Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.     

It's time to regulate: Coping with product‐induced nongenetic clonal instability in CHO cell lines via regulated protein expression

Clonal instability and titer loss during Chinese hamster ovary (CHO) cell line development (CLD) has several underlying causes, the most prominent of which are DNA copy number loss and DNA silencing. However, in some cases, clonal instability is due to the toxicity of the therapeutic protein(s) that clones express. Unlike DNA copy number loss, which may occur in some clones or DNA silencing that is prevalent in certain regions of the genome, the hallmark of product induced clonal instability is its manifestation in all the selected clones. To circumvent such product induced clonal instability, we have developed a vector construct that utilizes a regulated protein expression system in which the constitutive expression of the target protein(s) is prevented unless doxycycline is added to the culture. We have then successfully used this system to express, at high titers, an antibody for which constitutive expression results in clonal instability perhaps due to intracellular accumulation of the antibody. Our data shows that unlike the constitutively expressed or continuously induced clones, uninduced clones do not display instability. Furthermore, maintaining the uninduced clones in culture for months or subjecting them to freeze‐thaws did not have any effects on their titers. All together, our findings suggest that a regulated expression system could be suitable for production of difficult proteins that trigger instability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1432–1440, 2014     

Enhanced inducible mGlu1α receptor expression in Chinese hamster ovary cells

Inducible expression of the group-I metabotropic glutamate receptor (mGlu1alpha) in Chinese hamster ovary cells allows for the study of receptor density dependent effects. However, expression levels attainable with this system are lower than those reported for various brain regions and achieved by conventional (constitutive) transfection. Thus, direct comparison of mGlu1alpha receptor-mediated responses in this inducible expression system with those for receptors expressed heterologously or in vivo is compounded. We show here that inducible expression can be selectively augmented by butyrate pretreatment to levels approaching those reported for cerebral tissue. Enhanced mGlu1alpha receptor protein levels, agonist-induced inositol phosphate accumulation, as well as single-cell inositol 1,4,5-trisphosphate production and intracellular Ca(2+) mobilization occurred following co-induction with butyrate. In contrast, endogenous purinoceptor function was unaffected. Importantly, the ability to titrate receptor expression by varying isopropyl beta-thiogalactoside concentration was retained. Sodium butyrate thus offers a simple and convenient method to enhance inducible gene expression to levels found in vivo.     

Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.     

重组人尿激酶原在CHO细胞中的高效表达及其纯化

用鸡β- globin的MAR序列和人看家基因延伸因子1α(hEF-1α)的调控序列以及旱獭RNA稳定与输出序列,构建了重组人尿激酶原(recombinant human pro-urokinase,rhPro - UK)的高效表达载体,在CHO细胞中获得了rhPro - UK的高效稳定表达,rhPro - UK的表达水平达到1299 IU(以百万细胞1d的表达量计).采用阳离子交换层析、疏水层析和凝胶排阻层析的三步工艺纯化表达rhPro - UK的CHO细胞培养上清液,rhPro - UK的纯度达到98％、回收率为60％ ～70％.     

Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T₃ to T₆ generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.     

Testes‐specific transgene expression in insulin‐like growth factor‐I transgenic mice

Insulin‐like growth factor‐I (IGF‐I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF‐I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF‐I over‐expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF‐I cDNA (hIGF‐I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF‐I into a variety of tissues both reproductive and non‐reproductive. Yet expression of the foreign hIGF‐I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV‐hIGF‐I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF‐I serum levels, growth rates, or testicular histology have been observed between transgenic and non‐transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV‐hIGF‐I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP‐2 and IGFBP‐3. These results suggest that the testicular over‐expression of IGF‐I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV‐hIGF‐I transgenic mice, despite the presence of elevated testicular IGF‐I. The observation that testis specific IGF‐I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function. Mol. Reprod. Dev. 54:32–42, 1999. © 1999 Wiley‐Liss, Inc.     

rSJYB1 inhibits collagen type I protein expression in hepatic stellate cells via down‐regulating activity of collagen α1 (I) promoter

