Mutants of chinese hamster cells resistant to adenosine.

Chinese hamster cells do not grow in medium containing high concentrations of adenosine because pyrimidine biosynthesis is inhibited. Adenosine metabolism was examined in two mutant cell lines isolated on the basis of resistance to adenosine. One line was deficient in adenosine kinase suggesting that high intracellular AMP concentrations may block pyrimidine synthesis indirectly in wild type cells by inhibiting PRPP synthetase. Although no enzymatic defect could be identified in the other cell line, these cells inefficiently utilize adenosine supplied in the medium.     

Plasma membrane proteins and glycoproteins from chinese hamster cells sensitive and resistant to actinomycin D

Plasma membrane proteins and glycoproteins have been isolated from Chinese hamster cells of the spontaneously transformed DC-3F parental cell line and the DC-3F/AD X line with a high level of acquired resistance to actinomycin D. Plasma membrane preparations from both cell lines band at 1.16 g/ml after isopycnic centrifugation. We present evidence to indicate differences in the leucylpeptide backbones of the antibiotic-sensitive cells and the drug-resistant DC-3F/AD X cells. In addition, there are differences in the plasma membrane glycopeptides of the two cell lines as revealed by sodium dodecyl gel electrophoresis. Drug-resistant cells synthesize a surface glycopeptide which is much larger than the major one present on the drug-sensitive cells. Both of these cell lines are devoid of 5′-nucleotidase and alkaline phosphatase activities. The role of plasma membrane protein differences in drug-resistant cells is discussed.     

Temporal behavior of abrin in the intoxication of chinese hamster cells (line CHO)

Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [¹²⁵|] abrin. About 10% of the silver grains of internalized [¹²⁵|] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS-polyacrylamide gel electrophoresis of the internalized [¹²⁵|] abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.     

Selection and characterization of chinese hamster ovary cells resistant to the cytotoxicity of lectins

Summary Chinese hamster ovary (CHO) cells selected in a single step for resistance to the cytotoxicity of the lectin from red kidney beans (PHA) behave as authentic somatic cell mutants. The PHA-resistant (Pha^R) phenotype is stable in the absence of selection; its frequency in a sensitive population is increased several-fold by mutagenesis; and it behaves recessively in somatic cell hybrids. The activity of a specific glycosyl transferase which transfers N-acetylglucosamine (GlcNAc) to terminalα-mannose residues is dramatically reduced (⩽5% of the activity detected in wild-type CHO cells) in several independent Pha^R clones. These clones also exhibit (a) a decreased ability to bind [¹²⁵I]-PHA; (b) a marked resistance to the cytotoxicity of wheat germ agglutinin (WGA), Ricin (RIC) andLens culinaris agglutinin (LCA); (c) a 4- to 5-fold increased sensitivity to the cytotoxicity of concanavalin A (Con A); (d) an increased ability to bind¹²⁵I-Con A; and (e) decreased surface galactose residues—all properties consistent with the specific loss of the GlcNAc transferase activity. The lectins WGA, RIC, LCA and Con A have also been used to select, in a single step, resistant clones from each of two complementary CHO auxotrophic lines. These lectin-resistant clones have been characterized by their ability to survive cytotoxic doses of PHA, Con A, WGA, RIC or LCA, and 4–5 “lectin-resistance” phenotypes have been demonstrated. Complementation data is being sought by somatic cell hybridization. Preliminary results show that two phenotypically-distinct Con A^R mutants are complementary in that hybrid cells formed between them exhibit wild-type sensitivity to Con A. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Lipopolysaccharide effects on sensitive and resistant variant chinese hamster ovary cell lines

Chinese hamster ovary (CHO · K1 · PRO) cell growth was inhibited by addition of a gram-negative bacterial lipopolysaccharide (LPS) to the cell culture medium. Growth inhibition began after three or four days of incubation, was dose-dependent up to a maximum at an LPS concentration of 500 μg/ml and was accompanied by cell shape changes and enhanced cytoplasmic vacuolization. Formation of bizarre CHO · K1 · PRO cell shapes and vacuole formation were most pronounced after seven days of incubation with LPS and could be observed by light and electron microscopy. An LPS-resistant cell population was obtained by intermittent in vitro exposure to high levels of LPS; these variant cells or clones derived from them failed to display growth inhibition in the presence of LPS. A clone from the LPS-resistant variant population showed altered cell properties compared to the parental cell line which included changes in cell morphology, adhesion, and endocytosis. Parental cells were markedly density-inhibited, whereas the variant clone exhibited considerable growth after confluency. The LPS-resistant variant cells showed a more elongated morphology than the parental line. No significant differences were observed between rates of detachment of parental and variant cells when sparse cultures of either line were removed from tissue culture dishes by ethylenediaminetetracetate (EDTA). However, at confluency approximately 100% of the variant cells versus 35% of the parental cells were removed by EDTA in one hour. Measurements of ¹²⁵I-ferritin uptake by parental and variant cells showed approximately twenty-fold and twofold increases, respectively, in uptake induced by LPS when compared to untreated control cultures.     

