Growth Of NIH3T3 Cells Research Articles

Properties of chitin and chitosan extracted from silkworm pupae and egg shells

Chitin and chitosan from silkworm pupae and egg shells show distinct properties with excellent antimicrobial properties and cytocompatiblity. Spent silkworm pupae and hatched egg shells are discarded as waste but contain valuable carbohydrates, proteins and lipids. Chitosan has excellent antimicrobial properties and is widely used for food, medical and biotechnological applications. In this paper, we report the properties of chitin and chitosan from silkworm pupae and egg shells in comparison to commercially available chitosan. Defatted and deproteinated pupae and shells were demineralized and later deacetylated to form chitosan. Thermal behavior, physical structure, antimicrobial activity and ability to support the attachment and growth of NIH3T3 cells were studied. Chitin and chitosan from both pupae and shells had similar structure and composition. Crystallinity of the pupae chitosan was 48% compared to 38% for egg shell chitosan. Silkworm chitosan showed considerably higher antibacterial and antifungal activity compared to standard. Cells were viable in the presence of pupae and egg shell chitosan in all the concentrations tested. Based on these observations, it can be inferred that silkworm pupae and shells provide a renewable and sustainable source for chitosan with properties suitable for food and medical applications.

Characterization of TNNC1 as a Novel Tumor Suppressor of Lung Adenocarcinoma.

In this study, we describe a novel function of TNNC1 (Troponin C1, Slow Skeletal and Cardiac Type), a component of actin-bound troponin, as a tumor suppressor of lung adenocarcinoma (LUAD). First, the expression of TNNC1 was strongly down-regulated in cancer tissues compared to matched normal lung tissues, and down-regulation of TNNC1 was shown to be strongly correlated with increased mortality among LUAD patients. Interestingly, TNNC1 expression was enhanced by suppression of KRAS, and ectopic expression of TNNC1 in turn inhibited KRAS G12D-mediated anchorage independent growth of NIH3T3 cells. Consistently, activation of KRAS pathway in LUAD patients was shown to be strongly correlated with down-regulation of TNNC1. In addition, ectopic expression of TNNC1 inhibited colony formation of multiple LUAD cell lines and induced DNA damage, cell cycle arrest and ultimately apoptosis. We further examined potential correlations between expression levels of TNNC1 and various clinical parameters and found that low-level expression is significantly associated with invasiveness of the tumor. Indeed, RNA interference-mediated down-regulation of TNNC1 led to significant enhancement of invasiveness in vitro. Collectively, our data indicate that TNNC1 has a novel function as a tumor suppressor and is targeted for down-regulation by KRAS pathway during the carcinogenesis of LUAD.

