LAD Cells Research Articles

Long non-coding RNA LINC01116 is activated by EGR1 and facilitates lung adenocarcinoma oncogenicity via targeting miR-744-5p/CDCA4 axis

BackgroundLung adenocarcinoma (LAD) is one of the most frequently diagnosed pathological categories of human lung cancer. Nevertheless, the link between long non-coding RNA (lncRNA) LINC01116 and LAD remains poorly investigated.MethodsQRT-PCR and western blot were applied for quantifying the expression of RNAs and proteins. Both functional experiments assays in vitro and xenografts model in vivo were implemented for analyzing LINC01116 function in LAD while molecular relationship among RNAs was investigated via mechanism experiments.ResultsLINC01116 was expressed at an abnormally high level in LAD, which was induced by transcription activator EGR1. LINC01116 depletion restrained proliferation, migration and invasion, yet facilitated apoptosis of LAD cells. MiR-744-5p could bind to LINC01116. MiR-744-5p inhibitor reversed the inhibitory effects of silencing LINC01116 on LAD malignant behaviors. In addition, cell division cycle-associated protein 4 (CDCA4) shared binding sites with miR-744-5p. Silencing LINC01116 elicited decline in CDCA4 mRNA and protein levels. Moreover, CDCA4 up-regulation could counteract the biological effects of LINC01116 knockdown on LAD cells.ConclusionOur data revealed that LINC01116 promoted malignant behaviors of LAD cells by targeting miR-744-5p/CDCA4 axis, implying the theoretical potential of LINC01116 as a novel target for LAD treatment.

LINC02418 promotes malignant behaviors in lung adenocarcinoma cells by sponging miR-4677-3p to upregulate KNL1 expression

BackgroundLung adenocarcinoma (LAD) is a prevalent type of bronchogenic malignant tumor and one of the most critical factors related to human death. Long noncoding RNAs (lncRNAs) are involved in many complex biological processes and have been emerged as extremely important regulators of various cancers. LINC02418, a novel lncRNA, hasn’t been mentioned in previous studies on cancer development. Therefore, it’s important to define the potential function of LINC02418 in LAD.MethodsGene expression was examined by RT-qPCR or western blot. CCK-8, colony formation, TUNEL, and transwell assays were utilized to study the role of LINC02418 in LAD. The interaction of miR-4677-3p with LINC02418 (or KNL1) was verified through luciferase reporter, RIP and RNA pull-down assays.ResultsHigh expression of LINC02418 was observed in LAD specimens and cells. Downregulation of LINC02418 obstructed the proliferation and motility of LAD cells. Moreover, LINC02418 negatively modulated miR-4677-3p expression and miR-4677-3p overexpression could repress cell proliferation and migration. Moreover, kinetochore scaffold 1 (KNL1) expression was negatively modulated by miR-4677-3p but positively regulated by LINC02418. Furthermore, miR-4677-3p could bind with LINC02418 (or KNL1). Finally, KNL1 overexpression reversed the inhibitory function of LINC02418 deficiency in the malignant behaviors of LAD cells.ConclusionsLINC02418 contributes to the malignancy in LAD via miR-4677-3p/KNL1 signaling, providing a probable therapeutic direction for LAD.

DNMT1/miR-200a/GOLM1 signaling pathway regulates lung adenocarcinoma cells proliferation

