Immortalization Of Fibroblasts Research Articles

Human adipose-derived stromal cells as a feeder layer to improve keratinocyte expansion for clinical applications

The aim of this work is to propose a keratinocytes (KC) culture method for clinical practice with irradiated adipose-derived mesenchymal stromal cells (ASCs) as human feeder layer, avoiding murine immortalized fibroblasts, commonly request for producing skin substitutes. ASCs were isolated, expanded, irradiated, and co-cultured with autologous or allogeneic KC. All experiments were performed using murine fibroblasts as control. Cell counts, flow cytometric analysis and ELISA were carried out, in order to define cell yield, viability and cytokine secretion. Results indicate that the optimal X-ray dose for ASCs is 120 Gy and the optimal seeding density is 625 cells/cm2; moreover, flow cytometric analysis shows that the percentage of feeder layer cells reaches values lower than 1%, within 8 days of co-culture. KC reach confluence in 6.9 days on ASCs substrate and, after confluence, the number of live cells increases again in a multilayered structure. Moreover, results show higher levels of interleukin (IL)-1a in co-culture with ASCs compared with 3T3, while no differences were observed for IL-6 and IL-8. Therefore, human ASCs enable to obtain effectively in vitro expanded KC and represent a viable alternative to murine fibroblasts for the production of clinical use skin substitutes.

Novel 3D-microcarriers from recombinant spidroin for regenerative medicine.

Microcarriers generated from recombinant spidroin 1F9 are suitable for use as an injection material. The microcarriers were a heterogeneous mixture of microgel particles ranging from 50 to 300 µm in size with the predominance of particles of 50-150 µm. The surface of these microparticles had a complex topography and ensured efficient cultivation of primary and immortalized fibroblasts. Intradermal injections of microgel suspensions into the area of full-thickness skin wounds did not lead to the development of acute inflammation in mice; instead, they accelerated the recovery of skin tissue and stimulated neurogenesis and angiogenesis.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-β

Actions of transforming growth factor-β are largely context dependent. For instance, TGF-β is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-β also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-β would change in the context of transformation, we have compared the differential gene regulation by TGF-β in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-β in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-β is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-β. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-β in hFhTERT cells and TGF-β conferred action of these cells was mediated by WNT4. While TGF-β activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-β which matched with the response of HT1080 cells to TGF-β. These data suggest context dependent activation of non-canonical signaling by TGF-β.

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (review of the own data)

Expression of matrix metalloproteinases (MMPs) and their endogenous regulators has been investigated in squamous cervical carcinoma (SCC). The study included (i) immortalized fibroblasts (IF) and three clones of fibroblasts transformed by oncogene E7 HPV-16 (TF); (ii) cell lines associated with HPV-16 and HPV-18; (iii) tumor tissue samples from patients with SCC, associated with gene E7 HPV-16. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. Expression of MMP-9 was found only inTF. MMP-9 may serve as a TF marker. In TF expression mRNA TIMP-1 was decreased. The level of free endogenous inhibitors in TF was significantly lower then the level in IF. Expression MMP correlated with the tumorigenic potential of TF. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). The cell lines differed substantially in the level of expression of MMPI and their endogenous regulators. In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-9 expression in cell lines was not detected. Results of studies on these cell lines suggest existence of an imbalance in the system enzyme/inhibitor/activator, that increases destructive potential of these cells. The study of expression of MMP and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously determined by the increase of expression of collagenases MMP-1, MT1-MMP and gelatinase MMP-9, decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent to increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. In adjacent to the tumor normal tissue revealed a significant expression of MMP-1,-2,-9.

Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses.

In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP+) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP+ I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP+ and AP− I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP+ I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP+ I-MEFs. Together with sestrin 1 upregulation, we found that AP+ I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP+ I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP+ phenotype and achieve a quiescent state characterized by a new transcriptional network.

EMT or EMT-Promoting Transcription Factors, Where to Focus the Light?