Role of AML1-ETO Related Fusion Circular RNA in the Growth of Leukemia Cells

Introduction: The pathogenesis of acute myeloid leukemia (AML) is always associated with chromosomal translocation, such as t(8;21)(q21;q22), which results in the formation of AML1-ETOfusion gene. The AML1-ETO fusion protein leads to hematopoietic differentiation blocked and leukemogenesis. How genomic abnormities such as chromosomal translocation influence the production and biological function of noncoding RNAs is relatively less studied. With the application of high-throughput sequencing and bioinformatics, it is increasing clear that the circular RNAs (circRNA) played important role in multiple biological processes. However, circRNA is rarely studied in hematopoietic malignancies. Here we assessed the roles of AML1-ETO related fusion circRNA (F-CircAE) in AML1-ETO leukemia.Methods: Total RNA was extracted from leukemia cell lines Kasumi-1 and SKNO-1, then treated with RNaseR or not. PCR assay with divergent primers was performed to identify F-CircAEs. The F-CircAE retroviral expression vector was constructed using Gibson Assembly Cloning Kit. NIH3T3 cells were transduced with retrovirus, the proliferation and foci formation were detected by crystal violet staining. F-CircAE-expressingNIH3T3 cells were injected subcutaneously into nude mice. Tumor growth was monitored at indicated time. To knock down the expression of F-CircAEs, the short hairpin double-stranded oligo targeting the back-splice junction of the F-CircAEs was inserted into the pLKO.1 lentiviral vector. Kasumi-1 cells were transduced with lentivirus, then the proliferation and cell cycle distribution of Kasumi-1 cells was measured by MTS assay and PI staining. Western blot was used to detect the cell cycle associated protein expression. RNA pulldown assay was performed with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the instructions. The bound proteins in the pulldown assay were analyzed by spectrum analysis. RNA-seq were performed using the Illumina platform. Sequencing reads were analyzed by a standard RNA-Seq pipeline. Gene Set Enrichment Analysis (GSEA) were used to analyze the pathway enrichment.Results: Several F-CircAEswere identified in AML1-ETO leukemia cell lines (Kasumi-1, SKNO-1) and t(8;21) patients' bone marrow mononuclear cells (BMMCs), but not in K562 cells and donors' BMMCs (Figure 1). To further validate F-CircAEs, RNaseR treatment was conducted and amplification products of circular RNAs were still detectable in AML1-ETO positive cells (Figure 1). Subsequently, Sanger sequencing of these amplification circular products revealed the head to tail sites of F-CircAE.To examine the function of F-CircAE, overexprseeion of F-CircAE in NIH3T3 cells resulted in increased proliferation compared with mutated F-CircAE (F-CircAE-mut) overexprssion or control group (Figure 2). Meanwhile, more foci were formed in the overexpressed F-CircAE group. In vivo,F-CircAE overexpression also promoted the growth of NIH3T3 cells in the injection site of nude mice. Furthermore, silencing of two F-CircAEs reduced the growth rates of Kasumi-1 cells (Figure 3) and induced an increase in G1 phase and a decrease in S and G2/M phase compared with scramble group. The protein levels of CDK2 and CyclinD1, which were the cell cycle regulatory components at G1 boundary, were reduced in F-CircAE knockdown groups compared with scramble group. To explore the mechanism by which F-CircAE promoted leukemia cells proliferation, RNA-pulldown assay was performed and more than 70 proteins were found to bind to F-CircAE. Based on the sequencing reads of RNA-seq, GSEA revealed several pathways were enriched following F-CircAE knockdown in Kasumi-1 cells.Conclusions: F-CircAEs were existed in t (8;21) leukemia cell lines and primary AML leukemia patients' BMMCs. F-CircAE could bind to proteins involved in the growth of leukemia cells. Discovery of F-CircAEs in AML1-ETO leukemia could make an immense progress in understanding their pathogenic mechanism, indicating new marker for diagnosis and therapy target. DisclosuresNo relevant conflicts of interest to declare.

A Novel Approach via Surface Modification of Degradable Polymers With Adhesive DOPA-IGF-1 for Neural Tissue Engineering

The highly damaging state of spinal cord injuries has provided much inspiration for the design of surface modification of the implants that can promote nerve regeneration and functional reconstruction. DOPA-IGF-1, a new recombinant protein designed in our previous study, exhibited strong binding affinity to titanium and significantly enhanced the growth of NIH3T3 cells on the surface of titanium with the same biological activity as IGF-1. In this article, surface modification of poly(lactide-co-glycolide) (PLGA) films with recombinant DOPA-IGF-1 was performed to promote the paracrine activity of human umbilical cord mesenchymal stem cells (hUCMSCs) by secreting neurotrophic factors. DOPA-IGF-1 exhibited the strongest binding ability to PLGA films than commercial IGF-1 and nonhydroxylated YKYKY-IGF-1. In vitro cultures of hUCMSCs on the modified PLGA films showed that DOPA-IGF-1@PLGA substrates significantly improved the proliferation, adhesion, and neurotrophic factors secretion of hUCMSCs, especially for nerve growth factor, as confirmed by qRT-PCR and western blot analysis. Subsequently, the acquired neurotrophic factors secreted by the hUCMSCs cultured on the DOPA-IGF-1@PLGA films obviously enhanced neurite outgrowth of PC12 cells. Taken together, PLGA substrates with DOPA-IGF-1 immobilization is a promising platform for neural tissue engineering via neurotrophic factors secretion from MSCs and should be further tested in vivo.

