High-expressing Clones Research Articles

Antiapoptosis Engineering for Improved Protein Production from CHO Cells.

Improving the time integral of viable cell concentration by overcoming cell death, namely apoptosis, is one of the most widely used strategies for the efficient production of therapeutic proteins. By establishing stable cell lines that overexpress antiapoptotic genes or downregulate proapoptotic genes, the final product yields can be enhanced as cells become more resistant to environmental stresses. From the selection of high-expressing clones to verification of antiapoptotic activity, the method to construct a stable antiapoptotic cell line is discussed in this chapter.

Chromatin Accessibility Plays an Important Epigenetic Role on Antibody Expression From CMV Promoter and DNA Elements Flanking the CHO TI Host Landing-Pad.

Targeted integration (TI) Chinese hamster ovary (CHO) platforms are commonly used for protein expression. However, the impact of epigenetic modifications on protein expression in TI cell lines remains elusive since almost all the epigenetic studies focus on random integration (RI) of the gene of interest and only within the promoter region. To address the impact of epigenetic modifications on TI CHO cells, we utilized a standard mAb-1 to identify and characterize TI clones with the same transgene copy numbers but different levels of transgene transcription and titer. Surprisingly, while CMV promoters were not methylated and histone acetylation/methylation was present, these epigenetic markers did not trend with mRNA transcription and protein expression in our TI model. Instead, ATAC-seq data analysis revealed that differences in chromatin accessibility within the TI site could be a major factor impacting these observed differences. However, neither chromatin accessibility nor histone acetylation/methylation profiles in early cultures were predictive of high-expressing clones early during the CLD process. Finally, modulation of the histone profiles (H3K27ac and H3K4me3) at the CMV promoters within the TI integration site using dCas9 fusion proteins was not effective in further increasing mAb titers which could have been likely due to interference of the dCas9 fusion proteins with transcription from the CMV promoters. Overall, our data suggests increasing chromatin accessibility at the TI site is the most effective way to increase mRNA transcription and hence, productivity in TI cell lines.

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies.

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore "pandemic mAb" timelines need to be shortened. One acceleration tool is "deferred cloning" and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future "pandemic mAb."

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

Streptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug. Optimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity . Synthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard. Seed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10 6 IU/mg with 62.94 ± 2.3% clot lysis efficiency. A high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.

Toward Shortened the Time-to-Market for Biopharmaceutical Proteins: Improved Fab Protein Expression Stability Using the Cre/lox System in a Multi-Use Clonal Cell Line

Stable gene integration and rapid selection of high-expressing clones are important when developing biopharmaceutical systems to produce a protein of interest. According to regulatory guidelines, the final production clones should be stable through multiple cell generations. To achieve long-term stable expression of Fab genes via recombinase-mediated cassette exchange (RMCE), we modified mutual configurations of the lox sequences. By inversion of the spacer orientation, we avoided the loss of the integrated gene after several dozen cycles of cell division. This feature also prevents reversible transgene integration. Although the RMCE allows us to generate transgenic lines rapidly relative to current methods, it remains difficult to obtain stable industrial cell lines for long-term culturing and for the initial development stage. In this study, we present an approach to shortening the timeline for therapeutic protein development. Our approach provides easy access to the same clonal cell line in the initial development phase, and also for the production of biopharmaceutical proteins.

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae.

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3′-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

Early fate of exogenous promoters in E.coli.

Gene gain by horizontal gene transfer is a major pathway of genome innovation in bacteria. The current view posits that acquired genes initially need to be silenced and that a bacterial chromatin protein, H-NS, plays a role in this silencing. However, we lack direct observation of the early fate of a horizontally transferred gene to prove this theory. We combine sequencing, flow cytometry and sorting, followed by microscopy to monitor gene expression and its variability after large-scale random insertions of a reporter gene in a population of Escherichia coli bacteria. We find that inserted promoters have a wide range of gene-expression variability related to their location. We find that high-expression clones carry insertions that are not correlated with H-NS binding. Conversely, binding of H-NS correlates with silencing. Finally, while most promoters show a common level of extrinsic noise, some insertions show higher noise levels. Analysis of these high-noise clones supports a scenario of switching due to transcriptional interference from divergent ribosomal promoters. Altogether, our findings point to evolutionary pathways where newly-acquired genes are not necessarily silenced, but may immediately explore a wide range of expression levels to probe the optimal ones.

