Transfected CHO Cell Lines Research Articles

A preliminary study to establish the transfected CHO cell lines which highly express Trastuzumab - A biosimilar product of Herceptin

Human epidermal growth factor receptor 2 (HER2) has been identified as a molecular target for breast cancer therapy, such as Trastuzumab (Herceptin®). This has been shown to improve patient survival substantially. The current study is aiming to locally produce an anti-HER2 monoclonal antibody (named Trastuzumab) which has an equivalent biological properties in comparison with the original version, Herceptin®). In silico design and construction of recombinant vectors, as well as the establishment of transfected cell lines with high expression of Trastuzumab were performed. Based on the protein sequences obtained from the Drugbank, the DNA sequences encoding for the light chain (Tras-Lc) and heavy chain (Tras-Hc) of Trastuzumab were optimized and integrated into pNanogen-Hygro and pNanogen-Puro vectors, respectively. The Neon Transfection System was used to co-transfect the pNanogen-Tras-Lc-Hygro and pNanogen-Tras-Hc-Puro constructs into CHO cells. Different co-transfected single-cell-colonies selected on media supplemented with hygromycin and puromycin were used for ELISA and SDS-PAGE assays to identify the CHO cell lines which highly express Trastuzumab. Based on the present results, 30μg of both constructs were suitable for DNA co-transfection. After 07 days of culture, the highest amount of Trastuzumab (561 µg/ml) was obtained from the H06LD68 cell line.

Immature surfactant protein-B impairs the antioxidant capacity of HDL

Circulating immature surfactant protein B (proSP-B) forms emerged as the most reliable lung-specific circulating marker for alveolar-capillary membrane (ACM) dysfunction and for the overall clinical status of heart failure (HF). Notably, in terms of HF hospitalization, immature SP-B overwhelms the prognostic role of other most frequently used clinical parameters such as those related to lung dysfunction. The strong prognostic value of circulating proSP-B in HF suggests more widespread and possible systemic effects. Thus, we assessed the plasma distribution of proSP-B evaluating whether it exists in a lipoprotein-bound form and its impact on lipoprotein structure and function.ProSP-B forms were detectable in high-density lipoprotein (HDL) only. To assess the impact of proSP-B on HDL, HDL from healthy subjects were enriched with proSP-B produced by a stably transfected CHO cell line that specifically expresses and releases the human proSP-B. After enrichment, HDL size and lipoprotein electrophoretic mobility, and protein composition did not show apparent differences. HDL antioxidant capacity (HOI), assessed as their ability to inhibit air-induced LDL oxidation, was impaired after proSP-B enrichment. HOI was also higher in HF patients with respect to age-matched control healthy subjects (p = 0.013).Circulating proSP-B, besides its potential role as a specific marker for ACM dysfunction in HF patients with diagnostic and prognostic value, binds to human HDL impairing their antioxidant capacity. These findings shed light on proSP-B as a molecule that contributes to the reduction of the defense against oxidative stress, a key mediator in the pathogenesis of HF.

Acyclic Cucurbit[n]uril-Type Molecular Container Enables Systemic Delivery of Effective Doses of Albendazole for Treatment of SK-OV-3 Xenograft Tumors.

Approximately, 40-70% of active pharmaceutical ingredients (API) are severely limited by their extremely poor aqueous solubility, and consequently, there is a high demand for excipients that can be used to formulate clinically relevant doses of these drug candidates. Here, proof-of-concept studies demonstrate the potential of our recently discovered acyclic cucurbit[n]uril-type molecular container Motor1 (M1) as a solubilizing agent for insoluble drugs. M1 did not induce significant rates of mutations in various Salmonella typhimurium test strains during the Ames test, suggesting low genotoxicity. M1 also has low risk of causing cardiac toxicity in humans since it did not inhibit the human Ether-à-go-go-Related Gene channel as tested on transfected CHO cell lines via patch clamp analysis. Albendazole (ABZ) is a widely used antihelminthic agent but that has also shown promising efficacy against cancerous cells in vitro. However, due to its low aqueous solubility (2.7 μM) and poor pharmacokinetics, ABZ is clinically limited as an anticancer agent. Here we investigated the potential of M1 as a solubilizing excipient for ABZ formulation. A pharmacokinetic study indicated that ABZ escapes the peritoneal cavity resulting in 78% absolute bioavailability, while its active intermediate metabolite, albendazole sulfoxide, achieved 43% absolute bioavailability. The daily dosing of 681 mg/kg M1 complexed with 3.2 mg/kg of ABZ for 14 days did not result in significant weight loss or pathology in Swiss Webster mice. In vivo efficacy studies using this M1·ABZ inclusion complex showed significant decreases in tumor growth rates and increases in survival of mice bearing SK-OV-3 xenograft tumors. In conclusion, we provide substantial new evidence demonstrating that M1 is a safe and efficient excipient that enables in vivo parenteral delivery of poorly water-soluble APIs.

Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies.

The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by "self-cleaving" 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.

Developing an antibody targeting CεmX of mIgE for the treatment of allergic and other IgE-mediated diseases (VAC6P.954)

Abstract CεmX (also referred to as M1’) is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ε heavy chain of membrane-bound IgE (mIgE) on human B lymphocytes. Antibodies that target CεmX are potentially useful in controlling IgE production for treating allergic and other IgE-mediated diseases. Based on its abilities to bind to mIgE with high affinity and to lyse mIgE-expressing B lymphocytes by apoptosis, ADCC, and other cytolytic mechanisms, humanized 4B12 monoclonal antibody (h4B12 mAb) is now under preclinical development. In an allergic asthma animal model employing genetically modified mice that express mIgE containing human CεmX domain, we have demonstrated that mAb 4B12 was effective in reducing antigen-specific IgE, but not immunoglobulins of other isotypes. 4B12 could also markedly alleviate airway hyperresponsiveness (AHR) to inhaled methacholine in those mice. Furthermore, we have demonstrated that h4B12 could inhibit the production of human IgE in mice that had been reconstituted with human PBMCs. We have now developed a transfected CHO cell line capable of producing h4B12 at a high yield in preparation for human clinical trials.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

Effect of temperature, pH, dissolved oxygen, and hydrolysate on the formation of triple light chain antibodies in cell culture

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB-drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed-batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large-scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ∼50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels.

High‐level protein expression in scalable CHO transient transfection

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14-day fed-batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed.

FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.

Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts

BackgroundCytoadherence of Plasmodium falciparum-infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50–60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript.MethodsP. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBLα specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.ResultsEach cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.ConclusionTransfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.

Insensitivity to Aβ42-lowering Nonsteroidal Anti-inflammatory Drugs and γ-Secretase Inhibitors Is Common among Aggressive Presenilin-1 Mutations

Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.

Importance of NPA motifs in the expression and function of water channel aquaporin-1

The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and efficient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 deletion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water permeation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

Plasmodium falciparum: analysis of transcribed var gene sequences in natural isolates from the Brazilian Amazon region

Parasite isolates from Brazilian Western Amazonian patients suffering from uncomplicated falciparum malaria were matured in vitro and their var gene transcripts were analysed by RT-PCR and sequencing. Additionally, the cytoadherence patterns of these isolates were determined by panning techniques using transfected CHO cell lines expressing different surface receptors. All of the isolates tested showed between 4 and 13 different var gene transcripts per isolate. Several of these transcripts were present in more than one isolate and three sequences appeared to be preferentially expressed in natural infections. In most of the isolates, cytoadherence occurred to the receptors ICAM-1 and CD36. Several isolates showed a multiadherent profile. Analysis of MSP1 and MSP2 allelic polymorphism indicated polyclonal infections, that could be responsible for the multiadherent phenotype.

Sodium butyrate stimulates the synthesis of firefly luciferase in transfected CHO cells but levels of BiP chaperone are unaffected.

A stably transfected CHO cell line (LUCLEAD) was used where the coding region of native Firefly luciferase was linked to the 3'-UTR of the bovine growth hormone, and the 5'-nucleotides coding for the albumin signal peptide were linked to the N-terminal end of the luciferase coding region. Incubation of cells with 1 or 2 mM sodium butyrate (SB) for 72 h had no effect on cell growth since cultures reached confluency at the same time as control cells. Although cell cultures incubated with SB at a concentration of 4 mM were only about 60% confluent the luciferase content was about 5-fold higher than that in control cells. Cells incubated with either 1 or 2 mM SB showed intermediate levels of luciferase content. The amount of the chaperone BiP in the cells was not affected by incubation with SB. The results indicate that SB can be used to effectively promote synthesis of recombinant luciferase.

Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system

Lipopolysaccharide (LPS) is a key inflammatory mediator. Due to its ability to potently activate host inflammatory and innate defense responses, it has been proposed to function as an important molecule that alerts the host of potential bacterial infection. However, although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained from Porphyromonas gingivalis, an etiologic agent for periodontitis, causes a highly unusual host innate host response. It is an agonist for human monocytes and an antagonist for human endothelial cells. Correspondingly, although it activates p38 MAP kinase in human monocytes, P. gingivalis LPS does not activate p38 nor ERK MAP kinase in endothelial cells. In fact, P. gingivalis LPS is an effective inhibitor of Escherichia coli LPS induced p38 phosphorylation. These data show that P. gingivalis LPS modulates host defenses in endothelial cells by interfering with MAP kinase activation. In addition, P. gingivalis LPS is unusual in that it engages TLR-2 but not TLR-4 when examined in stably transfected CHO cell lines. We propose that, since LPS is a key ligand for the human innate host defense system, these unusual properties of P. gingivalis LPS are associated with the bacterium's role in the pathogenesis of periodontitis.

