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Over-expression of the X-linked inhibitor of apoptosis protein (XIAP) delays serum deprivation-induced apoptosis in CHO-K1 cells
Journal of Bioscience and Bioengineering VOL. 110 No. 3, 338 – 344, 2010 www.elsevier.com/locate/jbiosc
Over-expression of the X-linked inhibitor of apoptosis protein (XIAP) delays serum deprivation-induced apoptosis in CHO-K1 cells Jane C.J. Liew,1 Wen Siang Tan,1,2 Noorjahan Banu Mohamed Alitheen,3 Eng-Seng Chan,4 and Beng Ti Tey1,5,⁎ Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 1 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 2 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 3 Centre of Materials and Minerals, School of Engineering and IT, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia, 4 and Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 5 Received 10 December 2009; accepted 24 February 2010 Available online 29 March 2010
Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP overexpression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells overexpressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2 days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: XIAP; CHO-K1; Serum deprivation; Apoptosis; Mammalian cell culture; Flow cytometry]
Over the past decades, mammalian cells have been widely used to produce complex proteins for both therapeutic and diagnostic use. Chinese hamster ovary (CHO) cells, one of the commercially popular cell lines, are the choice for the mass production of various recombinant proteins. In general, cells face the risk of encountering numerous insults present in a bioreactor, such as mechanical agitation, nutrient depletion, hydrodynamic stress, toxin accumulation, hypoxia and viral infection (1). Upon these stresses, cells tend to undergo apoptosis cell death. Therefore, much effort has been expended to improve process efficiency via metabolic engineering, metabolic pathway analysis and optimization of the scale-up cultures (2–7). Mammalian cell culture requires growth factors and hormone that present in the serum to enhance cell growth and to retain its cellular functions (8). However, the use of serum has several disadvantages, including high cost, lot-to-lot variations, contamination of infectious agents, such as virus and prions. Also, the presence of various protein components in the serum complicates the downstream purification process, whereby the removal of serum proteins from secreted bioproducts can be laborious, time-consuming and costly. Hence, for effective downstream processing and purification of cellular products, ⁎ Corresponding author. Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89466289; fax: +60 3 86567120. E-mail address: [email protected] (B.T. Tey).
works on development of a serum-free culture that can be utilized for effective industrial-scale production of biopharmaceuticals are being explored continuously and extensively. Serum withdrawal not only inhibits cell growth, but also triggers apoptosis cell death. Apoptotic cell death causes premature termination of cell culture duration, particularly in high cell density culture (9). Studies have shown that serum deprivation induces apoptosis in a wide range of cell lines, including hybridomas, myelomas, CHO, and BHK cells (10–13). Much attention is now being devoted to strategies for controlling and limiting cell death, either through manipulation of media supplementation or alteration of intracellular biochemistry using genetic engineering (14,15). Researchers have found that the introduction of mitogenic factors, such as insulin, transferin and insulin-like growth factor, can prolong the viability of cells cultured in serum-free media. The addition of basic fibroblast growth factor and insulin synergistically promoted the growth of CHO cells under serumfree conditions (16). There have also been other attempts to reduce apoptosis in mammalian cell-culture systems by utilizing amino acid mixtures or chemical apoptosis inhibitors such as N-acetylcysteine (NAC), bonkrekic acid, z-VAD.fluoromethly ketone (FMK), or 7-amino4-trifluoromethyl coumarin (AFC) (17). Studies on anti-apoptotic proteins can suppress apoptosis effectively, thereby increasing the production of the bioproducts. Upregulation of anti-apoptosis engineering using anti-apoptotic proteins
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such as bcl-2 and bcl-xL had shown to improve the productivity of recombinant proteins by suppressing apoptotic cell death (11–13,18). Apoptosis is a programmed cell death mode where caspases are the key effector of apoptosis (19). Hence, inhibition of caspases activity is one of the effective methods to inhibit apoptosis cascade. The inhibitor of apoptosis protein (IAP) is a family of anti-apoptotic proteins that regulate apoptosis, where XIAP (X-linked IAP) is the star player of IAP family. XIAP inhibits the caspase activity through direct binding of its baculoviral IAP repeat (BIR) domains to caspases (20). It has been evaluated as one of the key regulators of apoptosis, not only that it is the most potent inhibitor of apoptosis, it is also the bestcharacterized member of IAP family. Originally, the function of XIAP was assigned to inhibit apoptosis, but it was then discovered to be involved in a number of diverse cellular processes, including cell cycling, ubiquitylation and receptor-mediated signaling (21), making it a multi-functional protein. Previously, Sauerwald et al. (22) reported that the over-expression of a XIAP variant, which retains the BIR domain but without the C-terminal RING domain, provided protection to CHO cells exposed to Sindbis virus, etoposide and spent medium. It was then found that this XIAP variant provides a consistent enhancement in the viable cell density in lactate supplemented cultures (23). While Kim et al. (24) reported that the over-expression of XIAP could not inhibit the sodium butyrate-induced apoptosis effectively in CHO cells, Kaufmann's group reported that co-expression of both XBP-1 and XIAP genes resulted in a higher titer of IgG in a serum-free medium (25). To date, there is no attempt to study the effect of XIAP over-expression on delaying the cell death and cell proliferation under serum deprived condition. In this study, we focused on the effect of the full length XIAP on the suppression of the cell proliferation and apoptosis under serum-deprived conditions. MATERIALS AND METHODS Cell line and cell maintenance The CHO-K1 cells were routinely cultured in Ham's F12 medium supplemented with 2 mM L-glutamine (Gibco, USA) and 10% FBS (Gibco, USA) and further cultured in serum-deprived medium. For MCF-7 cell line, cells were maintained in Dulbecco's modified Eagle's media containing 4.5 gm/l glucose (Sigma-Aldrich, UK) and 10% FBS (Gibco, USA). All cell lines were incubated at 37 °C in a humidified chamber at a fixed setting of 5% CO2. Transfection The pcDNA3-myc-XIAP mammalian expression vector was kindly provided by Dr. Takeo Namuro (Department of Urology, Oita Medical University, Japan). Construction of the plasmid, pcDNA3-myc-XIAP was described previously. The vector contains a 1.5 kb human XIAP coding region and resistance genes for ampicillin and neomycin. The myc tag was used for the detection of the XIAP protein expression (26). Transfection was conducted in a 6-well plate using the GeneJuice Transfection Reagent (Novagen, USA), as recommended by the manufacturer. Transfected cells were selected in 800 μg/ml G418 sulfate (Gibco, USA) selection medium for 2 weeks. Cell cloning by limiting dilution was conducted to select stable transfectant clones. MTT assay During screening of potential clones, relative cell viability was assessed by adding 20 μl of 0.5 g/l MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole] (Sigma, USA) solution to each well. After 4 hour of incubation, 100 μl of DMSO was added to the wells and further incubated for 30 min. Absorbance at 570 nm was measured by µ-Quant ELISA microplate reader (Bio-tek Instruments, USA). Analysis of XIAP expression Potential clones were propagated in 6-well culture plates and harvested when the cell density reached 90% confluent. Harvested cells were fixed with 100 μl Cytofix/Cytoperm Fixation/Permeabilization solution (BD, USA) and incubated on ice for 20 min. One μl of 150 μg/ml anti-XIAP antibody (BD, USA) was added and incubated on ice for 30 min. Cells were washed after incubation and 1 μl of 0.5 mg/ml FITC-conjugated goat anti-mouse antibody (BD, USA) was added and incubated on ice for 20 min in dark. XIAP-FITC fluorescence was measured by the FACS Calibur System (BD, USA), while XIAP expression was analyzed using the Cell Quest Software. Cell viability analysis Batch cultures were sacrificed at 24 h intervals and cell viability was determined by using the trypan blue exclusion method. Cell suspensions were mixed with 0.4% trypan blue solution (Sigma, USA) at a 1:1 dilution. Dead and viable cells were identified and the percentage was calculated. Cell density was measured using a haemacytometer slide and cell viability was determined by dividing the number of viable cells by the total number of cells. Fluorescent microscopic analysis of apoptosis Apoptosis/necrosis count was determined using acridine orange (AO)/propidium iodide (PI) dual stain fluorescent
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microscopy. Cells were stained with 10 μl of AO/PI dual dye solution (15 μg/ml AO, 15 μg/ml PI) (Sigma, USA) for 2 min. Approximately 10 μl of stained cells were loaded onto a glass slide and observed under an inverted fluorescence microscope (Nikon, Japan) using a 10x objective lens. Cell images were captured using the ESI-Element software. The cell population was classified into 4 categories: viable (green cells with diffused chromatin), early apoptotic (green cells with condensed chromatin), late apoptotic (red cells with condensed chromatin) and necrotic (red cells with noncondensed chromatin) (12). Measurement of Caspase-3 Activity Cell lysates were prepared from 6-well culture plates. In brief, caspase-3 activity in the extract of approximately 1 × 106 cells was measured using the Caspase-3/CPP32 colorimetric assay kit (Biovision, USA). The caspase substrate DEVD-pNA (4 mM, 5 μl) was added to the samples and incubated at 37 °C for 2 hr. The enzyme-catalyzed release of pNA was quantified at 400 nm using the µ-Quant ELISA microplate reader (Bio-tek Instruments, USA). Cell cycle analysis Harvested cells were centrifuged and pellets were resuspended thoroughly in remnant. Cells were then fixed with 500 μl of ice-cold 70% (v/v) ethanol and stored at -20 °C until analysis (at least 2 hours). Fixed cells were washed twice with ice-cold PBS washing buffer (2% BSA, 0.1% Azide-EDTA), resuspended in 1 ml of PBS staining buffer (0.1% Triton X-EDTA) containing 100 mg/ml RNase A, and incubated for 30 min. Cells were then stained with 200 μl of 50 μg/ml PI solution (Sigma, USA) in dark for 15 min. DNA fluorescence was measured by using the FACS Calibur System (BD, USA), while sub populations of DNA distribution histograms were analyzed using the Cell Quest Software. Cell debris and aggregates were excluded by appropriate gating.