STAT1 upregulates glutaminase and modulates amino acids and glutathione metabolism

We previously reported the upregulation of cellular Glu and glutathione levels in human ABCB5- and murine Abcb5-transfected cells. Here, we demonstrate the upregulation of STAT1 and glutaminase (GLS) in ABCB5/Abcb5-transfected cells. Among a total of four ABCB5/Abcb5 high-expressing clones with docetaxel resistance, three of the clones expressed STAT1 and GLS highly and showed resistance to docetaxel and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. Neither STAT1 nor GLS upregulation was observed in the remaining ABCB5 high-expressing clone, as well as in another two ABCB5 low-expressing clones; these three clones did not show BSO resistance. The ABCB5/STAT1 high-expressing clones showed higher cellular levels of Ala, Glu, and Asp and lower cellular levels of Phe, Trp, Leu, Ile, Gly, Met, Tyr, Val, and His compared to the ABCB5/STAT1 low-expressing clones. The former clones also showed a higher resistance to Glu. The STAT1-transfected clones expressed high levels of GLS and the corresponding mRNA, suggesting the transactivation of GLS by STAT1. These clones showed resistance to Glu and BSO, similar to the ABCB5/STAT1 high-expressing clones. The cellular glutathione levels of the STAT1-transfected clones were significantly higher than that of the control. The STAT1-transfected clones also showed greater resistance to the effect of BSO on the cellular glutathione depletion compared to the control. These results demonstrate that STAT1 upregulates GLS and modulates amino acids and glutathione metabolism. Although we were unable to directly prove STAT1 upregulation by ABCB5, our results suggest that ABCB5 expression, directly or indirectly, leads to the overexpression of STAT1.

Abstract P2-06-03: Insulin receptor isoform signaling in breast cancer

Abstract Recent data shows that insulin receptor (IR) and the type I insulin-like growth factor receptor (IGF1R) play important roles in breast cancer cell biology. Targeting only IGF1R has not been successful perhaps due to compensation by IR. IR exists in two isoforms, fetal isoform IR-A is a splice variant of IR which excludes exon 11. The adult/metabolic isoform IR-B is the predominant species expressed in normal tissues, while the fetal form IR-A is more highly expressed in breast cancer. IR-A mRNA in endocrine resistant cells is expressed at levels 24-fold higher than IGF1R expression (Gradishar, et al. Clin Cancer Res 22:301 2016 PMID: 26324738). In addition to its homodimer, IR-A can also dimerize with IGF1R to form a hybrid. Homodimer IR-A responds to IGF-II and insulin, while IGF1R/IR-A hybrid can also respond to IGF-I. Previous studies in our lab showed down regulation of IGFIR increases the sensitivity of breast cancer cells to insulin. To further investigate the roles of IR-A and IR-B in breast cancer biology, we cloned IR-A and IR-B isoforms into pLV-mCherry and pLJM1EGFP lentiviral expression vectors. Then MCF-7L breast cancer cells were infected with IR-A, IR-A vector control, IR-B and IR-B vector control. Both pooled and single clones were studied. Our results showed that both IR-A and IR-B were highly expressed in MCF-7L cells with the introduced IR tagged species present at a higher molecular weight confirmed by immunoprecipitation and immunoblot. The lower migrating species was endogenous IR. IR-A-pool/Clones had strong basal IR tyrosine phosphorylation, while IR-B-pool/clones did not. This basal phosphorylation did not activate downstream signaling as measured by pAKT and pErk1,2. IR-A basal phosphorylation was completely inhibited by BMS754807 (0.3μM), a dual IGFIR/IR tyrosine kinase inhibitor. Total levels of IRS1, IRS2, IGF1R, ERa, AKT and Erk1,2 levels were not changed in the over-expressing cells. Ligands-IGF-I (5nM), IGF-II (10nM) and Insulin (10nM) stimulated IGF1R/IR, IRS, AKT and Erk1,2 phosphorylation in IR-A-pool and high expression clone IR-A-G5. Cells expressing IR-A activated downstream signaling (IRS, AKT and Erk1,2) at 0.1nM insulin, while IR-A vector control cells required 1nM insulin. IR-B-pool overexpressing cells were not more sensitive to insulin compared to parental cells. Insulin significantly increased Erk1,2 phosphorylation in IR-A-G5 cell while IGF-I stimulation was minimal. In contrast, Erk1,2 phosphorylation in parental cells and vector control cells was primarily mediated by IGF-I, not insulin. IR-A overexpressing cells were stimulated in monolayer growth by insulin. These data show that IR-A expression, as seen in endocrine resistant breast cancer cells, sensitizes breast cancers to low concentrations of insulin. Thus, IR-A expression could serve as a target in breast cancer. Citation Format: Zhang X, Chan JY, Pan Y, Dong C, Yee D. Insulin receptor isoform signaling in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-03.

