共查询到20条相似文献,搜索用时 31 毫秒
1.
Bcl‐xL, 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‐xL overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐xL 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‐xL overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐xL 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‐xL 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. 相似文献
2.
Previously, the expression of caspase-3 siRNA could not effectively inhibit sodium butyrate (NaBu)-induced apoptotic cell death of recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Caspase-3 siRNA expressing cells appeared to compensate for the lack of caspase-3 by increasing active caspase-7 levels. For the successful inhibition of NaBu-induced apoptosis of rCHO cells, both caspase-3 and caspase-7 were down-regulated using the siRNA expression vector system. Co-down-regulation of caspase-3 and caspase-7 increased cell viability and extended culture longevity in serum-free culture in the presence or absence of 1mM NaBu addition. In the cultures with 1mM NaBu addition, the maximum hTPO concentration in rCHO cells with down-regulation of both caspases was approximately 55% higher than that in rCHO cells without down-regulation of caspases and approximately 16% higher than rCHO cells with down-regulation of only caspase-3. However, in the culture with 3mM NaBu, this strategy could not dramatically enhance the culture longevity and hTPO production, compared to Bcl-2 overexpression. The different result in hTPO production between down-regulation of caspases and Bcl-2 overexpression may be because the down-regulation of caspase-3 and caspase-7, unlike Bcl-2 overexpression, could not maintain mitochondrial membrane potential in the presence of 3mM NaBu. Taken together, co-down-regulation of caspase-3 and caspase-7 is effective in regard to extension of culture longevity and enhancement of hTPO production in a serum-free culture in the presence or absence of 1mM NaBu addition. 相似文献
3.
The effect of growth factor (GF) and sodium butyrate (NaBu) on Chinese hamster ovary (CHO) cell growth, cell viability and antibody production was investigated using shaking flasks in GF-containing and GF-deficient medium containing 0, 1 and 3 mM NaBu. The withdrawal of GF and the addition of NaBu suppressed cell growth, but they significantly increased specific antibody productivity, qAb. Interestingly, the withdrawal of GF in combination with the addition of NaBu markedly retarded cell death, leading to extended culture longevity. For instance, at 3 mM NaBu, cell viability fell below 80% after day 4 in GF-containing medium, but it remained over 80% until day 18 in GF-deficient medium. Due to the enhanced qAb and the extended culture longevity, approximately 2-fold increase in total antibody production was achieved in pseudo-perfusion culture with 1 mM NaBu in GF-deficient medium, compared to the culture in GF-containing medium. The effect of GF and NaBu on the change in the expression and activity of cellular proteins, c-Myc, Bcl-2 and pyruvate dehydrogenase (PDH), was also investigated. Both the withdrawal of GF and the addition of NaBu decreased the expression of c-Myc. The expression of Bcl-2 was enhanced by the addition of NaBu in a dose-dependent manner while it was not affected by the withdrawal of GF. In addition, both the withdrawal of GF and the addition of NaBu reduced metabolic rates, qGlc, qLac and YLac/Glc, and increased PDH activity while not affecting PDH expression, suggesting that they may reduce the glycolytic rates, but enhance the conversion rates of pyruvate to TCA intermediates. Taken together, the withdrawal of GF in combination with the addition of NaBu can be considered as a relevant strategy for alleviating NaBu-induced cell apoptosis and enhancing antibody production since it can be easily implemented as well as enhance qAb and extend culture longevity. 相似文献
4.
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 (qEPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The qEPO 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 qEPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the qEPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
5.
Previous research showed that co-down-regulation of caspase-3/7 in rCHO cells, unlike Bcl-2 overexpression, did not effectively block apoptotic cell death induced by 3mM sodium butyrate (NaBu). Here, it is found that the control of autophagy is also related to this different response to NaBu treatment. With NaBu treatment, co-down-regulation of caspase-3/7 enhanced autophagy induction, whereas Bcl-2 overexpression delayed onset of autophagy induction in a Beclin-1 independent manner. The blockage of autophagy showed a detrimental effect on cell viability even in the Bcl-2 overexpressing cells, which suggests the importance of autophagy control for successful anti-cell death engineering of rCHO cells. 相似文献
6.
When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO. 相似文献
7.