RESULTS Selection of stable XIAP expression clones The effect of XIAP over-expression on the suppression of apoptosis was investigated by transfecting the CHO-K1 cells with the pcDNA-myc-XIAP plasmid. Stable transfectants were selected in G418 selection medium and limiting dilution was performed to select high XIAP expresser clones. Further selection was conducted by exposing 30 isolated clones in medium without serum supplementation for 3 days. Ten most potential clones were then evaluated by MTT assay and the results are shown in Table 1. Three clones (Clones 3, 5 and 25) showing the highest viability were selected for the evaluation of XIAP expression. Flow cytometric analysis using anti-XIAP antibody conjugated to FITC was performed to confirm the XIAP expression in the selected clones. The evidence of the over-expression of XIAP in transfected population of CHO-K1 cells is shown in fluorescence histogram profiles (Fig. 1). This analysis gives a clear evidence that the selected clones (Clones 3, 5 and 25) (Fig. 1B-D) displayed higher levels of XIAP protein compared to the negative control (parental CHO-K1) (Fig. 1A). MCF-7 breast cancer cell was used as the positive control (Fig. 1E) in this analysis. An increase in fluorescence intensity was observed and the whole cell population of CHO-K1-XIAP cells was shifted to the right compared to the negative control. Stable CHO-K1-XIAP clone (Clone 5) displaying the highest expression level of XIAP was selected for further characterization. Approximately 84% of the cell population in clone 5 (Fig. 1C) was found to be expressing the XIAP stably after being passaged continuously in batch culture for 3 months. XIAP expression enhances survivability of CHO-K1 cells Both CHO-K1-XIAP and the control (parental CHO-K1) cell line were plated TABLE 1. Relative viability of the potential clones expressing XIAP. Clone
Relative Cell Viability (% of control)
1 2 3 5 7 11 12 15 20 25
3.4 38.3 141.3 119.3 95.9 91.1 56.7 57.1 97.1 98.2
After the clones were exposed in serum-deprived media for 3 days, the viability was measured using MTT assay. The relative cell viability (% of control) represents the percentage absorbance relative of the pcDNA-myc-XIAP transfected clones to the parental cells.
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FIG. 1. Analysis of XIAP expression in (A) CHO-K1, (B) CHO-K1-XIAP-3, (C) CHO-K1-XIAP-5 (D), CHO-K1-XIAP-25 and (E) MCF-7 cell lines.
and allowed to adhere for 24 h and then starved under serum-deprived condition. After 2 days of serum-deprivation, the viability of CHO-K1 cells expressing XIAP was still maintained above 90% (Fig. 2). In contrast, the control culture showed a rapid decrease in viability, a drop in viability to 40% was observed. By day 3, while the viability of CHO-K1-XIAP was still maintained at 75%, the control culture again was reduced to 30%, indicating a progressive loss of viability over times. As a result of serum deprivation, the control undergoes a more rapid apoptosis rate than CHO-K1-XIAP. This study reveals that XIAP provides a high level of protection throughout the duration of the experiment, with a viability of 43% by day 4 compared to only 20% for the control. Although expression of XIAP delayed the onset of
apoptosis, these cells eventually succumbed to cell death and the viability dropped to 30% at day 5. XIAP expression delays apoptosis induced by serum deprivation in CHO-K1 cells Fluorescence microscopy of AO and PI was employed to qualitatively visualize the distributions of viable, early apoptotic, late apoptotic and necrotic cells. The cells were classified accordingly to that described in Tey et al. (12) and the results were expressed as percentages of the total cells. At days 2 and 3, while the viability of CHO-K1-XIAP population maintain at 85% and 72% (Fig. 3A), the control culture showed a lower viability at about 55% and 38%, respectively (Fig. 3B). Throughout day 2 to day 5, apoptosis was found to progress gradually in both cell line, but with CHO-K1-
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FIG. 2. Viability analysis of CHO-K1 and CHO-K1-XIAP cells cultured under serumdeprived condition. Cell viability was determined by dividing the number of viable cells by the total number of cells in terms of percentage. Error bars represent standard deviation (n = 3).
FIG. 3. Apoptosis/ necrosis analysis of (A) CHO-K1-XIAP and (B) CHO-K1 cells. Acridine orange and propidium iodide were used in the dual staining analysis. In the figure, the term Early ap and Late ap denotes early apoptotic cells and late apoptotic cells, respectively. Error bars represent standard deviation (n = 3).