P1-175 - Heterogeneity of EGFR-mutant and PD-L1 high-expressing clones affects treatment efficacy of EGFR-TKI and PD-1 inhibitor

P1-175 - Heterogeneity of EGFR-mutant and PD-L1 high-expressing clones affects treatment efficacy of EGFR-TKI and PD-1 inhibitor

A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.

Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.

Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 × 10−5% w/v biotin) which not only simplified the protein expression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both MutS and Mut+ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.

Anti-Apoptosis Engineering for Improved Protein Production from CHO Cells.

Improving the time integral of viable cell concentration by overcoming cell death, namely apoptosis, is one of the widely used strategies for efficient production of therapeutic proteins. By establishing stable cell lines that overexpress anti-apoptotic genes or down-regulate pro-apoptotic genes, the final product yields can be enhanced as cells become more resistance to environmental stresses. From the selection of high-expressing clones to verification of anti-apoptotic activity, the method to construct a stable anti-apoptotic cell line is discussed in this chapter.

Codon Optimizing for Increased Membrane Protein Production: A Minimalist Approach.

Reengineering a gene with synonymous codons is a popular approach for increasing production levels of recombinant proteins. Here we present a minimalist alternative to this method, which samples synonymous codons only at the second and third positions rather than the entire coding sequence. As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification of a mini-library by PCR; and (3) screening for high-expressing clones.

Abstract 3013: Different tumor-initiating cells from a IDH1 wt glioblastoma patient

Abstract Glioblastoma multiforme, as its name suggests, composes pathologically heterogeneous mixed cells, such as variable degree of cellular and nuclear polymorphism, or so called “gemistocytes” or “giant cells”. Cancer cell regulates dynamic cell-cell interactions, thus, cell-cell dynamic heterogeneity is fundamental questions to know which cells are origin, therapeutic targets or source of recurrences for these decades. Therefore, functional analysis of each heterogenous cells and their role in tumor pathogenesis are critical. To investigate the cellular origin of population diversity, we established 4 subclones from a IDH1 wild type GBM patient. These subclones were subsequently propagated and analyzed. All clones expressed stem cell makers (nestin, sox2, musashi) heterogeneously. The morphology, self-renewal and proliferative capacities of the subclones were varying. FACS analysis showed that high CD133 expression clones tended to have high proliferation and invasive capacities. Most of these clones’ cells expressed high level of CD44, an adhesion molecule in cancer stem cells regulating redox status in cancer cells. Surface expression of CD24 and CD56 showed statistically different pattern. However, these 2 did not enhance their morphology, proliferation capacities and tumorigenesis. On the other hand, cDNA analysis with using 58721 probes showed that the level of COL1A1 and IGFBP7 were matched their morphology and tumorigenesis character. In animal models, xenografts from subclones, tumor-progression/invasion and animal survivals were also different. Some clones invaded contra-lateral hemisphere via tract fiber. Our observations suggest that single cell derived subclones from a patient is capable of producing phenotypically heterogeneous self-renewing progenies in an in vitro and in vivo setting. The functional analysis of the heterogeneous GSC clones may provide a better understanding of GSC biology and novel means for testing of new treatment strategies that focus on the eradication of the true GSCs. Citation Format: Akio Soeda, Akira Hara, Takahiro Kunisada, Toru Iwama. Different tumor-initiating cells from a IDH1 wt glioblastoma patient. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3013. doi:10.1158/1538-7445.AM2014-3013

High-throughput ClonePix FL analysis of mAb-expressing clones using the UCOE expression system

Therapeutic recombinant monoclonal antibodies (mAbs) are commonly produced by high-expressing, clonal, mammalian cells. Creation of these clones for manufacturing remains heavily reliant on stringent selection and gene amplification, which in turn can lead to genetic instability, variable expression, product heterogeneity and prolonged development timelines. Inclusion of cis-acting ubiquitous chromatin opening elements (UCOE™) in mammalian expression vectors has been shown to improve productivity and facilitate high-level gene expression irrespective of the chromosomal integration site without lengthy gene amplification protocols. In this study we have used high-throughput robotic clone selection in combination with UCOE™ containing expression vectors to develop a rapid, streamlined approach for early-stage cell line development and isolation of high-expressing clones for mAb production using Chinese hamster ovary (CHO) cells. Our results demonstrate that it is possible to go from transfection to stable clones in only 4 weeks, while achieving specific productivities exceeding 20 pg/cell/day. Furthermore, we have used this approach to quickly screen several process-crucial parameters including IgG subtype, enhancer-promoter combination and UCOE™ length. The use of UCOE™-containing vectors in combination with automated robotic selection provides a rapid method for the selection of stable, high-expressing clones.