Differential Effects of Estrogen Receptor β Isoforms on Glioblastoma Progression

The estrogen receptor β (ERβ) functions as a tumor suppressor in glioblastoma (GBM) cells. However, the in vivo significance of endogenous ERβ and the roles of its isoforms in GBM are incompletely understood. Using ERβ isoform-specific PCR screening, we found that GBM cells predominantly express ERβ1 and ERβ5, along with low levels of ERβ2 and ERβ4. We observed greater ERβ5 expression in higher grades of glioma than in lower grades. In CRISPR-based ERβ knockout (KO) cells and ERβ KO cells uniquely expressing ERβ1 or ERβ5 only, ERβ1 significantly reduced proliferation. Compared with parental GBM cells, ERβ KO cells exhibited high migratory and invasive potentials, and reexpression of ERβ1 resulted in the reduction of this phenotype. Interestingly, ERβ5 expression increased foci formation and anchorage-independent growth of NIH3T3 cells and increased motile structure formation, including filopodia and ruffles in GBM cells. Only ERβ1-expressing tumors resulted in longer mouse survival. RNA-Seq analysis revealed unique pathways modulated by ERβ1 and ERβ5. Compared with ERβ KO cells, ERβ1 cells exhibited lower activation of mTOR signaling molecules, including p-mTOR, p-S6K, and p-S6, and ERβ5-expressing cells had enhanced mTOR downstream signaling. Unique proteins including several that function as regulators of mTOR, immunomodulatory, and apoptosis pathways bound to ERβ1 and ERβ5 isoforms. Our work confirms the tumor-suppressive potential of ERβ1 and reveals the acquired oncogenic ability of ERβ5 in GBM cells. ERβ isoform status and their unique interactions with oncogenic pathways may have important implications in GBM progression.Significance: These findings suggest that only ERβ isoform 1 has tumor suppressor function in GBM and that ERβ isoform switching contributes to GBM progression. Cancer Res; 78(12); 3176-89. ©2018 AACR.

In vitro release of theophylline and cytotoxicity of two new metal–drug complexes

Two new metal–drug complexes ([Mg(TPL)2(H2O)4]) (1) and ([Ca(TPL)2(H2O)4]·(TPL)2 (2) (TPL = theophylline) were synthesized under hydrothermal conditions. Their structures were characterized by single crystal X-ray diffraction analysis, infrared spectrophotometry (IR) analysis, elemental analysis, X-ray powder diffraction (XPRD) analysis and thermogravimetric analysis (TGA). The TPL’s release rates of two complexes tablets were both obviously slower than the free TPL tablets under pure water or phosphate-buffered solution due to the coordination bonds and hydrogen bonds existence in the complexes. Compared with the cytotoxicity of TPL, two complexes showed better cytocompatibility toward NIH-3 T3 cells using an MTT assay, attributing to magnesium and calcium ions that they may have important functions for promoting the growth of NIH-3 T3 cells.

SY-10-5 - Fibroblast growth factor receptors as a potential therapeutic target in patients with intrahepatic cholangiocarcinoma

Gemcitabine + cisplatin combination therapy has become a standard regimen for advanced biliary tract cancer, based on the positive results of phase III clinical trials. However, the overall treatment outcomes for patients remain dismal. A better understanding of the molecular basis of cancer would help develop targeted therapeutic agents against druggable genetic aberrations identified in cancer genomes. For example, the EML4-ALK fusion gene has recently been identified in a small subset of non-small cell lung cancer patients, and tyrosine kinase inhibitors that target ALK are particularly effective. Recently, a FGFR2 fusion gene in patients with intrahepatic cholangiocarcinoma (ICC) was identified by comprehensive genomic analysis, with a positive rate of approximately 6%–14%. In in vivo and in vitro settings, the oncogenic activity of FGFR2 fusion proteins was identified, and FGFR kinase inhibitors were found to effectively suppress anchorage-independent growth of NIH3T3 cells expressing FGFR2 fusions. Under such conditions, several FGFR kinase inhibitors are currently under early stage clinical investigation. Therefore, FGFR2 fusion gene is one of the promising therapeutic targets in patients with ICC. We have conducted the multicenter screening program in biliary tract cancer. Advanced biliary tract cancer patients were enrolled and the rearrangement of the FGFR2 gene locus verified by fluorescence in situ hybridization with break-apart probes. In addition, the nationwide cancer genome screening project using next generation sequencing technology (SCRUM-Japan) is ongoing in Japan. The target diseases in this project are cancer of lung and digestive system including ICCs. Such approaches to identify driver mutations in cancer patients will enable more efficient drug development and precision medicine.

New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells.

BackgroundEGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2–7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation.MethodsWe constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis.ResultsIn the course of screening 30,000 substances, a reagent, “Ertredin” was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis.ConclusionWe developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2521-9) contains supplementary material, which is available to authorized users.