The epithelial-to-mesenchymal transition (EMT) is commonly considered as a main driving force of the metastatic cascade. An impressive series of experiments and observations worldwide supports its pivotal role in promoting cancer cell dissemination at the invasive fronts of tumors, intravasation, cell survival in fluids, and extravasation, with secondary site colonization being the only step requiring a return to an epithelial phenotype (1). Beyond this, commitment of epithelial cells into an EMT program has been associated with resistance to chemo, radio-, and hormono-therapies, definitively extending the interest of studying EMT from the embryologist community to the oncology and medical fields. Obviously, EMT turns out to constitute a transdifferentiation program, allowing epithelial cells to escape from numerous stresses, including mechanical constrains, hypoxia, nutrient depletion, and unfortunately therapeutic treatments (1). EMT is orchestrated by a restricted number of transcription factors mainly the three Snail, Twist, and Zeb families (EMT-TFs). With inclusion of miRNAs, these factors constitute a complex interactome, able to sensor multiple signals received from the proximal microenvironment and relay them onto gene expression (2, 3). Generally undetectable in adult epithelial cells in homeostatic conditions, EMTTFs were found to be recurrently expressed in various types of cancers including multiple carcinomas, an expression often associated with a high metastatic risk. An outstanding amount of work has been performed over the last years evaluating their relative contribution to the initiation and maintenance of the EMT process, with a significant impact on the prognostic value occurring following to their detection in primary tumors or in disseminated cancer cells (4, 5). Does this mean that the oncogenic potential of these factors in epithelial cells only rely on their ability to promote EMT or does their oncogenic activity extend further than EMT induction? By performing in vitro functional assays, we and others firstly demonstrated that several of these embryonic transcription factors, including the TWIST proteins, alleviated fail-safe program induction. Through this, they were shown to cooperate with mitogenic oncoproteins such as RAS and N-MYC in promoting cell transformation in vitro, as well as in lung and breast carcinoma development in transgenic mouse models (4, 6–9). The underlying mechanisms have been largely explored. The TWIST1 protein was found to directly interact with p53 and to destabilize the oncosuppressive protein by altering specific post-translational modifications (10). Furthermore, TWIST1 alleviates induction of cyclin-dependent kinase inhibitors (CDKN1A,CDKN2A, and CDKN2B), thereby sustaining cell proliferation (8, 11). These pleiotropic properties probably provide TWIST proteins unique properties. As TWIST proteins experimentally are inefficient in triggering EMT as compared with SNAIL and ZEB proteins, their main functions may consist in protecting cells from fail-safe programs during the EMT-associated genetic reprograming. In this respect, they might be considered as survival factors rather than EMT inducers. Although SNAIL and ZEB proteins, unlike the TWIST transcription factors, fail to prevent HRASG12Vinduced senescence in murine embryonic fibroblasts (B. Gras, unpublished data), we obviously cannot exclude that they facilitate the escape from oncogene-induced fail-safe programs in other cellular settings and/or experimental conditions. In support of this assumption, ZEB proteins were indeed reported to protect lung cancer cells from EGFR-induced senescence through their ability to down-modulate CDKN1A and CDKN2B (12). In line with this observation, ZEB1 was demonstrated to be positioned downstream of RB and to contribute to fibroblast immortalization induced by RB and RB-like protein depletion (13). Enforced expression of SNAIL or ZEB proteins in non-transformed mammary epithelial cells, and the consequent activation of RAS-downstream pathways, predominantly triggers EMT. Nonetheless, it also accidentally promotes the commitment of cells into a senescence program, unveiled by their SA-β-galactosidase activity (B Gras and SA, unpublished data). This observation is consistent with the reported anti-proliferative properties of SNAIL and ZEB proteins in epithelial cells (14) and with the recognized antagonism between cell proliferation and dissemination. It additionally gives a rationale to the restricted staining of these transcription factors to the tumor-stromal interface, stabilized by microenvironmental EMTpermissive conditions (15–17). The need to maintain ZEB and SNAIL proteins at

Structural Analysis, Electrochemical Behavior, and Biocompatibility of Novel Quaternary Titanium Alloy with near β Structure

