Reduction of the Amount of Mrna Relative to Total Protein

Journal of Bioscience and Bioengineering VOL. 129 No. 1, 121e128, 2020 www.elsevier.com/locate/jbiosc

Characterization of Chinese hamster ovary cells with disparate chromosome numbers: Reduction of the amount of mRNA relative to total protein

1, 2, 1, , 3 1 Noriko Yamano-Adachi, Norichika Ogata, z Sho Tanaka, z x Masayoshi Onitsuka, and Takeshi Omasa

Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan,1 Nihon BioData Corporation, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa 213- 0012, Japan,2 and Graduate School of Technology, Industrial and Social Sciences, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima, Tokushima 770-8506, Japan3

Received 9 April 2019; accepted 17 June 2019 Available online 11 July 2019 Chromosomes in Chinese hamster ovary (CHO) cells are labile. We have shown that high-chromosome-number CHO cells have greater potential to become robust producers of recombinant proteins. One explanation being the increase in transgene integration sites. However, high-chromosome-number cell clones produce more IgG3 following culture of single-cell clones, even under conditions that yield the same number of integrations as cells with normal chromosome numbers. Here, we characterized high-chromosome-number cells by transcriptome analysis. RNA standards were used to normalize transcriptomes of cells that had different chromosome numbers. Our results demonstrate that the mRNA ratio of b-actin and many other genes in high-chromosome-number cells to that in normal-chromosome-number cells per cell (normalized to RNA standards) was smaller than the equivalent genomic size and cell volume ratios. Many genes encoding membrane proteins are more highly expressed in high-chromosome-number cells, probably due to differences in cell size caused by the increase in chromosomes. In addition, genes related to histone modiﬁcation and lipid metabolism are differentially expressed. The reduced transcript level required per protein produced in total and the different intracellular signal transductions might be key factors for antibody production. Ó 2019, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Aneuploidy; Antibody; Chinese hamster ovary cell; Chromosomal aberration; Omics]

The demand for biopharmaceutical products (and in particular, aberrations in CHO cells result in high recombinant protein- therapeutic antibodies) is rising (1,2). Chinese hamster ovary (CHO) producing lines. Relevant chromosomal aberrations include a cells account for 33% of all host cells approved for biopharmaceu- deletion in the telomeric region of chromosome 8 of CHO-K1- tical use in the United States and European Union from 2010 to July derived cells (16e18) and high chromosome number cells, which 2014 (2). CHO cells are advantageous for establishing human-like we isolated from a CHO-DG44 line (19). post-translational modifications such as glycosylation, folding, Indeed, the CHO-DG44 (20) line is a commonly used host cell for and assembly of recombinant proteins (3). manufacturing recombinant proteins. CHO-DG44 cells are dihy- The genomes of CHO cells and female Chinese hamsters have drofolate reductase (Dhfr)-deficient, and cannot reduce dihy- been sequenced using next-generation sequencing technologies drofolic acid to tetrahydrofolic acid. Dhfr genes can be transfected (4,5), with CHO-omics data available online (6). Moreover, further and exogenous Dhfr and its surrounding genes amplified under the analyses of the CHO transcriptome, microRNA transcriptome, and pressure of a Dhfr inhibitor, methotrexate (MTX) (21). Based on the proteome under several conditions are being performed (7e9). distribution of chromosome number in the CHO-DG44 line, we Chromosomes in CHO cells frequently undergo genomic variations have shown that most cells contain approximately 21 chromo- due to genetic instability (5,10e13). Aneuploidy underlies rapid somes, whereas 4% harbor more than 30 chromosomes (19). adaptive evolution in yeast cells, and may account for gene Notably, CHO-DG44 cells with high chromosome numbers main- expression changes due to altered chromosomal stoichiometry and tain this count for at least several months (19). downstream effects of beneficial mutations (14,15). In general, Subsequent studies have shown that high chromosome number chromosomal aneuploidy is thought to be caused by genetic cells show greater potential for becoming high protein producers. nondisjunction. Recent studies have shown that chromosomal Two cell lines isolated from CHO-DG44, namely DG44-SC20 and DG44-SC39, with modal chromosome numbers of 20 and 39, respectively, were stably transfected with an expression vector containing immunoglobulin G3 (IgG3) and Dhfr. These cell lines Corresponding author. Tel./fax: 81 6 6879 4157. þ were subsequently termed IgG3-SC20 and IgG3-SC39 (19). The E-mail addresses: [email protected] (N. Yamano-Adachi), IgG3-SC39 cell pool showed a considerably higher IgG3-specific [email protected] (N. Ogata), [email protected] (S. Tanaka), [email protected] (M. Onitsuka), [email protected] production rate than the IgG3-SC20 cell pool, with or without (T. Omasa). gene amplification by MTX (19). However, there were no differ- z These authors contributed equally to this work. ences in specific growth rate between IgG3-SC20 and IgG3- x Present address: API Process Development Dept. (Biotechnology), Chugai SC39 cell pools (19). Clonal analysis revealed that half of IgG3- Pharmaceutical Co., Ltd., 5-1 Ukima 5-chome, Kita-ku, Tokyo 115-8543, Japan.