A Bioorthogonal Approach for the Preparation of a Titanium‐Binding Insulin‐like Growth‐Factor‐1 Derivative by Using Tyrosinase

AbstractThe generation of metal surfaces with biological properties, such as cell‐growth‐enhancing and differentiation‐inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel‐inspired bioorthogonal approach to design a 3,4‐hydroxyphenalyalanine‐containing recombinant insulin‐like growth‐factor‐1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.

A Bioorthogonal Approach for the Preparation of a Titanium-Binding Insulin-like Growth-Factor-1 Derivative by Using Tyrosinase.

The generation of metal surfaces with biological properties, such as cell-growth-enhancing and differentiation-inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel-inspired bioorthogonal approach to design a 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth-factor-1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.

Proliferative constituents from Selaginella moellendorffii Hieron

Two new compounds, 7,3⿲-di-O-methylquercetin-4⿲-O-[β-galactosyl-(1⿿3)-β-glucoypranactosyl]-3-O-β- glucopyranoside (1) and (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (2), were isolated from Selaginella moellendorffii Hieron. Their structures were determined by spectroscopic methods. Compound 1 stimulated the growth of NIH3T3 cells and promoted the cell migration through increasing the gene and protein expression of connective tissue growth factor.

Proliferative Constituents from the Leaves of Micromelum integerrimum

Two new compounds, 5-O-methyl-4-desmethyl-myricanol (1) and 6-formyl-5-isopropyl-3-hydroxymethyl-7-methyl-1H-indene (2), were isolated from the leaves of Micromelum integerrimum. Their structures were determined by spectroscopic methods. Additionally, compound 1 could stimulate the growth of NIH3T3 cells and promote cell migration. Compound 1 might exert its effects through increasing the protein expression of connective tissue growth factor.

Abstract 1917: Targeting wildtype and L858R/T790M mutant EGFR by isoliquiritigenin induces apoptosis and Inhibits tumor growth of NSCLC

Abstract Genetic alteration of epidermal growth factor receptor (EGFR) is a high risk factor of non-small-cell lung cancer (NSCLC). Isoliquiritigenin (ILQ), a chalcone derivative, reported anti-cancer activities. In this study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor -sensitive and -resistant NSCLC cells and elucidated its molecular mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both tyrosine kinase inhibitor-sensitive (HCC827, H1650; delE746-A750 EGFR) and -resistant (H1975; L858R/T790M EGFR) NSCLC cells. However, ILQ didn't showed the inhibitory effects of cell growth and induction of apoptosis in Met amplified-HCC827GR or K-ras mutated-A549 cells. ILQ induced apoptotic markers, the cleavage of caspase-3 and poly-(ADP ribose)-polymerase, increased expression of Bim and reduced expression of Bcl-2. ILQ inhibited the catalytic activity of both wildtype and double mutant (L858R/T790M) EGFR by kinase assay. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wildtype or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells in the western blotting analysis. ILQ directly bound both wildtype and double-mutant EGFR by ATP-competitive manner. Computational docking model expected that ILQ interacted with Glu762 and Met793 sites of wildtype EGFR and Lys745, Met793 and Asp855 of mutant EGFR. ILQ attenuated the tumor growth of H1975 cell xenografted athymic mice in vivo, which was caused by decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wildtype or mutant EGFR. Citation Format: Mee-Hyun Lee, Sung Keun Jung, Do Young Lim, Yong-Yeon Cho, Cheol-Jung Lee, Ji-Hong Song, Myoung Ok Kim, Sung-Hyun Kim, Ann M. Bode, Zigang Dong. Targeting wildtype and L858R/T790M mutant EGFR by isoliquiritigenin induces apoptosis and Inhibits tumor growth of NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1917. doi:10.1158/1538-7445.AM2015-1917

Characterization of SLC22A18 as a tumor suppressor and novel biomarker in colorectal cancer