Rapid Generation of Stable Cell Lines Expressing High Levels of Erythropoietin, Factor VIII, and an Antihuman CD20 Antibody Using Lentiviral Vectors

Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5½ months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules.

Development of a novel ER stress based selection system for the isolation of highly productive clones

Most biotherapeutic drugs are recombinant monoclonal antibodies which are mostly produced in monoclonal cell lines derived from Chinese hamster ovary (CHO) cells. Various clones expressing a monoclonal recombinant antibody were analyzed and a correlation of the antibody concentration and the relative mRNA level of calreticulin (CALR), glucose-regulated protein 78 and 94 kDa (GRP78, GRP94) and spliced X-box binding protein 1 (XPB1) was observed. By means of these results we were motivated to establish a novel selection system based on endoplasmic reticulum (ER) stress, which allows the rapid identification and isolation of high-expressing clones out of a pool mainly consisting of low- and medium-producing cells. Several ER stress responsive elements were tested with the aid of a recombinase mediated cassette exchange (RMCE) procedure. Very surprisingly, only GRP78 reporter constructs were strongly stimulated upon antibody expression. Furthermore we found that GRP78 reporter constructs are very suitable to reflect the level of antibody expression (IgG) in recombinant CHO cells. Based on these results, it is concluded, that the novel ER stress based selection system developed during this study is suitable to identify and isolate clones with a high level of antibody expression.

Structural and Functional Characterization of Monomeric EphrinA1 Binding Site to EphA2 Receptor

The EphA2 receptor is overexpressed in glioblastoma multiforme and has been to shown to contribute to cell transformation, tumor initiation, progression, and maintenance. EphrinA1 (eA1) is a preferred ligand for the receptor. Treatment with monomeric eA1, the form of eA1 found in the extracellular environment, causes receptor phosphorylation, internalization, and down-regulation with subsequent anti-tumor effects. Here, we investigated the structure-function relationship of a monomeric eA1 focusing on its G-H loop ((108)FQRFTPFTLGKEFKE(123)G), a highly conserved region among eAs that mediates binding to their receptors. Alanine substitution mutants of the G-H loop amino acids were transfected into U-251 MG glioblastoma multiforme cells, and functional activity of each mutant in conditioned media was assessed by EphA2 down-regulation, ERK and AKT activation and cellular response assays. Alanine substitutions at positions Pro-113 Thr-115, Gly-117, Glu-122, and also Gln-109 enhanced the EphA2 receptor down-regulation and decreased p-ERK and p-AKT. Substitution mutants of eA1 at positions Phe-108, Arg-110, Phe-111, Thr-112, Phe-114, Leu-116, Lys-118, Glu-119, and Phe-120 had a deleterious effect on EphA2 down-regulation when compared with eA1-WT. Mutants at positions Phe-108, Lys-18, Lys-121, Gly-123 retained similar properties to eA1-WT. Recombinant eA1-R110A, -T115A, -G117A, and -F120A have been found to exhibit the same characteristics as the ligands contained in the conditioned media mainly due to the differences in their binding to the receptor. Thus, we have identified variants of eA1 that possess either superagonistic or antagonistic properties. These new findings will be important in the understanding of the receptor/ligand interactions and in further design of anti-cancer therapies targeting the eA/EphA system.

Lysyl oxidase and enhancement of cell proliferation and angiogenesis in oral squamous cell carcinoma

Lysyl oxidase (LOX) is a copper-dependent enzyme that cross-links collagen and elastin in the extracellular matrix. LOX overexpressed in various tumors. The manner in which LOX affects tumor growth remains controversial. Chemical treatment and gene transfection were used to induce LOX overexpression or inhibition in cell lines SAS and SVEC4-10. LOX mRNA, protein, and activity were confirmed before tube formation assay and tumorigenesis. The microvessels in the tumor section were detected by immunostaining CD31-positive endothelial cells. LOX overexpression and copper induction of LOX activity increased SVEC4-10 tube formation. LOX silencing and β-aminopropionitrile inhibition of LOX activity had opposite effects. LOX overexpression increased proliferation and proliferating cell nuclear antigen expression. High LOX expression clones increased tumor size in a tumorigenesis model. The microvascular numbers were higher in LOX overexpression tumors than in control tumors. LOX can induce cell proliferation and angiogenesis in oral squamous cell carcinoma.