Finbarr O'Sullivan

Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7

The efficient production of recombinant proteins by Chinese Hamster Ovary (CHO) cells in modern bioprocesses is often augmented by the use of proliferation control strategies. The most common method is to shift the culture temperature from 37 °C to 28-33 °C though genetic approaches to achieving the same effect are also of interest. In this work we used qRT-PCR-based expression profiling using TLDA™ cards to identify miRNAs displaying differential expression 24h after temperature-shift (TS) from 37 °C to 31 °C. Six miRNAs were found to be significantly up-regulated (mir-219, mir-518d, mir-126, mir-30e, mir-489 and mir-345) and four down-regulated (mir-7, mir-320, mir-101 and mir-199). Furthermore, qRT-PCR analysis of miR-7 expression over a 6 day batch culture, with and without TS, demonstrated decreased expression over time in both cultures but to a significantly greater extent in cells shifted to a lower culture temperature. Unexpectedly, when miR-7 levels were increased transiently by transfection with miR-7 mimic in CHO-K1 cells, cell proliferation at 37 °C was effectively blocked over a 96 h culture period. On the other hand, transient inhibition of endogenous miR-7 levels using antagonists had no impact on cell growth. The exogenous overexpression of miR-7 also resulted in increased normalised (per cell) production at 37 °C, though the yield was lower than cells grown at reduced temperature. This is the first report demonstrating a functional impact of specific miRNA disregulation on CHO cell behavior in batch culture and provides some evidence of the potential which these molecules may have in terms of engineering targets in CHO production clones. Finally, we report the cloning and sequencing of the hamster-specific cgr-miR-7.

Large scale microarray profiling and coexpression network analysis of CHO cells identifies transcriptional modules associated with growth and productivity

Weighted gene coexpression network analysis (WGCNA) was utilised to explore Chinese hamster ovary (CHO) cell transcriptome patterns associated with bioprocess relevant phenotypes. The dataset set used in this study consisted of 295 microarrays from 121 individual CHO cultures producing a range of biologics including monoclonal antibodies, fusion proteins and therapeutic factors; non-producing cell lines were also included. Samples were taken from a wide range of process scales and formats that varied in terms of seeding density, temperature, medium, feed medium, culture duration and product type. Cells were sampled for gene expression analysis at various stages of the culture and bioprocess-relevant characteristics including cell density, growth rate, viability, lactate, ammonium and cell specific productivity (Qp) were determined. WGCNA identified six distinct clusters of co-expressed genes, five of which were found to have associations with bioprocess variables. Two coexpression clusters were found to be associated with culture growth rate (1 positive and 1 negative). In addition, associations between a further three coexpression modules and Qp were observed (1 positive and 2 negative). Gene set enrichment analysis (GSEA) identified a number of significant biological processes within coexpressed gene clusters including cell cycle, protein secretion and vesicle transport. In summary, the approach presented in this study provides a novel perspective on the CHO cell transcriptome.

Predicting cell-specific productivity from CHO gene expression.

Improving the rate of recombinant protein production in Chinese hamster ovary (CHO) cells is an important consideration in controlling the cost of biopharmaceuticals. We present the first predictive model of productivity in CHO bioprocess culture based on gene expression profiles. The dataset used to construct the model consisted of transcriptomic data from 70 stationary phase, temperature-shifted CHO production cell line samples, for which the cell-specific productivity had been determined. These samples were utilised to investigate gene expression over a range of high to low monoclonal antibody and fc-fusion-producing CHO cell lines. We utilised a supervised regression algorithm, partial least squares (PLS) incorporating jackknife gene selection, to produce a model of cell-specific productivity (Qp) capable of predicting Qp to within 4.44 pg/cell/day root mean squared error in cross model validation (RMSE(CMV)). The final model, consisting of 287 genes, was capable of accurately predicting Qp in a further panel of 10 additional samples which were incorporated as an independent validation. Several of the genes constituting the model are linked with biological processes relevant to protein metabolism.

The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.

Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.

Demodex-Associated Bacillus Proteins Induce an Aberrant Wound Healing Response in a Corneal Epithelial Cell Line: Possible Implications for Corneal Ulcer Formation in Ocular Rosacea

PURPOSE The aim of the work presented here was to establish the response of a corneal epithelial cell line (hTCEpi) to protein extracted from a bacterium (Bacillus oleronius) previously isolated from a Demodex mite from a rosacea patient. METHODS The response of the corneal epithelial cell line to Bacillus proteins was measured in terms of alterations in cell migration and invasiveness. Changes in the expression of metalloproteinase genes and proteins were also assessed. RESULTS The results indicated increased cell migration (14.5-fold, P = 0.001) as measured using 8-μm PET inserts (BD Falcon) in a transwell assay and invasiveness (1.7-fold, P = 0.003) as measured using 8-μm Matrigel (BD Biocoat) invasion inserts in a 24-well plate assay format, following exposure to the Bacillus proteins. Cells exposed to the Bacillus protein showed a dose-dependent increase in expression of genes coding for matrix metalloprotease (MMP)-3 (61-fold) and MPP-9 (301-fold). This dose-dependent increase in gene expression was also reflected in elevated levels of MMP-9 protein (1.34-fold, P = 0.033) and increased matrix metalloprotease activity (1.96-fold, P = 0.043) being present in the culture supernatant. Cells also displayed reduced levels of β-integrin (1.25-fold, P = 0.01), indicative of increased motility and elevated levels of vinculin (2.7-fold, P = 0.0009), suggesting altered motility. CONCLUSIONS The results indicate that exposure of corneal epithelial cells to Bacillus proteins results in an aberrant wound healing response as visualized using a scratch wound assay. These results suggest a possible link between the high density of Demodex mites on the eyelashes of ocular rosacea patients and the development of corneal ulcers.

Microarray expression profiling identifies genes regulating sustained cell specific productivity (S‐Qp) in CHO K1 production cell lines

Fed batch culture processes are often characterized by decreasing cell culture performance as the process continues, presumably through the depletion of vital nutrients and the accumulation of toxic byproducts. We have similarly observed that cellular productivity (Qp) often declines during the course of a fed batch process; however, it is not clear why some cell lines elicit this behavior, while others do not. We here present a transcriptomic profiling analysis of a phenotype of sustained Qp (S‐Qp) in production Chinese hamster ovary (CHO) culture, in which a marked drop in Qp levels (“non‐sustained” (NS) phenotype) in two cell lines irrespective of viability levels was compared to two cell lines that consistently displayed high Qp throughout the culture (“sustained” (S) phenotype). Statistical analysis of the microarray data resulted in the identification of 22 gene transcripts whose expression patterns were either significantly negatively or positively correlated with long‐term maintenance of Qp over the culture lifespan. qPCR analysis of four of these genes on one of each (NS2, S2) of the cell lines examined by microarray analysis confirmed that two genes (CRYAB and MGST1) both replicated the microarray results and were differentially regulated between the NS and S phenotypes.

Activation of liver X receptor suppresses the production of the IL-12 family of cytokines by blocking nuclear translocation of NF-κBp50

There is now convincing evidence that liver X receptor (LXR) is an important modulator of the inflammatory response; however, its mechanism of action remains unclear. This study aimed to examine the effect of LXR on the IL-12 family of cytokines and examined the mechanism by which LXR exerted this effect. We first demonstrated that activation of murine-derived dendritic cells (DC) with a specific agonist to LXR enhanced expression of LXR following activation with LPS, suggesting a role in inflammation. Furthermore, we showed LXR expression to be increased in vivo in dextrane sulphate sodium-induced colitis. LXR activation also suppressed production of IL-12p40, IL-12p70, IL-27 and IL-23 in murine-derived DC following stimulation with LPS, and specifically targeted the p35, p40 and EBI3 subunits of the IL-12 cytokine family, which are under the control of the NF-κB subunit p50 (NF-κBp50). Finally, we demonstrated that LXR can associate with NF-κBp50 in DC and that LXR activation prevents translocation of the p50 subunit into the nucleus. In summary, our study indicates that LXR can specifically suppress the IL-12 family of cytokines though its association with NF-κBp50 and highlights its potential as a therapeutic target for chronic inflammatory diseases.

Exposure of a corneal epithelial cell line (hTCEpi) to Demodex-associated Bacillus proteins results in an inflammatory response.