Reproduced from Journal of Bioscience and Bioengineering 129: 121-128 (2020).

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SC39 cells contained more vector integration sites than IgG3- mRNA-Seq analysis of DG44-SC20 and DG44-SC39 and RNA standards For SC20 cells, explaining the high production rate. Nonetheless, the RNA-Seq with or without poly-A RNA extraction, a library was prepared using the TruSeq RNA Sample Kit (Illumina, San Diego, CA, USA). For RNA-Seq with poly-A remaining half (with the same number of vector integration sites as RNA extraction (mRNA), samples were extracted according to the manufacturer’s IgG3-SC20, i.e., a single integration site) also produced more IgG3 protocols. For RNA-Seq without poly-A RNA extraction (total RNA), the purify and following culture of single-cell clones than any of the IgG3-SC20 fragment mRNA method (in which polyA-containing mRNA molecules are purified clones, regardless of gene amplification (19). In this study, we over two rounds using oligo-dT-coated magnetic beads) was omitted and 0.5 mL performed transcriptome analyses on the DG44-SC20 and DG44- extracted total RNA solution added to 19.5 mL of elute, prime, and fragment mixture in the make RNA fragmentation plate step. Libraries for RNA-Seq were SC39 cell lines, and antibody-producing IgG3-SC20 and IgG3- sequenced on a NextSeq 500 (Illumina). Short read data have been deposited into SC39 hetero-cell pools, to characterize antibody production in the Short Read Archive of the DNA Data Bank of Japan (DDBJ) under project ID high chromosome number CHO cells at the cellular level. DRA005919. Normalization is essential for discovering biologically important All raw sequencing reads were mapped to the CHO-K1 RefSeq assembly (ID GCF_000223135.1) and NMIJ CRM 6204-a using a short read aligner, Bowtie (version changes in expression in such analyses (22). As the expression of 0.12.8) (33), with modified parameters (-l 75 -n 2 -p 4). All data were processed housekeeping genes can differ between cells, gene expression pro- using Bash (version 3.2, https://www.gnu.org/software/bash/) and visualized using files between cells with disparate chromosome numbers are R (3.0.2) (https://www.r-project.org/). Differentially expressed genes (DEGs) were compared by mRNA-Seq analysis using RNA standards per cell analyzed using R and the TCC package (version 1.1.99) (24). Read counts for RNA (22,23). When gene expression is adjusted for RNA amount, it is standards were corrected by actual cell number, actual RNA standard stock solution fi fi volume (as measured on an electronic scale), and actual concentration of RNA dif cult to determine the characteristics of speci c cells, in any case standard stock solutions in cell lysates, as calculated by the weight of solution to be RNA amount differs between cells. The trimmed mean of M values diluted, and that of the diluent. (TMM) method is the most widely accepted normalization method Sample preparation for mRNA-Seq and HiCEP analyses To prepare RNA for comparison of transcriptome data (24). This method focuses on samples for mRNA-Seq and HiCEP analyses of DG44-SC20, DG44-SC39, IgG3-SC20, and IgG3-SC39 cells, 7.5 105 cells in 20 mL medium were used to seed a T-75 the fact that most genes, such as housekeeping genes, show no � flask. Cells were harvested after 4 days of culture and total RNA extracted from expression variation. According to this definition, TMM-based 2.5 106 cells using the RNeasy Mini Kit (Qiagen). They were confluent at the � normalization fails on cells of different genome sizes. Spiked-in time of cell harvesting. The same total RNA samples were used in mRNA-Seq and standards have been used to develop a reliable gene expression HiCEP analyses of DG44-SC20, DG44-SC39, IgG3-SC20, and IgG3-SC39 cells. analysis protocol (25e29). Here, we examined the use of spiked-in mRNA-Seq analysis mRNA-Seq analysis (target number of bases: 6G) of standards in addition to TMM-based normalization for mRNA-Seq DG44-SC20, DG44-SC39, IgG3-SC20, and IgG3-SC39 was performed by Macrogen analysis to compare aneuploid cells. Moreover, in our tran- Japan (Kyoto, Japan) on an Illumina HiSeq 2000 with a 100 bp paired-end. Sequencing libraries were prepared using the TruSeq RNA Sample Prep Kit scriptome analysis, we also performed high-coverage expression (Illumina). Sequence data were preprocessed using Prinseq-LITE (34). The profiling (HiCEP) analysis (30) based on amplified fragment length preprocessed sequence of each sample was first assembled de novo by Trinity (35) polymorphism (AFLP) gene expression profiling (31,32). HiCEP and then by TGICL (36). Next, transcripts were quantified by RSEM (37). Finally, measures comprehensive gene expression profiles without sequence mRNA-Seq data were analyzed using Subio Platform (Subio Inc., Kagoshima, Japan), and an annotation database constructed by Maze, Inc. (Tokyo, Japan). information (30), and is a powerful tool to detect unknown tran- Protein subcellular localization and pathways of differentially expressed genes scripts, particularly in species for which entire full-length products of were identified using a comprehensive information platform, KeyMolnet (KM transcripts are not well known, such as Chinese hamster. Our Data Inc., Tokyo, Japan) (38). Cell number-corrected mRNA-Seq analysis data were research provides new insights into the transcriptome of CHO cells used for KeyMolnet analysis. Short read data have been deposited into the Short with various karyotypes, particularly those with different chromo- Read Archive of the DNA Data Bank of Japan (DDBJ) under project ID DRA005860. Assembled data have been deposited in the DNA Data Bank of Japan’s some numbers. Transcriptome Shotgun Assembly (IACG01000000). HiCEP analysis HiCEP analysis was performed by the National Institutes for Quantum and Radiological Science and Technology (Chiba, Japan). In brief, double- stranded cDNA was synthesized using a biotinylated oligo-dT primer (30). cDNA was MATERIALS AND METHODS digested with MspI followed by ligation to MspI adapter (30). Ligated products with biotin at the 50-terminus were purified by binding to magnetic beads coated with streptavidin (30). cDNA fragments were digested on the magnetic beads with Cell lines The CHO DG44 (Dhfr-) cell line was provided by Dr. L. Chasin of MseI, and ligation performed with MseI adapter (30). For selective PCR, the primer Columbia University (New York, NY, USA). Two subclones of the CHO DG44 line with was designed to match the adapters: 16 sequences of MspI-NN primers and 16 modal chromosome numbers of 20 and 39, were termed DG44-SC20 and DG44- sequences of MseI-NN primers labeled with fluorescent dyes (30). Overall, 256 SC39, respectively. Cells transfected with an expression vector containing IgG3 and (16 16) PCR products were denatured and individually loaded on an ABI Prism � Dhfr were named IgG3-SC20 and IgG-SC39 (19). 310 (Thermo Fisher Scientific, Waltham, MA, USA) for electrophoresis (30). HiCEP Cell culture DG44-SC20 and DG44-SC39 cells were cultured in Iscove’s fragment database was generated by Maze, Inc. fi ’ modi ed Dulbecco s medium (IMDM) (Sigma Aldrich, St. Louis, MO, USA) Protein quantification Cells were homogenized in iced RIPA buffer with a supplemented with 10 mM thymidine, 100 mM hypoxanthine, and 10% dialyzed protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan) and a phosphatase fetal bovine serum (FBS) (SAFC Biosciences, Lenexa, KS, USA). IgG3-SC20 and IgG- inhibitor cocktail (Nacalai Tesque, Inc.) at 1.0 106 cells/90 mL. After centrifugation at � SC39 cells were cultured in IMDM containing 300 nM MTX (Sigma Aldrich) and 10,000 g for 20 min at 4 C, the total protein content in each supernatant was � � 10% dialyzed FBS without thymidine and hypoxanthine. They were cultured at measured using a BCA protein assay kit (Thermo Fisher Scientific). 37�C with 5% CO2. A Vi-cell XR automated cell analyzer (Beckman Coulter, Brea, Measurement of intracellular ATP level Cell samples were collected 48, 72, CA, USA) was used to count viable cell number. 96, 120, and 144 h after seeding cells at 1 105 cells/well of a six-well plate. AMERIC- � Sample preparation for mRNA-Seq analysis and cell number ATP Kit (Applied Medical Enzyme Research Institute Corporation, Tokushima, Japan) normalization To prepare RNA samples for mRNA-Seq analysis of DG44-SC20 was used in accordance with the manufacturer’s protocols. Relative luminescent and DG44-SC39 cells, 2 105 cells in 2 mL medium were used to seed one well of � units were measured using a white colored 96-well half-area plate and an Enspire a 6-well plate. Cells were harvested after reaching 2 106 cells. They were � Multimode Plate Reader (PerkinElmer, Inc., Waltham, MA, USA). confluent at the time of cell harvesting. RNA standards were added to cell lysates Western blotting Cell samples were collected 48, 96, and 144 h after seeding of 1 105 cells. These were a set of aqueous solutions of RNAs of five different � cells at 1 105 cells/well of a six-well plate. Western blotting was performed with an nucleotide sequences based on management systems conforming to ISO GUIDE � SDSePAGE electrophoresis system. The proteins separated in a polyacrylamide gel 34: 2009 and ISO/IEC 17025: 2005 (NMIJ CRM 6204-a; National Institute of were blotted onto a PVDF membrane by a semi-dry blotting system. The Advanced Industrial Science and Technology, Tokyo, Japan). Specifically, five membranes were incubated with 5% BSA/TBS with 0.1% Tween20 blocking buffer original RNA standard stock solutions (RNA 500-A, RNA 500-B, RNA-500-C, RNA- at room temperature for 1 h. Then, each primary antibody was incubated with 1000A, and RNA-1000 B) were diluted with water at 1/10, 1/100, 1/1000, 1/10,000, membrane at 4 C overnight. The primary antibodies used were as follows: 4E-BP1 and 1/1,000,000. Solutions were weighed, and the actual dilution ratio calculated. � antibody (Cell Signaling Technology, Inc., Danvers, MA, USA; 9452) and phospho- Cells were lysed on a QIAshredder (Qiagen, Venlo, Netherlands) and stored 4E-BP1 Ser65 antibody (Cell Signaling Technology, Inc.; 9451). The next day, the at 80�C until use. Diluted solutions of RNA standards were added to cell lysates, � membranes were incubated with horseradish peroxidase (HRP)-conjugated and total RNA extracted using the RNeasy Mini Kit (Qiagen).