Fangfang Zheng Kaiwen He Xin Li Dan Zhao Fei Sun Yu Zhang Dan Nie Xingda Li Wenfeng Chu Yan Sun Yanjie Lu 《Bioscience reports》2013,33(2)
NPC (nasopharyngeal carcinoma) is a common malignancy in southern China without defined aetiology. Recent studies have shown that TGFBR3 (transforming growth factor type III receptor, also known as betaglycan), exhibits anticancer activities. This study was to investigate the effects of TGFBR3 on NPC growth and the mechanisms for its actions. Effects of TGFBR3 overexpression on cell viability and apoptosis were measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], AO/EB (acridine orange/ethidium bromide) staining and electron microscopy in human NPC CNE-2Z cells. The expression of apoptosis-related proteins, p-Bad, Bad, XIAP (X-linked inhibitor of apoptosis), AIF (apoptosis-inducing factor), Bax and Bcl-2, was determined by Western blot or immunofluorescence analysis. Caspase 3 activity was measured by caspase 3 activity kit and [Ca2+]i (intracellular Ca2+ concentration) was detected by confocal microscopy. Transfection of TGFBR3 containing plasmid DNA at concentrations of 0.5 and 1 μg/ml reduced viability and induced apoptosis in CNE-2Z in concentration- and time-dependent manners. Forced expression of TGFBR3 up-regulated pro-apoptotic Bad and Bax protein, and down-regulated anti-apoptotic p-Bad, Bcl-2 and XIAP protein. Furthermore, transient overexpression of TGFBR3 also enhanced caspase 3 activity, increased [Ca2+]i and facilitated AIF redistribution from the mitochondria to the nucleus in CNE-2Z cells, which is independent of the caspase 3 pathway. These events were associated with TGFBR3-regulated multiple targets involved in CNE-2Z proliferation. Therefore transient overexpression of TGFBR3 may be a novel strategy for NPC prevention and therapy. 相似文献
8.
Sodium butyrate (NaBu) can enhance the expression of genes controlled by some of the mammalian promoters, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on a foreign protein expression is compromised by its cytotoxic effect on cell growth. To overcome this cytotoxic effect of NaBu, the expression vector of antisense RNA of caspase-3 was constructed and transfected to recombinant Chinese hamster ovary (rCHO) cells producing a humanized antibody. Using this antisense RNA strategy, rCHO cells (B3) producing a low level of caspase-3 proenzyme were established. When batch cultures of both B3 cells and control cells transfected with antisense RNA-deficient plasmid were performed in the absence of NaBu, both cells showed similar profiles of cell growth and antibody production. Compared with control cell culture, under the condition of 5 mM NaBu addition at the exponential growth phase, expression of antisense RNA of caspase-3 significantly suppressed the NaBu-induced apoptosis of B3 cells and extended culture longevity by >2 days if the culture was terminated at cell viability of 50%. However, compared with control cell culture, the final antibody concentration of B3 cell culture was not increased in the presence of NaBu, which may be due to the loss of cellular metabolic capability resulted from the depolarization of mitochondrial membrane. Taken together, this study suggests that, although expression of antisense RNA of caspase-3 does not improve antibody productivity of rCHO cells, it can suppress NaBu-induced apoptotic cell death of rCHO cells and thereby may reduce problems associated with cellular disintegration. 相似文献
9.
10.
Yeon‐Gu Kim Jee Yon Kim Chaya Mohan Gyun Min Lee 《Biotechnology and bioengineering》2009,103(4):757-766
Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐xL overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐xL overexpression (EPO‐off‐Bcl‐xL) was established using the Tet‐off system. The expression level of Bcl‐xL in EPO‐off‐Bcl‐xL cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐xL overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐xL overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐xL overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐xL revealed that Bcl‐xL interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐xL overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc. 相似文献
11.
Fei Chen Tianci Kou Li Fan Yan Zhou Zhaoyang Ye Liang Zhao Wen-Song Tan 《Biotechnology and Bioprocess Engineering》2011,16(6):1157-1165
Cell cultures containing 0 ∼ 5 mM sodium butyrate (NaBu) and grown at 30 and 37°C were conducted to investigate the combined
effect of NaBu and low temperature on the quantity and quality of an antibody production in CHO cells. Although NaBu addition
decreased cell viability by apoptosis in a dose-dependent manner at both 30 and 37°C, the onset of significant apoptosis induced
by NaBu was delayed by lowering culture temperature. The highest specific antibody productivity (q
p) of 23.26 pg/cell/day was obtained in the culture containing 2 mM NaBu at 30°C; however, the highest antibody concentration
of 167.84 mg/L was achieved in the culture containing 1 mM NaBu at 30°C, as the detrimental effect of further NaBu addition
on cell growth compromised its beneficial effect on q
p. Moreover, protein quality with respect to the total sialic acid content and Nglycolylneuraminic acid (Neu5Gc) level was
evaluated. There were no apparent changes regarding the total sialic acid content of the antibody, but manipulation of cultures
with NaBu treatment or (and) low culture temperature did decrease Neu5Gc levels by 5 ∼ 10%. Biological activity of the antibody
was also assessed, and no obvious changes were observed. Collectively, the simultaneous application of NaBu and low culture
temperature was an effective way to extend culture period and enhance final antibody concentration, without compromising the
sialic acid content or biological activity. 相似文献
12.
