was removed, and dynamic positioning of Rap2 at the intercellular bridge in respect to α-tubulin was monitored. A time-dependent relocalization of Rap2 from peripheral flanking regions to the midbody was detected. Intercellular bridge localizations of Rap2 were concluded with observations from a sample of ∼50 cells in which over 75% showed similar positioning patterns. Two sets of representative micrographs were displayed. Bars represent 10 µm.</p

<i>CCDC124</i> mRNA is ubiquitously expressed in human tissues, and it encodes a 32 kDa protein.

<p>(A) Hybridization of part of the coding region of <i>CCDC124</i> to an adult human multiple tissues Northern blot containing 2 µg of polyA-mRNA each lane. A single transcript of ∼1061 nucleotides was detectable in all human tissues analyzed, except the placenta with a second smaller transcript variant. The same blot was rehybridized with probes corresponding to two differentially expressed genes, β-actin and GAPDH, to monitor blotting quality. (<b>B</b>) Specific detection of ectopically expressed Ccdc124 by anti-Ccdc124 antibodies. HEK-293 cells, either non-transfected, or transfected with CMV-promoter controlled Ccdc124 were lysed, protein lysates were separated by SDS-PAGE, and immunoblot was performed either with anti-Ccdc124 antibodies alone, or same antibodies pre-incubated with 100 ng of competing peptide epitope corresponding to N-terminus 24mer peptide of Ccdc124. (<b>C</b>) Expression of Flag-tagged Ccdc124 protein was specifically detected by the anti-Ccdc124 or with anti-Flag antibodies, as indicated. Asterisk (*) indicates C-terminus flag-tag insertion dependent N-terminus cleaved form of Ccdc124. The expression of calnexin was confirmed in all cell lysates as an equal loading control.</p

Depletion of Ccdc124 in HeLa cells by RNAi leads to cytokinesis failure.

<p>(A) HeLa cells were transfected with either esiRNAs or shRNA vectors (Sh1, Sh2, Sh3) targeting Ccdc124, cell lysates were collected at 48 hrs post-transfection, and immunoblotted with antisera to Ccdc124. Where indicated, Ccdc124 expression vector (CMV-Ccdc124) was cotransfected with gene-specific esiRNAs in order to rescue the cellular effect of Ccdc124 depletion. Scrambled control transfections were indicated (Scr). Calnexin expression was monitored as loading control. (<b>B</b>) Immunostainings of endogenous Ccdc124 in cells transfected with Ccdc124-specific esiRNA, or with scrambled control esiRNA were carried out with anti-Ccdc124 Ab. Costainings with γ-tubulin antisera have indicated subcellular positions of MTOCs (<b>C</b>) Cells described and analyzed in (A) were scored for bi- and multinucleation (n = 5± SD). (<b>D</b>) Representative micrographs of Ccdc124 depleted multinuclear or control esiRNA treated normal dividing cells described in (C). Bars represent 10 µm.</p

Rap2 effector proteins translocate to the midbody depending on the signal transduction activity of Rap2.

<p>(A) HeLa cells were transfected with the vector containing GFP-labeled Rap Binding Domain of RalGDS [GFP-RBD(RalGDS)] which interacts only with the GTP-bound active form of Rap2, and cells were monitored at interphase and at cytokinetic abscission following immunostainings involving anti-Rap2 monoclonal Abs and Alexa568-red labeled anti-mouse secondary Abs. Colocalization of GFP-RBD(RalGDS) with Rap2 indicated that at the midbody Rap2 is in its active (Rap2<b>^.</b>GTP) form. (<b>B</b>) HeLa cells were cotransfected with GFP-RBD(RalGDS) either together with HA-Rap2-WT, or with HA-Rap2-S17N (inactive dominant negative form) and their localizations were monitored with anti-HA-epitope Abs. Positioning of GFP-RBD(RalGDS) were assessed by monitoring GFP-signal observed at the midbody. Localization of the GFP-RBD(RalGDS) depended on the presence of active Rap2 in cells. As in (A), at least 50 cells were monitored in each experiment, and representative pictures display Rap2 and GFP-RBD(RalGDS) localizations observed at least ∼90% of cells cotransfected with indicated vectors. Bars represent 10 µm.</p

RasGEF1B and Ccdc124 colocalize at the midbody.

<p>(A–B) Subcellular localizations of RasGEF1B proteins in synchronously dividing HeLa cells were detected with specific anti-RasGEF1B antibodies. Cell divisions were synchronized as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069289#pone-0069289-g002" target="_blank">Figure 2</a>, above. Representative immunofluorescence microscopy images of HeLa cells costained with anti-RasGEF1B, and either anti-γ-tubulin (A) or α-tubulin (B) antibodies illustrating the position of MTOCs and the midbody at cytokinesis. Arrowheads show RasGEF1B detected at the midzone and the midbody. (C) Immunofluorescence signals observed at midbody were significantly decreased when endogenous RasGEF1B were depleted by transfections with specific shRNA vectors (Sh-C or Sh-D, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069289#pone.0069289.s004" target="_blank">Fig. S4</a>) and representative micrographs were shown. (D) HeLa cells transiently transfected with GFP-RasGEF1B were fixed and stained using anti-Ccdc124 Abs. Arrowheads indicate midbody positions of GFP-RasGEF1B, Ccdc124, and their colocalizations at the midbody. Bars represent 10 µm.</p

Rap2 colocalize with Ccdc124 and RasGEF1B at the subcellular level.

<p>Subcellular localizations of endogenous Rap2 and Ccdc124 or RasGEF1B proteins were studied in HeLa cells by immunofluorescence methods. (<b>A</b>) At anaphase, Rap2 was clearly localized at the midzone, while Ccdc124 concentration at the same localization was less pronounced. However, at telophase, both proteins were concentrated at the puncta characterizing the midbody, Rap2 rather surrounding Ccdc124. During cytokinetic abscission, a clear colocalization of both Rap2 and Ccdc124 were observed at the midbody. (<b>B</b>) Similar to panel (A), Rap2 translocation to the midzone has started during anaphase. Two representative images of anaphase cells were shown in the corresponding panel. Both Rap2 and RasGEF1B proteins colocalized at the midbody during cytokinetic abscission. Arrowheads either indicate subcellular localization of Rap2 at anaphase and telophase, or they indicate the colocalization of Rap2 with RasGEF1B/Ccdc124 at the midbody during cytokinesis. (<b>C</b>) HeLa cells were transfected with shRNA vectors (Sh1, Sh2, Sh3) targeting Rap2, then cell lysates were collected at 48 hrs post-transfection, and immunoblotted with anti-Rap2 Ab. Scrambled control transfection was indicated (Scr). Calnexin expression was monitored as loading control. In parallel experiments, similarly treated cells were immunostained with anti-Rap2 and anti-α-tubulin antibodies, followed by scorings for multinucleation (n = 5± SD), as reported on the graph above the immunoblot. (<b>D</b>) Representative micrographs of midbody stage cells depleted in endogenous Rap2 (C). Bars represent 10 µm.</p