Isolation of Chinese hamster ovary ribosomal mutants differentially resistant to ricin, abrin, and modeccin

The molecular action of ricin A chain involves cleavage of the N-glycosidic bond between ribose and the adenine 4324 nucleotides from the 5' end of mammalian 28 S rRNA (Endo, Y., and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130). In this paper, four ricin- and abrin-resistant Chinese hamster ovary cell mutants that possess ribosomes resistant to this N-glycosidase action are described. Three of the mutant phenotypes, Lec26, Lec27, and Lec28, were recessive in somatic cell hybrids and define at least two new lectin-resistant complementation groups. The most extensively characterized mutant type, LEC17, was dominant in such hybrids. None of the mutants were cross-resistant to modeccin. Post-mitochondrial supernatants from each of the four mutants were resistant to inhibition of cell-free protein synthesis by ricin, ricin A chain, and abrin. In addition, polysomes isolated from mutant cells were resistant to cleavage of the adenine-ribose N-glycosidic bond by ricin A chain or abrin, as assayed by the release of an approximately 470-nucleotide fragment following aniline treatment of ribosomal RNA extracted from toxin-treated polysomes. The unique lectin-resistance properties of the different mutants suggests that the accessibility of adenine 4324 to each toxin differs. It seems likely that the recessive Chinese hamster ovary ribosomal mutants reflect structural changes in different ribosomal proteins while the dominant phenotype may be due to the modification of protein(s) or rRNA involved in toxin-ribosome interaction. Further analysis of these cell lines should provide new insights into the structure/function relationships of eukaryotic ribosomes.     

Chinese hamster cell variants resistant to the A chain of ricin carry altered ribosome function

Ricin, a toxic lectin from Ricinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 microgram/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.     

Stable alterations at the cell membrane of Chinese hamster ovary cells resistant to the cytotoxicity of phytohemagglutinin.

Chinese hamster ovary (CHO) cells selected for resistance to the cytotoxicity of phytohemagglutin (PHA) have been found to exhibit stable alterations at their plasma membranes. The PHA-resistant (PhaR) cells bind markedly less 125I-PHA than do sensitive CHO cells and also exhibit an increased sensitivity to the cytotoxicity of concanavalin A, a lectin of different receptor specificity. Mutagenesis with ethylmethanesulfonate increases the proportion of PhaR cells 20- to 100-fold. PHA-resistant cells maintained for up to 8 months in continuous culture in the absence of the selective agent have retained the PhaR phenotype. These and other characteristics of the experimental system suggest that CHO cells selected for PHA resistance are authentic somatic cell mutants. The Pha marker appears to behave recessively in hybrids formed between PhaR and PhaS cells.     

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.     

Identifying bottlenecks in transient and stable production of recombinant monoclonal-antibody sequence variants in chinese hamster ovary cells

The increasing demand for antibody-based therapeutics has emphasized the need for technologies to improve recombinant antibody titers from mammalian cell lines. Moreover, as antibody therapeutics address an increasing spectrum of indications, interest has increased in antibody engineering to improve affinity and biological activity. However, the cellular mechanisms that dictate expression and the relationships between antibody sequence and expression level remain poorly understood. Fundamental understanding of how mammalian cells handle high levels of transgene expression and of the relationship between sequence and expression are vital to the development of new antibodies and for increasing recombinant antibody titers. In this work, we analyzed a pair of mutants that vary by a single amino acid at Kabat position 49 (heavy-chain framework), resulting in differential transient and stable titers with no apparent loss of antigen affinity. Through analysis of mRNA, gene copy number, intracellular antibody content, and secreted antibody, we found that while translational/post-translational mechanisms are limiting in transient systems, it appears that the amount of available transgenic mRNA becomes the limiting event on stable integration of the recombinant genes. We also show that amino acid substitution at residue 49 results in production of a non-secreted HC variant and postulate that stable antibody expression is maintained at a level which prevents toxic accumulation of this HC-related protein. This study highlights the need for proper sequence engineering strategies when developing therapeutic antibodies and alludes to the early analysis of transient expression systems to identify the potential for aberrant stable expression behavior.     

Isolation and cell cycle analysis of temperature-sensitive mutants from chinese hamster cells

HeLa cell mitochondria were allowed to incorporate ³H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.     