ObjectivesLung adenocarcinoma (LAD) comprises about 80% of all diagnosed lung cancers. However, the underlying regulatory mechanism of LAD cell proliferation is largely unclear. The emergence of microRNAs and molecular-targeted therapies adds a new dimension in our efforts to combat this deadly disease. MethodIn this work, the A549 and H1650 human lung cancer cell lines were used in this study. The proliferation was evaluated by the MTT and BrdU assay. The expression level of related proteins was detected by western blot. ResultWe reported GOLM1 was highly expressed in LAD cells and associated with low survival ratio and higher grade malignancy. Knockdown of GOLM1 repressed the LAD cell proliferation. Overexpression of GOLM1 promoted the cell proliferation. Further we found that the level of microRNA-200a (miR-200a) expression was low in LAD cells. miR-200a repress GOLM1 expression by directly targeting its 3′ UTR. Overexpression of miR-200a repressed the cell proliferation and blocked the increase of LAD cell proliferation caused by GOLM1 overexpression. Further, we found that miR-200 was downregulated by DNMT1.Overexpression of DNMT1 blocked the function of miR-200a on repressing proliferation. We then found that knockdown of DNMT1 repressed LAD cell proliferation, which could be rescued by GOLM1 overexpression. ConclusionThis work revealed the critical function of GOLM1/miR-200a/DNMT1 signaling pathway on regulating LAD cell proliferation, and might lay the foundation for further clinical treatment of LAD.

LincRNA FEZF1-AS1 contributes to the proliferation of LAD cells by silencing p57 expression.

LincRNA FEZF1-AS1 has been identified to exert oncogenic functions in various biological processes of tumorigenesis. However, the function of FEZF1-AS1 in lung adenocarcinoma still remains unclear. Our findings revealed that FEZF1-AS1 was increased in lung adenocarcinoma tissues and cell lines and high level of FEZF1-AS1 was associated with poor prognosis of lung adenocarcinoma. Functional experiments and mechanistic investigations demonstrated that knockdown of FEZF1-AS1 significantly repressed proliferation through influencing the distribution of cell cycle. Besides, we also uncovered that FEZF1-AS1 could suppress p57 expression through recruiting EZH2 and LSD1 to the promoter of p57, thus influenced the cell cycle and proliferation. Collectively, our results suggested that FEZF1-AS1 was involved in the progression of lung adenocarcinoma and might be as a potential therapy target for human lung adenocarcinoma.

HMGB1-mediated autophagy promotes docetaxel resistance in human lung adenocarcinoma.

BackgroundDocetaxel resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). High-mobility group box 1 (HMGB1) has been shown to promote autophagy protection in response to antitumor therapy, but the exact molecular mechanism underlying HMGB1-mediated autophagy has not been clearly defined.MethodsLung adenocarcinoma (LAD) cells were transfected with pcDNA3.1-HMGB1 or HMGB1 shRNA, followed by docetaxel treatment. Cell viability and proliferation were tested by MTT assay and colony formation assay, respectively. Annexin V flow cytometric analysis and western blot analysis of activated caspase3 and cleaved PARP were used to evaluate apoptosis, while immunofluorescence microscopy and transmission electron microscopy were applied to assess autophagy activity. The formation of the Beclin-1-PI3K-III complex was examined by immunoprecipitation analysis. NOD/SCID mice were inoculated with docetaxel-resistant SPC-A1/DTX cells transfected with control or HMGB1 shRNA.ResultsHMGB1 translocated from the nucleus to the cytoplasm in LAD cells exposed to docetaxel and acted as a positive regulator of autophagy, which inhibited apoptosis and increased drug resistance. Suppression of HMGB1 restored the sensitivity of LAD cells to docetaxel both in vivo and in vitro. Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.ConclusionsOur results demonstrated that HMGB1-regulated autophagy is a significant contributor to docetaxel resistance in LAD cells. Suppression of HMGB1 or limiting HMGB1 cytosolic translocation diminished autophagic protection in response to docetaxel in LAD cells.

MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

Increasing evidence shows that dysregulation of microRNAs (miRNAs) is involved in malignant transformation. We investigated the clinical significance of miR-650 and its involvement in chemoresistance to docetaxel. Our results showed that the relative expression level of miR-650 was significantly higher in LAD tissues than in corresponding nontumor tissues and high level of miR-650 expression was found to be significantly associated with high incidence of lymph node metastasis, advanced clinical stage and poor prognosis of LAD patients. Univariate and multivariate analyses indicated that high miR-650 expression was an independent prognostic factor for survival. Also, we found that the level of miR-650 in LAD tissues was correlated with the response of patients to docetaxel-based chemotherapy. Silencing of miR-650 could increase the in vitro sensitivity of docetaxel-resistant LAD cells to docetaxel, while upregulation of miR-650 decreased the sensitivity of parental LAD cells to docetaxel both in vitro and in vivo. Additionally, silencing of miR-650 could enhance the caspase-3-dependent apoptosis, which might be correlated with the decreased ratio of Bcl-2/Bax. Further researches suggested that inhibitor of growth 4 (ING4) was a direct target of miR-650. Downregulated or upregulated ING4 expression could partially rescue the effects of miR-650 inhibitor or mimics in docetaxel-resistant or parental LAD cells. Furthermore, we found that ING4 was upregulated in docetaxel-responding LAD tissues, and its expression was inversely correlated with miR-650. Thus, miR-650 is a novel prognostic marker in LAD and its expression is a potential indicator of chemosensitivity to docetaxel-based chemotherapy regimen.

Neuropeptides activate human mast cell degranulation and chemokine production

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcepsilonRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34(+) progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-gamma (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte-macrophage colony-stimulating factor, but not IL-4, interferon-gamma or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases.

IgE mediated release of 3H-5HT from human mast cells (LAD cells)

RATIONALE: The availability of human mast cell lines offers the possibility that antigen-specific IgEs in body fluids may be examined for their ability to activate human mast cells. The confounding factor is that β-hexosaminidase (β-hex) and histamine assays are difficult to employ because of the natural presence of these mediators in serum. We therefore determined whether the LAD human mast cell lines would take up serotonin (3H-5HT) and release this molecule following FcεRI cross linking. METHODS: LAD cells were incubated with 3H-5HT, followed by sensitization with biotinylated IgE (bIgE) and cross linking with streptavidin (SA). 3H-5HT release was compared with β-hex release. In some experiments, cells were incubated with dialyzed serum from ragweed sensitive individuals followed by activation with ragweed antigen E. In other experiments, cells were sensitized with bIgE in the presence of serum known to contain IgE by RAST and checked for release after SA cross linking. RESULTS: Uptake of 3H-5HT by mast cells reached a plateau by 48 hours. Percent IgE mediated 3H-5HT release paralleled % β-hex release. Highest % release for LAD cells was 60%. Release from LAD cells using 1:25-1:50 dilutions of dialyzed ragweed serum followed by the addition of ragweed antigen E reached over 21%. LAD cells could be sensitized in the presence of human sera. CONCLUSIONS: Human mast cells may be used in a 3H-5HT release assay to measure activation with bIgE/SA or human serum from ragweed sensitive individuals. The presence of serum containing IgE does not inhibit bIgE-mediated release of 3H-5HT.

CD52 Is Expressed on Human Mast Cells and Is a Therapeutic Target for the Anti-CD52 Monoclonal Antibody Campath-1H in Waldenstrom's Macroglobulinemia and Mast Cell Disorders.

Campath-1H is a monoclonal antibody used in the treatment of CD52 expressing B-cell malignancies. Recently, we and others have observed that Campath-1H shows unusually high activity in patients with relapsed/refractory Waldenstrom's macroglobulinemia (WM), a B-cell malignancy with excess bone marrow mast cells (BMMC). Importantly, as we have recently shown, BMMC appear to provide direct support for WM tumor cell growth (Tournilhac et al, JCO 2004 22:571S), and are therefore a potential therapeutic target in WM. We therefore examined BMMC from patients with WM and other mast cell disorders for cell surface expression of CD52, and targeting by Campath-1H in preclinical studies. Multicolor flow cytometric analysis demonstrated CD52 expression on BMMC (FceRI+, CD117+) from 13/15 WM; 2/2 systemic mastocytosis (SM) patients; 2/4 healthy donors; as well as on the LAD and HMC MC lines. Moreover, RT-PCR analysis confirmed CD52 expression in sorted BMMC from 6/7 WM, along with 6/6 healthy donors. Importantly, Campath-1H induced high levels of antibody dependent cell mediated cytotoxicity (ADCC) actvity against LAD mast cells using activated NK effector cells. No direct cytotoxicity or antiproliferative activity by Campath-1H on LAD cells was observed. These studies demonstrate that CD52 is widely expressed on human MC and provide support for the use of Campath-1H in the treatment of WM and other systemic mast cell disorders.