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XIAP cells progressing at a slower rate as compared to the control culture. XIAP has been reported as an effective inhibitor to caspase-3 (20). Therefore, caspase-3 activity assay was performed to investigate the effect of the expression of XIAP on the inhibition of caspase-3 activities. The total enzymatic activity of caspase-3 was measured by adding DEVD-pNA, a caspase substrate which can be cleaved by caspase-3. Fig. 4 shows the caspase-3 activity in cells sampled from day 1 to day 3. CHO-K1-XIAP showed an overall decrease in the caspase-3 activity when compared to that of the control. Notably on day 2, a rapid increase in caspase-3 was observed in the control culture, while CHO-XIAP-K1 showed no significant increase of the caspase-3 activity when compared to day 1. This result shows that over-expression of XIAP inhibits the induction of caspase-3 activity, which attenuates apoptosis in response to serum withdrawal. Flow cytometry was performed to investigate the cell cycle profile of both cell lines under serum-deprived condition. The results illustrated in Fig. 5 show that while majority of CHO-K1-XIAP population maintained at the G0/G1 peak on day 2 (Fig. 5A), the control culture peaks collapse with half of the population in the sub-G1 or apoptotic region (Fig. 5B), where XIAP expressing cell culture peaks were less affected. As illustrated in Table 2, the sub-G1 population was 6.6% for CHO-K1-XIAP and 50.3% for the control at day 2. By day 3, while the control culture reached 61.8% of apoptosis (sub-G1 population), CHO-K1-XIAP still maintained a higher viability by only showing 25% of cell death. Additionally, after 3 days of serumdeprivation, XIAP over-expression caused a decrement in the percentage of cells in the S phase and always maintained a higher percentage of cells in G0/G1 compared to the control cells. XIAP expression attenuates cell proliferation in CHO-K1 cells in serum-deprived medium The growth profiles and viable cell densities of both CHO-K1-XIAP and control cells in serum deprived medium are shown in Fig. 6. The viable cell density of the control cells decreased from day 2 onwards. In contrast, the induction of cell death of CHO-K1-XIAP cells was delayed as a result of XIAP expression. However, we observed an overall increase of total cell density in the control cells. Fig. 6B shows a 30% increment in total cell density in the control cells from days 0 to 2, while only 13% of increment in total cell density of CHO-K1-XIAP was observed at the same period of time. Even under serum-supplemented condition, CHO-K1-XIAP also exhibited a slower cell growth pattern as compared to the control (Fig. 7). Throughout days 2 to 5, CHO-K1-XIAP exhibited a higher percentage of cell population in the G0/G1 phase (Table 2), where this observation clearly suggests a difference in cell proliferation between CHO-K1-XIAP and the control. This result shows that over-expression of XIAP induces G0/G1 growth arrest, where proliferation was
FIG. 4. Measurement of caspase-3 activity in CHO-K1 and CHO-K1-XIAP cells cultured under serum-deprived medium. Relative caspase-3 activity was estimated as a ratio of the activity in cells irrespective conditions to that in cells cultured under complete growth medium (10% serum). Error bars represent standard deviation (n = 3).
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FIG. 5. Flow cytometric DNA analysis data showing cell cycle distribution for (A) CHO-K1-XIAP and (B) CHO-K1 cells cultured under serum-deprived condition.
retracted and thus resulting a 50% reduction in maximum cell density. The inhibitory effect was already visible after 2 days of serum deprivation. DISCUSSION Serum deprivation has been considered as one of the environmental stresses that can induce apoptosis cell death in mammalian cells cultured in bioreactors (8). Therefore, removal of serum usage in cell culture processes while delaying apoptosis represents a potentially major biotechnology challenge. Previous studies have shown
that by over-expressing one or more anti-apoptotic gene in mammalian cell cultures, apoptosis can be delayed and cell survival rate can be extended (8–14). Bcl-2 is one of the early anti-apoptotic genes that inhibit the release of pro-apoptotic molecules from mitochondria. It has been studied extensively in culture processes and has been shown to provide significant protection to various assaults including, hyperoxia and hypoxia conditions, depletion of nutrients, ammonia exposure and viral infection (11–13,18). While over-expression of bcl-2 maintains mitochondrial integrity by inhibiting mitochondrial apoptosis and ensuing caspase activation, XIAP acts late in the apoptotic pathway by inhibitory binding to
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TABLE 2. Cell cycle distributions for CHO-K1-XIAP and CHO-K1 cell lines in batch culture during serum deprivation-induced apoptosis. Day
2 3 4 5
Sub-G1/Apoptotic (%) CHO-K1XIAP
CHOK1
6.6 25.2 59.1 68.9
50.3 61.8 72.0 80.1
G0/G1 (%) CHO-K1XIAP 77.0 63.5 39.0 29.2
CHOK1 44.1 34.1 26.1 18.0
S (%) CHO-K1XIAP 9.0 9.0 1.6 1.5
G2/M (%) CHOK1
CHO-K1XIAP
CHOK1
4.7 3.2 1.5 1.6
7.4 2.5 0.4 0.4
0.9 0.9 0.4 0.4
downstream caspases. Therefore, XIAP may provide better protection when the mitochondrial cascade is overwhelmed, where caspases will be activated thereafter. In support of this view, we decided to investigate the ability of XIAP on the extension of the life span of CHO-K1 mammalian cells cultured in a serum-deprived environment. Our findings demonstrated that the expression of exogenous XIAP reduced apoptosis, prolonged culture and slowed down proliferation. In the absence of serum, the control culture was unable to survive for a prolonged period, however, CHO-K1-XIAP cells were found to be more effective in enhancing survivability under such harsh condition. Light micrograph images also revealed rounding and detachment of the control
FIG. 6. (A) Viable cell density and (B) growth profile of cells in batch cultures under serum-deprived condition. Cells were seeded at a concentration of 1.5 × 105 cells/ml.
FIG. 7. Total cell density of CHO-K1 and CHO-K1-XIAP cells cultured under 10% of serumsupplemented condition. Cells were seeded at a concentration of 1.5 × 105 cells/ml.
culture after 2 days of serum starvation; whereas, CHO-K1-XIAP cells remained healthy, elongated and adhered at this time point (data not shown). The control culture was affected determinately by the stressful condition of serum deprivation and within 2 days, their cell viability was reduced to 40%, with the XIAP clones showing an average cell viability of more than 90%. However, during the fourth day of serum-starvation, a sudden drop in the viability of CHO-K1-XIAP cells was observed. By that time point, cell death might be caused by the severe conditions of nutrient deprivation and accumulation of toxic metabolites in the batch cultures. Flow cytometric analysis was used to determine whether the rapid decrease in cell viability in serum-deprived media was caused by apoptosis. Apoptotic cells were presented as sub-Gl population as cells undergoing apoptosis have a lower DNA content. The results showed that although CHO-K1-XIAP cells exhibited a slower growth pattern, most of the cells were still located in the G0/G1 phase. Tey and Al-Rubeai (27) and Simpson et al. (18) reported that under a suboptimal culture condition, NS0 and hybridoma cells expressing the bcl-2 were accumulated in the G1-phase. Exit of cells from the normal cell cycle into a “quiescent state” (arrested at G1 phase) is a common strategy for some cancer cell lines in response to growth factor deprivation (28). Previous studies have demonstrated that antiapoptotic members of the bcl-2 family proteins suppressed proliferation and findings on bcl-2 expression proved that this protein does slow down cell growth (29). Studies by Mazur et al. (30) also demonstrated that expression of cyclin-dependent kinase inhibitor p27 resulted in growth arrest and a substantial improvement in protein productivity. XIAP has been identified as an active repressor of the cell cycle (31). Studies have shown that XIAP exhibits its anti-proliferative effect in the G1/S boundary of the cell cycle through its ubiquitin ligase activity (32), where XIAP may target individual cell-cycle progression elements, such as cyclin A and D1 for degradation (31), resulting an increase in the percentage of cells in G0/G1 phase. It has been documented that cells under growth arrested condition switches their cellular biosynthetic machinery from proliferation to product synthesis (10). Various studies demonstrated that the decrease in proliferation increases protein productivity. For instance, Fusseneggar et al. (33) demonstrated that by over-expressing a tumor suppressor gene to arrest the cells in G1 phases has resulted in four-fold increase in human alkaline phosphatase production. While Fox et al. (34) showed that growth arrest in G0/ G1 phase increases the productivity of IFN-γ when exposed to low temperature. Their study demonstrated
