Peter Morin Nissom

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (∼20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336.

Transcriptome and Proteome Profiling to Understanding the Biology of High Productivity CHO Cells

A combined transcriptome and proteome analysis was carried out to identify key genes and proteins differentially expressed in Chinese hamster ovary (CHO) cells producing high and low levels of dhfr-GFP fusion protein. Comparison of transcript levels was performed using a proprietary 15 K CHO cDNA microarray chip, whereas proteomic analysis was perfomed using iTRAQ quantitative protein profiling technique. Microarray analysis revealed 77 differentially expressed genes, with 53 genes upregulated and 24 genes downregulated. Proteomic analysis gave 75 and 80 proteins for the midexponential and stationary phase, respectively. Although there was a general lack of correlation between mRNA levels and quantitated protein abundance, results from both datasets concurred on groups of proteins/genes based on functional categorization. A number of genes (20%) and proteins (45 and 23%) were involved in processes related to protein biosynthesis. We also identified three genes/proteins involved in chromatin modification. Enzymes responsible for opening up chromatin, Hmgn3 and Hmgb1, were upregulated whereas enzymes that condense chromatin, histone H1.2, were downregulated. Genes and proteins that promote cell growth (Igfbp4, Ptma, S100a6, and Lgals3) were downregulated, whereas those that deter cell growth (Ccng2, Gsg2, and S100a11) were upregulated. Other main groups of genes and proteins include carbohydrate metabolism, signal transduction, and transport. Our findings show that an integrated genomic and proteomics approach can be effectively utilized to monitor transcriptional and posttranscriptional events of mammalian cells in culture.

A novel normalization method for effective removal of systematic variation in microarray data

Normalization of cDNA and oligonucleotide microarray data has become a standard procedure to offset non-biological differences between two samples for accurate identification of differentially expressed genes. Although there are many normalization techniques available, their ability to accurately remove systematic variation has not been sufficiently evaluated. In this study, we performed experimental validation of various normalization methods in order to assess their ability to accurately offset non-biological differences (systematic variation). The limitations of many existing normalization methods become apparent when there are unbalanced shifts in transcript levels. To overcome this limitation, we have proposed a novel normalization method that uses a matching algorithm for the distribution peaks of the expression log ratio. The robustness and effectiveness of this method was evaluated using both experimental and simulated data.

Developing genomic platforms for Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used in recombinant protein production, yet despite their importance in bioprocessing, few genomic resources have been developed for this cell line. Over the past several years, we have made considerable progress in the development of genomic tools for CHO. Using Sanger-based sequencing technology, we have accrued a sequence repertoire of more than 68,000 expressed sequence tags (ESTs), representing more than 28,000 unique CHO transcripts. Using closely related species, we have functionally annotated this sequence set and have currently achieved significant representation in a number of functional classes, including some closely tied to recombinant protein production. This sequence repository has been used to design custom CHO Affymetrix arrays for transcriptome analysis. Illumina Solexa deep sequencing technology was also applied to study the CHO cell transcriptome and survey the identity and expression of small RNAs. These applications demonstrate the utility of genomic tools, and illustrate the applicability of emerging next-generation sequencing technologies.

Conserved microRNAs in Chinese hamster ovary cell lines.

MicroRNAs (miRNAs), a class of short (20-24 nt) non-coding RNAs that direct post-transcriptional repression of messenger RNAs, increasingly have been shown to play a key role in regulating cellular physiology. We investigated the prevalence of miRNAs in Chinese hamster ovary (CHO) cells by high-throughput sequencing. Six cDNA libraries of small RNAs from four CHO cell lines were constructed and sequenced by Illumina sequencing. Three hundred fifty distinct miRNA and miRNA* sequences were identified through homology with other species, including mouse, rat, and human. While the majority of the identified miRNAs appear to be expressed ubiquitously, many miRNAs were found to have a wide range of expression levels between cell lines. The identification of these miRNAs will facilitate investigations of their contribution to the hyperproductivity trait.