This article analyses the microstructure, electrochemical behavior, and biocompatibility of a novel Ti-20Nb-10Zr-5Ta alloy with low Young’s modulus (59 GPa) much closer to that of bone, between 10 and 30 GPa, than Ti and other Ti alloys used as implant biomaterial. XRD and SEM measurements revealed a near β crystalline microstructure containing β phase matrix and secondary α phase, with a typical grain size of around 200 μm. The corrosion behavior in neutral Ringer solution evidenced: self-passivation behavior characterizing a very resistant passive film; an easy passivation as a result of favorable influence of the alloying elements Nb, Zr, and Ta that participate with their passive oxides to the formation of the alloy passive film; low corrosion and ion release rates corresponding with very low toxicity. In MEM solution, the novel alloy demonstrated very high corrosion resistance and no susceptibility to localized corrosion. Biocompatibility was evaluated on in vitro human osteoblast-like and human immortalized pulmonary fibroblast cell (Wi-38) lines and the new Ti-20Nb-10Zr-5Ta alloy exhibited no cytotoxicity. The new Ti-20Nb-10Zr5Ta alloy is a promising material for implants due to combined properties of low elastic modulus, very low corrosion rate, and good biocompatibility.

Long-term corrosion behavior and biocompatibility testing of titanium-based alloy covered with nano-crystalline hydroxyapatite

In this paper, we applied on the surface of the new Ti%20Nb%10Zr%5Ta alloy nano-crystalline hydroxyapatite (HA) coating by the method of the chemical deposition in a solution supersaturated with Ca2+ and inline image ions. The composition and crystallinity of the obtained coating was determined by X-ray diffraction, micro-Raman, infrared, and energy dispersive X-ray spectroscopies and its morphology and porosity by scanning electron microscopy. The coating long-term corrosion behavior for 3000 soaking hours in simulated human fluids (Ringer's solutions of different pH values) was studied by cyclic potentiodynamic and linear polarization, electrochemical impedance spectroscopy and monitoring of the open circuit potentials and corresponding open circuit potential gradient due to the pH non-uniformity of the biofluid. Also, the coating biocompatibility was performed by cells (human osteoblast % HOS and human immortalized pulmonary fibroblast % Wi-38) adhesion and proliferation testing in the HA coated bioalloy extract compared to respective effects of the un-coated bioalloy extract. The initial HA nano-coating promoted the deposition of new HA layers, thickening itself and showing its ability to induce the formation of this main inorganic component of the human bone, therefore denoting bioactivity of the HA nano-coated alloy.

Role of miR-26b in carcinoma-associated fibroblasts and effect on migration and invasion of breast cancer epithelial cells

