Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

Background  

The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of fut8 gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat fut8 gene.     

Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state

gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.     

Methylation mediated Gadd45β enhanced the chemosensitivity of hepatocellular carcinoma by inhibiting the stemness of liver cancer cells

Background

Defects of the growth arrest DNA damage-inducible gene 45β (Gadd45β) play an important role in the progression of tumor and confer resistance to chemotherapy. However, the role of Gadd45β in the apoptosis of hepatocellular carcinoma is still not clear. Purpose of this study was to explore the effect of Gadd45β on the apoptosis of liver cancer cells, and the possible mechanism was examined.

Result

In this study, we first confirmed the decreased expression of Gadd45β in human liver cancer tissues and human liver cancer cell lines, when compared to the peri-tumor liver tissue and normal liver cells. And, it was found that Gadd45β could inhibit the stemness of liver cancer cells, enhancing the apoptosis of cancer cells induced by chemotherapy. Furthermore, the results showed that HCC tissues and cell lines showed a higher methylation status in Gadd45β promoter than that in peri-tumor tissues and normal liver cells. Methylation was then reversed by pretreatment of SMMC-7721 and Hep-3B with 5-azacytidine which is the DNA methyltransferase inhibitor. And the 5-azacytidine decreased the stemness of SMMC-7721 and Hep-3B, enhanced the sensitivity of SMMC-7721 and Hep-3B to cisplatin.

Conclusions

Methylation mediated Gadd45β expression inhibited the stemness of liver cancer cells, promoting the chemotherapy-induced apoptosis. Thus Gadd45β may be the potential target for enhancing the chemosensitivity of human hepatocellular carcinoma.

     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand

Background

The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production.

Results

In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water.

Conclusion

Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.     

Optimization of the <Emphasis Type="Italic">Lactococcus lactis</Emphasis> nisin-controlled gene expression system NICE for industrial applications

Background  

The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein.     

Hepatocyte proliferation/growth arrest balance in the liver of mice during E. multilocularis infection: a coordinated 3-stage course

Background

Alveolar echinococcosis (AE) is characterized by the tumor-like growth of Echinococcus (E.) multilocularis. Very little is known on the influence of helminth parasites which develop in the liver on the proliferation/growth arrest metabolic pathways in the hepatocytes of the infected liver over the various stages of infection.

Methodology/Principal Findings

Using Western blot analysis, qPCR and immunohistochemistry, we measured the levels of MAPKs activation, Cyclins, PCNA, Gadd45β, Gadd45γ, p53 and p21 expression in the murine AE model, from day 2 to 360 post-infection. Within the early (day 2–60) and middle (day60–180) stages, CyclinB1 and CyclinD1 gene expression increased up to day30 and then returned to control level after day60; Gadd45β, CyclinA and PCNA increased all over the period; ERK1/2 was permanently activated. Meanwhile, p53, p21 and Gadd45γ gene expression, and caspase 3 activation, gradually increased in a time-dependent manner. In the late stage (day180–360), p53, p21 and Gadd45γ gene expression were significantly higher in infected mice; JNK and caspase 3 were activated. TUNEL analysis showed apoptosis of hepatocytes. No significant change in CyclinE, p53 mRNA and p-p38 expression were observed at any time.

Conclusions

Our data support the concept of a sequential activation of metabolic pathways which 1) would first favor parasitic, liver and immune cell proliferation and survival, and thus promote metacestode fertility and tolerance by the host, and 2) would then favor liver damage/apoptosis, impairment in protein synthesis and xenobiotic metabolism, as well as promote immune deficiency, and thus contribute to the dissemination of the protoscoleces after metacestode fertility has been acquired. These findings give a rational explanation to the clinical observations of hepatomegaly and of unexpected survival of AE patients after major hepatic resections, and of chronic liver injury, necrosis and of hepatic failure at an advanced stage and in experimental animals.     