152 153 VOL. 129, 2020 CHANGES IN MRNA LEVELS OF ANEUPLOID CHO CELLS 123 secondary antibody, rabbit anti-mouse IgG H&L (Abcam, Cambridge, UK; ab6728), at of samples (A: [log DG44-SC39 log DG44-SC20]/2). Values 2 þ 2 room temperature for 4 h. Immunoreactive bands were visualized using Immobilon were normalized using the TMM-based TMM-baySeq-TMM Western Chemiluminescent HRP substrate (Merck Millipore, Darmstadt, Germany) pipeline with R and the TCC package (24). Light green dots and detected by WSE-6100 LuminoGraph I (ATTO Corporation, Tokyo, Japan). fi Signal intensities were measured by CS Analyzer4 software (ATTO Corporation). represent the ve RNA standards. Because M values of RNA standards were nearly equal between samples for which mRNA was not selected (Fig. 1A and Table S1) and selected (Fig. 1B and Table S2), the binding efficiency of RNA standards to poly-T RESULTS magnetic beads can be assumed to be the same as mRNA. The b- actin ratio in DG44-SC39 to DG44-SC20 cells per cell, normalized Normalization of mRNA levels to cell number in cell lines to RNA standards, was 1.223. This is the same as with other genes with different chromosome numbers Expression levels of that show a propensity to remain constant (Fig. 1B), so this cell housekeeping genes (e.g., b-actin) may differ between CHO cells number-corrected mRNA-Seq data were used in subsequent due to aneuploidy. Therefore, existing mRNA levels per cell were analyses. Expression ratio of other representative housekeeping first examined. RNA standards (NMIJ CRM 6204-a) were mixed genes in DG44-SC39 to DG44-SC20 cells per cell is 1.350 for with cells depending on cell number. It was not known if the glyceraldehyde 3-phosphate dehydrogenase and 1.338 for poly-A sequence of RNA standards had the same binding ribosomal protein L30. Histograms of mRNA expression ratios for efficiency to poly-T magnetic beads as mRNA. Consequently, we DG44-SC39:DG44-SC20 per cell are shown in Fig. 1C. performed reverse transcription using random primers on total RNA (of DG44-SC20 and DG44-SC39 cells) and RNA standards Number of differentially expressed genes classified by under two conditions: with mRNA either selected or not by poly- subcellular localization mRNA-Seq analysis was performed on T magnetic beads. Next, the sequences and amount of reverse DG44-SC20, DG44-SC39, IgG3-SC20, and IgG3-SC39 cells. transcribed RNA were determined by RNA-Seq. The MA plot Differentially expressed genes per cell, as identified by mRNA- showing differences in levels of reverse transcribed RNA between Seq, had levels more than twice or less than half those in cells DG44-SC20 and DG44-SC39 cells is shown in Fig. 1. The vertical with a different chromosome number. Differentially expressed axis represents differences between samples in binary log- genes were classified by subcellular localization of the protein by transformed values (M: log2 DG44-SC39 e log2 DG44-SC20), and KeyMolnet, a bioinformatics tool for analyzing protein networks the horizontal axis shows average binary log-transformed values (38). More than 75% of genes that were classified to the cell