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that mRNA levels were elevated in G0/G1 phase, and a cell line can still exhibit growth-associated productivity. In addition, Liu and Chen (35) reported that the productivity of the recombinant protein was enhanced by 57% as a result of the addition of DMSO to arrest the cells at phase G0/G1, owing to the fact that growth-arrested cells do not need to devote cellular resources to biomass production. Therefore, blocking apoptosis while inducing cell-cycle arrest may be a worthwhile approach in developing a more economical and efficient process. ACKNOWLEDGMENTS Special thanks to Dr. T. Namuro (Oita Medical University, Japan) for providing the pcDNA3-myc-XIAP vector. This study was supported by the FRGS Grant (01-01-07-162 FR) from the Ministry of Higher Education, Malaysia and the Science Fund (02-01-04 SF0763) from the Ministry of Science, Technology and Innovation, Malaysia. J.C.J. Liew is supported by the Graduate Research Fellowship from Universiti Putra Malaysia. References 1. Tey, B. T., Singh, R. P., and Al-Rubeai, M.: Programmed cell death – an overview of apoptosis in cell culture, Asia Pac. J. Mol. Biol. Biotechnol., 9, 79–106 (2001). 2. Omasa, T., Tanaka, R., Doi, T., Ando, M., Kitamoto, Y., Honda, K., Kishimoto, M., and Ohtake, H.: Decrease in Antithrombin III Fucosylation by expressing GDPfucose transporter siRNA in Chinese hamster ovary cells, J. Biosci. Bioeng., 106, 168–173 (2008). 3. Omasa, T., Takami, T., Ohya, Kiyama, E., Hayashi, T., Nishii, H., Miki, H., Kobayashi, K., Honda, K., and Ohtake, H.: Overexpression of GADD34 ehnaces production of recombinant human antithrombin III in Chinese hamster ovary cells, J. Biosci. Bioeng., 106, 568–573 (2008). 4. Matsunaga, N., Kano, K., Maki, Y., and Dobashi, T.: Culture scale-up studies as seen from the viewpoint of oxygen supply and dissolved carbon dioxide stripping, J. Biosci. Bioeng., 107, 412–418 (2009). 5. Matsunaga, N., Kano, K., Maki, Y., and Dobashi, T.: Estimation of dissolved carbon dioxide stripping in a large bioreactor using model medium, J. Biosci. Bioeng., 107, 419–424 (2009). 6. Mc-Murray-Beaulieu, V., Hisiger, S., Durand, C., Perrier, M., and Jolicoeur, M.: Na-butyrate sustains energetic states of metabolism and t-PA productivity of CHO cells, J. Biosci. Bioeng., 108, 160–167 (2009). 7. Zhao, Q. and Kurata, H.: Maximum entropy decomposition of flux distribution at steady state to elementary modes, J. Biosci. Bioeng., 107, 84–89 (2009). 8. Tey, B. T. and Al-Rubeai, M.: Bcl-2 over-expression reduced the serumdependency and improved the nutrient metabolism in NS0 cell culture, Biotechnol. Bioprocess Eng., 10, 254–261 (2005). 9. Tey, B. T. and Al-Rubeai, M.: Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity, Apoptosis, 9, 843–852 (2004). 10. Singh, P. R., Al-Rubeai, M., Gregory, C. D., and Emery, A. N.: Cell death in bioreactors – a role for apoptosis, Biotechnol. Bioeng., 44, 720–726 (1994). 11. Tey, B. T., Singh, R. P., Piredda, L., and Al-Rubeai, M.: Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures, J. Biotechnol., 79, 147–159 (2000). 12. Tey, B. T., Singh, R. P., Piredda, L., and Al-Rubeai, M.: Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody, Biotechnol. Bioeng., 68, 31–43 (2000). 13. Mastrangelo, A. J., Hardwick, J. M., Zou, S., and Betenbaugh, M. J.: Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults, Biotechnol. Bioeng., 67, 555–564 (2000).
14. Sakai, K., Matshunaga, T., Hayashi, C., Yamaji, H., and Fukada, H.: Effect of phosphatidic acid on recombinant protein production by Chinese hamster ovary cells in serum-free culture, Biochem. Eng. J., 10, 85–92 (2002). 15. Arden, N. and Betenbaugh, M. J.: Life and death in mammalian cell culture: strategies for apoptosis inhibition, Trends Biotechnol., 22, 174–180 (2004). 16. Liu, C. H. and Wu, K. W.: Synergistic effects of basic fibroblast growth factor and insulin on Chinese hamster ovary cells under serum-free condition, J. Biosci. Bioeng., 107, 312–317 (2009). 17. Mastrangelo, A. J., Zou, S., Hardwick, J. M., and Betenbaugh, M. J.: Anti-apoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection, Biotechnol. Bioeng., 65, 298–305 (1999). 18. Simpson, N. H., Milner, A. E., and Al-Rubeai, M.: Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions, Biotechnol. Bioeng., 54, 1–16 (1997). 19. Verhagen, A. M., Coulson, E. J., and Vaux, D. L.: Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs, Genome Biol., 2, 3009.1–3009.10 (2001). 20. Zhang, A., Wu, Y., Lai, H. W. L., and Yew, D. T.: Apoptosis – a brief review, Neuroembryol., 3, 47–59 (2004). 21. Holcik, M., Gibson, H., and Komeluk, R. G.: XIAP: apoptotic brake and promising therapeutic target, Apoptosis, 6, 253–261 (2001). 22. Sauerwald, T. M., Figueroa Jr., B., Jr., Oyler, G. A., Hardwick, J. M., and Betenbaugh, M. J.: Combining caspase and mitochondrial dysfunction inhibitors of apoptosis to limit cell death in mammalian cell cultures, Biotechnol. Bioeng., 94, 362–372 (2006). 23. Dorai, H., Kyung, Y. S., Ellis, D., Kinney, C., Lin, C., Jan, D., Moore, G., and Betenbaugh, M. J.: Expression of anti-apoptosis genes alters lactate metabolism of Chinese hamster ovary cells in culture, Biotechnol. Bioeng., 103, 592–608 (2009). 24. Kim, Y. G., Kim, J. Y., and Lee, G. M.: Effect of XIAP overexpression on sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells producing erythropoietin, J. Biotechnol., 144, 299–303 (2009). 25. Becker, E., Lore, F., Pfizenmaier, K., and Kaufmann, H.: Evaluation of a combinatorial cell engineering approach to overcome apoptotic effects in XBP-1 (s) expressing cells, J. Biotechnol., 146, 198–206 (2010). 26. Nomura, T., Mimata, H., Takeuchi, Y., Yamamoto, H., Miyamoto, E., and Nomura, Y.: The X-linked inhibitor of apoptosis protein inhibits taxol-induced apoptosis in LNCaP cells, Urol. Res., 31, 37–44 (2003). 27. Tey, B. T. and Al-Rubeai, M.: Effect of bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells, J. Biosci. Bioeng., 100, 303–310 (2005). 28. Linette, G. P., Li, Y., Roth, K., and Korsmeyer, S. J.: Cross talk between cell death and cell cycle progression: Bcl-2 regulates NFAT-mediated activation, Proc. Natl. Acad. Sci. U. S. A., 93, 9545–9552 (1996). 29. Deng, X., Gao, F., and May Jr., W. S., Jr.: Bcl-2 retards G1/S cell cycle transition by regulating intracellular ROS, Blood, 102, 3179–3185 (2003). 30. Mazur, X., Fussenegger, M., Renner, W. A., and Bailey, J. M.: Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27, Biotechnol. Prog., 14, 705–713 (1998). 31. Levkau, B., Garton, K. J., Ferri, N., Kloke, K., Nofer, J., Baba, H. A., Raines, E. W., and Breithardt, G.: XIAP induces cell-cycle arrest and activates nuclear factor-κB, Circ. Res., 88, 282–290 (2001). 32. Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., and Ashwel, A. D.: Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli, Science, 288, 874–877 (2000). 33. Fussenegger, M., Mazur, X., and Bailey, J. E.: Novel cytostatic process enhances the productivity of Chinese hamster ovary cells, Biotechnol. Bioeng., 55, 927–939 (1997). 34. Fox, S. R., Tan, H. K., Tan, M. C., Niki, S. C., Wong, C., Yap, M. G. S., and Wang, D. I. C.: A detailed understanding of the enhanced hypothermic productivity of interferon-γ by Chinese-hamster ovary cells, Biotechnol. Appl. Biochem., 41, 255–264 (2005). 35. Liu, C. H. and Chen, L. H.: Promotion of recombinant macrophage colony stimulating factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells, J. Biosci. Bioeng., 103, 45–49 (2007).
