Morphological alteration of X-ray induced partially transformed human cells by transfection with a small c-myc DNA sequence

During attempts to transform a normal human fibroblast strain (GM730) by X-irradiation, we obtained a partially transformed cell strain (GM730pt) which demonstrates several aspects of the transformed phenotype including morphological changes, increased saturation density, growth in soft agar, and focus formation in long-term cultures. When GM730pt cells were transfected with the feline c-myc gene, morphology of the cells changed dramatically following seven days of expression. Transfection of other plasmid DNAs or oncogenes such as pUC8, pSV2neo, src, sis, and H-ras had little or no effects on the phenotype of GM730pt cells. On the other hand, a gel purified, small fragment of c-myc DNA had a complete cell alteration activity. Furthermore, Bal 31 deletion and M13 sequencing experiments showed that the alteration seen in GM730pt cells is delimited to a 24 nucleotide stretch (active myc element) from the second intron of the feline c-myc gene that contains a T-rich sequence.     

Proteome annotations and identifications of the human pulmonary fibroblast

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray-mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-beta, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed.     

Proteome analysis of human mesothelial cells during epithelial to mesenchymal transitions induced by shed menstrual effluent

Peritoneal endometriosis is the result of ectopic implantation and growth of endometrium tissue that has been regurgitated into the abdominal cavity during menstruation. We have previously shown that menstrual effluent induces epithelial to mesenchymal transitions (EMT) in mesothelial cells, which results in cell retraction and exposure of submesothelial extracellular matrix. Since endometrial tissue preferentially adheres to the extracellular matrix, adhesion of endometrial tissue to the peritoneum is facilitated. The EMT were shown to be associated with differential expression and phosphorylation of mesothelial proteins. Using radiolabeling and proteomics we detected changes in protein expression and phosphorylation that occur in mesothelial cells during the EMT process. The identity of 73 proteins, which were obtained from 324 analyzed spots, was confirmed. The expression of 35 proteins involved in organization of the cytoskeleton, signal transduction, regulation of the redox state, and production of ATP, was altered during the EMT process. Four of the identified proteins were differentially phosphorylated: annexin-1, an actin-binding protein and a substrate for receptor tyrosine kinases; tropomyosin-alpha, a regulator of actin filament stability and cell shape; elongation factor 1 delta; ATP synthase beta-chain. In conclusion, factors from menstrual effluent induce specific changes in the expression and phosphorylation status of structural, regulatory and metabolic proteins relevant to the complex process of EMT in mesothelial cells.     

Differential initiation of innate immune responses induced by human cytomegalovirus entry into fibroblast cells

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (alphabeta IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-kappaB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with beta(1) integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.     

A comparison of markers of human fibroblast transformation induced by chemical carcinogen treatment or by transfection of an origin-defective SV40-containing plasmid

We have investigated the sensitivity to oncogenic transformation by an origin-defective SV40-containing plasmid, '8-16' (ori-SV40), of skin fibroblasts from normal individuals (NF), and from patients with 2 hereditary diseases characterized by an increased cancer risk, Bloom's syndrome (BS) and Fanconi's anemia (FA). It was hypothesized that perhaps these cells had already undergone some stage, or stages, of the progression to neoplasia, and that as a consequence of these changes, one could observe differential expression of characteristics of the transformed phenotype in these cells compared to normal, or perhaps they would behave differently in vivo. The data showed that FA cells and NF possessed comparable sensitivities to transformation by ori-SV40 DNA transfection, as measured either by focus formation above a confluent monolayer, or anchorage-independent growth. The BS cells, on the other hand, were 5-10 times less sensitive to this method of transformation, and further, the transformed phenotype was unstable. The resistance of BS cells to transformation by the 8-16 plasmid may be a reflection of their inherent genetic instability which affects stable integration and expression of the transfected plasmid DNA, since no differences in initial uptake of transfected DNA were observed between the various cell strains. Immortality and tumorigenicity were not readily demonstrated in this ori-SV40 transformation model. The results are discussed in relationship to the characteristics of the transformed phenotype of chemically treated normal human fibroblasts. SV40, an agent known to transform human cells, can be cast in a positive control role with respect to the appropriateness of the assays, the frequency of appearance of various markers, immortality and tumorigenicity. The tumorigenicity results are further compared to results obtained during the establishment of a wide range of fresh human tumor biopsies as xenograft lines in athymic nude mice, with particular emphasis on the sarcoma data.     

