Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

Cell Origami: Self-Folding of Three-Dimensional Cell-Laden Microstructures Driven by Cell Traction Force

This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.     

Combined experimental and computational analysis of DNA damage signaling reveals context‐dependent roles for Erk in apoptosis and G1/S arrest after genotoxic stress

Following DNA damage, cells display complex multi‐pathway signaling dynamics that connect cell‐cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell‐cycle re‐entry and proliferation, permanent cell‐cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time‐resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin‐induced DNA damage in the presence or absence of TNFα co‐treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell‐cycle regulatory responses. Two relational modeling approaches were then used to identify network‐level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term ‘time‐interval stepwise regression.’ Taken together, the results from these analysis methods revealed complex, cytokine‐modulated inter‐relationships among multiple signaling pathways following DNA damage, and identified an unexpected context‐dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug.     

Host‐parasite coevolution can promote the evolution of seed banking as a bet‐hedging strategy

Seed (egg) banking is a common bet‐hedging strategy maximizing the fitness of organisms facing environmental unpredictability by the delayed emergence of offspring. Yet, this condition often requires fast and drastic stochastic shifts between good and bad years. We hypothesize that the host seed banking strategy can evolve in response to coevolution with parasites because the coevolutionary cycles promote a gradually changing environment over longer times than seed persistence. We study the evolution of host germination fraction as a quantitative trait using both pairwise competition and multiple mutant competition methods, while the germination locus can be genetically linked or unlinked with the host locus under coevolution. In a gene‐for‐gene model of coevolution, hosts evolve a seed bank strategy under unstable coevolutionary cycles promoted by moderate to high costs of resistance or strong disease severity. Moreover, when assuming genetic linkage between coevolving and germination loci, the resistant genotype always evolves seed banking in contrast to susceptible hosts. Under a matching‐allele interaction, both hosts’ genotypes exhibit the same seed banking strategy irrespective of the genetic linkage between loci. We suggest host–parasite coevolution as an additional hypothesis for the evolution of seed banking as a temporal bet‐hedging strategy.     

Characterization of cell‐banking parameters for the cryopreservation of mammalian cell lines in 100‐mL cryobags

This article describes a cell banking process for rBHK cell lines in 100‐mL cryobags. As the use of larger volume cell banks requires greater cell numbers and longer preparation time, extensive characterization of key process parameters beyond the conventional ranges was performed to support a cGMP banking process. All experiments were conducted using two recombinant BHK21 cell lines, one of them cotransfected with Hsp70. The results show that the entire cell banking process for these BHK cell lines can be performed at room temperature. A DMSO exposure time up to 5 h either directly in a bioreactor or in shaker flasks did not result in any significant negative effect after cell thaw, when the cryocontainers were frozen immediately after filling. Extensive characterization did not indicate any significant apoptotic effects after thaw. However, the Hsp70 cotransfected cell line did show a slightly better protection from potential cryopreservation‐induced apoptosis. Surprisingly, it was found that cells transferred into cryobags showed a low recovery rate after thaw if the incubation time exceeded 1.5 h before freezing. Additional experiments confirmed that the DMSO exposure time inside the cryocontainer in contrast to the DMSO exposure in a reactor or shaker flasks is much more critical. The cryobag cell banking process should therefore be performed within a 1½–2 h window; a banking process for vials should not exceed 2½ h. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effects of different detachment procedures on viability,nitroxide reduction kinetics and plasma membrane heterogeneity of V‐79 cells

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G₁). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.     

Development of small scale cell culture models for screening poloxamer 188 lot‐to‐lot variation

Shear protectants such as poloxamer 188 play a critical role in protecting cells during cell culture bioprocessing. Lot‐to‐lot variation of poloxamer 188 was experienced during a routine technology transfer across sites of similar scale and equipment. Cell culture medium containing a specific poloxamer 188 lot resulted in an unusual drop in cell growth, viability, and titer during manufacturing runs. After switching poloxamer lots, culture performance returned to the expected level. In order to control the quality of poloxamer 188 and thus maintain better consistency in manufacturing, multiple small scale screening models were developed. Initially, a 5L bioreactor model was established to evaluate cell damage by high sparge rates with different poloxamer 188 lots. Subsequently, a more robust, simple, and efficient baffled shake flask model was developed. The baffled shake flask model can be performed in a high throughput manner to investigate the cell damage in a bubbling environment. The main cause of the poor performance was the loss of protection, rather than toxicity. It was also suggested that suspicious lots can be identified using different cell line and media. The screening methods provide easy, yet remarkable models for understanding and controlling cell damage due to raw material lot variation as well as studying the interaction between poloxamer 188 and cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1411–1418, 2014     

An ultra scale‐down methodology to characterize aspects of the response of human cells to processing by membrane separation operations

Tools that allow cost‐effective screening of the susceptibility of cell lines to operating conditions which may apply during full scale processing are central to the rapid development of robust processes for cell‐based therapies. In this paper, an ultra scale‐down (USD) device has been developed for the characterization of the response of a human cell line to membrane‐based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. The cell line used to develop the measurements was a clinically relevant human fibroblast cell line. The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. Transmission of LDH was investigated with time of operation and for the two disc speeds investigated (6,000 and 10,000 rpm or ? _max ≈ 1.9 and 13.5 W mL^?1, respectively). As expected, increased energy dissipation rate led to increased transmission as well as significant increases in rate and extent of cell damage. The method developed can be used to test the impact of varying operating conditions and cell lines on cell damage and morphological changes. Biotechnol. Bioeng. 2017;114: 1241–1251. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