Lymphoplasmacytic Cells and Mast Cells Are Targets for Imatinib Mesylate (Gleevec, Glivec) in Waldenstrom's Macroglobulinemia.

Recently, we demonstrated that in Waldenstrom's macroglobulinemia (WM), clonal lymphoplasmacytic cells (LPC) appear to derive growth and survival signals from excess mast cells (MC) present in the bone marrow (BM). (Tournilhac et al, JCO 2004 22:571S). We therefore have sought agents which could target both LPC and MC. One such candidate is imatinib mesylate which has shown activity in certain mast cell disorders likely on the basis of its ability to target the tyrosine kinases, CD117 (Stem Cell Factor Receptor, c-kit), and Platelet Derived Growth Factor Receptor (PDGFa-R). By flow cytometric analysis, we demonstrated CD117 expression on sorted LPC from 17/22 (76.2%) WM patients, above 20% in 13 cases, whose expressionwe confirmed by RT-PCR analysis. Moreover, we also demonstrated expression of PDGFa-R along with its ligand PDGFa in BM LPC from 16/18 (88.9%) and 7/10 (70%) WM patients.. In addition to CD117, we found by RT-PCR analysis a PDGFa expression in LAD and HMC-1 MC lines and in KU the basophilic cell lines KU as well as in sorted BM MC (FcER1+ CD117+) from 3/10 (30%) WM patients. Importantly, co-culture of sorted LPC from WM patients along with 0.5% paraformaldehyde fixed LAD cells or ex vivo expanded (EVE) BM MC induced proliferation of sorted LPC from WM patients, which was inhibited at pharmacologically achievable levels of imatinib mesylate with an IC50 of 0.5–10 μmoles/L. Moreover, imatinib mesylate inhibited the LAD, HMC-1 and KU cell lines as well as EVE cord blood MC or EVE BM MC at an IC50 of 0.01–1 μmoles/L. Lastly, imatinib mesylate also inhibited proliferation of both MC and LPC when unfixed LAD or ex vivo MC were incubated with WM LPC with an IC50 of 1–10 μmoles/L. These studies therefore demonstrate that both LPC and MC are therapeutic targets for imatinib mesylate, and provide the framework for clinical studies evaluating its use in WM.

CR3 (Mac-1, alpha M beta 2, CD11b/CD18) and Fc gamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and tyrosine phosphorylation.

Cooperation among plasma membrane receptors in activating signal transduction cascades is not well understood. For almost 20 years, it has been clear that when a particulate foreign body is opsonized with complement as well as IgG, the efficiency of IgG effector functions is markedly enhanced. However, the molecular mechanisms involved in cooperation between IgG Fc receptors and complement receptors have not been elucidated. In this work, we show that when human neutrophils (PMN) are plated on a surface coated with both anti-CR3 and anti-Fc gamma RIII antibodies, the respiratory burst which occurs is equivalent to that stimulated by anti-Fc gamma RII. The CR3 ligand iC3b is as effective as anti-CR3 for cooperating with anti-Fc gamma RIII in generation of a respiratory burst. The synergy between CR3 and Fc gamma RIII for activating the NADPH oxidase is abolished by Fab of anti-Fc gamma RII. Nonetheless, the observed synergy is not an artifact of unintended Fc gamma RII ligation, since (a) only this combination of antibodies works to generate H2O2; (b) coating plates with either of the antibodies alone cannot activate the respiratory burst at any dose; (c) LAD (CR3 deficient) cells, which are perfectly competent to mount a respiratory burst when Fc gamma RII is engaged, are incapable of activating the respiratory burst when adherent to wells coated with anti-Fc gamma RIII and anti-CR3; (d) direct engagement of Fc gamma RII activates the respiratory burst by a pathway pharmacologically distinguishable from the synergistic respiratory burst. Fc gamma RIII/CR3 synergy is abolished by cytochalasin B and herbimicin, suggesting that both the actin cytoskeleton and tyrosine phosphorylation are necessary for activation of the synergistic respiratory burst. Further analysis shows that CR3 and Fc gamma RIII have distinct roles in activation of this Fc gamma RII-dependent assembly of the NADPH oxidase. Ligation of CR3 is sufficient to lead to Fc gamma RII association with the actin cytoskeleton on the adherent PMN surface. Coligation of Fc gamma RIII is required for tyrosine phosphorylation of Fc gamma RII. These data are consistent with a model in which phosphorylation of Fc gamma RII or a closely associated substrate initiates activation of a signal transduction pathway leading to oxidase assembly. These are the first data to demonstrate a molecular mechanism for synergy between IgG Fc and complement receptors in activation of phagocyte effector functions.