Protein alteration of HepG2.2.15 cells induced by iron overload

Hepatitis B can progress into hepatocellular carcinoma. Body irons may interfere with the clearance of hepatitis B virus (HBV) and contribute to genesis of tumor. To investigate the role of iron played in HBV-related pathogenesis, here we studied the effect of iron with different concentrations and valence states on growth of HepG2.2.15 cells and secretion of virus proteins. A strong tolerance of HepG2.2.15 cells to iron challenge was found. The concentration of hepatitis B surface antigen in cell culture medium was decreased after iron stimulation. Lower concentrations of iron facilitated hepatitis B e-antigen (HBeAg) secretion. Fe(2+) appeared more effective on HBeAg secretion than Fe(3+) did. In parallel, the differential protein profiles in HepG2.2.15 cells were studied by iTRAQ and LC-MS/MS. The differentially expressed proteins were mainly involved in stress response, signal transduction, apoptosis, etc. Four proteins (14-3-3 β/α, VCP, migration inhibitory factor, and Nup153) were verified by Western-blotting and found to be consistent with the iTRAQ data. Interestingly, nuclear import of Nuclear factor kappa B (NFκB) and its activity were found to be affected by the decreased Nup153 in iron stimulated HepG2.2.15 cells. The results may indicate possible molecular mechanism how the synergism of HBV and iron stimulation damages host liver cells.     

Differential gene expression of human keratinocyte HaCaT cells induced by fibroblast growth factor 10 treatment

Fibroblast growth factor 10 (FGF10) has multiple biological activities involved in angiogenesis, mitogenesis, cellular differentiation, development, and tissue injury repair. Our previous studies revealed that treatment of FGF10 remarkably stimulated HaCaT cell proliferation and abbreviated cell apoptosis. However, the molecular mechanisms remain largely unknown. The aim of this study was to investigate FGF10-induced modifications in gene expression in the HaCaT cells by using the cDNA microarray technique. The microarray data showed that FGF10 modified the expression of 2117 genes, 861 being up-regulated and 1256 down-regulated, using a threshold of twofold. Eight of nine candidate genes, validated by real-time quantitative polymerase chain reaction (qPCR), were correlated well with the array data. The GenMAPP and MappFinder software packages were further used for pathway analysis of these significantly altered genes. In support of multiple biological functions for FGF10, several gene pathways were found to be involved in processes of cell cycle, DNA repair, apoptosis, development, and wound healing. These data also provide a basis to further investigation of FGF10 molecular mechanisms.     

Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

Background  

The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.     

Proteome alteration of U251 human astrocytoma cell after inhibiting retinoic acid synthesis

Retinoic acid (Ra) is crucial for the patterning and neuronal differentiation in the central nervous system (CNS). Ra deficiency in animals disrupts the motor activities and memory abilities. The molecular mechanisms underlying these behavior abnormalities remain largely unknown. In the current study, we treated the astrocytoma cells with citral, an inhibitor of Ra synthesis. We analyzed the differences in the protein concentrations between the treated and untreated astrocytoma cells by two-dimensional gel electrophoresis (2-DE), Imagemaster software, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In total, 39 of 46 altered protein spots with significant mascot scores were identified representing 36 proteins, that were involved in significantly altered glutamate metabolism, lipid metabolism, mitochrondrial function, and oxidative stress response by Ingenuity Pathway Analysis (IPA). Altered 3-phosphoglycerate dehydrogenase (PHGDH) was also observed in western blot. These data provide some clues for explaining the behavioral changes caused by Ra deficiency, and support the hypothesis that Ra signaling is associated with some symptoms of neurodegenerative disorders and schizophrenia.     

Functional characterization of human T cells immortalized by oncogene transfection

We have succeeded in immortalizing human lymphocytes derived from the peripheral blood of a healthy donor and of an atopic patient, and from the lymph node of a cancer patient by oncogene transfection (Alam et al., 1996). All immortalized human lymphocytes were shown to be CD3+ and CD19–, indicating that these immortalized human lymphocytes were all T cells. We established 317, 154 and 692 individual immortalized human T cell lines derived from the healthy donor, the atopic patient and the cancer patient, respectively. The ratios of CD4+ and CD8+ subpopulations within the set containing immortalized T cells derived from the healthy donor were shown to be varied depending on the combinations of transfected oncogenes used. However, CD8+ cells were found to be the dominant subpopulation of immortalized T cells derived from the atopic patient and the cancer patient. These immortalized T cells showed different proliferative responses in the presence of exogenous human IL–2 depending on their origin, and was consistent with the surface expression of the IL–2 receptor. Furthermore, the cytokine secretion patterns of these immortalized T cells stimulated with mitogen were investigated. The results showed that the immortalized T cells from the healthy donor is able to secrete various kinds of cytokines such as IL–2, IL–10, -IFN and GM-CSF. However, immortalized T cells from the cancer patient was shown to only secrete IL–2 and GM-CSF. These results suggest that depending on the origin, the immortalized T cells came from different subsets or from cells in different activated states. Mixed lymphocytes reactions demonstrated that these immortalized T cells are able to proliferate in the presence of allogenic or xenogenic stimulator cells, suggesting that they maintain the ability to recognize specific antigens on the stimulator cells and can proliferate even after the immortalization. Furthermore, immortalized T cells derived from the healthy donor and the cancer patient strongly responded to K562 cells, suggesting that MHC-nonrestricted killer T cells were also immortalized.Abbreviations IL–2R – interleukin 2 receptor; MLR – mixed lymphocyte reaction     

High-efficiency transfection of human endothelial cells mediated by cationic lipids

