HMEC Cell Research Articles

P-303 - Nitrite-dependent nitric oxide formation in humans: role of molybdoenzymes

Nitric oxide radical (NO) is a signaling molecule involved in several physiological and pathological processes in humans and a new nitrate-nitrite-NO pathway has emerged as a physiological alternative to the classic pathway of NO formation from L-arginine under hypoxic conditions. In this presentation, the nitrite-dependent NO formation by the rat and human molybdoenzymes xanthine dehydrogenase, oxidase (XD, XO) and aldehyde oxidase (AO) will be discussed. The ability of XD, XO and AO to trigger the NO formation will be shown to be fine-tuned by the dioxygen availability and greatly amplified under the acidic conditions characteristic of ischemia. The potential in vivo physiological relevance of these enzymes for the NO formation (evaluated using HepG2 and HMEC cell lines subjected to hypoxia) will be shown to be significant, with the XD/XO plus AO accounting to as much as 50% of the nitrite-dependent NO formation under hypoxia. Collectively, our results supported that XD/XO and AO can contribute to NO generation under hypoxia inside a living human cell.

Cytoplasmic PELP1 and ERRgamma protect human mammary epithelial cells from Tam-induced cell death.

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

Nitrite reductase activity of rat and human xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: evaluation of their contribution to NO formation in vivo.

Nitrite is presently considered a NO "storage form" that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme's specificity constant for nitrite increase 8-fold, while the Km(NO2(-)) decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the Ki(O2) values found (24.3-48.8 μM) suggest that in vivo NO formation would be fine-tuned by dioxygen availability. The potential in vivo relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised.

Genome wide mapping of UBF binding-sites in mouse and human cell lines.

The upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. We recently reported in Sanij et al. (2014) [1] that the two isoforms of UBF (UBF1/2) are enriched at Pol II-transcribed genes throughout the mouse and human genomes. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) of UBF1/2, Pol I, Pol II, H3K9me3, H3K4me4, H3K9ac and H4 hyperacetylation, we reported a correlation of UBF1/2 binding with enrichments in Pol II and markers of active chromatin. In addition, we examined a functional role for UBF1/2 in mediating Pol II transcription by performing expression array analysis in control and UBF1/2 depleted NIH3T3 cells. Our data demonstrate that UBF1/2 bind highly active Pol II-transcribed genes and mediate their expression without recruiting Pol I. Furthermore, we reported ChIP-sequencing analysis of UBF1/2 in immortalized human epithelial cells and their isogenically matched transformed counterparts. Here we report the experimental design and the description of the ChIP-sequencing and microarray expression datasets uploaded to NCBI Sequence Research Archive (SRA) and Gene Expression Omnibus (GEO).

Biocompatibility of a self-assembled glycol chitosan nanogel

The research of chitosan-based nanogel for biomedical applications has grown exponentially in the last years; however, its biocompatibility is still insufficiently reported. Hence, the present work provides a thorough study of the biocompatibility of a glycol chitosan (GC) nanogel. The obtained results showed that GC nanogel induced slight decrease on metabolic activity of RAW, 3T3 and HMEC cell cultures, although no effect on cell membrane integrity was verified. The nanogel does not promote cell death by apoptosis and/or necrosis, exception made for the HMEC cell line challenged with the higher GC nanogel concentration. Cell cycle arrest on G1 phase was observed only in the case of RAW cells. Remarkably, the nanogel is poorly internalized by bone marrow derived macrophages and does not trigger the activation of the complement system. GC nanogel blood compatibility was confirmed through haemolysis and whole blood clotting time assays. Overall, the results demonstrated the safety of the use of the GC nanogel as drug delivery system.

ERK5/BMK1 Is a Novel Target of the Tumor Suppressor VHL: Implication in Clear Cell Renal Carcinoma

Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas.