Modeling shear‐induced CHO cell damage in a rotary positive displacement pump

Rotary lobe pumps are commonly used in the biotechnology industry for a variety of purposes. Shear damage to animal cells within the rotary lobe pump can adversely affect the product yield or purity during, for example, cell concentration via cross‐flow filtration. In this research, CHO cells grown in 20‐L bioreactors were fed to a rotary lobe pump in both single pass and recycle experiments were conducted at different RPMs and “slip” conditions. The results indicate that the slip flow rate more severely impacts the viability of the CHO cells than the pump RPM. A novel mathematical modeling approach is presented that predicts shear rates in all of the positive displacement pump's slip regions, and then predicts cell death vs. operating conditions. This model accounts for the complex flow situation that results from changes to RPM, backpressure and pump geometry (i.e., clearances). © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

Dovitinib (TKI258; formerly CHIR‐258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G₂/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single‐cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G₂ arrest similar to the G₂ DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ‐H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G₂ arrest could be overcome by abrogation of the G₂ DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib‐mediated G₂ cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP‐ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.     

A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO‐SEAP cell line by means of a genome‐wide high‐content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell‐specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR‐30 family substantially improves bioprocess performance of CHO cells. Stable miR‐30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR‐30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.     

Creating a completely “cell‐free” system for protein synthesis

Cell‐free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell‐free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell‐free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell‐free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell‐free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems’ protein synthesis capabilities. Lyophilization provides the additional benefit of long‐term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely “cell‐free” systems. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1716–1719, 2015     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

On‐demand killing of adherent cells on photo‐acid‐generating culture substrates

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro‐projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro‐projection systems currently available, we introduced the assist of the photo‐responsive culture substrates functionalized with a photo‐acid‐generating polymer. In clear contrast to the existing laser‐based methods requiring point scanning, areal micro‐prjection of blue light with the wavelength 436 nm killed many CHO‐K1 cells at a time in the irradiated area on the substrate. The effect of the photo‐generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo‐selective killing of the adherent cells after preliminarily patterning through the photo‐induced removal of cell adhesion‐inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348–352. © 2012 Wiley Periodicals, Inc.     

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing practices for recombinant adeno‐associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10⁴–10⁵ vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small‐scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol‐step gradient whereas large‐scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead‐end filtration) and chromatography based‐methods are used for large‐scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.     

Human mesenchymal stem cells: A bank perspective on the isolation,characterization and potential of alternative sources for the regeneration of musculoskeletal tissues

The continuous discovery of human mesenchymal stem cells (hMSCs) in different tissues is stirring up a tremendous interest as a cell source for regenerative medicine therapies. Historically, hMSCs have been always considered a sub‐population of mononuclear cells present in the bone marrow (BM). Although BM‐hMSCs are still nowadays considered as the most promising mesenchymal stem cell population to reach the clinics due to their capacity to differentiate into multiple tissues, hMSCs derived from other adult and fetal tissues have also demonstrated to possess similar differentiation capacities. Furthermore, different reports have highlighted a higher recurrence of hMSCs in some of these tissues as compared to BM. This offer a fascinating panorama for cell banking, since the creation of a stem cell factory could be envisioned where hMSCs are stocked and used for ad hoc clinical applications. In this review, we summarize the main findings and state of the art in hMSCs isolation, characterization, and differentiation from alternative tissue sources and we attempt to compare their potency for musculoskeletal regeneration. J. Cell. Physiol. 228: 680–687, 2013. © 2012 Wiley Periodicals, Inc.     

Biotechnology: Health care,agriculture, industry,environment

New developments in different branches of biotechnology are discussed. The production of peptide hormones, new interferons and other lymphokines by the microbial and cell cultures, and new enzyme inhibitors of microbial origin are the most important for health care and pharmacy. The main direction in research in the agriculture represents the development of the new, very effective methods of nitrogen fixation and the production of animal growth hormones by genemanipulated microorganisms. One of the most important field of application of biotechnology is the chemical industry, c.f. microbial production of polymers and biotransformation of compounds previously produced by chemical methods (acrylamide, adipic acid, naphtalen conversion, etc.). Several novel methods of degradation of the cellulosic materials are mentioned and exploitation of biotechnology in environmental protection is also discussed.     

Targeting the lysosome by an aminomethylated Riccardin D triggers DNA damage through cathepsin B‐mediated degradation of BRCA1

RD‐N, an aminomethylated derivative of riccardin D, is a lysosomotropic agent that can trigger lysosomal membrane permeabilization followed by cathepsin B (CTSB)‐dependent apoptosis in prostate cancer (PCa) cells, but the underlying mechanisms remain unknown. Here we show that RD‐N treatment drives CTSB translocation from the lysosomes to the nucleus where it promotes DNA damage by suppression of the breast cancer 1 protein (BRCA1). Inhibition of CTSB activity with its specific inhibitors, or by CTSB‐targeting siRNA or CTSB with enzyme‐negative domain attenuated activation of BRCA1 and DNA damage induced by RD‐N. Conversely, CTSB overexpression resulted in inhibition of BRCA1 and sensitized PCa cells to RD‐N‐induced cell death. Furthermore, RD‐N‐induced cell death was exacerbated in BRCA1‐deficient cancer cells. We also demonstrated that CTSB/BRCA1‐dependent DNA damage was critical for RD‐N, but not for etoposide, reinforcing the importance of CTSB/BRCA1 in RD‐N‐mediated cell death. In addition, RD‐N synergistically increased cell sensitivity to cisplatin, and this effect was more evidenced in BRCA1‐deficient cancer cells. This study reveals a novel molecular mechanism that RD‐N promotes CTSB‐dependent DNA damage by the suppression of BRCA1 in PCa cells, leading to the identification of a potential compound that target lysosomes for cancer treatment.