Over-expression of the X-linked inhibitor of apoptosis protein (XIAP) delays serum deprivation-induced apoptosis in CHO-K1 cells Jane C.J. Liew,1 Wen Siang Tan,1,2 Noorjahan Banu Mohamed Alitheen,3 Eng-Seng Chan,4 and Beng Ti Tey1,5,⁎ Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 1 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 2 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, 3 Centre of Materials and Minerals, School of Engineering and IT, Universiti Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia, 4 and Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 5 Received 10 December 2009; accepted 24 February 2010 Available online 29 March 2010
Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP overexpression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells overexpressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2 days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: XIAP; CHO-K1; Serum deprivation; Apoptosis; Mammalian cell culture; Flow cytometry]
Over the past decades, mammalian cells have been widely used to produce complex proteins for both therapeutic and diagnostic use. Chinese hamster ovary (CHO) cells, one of the commercially popular cell lines, are the choice for the mass production of various recombinant proteins. In general, cells face the risk of encountering numerous insults present in a bioreactor, such as mechanical agitation, nutrient depletion, hydrodynamic stress, toxin accumulation, hypoxia and viral infection (1). Upon these stresses, cells tend to undergo apoptosis cell death. Therefore, much effort has been expended to improve process efficiency via metabolic engineering, metabolic pathway analysis and optimization of the scale-up cultures (2–7). Mammalian cell culture requires growth factors and hormone that present in the serum to enhance cell growth and to retain its cellular functions (8). However, the use of serum has several disadvantages, including high cost, lot-to-lot variations, contamination of infectious agents, such as virus and prions. Also, the presence of various protein components in the serum complicates the downstream purification process, whereby the removal of serum proteins from secreted bioproducts can be laborious, time-consuming and costly. Hence, for effective downstream processing and purification of cellular products, ⁎ Corresponding author. Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89466289; fax: +60 3 86567120. E-mail address: [email protected] (B.T. Tey).
works on development of a serum-free culture that can be utilized for effective industrial-scale production of biopharmaceuticals are being explored continuously and extensively. Serum withdrawal not only inhibits cell growth, but also triggers apoptosis cell death. Apoptotic cell death causes premature termination of cell culture duration, particularly in high cell density culture (9). Studies have shown that serum deprivation induces apoptosis in a wide range of cell lines, including hybridomas, myelomas, CHO, and BHK cells (10–13). Much attention is now being devoted to strategies for controlling and limiting cell death, either through manipulation of media supplementation or alteration of intracellular biochemistry using genetic engineering (14,15). Researchers have found that the introduction of mitogenic factors, such as insulin, transferin and insulin-like growth factor, can prolong the viability of cells cultured in serum-free media. The addition of basic fibroblast growth factor and insulin synergistically promoted the growth of CHO cells under serumfree conditions (16). There have also been other attempts to reduce apoptosis in mammalian cell-culture systems by utilizing amino acid mixtures or chemical apoptosis inhibitors such as N-acetylcysteine (NAC), bonkrekic acid, z-VAD.fluoromethly ketone (FMK), or 7-amino4-trifluoromethyl coumarin (AFC) (17). Studies on anti-apoptotic proteins can suppress apoptosis effectively, thereby increasing the production of the bioproducts. Upregulation of anti-apoptosis engineering using anti-apoptotic proteins
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such as bcl-2 and bcl-xL had shown to improve the productivity of recombinant proteins by suppressing apoptotic cell death (11–13,18). Apoptosis is a programmed cell death mode where caspases are the key effector of apoptosis (19). Hence, inhibition of caspases activity is one of the effective methods to inhibit apoptosis cascade. The inhibitor of apoptosis protein (IAP) is a family of anti-apoptotic proteins that regulate apoptosis, where XIAP (X-linked IAP) is the star player of IAP family. XIAP inhibits the caspase activity through direct binding of its baculoviral IAP repeat (BIR) domains to caspases (20). It has been evaluated as one of the key regulators of apoptosis, not only that it is the most potent inhibitor of apoptosis, it is also the bestcharacterized member of IAP family. Originally, the function of XIAP was assigned to inhibit apoptosis, but it was then discovered to be involved in a number of diverse cellular processes, including cell cycling, ubiquitylation and receptor-mediated signaling (21), making it a multi-functional protein. Previously, Sauerwald et al. (22) reported that the over-expression of a XIAP variant, which retains the BIR domain but without the C-terminal RING domain, provided protection to CHO cells exposed to Sindbis virus, etoposide and spent medium. It was then found that this XIAP variant provides a consistent enhancement in the viable cell density in lactate supplemented cultures (23). While Kim et al. (24) reported that the over-expression of XIAP could not inhibit the sodium butyrate-induced apoptosis effectively in CHO cells, Kaufmann's group reported that co-expression of both XBP-1 and XIAP genes resulted in a higher titer of IgG in a serum-free medium (25). To date, there is no attempt to study the effect of XIAP over-expression on delaying the cell death and cell proliferation under serum deprived condition. In this study, we focused on the effect of the full length XIAP on the suppression of the cell proliferation and apoptosis under serum-deprived conditions. MATERIALS AND METHODS Cell line and cell maintenance The CHO-K1 cells were routinely cultured in Ham's F12 medium supplemented with 2 mM L-glutamine (Gibco, USA) and 10% FBS (Gibco, USA) and further cultured in serum-deprived medium. For MCF-7 cell line, cells were maintained in Dulbecco's modified Eagle's media containing 4.5 gm/l glucose (Sigma-Aldrich, UK) and 10% FBS (Gibco, USA). All cell lines were incubated at 37 °C in a humidified chamber at a fixed setting of 5% CO2. Transfection The pcDNA3-myc-XIAP mammalian expression vector was kindly provided by Dr. Takeo Namuro (Department of Urology, Oita Medical University, Japan). Construction of the plasmid, pcDNA3-myc-XIAP was described previously. The vector contains a 1.5 kb human XIAP coding region and resistance genes for ampicillin and neomycin. The myc tag was used for the detection of the XIAP protein expression (26). Transfection was conducted in a 6-well plate using the GeneJuice Transfection Reagent (Novagen, USA), as recommended by the manufacturer. Transfected cells were selected in 800 μg/ml G418 sulfate (Gibco, USA) selection medium for 2 weeks. Cell cloning by limiting dilution was conducted to select stable transfectant clones. MTT assay During screening of potential clones, relative cell viability was assessed by adding 20 μl of 0.5 g/l MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole] (Sigma, USA) solution to each well. After 4 hour of incubation, 100 μl of DMSO was added to the wells and further incubated for 30 min. Absorbance at 570 nm was measured by µ-Quant ELISA microplate reader (Bio-tek Instruments, USA). Analysis of XIAP expression Potential clones were propagated in 6-well culture plates and harvested when the cell density reached 90% confluent. Harvested cells were fixed with 100 μl Cytofix/Cytoperm Fixation/Permeabilization solution (BD, USA) and incubated on ice for 20 min. One μl of 150 μg/ml anti-XIAP antibody (BD, USA) was added and incubated on ice for 30 min. Cells were washed after incubation and 1 μl of 0.5 mg/ml FITC-conjugated goat anti-mouse antibody (BD, USA) was added and incubated on ice for 20 min in dark. XIAP-FITC fluorescence was measured by the FACS Calibur System (BD, USA), while XIAP expression was analyzed using the Cell Quest Software. Cell viability analysis Batch cultures were sacrificed at 24 h intervals and cell viability was determined by using the trypan blue exclusion method. Cell suspensions were mixed with 0.4% trypan blue solution (Sigma, USA) at a 1:1 dilution. Dead and viable cells were identified and the percentage was calculated. Cell density was measured using a haemacytometer slide and cell viability was determined by dividing the number of viable cells by the total number of cells. Fluorescent microscopic analysis of apoptosis Apoptosis/necrosis count was determined using acridine orange (AO)/propidium iodide (PI) dual stain fluorescent
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microscopy. Cells were stained with 10 μl of AO/PI dual dye solution (15 μg/ml AO, 15 μg/ml PI) (Sigma, USA) for 2 min. Approximately 10 μl of stained cells were loaded onto a glass slide and observed under an inverted fluorescence microscope (Nikon, Japan) using a 10x objective lens. Cell images were captured using the ESI-Element software. The cell population was classified into 4 categories: viable (green cells with diffused chromatin), early apoptotic (green cells with condensed chromatin), late apoptotic (red cells with condensed chromatin) and necrotic (red cells with noncondensed chromatin) (12). Measurement of Caspase-3 Activity Cell lysates were prepared from 6-well culture plates. In brief, caspase-3 activity in the extract of approximately 1 × 106 cells was measured using the Caspase-3/CPP32 colorimetric assay kit (Biovision, USA). The caspase substrate DEVD-pNA (4 mM, 5 μl) was added to the samples and incubated at 37 °C for 2 hr. The enzyme-catalyzed release of pNA was quantified at 400 nm using the µ-Quant ELISA microplate reader (Bio-tek Instruments, USA). Cell cycle analysis Harvested cells were centrifuged and pellets were resuspended thoroughly in remnant. Cells were then fixed with 500 μl of ice-cold 70% (v/v) ethanol and stored at -20 °C until analysis (at least 2 hours). Fixed cells were washed twice with ice-cold PBS washing buffer (2% BSA, 0.1% Azide-EDTA), resuspended in 1 ml of PBS staining buffer (0.1% Triton X-EDTA) containing 100 mg/ml RNase A, and incubated for 30 min. Cells were then stained with 200 μl of 50 μg/ml PI solution (Sigma, USA) in dark for 15 min. DNA fluorescence was measured by using the FACS Calibur System (BD, USA), while sub populations of DNA distribution histograms were analyzed using the Cell Quest Software. Cell debris and aggregates were excluded by appropriate gating.