FIG. 1. Comparison of RNA amount per cell in cells with different chromosome numbers. Differences in RNA amount between RNA-Seq measurements of DG44-SC20 and DG44- SC39 cells were visualized using MA plots. Total RNA (A) or mRNA selected from total RNA by poly-T magnetic beads (B) was reverse transcribed using random primers. The MA plot for group G1 corresponds to DG44-SC20, and group G2 corresponds to DG44-SC39. Values were normalized using a TMM-based TMM-baySeq-TMM pipeline. Light green dots represent each of the five RNA standards diluted to various concentrations. The light green line reflects the average of RNA standards. Magenta dots are differentially expressed genes identified by the BenjaminieHochberg method (false discovery rate, q-value < 0.05). Each RNA-Seq was performed in three independent measurements. (C) Histograms of mRNA ratios in cells with different chromosome numbers DG44-SC39:DG44-SC20 as measured by mRNA-Seq. mRNA-Seq data were corrected by cell number according to the RNA standard results. Black bars indicate predicted cell volume ratios.

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membrane, and all genes that were classified to the nuclear products in IgG3-SC20, and 35,133 products in IgG3-SC39 cells membrane, being higher in DG44-SC39 and IgG3-SC39 cells (Table S4). In total, 42,231 expression products were observed in compared with DG44-SC20 and IgG3-SC20 cells, respectively these four CHO-DG44 lines (Table S4). In principle, only one (Table 1). Proteins that localized to the cytoplasm also had a target fragment is obtained from a single transcription product by higher number of classified genes upregulated in DG44-SC39 and HiCEP. Thus, the total number of expression products detected IgG3-SC39 cells compared with DG44-SC20 and IgG3-SC20 cells, was larger than the currently predicted number of genes in the respectively (Table 1). Conversely, the number of classified genes ancestral CHO-K1 cell line (4) and female Chinese hamster (5). for intranuclear proteins that had lower levels in IgG3-SC39 However, this is reasonable because both coding and non-coding versus IgG3-SC20 cells, exceeded that of more highly expressed RNA are detected by the HiCEP method. In addition, HiCEP can genes (Table 1). In DG44-SC39 cells, the levels of more than two- quantitatively detect transcripts in a range as low as 1e500 thirds of classified genes coding for intranuclear proteins were copies/cell (30). Of 42,231 expression products, 33,037 products greater than those of DG44-SC20 cells (Table 1). showed similar expression (expression ratios between 0.5 and 2, and including undetected products) among the four samples Pathways related to differentially expressed (Table S4). These numbers were obtained under conditions with genes Pathways related to differentially expressed genes per equal amounts of total RNA at the first reverse transcription step. cell, as identified by mRNA-Seq, were searched using the Unfortunately, we failed to obtain results of a satisfactory quality KeyMolnet network. The pathways identified are shown in for product sequences. Table 2 by chromosome number (DG44-SC20 vs. DG44-SC39, and The number of genes with expression levels more than twice IgG3-SC20 vs. IgG3-SC39). Selected important genes are listed in or less than half between cells with or without an IgG3 expression Table S3. Differentially expressed genes were primarily related to vector was examined in cells with the same number