In Vitro and In Vivo Evidence for Lack of Endovascular Remodeling by Third Trimester Trophoblasts

The placental–decidual interaction through invading trophoblasts determines whether a physiological transformation of the uterine spiral arteries is established or not. Trophoblast-orchestrated artery remodeling is central to normal placentation. Dysregulated uteroplacental interaction and vascular remodeling are thought to be associated with the molecular events underlying the pathology of late pregnancy anomalies including preeclampsia. Although the exact gestational age at which trophoblast invasion ceases is not known, it remains unclear whether late pregnancy trophoblasts retain the ability to transform the uterine arteries. Here, we have developed a dual cell, in vitro culture system that mimics the vascular remodeling events during normal pregnancy. We demonstrate that first and third trimester trophoblasts respond differentially to interactive signals from endothelial cells when cultured on matrigel. Term primary trophoblasts or immortalized third trimester extravillous TCL1 trophoblasts not only fail to respond to signals from endothelial cells but also inhibit endothelial cell tube formation. In contrast, HTR8 cells, representing a first trimester trophoblast cell line with invasive properties, undergo spontaneous migration and synchronize with the endothelial cells in a capillary network. This disparity in behavior was confirmed in vivo using a matrigel plug assay. Poor expression of VEGF C and VEGF receptors coupled with high E-cadherin expression by term primary trophoblasts and TCL1 cells contributed to their restricted interactive and migratory properties. We further show that the kinase activity of VEGF R2 is essential for proactive crosstalk by HTR8 cells. This unique behavior of first trimester trophoblasts in the presence of endothelial cells offers a potential approach to study cell–cell interactions and to decipher modulatory components in the serum samples from adverse pregnancy outcomes

Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin

The development of cisplatin resistance in human cancers is controlled by multiple genes and leads to therapeutic failure. Hypermethylation of specific gene promoters is a key event in clinical resistance to cisplatin. Although the usage of multiple promoters is frequent in the transcription of human genes, the role of alternative promoters and their regulatory sequences have not yet been investigated in cisplatin resistance genes. In a new approach, we hypothesized that human cancers exploit the specific transcription factor-binding sites (TFBS) and CpG islands (CGIs) located in the alternative promoters of certain genes to acquire platinum drug resistance. To provide a useful resource of regulatory elements associated with cisplatin resistance, we investigated the TFBS and CGIs in 48 alternative promoters of 14 hypermethylated cisplatin resistance genes previously reported. CGIs prone to methylation were identified in 28 alternative promoters of 11 hypermethylated genes. The majority of alternative promoters harboring CGIs (93%) were clustered in one phylogenetic subclass, whereas the ones lacking CGIs were distributed in two unrelated subclasses. Regulatory sequences, initiator and TATA-532 prevailed over TATA-8 and were found in all the promoters. B recognition element (BRE) sequences were present only in alternative promoters harboring CGIs, but CCAAT and TAACC were found in both types of alternative promoters, whereas downstream promoter element sequences were significantly less frequent. Therefore, it was hypothesized that BRE and CGI sequences co-localized in alternative promoters of cisplatin resistance genes may be used to design molecular markers for drug resistance. A more extensive knowledge of alternative promoters and their regulatory elements in clinical resistance to cisplatin is likely to usher novel avenues for sensitizing human cancers to treatment.Cancer Prevention and Research Institute of Texas (CPRIT; no. RP130266

Transthyretin Is Dysregulated in Preeclampsia, and Its Native Form Prevents the Onset of Disease in a Preclinical Mouse Model

Preeclampsia is a major pregnancy complication with potential short- and long-term consequences for both mother and fetus. Understanding its pathogenesis and causative biomarkers is likely to yield insights for prediction and treatment. Herein, we provide evidence that transthyretin, a transporter of thyroxine and retinol, is aggregated in preeclampsia and is present at reduced levels in sera of preeclamptic women, as detected by proteomic screen. We demonstrate that transthyretin aggregates form deposits in preeclampsia placental tissue and cause apoptosis. By using in vitro approaches and a humanized mouse model, we provide evidence for a causal link between dysregulated transthyretin and preeclampsia. Native transthyretin inhibits all preeclampsia-like features in the humanized mouse model, including new-onset proteinuria, increased blood pressure, glomerular endotheliosis, and production of anti-angiogenic factors. Our findings suggest that a focus on transthyretin structure and function is a novel strategy to understand and combat preeclampsia

Isothiocyanate NB7M causes selective cytotoxicity, pro-apoptotic signalling and cell-cycle regression in ovarian cancer cells

The present report identifies indole-3-ethyl isothiocyanate NB7M as a potent cytotoxic agent with selective activity against cell lines derived from various tumour types. Ovarian cancer cell lines showed sensitivity to NB7M (60–70% cytotoxicity at 2.5 μM), in contrast to control cells (TCL-1 and HTR-8; IC50 ∼15 μM). In a screen performed by the National Cancer Institute (NCI) (NCI60 cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC50 between 0.1 and 10 μM depending on the cell line studied. Using SKOV-3 ovarian cancer cells as a model, mechanisms of cytotoxicity were analysed. NB7M caused hallmarks of apoptosis such as PARP-1 deactivation, chromatin condensation, DNA nicks, activation of caspases-9, -8, -3, loss of mitochondrial transmembrane depolarisation potential and upregulation of pro-apoptotic mitogen activated protein kinases (p38, SAP/JNK). NB7M downregulated phosphorylation of prosurvival kinases (PI-3K, AKT, IKKα), transcription factor NF-κB, and expression of DNA-Pk and AXL receptor tyrosine kinase. Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression. The present report suggests that NB7M is a selective cytotoxic agent in vitro for cell lines derived from ovarian and certain other tumours. In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice

<div><p>Objective</p><p>Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring.</p><p>Methods</p><p>Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.</p><p>Results</p><p>Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10^-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10^-2). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR).</p><p>Conclusions</p><p>A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the <i>in vivo</i> pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.</p></div

Enzymic Conversion of Agmatine to Putrescine inL athyrms satiuus seedlings - Purification and properties of a multifunctional enzyme (putrescine synthase)

The participation of a multifunctional enzy(am sein - gle polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate has been purified to homogeneity anhda s M, = 55,000

Cancer-Specific Biomarker hNQO1-Activatable Fluorescent Probe for Imaging Cancer Cells In Vitro and In Vivo

Human NAD(P)H quinone oxidoreductase-1 (hNQO1) is an important cancer-related biomarker, which shows significant overexpression in malignant cells. Developing an effective method for detecting NQO1 activity with high sensitivity and selectivity in tumors holds a great potential for cancer diagnosis, treatment, and management. In the present study, we report a new dicyanoisophorone (DCP) based fluorescent probe (NQ-DCP) capable of monitoring hNQO1 activity in vitro and in vivo in both ratiometric and turn-on model. NQ-DCP was prepared by conjugating dicyanoisophorone fluoroprobe with hNQO1 activatable quinone propionic acid (QPA), which remain non-fluorescent until activation by tumor-specific hNQO1. NQ-DCP featured a large Stokes shift (145 nm), excellent biocompatibility, cell permeability, and selectivity towards hNQO1 allowed to differentiate cancer cells from healthy cells. We have successfully employed NQ-DCP to monitor non-invasive endogenous hNQO1 activity in brain tumor cells in vitro and in xenografted tumors developed in nude mice

S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process