BackgroundIn recent years there has been an increasing awareness of the role of the microenvironment surrounding breast cancer epithelium, particularly the carcinoma-associated fibroblast (CAF), in modulating the behaviour of breast tumours. MicroRNAs, a family of small non-coding RNAs that are key in the post-transcriptional regulation of mRNAs, have a role in controlling the behaviour of cancers and have been studied extensively in breast cancer epithelial cells. We hypothesised that miRNAs have important roles in the behaviour of breast CAFs and, in turn, affect the behaviour of malignant breast epithelial cells. MethodsWe used laser capture microdissected normal fibroblast and CAFs from clinical samples, and a tissue co-culture model, to examine expression of miRNAs in breast CAFs. Deregulated miRNAs that were identified with this screening strategy were further assessed in a larger set of paired laser capture microdissected normal fibroblasts and CAFs. We assessed functional effects on fibroblasts by overexpressing, or knocking down miRNAs (or both) in immortalised fibroblast cell lines and using various growth, migration, and invasion assays. Functional effects of fibroblasts with reduced miRNA on breast cancer epithelial cells were examined in co-cultures. To identify potential miRNA targets and pathways downstream, we used mass spectrometry and in-silico analysis. The clinical relevance of these targets was examined by interrogating publicly available datasets. FindingsSix miRNAs were consistently downregulated in CAFs compared with normal fibroblasts with a fold change of more that ten in both the tissue co-culture model and in patient samples. Of these miRNAs, miR-26b was downregulated in 12 of 14 cases of microdissected matched normal fibroblasts and CAFs from clinical formalin fixed paraffin embedded samples (Wilcoxon signed rank test, p=0·04), and consistently in a further four of four cases of matched primary cultures of normal fibroblasts and CAFs. Reduction of the level of miR-26b in immortalised breast fibroblasts showed a small decrease in proliferation but a very notable increase in migration (p<0·01) and invasion of fibroblasts (p=0·03). There was no difference in the growth of cancer epithelial cells when they were co-cultured with fibroblasts with low miR-26b compared with controls. However, these fibroblasts increased the migration (p<0·01) and invasion (p<0·01) of breast cancer epithelial cells. We identified numerous proteins that were differentially expressed between miR-26b knockdown and control fibroblasts. Pathway analyses of the differentially expressed proteins (p<0·05) were enriched for components of the glycolytic pathway and the cytoskeleton regulation by RHO GTPase pathway. COL12A1 was identified as a target of miR-26b. Increased stromal mRNA expression of COL12A1 was significantly associated with increased recurrence of breast cancer on multivariate analysis in publically available datasets (hazard ratio 17, 95% CI 1·9–159; p=0·01). InterpretationWe have shown that downregulation of miR-26b in breast cancer fibroblasts increases the migration and invasion of breast cancer epithelial cells, and we have identified molecular changes that are downstream of miR-26b. These findings add to the growing body of evidence that epithelial-stromal interactions are important in modulating the behaviour of breast cancer. FundingUK Medical Research Council, Pathological Society of Great Britain and Ireland, Breast Cancer Campaign, National Institute of Health Research.

Internalized PCSK9 dissociates from recycling LDL receptors in PCSK9-resistant SV-589 fibroblasts

Secreted PCSK9 binds to cell surface LDL receptor (LDLR) and directs the receptor for lysosomal degradation. PCSK9 is potent at inducing LDLR degradation in cultured liver-derived cells, but it is considerably less active in immortalized fibroblasts. We examined PCSK9 trafficking in SV-589 human skin fibroblasts incubated with purified recombinant wild-type PCSK9 or gain-of-function mutant PCSK9-D374Y with increased LDLR binding affinity. Despite LDLR-dependent PCSK9 uptake, cell surface LDLR levels in SV-589 fibroblasts were only modestly reduced by wild-type PCSK9, even at high nonphysiological concentrations (20 µg/ml). Internalized 125I-labeled wild-type PCSK9 underwent lysosomal degradation at high levels, indicating its dissociation from recycling LDLRs. PCSK9-D374Y (2 µg/ml) reduced cell surface LDLRs by approximately 50%, but this effect was still blunted compared with HepG2 hepatoma cells. Radioiodinated PCSK9-D374Y was degraded less efficiently in SV-589 fibroblasts, and Alexa488-labeled PCSK9-D374Y trafficked to both lysosomes and endocytic recycling compartments. Endocytic recycling assays showed that more than 50% of internalized PCSK9-D374Y recycled to the cell surface compared with less than 10% for wild-type PCSK9. These data support that wild-type PCSK9 readily dissociates from the LDLR within early endosomes of SV-589 fibroblasts, contributing to PCSK9-resistance. Although a large proportion of gain-of-function PCSK9-D374Y remains bound to LDLR in these cells, degradative activity is still diminished.

The stem cell transcription factor ZFP57 induces IGF2 expression to promote anchorage-independent growth in cancer cells

Several common biological properties between cancer cells and embryonic stem (ES) cells suggest the possibility that some genes expressed in ES cells might have important roles in cancer cell growth. The transcription factor ZFP57 is expressed in self-renewing ES cells and its expression level decreases during ES cell differentiation. This study showed that ZFP57 is involved in the anchorage-independent growth of human fibrosarcoma HT1080 cells in soft agar. ZFP57 overexpression enhanced, whereas knockdown suppressed, HT1080 tumor formation in nude mice. Furthermore, ZFP57 regulates the expression of insulin-like growth factor 2 (IGF2), which has a critical role in ZFP57-induced anchorage-independent growth. ZFP57 also promotes anchorage-independent growth in ES cells and immortal fibroblasts. Finally, immunohistochemical analysis revealed that ZFP57 is overexpressed in human cancer clinical specimens. Taken together, these results suggest that the ES-specific transcription factor ZFP57 is a novel oncogene.