of chromo- cell growth, differentiation, and apoptosis. In addition, among somes. The number of differentially expressed products deter- histone modification pathways, histone acetyltransferase (HAT) mined by HiCEP or mRNA-Seq are shown in Fig. 2. Notably, the signaling was lower in IgG3-SC39 cells, as was breast cancer number of genes with decreased expression in IgG3-SC39 versus (BRCA) signaling in the DG44-SC39 and IgG3-SC39 lines (Table 2). DG44-SC39 cells exceeded those with increased expression Moreover, transcriptional regulation by sterol regulatory element- (Fig. 2). binding protein (SREBP), which governs cholesterol synthesis (39,40), and peroxisome proliferator-activated receptor gamma Protein expression and intracellular ATP levels of cells with (PPARg), which controls fatty acid metabolism and adipocyte different chromosome numbers We previously reported that differentiation (41), were increased in IgG3-SC39 cells (Table 2). DG44-SC39 and IgG3-SC39 cells have larger diameters than Beta-amyloid signaling, which is involved in lipid metabolism DG44-SC20 and IgG3-SC20 cells (19). The predicted volume (42,43), was increased in DG44-SC39 and IgG3-SC39 cells (Table 2). ratios based on these diameters are 2.24 (DG44-SC39:DG44- HiCEP and mRNA-Seq analyses of gene expression frequency SC20) and 1.59 (IgG3-SC39:IgG3-SC20). Compared with the with or without IgG3 expression vector An AFLP-based gene modest increase of mRNA in high-chromosome-number cells, expression profiling method, called HiCEP (30), was used to the protein expression ratios are 1.92 (DG44-SC39:DG44-SC20) detect transcription products in DG44-SC20, DG44-SC39, IgG3- and 1.62 (IgG3-SC39:IgG3-SC20), which are similar to the cell SC20, and IgG3-SC39 cells. By HiCEP analysis, we detected 35,628 volume ratios (Fig. 3A). The amount of intracellular ATP, energy products in DG44-SC20, 35,617 products in DG44-SC39, 34,513 for protein synthesis which is thought to be the most consumed

TABLE 1. Subcellular localization of proteins encoded by differentially expressed genes.

DG44-SC20 vs. DG44-SC39 IgG3-SC20 vs. IgG3-SC39

High in SC39 Low in SC39 High in SC39 Low in SC39

Cell membrane 32 10 41 11 Nuclear membrane 8 0 8 0 Cytoplasm 9 1 12 8 Intranuclear 84 47 105 143 Number of genes searched in KeyMolnet 431 188 459 417 (number of annotated genes)

TABLE 2. Differently regulated pathways in high chromosome number cells.

High in SC39 Low in SC39

CHO-DG44 LHRa RIP kinasea GnRHa Transcriptional regulation by RB/E2Fa Chemokine (CX3C, XC)a P2Ya Both CHO-DG44 and IgG3-expressing CHO-DG44 Transcriptional regulation by AP-1a Caspasea Beta-amyloidb BRCAc IgG3-expressing CHO-DG44 Spliceosome assembly Spliceosome assembly Transcriptional regulation by SREBPb Transcriptional regulation by Myca TG2a HATc Transcriptional regulation by PPARgb Transcriptional regulation by SMADa LHR, luteinizing hormone/choriogonadotropin receptor; GnRH, gonadotropin releasing hormone; AP-1, activator protein-1; SREBP, sterol regulatory element-binding protein; TG2, tissue transglutaminase; PPARg, peroxisome proliferator-activated receptor gamma; RIP, Receptor interacting protein; RB/E2F, retinoblastoma/E2 pro- moter binding factor; BRCA, breast cancer; Myc, myelocytomatosis oncogene; HAT, histone acetyltransferase; SMAD, mothers against decapentaplegic homolog. a Gene related to cell growth, differentiation, and apoptosis. b Gene related to lipid metabolism. c Gene related to histone modiﬁcation.

