Amplification of ornithine decarboxylase gene in response to polyamine deprivation in Chinese hamster ovary cells

We have isolated from an arginase-deficient polyamine-dependent Chinese hamster ovary cell line a new mutant strain that has greatly increased ornithine decarboxylase activity. This enables the cells, in the absence of ornithine, to decarboxylate lysine into cadaverine (diaminopentane) that is further converted into N-(3-aminopropyl)cadaverine and N,N'-bis(3-aminopropyl)cadaverine. These unusual polyamines can support the growth of the cells without added polyamines derived from ornithine. Immunoreactive ornithine decarboxylase-like protein was clearly increased in the mutant cells but could not solely account for the greatly increased enzyme activity. Southern blot analysis of DNA hybridized to a plasmid carrying ornithine decarboxylase-cDNA revealed at least a 32-fold amplification of the ornithine decarboxylase gene. Ornithine decarboxylase-mRNA concentration was also highly increased in the cells. The half-life of the enzyme and the Km for ornithine were not altered from those of the parental cell line.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Summary Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B. This investigation was supported by PHS Grant CA32444, awarded by the National Cancer Institute. A. R. L. G. is a recipient of a USPHS fellowship, GM09226-01, and S. M. T. was supported by NIH training Grant AMO 7282.     

Lipopolysaccharide and serum synergistically stimulate ornithine decarboxylase in Chinese hamster ovary cells

Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B.     

Amplification and expression of heterologous ornithine decarboxylase in Chinese hamster cells.

We have developed an amplifiable mammalian expression vector based on the enzyme ornithine decarboxylase (ODC). We show greater than 700-fold amplification of this vector in ODC-deficient Chinese hamster ovary cells. A passive coamplified marker, dihydrofolate reductase (dhfr), was amplified and overexpressed 1,000-fold. This ODC vector was a dominant marker in a variety of cell types and displayed at least 300-fold amplification in wild-type Chinese hamster ovary cells.     

Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylase-like protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10(-12) M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.     

Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell line.     

The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells

We have constructed a cDNA library from the highly hydroxyurea-resistant hamster cell line 600H in which the activity of ribonucleotide reductase is elevated more than 80-fold. Using the technique of differential hybridization, we have isolated a number of cDNA clones from this library which are homologous to genomic DNA sequences amplified in the 600H cell line compared to the V79 parental line. One of these cDNA clones by sequence analysis was found to code for ornithine decarboxylase. This was confirmed by in vitro translation of poly(A+) RNA isolated by hybridization-selection followed by immunoprecipitation with antiserum specific for mouse ornithine decarboxylase. Genomic sequences homologous to the cDNA clone were shown to be sequentially amplified 6-20-fold in hamster cell lines selected stepwise for resistance to increasing concentrations of hydroxyurea. Genomic sequences homologous to a cDNA for the M2 subunit of ribonucleotide reductase were also amplified in these cell lines, and the degree of M2 sequence amplification corresponded to the degree of amplification of ornithine decarboxylase sequences, suggesting that the two genes had been co-amplified during the selection of the hydroxyurea-resistant phenotype.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium

Recent advances in transient transfection protocols using polyethylenimine (PEI) as a transfection reagent have led to the development of economical methods that provide yields sufficient for industrial production of proteins for many preclinical needs. There are many variables that can be optimized to improve protein expression in transient transfection, and one of the most critical is the medium in which the cells are grown. While transfection with PEI works well in media containing serum, the biopharmaceutical industry is moving away from animal-derived components in media. A number of serum-free media have been found to allow transient transfection, but many others do not for reasons that are not clear. Thus, knowledge of the components of serum-free media that can cause inhibition of PEI-mediated transient transfection would be useful for media development. In this study, an analysis was performed of various components of a serum-free medium used for Chinese hamster ovary cells in which PEI-mediated transient transfection was inhibited. We found that an iron supplement added to the medium was responsible for the inhibition. Further investigation showed that iron (III) citrate, a common iron chelator found in serum-free medium, was the specific component that caused the effect. Further, we showed that inhibition of transient transfection was caused by iron (III) citrate specifically, rather than citrate or iron alone. Finally, we showed that various iron chelators in serum-free media other than iron (III) citrate do not inhibit antibody expression.     

Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.     

Monitoring gene expression in Chinese hamster ovary cells using secreted apoaequorin.

A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by fusing the DNA fragment of the signal peptide sequence of human follistatin to the apoaequorin gene. Transfection of Chinese hamster ovary cells with the vector causes the apoaequorin to be secreted directly into the culture medium. Assay is carried out by removing a small aliquot of the culture medium, incubating it with coelenterazine, and adding Ca2+ to trigger light emission from the regenerated aequorin. The light intensity is measured with a photomultiplier photometer and is proportional to the amount of apoaequorin present. The method is highly specific and sensitive and can be carried out in a relatively short period of time.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Cell-cycle-dependent expression of human ornithine decarboxylase

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T-cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density-arrested W138 fibroblasts. Following stimulation of T-lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density-arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent W138 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.     

Hyperosmolarity enhances transient recombinant protein yield in Chinese hamster ovary cells

The effect of hyperosmolarity on transient recombinant protein production in Chinese hamster ovary (CHO) cells was investigated. Addition of 90 mM NaCl to the production medium ProCHO5 increased the volumetric yield of recombinant antibody up to 4-fold relative to transfection in ProCHO5 alone. Volumetric yields up to 50 mg l⁻¹ were achieved in a 6 day batch culture of 3 l. In addition, hyperosmolarity reduced cell growth and increased cell size. The addition of salt to cultures of transiently transfected CHO cells is a simple and cost-effective method to increase TGE yields in this host.     

Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells.

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.