SLC22A18, solute carrier family 22, member 18, has been proposed to function as a tumor suppressor based on its chromosomal location at 11p15.5, mutations and aberrant splicing in several types of cancer and down-regulation in glioblastoma. In this study, we sought to demonstrate the significance of SLC22A18 as a tumor suppressor in colorectal cancer (CRC) and provide mechanistic bases for its function. We first showed that the expression of SLC22A18 is significantly down-regulated in tumor tissues using matched normal-tumor samples from CRC patients. This finding was also supported by publically accessible data from The Cancer Genome Atlas (TCGA). Functionally, SLC22A18 inhibits colony formation and induces of G2/M arrest consistent with being a tumor suppressor. Interestingly, suppression of KRAS by RNA interference promotes SLC22A18 expression, and expression of SLC22A18 in turn inhibits KRAS(G12D)-mediated anchorage independent growth of NIH3T3 cells indicating a mutual negative interaction. Finally, we evaluated diagnostic and prognostic values of SLC22A18 using clinical and gene expression data from TCGA which revealed a significantly worse long-term prognosis for patients with low level SLC22A18 expression. In sum, we established SLC22A18 as a tumor suppressor in colon epithelial cells and propose that SLC22A18 is potentially a marker of diagnostic and prognostic values.

Decreasing Phosphatidylcholine on the Surface of the Lipid Droplet Alters Protein Binding

Excess lipid is stored in an intracellular organelle known as the lipid droplet. While the core of the lipid droplet is rich in neutral lipids, the surface is primarily comprised of two phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The lipid droplet surface is further decorated with proteins, such as perilipin 2, which facilitate lipid storage and utilization. Here, we show that changes to the phospholipid composition of the lipid droplet surface alters protein binding to the lipid droplet. We utilize studies with synthetic lipid droplets to demonstrate that reduced ratios of PC:PE increase the binding of perilipin 2 in vitro. Moreover, altering the availability of intracellular PC by growing NIH 3T3 cells in choline deficient media decreases the PC:PE ratio on lipid droplets and increases intracellular perilipin 2 association with the lipid droplet. While total levels of lipid droplet‐associated proteins are not changed, choline deficiency alters the ability of other unidentified proteins to associate with the lipid droplet. Future experiments will seek to identify these currently unknown proteins, and continue to explore the connections between the lipid droplet phospholipid composition, protein binding, and the ability of the lipid droplet to store and utilize fat.

Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR

Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.

Transformation of NIH3T3 mouse fibroblast cells by MUC16 mucin (CA125) is driven by its cytoplasmic tail.

MUC16 (CA125) is a transmembrane mucin that contributes to the progression of epithelial ovarian cancer (EOC). Expression of MUC16 is not detectable in the epithelial surface of normal ovaries. MUC16 expression is, however, common in serous EOC as well as in metastatic and recurrent tumors. Despite these observations, its contribution to the development of EOC is unknown. We stably expressed either empty vector, MUC16 C-terminal domain (MUC16 CTD) or MUC16 TMU (a construct that lacks the cytoplasmic tail) in NIH3T3 mouse fibroblast cells. In this study, we provide evidence for the role of MUC16 CTD in oncogenic transformation. We show that ectopic expression of MUC16 CTD enhances the growth of NIH3T3 cells under normal and low serum conditions, and promotes anchorage-dependent colony formation. The deletion of the cytoplasmic tail abrogated these effects. MUC16 CTD expression in NIH3T3 cells also enhances the formation of colony in soft agar as compared to MUC16 TMU. MUC16 CTD expression enhances tumor formation in nude mice. Our findings provide the first evidence that MUC16 induces the transformation of NIH3T3 cells and indicate that MUC16 functions as an oncogene. Furthermore, our data suggest that the cytoplasmic tail is critical for MUC16 oncogenic properties.

NR4A1 promotes PDGF-BB-induced cell colony formation in soft agar.

The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth.

Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells

CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells.

Differential cytotoxic effects of gold nanoparticles in different mammalian cell lines

Gold nanoparticles (AuNPs) possess unique properties that have been exploited in several medical applications. However, a more comprehensive understanding of the environmental safety of AuNPs is imperative for use of these nanomaterials. Here, we describe the impacts of AuNPs in various mammalian cell models using an automatic and dye-free method for continuous monitoring of cell growth based on the measurement of cell impedance. Several well-established cytotoxicity assays were also used for comparison. AuNPs induced a concentration-dependent decrease in cell growth. This inhibitory effect was associated with apoptosis induction in Vero cells but not in MRC-5 or NIH3T3 cells. Interestingly, cDNA microarray analyses in MRC-5 cells supported the involvement of DNA damage and repair responses, cell-cycle regulation, and oxidative stress in AuNP-induced cytotoxicity and genotoxicity. Moreover, autophagy appeared to play a role in AuNPs-induced attenuation of cell growth in NIH3T3 cells. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles.