Murine Fibroblast Cell Line NIH3T3 Research Articles

Extracellular biopolymers produced by Dictyosphaerium family - Chemical and immunomodulative properties

Many microalgal species produce a wide range of highly-value products which are interesting for biotechnological applications. Cultivation of microalgal species Dictyosphaerium pulchellum and Dictyosphaerium tetrachotomum, strains Růžicka and Fott resulted yields of 0.2, 0.7 and 1.8 g/L of extracellular biopolymers (EPSs), respectively. All biopolymers were shown to be anionic proteoglycans. The sugar composition analyses of all EPSs showed high contents of hexoses and the presence of partially methylated monosaccharide residues, i.e. hexoses, and deoxy hexoses. The dominant sugar component of all EPSs was found to be galactose. Extracellular microalgal biopolymers were subjected to immunobiological and immunotoxicological evaluation using murine melanoma cancer cells B16, murine fibroblast cell line NIH-3T3, murine macrophages cell line RAW 264.7 and skin construct EpiDerm™ (EPI-200). The EPSs exerted the antiproliferative effectivity; treatment of EPS induced proinflammatory cytokines TNF-α, IL-6, IL-12, IL-1β and IL-17, also engaged in anti-cancer immunity. Immunotoxicological studies revealed their non-toxic character and safe application on EpiDerm™.

Osteosarcoma-derived extracellular vesicles induce a tumor-like phenotype in normal recipient cells.

Osteosarcoma is the most common primary bone cancer and the most frequent cause of bone cancer-related deaths in children and adolescents. Osteosarcoma cells are able to establish a crosstalk with resident bone cells leading to the formation of a deleterious vicious cycle. We hypothesized that osteosarcoma cells can release, in the bone microenvironment, transforming Extracellular Vesicles (EVs) involved in regulating bone cell proliferation and differentiation, thereby promoting tumor growth. We assessed EV production by three osteosarcoma cell lines with increasing aggressiveness in order to investigate their roles in the communication between osteosarcoma cells and normal recipient cells. Osteosarcoma-derived EVs were used to treat the murine fibroblast cell line NIH3T3 and to study the induction of tumor-like phenotypes. Our results showed that osteosarcoma cell lines are able to produce EVs that fuse to recipient cells, with a very high uptake efficiency. The treatment of recipient NIH3T3 with osteosarcoma-derived EVs induced substantial biological and functional effects, as an enhanced proliferation and survival capability under starved conditions, high levels of activated survival pathways, an increased migration, adhesion, and 3D sphere formation and the acquired capability to grow in an anchorage-independent manner. Moreover, in murine NIH3T3 we found human mRNAs of TNF-α, IL-6, and TGF-β, as well as a de novo expression of murine MMP-9 and TNF-α following the treatment of human osteosarcoma-derived EVs.

Electrical Stimulation of NIH-3T3 Cells with Platinum-PEDOT-Electrodes Integrated in a Bioreactor

The objective of this work involves the development and integration of electrodes for the electrical stimulation of cells within a bioreactor. Electrodes need to fit properties such as biocompatibility, large reversible charge transfer and high flexibility in view of their future application as implants on the tympanic membrane. Flexible thin-film platinum-poly(3,4-ethylene-dioxythiophene)-electrodes on a poly(ethylene terephthalate)-foil manufactured using microsystems technology were integrated into a bioreactor based on the design of a 24 well plate. The murine fibroblast cell line NIH-3T3 was cultured on the foil electrodes and the cells were stimulated with direct voltage and unipolar pulsed voltage. The amplitude, the pulse length and the ratio of pulse to pause were varied. The stimulated cells were stained in order to determine the angle between the cell cleavage plane of the dividing cells and the vector of the electric field. These angles were subsequently used to calculate the polarization index, which is a measure of the orientation of the metaphase plane of dividing cells that occurs for example during wound healing or embryonic morphogenesis.

MRGD, a MAS-related G-protein Coupled Receptor, Promotes Tumorigenisis and Is Highly Expressed in Lung Cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target.

Inhibition of hyaluronan synthesis in breast cancer cells by 4‐methylumbelliferone suppresses tumorigenicity in vitro and metastatic lesions of bone in vivo

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Previous studies demonstrated that inhibition of HA suppressed the tumorigenicity of various malignant tumors including breast cancer. 4-methylumbelliferone (MU) has been reported to inhibit HA synthesis in several cell types. However, few studies have focused on the effects of HA inhibition in breast cancer cells by MU, nor the effects on bone metastasis. We hypothesized that MU would suppress the progression of bone metastasis via inhibition of HA synthesis. Here, we investigated the effects of MU on HA expression in MDA-MB-231 breast cancer cell line in addition to their tumorigenicity in vitro and in vivo. HAS2 mRNA expression was downregulated after 6 and 24 hr treatment with MU. Quantitative analysis of HA revealed that MU significantly inhibited the intracellular and cell surface HA. MU significantly inhibited cell growth and induced apoptosis as determined by cell proliferation and TUNEL assays, respectively. Phosphorylation of Akt was suppressed after 12 and 24 hr treatment with MU. MU treatment also inhibited cell motility as well as cell invasiveness. MU also inhibited cell growth and motility in murine fibroblast cell line NIH3T3. In vivo, administration of MU inhibited the expansion of osteolytic lesions on soft X-rays in mouse breast cancer xenograft models. HA accumulation in bone metastatic lesions was perturbed peripherally. These data suggest that MU might be a therapeutic candidate for bone metastasis of breast cancer via suppression of HA synthesis and accumulation.

Lidocaine Inhibits NIH-3T3 Cell Multiplication by Increasing the Expression of Cyclin-Dependent Kinase Inhibitor 1A (p21)

We explored molecular mechanisms by which lidocaine inhibits growth in the murine embryonic fibroblast cell line NIH-3T3. Local anesthetics can adversely affect cell growth in vitro. Their effects on wound healing are controversial. We examined the effects and novel mechanisms by which lidocaine affects in vitro multiplication of the murine fibroblast cell line NIH-3T3. NIH-3T3 cells were grown in culture with lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM]. Cell multiplication was assessed by determining cell counts on subsequent days, while mechanisms by which inhibition occurred were evaluated by bromodeoxyuridine uptake, gene expression using polymerase chain reaction array, and Western blot analysis to verify increased levels of affected proteins. Lidocaine caused dose-dependent inhibition of multiplication of NIH-3T3 cells. Effects ranged from no inhibition [0.05 and 0.5 mM] and mild inhibition [1 mM], to severe inhibition [2 and 5 mM] [P = 0.006]. Lidocaine 2 mM inhibited bromodeoxyuridine uptake at day 3.5 [P = 0.02 versus control, and P = 0.0495 vs 1 mM lidocaine]. On day 1.5, lidocaine upregulated expression of cyclin-D1 and cyclin-dependent kinase inhibitor 1A [p21]. On day 2.5, lidocaine increased the levels of p21 protein. Low concentrations of lidocaine, as would be seen in plasma after spinal, epidural, or plexus anesthesia, do not significantly affect multiplication of fibroblasts. Higher doses of lidocaine arrest cell multiplication at the S-phase of the growth cycle by upregulation of p21, an extremely potent inhibitor of cell multiplication. Higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing.

Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes

BackgroundSafe and efficient vector systems for delivering antigens or immunomodulatory molecules to dendritic cells (DCs), T lymphocytes or both are considered effective means of eliciting adaptive immune responses and modulating their type, extent, and duration. As a possible tool toward this end, we have developed a self-inactivating vector derived from feline immunodeficiency virus (FIV) showing performance characteristics similar to human immunodeficiency virus-derived vectors but devoid of the safety concerns these vectors have raised.MethodsThe pseudotyped FIV particles were generated with a three-plasmid system consisting of: the packaging construct, providing Gag, Pol and the accessory proteins; the vector(s), basically containing FIV packaging signal (ψ), Rev responsive element, R-U5 region at both ends, and the green fluorescent protein as reporter gene; and the Env plasmid, encoding the G protein of vesicular stomatitis virus (VSV-G) or the chimeric RD114 protein. Both packaging and vector constructs were derived from p34TF10, a replication competent molecular clone of FIV. The pseudotyped particles were produced by transient transfection in the Crandell feline fibroblast kidney (CrFK) or the human epithelial (293T) cell line.ResultsTo broaden its species tropism, the final vector construct was achieved through a series of intermediate constructs bearing a longer ψ, the FIV central polypurin tract sequence (cPPT), or the woodchuck hepatitis post-regulatory element (WPRE). These constructs were compared for efficiency and duration of transduction in CrFK or 293T cells and in the murine fibroblast cell line NIH-3T3. Whereas ψ elongation and cPPT addition did not bring any obvious benefit, insertion of WPRE downstream GFP greatly improved vector performances. To maximize the efficiency of transduction for ex-vivo murine DCs and T-lymphocytes, this construct was tested with VSV-G or RD114 and using different transduction protocols. The results indicated that the FIV construct derived herein stably transduced both cell types, provided that appropriate vector makeup and transduction protocol were used. Further, transduced DCs underwent changes suggestive of an induced maturation.ConclusionIn contrast to previously described FIV vectors that were poorly efficient in delivering genetic material to DCs and T lymphocytes, the vector developed herein has potential for use in experimental immunization strategies.

8. Adeno-Associated Virus 2-Mediated Gene Transfer: A Complex Interaction between Epidermal Growth Factor Receptor Protein Tyrosine Kinase Signaling and Ubiquitin/Proteasome Pathway in Transgene Expression

The transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein, FKBP52, inhibits the viral second-strand DNA synthesis, and consequently, transgene expression (J. Virol., 75: 9818, 2001). FKBP52 is phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), which inhibits AAV second-strand DNA synthesis by greater than 90% (J. Virol. 72: 9835, 1998; J. Virol., 75: 9818, 2001). Tyrosine-phosphorylated FKBP52 is dephosphorylated by T-cell protein tyrosine phosphatase (TC-PTP), which negatively regulates EGFR-PTK signaling, and leads to a significant increase in AAV transduction (J. Virol. 77: 2741, 2003). We have documented high-efficiency transduction of hepatocytes in TC-PTP-transgenic mice (Gene Ther., 11: 1165, 2004). We have also documented that intracellular trafficking of AAV from cytosol to nucleus is improved in hematopoietic cells from TC-PTP transgenic mice (Hum. Gene Ther., 15: 1207, 2004). In the present studies, we studied the role of EGFR-PTK signaling in AAV-mediated transgene expression. Consistent with our previous studies, less than 5% of HeLa cells were transduced by single-stranded (ss) AAV-EGFP vectors, and pretreatment of cells with Tyrphostin 23 (Tyr23), a specific inhibitor of EGFR-PTK, or stable expression of TC-PTP resulted in respectively |[sim]|12-fold and |[sim]|15-fold increase in AAV transduction. Although transduction by self-complementary (sc) AAV2-EGFP vectors was |[sim]|8-fold higher compared with their ss counterparts, pretreatment with Tyr23 or stable expression of TC-PTP resulted in a further |[sim]|10-fold increase in transduction. These data suggested that Tyr23 and TC-PTP affected other aspects of EGFR-PTK signaling involved in regulating vector transduction. We next examined whether Tyr23 or TC-PTP modulated the ubiquitin/proteasome pathway, both because proteasomes have been implicated in the regulation of EGFR endocytosis, and proteasome inhibitors have been shown to augment AAV transduction. Treatment of HeLa cells with MG132, a specific inhibitor of proteasome, resulted in respectively |[sim]|6-fold and |[sim]|8-fold increase in transduction efficiency of ss and scAAV vectors, However, neither a synergistic nor an additive effect was observed following pretreatment with MG132+Tyr23 or MG132+TC-PTP with either ss or scAAV vectors in HeLa cells. Similar results were obtained with murine fibroblast cell line NIH3T3, adult mouse hepatocyte cell line H2.35, and fetal mouse hepatocyte cell line FL83B. Taken together, these data indicate that complex interactions between EGFR-PTK signaling and ubiquitin/ proteasome pathway affect various aspects of intracellular trafficking and second-strand DNA synthesis of AAV vectors. A better understanding of these interactions is likely to be important in yielding new insights in the optimal use of recombinant AAV vectors in human gene therapy.

COMPARISON THE CYTOTOXICITY OF HYDROXYAPATITE MEASURED BY DIRECT CELL COUNTING AND MTT TEST IN MURINE FIBROBLAST NIH-3T3 CELLS

The worldwide growing interest to biomaterials over the last years results from their irreplaceable role in medical clinic. Hydroxyapatite is used in bone reconstruction because of its similar chemical structure compared to the inorganic composition of human bone and it is basic building component of many newly prepared biomaterials. In this study, we evaluated cytotoxic/antiproliferative activity of hydroxyapatite extract using murine fibroblast cell line NIH-3T3 and two in vitro different cytotoxic assays: growth inhibition assay and MTT assay. Hydroxyapatite extract after 72 h of incubation manifested the significant in vitro cytotoxic/antiproliferative effect only at the highest concentration tested (100 %). The antiproliferative effect of hydroxyapatite extract at the other concentrations tested (75 %, 50 %, 25 %, 10 %, 5 % and 1 %) was directly proportional to the concentration and the time of influence. The inhibition of cell proliferation was 86.8 - 0 %. The sensitivity of cell growth inhibition assay (direct counting of viable cells) to the extract influence was higher than that of MTT test.

Triptolide, an active component of the Chinese herbal remedy Tripterygium wilfordii Hook F, inhibits production of nitric oxide by decreasing inducible nitric oxide synthase gene transcription.

The ethyl acetate (EA) extract of Tripterygium wilfordii Hook F (TWHF) and its major active component, triptolide, have been reported to be effective in the treatment of rheumatoid arthritis and other autoimmune inflammatory diseases. Nitric oxide (NO) has been recognized as an important mediator of inflammation. This study was therefore undertaken to examine the effects of the EA extract and triptolide on the production of NO and inducible NO synthase (iNOS) gene expression and transcription in vivo and in vitro. Peritoneal macrophages from C57BL/6J mice treated orally with the EA extract of TWHF were assayed for NO production and iNOS messenger RNA (mRNA) expression by reverse transcriptase-polymerase chain reaction. The murine fibroblast cell line NIH3T3 was also assessed for NO production and iNOS mRNA expression, as well as for iNOS promoter activation, Oct-1 nuclear binding capacity, and Oct-1 protein content by transient transfection, electrophoretic mobility shift assay, and immunoblotting, respectively. NO production and iNOS mRNA expression by macrophages from C57BL/6J mice immunized with trinitrophenyl-bovine serum albumin in Freund's complete adjuvant were significantly inhibited by oral administration of the EA extract (52.3% and 59.8% of control, respectively, at one-eighth of the dose that is lethal for 50% of the animals [LD(50)] and 21.0% and 38.1% of control, respectively, at one-fourth the LD(50)). Moreover, the EA extract and triptolide significantly inhibited NO production in vitro in activated peritoneal macrophages, which reflected a decreased level of iNOS mRNA. Finally, triptolide inhibited promoter activity of the iNOS gene and induction of the activity of the regulator of iNOS transcription, Oct-1. The EA extract of TWHF and triptolide inhibit transcription of the iNOS gene. This may contribute to the antiinflammatory effects of this traditional herbal remedy.

Role of osteopontin in the pathogenesis of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is initiated by migration, adhesion, and proliferation of fibroblasts. Osteopontin (OPN) is one of the cytokines produced by activated macrophages and mediates various functions, including cell attachment and migration, by interacting with alphav integrin. In this study, we have investigated the role of OPN in the pathogenesis of pulmonary fibrosis. We developed a mouse model for pulmonary fibrosis by intratracheal instillation of bleomycin (BLM). OPN was strongly expressed in alveolar macrophages accumulating in the fibrotic area of the lung. OPN messenger RNA (mRNA) in the lung was notably induced by BLM instillation, and the development of the fibrotic process was associated with an increase in the expression of OPN mRNA and protein. In vitro, recombinant OPN enhanced migration, adhesion, and platelet-derived growth factor (PDGF)-mediated DNA synthesis of murine fibroblast cell line NIH3T3. These effects of OPN on fibroblasts were significantly suppressed by addition of antimouse alphav integrin monoclonal antibody (RMV-7). Furthermore, treatment of mice with RMV-7 repressed the extent of pulmonary fibrosis in this model. Conclusively, these data suggest that OPN produced by alveolar macrophages functions as a fibrogenic cytokine that promotes migration, adhesion, and proliferation of fibroblasts in the development of BLM-induced pulmonary fibrosis.