RESULTS Selection of stable XIAP expression clones The effect of XIAP over-expression on the suppression of apoptosis was investigated by transfecting the CHO-K1 cells with the pcDNA-myc-XIAP plasmid. Stable transfectants were selected in G418 selection medium and limiting dilution was performed to select high XIAP expresser clones. Further selection was conducted by exposing 30 isolated clones in medium without serum supplementation for 3 days. Ten most potential clones were then evaluated by MTT assay and the results are shown in Table 1. Three clones (Clones 3, 5 and 25) showing the highest viability were selected for the evaluation of XIAP expression. Flow cytometric analysis using anti-XIAP antibody conjugated to FITC was performed to confirm the XIAP expression in the selected clones. The evidence of the over-expression of XIAP in transfected population of CHO-K1 cells is shown in fluorescence histogram profiles (Fig. 1). This analysis gives a clear evidence that the selected clones (Clones 3, 5 and 25) (Fig. 1B-D) displayed higher levels of XIAP protein compared to the negative control (parental CHO-K1) (Fig. 1A). MCF-7 breast cancer cell was used as the positive control (Fig. 1E) in this analysis. An increase in fluorescence intensity was observed and the whole cell population of CHO-K1-XIAP cells was shifted to the right compared to the negative control. Stable CHO-K1-XIAP clone (Clone 5) displaying the highest expression level of XIAP was selected for further characterization. Approximately 84% of the cell population in clone 5 (Fig. 1C) was found to be expressing the XIAP stably after being passaged continuously in batch culture for 3 months. XIAP expression enhances survivability of CHO-K1 cells Both CHO-K1-XIAP and the control (parental CHO-K1) cell line were plated TABLE 1. Relative viability of the potential clones expressing XIAP. Clone
Relative Cell Viability (% of control)
1 2 3 5 7 11 12 15 20 25
3.4 38.3 141.3 119.3 95.9 91.1 56.7 57.1 97.1 98.2
After the clones were exposed in serum-deprived media for 3 days, the viability was measured using MTT assay. The relative cell viability (% of control) represents the percentage absorbance relative of the pcDNA-myc-XIAP transfected clones to the parental cells.
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FIG. 1. Analysis of XIAP expression in (A) CHO-K1, (B) CHO-K1-XIAP-3, (C) CHO-K1-XIAP-5 (D), CHO-K1-XIAP-25 and (E) MCF-7 cell lines.
and allowed to adhere for 24 h and then starved under serum-deprived condition. After 2 days of serum-deprivation, the viability of CHO-K1 cells expressing XIAP was still maintained above 90% (Fig. 2). In contrast, the control culture showed a rapid decrease in viability, a drop in viability to 40% was observed. By day 3, while the viability of CHO-K1-XIAP was still maintained at 75%, the control culture again was reduced to 30%, indicating a progressive loss of viability over times. As a result of serum deprivation, the control undergoes a more rapid apoptosis rate than CHO-K1-XIAP. This study reveals that XIAP provides a high level of protection throughout the duration of the experiment, with a viability of 43% by day 4 compared to only 20% for the control. Although expression of XIAP delayed the onset of
apoptosis, these cells eventually succumbed to cell death and the viability dropped to 30% at day 5. XIAP expression delays apoptosis induced by serum deprivation in CHO-K1 cells Fluorescence microscopy of AO and PI was employed to qualitatively visualize the distributions of viable, early apoptotic, late apoptotic and necrotic cells. The cells were classified accordingly to that described in Tey et al. (12) and the results were expressed as percentages of the total cells. At days 2 and 3, while the viability of CHO-K1-XIAP population maintain at 85% and 72% (Fig. 3A), the control culture showed a lower viability at about 55% and 38%, respectively (Fig. 3B). Throughout day 2 to day 5, apoptosis was found to progress gradually in both cell line, but with CHO-K1-
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FIG. 2. Viability analysis of CHO-K1 and CHO-K1-XIAP cells cultured under serumdeprived condition. Cell viability was determined by dividing the number of viable cells by the total number of cells in terms of percentage. Error bars represent standard deviation (n = 3).
FIG. 3. Apoptosis/ necrosis analysis of (A) CHO-K1-XIAP and (B) CHO-K1 cells. Acridine orange and propidium iodide were used in the dual staining analysis. In the figure, the term Early ap and Late ap denotes early apoptotic cells and late apoptotic cells, respectively. Error bars represent standard deviation (n = 3).
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XIAP cells progressing at a slower rate as compared to the control culture. XIAP has been reported as an effective inhibitor to caspase-3 (20). Therefore, caspase-3 activity assay was performed to investigate the effect of the expression of XIAP on the inhibition of caspase-3 activities. The total enzymatic activity of caspase-3 was measured by adding DEVD-pNA, a caspase substrate which can be cleaved by caspase-3. Fig. 4 shows the caspase-3 activity in cells sampled from day 1 to day 3. CHO-K1-XIAP showed an overall decrease in the caspase-3 activity when compared to that of the control. Notably on day 2, a rapid increase in caspase-3 was observed in the control culture, while CHO-XIAP-K1 showed no significant increase of the caspase-3 activity when compared to day 1. This result shows that over-expression of XIAP inhibits the induction of caspase-3 activity, which attenuates apoptosis in response to serum withdrawal. Flow cytometry was performed to investigate the cell cycle profile of both cell lines under serum-deprived condition. The results illustrated in Fig. 5 show that while majority of CHO-K1-XIAP population maintained at the G0/G1 peak on day 2 (Fig. 5A), the control culture peaks collapse with half of the population in the sub-G1 or apoptotic region (Fig. 5B), where XIAP expressing cell culture peaks were less affected. As illustrated in Table 2, the sub-G1 population was 6.6% for CHO-K1-XIAP and 50.3% for the control at day 2. By day 3, while the control culture reached 61.8% of apoptosis (sub-G1 population), CHO-K1-XIAP still maintained a higher viability by only showing 25% of cell death. Additionally, after 3 days of serumdeprivation, XIAP over-expression caused a decrement in the percentage of cells in the S phase and always maintained a higher percentage of cells in G0/G1 compared to the control cells. XIAP expression attenuates cell proliferation in CHO-K1 cells in serum-deprived medium The growth profiles and viable cell densities of both CHO-K1-XIAP and control cells in serum deprived medium are shown in Fig. 6. The viable cell density of the control cells decreased from day 2 onwards. In contrast, the induction of cell death of CHO-K1-XIAP cells was delayed as a result of XIAP expression. However, we observed an overall increase of total cell density in the control cells. Fig. 6B shows a 30% increment in total cell density in the control cells from days 0 to 2, while only 13% of increment in total cell density of CHO-K1-XIAP was observed at the same period of time. Even under serum-supplemented condition, CHO-K1-XIAP also exhibited a slower cell growth pattern as compared to the control (Fig. 7). Throughout days 2 to 5, CHO-K1-XIAP exhibited a higher percentage of cell population in the G0/G1 phase (Table 2), where this observation clearly suggests a difference in cell proliferation between CHO-K1-XIAP and the control. This result shows that over-expression of XIAP induces G0/G1 growth arrest, where proliferation was
FIG. 4. Measurement of caspase-3 activity in CHO-K1 and CHO-K1-XIAP cells cultured under serum-deprived medium. Relative caspase-3 activity was estimated as a ratio of the activity in cells irrespective conditions to that in cells cultured under complete growth medium (10% serum). Error bars represent standard deviation (n = 3).