Cytogenetic characterization of a mouse cell line transformed by a bacterial plasmid

A series of mouse lines have been obtained by cotransformation of LTA mouse cells (tkaprt-) with the ptkl plasmid as selectable marker and pR plasmid (M.W 23 Kb). This latter one confers a stable genetic change in the expression of UV-and 4NQOresistance both to bacterial and mammalian cells (Elli et al., 1983; Marcucci et al., 1986). The cytogenetic characterization of pR transformed lines is of value in ascertaining whether the transformation event has in some way modified the genome of the parental line and whether the pR integration occurs at specific chromosomal sites. One of the cotransformed lines, the LA-D line, has been cytogenetically characterized and compared with the untransformed LTA line, with a clonal line derived from LTA and with the LAtkO line, transformed only with the tk gene. The metaphases were obtained by standard methods after cell synchronization with BudR (17hrs) followed by a 7 hrs thymidine pulse. The preparations were stained by 33258 Hoechst + Giemsa or by acridine orange followed by CBG banding. The karyotype of LAD cells (Fig. 1) is characterized by the presence of 15 different nonacrocentric chromosomes [1-15], 23 different acrocentric chromosomes (A1-A23), some of which are represented only once, and 1 subteloncentric chromosome (S1), whose long arm is homologous to one of the acrocentric chromosomes. The analysis of constitutive heterochromatin distribution shows that: 12 out of the 15 nonacrocentric chromosomes have two adjacent pericentromeric regions of heterochromatin (suggesting their origin by Robertsonian fusion); the chr.12 has only one centromeric region of heterochromatin; the chr.13 has three heterochromatic regions intercalated by euchromatin; t h e chr.14 has one C positive centromeric region and an additional one on its short

The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells

The LA-D cells, obtained by cotransformation of LTA mouse cells (tk − aprt −) with pR plasmid and with tk gene as selective marker, are significantly more resistant to UV light and 4-nitroquinoline- N-1-oxide than LTA control cells. In this work, we report that the LA-D cells exhibit different degrees of response to various DNA-damaging agents: wild-type survival to mitomycin, increased sensitivity to bleomycin, cis-diamminedichloroplatinum and N-methyl- N′-nitro- N-nitrosoguadine. The pR plasmid could, therefore, play an important role in the DNA-repair mechanisms that modulate the cytotoxic effect of the DNA-inhibitory agents. The possible interactions between pR plasmid products and the different repair enzymes involved are discussed.

Mutant of Escherichia coli that instantaneously loses the ability to adsorb lambda bacteriophage upon exposure to high temperature.

lad (lambda adsorption), an Escherichia coli mutant that loses the ability to adsorb lambda phage immediately after a shift to high temperature (e.g., 42 C), was isolated. This property for phage adsorption is irreversible and has been observed with phage lambda and 21 but not with phages 434, phi 170, and phi 80. A crude receptor preparation, extracted from lad cells will cholate-ethylenediaminetetraacetic acid by the procedure of Randall-Hazelbauer and Schwartz (1973), inactivated the phage lambda only at low temperature.