PURPOSE A role for a bacterium, Bacillus oleronius, originally isolated from a Demodex mite, in the induction of ocular rosacea has been proposed. The aim of this work was to characterize the response of a corneal epithelial cell line to Bacillus proteins, as this might give an insight into how such proteins contribute to the symptoms of ocular rosacea in vivo. METHODS The effect of exposing Bacillus protein preparation on human telomerase-immortalized corneal epithelial cells (hTCEpi) was measured by monitoring changes in cell proliferation and the expression of a number of genes associated with inflammation. The production of inflammatory cytokines was measured and the expression and activity of MMP-9 was quantified. RESULTS Exposure of hTCEpi cells to 2 or 6 μg/mL Bacillus protein resulted in a dose-dependent reduction in cell proliferation. Exposure of cells to 6 μg/mL Bacillus protein did not induce apoptosis, but there was an increase in the expression of genes coding for IL-6 (13.8-fold), IL-1β (4.0-fold), IL-8 (11.1-fold), and TNF-α (4.1-fold). Increased expression of genes coding for the defensins, CCL20 (4.5-fold) and S100A7 (6.8-fold) also was observed. Elevated production of IL-6 and IL-8 was evident from cells exposed to 2 and 6 μg/mL Bacillus protein. The hTCEpi cells demonstrated increased MMP-9 expression (3.2-fold, P = 0.003) and activity (2.2-fold, P = 0.0186) after 48 hours of exposure to 6 μg/mL Bacillus protein preparation. CONCLUSIONS The results suggest that interaction of Demodex-associated Bacillus proteins with the corneal surface could lead to tissue degradation and inflammation, possibly leading to corneal scarring.

Comparative Transcriptomic Analysis of Cultivated Limbal Epithelium and Donor Corneal Tissue Reveals Altered Wound Healing Gene Expression

PURPOSE The improved surgical outcomes associated with transplantation of cultivated amniotic membrane expanded limbal epithelium (AMLE) compared to traditional donor methods has led to substantial adoption of this technique for treatment of limbal stem cell deficiency. METHODS The mRNA expression profiles of AMLE and CE were assayed using microarrays. Transcripts with a 1.5-fold change in either direction in addition to a Bonferroni adjusted P value < 0.05 were considered to be differentially expressed. Expression changes detected by microarray profiling and important corneal-limbal markers were assessed using quantitative real-time PCR (qRT-PCR) and immunofluorescence staining. RESULTS A total of 487 probe sets (319 upregulated and 168 downregulated) were found to be differentially expressed between AMLE and CE. Enrichment analysis revealed significant overrepresentation of multiple biological processes (e.g., response to wounding, wound healing, and regulation of cell morphogenesis) within the differentially expressed gene list. The expression of a number of genes that were upregulated (ABCG2, S100A9, ITGA5, TIMP2, FGF5, PDGFC, SEMA3A) and downregulated (KLF4, P63α) in AMLE was confirmed using qRT-PCR. Immunofluorescence confirmed that AMLE cultures were P63α, ABCG2, CK3, CK12, and E-cadherin (E-cad) positive. CONCLUSIONS In this study, we have shown that genes associated with wound healing processes are upregulated in AMLE. These gene expression changes may contribute to corneal restoration and the positive outcomes associated with transplantation.

Acute exposure to organic and inorganic sources of copper: Differential response in intestinal cell lines

Abstract Scope Copper supplementation in nutrition has evolved from using inorganic mineral salts to organically chelated minerals but with limited knowledge of the impact at the cellular level. Methods Here, the impact of inorganic and organic nutrient forms (glycinate, organic acid, and proteinate) of copper on the cellular level is investigated on intestinal cell lines, HT29 and Caco‐2, after a 2‐hr acute exposure to copper compounds and following a 10‐hr recovery. Results Following the 10‐hr recovery, increases were observed in proteins involved in metal binding (metallothioneins) and antioxidant response (sulfiredoxin 1 and heme oxygenase 1), and global proteomic analysis suggested recruitment of the unfolded protein response and proteosomal overloading. Copper organic acid chelate, the only treatment to show striking and sustained reactive oxygen species generation, had the greatest impact on ubiquitinated proteins, reduced autophagy, and increased aggresome formation, reducing growth in both cell lines. The least effect was noted in copper proteinate with negligible impact on aggresome formation or extended growth for either cell line. Conclusion The type and source of copper can impact significantly at the cellular level.