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FIG. 5. Flow cytometric DNA analysis data showing cell cycle distribution for (A) CHO-K1-XIAP and (B) CHO-K1 cells cultured under serum-deprived condition.
retracted and thus resulting a 50% reduction in maximum cell density. The inhibitory effect was already visible after 2 days of serum deprivation. DISCUSSION Serum deprivation has been considered as one of the environmental stresses that can induce apoptosis cell death in mammalian cells cultured in bioreactors (8). Therefore, removal of serum usage in cell culture processes while delaying apoptosis represents a potentially major biotechnology challenge. Previous studies have shown
that by over-expressing one or more anti-apoptotic gene in mammalian cell cultures, apoptosis can be delayed and cell survival rate can be extended (8–14). Bcl-2 is one of the early anti-apoptotic genes that inhibit the release of pro-apoptotic molecules from mitochondria. It has been studied extensively in culture processes and has been shown to provide significant protection to various assaults including, hyperoxia and hypoxia conditions, depletion of nutrients, ammonia exposure and viral infection (11–13,18). While over-expression of bcl-2 maintains mitochondrial integrity by inhibiting mitochondrial apoptosis and ensuing caspase activation, XIAP acts late in the apoptotic pathway by inhibitory binding to
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TABLE 2. Cell cycle distributions for CHO-K1-XIAP and CHO-K1 cell lines in batch culture during serum deprivation-induced apoptosis. Day
2 3 4 5
Sub-G1/Apoptotic (%) CHO-K1XIAP
CHOK1
6.6 25.2 59.1 68.9
50.3 61.8 72.0 80.1
G0/G1 (%) CHO-K1XIAP 77.0 63.5 39.0 29.2
CHOK1 44.1 34.1 26.1 18.0
S (%) CHO-K1XIAP 9.0 9.0 1.6 1.5
G2/M (%) CHOK1
CHO-K1XIAP
CHOK1
4.7 3.2 1.5 1.6
7.4 2.5 0.4 0.4
0.9 0.9 0.4 0.4
downstream caspases. Therefore, XIAP may provide better protection when the mitochondrial cascade is overwhelmed, where caspases will be activated thereafter. In support of this view, we decided to investigate the ability of XIAP on the extension of the life span of CHO-K1 mammalian cells cultured in a serum-deprived environment. Our findings demonstrated that the expression of exogenous XIAP reduced apoptosis, prolonged culture and slowed down proliferation. In the absence of serum, the control culture was unable to survive for a prolonged period, however, CHO-K1-XIAP cells were found to be more effective in enhancing survivability under such harsh condition. Light micrograph images also revealed rounding and detachment of the control
FIG. 6. (A) Viable cell density and (B) growth profile of cells in batch cultures under serum-deprived condition. Cells were seeded at a concentration of 1.5 × 105 cells/ml.
FIG. 7. Total cell density of CHO-K1 and CHO-K1-XIAP cells cultured under 10% of serumsupplemented condition. Cells were seeded at a concentration of 1.5 × 105 cells/ml.
culture after 2 days of serum starvation; whereas, CHO-K1-XIAP cells remained healthy, elongated and adhered at this time point (data not shown). The control culture was affected determinately by the stressful condition of serum deprivation and within 2 days, their cell viability was reduced to 40%, with the XIAP clones showing an average cell viability of more than 90%. However, during the fourth day of serum-starvation, a sudden drop in the viability of CHO-K1-XIAP cells was observed. By that time point, cell death might be caused by the severe conditions of nutrient deprivation and accumulation of toxic metabolites in the batch cultures. Flow cytometric analysis was used to determine whether the rapid decrease in cell viability in serum-deprived media was caused by apoptosis. Apoptotic cells were presented as sub-Gl population as cells undergoing apoptosis have a lower DNA content. The results showed that although CHO-K1-XIAP cells exhibited a slower growth pattern, most of the cells were still located in the G0/G1 phase. Tey and Al-Rubeai (27) and Simpson et al. (18) reported that under a suboptimal culture condition, NS0 and hybridoma cells expressing the bcl-2 were accumulated in the G1-phase. Exit of cells from the normal cell cycle into a “quiescent state” (arrested at G1 phase) is a common strategy for some cancer cell lines in response to growth factor deprivation (28). Previous studies have demonstrated that antiapoptotic members of the bcl-2 family proteins suppressed proliferation and findings on bcl-2 expression proved that this protein does slow down cell growth (29). Studies by Mazur et al. (30) also demonstrated that expression of cyclin-dependent kinase inhibitor p27 resulted in growth arrest and a substantial improvement in protein productivity. XIAP has been identified as an active repressor of the cell cycle (31). Studies have shown that XIAP exhibits its anti-proliferative effect in the G1/S boundary of the cell cycle through its ubiquitin ligase activity (32), where XIAP may target individual cell-cycle progression elements, such as cyclin A and D1 for degradation (31), resulting an increase in the percentage of cells in G0/G1 phase. It has been documented that cells under growth arrested condition switches their cellular biosynthetic machinery from proliferation to product synthesis (10). Various studies demonstrated that the decrease in proliferation increases protein productivity. For instance, Fusseneggar et al. (33) demonstrated that by over-expressing a tumor suppressor gene to arrest the cells in G1 phases has resulted in four-fold increase in human alkaline phosphatase production. While Fox et al. (34) showed that growth arrest in G0/ G1 phase increases the productivity of IFN-γ when exposed to low temperature. Their study demonstrated