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during protein translation in the cellular process (44,45), was levels, which were similar to the cell volume ratios. One hypothesis measured and compared between cells with different to explain this is that the level of transcripts required per protein chromosome numbers; it was higher in DG44-SC39 and IgG3- produced is reduced in high-chromosome-number cells. From our SC39 cells than in DG44-SC20 and IgG3-SC20 cells, respectively, results, 4E-BP1, which requires phosphorylation for translation at multiple time points (Fig. 3B). In addition, the relative initiation, is more phosphorylated, and the intracellular ATP level, amounts of total (detected independent of phosphorylation) which indicates the stored energy for the cell, is increased in cells eukaryotic translation initiation factor 4E (eIF-4E)-binding with high chromosome numbers. According to the pathway anal- protein 1 (4E-BP1) and phosphorylated 4E-BP1 (p4E-BP1) in ysis, many pathways related to cell growth, differentiation, and DG44-SC39 and IgG3-SC39 cells exceeded those of DG44-SC20 apoptosis were altered, while lipid metabolism genes were upre- and IgG3-SC20 cells, respectively, at multiple time points gulated in high-chromosome-number cells. Beta-amyloid (Fig. 4A,B). p4E-BP1 dissociates from eIF-4E and activates signaling, which was increased in DG44-SC39 and IgG3- protein synthesis (46). SC39 cells, might be associated with an increase in nuclear and cell membranes (43). Our results demonstrate that many genes DISCUSSION encoding membrane proteins are more highly expressed in high chromosome number cells, probably due to differences in cell size In this study, there was a nearly 2-fold difference in chromo- caused by the increase in chromosomes. Conversely, SREBP and some number between DG44-SC20 and DG44-SC39 cells, whereas PPARg, which are also involved in lipid metabolism, were upregu- the levels of b-actin and most other genes detected by mRNA-Seq lated in IgG3-SC39 versus IgG3-SC20 cells, but not between DG44- differed by approximately 1.2-fold. In contrast, the equivalent SC20 and DG44-SC39 cells. The CHO consortium has highlighted an protein expression ratios were greater than the transcriptome interest in lipid metabolism and membrane biogenesis because the

Number of genes Number of genes increased by IgG3 expression decreased by IgG3 expression

DG44-SC20 DG44-SC39 DG44-SC20 DG44-SC39 < IgG3-SC20 < IgG3-SC39 > IgG3-SC20 > IgG3-SC39 HiCEP 306 158 640 231 74 755

DG44-SC20 DG44-SC39 DG44-SC20 DG44-SC39 < IgG3-SC20 < IgG3-SC39 > IgG3-SC20 > IgG3-SC39 mRNA-seq 222 89 309 173 63 602

FIG. 2. Number of differentially expressed products determined by HiCEP or mRNA-Seq. The number of differentially expressed products (more than two-fold or less than half) between cells in the presence or absence of an IgG3 expression vector in cells with the same chromosome number are shown using Venn diagrams.

A B

Total protein (ng/cell) Intracellular** ATP (pmol/cell)

0.3 35 *

***

30 ***

** ** 25 *

0.2 20 ** 15 0.1 10 5 0 0 DG44-SC20 DG44-SC39 IgG3-SC20 IgG3-SC39 48 72 96 120 144 (h) DG44-SC20 DG44-SC39 IgG3-SC20 IgG3-SC39

FIG. 3. The amounts of total protein and intracellular ATP of cells with different chromosome numbers. The amounts of total protein (A) and intracellular ATP (B) were measured and compared between cells with different chromosome numbers. The amount of total protein was determined by the bicinchoninic acid method. Intracellular ATP level was determined by ATP-dependent luciferin oxidation catalyzed by luciferase. Horizontal axis shows the amount of time elapsed since the seeding of cells. Analysis of variance (ANOVA) was used to compare the difference among four groups. As a post hoc test, multiple comparisons with Scheffe’s method were performed. Values are expressed as mean standard � deviation (n 3), *p < 0.05, **p < 0.01, ***p < 0.001 vs. corresponding SC20. ¼