Chromosome and chromatid exchanges in chinese hamster cells

Chromosomes were studied by autoradiography in a mixed culture of diploid and tetraploid cells, after having induced fusion with Sendai virus between two Chinese hamster cell populations, one labelled with ³H-, the other with ¹⁴C-thymidine; sister chromatid exchanges were studied in the ³H diploid cells and exchanges between chromosomes in the ³H-¹⁴C tetraploid synkaryons. In both cases, the frequency of exchanges increases after U. V. irradiation.Aspirant du Fonds National de la Recherche Scientifique.     

Purine uptake by azaguanine-resistant chinese hamster cells

In this study the resistance of a number of lines of Chinese hamster ovary cells to azaguanine is examined. Those which are drug resistant by virtue of a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) fail to take up any exogenous hypoxanthine or azaguanine. A second class of drug resistant cells which grow in the reverse selective HAT medium and have levels of HPRT in the range of the wild type parent line take up these purines at lower rates than the non-resistant cells and incorporate smaller amounts of them into trichloracetic acid-insoluble constituents. The results suggest that their basis for resistance resides in lowered incorporation of azaguanine into DNA and RNA, possibly due to a mofified HPRT molecule which accepts hypoxanthine, but not azaguanine as a substrate.     

Selection of chinese hamster somatic cell mutants after irradiation of BUdR-labelled cells.

The selection of somatic cell mutants after irradiation of BUdR-labelled cells is improved with the use of a mercury lamp combined with a plastic sheet which filters the unwanted wave lengths below 303 nm. New somatic cell mutants defective for galactose metabolism or auxotrophe for fetal calf serum were thus isolated.     

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan- deficient cells did not attach to substrate composed of a heparin- binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.     

The isolation and characterization of the plasma membrane of cultured chinese hamster ovary cells.

Plasma membranes were prepared from cultured Chinese hamster ovary cells utilizing a two-phase polymer system and were characterized by enzymatic and chemical assay, and by electron microscopy. The usual degree of purification of presumptive membrane markers such as Na+-K+ ATPase (ATP phosphohydrolase, EC 3.6.1.3) ranged from three-to eightfold. Gel electrophoresis in SDS revealed several polypeptides and two glycopeptides which were enriched in the plasma membrane fraction.     

Reversible inactivation of autosomal alleles in chinese hamster cells

Evidence is presented which suggests that a CHO-derived thymidine kinase (Tk) heterozygote, ts201, can reversibly inactivate the wild type Tk allele, and that this event may result in simultaneous inactivation of a linked allele, galactokinase (Glk). The clone ts201 was isolated as a revertant of a stable Tk^? 5-bromodeoxyuridine (BrdU)-resistant mutant of CHO. The reacquired Tk activity differed from that of the wild type with respect to K_m and heat resistance, supporting the contention that ts201 was genetically heterozygous (Tk^+/?). At frequencies varying from 0.02–0.5, segregants of ts201 could be isolated by cloning in BrdU at 39°. These derivatives were phenotypically Tk^? (by enzymology and by autoradiography of ³H-dT labeled cells), but after removal of BrdU, reacquired the Tk⁺ phenotype at frequencies varying randomly from clone to clone. From mutagenized populations of Ts201 two variants were isolated, 71t and 72c, by selection at 39° in 2-deoxygalactose (2-dgal). Resistance to 2-dgal has been correlated with a mutation in the gene for Glk, which is syntenic with that for Tk and evidence is presented suggesting that 71t and 72c are Glk^+/?. Cloning efficiencies in doubly selective media and autoradiographic data showed that at 39° a coordination existed between the levels of Tk and Glk, which in 71t was inverse, and in 72c, direct. These data led to the hypothesis that pairs of linked alleles can be inactivated or re-expressed simultaneously. The inactivating effect was temperature-sensitive, since at 33°, the cloning efficiency in BrdU and in 2-dgal was very low.     

Induction of thymidine kinase in enzyme-deficient chinese hamster cells

Previous work with Chinese hamster cells suggests that thymidine kinase deficiency and loss of potential for plating in HAT medium may arise by a process of mutation coupled with site-specific repression by bromodeoxyuridine at the tk locus. In this study, tk^? Chinese hamster cells were exposed to a series of inductors to determine whether revertants for the putative second stage originate by genetic or epigenetic change. Brief exposure to 5-azacytidine resulted in massive conversion to the HAT⁺ state, and revertants showed levels of thymidine kinase activity intermediate between those of tk^? and wild-type cells. By contrast, incidence of HAT⁺ cells rose only slightly in populations mutagenized with ethyl methanesulfonate. Large increases in frequency of HAT⁺ cells were obtained by treatment with n-butyrate and L-ethionine, which affect gene expression in other cell systems but have no known mutagenic potential. Induction of HAT⁺ revertants seems to be mediated by a stable epigenetic shift, which reverses the gradual extinction of thymidine kinase activity in the parent cells. The data support the view that induction in Chinese hamster cells results from changes in DNA methylation patterns, and suggest studies to define the process in molecular terms.