Propofol alleviates hypoxia-induced nerve injury in PC-12 cells by up-regulation of microRNA-153

Background

Although the neuroprotective role of propofol has been identified recently, the regulatory mechanism associated with microRNAs (miRNAs/miRs) in neuronal cells remains to be poorly understood. We aimed to explore the regulatory mechanism of propofol in hypoxia-injured rat pheochromocytoma (PC-12) cells.

Methods

PC-12 cells were exposed to hypoxia, and cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry assay/Western blot analysis, respectively. Effects of propofol on hypoxia-injured cells were measured, and the expression of miR-153 was determined by stem-loop RT-PCR. After that, whether propofol affected PC-12 cells under hypoxia via miR-153 was verified, and the downstream protein of miR-153 as well as the involved signaling cascade was finally explored.

Results

Hypoxia-induced decrease of cell viability and increase of apoptosis were attenuated by propofol. Then, we found hypoxia exposure up-regulated miR-153 expression, and the level of miR-153 was further elevated by propofol in hypoxia-injured PC-12 cells. Following experiments showed miR-153 inhibition reversed the effects of propofol on hypoxia-treated PC-12 cells. Afterwards, we found BTG3 expression was negatively regulated by miR-153 expression, and BTG3 overexpression inhibited the mTOR pathway and AMPK activation. Besides, hypoxia inhibited the mTOR pathway and AMPK, and these inhibitory effects could be attenuated by propofol.

Conclusion

Propofol protected hypoxia-injured PC-12 cells through miR-153-mediataed down-regulation of BTG3. BTG3 could inhibit the mTOR pathway and AMPK activation.

     

Syncytins expression in cultured trophoblast cells according to differentiation status

Background  

Data of syncytin 1 and 2 env gene expression in human placenta and participation in the syncytialisation phenomena has been reported. However, there are not many studies on simultaneous changes in expression of both syncytins in culture. We sought evidence on the relative expression of syncytins and syncytin 1 receptors in trophoblast cell culture treated with a differentiation inducing factor (forskolin).     

PI3-K/Akt pathway contributes to IL-6-dependent growth of 7TD1 cells

Background  

Recently, growing evidence suggests the involvement of PI 3-K/Akt in IL-6-dependent survival and proliferative responses in several types of cells. However, whether PI 3-K/Akt plays the same role in IL-6-dependent growth of 7TD1 mouse-mouse B cell hybridoma is not known.     

Open field trial of genetically modified parthenocarpic tomato: seedlessness and fruit quality

Background

Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state.

Results

As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin μ heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer.

Conclusion

This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.     

Gadd45a is an RNA binding protein and is localized in nuclear speckles

Background

The Gadd45 proteins play important roles in growth control, maintenance of genomic stability, DNA repair, and apoptosis. Recently, Gadd45 proteins have also been implicated in epigenetic gene regulation by promoting active DNA demethylation. Gadd45 proteins have sequence homology with the L7Ae/L30e/S12e RNA binding superfamily of ribosomal proteins, which raises the question if they may interact directly with nucleic acids.

Principal Findings

Here we show that Gadd45a binds RNA but not single- or double stranded DNA or methylated DNA in vitro. Sucrose density gradient centrifugation experiments demonstrate that Gadd45a is present in high molecular weight particles, which are RNase sensitive. Gadd45a displays RNase-sensitive colocalization in nuclear speckles with the RNA helicase p68 and the RNA binding protein SC35. A K45A point mutation defective in RNA binding was still active in DNA demethylation. This suggests that RNA binding is not absolutely essential for demethylation of an artificial substrate. A point mutation at G39 impared RNA binding, nuclear speckle localization and DNA demethylation, emphasizing its relevance for Gadd45a function.

Significance

The results implicate RNA in Gadd45a function and suggest that Gadd45a is associated with a ribonucleoprotein particle.     

Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts

Introduction  

Rheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation.     

Sources of variability and effect of experimental approach on expression profiling data interpretation

Background  

We provide a systematic study of the sources of variability in expression profiling data using 56 RNAs isolated from human muscle biopsies (34 Affymetrix MuscleChip arrays), and 36 murine cell culture and tissue RNAs (42 Affymetrix U74Av2 arrays).