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A Relative amount of 4E-BP1/cell B Relative amount of p4E-BP1/cell 5 8 7 4 6 5 3 DG44-SC20 4 DG44-SC39 2 3 IgG3-SC20 2 IgG3-SC39 1 1 0 0 48 96 144 (h) 48 96 144 (h)

FIG. 4. 4E-BP1 protein expression levels of cells with different chromosome numbers. Relative amounts of total 4E-BP1 (detected independent of phosphorylation) (A) and phosphorylated 4E-BP1 (p4E-BP1) (B) per cell were determined by western blotting. Signal intensities were measured using image analysis software, CS Analyzer4. Horizontal axis shows the amount of time elapsed since the seeding of cells. These are results of independent experiments of three points, 48, 96, and 144 h after seeding cells, but one result for each time point.

membrane turnover rate in protein-secreting cells is likely to be unpublished data demonstrate many chromosomal rearrange- high (47). In addition, BRCA signaling was found to be restricted in ments in CHO cells regardless of chromosome number. Inserted high-chromosome-number DG44-SC39 and IgG3-SC39 cells exogenous genes might be deleted by chromosomal rearrange- compared with that in DG44-SC20 and IgG3-SC20 lines. We have ments (67). According to previous results, the stability of each shown that, in contrast to a control cell line, downregulation of chromosome in a CHO cell differs (11). To exploit high chromosome BRCA1 in CHO cells maintained H3K4 trimethylation levels (a number CHO cells, particularly those with chromosome aberra- hallmark of transcriptionally active chromatin) around transcrip- tions, it is speculated to be important to introduce genes into tion start sites of transgenes for over 20 passages, thereby relatively stable chromosomes. Using high chromosome number extending the period of high-yield production of antibodies (48). It cells that are more efficient translatable cells and larger scale is an intriguing question why the number of genes with decreased antibody manufacturing cells will become a powerful tool, if long- mRNA expression was increased by IgG expression in high- term stable expression is guaranteed. chromosome-number cells, but this remains unresolved. Under- Supplementary data to this article can be found online at standably, these effects are synergistic and complex, necessitating https://doi.org/10.1016/j.jbiosc.2019.06.012. functional studies of these pathways in recombinant protein production. An increased set of chromosomes is known as polyploidy, and ACKNOWLEDGMENTS can occur during evolution and development (15,49,50). Whole- genome duplication (WGD) reduces the risk of extinction through This work was funded partly by the Ministry of Economy, Trade functional redundancy, mutational robustness, and increased rates and Industry (METI) of Japan and the Japan Agency for Medical of evolution and adaptation (51,52). There is also the possibility that Research and Development (AMED) for the “Developing key tech- WGDs are beneficial, independent of changes in genomic se- nology for discovering and manufacturing pharmaceuticals used quences. The skeletal DNA theory has long been discussed (53e56), for next-generation treatments and diagnoses’’ (JP17ae0101003 arguing that nuclear volumes are genetically determined primarily and JP18ae0101054), and partly by a Japan Society for the Promo- by the amount of nuclear DNA, and modulated somewhat by genes tion of Science (JSPS) KAKENHI Grant (JP26630433, JP26249125, that affect the degree of DNA packing or unfolding (57). Also, the JP17H06157, and JP18H05940). We thank Rachel James, Ph.D., from mechanical properties of nuclei are highly rigid, and can be Edanz Group for editing a draft of this manuscript. We also thank examined using microneedle-based force measurements (58). In Mr. Christopher Quach and Mr. Jun Ho Lee for their language advice. fi fl our study, the nearly 2-fold increase in genomic DNA volume The authors declare no nancial or commercial con ict of interest, enlarged cell volume by 2.24-times, supporting the skeletal DNA and this manuscript does not contain human studies or experi- hypothesis. Notably, the difference in size between IgG3-producing ments using animals. SC39 and IgG3-producing SC20 cells (1.59-fold) was smaller than between DG44-SC39 cells and DG44-SC20 cells (2.24-fold), sug- References gesting that a well-developed cell skeleton (skeletal DNA) prevents fl cell in ation and organelle dislocation in CHO cells because high- 1. Walsh, G.: Biopharmaceutical benchmarks 2010, Nat. 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