Amyloid beta peptide is recognized as the main constituent of the extracellular amyloid plaques, the major neuropathological hallmark of Alzheimer's disease. Abeta is a small peptide constitutively expressed in normal cells, not toxic in the monomeric form but aggregated Abeta is assumed to be the main if not the only factor causing Alzheimer's disease. Interestingly, the new reports suggest neurotoxicity of soluble Abeta oligomers rather than amyloid fibrils. Because of the fact that fibrils were thought to be the main toxic species in AD, early structural studies focused on fibrils themselves and Abeta monomers, as their building blocks while there is practically no data on oligomer structure and mechanism of neurotoxicity. Using a model peptide spanning residues 10–30 of Abeta, obtained by overexpression in bacteria, we have employed mass spectrometry of noncovalent complexes and disulfide rearrangement assay to gain new insight into structure and dynamics of a prenucleation step of Abeta peptide oligomerisation.     

Fatty acids and the selective alteration of in vitro proliferation in human fibroblast and guinea-pig smooth-muscle cells

Summary Human-foreskin fibroblast (HF) and guinea-pig aorta smooth-muscle (SM) cultures were treated with several saturated and unsaturated fatty acids. Relative plating efficiencies were used to determine the proliferative response to each treatment. At low concentrations (16 to 18 μm), proliferation in HF cultures was inhibited by 8,11,14-eicosatrienoic acid (20:3), and stimulated by both 5,8,11,14-eicosatetraenoic acid (20:4) and 9-octadecenoic acid (18:1). At these levels, proliferation in SM cultures was unchanged by 20:3, inhibited by 20:4, and enhanced by 18:1. At higher concentrations (80 to 90 μm), HF cultures were inhibited by all three unsaturated fatty acids. At these same concentrations, proliferation in SM cultures was inhibited by 20:3 and 20:4, whereas 18:1 continued to stimulate proliferation. Thus proliferative response was a specific effect of the fatty acid used, its concentration, and the cell line involved. Further treatment of SM cultures by tetradecanoic acid (14:0), hexadecanoic acid (16:0), and octadecanoic acid (18:0) showed that their relative abilities to inhibit cell proliferation increased with increasing chain length. Concentrations required for the effective inhibition of proliferation in SM cultures by 14:0, 16:0 and 18:0 were 220 μm, 95μm and 18μm, respectively. The fatty acids used in these studies are all endogenous components of sera used as growth supplements in in vitro systems. Their roles as prostaglandin and hydroperoxy fatty-acid precursors (20:3 and 20:4), inhibitors of prostaglandin biosynthesis (18:1), or as calcium ionophores (14:0, 16:0, and 18:0) may allow them to function as endogenous controls of cell proliferation. This work was supported in part by National Heart and Lung Institute Grant HL-11897.     

Current standards and pitfalls associated with the transfection of primary fibroblast cells

Cultured fibroblast cells, especially dermal cells, are used for various types of scientific research, particularly within the medical field. Desirable features of the cells include their ease of isolation, rapid cellular growth, and high degree of robustness. Currently, fibroblasts are mainly used to obtain pluripotent cells via a reprogramming process. Dermal fibroblasts, are particularly useful for gene therapies used for promoting wound healing or minimizing skin aging. In recent years, fibroblast transfection efficiencies have significantly improved. In order to introduce molecules (most often DNA or RNA) into cells, viral-based systems (transduction) or non-viral methods (transfection) that include physical/mechanical processes or lipid reagents may be used. In this article, we describe critical points that should be considered when selecting a method for transfecting fibroblasts. The most effective methods used for the transfection of fibroblasts include both viral-based and non-viral nucleofection systems. These methods result in a high level of transgene expression and are superior in terms of transfection efficacy and viability.     

A cell surface alteration in mouse L cells induced by interferon

Mouse L cells grown in suspension culture when treated with L cell interferon have a greater electrophoretic mobility toward the anode than control cells. This change in electrophoretic mobility depends on the concentration of interferon in the medium and the duration of interferon interaction with the cells. It is concluded that the interferon-treated cells have a greater net negative charge on the cell surface than control cells and it is suggested that the cell surface is altered because of the interaction with interferon.     

Proteome analysis of X-ray irradiated human erythroleukemia cells

In order to discover molecular biomarkers in radiation response we investigated the effects of X-radiation on radioresistant K562 cells by using a comparative proteomic analysis. In treated cells 29 up-regulated and 10 down-regulated proteins were detected by image analysis and identified by mass spectrometry. Elongation factor 1 alpha 1 and stress-70 protein showed a 6.2 and 5.4 fold increase respectively in treated cells. Additional proteins such us pi and omega classes glutathione transferases, ATP synthase D chain, were also found to be up-regulated, suggesting that the enzyme belonging to the cellular detoxification system against oxidative stress and energetic metabolism may have a key role in the cellular response to radiation injury. This data set may provide a useful tool to design a combined chemo- and radiotherapic strategy against leukemia disease.     

Improved transfection efficiency of cultured human cells

We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine (Life Technologies) and Effectene (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5-3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.