Isolation and Immortalization of Fibroblasts from Different Tumoral Stages

Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is essential to fully understand their role in tumour progression. Previous studies have addressed this issue using human fibroblasts and comparing normal and adjacent fibroblasts to tumour-associated fibroblasts (Kalluri and Zeisberg, 2006). However, these studies are hampered by the intrinsic variability of human samples (e.g. pairing, age, genomic landscape, etc). In order to overcome these issues, we used a fully characterised mouse breast cancer model, MMTV-PyMT (Guy et al., 1992; Lin et al., 2003). MMTV-PyMT transgenic mice express the Polyoma Virus middle T antigen under the direction of the mouse mammary tumor virus promoter/enhancer. This is a multifocal luminal breast cancer model that goes through well defined and characterised stages (namely, hyperplasia, adenoma, carcinoma and invasive carcinoma). Interestingly, this model has a 100% incidence, is very desmoplastic (presenting high concentration of fibroblasts) and gives raise to spontaneous metastasis in the lung with 80-94% incidence. Importantly, at least for the inguinal mammary glands (glands 4 and 9), the different tumoral stages are well correlated to the age of the mouse: hyperplasia arising at 6 weeks of age, adenoma between 6-8 weeks of age, carcinoma and invasive carcinoma from 8 weeks onwards. This model allowed us to confidently isolate fibroblasts from different tumoral stages and carefully characterise their functional and molecular properties (Calvo et al., 2013).

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.

Establishment of an immortal chicken embryo liver-derived cell line

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

The proteome of Toll-like receptor 3–stimulated human immortalized fibroblasts: Implications for susceptibility to herpes simplex virus encephalitis

Inborn errors in Toll-like receptor 3 (TLR3)-IFN type I and III pathways have been implicated in susceptibility to herpes simplex virus encephalitis (HSE) in children, but most patients studied do not carry mutations in any of the genes presently associated with HSE susceptibility. Moreover, many patients do not display any TLR3-IFN-related fibroblastic phenotype. To study other signaling pathways downstream of TLR3 and/or other independent pathways that may contribute to HSE susceptibility. We used the stable isotope labeling of amino acids in cell culture proteomics methodology to measure changes in the human immortalized fibroblast proteome after TLR3 activation. Cells from healthy controls were compared with cells from a patient with a known genetic etiology of HSE (UNC-93B-/-) and also to cells from an HSE patient with an unknown gene defect. Consistent with known variation in susceptibility of individuals to viral infections, substantial variation in the response level of different healthy controls was observed, but common functional networks could be identified, including upregulation of superoxide dismutase 2. The 2 patients with HSE studied show clear differences in functional response networks when compared with healthy controls and also when compared with each other. The present study delineates a number of novel proteins, TLR3-related pathways, and cellular phenotypes that may help elucidate the genetic basis of childhood HSE. Furthermore, our results reveal superoxide dismutase 2 as a potential therapeutic target for amelioration of the neurologic sequelae caused by HSE.

Creating a tumor-resistant microenvironment: Cell-mediated delivery of TNFα completely prevents breast cancer tumor formation in vivo

