Paracrine factors of human mesenchymal stem cells increase wound closure and reduce reactive oxygen species production in a traumatic brain injury in vitro model.

Traumatic brain injury (TBI) consists of a primary and a secondary insult characterized by a biochemical cascade that plays a crucial role in cell death in the brain. Despite the major improvements in the acute care of head injury victims, no effective strategies exist for preventing the secondary injury cascade. This lack of success might be due to that most treatments are aimed at targeting neuronal population, even if studies show that astrocytes play a key role after a brain damage. In this work, we propose a new model of in vitro traumatic brain-like injury and use paracrine factors released by human mesenchymal stem cells (hMSCs) as a neuroprotective strategy. Our results demonstrate that hMSC-conditioned medium increased wound closure and proliferation at 12 h and reduced superoxide production to control conditions. This was accompanied by changes in cell morphology and polarity index, as both parameters reflect the ability of cells to migrate toward the wound. These findings indicate that hMSC is an important regulator of oxidative stress production, enhances cells migration, and shall be considered as a useful neuroprotective approach for brain recovery following injury.

HDAC inhibition by SNDX-275 (Entinostat) restores expression of silenced leukemia-associated transcription factors Nur77 and Nor1 and of key pro-apoptotic proteins in AML.

Nur77 and Nor1 are highly conserved orphan nuclear receptors. We have recently reported that nur77(-/-)nor1(-/-) mice rapidly develop acute myeloid leukemia (AML) and that Nur77 and Nor1 transcripts were universally downregulated in human AML blasts. These findings indicate that Nur77 and Nor1 function as leukemia suppressors. We further demonstrated silencing of Nur77 and Nor1 in leukemia stem cells (LSCs). We here report that inhibition of histone deacetylase (HDAC) using the specific class I HDAC inhibitor SNDX-275 restored the expression of Nur77/Nor1 and induced expression of activator protein 1 transcription factors c-Jun and JunB, and of death receptor TRAIL, in AML cells and in CD34(+)/38(-) AML LSCs. Importantly, SNDX-275 induced extensive apoptosis in AML cells, which could be suppressed by silencing nur77 and nor1. In addition, pro-apoptotic proteins Bim and Noxa were transcriptionally upregulated by SNDX-275 in AML cells and in LSCs. Our present work is the first report of a novel mechanism of HDAC inhibitor-induced apoptosis in AML that involves restoration of the silenced nuclear receptors Nur77 and Nor1, activation of activator protein 1 transcription factors, a death receptor and pro-apoptotic proteins.

The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction by Mdm2 inhibition but enhances p53-mediated mitochondrial apoptosis in p53 wild-type AML.

Activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway and inactivation of wild-type p53 by murine double minute 2 homologue (Mdm2) overexpression are frequent molecular events in acute myeloid leukemia (AML). We investigated the interaction of PI3K/Akt/mTOR and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 and the Mdm2 inhibitor Nutlin-3. We found that PI-103, which itself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformational change and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3 combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability. We suggest that PI-103 actively enhances downstream p53 signaling and that a combination strategy aimed at inhibiting PI3K/Akt/mTOR signaling and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

Transcriptional activation of rat creatine kinase B by 17beta-estradiol in MCF-7 cells involves an estrogen responsive element and GC-rich sites.

The rat creatine kinase B (CKB) gene is induced by estrogen in the uterus, and constructs containing rat CKB gene promoter inserts are highly estrogen-responsive in cell culture. Analysis of the upstream -568 to -523 region of the promoter in HeLa cells has identified an imperfect palindromic estrogen response element (ERE) that is required for hormone inducibility. Analysis of the CKB gene promoter in MCF-7 breast cancer cells confirmed that pCKB7 (containing the -568 to -523 promoter insert) was estrogen-responsive in transient transfection studies. However, mutation and deletion analysis of this region of the promoter showed that two GC-rich sites and the concensus ERE were functional cis-elements that bound estrogen receptor alpha (ERalpha)/Sp1 and ERalpha proteins, respectively. The role of these elements was confirmed in gel mobility shift and chromatin immunoprecipitation assays and transfection studies in MDA-MB-231 and Schneider Drosophila SL-2 cells. These results show that transcriptional activation of CKB by estrogen is dependent, in part, on ERalpha/Sp1 action which is cell context-dependent.

Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements.