Abstract 4926: Modeling the transcriptome landscape of HER2+ breast cancer

Abstract Motivation: Overexpression of HER2 (the product of the ERBB2 gene) occurs in about 15% of all breast tumors. We have undertaken to use next generation transcriptome sequencing technology to identify genomic features that are unique to HER2+ tumors. Interactome mapping was then used to integrate the genes associated with these features into a transcriptome landscape model, with a view towards identifying nodes of interaction that might be targeted in HER2+ tumors. Methods: We performed 50nt paired-end RNA-sequencing for 24 breast tumors: 8 each HER2+, ER+, triple negatives (TN). In addition to breast adenocarcinomas, we also sequenced 8 early passage non-transformed HMEC cell lines as normal controls. Reads from RNA sequencing were aligned to the genome and exon junctions using TopHat software. Gene counts were summarized and annotations were performed using our in-house programs. Tukey's test was used to obtain genes or transcripts that are specific to HER2 tumor group compared to other tumors/normal. A combination of bioinformatics softwares and algorithms were used to identify SNVs. Results: Only 13527 genes with median gene count greater than 16 reads in at least one of the 4 groups were considered for differential gene expression and splicing analysis. Some 696 genes were differentially expressed in HER2+ tumors compared to ER+, TN tumors and HMECs. We identified 272 alternately spliced transcripts for which the HER2+ tumors exhibited a mean transcript expression ratio significantly different from the means of other tumor groups. We also identified 4735 expressed single nucleotide variants that are uniquely associated with HER2+ tumors compared to other tumors/normal groups. Among these 3579/4735 sequence polymorphisms were not present in the 1000 genome germline sequence database or in the dbSNP132 database of naturally occurring germline polymorphisms. Integration of all the genes obtained from genomic feature analyses (differential expression, alternative splicing, single nucleotide variance) has been carried out to indentify biological processes that are specific to the HER2+ tumor subtype. Key nodes and pathways that are specific to HER2+ tumors will be evaluated for association with clinical outcome in a large series of patients who have received HER2-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4926. doi:1538-7445.AM2012-4926

Nano Barcoding Cell-Based Biosensor Using Fluorophor-Embedded Silica Nanotubes

A simple and reliable drug screening method was developed using peptide hydrogel cell beads coded by quantum dot-embedded silica nanotubes. Very long silica nanotubes were fabricated upon a nanoporous alumina template using sol-gel techniques. The physical shapes of the nanotubes were measured by TEM (Transmission Electron Microscope). Green and red quantum dots embedded in silica nanotubes were applied to peptide hydrogel cell beads as coding materials. This was confirmed by confocal microscopy that examined fluorescence levels and quantum dot shapes. The peptide hydrogel cell beads coded with silica nanotubes were loaded into a PDMS single chamber in order to assess the effect of doxorubicin on HMEC and MCF-7 cells, which was measured in hydrogel cell beads by live and dead cell staining using coding materials. As a result, MCF-7 cancer cells were more affected by doxorubicin than HMEC; however, doxorubicin induced HMEC cell death at a relatively high concentration (> 5 microg/ml).

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

Introduction:Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells.Materials and Methods:In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells.Results:The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC.Conclusions:Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

Cancer‐specific targeting of an adenovirus‐delivered herpes simplex virus thymidine kinase suicide gene using translational control

Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.

SELDI-MS-based expression profiling of ductal invasive and lobular invasive human breast carcinomas

Expression profiling using proteomic techniques has a great potential to identify new biomarkers that might help to better diagnose and treat diseases such as breast cancer, which is one of the leading causes of cancer death in women. Surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS) combines chromatographic separation of peptides and proteins with mass spectrometry and is a fast, user-friendly tool to analyze protein and peptide profiles. SELDI-MS was employed for a comparative analysis of lobular invasive versus ductal invasive breast tumors to find differentially expressed proteins and peptides, and to validate this technique for biomarker identification using complex samples such as tissue. After optimization of sample preparation using HMEC and MCF-7 cell lines, 20 breast tumors were analyzed, and about 550 mass signals corresponding to an estimated 140 native peptides and proteins were detected in each tumor. Only 14% of the mass signals were present in more than six tumors of one subgroup or in more than 12 tumors of both groups showing a great overall heterogeneity of the peptide and protein profiles obtained. Peptide mass signals specific for each of the analyzed groups were identified. In addition, we detected peptides from laser-microdissected ductal invasive and intraductal tumor parts corresponding to peptides present in whole tumors. The low amount of identified peptides and proteins and the observed heterogeneity suggest that SELDI-MS is not well suited for biomarker identification of and profiling experiments on complex samples such as tumor tissue.

Inhibition of radiation-induced invasiveness and endothelial sustain by an MMP-inhibitor through interaction with radiation-enhanced MMP-system and VEGF release

