Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation

The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of various activated caspases demonstrated the onset of apoptosis in Chinese hamster ovary cells during prolonged cultivation was primarily through the intrinsic pathway. Differential in gel electrophoresis proteomic study comparing protein samples collected during cultivation resulted in the identification of 40 differentially expressed proteins, including four cytoskeletal proteins, ten chaperone and folding proteins, seven metabolic enzymes and seven other proteins of varied functions. The induction of seven ER chaperones and foldases is a solid indication of the onset of the unfolded protein response, which is triggered by cellular and ER stresses, many of which occur during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes that a change in the energy metabolism likely occurred as culture conditions degraded and apoptosis advanced. By identifying the intracellular changes during cultivation, this study provides a foundation for optimizing cell line-specific cultivation processes, prolonging longevity and maximizing protein production.     

Genetically engineering mammalian cell lines for increased viability and productivity

The generation of new host cell lines for the production of foreign proteins can be achieved by cell engineering. This approach can be used to enhance the cell's ability to produce proteins that are properly processed and secreted at elevated levels and consequently can increase the overall productivity of an expression system. One potential target for cell engineering is the modification of the cell's protein folding capacity. The appropriate folding, assembly, localization and secretion of newly synthesized proteins is dependent upon the action of a group of proteins known as molecular chaperones. Improving the host cell's chaperoning capacity might increase the yield of properly folded recombinant proteins by preventing the formation of insoluble aggregates. Another potentially beneficial cell engineering goal is the inhibition of physiological cell death. The productivity of genetically engineered cells is dependent upon the maintenance of high levels of cell viability throughout the bioprocess period. Fluctuations in a cell's environment can trigger a deliberate form of cell death known as apoptosis. The proteins that mediate this self-destruction are currently being characterized. Regulating the expression of these death genes by cellular engineering could limit the loss of productivity that results from the physiological death of the recombinant cell line.     

提高哺乳动物工程细胞抗凋亡能力的基因策略

利用哺乳动物细胞发酵生产重组蛋白药物具有细菌、酵母等表达系统所不具备的显著优势,因此在生物制药工程中的重要性越来越突出。哺乳动物细胞对工业生产环境下的各种应激环境耐受能力差,易发生细胞凋亡,严重阻碍了大规模生产,降低了生产效率。细胞凋亡是细胞必经的生物学过程,随着对凋亡机制的深入了解,发展出各种抗凋亡策略,并有望应用于重构更适合工业生产的工程细胞。常用的抗凋亡策略包括:下调凋亡蛋白、上调抗凋亡蛋白、增强生长因子自表达、减少有毒代谢产物生成等。以上策略虽然能在一定程度上提高细胞的抗凋亡能力,但距离满足生产的工程细胞重构还有距离,围绕提高工程细胞的抗凋亡能力,已发展出"凋亡工程"这一重要的技术领域。     

Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells

Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.     

Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection.

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy. Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade. In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells. In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls. Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases. Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures. Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs.     

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl‐2 family proteins such as Bcl‐2 and Bcl‐x_L potently inhibit apoptosis, no studies have examined the use of the Bcl‐2 family protein, Mcl‐1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl‐1 protein and a Mcl‐1 mutant protein that is not degraded by the proteasome in a serum‐free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl‐1 led to increased viabilities in fed‐batch culture, with cell lines expressing the Mcl‐1 mutant maintaining ～90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl‐1‐expressing cell lines were isolated that consistently showed increases in antibody production of 20–35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl‐1‐expressing cell lines, and Mcl‐1‐expressing cells exhibited 3‐fold lower caspase‐3 activation when compared with the control cell lines. Altogether, the expression of Mcl‐1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet

Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.     

Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry

Recently, autophagy, which is a degradative process, has drawn attention as an anti-cell death engineering target in addition to apoptosis in recombinant Chinese hamster ovary (rCHO) cell cultures for enhanced production of therapeutic proteins. Appropriate autophagy monitoring methods, that are suitable for long term CHO cell cultures, are necessary in order to investigate the culture conditions that affect the autophagy pathway and to select appropriate engineering targets for autophagy control. Herein, detailed protocols for autophagy monitoring methods based on flow cytometry are provided using the GFP-LC3-overexpressing CHO DG44 host cell line or MDC-like molecules in rCHO cells grown as an adherent culture with serum-containing medium or suspension culture with serum-free medium. Furthermore, combined with the apoptosis detection based on the Annexin V-PS interaction, the simultaneous detection of autophagy and apoptosis is also described. It is anticipated that the protocols described herein will assist in the fast, high throughput monitoring of autophagy that can support other existing autophagy assays.     