Cyclin D1 gene expression is induced by 17beta-estradiol (E2) in human breast cancer cells and is important for progression of cells through the G(1) phase of the cell cycle. The mechanism of activation of cyclin D1 is mitogen- and cell context-dependent, and this study describes the role of multiple promoter elements required for induction of cyclin D1 by E2 in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Transcriptional activation of cyclin D1 by E2 was dependent, in part, on a proximal cAMP-response element at -66, and this was linked to induction of protein kinase A-dependent pathways. These results contrasted to a recent report showing that induction of cyclin D1 by E2 in ER-positive MCF-7 and HeLa cells was due to up-regulation of c-jun and subsequent interaction of c-Jun-ATF-2 with the CRE. Moreover, further examination of the proximal region of the cyclin D1 promoter showed that three GC-rich Sp1-binding sites at -143 to -110 were also E2-responsive, and interaction of ERalpha and Sp1 proteins at these sites was confirmed by electromobility shift and chromatin immunoprecipitation assays. Thus, induction of cyclin D1 by E2 in ZR-75 cells is regulated through nuclear ERalpha/Sp1 and epigenetic protein kinase A activation pathways, and our results suggest that this mechanism may be cell context-dependent even among ER-positive breast cancer cell lines.

Transcriptional activation of deoxyribonucleic acid polymerase alpha gene expression in MCF-7 cells by 17 beta-estradiol.

Treatment of MCF-7 human breast cancer cells with 17beta-estradiol (E(2)) results in increased DNA synthesis and cell proliferation and enhanced enzyme activities associated with purine/pyrimidine biosynthesis. The mechanism of enhanced DNA polymerase alpha activity was investigated by analysis of the promoter region of this gene. E(2) induced luciferase (reporter gene) activity in MCF-7 cells transfected with pDNAP1, pDNAP2, and pDNAP3 containing -1515 to +45, -248 to +45 and -116 to +45 inserts from the DNA polymerase alpha gene promoter, whereas no induction was observed with pDNAP4 (-65 to +45 insert). The induction response was dependent on cotransfection with estrogen receptor alpha (ER(alpha)), and transactivation was also observed with a mutant ER(alpha) that did not express the DNA-binding domain. Subsequent functional, DNA binding, and DNA footprinting studies showed that a GC-rich region at -106 to -100 was required for E(2)-mediated transactivation, and Sp1 protein, but not ER(alpha), bound this sequence. Transcriptional activation of DNA polymerase alpha by E(2) is associated with ER(alpha)/Sp1 action at a proximal GC-rich promoter sequence, and this gene is among a growing list of E(2)-responsive genes that are induced via ER(alpha)/Sp1 protein interactions that do not require direct binding of the hormone receptor to DNA.

Transcriptional activation of cathepsin D gene expression by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition.

17beta-estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells and this response is inhibited by aryl hydrocarbon receptor (AhR) agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Analysis of the cathepsin D gene promoter initially identified a pentanucleotide GCGTG core dioxin responsive element (DRE) that blocked E2 action by inhibiting formation of a transcriptionally active estrogen receptor (ER)-Sp1 complex. A second functional downstream inhibitory DRE (iDRE2) (-130 to -126) has now been identified in the cathepsin D gene promoter and inhibition of E2-induced transactivation involves inhibitory AhR crosstalk with the E2-responsive adenovirus major late promoter element (MLPE) at -124 to -104 in the cathepsin D gene promoter. The MLPE site primarily binds USF1/USF2 and ERalpha, and gel mobility shift and DNA footprinting assays show that the AhR complex decreases binding of these transcription factors to the MLPE.

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site.

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.

Transcriptional activation of thymidylate synthase by 17beta-estradiol in MCF-7 human breast cancer cells.

Thymidylate synthase (TS) catalyzes methylation of deoxyuridine phosphate to give deoxythymidine phosphate, and 17beta-estradiol (E2) induces TS gene expression in MCF-7 human breast cancer cells. Analysis of the TS gene promoter showed that E2-responsiveness required the -229 to -140 promoter region containing a G-rich sequence and CACCC box. Subsequent mutational analysis of this region indicated that only the G-rich motif (-150 to -142) was required for E2 action. Results of gel mobility shift and in vitro DNA footprinting assays showed that both estrogen receptor alpha (ERalpha) and Sp1 proteins were required for hormone-induced trans-activation that involved ERalpha/Sp1 binding to the G-rich site in which only Sp1 protein bound DNA. Both proteins also interacted in Drosophila cells in functional assays, confirming the transcriptional activation of TS-involved ERalpha/Sp1, and this adds to the increasing number of genes that are activated through this pathway in breast cancer cells.

Inhibition of vascular endothelial growth factor expression in HEC1A endometrial cancer cells through interactions of estrogen receptor alpha and Sp3 proteins.

Treatment of HEC1A endometrial cancer cells with 10 nm 17beta-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from -2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor alpha (ERalpha) and reversed by the anti-estrogen ICI 182, 780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between -66 to -47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERalpha proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERalpha proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERalpha-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.