Ras mutation, irrespective of cell type and p53 status, determines a cell's destiny to undergo apoptosis by okadaic acid, an inhibitor of protein phosphatase 1 and 2A.

Okadaic acid (OA), a toxin from the black sponge Halicondria okadai, is a specific inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). OA is a tumor promoter but also induces apoptosis in some tumor cell lines. In this study, we determined whether ras mutation and/or p53 status are characteristics associated with the cell's sensitivity to the induction of apoptosis by OA. Several cell lines that differed in ras and p53 mutations were treated with OA (10-100 nM). At 24 to 48 h after treatment, the percentage of cells undergoing apoptosis was quantitated. The cell lines with mutations in either H-ras (human bladder carcinoma cell line T24 and mouse keratinocyte cell line 308), or K-ras (human colon carcinoma cell lines DLD-1 and HCT116; human prostate cancer cell lines LNCaP and PC-3; human lung cancer cell lines Calu-6 and SKLU-1; and human pancreatic cancer cell line MIAPaCa2) were more sensitive to OA-induced apoptosis (3- to 10-fold) than the cell lines that lacked the ras mutation (mouse epidermal cell lines C50 and JB6; murine fibroblast cell line NIH3T3; human colon cancer cell line HT29; human kidney epithelial cell line Hs715.K; and human pancreatic cancer cell line Bx-PC3). Similarly, using isogenic cell lines we found that overexpression of mutated H-ras in NIH3T3 and in SV40 immortalized human uroepithelial cells (SVHUC) enhanced their sensitivity to undergo apoptosis in response to OA treatment. The T24, DLD-1, SKLU-1, Calu-6, and MIAPaCa2 cell lines express mutated p53. The SVHUC as well as their ras-transfected counterparts have inactive p53 due to complex formation between large "T" antigen and p53. Taken together, these results imply that OA-induced apoptosis may involve a p53-independent pathway. The transfectants (NIH3T3-ras and SVHUC-ras), which express mutated H-ras, have up-regulated PP2A activity. OA treatment inhibited in vivo the levels of PP1 and PP2A activity, and induced apoptosis in SVHUC-ras and other cell lines. We conclude that OA-induced cell death pathway in ras-activated cell lines may involve a cross talk between PP1 and PP2A and ras signaling pathways. In light of the present results, the current theory that OA promotes mouse skin tumor formation by selective expansion of initiated cells that harbor ras mutations needs reevaluation.

Experimental studies on radiation-inducible human TNF gene therapy for cancer

Tumor necrosis factor (TNF) has certain radioprotective effect on host tissue and is capable of enhancing the antitumor effect of radiotherapy. In addition, the transcriptional regulation of the promoter region of Egr-1 gene is activated by ionizing radiation. So we fused Egr-1 promoter with hTNF-α cDNA, and resultantly constructed a double-copy and radiationinducible retroviral vector named as pETDC. After packaged with Psi-2 and Crip cellsin vitro, the hTNF recombinant retroviruses were in the titers of 4×105 CFU/ml. By infection of murine fibroblast cell line NIH3T3 and murine melanoma cell line B16.F10 with the recombinant retroviruses and followed by G418 resistant selection, two positive clones secreting TNF at the levels of 2.1 ng/ml and 1.1 ng/ml respectively were generated. After exposure to 20 Gy ionizing radiation, TNF secretions from the two positive clones were elevated to 13.8 ng/ml (6.6-fold) and 5.7 ng/ml (5.2-fold) respectively. Furthermore, hTNF-α expression in pETDC-transfected cells was confirmed by RT-PCR. These data provide an experimental bases for the application of TNF gene therapy combined with local radiotherapy in cancer patients.