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that mRNA levels were elevated in G0/G1 phase, and a cell line can still exhibit growth-associated productivity. In addition, Liu and Chen (35) reported that the productivity of the recombinant protein was enhanced by 57% as a result of the addition of DMSO to arrest the cells at phase G0/G1, owing to the fact that growth-arrested cells do not need to devote cellular resources to biomass production. Therefore, blocking apoptosis while inducing cell-cycle arrest may be a worthwhile approach in developing a more economical and efficient process. ACKNOWLEDGMENTS Special thanks to Dr. T. Namuro (Oita Medical University, Japan) for providing the pcDNA3-myc-XIAP vector. This study was supported by the FRGS Grant (01-01-07-162 FR) from the Ministry of Higher Education, Malaysia and the Science Fund (02-01-04 SF0763) from the Ministry of Science, Technology and Innovation, Malaysia. J.C.J. Liew is supported by the Graduate Research Fellowship from Universiti Putra Malaysia. References 1. Tey, B. T., Singh, R. P., and Al-Rubeai, M.: Programmed cell death – an overview of apoptosis in cell culture, Asia Pac. J. Mol. Biol. Biotechnol., 9, 79–106 (2001). 2. Omasa, T., Tanaka, R., Doi, T., Ando, M., Kitamoto, Y., Honda, K., Kishimoto, M., and Ohtake, H.: Decrease in Antithrombin III Fucosylation by expressing GDPfucose transporter siRNA in Chinese hamster ovary cells, J. Biosci. Bioeng., 106, 168–173 (2008). 3. Omasa, T., Takami, T., Ohya, Kiyama, E., Hayashi, T., Nishii, H., Miki, H., Kobayashi, K., Honda, K., and Ohtake, H.: Overexpression of GADD34 ehnaces production of recombinant human antithrombin III in Chinese hamster ovary cells, J. Biosci. Bioeng., 106, 568–573 (2008). 4. Matsunaga, N., Kano, K., Maki, Y., and Dobashi, T.: Culture scale-up studies as seen from the viewpoint of oxygen supply and dissolved carbon dioxide stripping, J. Biosci. Bioeng., 107, 412–418 (2009). 5. Matsunaga, N., Kano, K., Maki, Y., and Dobashi, T.: Estimation of dissolved carbon dioxide stripping in a large bioreactor using model medium, J. Biosci. Bioeng., 107, 419–424 (2009). 6. Mc-Murray-Beaulieu, V., Hisiger, S., Durand, C., Perrier, M., and Jolicoeur, M.: Na-butyrate sustains energetic states of metabolism and t-PA productivity of CHO cells, J. Biosci. Bioeng., 108, 160–167 (2009). 7. Zhao, Q. and Kurata, H.: Maximum entropy decomposition of flux distribution at steady state to elementary modes, J. Biosci. Bioeng., 107, 84–89 (2009). 8. Tey, B. T. and Al-Rubeai, M.: Bcl-2 over-expression reduced the serumdependency and improved the nutrient metabolism in NS0 cell culture, Biotechnol. Bioprocess Eng., 10, 254–261 (2005). 9. Tey, B. T. and Al-Rubeai, M.: Suppression of apoptosis in perfusion culture of Myeloma NS0 cells enhances cell growth but reduces antibody productivity, Apoptosis, 9, 843–852 (2004). 10. Singh, P. R., Al-Rubeai, M., Gregory, C. D., and Emery, A. N.: Cell death in bioreactors – a role for apoptosis, Biotechnol. Bioeng., 44, 720–726 (1994). 11. Tey, B. T., Singh, R. P., Piredda, L., and Al-Rubeai, M.: Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures, J. Biotechnol., 79, 147–159 (2000). 12. Tey, B. T., Singh, R. P., Piredda, L., and Al-Rubeai, M.: Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody, Biotechnol. Bioeng., 68, 31–43 (2000). 13. Mastrangelo, A. J., Hardwick, J. M., Zou, S., and Betenbaugh, M. J.: Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults, Biotechnol. Bioeng., 67, 555–564 (2000).
14. Sakai, K., Matshunaga, T., Hayashi, C., Yamaji, H., and Fukada, H.: Effect of phosphatidic acid on recombinant protein production by Chinese hamster ovary cells in serum-free culture, Biochem. Eng. J., 10, 85–92 (2002). 15. Arden, N. and Betenbaugh, M. J.: Life and death in mammalian cell culture: strategies for apoptosis inhibition, Trends Biotechnol., 22, 174–180 (2004). 16. Liu, C. H. and Wu, K. W.: Synergistic effects of basic fibroblast growth factor and insulin on Chinese hamster ovary cells under serum-free condition, J. Biosci. Bioeng., 107, 312–317 (2009). 17. Mastrangelo, A. J., Zou, S., Hardwick, J. M., and Betenbaugh, M. J.: Anti-apoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection, Biotechnol. Bioeng., 65, 298–305 (1999). 18. Simpson, N. H., Milner, A. E., and Al-Rubeai, M.: Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions, Biotechnol. Bioeng., 54, 1–16 (1997). 19. Verhagen, A. M., Coulson, E. J., and Vaux, D. L.: Inhibitor of apoptosis proteins and their relatives: IAPs and other BIRPs, Genome Biol., 2, 3009.1–3009.10 (2001). 20. Zhang, A., Wu, Y., Lai, H. W. L., and Yew, D. T.: Apoptosis – a brief review, Neuroembryol., 3, 47–59 (2004). 21. Holcik, M., Gibson, H., and Komeluk, R. G.: XIAP: apoptotic brake and promising therapeutic target, Apoptosis, 6, 253–261 (2001). 22. Sauerwald, T. M., Figueroa Jr., B., Jr., Oyler, G. A., Hardwick, J. M., and Betenbaugh, M. J.: Combining caspase and mitochondrial dysfunction inhibitors of apoptosis to limit cell death in mammalian cell cultures, Biotechnol. Bioeng., 94, 362–372 (2006). 23. Dorai, H., Kyung, Y. S., Ellis, D., Kinney, C., Lin, C., Jan, D., Moore, G., and Betenbaugh, M. J.: Expression of anti-apoptosis genes alters lactate metabolism of Chinese hamster ovary cells in culture, Biotechnol. Bioeng., 103, 592–608 (2009). 24. Kim, Y. G., Kim, J. Y., and Lee, G. M.: Effect of XIAP overexpression on sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells producing erythropoietin, J. Biotechnol., 144, 299–303 (2009). 25. Becker, E., Lore, F., Pfizenmaier, K., and Kaufmann, H.: Evaluation of a combinatorial cell engineering approach to overcome apoptotic effects in XBP-1 (s) expressing cells, J. Biotechnol., 146, 198–206 (2010). 26. Nomura, T., Mimata, H., Takeuchi, Y., Yamamoto, H., Miyamoto, E., and Nomura, Y.: The X-linked inhibitor of apoptosis protein inhibits taxol-induced apoptosis in LNCaP cells, Urol. Res., 31, 37–44 (2003). 27. Tey, B. T. and Al-Rubeai, M.: Effect of bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells, J. Biosci. Bioeng., 100, 303–310 (2005). 28. Linette, G. P., Li, Y., Roth, K., and Korsmeyer, S. J.: Cross talk between cell death and cell cycle progression: Bcl-2 regulates NFAT-mediated activation, Proc. Natl. Acad. Sci. U. S. A., 93, 9545–9552 (1996). 29. Deng, X., Gao, F., and May Jr., W. S., Jr.: Bcl-2 retards G1/S cell cycle transition by regulating intracellular ROS, Blood, 102, 3179–3185 (2003). 30. Mazur, X., Fussenegger, M., Renner, W. A., and Bailey, J. M.: Higher productivity of growth-arrested Chinese hamster ovary cells expressing the cyclin-dependent kinase inhibitor p27, Biotechnol. Prog., 14, 705–713 (1998). 31. Levkau, B., Garton, K. J., Ferri, N., Kloke, K., Nofer, J., Baba, H. A., Raines, E. W., and Breithardt, G.: XIAP induces cell-cycle arrest and activates nuclear factor-κB, Circ. Res., 88, 282–290 (2001). 32. Yang, Y., Fang, S., Jensen, J. P., Weissman, A. M., and Ashwel, A. D.: Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli, Science, 288, 874–877 (2000). 33. Fussenegger, M., Mazur, X., and Bailey, J. E.: Novel cytostatic process enhances the productivity of Chinese hamster ovary cells, Biotechnol. Bioeng., 55, 927–939 (1997). 34. Fox, S. R., Tan, H. K., Tan, M. C., Niki, S. C., Wong, C., Yap, M. G. S., and Wang, D. I. C.: A detailed understanding of the enhanced hypothermic productivity of interferon-γ by Chinese-hamster ovary cells, Biotechnol. Appl. Biochem., 41, 255–264 (2005). 35. Liu, C. H. and Chen, L. H.: Promotion of recombinant macrophage colony stimulating factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells, J. Biosci. Bioeng., 103, 45–49 (2007).