Here, we provide the necessary proof of concept, that it is possible to metabolically create a non-permissive or “hostile” stromal microenvironment, which actively prevents tumor engraftment in vivo. We developed a novel genetically engineered fibroblast cell line that completely prevents tumor formation in mice, with a 100% protection rate. No host side effects were apparent. This could represent a viable cellular strategy for preventing and treating a variety of human cancers. More specifically, we examined the autocrine and paracrine effects of the cellular delivery of TNFα on breast cancer tumor growth and cancer metabolism. For this purpose, we recombinantly overexpressed TNFα in human breast cancer cells (MDA-MB-231) or human immortalized fibroblasts (hTERT-BJ1). Our results directly show that TNFα functions as a potent tumor suppressor. Remarkably, TNFα-expressing breast cancer cells were viable, without any significant increases in their basal apoptotic rate. However, after 4 weeks post-implantation, TNFα-expressing breast cancer cells failed to form any tumors in xenografted mice (0 tumors/10 injections), ultimately conferring 100% protection against tumorigenesis. Similarly, TNFα-overexpressing fibroblasts were also viable, without any increases in apoptosis. Significantly, complete tumor suppression was obtained by co-injecting TNFα expressing stromal fibroblasts with human breast cancer cells, indicating that paracrine cell-mediated delivery of TNFα can also prevent tumor engraftment and growth (0 tumors/10 injections). Mechanistically, TNFα induced autophagy and mitochondrial dysfunction in both epithelial cancer cells and stromal fibroblasts, preventing energy transfer from the tumor microenvironment, likely “starving” the cancer cells to death. In addition, via qRT-PCR analysis of MDA-MB-231 cells, we observed that TNFα mediated the upregulation of gene transcripts associated with inflammation and senescence [IL-1-β, IL-6, IL-8, MCP-1, COX-2, p21(WAF1/CIP1)] and downregulated known tumor-promoting genes (collagen VI and MMP2). Recombinant overexpression of TNFα receptor(s) in MDA-MB-231 cells also significantly reduced tumor growth, but was not as effective as the TNFα ligand itself in preventing tumor growth. Thus, we propose that stromal cell-mediated delivery of TNFα to human tumors [using transfected fibroblasts or mesenchymal stem cells (hMSCs)] may be a novel and effective strategy for the prevention and treatment of human cancers.

Establishment of an immortal cynomolgus macaque fibroblast cell line for propagation of cynomolgus macaque cytomegalovirus (CyCMV)

Cynomolgus macaques are widely used as an animal model in biomedical research. We have established an immortalized cynomolgus macaque fibroblast cell line (MSF-T) by transducing primary dermal fibroblasts isolated from a 13-year-old male cynomolgus macaque with a retrovirus vector expressing human telomerase reverse transcriptase (hTERT). The MSF-T cells showed increased telomerase enzyme activity and reached over 200 in vitro passages compared to the non-transduced dermal fibroblasts, which reached senescence after 43 passages. The MSF-T cell line is free of mycoplasma contamination and is permissive to the newly identified cynomolgus macaque cytomegalovirus (CyCMV). CyCMV productively infects MSF-T cells and induces down-regulation of MHC class I expression. The MSF-T cell line will be extremely useful for the propagation of CyCMV and other cynomolgus herspesviruses in host-derived fibroblast cells, allowing for the retention of host-specific viral genes. Moreover, this cell line will be beneficial for many in vitro experiments related to this animal model.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3–18) and its analog [Phe8]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity.

Functional study of Villin 2 protein expressed in longissimus dorsi muscle of Korean native cattle in different growth stages

The aim of this study was to investigate protein profiles related to the induction of adipogenesis within the bovine longissimus dorsi muscle (BLDM) by proteomic analysis. We analyzed BLDM proteins at different growth stages to clarify the physiological mechanisms of marbled muscle development in 20 head of Korean native cattle (11 month: 10 head, 17 month: 10 head). BLDM proteins were analyzed by two-dimensional electrophoresis and image analysis. Villin 2 was specifically identified by mass spectrometry and a protein search engine. Villin 2 protein expression in BLDM decreased during the fat development stage in test steers. In a Western blot cell culture study of spontaneously immortal bovine muscle fibroblasts, the abundance of Villin 2 was shown to be down-regulated during differentiation into muscle. In 3T3-L1 mouse embryonic fibroblasts, Villin 2 was decreased during differentiation into adipocytes. The results suggest that Villin 2 may be related to the induction of transdifferentiation and adipogenesis in bovine longissimus dorsi muscle.