PUMA-BH3结构域短肽的原核表达、纯化及促凋亡活性鉴定

Bcl-2家族蛋白质在线粒体途径凋亡的调控机制中起着重要的作用,p53正向细胞凋亡调控因子（p53 up-regulated modulator of apoptosis protein,PUMA)是该家族的一种只含有BH3同源区域的促凋亡蛋白。为得到PUMA的BH3结构域短肽并检测其生物学活性,将人工合成的编码PUMA-BH3肽的DNA片段克隆到质粒pTYB2上,构建出表达PUMA-BH3-内含肽-几丁质结合域融合蛋白的原核表达载体pTYB2-PUMA-BH3,转化大肠杆菌BL-21(DE3)中IPTG诱导表达。表达的融合蛋白经几丁质亲和层析、二硫苏糖醇（DTT）的柱内还原,直接获得可溶性PUMA-BH3肽。通过研究重组PUMA-BH3肽在体外条件下对线粒体活力、线粒体肿胀度以及细胞色素c释放的影响来鉴定其生物学活性。结果表明,获得的可溶性PUMA-BH3肽能作用于离体线粒体,引起线粒体活力降低,线粒体肿胀并能诱导细胞色素c释放。环孢菌素A对此有一定的抑制作用,提示PUMA-BH3肽对线粒体的上述作用是通过促进通透性转运孔( PTP)开放实现的。经原核表达及纯化,获得了具有促凋亡活性的PUMA-BH3肽,为进一步研制控制凋亡过程的药物奠定了基础。     

Ribosome inactivating proteins and apoptosis

Ribosome inactivating proteins (RIPs) are protein toxins that are of plant or microbial origin that inhibit protein synthesis by inactivating ribosomes. Recent studies suggest that RIPs are also capable of inducing cell death by apoptosis. Though many reports are available on cell death induced by RIPs, the mechanism involved is not well studied. Comparison of pathways of apoptosis and cellular events induced by various RIPs suggests a central role played by mitochondria, probably acting as an integrator of cellular stress and cell death. The purpose of this review is to compare the various apoptotic pathways that may be involved and propose a general pathway in RIP-induced cell death.     

‘Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. ‘Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis‐related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.     

Cell metabolism: An essential link between cell growth and apoptosis

Growth factor-stimulated or cancerous cells require sufficient nutrients to meet the metabolic demands of cell growth and division. If nutrients are insufficient, metabolic checkpoints are triggered that lead to cell cycle arrest and the activation of the intrinsic apoptotic cascade through a process dependent on the Bcl-2 family of proteins. Given the connections between metabolism and apoptosis, the notion of targeting metabolism to induce cell death in cancer cells has recently garnered much attention. However, the signaling pathways by which metabolic stresses induce apoptosis have not as of yet been fully elucidated. Thus, the best approach to this promising therapeutic avenue remains unclear. This review will discuss the intricate links between metabolism, growth, and intrinsic apoptosis and will consider ways in which manipulation of metabolism might be exploited to promote apoptotic cell death in cancer cells. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

VDAC, a multi-functional mitochondrial protein as a pharmacological target

Regulation of mitochondrial physiology requires an efficient exchange of molecules between mitochondria and the cytoplasm via the outer mitochondrial membrane (OMM). The voltage-dependent anion channel (VDAC) lies in the OMM and forms a common pathway for the exchange of metabolites between the mitochondria and the cytosol, thus playing a crucial role in the regulation of metabolic and energetic functions of mitochondria. VDAC is also recognized to function in mitochondria-mediated apoptosis and in apoptosis regulation via interaction with anti-apoptotic proteins, namely members of Bcl-2 family, and the pro-survival protein, hexokinase, overexpressed in many cancer types. Thus, VDAC appears to be a convergence point for a variety of cell survival and cell death signals, mediated by its association with various ligands and proteins. In this article, we review mammalian VDAC, specifically focusing on VDAC1, addressing its functions in cell life and the regulation of apoptosis and its involvement in several diseases. Additionally, we provide insight into the potential of VDAC1 as a rational target for novel therapeutics.     

Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acyl-modified N-acetylmannosamine analogs in Jurkat cells

The supplementation of the sialic acid biosynthetic pathway with exogenously supplied N-acetylmannosamine (ManNAc) analogs has many potential biomedical and biotechnological applications. In this work, we explore the structure-activity relationship of Man-NAc analogs on cell viability and metabolic flux into the sialic acid biosynthetic pathway to gain a better understanding of the fundamental biology underlying "glycosylation engineering" technology. A panel of ManNAc analogs bearing various modifications on the hydroxyl groups as well as substitutions at the N-acyl position was investigated. Increasing the carbon chain length of ester derivatives attached to the hydroxyl groups increased the metabolic efficiency of sialic acid production, whereas similar modification to the N-acyl group decreased efficiency. In both cases, increases in chain length decreased cell viability; DNA ladder formation, Annexin V-FITC two-dimensional flow cytometry assays, caspase-3 activation, and down-regulation of sialoglycoconjugate-processing enzymes established that the observed growth inhibition and toxicity resulted from apoptosis. Two of the panel of 12 analogs tested, specifically Ac(4)ManNLev and Ac(4) ManNHomoLev, were highly toxic. Interestingly, both of these analogs maintained a ketone functionality in the same position relative to the core monosaccharide structure, and both also inhibited flux through the sialic acid pathway (the remainder of the less toxic analogs either increased or had no measurable impact on flux). These results provide fundamental insights into the role of sialic acid metabolism in apoptosis by demonstrating that ManNAc analogs can modulate apoptosis both indirectly via hydroxylgroup effects and directly through N-acyl-group effects.     

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of <Emphasis Type="Italic">Lonomia obliqua</Emphasis>

Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system.     

Regulation of macrophage physiology by L-arginine: role of the oxidative L-arginine deiminase pathway

The L-arginine content of the extracellular fluid in sites of predominant macrophage infiltration is reduced below plasma levels due to the activity of macrophage-derived arginase. Investigation of the effects of altered L-arginine availability on macrophage physiology reveals that culture of rat peritoneal macrophages in media containing L-arginine in the concentrations present in inflammatory lesions (less than 0.1 mM) enhances activation-associated functions. In contrast, culture in the higher L-arginine concentrations found in standard tissue culture media (0.4 to 1.2 mM) suppresses most macrophage functions (superoxide production, phagocytosis, and protein synthesis). An exception is the tumor cytotoxicity of Corynebacterium parvum-elicited macrophages which is enhanced by culture in supraphysiologic concentrations of L-arginine. Work reported here investigated the mechanisms for these L-arginine-dependent effects and, more specifically, the role of the recently described oxidative L-arginine deiminase pathway in the regulation of macrophage physiology. Overnight culture of resident or C. parvum-elicited peritoneal macrophages in media containing increasing concentrations of L-arginine (6 microM to 1 mM) resulted in: inhibition of electron transport chain activity (resident and C. parvum-elicited macrophages), increased lactate production (resident macrophages), and decreased ATP content (resident and C. parvum-elicited macrophages). In line with these findings, viability was markedly decreased after 2 days of culture when the initial L-arginine concentration was greater than or equal to 0.1 mM. As shown before, increasing media concentrations of L-arginine were associated with suppression of superoxide production and cytotoxicity in resident macrophages, and with reduced superoxide production and increased cytotoxicity in C. parvum-elicited macrophages. All L-arginine-dependent metabolic and functional alterations, as well as the loss of viability, were prevented by NG-monomethyl-L-arginine, a specific inhibitor of the oxidative L-arginine deiminase pathway. These results demonstrate that flux of L-arginine through the oxidative L-arginine deiminase pathway results in the inhibition of oxidative metabolism in rat macrophages. This metabolic inhibition may, through alterations in the macrophage high energy phosphate stores, mediate the suppression of cell functions and result ultimately in cell death.     

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.     

线粒体在神经元程序性死亡中的作用新进展

神经元的死亡是许多神经系统疾病如阿尔茨海默病、帕金森病、急性青光眼等发生发展过程中的重要事件,传统认为,细胞死亡有凋亡、自噬、坏死三种方式,凋亡和自噬为程序性的细胞死亡,坏死为非程序性的死亡途径。而近年来的研究发现了一种名为程序性坏死(necroptosis)的可调控的坏死,因此,对这些可调控的细胞死亡的研究对治疗这类神经系统疾病有重要的意义。大量研究发现,在能量代谢和自由基代谢中占据着重要地位的线粒体在细胞死亡过程中也发挥重要作用。本文对线粒体在神经元凋亡、自噬和程序性坏死中的生物学作用的最新进展做一综述。