Given the important role of radiotherapy in cancer cure, few recent observations showed that tumor-cell-compartment and stromal-compartment interplay could be involved in treatment failure. We show that cultured B16F10 melanoma cells displayed a radioinduced invasive phenotype and an activated endothelial protective paracrine pathway, and that coadministration of an MMP-inhibitor, metastat, could reverse these enhanced invasive capabilities possibly involving VEGF release. B16F10 melanoma and HMEC endothelial cells underwent IR at 2 Gy, 4 Gy and 8 Gy ± Metastat ± aVEGFMoAb (specific neutralizing VEGF-inhibitor monoclonal antibody), before clonogenic/proliferation assays, zymography, matrigel assays, and condition medium collection for VEGF-ELISA, or used to interact in HMEC matrigel assays. In brief, for testing endothelial cell invasion, HMEC were first diluted in irradiated-melanoma-conditioned-medium (B16CM) and added to the upper chambers before same treatments conditions were applied. 1.No enhancement of radiation response on clonogenic survival and cell proliferation by Metastat: No difference in surviving fractions or in proliferation is observed when non cytotoxic metastat is combined to IR neither for B16, nor for HMEC. 2.Metastat abrogates irradiation-induced melanoma cell invasiveness: IR increases B16-invasion, in a time and dose-dependent manner, up to 3 fold. Metastat brought invasion rate under basal control level. 3.Conditioned-medium from 8Gy irradiated B16 cells (B16CM) promotes HMEC cell invasiveness which can be reversed by metastat: Exposed to conditioned medium issued from irradiated B16F10 cells (8Gy/48h), invasive potentials of the endothelial cells significantly increase (>2fold). Metastat addition again decreased this B16CM-induced invasion by 4 fold. Unlike the B16F10 cells, irradiated HMEC at 4 Gy did not display a significant change in their invasiveness compared to the unirradiated HMEC. 4.Irradiation increases MMP-2 activity in vitro on B16 melanoma cells: MMP activity is induced by irradiation, in a time and dose-dependent-manner on zymography tests: (maximum at 48h/ 8Gy) both for active form and the MMP2-proenzyme. 5.Irradiation decreases MMP-2 activity in vitro on endothelial HMEC cells: MMP activity is inhibited, in a time and dose-dependent-manner (minimum 48h/8Gy) 6.Irradiation increases VEGF-expression in B16F10 and is reversed by metastat: the melanoma cells display a time-dependent increase in VEGF secretion at 8Gy of IR: conditioned medium showed significant elevation (P < 0.05) of VEGF relative expression at 48h (more than 3 fold). Addition of Metastat almost completely abrogated these changes. For endothelial cells, no VEGF could be detected in any condition except, although at low levels, after 48h. 7.Irradiation-induced melanoma-cell-invasiveness and HMEC-B16-conditioned-medium-induced-invasiveness are specifically but partially inhibited by a specific neutralising anti-VEGF monoclonal antibody: a 2 fold inhibition of cell invasion in response to 8Gy-IR induction of B16 invasive potential is reached, and a 2 fold HMEC inhibition of the invasion induced by the B16CM. Nor for the irradiated melanoma cells, neither for the irradiated endothelial cells the degree of inhibition provided by the aVEGFMoAb could reach the one achieved by Metastat. These data, added to our in-vivo results, are suggesting the existence of a radiation-induced tumor-surviving pathway involving at least the MMP proteolytic system, which was impeded by an MMP inhibitor. In addition, these results underscore that this radio-induced paracrine protective pathway, partially involving VEGF release by the tumor cells, is also sustaining endothelial cells and that other MMP-downstream compound should be unfolded.

Halophilols A and B, two new stilbenes from Iris halophila.

A new monomeric stilbene, halophilol A (1), and a new tetrastilbene, halophilol B (2), along with three known oligostilbenes were isolated from the seeds of Iris halophila. Their structures were established on the basis of the spectral data. The oligostilbene skeleton is encountered for the first time in the Iridaceae family. Bioactivity tests showed that 1 had moderate cytotoxicity against KB and HMEC cell lines (IC50 = 17.28 microM, 22.47 microM respectively), while 2 was inactive.

Differential activation of nuclear transcription factor κB, gene expression, and proteins by amifostine's free thiol in human microvascular endothelial and glioma cells

The effects of WR1065 (SH), the free thiol form of amifostine, on nuclear transcription factor kappaB (NFkappaB) activation, manganese superoxide dismutase (MnSOD) gene expression, and secretion of human vascular endothelial cell growth factor (hVEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, P-selectin, and interleukins IL-1alpha, IL-6, and IL-8 were investigated and compared in human microvascular endothelial (HMEC) and human glioma cells. WR1065 was evaluated at 2 concentrations, 4 mmol/L, ie, its most effective cytoprotective dose, and 40 micromol/L, a noncytoprotective but highly effective dose capable of preventing radiation and chemotherapeutic drug-induced mutations in exposed cells. A 30-minute exposure of HMEC and glioma cell lines U87 and U251 to WR1065 at either of the concentrations resulted in a marked activation of NFkappaB as determined by a gel shift assay, with the maximum effect observed between 30 minutes and 1 hour after treatment. Using a supershift assay, WR1065 exposure was observed to affect only the p50-p65 heterodimer, and not the homodimers or heterodimers containing p52 or c-Rel subunits of NFkappaB. WR1065 was also found to enhance MnSOD gene expression in both HMEC and glioma cells. Gene expression was enhanced 1.8-fold over control levels in HMEC over a period ranging from 12 to 24 hours after the time of maximum activation of NFkappaB. In contrast, MnSOD gene expression in U87 cells rose 3.5 times above control levels over this same period. WR1065 had no effect on the levels of adhesion molecules, cytokines, and growth factors secreted by cells exposed for up to 24 hours as measured by enzyme-linked immunosorbent assay.