Diego Sobradillo Miriam Hernández-Morales Daniel Ubierna Mary P. Moyer Lucía Nú?ez Carlos Villalobos 《The Journal of biological chemistry》2014,289(42):28765-28782
We have investigated the molecular basis of intracellular Ca2+ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca2+ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca2+ levels, agonist-induced Ca2+ increases, store-operated Ca2+ entry (SOCE), and store-operated currents (ISOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca2+ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca2+ release-activated Ca2+ currents (ICRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced ICRAC plus a nonselective ISOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca2+ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca2+ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca2+ remodeling and tumor features in colon cancer. 相似文献
13.
14.
MicroRNA‐328 is involved in the effect of selenium on hydrogen peroxide‐induced injury in H9c2 cells
Xiaolin Zheng Xiaoyan Hu Tangdong Ge Mengdi Li Minxia Shi Jincheng Luo Hehuan Lai Tingting Nie Fenglan Li Hui Li 《Journal of biochemical and molecular toxicology》2017,31(8)
Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress‐induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura‐2AM was used to calculate intracellular Ca2+ concentration. Sodium selenite could ameliorate hydrogen peroxide (H2O2)‐induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H2O2 or sodium selenite decreased miR‐328 levels. MiR‐328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca2+ concentration, while inhibition produced opposite effects. MiR‐328 might be involved in the effect of sodium selenite on H2O2‐induced cell death in H9c2 cells. 相似文献
15.
16.
17.
Sodium butyrate (NaBu) can enhance the expression of foreign protein of recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. Thus, the beneficial effect of using a higher concentration of NaBu on foreign protein expression in rCHO cells is compromised by its growth inhibitory and cytotoxic effects. To overcome this cytotoxic effect of NaBu, an expression vector of small interfering RNAs (siRNAs) targeting against caspase-3, a key effector component in apoptosis, was constructed and transfected into rCHO cells producing human thrombopoietin (hTPO). Using this siRNA strategy, rCHO cells (F21 cells) expressing a low level of caspase-3 proenzyme determined by RT-PCR and Western blot analysis were established. Under the condition of 1-5 mM NaBu addition at the exponential growth phase, down-regulation of caspase-3 in F21 cells could not effectively inhibit NaBu-induced apoptotic cell death. This NaBu-induced apoptotic cell death occurred because F21 cells appeared to compensate for the lack of caspase-3 by increasing the active caspase-7 level. These results suggest that the intracellular caspase's interconnectivity should be taken into consideration for the successful inhibition of apoptosis of rCHO cells. 相似文献
18.
19.
Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments. 相似文献
20.
Previously, overexpression of anti‐apoptotic proteins, such as E1B‐19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl‐xL, a well‐known anti‐apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody‐producing rCHO cell lines with regulated Bcl‐xL overexpression (CS13*‐0.02‐off‐Bcl‐xL and CS13*‐1.00‐off‐Bcl‐xL) were established using the Tet‐off system. When cells were cultivated without Bcl‐xL overexpression, the specific lactate production rate (qLac) of CS13*‐0.02‐off‐Bcl‐xL and CS13*‐1.00‐off‐Bcl‐xL were 7.32 ± 0.37 and 6.78 ± 0.56 pmol/cell/day, respectively. Bcl‐xL overexpression, in the absence of doxycycline, did not affect the qLac of either cell line, though it enhanced the viability during cultures. Furthermore, activities of the enzymes related to glucose and lactate metabolism, such as hexokinase, glucose‐6‐phosphate dehydrogenase, lactate dehydrogenases, and alanine aminotransferase, were not affected by Bcl‐xL overexpression either. Taken together, Bcl‐xL overexpression showed no significant effect on the lactate metabolism of rCHO cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1594–1598, 2013 相似文献