Mutation of Any Site of N-Linked Glycosylation Accelerates the In Vivo Clearance of Recombinant Rabbit Antithrombin

Antithrombin (AT) is a plasma protein with four sites of N-linked glycosylation. Asn 135 is incompletely glycosylated, and the resulting 3-glycan AT is cleared more rapidly in vivo than the 4-glycan form. The Asn codons in each of the four sites of glycosylation were altered in turn, to create four mutant rabbit AT cDNAs. Permanently transfected CHO cell lines were generated following transfection of the resulting constructs, encoding either the wild-type rabbit AT (AT-WT) or one of the four underglycosylated variants (AT-N96Q, AT-N135Q, AT-N155Q, and AT-N155Q). Comparison of the five resulting recombinant AT proteins revealed that the major AT species of each variant co-migrated on SDS gels, and migrated more rapidly than the major form of AT-WT. The shift in mobility, from 60 to 57 kDa, was consistent with the loss of one fully sialylated complex N-linked glycan. Neither the amount of AT secreted (range: 1.25 to 4.2 μg/10 6 cells/day) nor the kinetics of secretion differed significantly between cell lines expressing AT-WT or any of the AT variants. All forms of recombinant rabbit AT were capable of forming denaturation-resistant complexes with thrombin. Purification and radioiodination of each of the five recombinant AT proteins permitted pharmacokinetic analysis of their individual clearance in rabbits. While neither the equilibration half-life (t 0.5α) nor the terminal catabolic half-life (t 0.5β) differed significantly between plasma-derived rabbit AT and AT-WT, the t 0.5β of all the underglycosylated variants was decreased relative to that of AT-WT (maximum reduction in mean: from 70.1±3.2 h to 52.4±2.5 h). These results suggest that the overall extent of glycosylation, rather than the location within AT of the glycan chains, is a primary determinant of AT clearance.

Evidence for the participation of beta-hexosaminidase in human sperm-zona pellucida interaction in vitro.

Mammalian sperm-zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (beta-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm-ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.

Interaction of CD46 with measles virus: accessory role of CD46 short consensus repeat IV.

To define further the accessory role(s) of the CD46 (membrane cofactor protein) short consensus repeat (SCR) III and IV domains in the interaction of CD46 with measles virus (MV), chimeric proteins were generated by substituting domains from the structurally related protein decay accelerating factor (DAF, CD55): x3DAF (exchange of CD46 SCR III) and x4DAF (exchange of SCR IV). Transfected CHO cell lines that stably expressed these chimeric proteins were compared for MV binding and infection. Compared with wild-type CD46 (I-II-III-IV), a significant decrease in MV binding was observed with x4DAF. Despite this limited binding, these cells were still capable of supporting virus entry. In a quantitative fusion assay, no significant differences in fusion were observed as a result of the exchange of either CD46 SCR III or IV. However, the down-regulation of cell surface CD46 typically observed following MV infection was abolished with x4DAF, as was the redistribution of CD46 on the cell surface. Thus, CD46 SCR IV appears to be required for optimal virus binding and receptor down-regulation, although importantly, in spite of these functional limitations, x4DAF can still be used for MV entry.

Role of Antioxidant Enzymes on Ionizing Radiation Resistance

Exposure of cells to ionizing radiation leads to formation of reactive oxygen species (ROS) that are associated with radiation-induced cytotoxicity. The antioxidant enzymes manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), and glutathione peroxidase (GPx), are key intracellular antioxidants in the metabolism of ROS. We investigated the potential role of the three antioxidant enzymes in radioresistance by analyzing cell morphology, cell viability, and cellular radioresistance in stably transfected CHO cell lines overexpressing MnSOD, CuZnSOD, and GPx, respectively. We found that overexpression of human MnSOD substantially protected cells from ionizing radiation injury. Relative to MnSOD, GPx could slightly reduce the radiation sensitivity while the radioresistance in CuZnSOD expressing cells did not change significantly. The results suggested that MnSOD may play a central role in protecting cells against reactive oxygen species injury during ionizing radiation exposure among MnSOD, CuZnSOD, and GPx.