YB1 is a negative regulator in liver fibrosis. We wondered whether SJYB1, a homologous protein of YB1 from Schistosoma japonicum, has an effect on liver fibrosis in vitro. Recombinant SJYB1 (rSJYB1) protein was expressed in a bacterial system and purified by Ni‐NTA His·Bind Resin. A human hepatic stellate cell line, the LX‐2 cell line, was cultured and treated with rSJYB1. The role of rSJYB1 on LX‐2 cells was then analysed by Western blot and luciferase assay. We succeeded in expressing and purifying SJYB1 in a bacterial system and the purified rSJYB1 could be recognized by S japonicum‐infected rabbit sera. Western bolt analysis showed that rSJYB1 inhibited the expression of collagen type I, but had little effect on α‐smooth muscle actin (α‐SMA). Further analysis revealed that rSJYB1 inhibited the activity of collagen α1 (I) (COL1A1) promoter and functioned at ?1592/?1176 region of COL1A1 promoter. Our data demonstrate that rSJYB1‐mediated anti‐fibrotic activity involves inhibiting the activity of COL1A1 promoter and subsequently suppressing the expression of collagen type I in hepatic stellate cells.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

人sApo2L-Fc的分子构建及其在CHO细胞中的表达

目的：构建人sApo2L-Fc分子,在中国仓鼠卵巢细胞（CHO）中表达有生物学活性的人Apo2L-Fc融合蛋白。 方法：将sApo2L-Fc基因克隆入pcDNA3.1（+）表达载体,重组质粒转化大肠杆菌DH5α。挑取阳性克隆扩大培养,提取质粒进行酶切鉴定;采用脂质体法将重组质粒转入CHO细胞,经G418加压筛选、ELISA检测,挑选表达较高的阳性转化子扩大培养;表达的sApo2L-Fc融合蛋白经Protein A亲和柱纯化,纯化产物用SDS-PAGE、Western Blotting检测样品的分子量及免疫原性,用L929细胞进行生物活性测定。 结果：酶切鉴定及测序显示重组子构建与预想一致;ELISA证实了sApo2L-Fc融合蛋白在CHO细胞中的表达;SDS-PAGE检测到纯化产物的分子量与理论分子量相符;在同样的位置,Western Blotting显示阳性;L929细胞测定：纯化产物的生物学活性达1.0×105IU/mg。 结论：构建了sApo2L-Fc的表达载体,并成功地在CHO中表达,表达的sApo2L-Fc融合蛋白具有生物学活性。     

A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase?. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase?), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

Influence of type‐I Interferon receptor expression level on the response to type‐I Interferons in human pancreatic cancer cells

Pancreatic cancer is a highly aggressive malignancy with limited treatment options. Type‐I interferons (e.g. IFN‐α/‐β) have several anti‐tumour activities. Over the past few years, clinical studies evaluating the effect of adjuvant IFN‐α therapy in pancreatic cancer yielded equivocal results. Although IFN‐α and ‐β act via the type‐I IFN receptor, the role of the number of receptors present on tumour cells is still unknown. Therefore, this study associated, for the first time, in a large panel of pancreatic cancer cell lines the effects of IFN‐α/‐β with the expression of type‐I IFN receptors. The anti‐tumour effects of IFN‐α or IFN‐β on cell proliferation and apoptosis were evaluated in 11 human pancreatic cell lines. Type‐I IFN receptor expression was determined on both the mRNA and protein level. After 7 days of incubation, IFN‐α significantly reduced cell growth in eight cell lines by 5–67%. IFN‐β inhibited cell growth statistically significant in all cell lines by 43–100%. After 3 days of treatment, IFN‐β induced significantly more apoptosis than IFN‐α. The cell lines variably expressed the type‐I IFN receptor. The maximal inhibitory effect of IFN‐α was positively correlated with the IFNAR‐1 mRNA (P < 0.05, r = 0.63), IFNAR‐2c mRNA (P < 0.05, r = 0.69) and protein expression (P < 0.05, r = 0.65). Human pancreatic cancer cell lines variably respond to IFN‐α and ‐β. The expression level of the type‐I IFN receptor is of predictive value for the direct anti‐tumour effects of IFN‐α treatment. More importantly, IFN‐β induces anti‐tumour effects already at much lower concentrations, is less dependent on interferon receptor expression and seems, therefore, more promising than IFN‐α.     

Triptolide inhibits cyclooxygenase-2 and inducible nitric oxide synthase expression in human colon cancer and leukemia cells

Triptolide (TP),a traditional Chinese medicine,has been reported to be effective in thetreatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines.Thisstudy investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia(K562),and elucidates the possible molecular mechanism involved.SW114 and K562 cells were treatedwith different doses of TP (0,5,10,20,or 50 ng/ml).The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation ofboth tumor cell lines in a dose-dependent manner.To further investigate its mechanisms,the productsprostaglandin E_2 (PGE2) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay(ELISA).Our data showed that TP strongly inhibited the production of NO and PGE_2. Consistent with theseresults,the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulatedboth at the mRNA level and the protein expression level,as shown by real-time RT-PCR and Westernblotting.These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activitycould be involved in the antitumor mechanisms of TP.