Mechanisms of inhibitory aryl hydrocarbon receptor-estrogen receptor crosstalk in human breast cancer cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that forms a functional heterodimeric complex with the AhR nuclear translocator (Arnt) protein. The environmental toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a high affinity ligand for the AhR and has been extensively used to investigate AhR-mediated biochemical and toxic responses. TCDD modulates several endocrine pathways including inhibition of 17beta-estradiol-induced responses in the immature and ovariectomized rodent uterus and mammary gland and in human breast cancer cell lines. TCDD inhibits formation and growth of mammary tumors in carcinogen-induced rodent models and relatively nontoxic selective AhR modulators (SAhRMs) are being developed for treatment of breast cancer. The mechanisms of inhibitory AhR-estrogen receptor (ER) crosstalk have been investigated in MCF-7 breast cancer cells by analysis of promoter regions of genes induced by E2 and inhibited by TCDD. AhR-mediated inhibition of E2-induced cathepsin D, pS2, c-fos, and heat shock protein 27 gene expression involves direct interaction of the AhR complex with inhibitory pentanucleotide (GCGTG) dioxin responsive elements (iDREs) resulting in disruption of interactions between proteins binding DNA elements required for ER action and the basal transcription machinery. Mechanisms of inhibitory AhR-ER crosstalk indicate that functional iDREs are required for inhibition of some genes; however, results indicate that other interaction pathways are important including AhR-mediated proteasome-dependent degradation of the ER.

Mechanisms of transcriptional activation of bcl-2 gene expression by 17beta-estradiol in breast cancer cells.

bcl-2 gene expression is induced by 17beta-estradiol (E2) in T47D and MCF-7 human breast cancer cells, and the mechanism of E2 responsiveness was further investigated by analysis of the bcl-2 gene promoter. The -1602 to -1534 distal region (bcl-2j) of the promoter was E2-responsive; however, in gel mobility shift assays, the estrogen receptor alpha (ER(alpha)) did not bind [(32)P]bcl-2j, whereas Sp1 protein formed a retarded band complex. Further analysis demonstrated that the upstream region (-1603 to -1579) of the bcl-2 gene promoter contained two GC/GA-rich sites at -1601 (5'-GGGCTGG-3') and -1588 (3'-GGAGGG-5') that bound Sp1 protein. Subsequent studies confirmed that transactivation by E2 was dependent on ER(alpha)/Sp1 interactions with both GC-rich sites, and this was confirmed by in vitro footprinting. In contrast, a 21-base pair E2-responsive downstream region (-1578 to -1534) did not bind Sp1 or ER(alpha) protein; however, analysis of a complex binding pattern with nuclear extracts showed that ATF-1 and CREB-1 bound to this motif. These data coupled with results of transient transfection studies demonstrated that transcriptional activation by E2 of the -1578 to -1534 region of the bcl-2 gene promoter was dependent on induction of cAMP and subsequent activation through a cAMP response element. Thus, hormone regulation of bcl-2 gene expression in breast cancer cells involves multiple enhancer elements and E2-mediated transactivation does not require direct binding of the estrogen receptor with promoter DNA.

Transcriptional activation of c-fos protooncogene by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition.

17Beta-estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human breast cancer cells, and maximal induction was observed within 1 h after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced response within 2 h. The molecular mechanism of this response was further investigated using pFC2-CAT, a construct containing a -1400 to +41 sequence from the human c-fos protooncogene linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected with pFC2-CAT, 10 nM E2 induced an 8.5-fold increase of CAT activity, and cotreatment with 10 nM TCDD decreased this response by more than 45%. Alpha-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the inhibitory effects of TCDD; moreover, the inhibitory response was not observed in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was required for estrogen receptor cross-talk. The E2-responsive sequence (-1220 to -1155) in the c-fos gene promoter contains two putative core pentanucleotide dioxin-responsive elements (DREs) at -1206 to -1202 and -1163 to -1159. In transient transfection assays using wild-type and core DRE mutant constructs, the downstream core DRE (at -1163 to -1159) was identified as a functional inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift, and in vitro DNA footprinting assays showed that the AhR complex interacted with the core DRE that also overlapped the E2-responsive GC-rich site (-1168 to -1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be due to quenching or masking at the Sp1-binding site.

[Ogilvie syndrome or acute colonic pseudo-obstruction. Current concepts in diagnosis and treatment]. / Síndrome de Ogilvie o pseudo-obstrucción colónica aguda. Conceptos actuales en diagnóstico y tratamiento.

Ogilvie's syndrome or acute colonic pseudo-obstruction is a motility disorder characterized by acute and progressive colonic distension. This syndrome occurs in hospitalized patients with several medical or surgical diseases with an unclear pathophysiology. Diagnosis is established by the clinical history, physical examination and radiological findings on plain abdominal X-ray. Treatment includes: 1. general measures to reduce colonic distension, 2. drugs that improve colon motility, 3. endoscopic colonic decompression and 4. surgery. Age, associated diseases, elapsed time and diameter of cecal dilatation, presence of necrosis and perforation are the main prognostic factors. Recurrence after medical treatment is 20-50 percent; intrahospital mortality is 30 percent. A practical algorithm for the management of these patients is proposed.