Quality assessment of cross-species hybridization of CHO transcriptome on a mouse DNA oligo microarray

DNA microarray technology has been widely utilized for species with extensive genome sequence information available. Given the limited genomic information pertaining to Chinese hamster ovary (CHO) cell line, cross-species hybridization using mouse microarrays provides a viable alternative. In this study, the utility of mouse Affymetrix microarrays for transcriptome profiling in CHO cells was assessed by hybridizing identical sets of cRNA samples from CHO cells on both mouse and CHO Affymetrix microarrays. Expression level measured by probe sets for orthologous transcripts on the two microarrays was compared. Only a fraction of the orthologous probes which detected expression calls in same species hybridization were similarly called present in cross species hybridization. In further analysis at the 25-mer probe level, it was revealed that specific hybridization signals were detectable by the subset of mouse probes that have a high degree of homology to the corresponding CHO sequences. The feasibility of cross species hybridization for quantifying the extent of differential expression was assessed by comparing transcript levels of CHO cells cultivated with and without sodium butyrate. While same species hybridization gave consistent degree of differential expression calls in replicated runs, a much inferior ability in quantifying differential expression was seen with cross species hybridization. Our results demonstrate that through detailed analysis of homology at the probe pair level, a subset of probes on existing mouse Affymetrix oligo-array can be used successfully for transcriptome profiling of CHO cells.

Annexin-A1 regulates microRNA-26b* and microRNA-562 to directly target NF-κB and angiogenesis in breast cancer cells

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3′ UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer.

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

BackgroundThe overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression.ResultsThe periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes.ConclusionsThe simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.

Hcc-2, a novel mammalian ER thioredoxin that is differentially expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. Thus there is great interest to identify novel HCC diagnostic markers for early detection of the disease and tumour specific associated proteins as potential therapeutic targets in the treatment of HCC. Currently, we are screening for early biomarkers as well as studying the development of HCC by identifying the differentially expressed proteins of HCC tissues during different stages of disease progression. We have isolated, by reverse transcriptase and polymerase chain reaction (RT‐PCR), a 1741 bp cDNA encoding a protein that is differentially expressed in HCC. This novel protein was initially identified by proteome analysis and we designate it as Hcc‐2. The protein is upregulated in poorly‐differentiated HCC but unchanged in well‐differentiated HCC. The full‐length transcript encodes a protein of 363 amino acids that has three thioredoxin (Trx) (CGHC) domains and an ER retention signal motif (KDEL). Fluorescence GFP tagging to this protein confirmed that it is localized predominantly to the cytoplasm when expressed in mammalian cells. Protein alignment analysis shows that it is a variant of the TXNDC5 gene, and the human variants found in Genbank all show close similarity in protein sequence. Functionally, it exhibits the anticipated reductase activity in the insulin disulfide reduction assay, but its other biological role in cell function remains to be elucidated. This work demonstrates that an integrated proteomics and genomics approach can be a very powerful means of discovering potential diagnostic and therapeutic protein targets for cancer therapy.

Identification and expression analysis of miRNAs during batch culture of HEK-293 cells

MicroRNAs (miRNAs) are small non-coding RNAs of about 20-24 nucleotides in length. They regulate gene expression negatively and have been implicated in a wide variety of biological processes. To identify potential miRNAs that may influence the growth and proliferation of mammalian cells cultured in bioreactors, we applied miRNA microarray expression profiling technology to batch cultures of HEK293 cells in protein free media. In our study, we identified miRNAs that were differentially expressed during the exponential and stationary phases, 13 of these showed distinct up regulation trends while 1 exhibited down regulation. These miRNAs have been implicated in cellular differentiation, growth arrest and apoptosis. Specifically, miR-16 and let-7b are potentially useful in the enhancement of bioreactor cell cultures.