scholarly journals DNA amplification in multidrug, cross-resistant Chinese hamster ovary cells: molecular characterization and cytogenetic localization of the amplified DNA.

1986 ◽  
Vol 103 (4) ◽  
pp. 1159-1166 ◽  
Author(s):  
L D Teeter ◽  
S Atsumi ◽  
S Sen ◽  
T Kuo
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dna Amplification ◽  
Chromosome 1 ◽  
Cosmid Library ◽  
Cross Resistance ◽  
Northern Hybridization ◽  
Animal Cells ◽  
Ovary Cells

Vincristine-resistant (VCR) Chinese hamster ovary (CHO) cells have been established by stepwise selection in increasing concentrations of vincristine. These cells exhibit multidrug cross-resistance to a number of drugs that have no structural or functional similarities. Cytogenetic analyses of resistant cells revealed the presence of double minutes and expanded chromosomal segments, thus implicating gene amplification as a possible mechanism of resistance. An amplified DNA segment isolated from other multidrug cross-resistant CHO cell lines (Roninson, I. B., H. T. Abelson, D. E. Housman, N. Howell, and A. Varshavsky, 1984, Nature (Lond.), 309:626-628) is also amplified in our VCR lines. This DNA segment was used as a probe to screen a cosmid library of VCR genomic DNA, and overlapping clones were retrieved. All of these segments, totaling approximately 45 kilobases (kb), were amplified in VCR cells. Using in situ hybridization, we localized the amplification domain to the long arm of CHO chromosome 1 or Z1. Northern hybridization analysis revealed that a 4.3-kb mRNA was encoded by this amplified DNA domain and was over-produced in the VCR cells. Suggestions for the involvement of these amplified DNA segments in the acquisition of multidrug cross-resistance in animal cells are also presented.

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

scholarly journals Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477 ◽  
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

scholarly journals Cytoplasmic inheritance of oligomycin resistance in Chinese hamster ovary cells.

1980 ◽  
Vol 86 (3) ◽  
pp. 723-729 ◽  
Author(s):  
G A Breen ◽  
I E Scheffler
Keyword(s):  
Chinese Hamster Ovary ◽  
Doubling Time ◽  
Chinese Hamster Ovary Cells ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Cross Resistance ◽  
Mitochondrial Atpase ◽  
Wild Type ◽  
Ovary Cells

Oligomycin-resistant clones were isolated from Chinese hamster ovary cells by treatment of cells with ethidium bromide, followed by mutagenesis with ethylmethane sulfonate and selection in oligomycin. One clone (Olir 8.1) was chosen for further study. Olir 8.1 cells grow with doubling time similar to that of wild-type cells, whether grown in the presence or absence of drug (doubling time of 13-14 h). In plating efficiency experiments, Olir 8.1 cells are approximately 100-fold more resistant to oligomycin than are wild-type cells. There is approximately a 32-fold increase in the resistance to inhibition by oligomycin of the mitochondrial ATPase from Olir 8.1 cells. The electron transport chain is functional in Olir 8.1 cells. Oligomycin resistance is stable in the absence of selective pressure. There is little or no cross-resistance of Olir 8.1 cells to venturicidin and dicyclohexylcarbodiimide, other inhibitors of the mitochondrial ATPase, or to chloramphenicol, an inhibitor of mitochondrial protein synthesis. Oligomycin resistance is dominant in hybrids between Olir 8.1 cells and wild-type cells. Fusions of enucleated Olir 8.1 cells with sensitive cells and characterization of the resulting "cybrid" clones indicates that oligomycin resistance in Olir 8.1 cells is cytoplasmically inherited.

DNA Amplification in Histidinol-Resistant Chinese Hamster Ovary Cells

2020 ◽  
pp. 275-286
Author(s):  
Florence W. L. Tsui ◽  
Louis Siminovitch
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dna Amplification ◽  
Ovary Cells

scholarly journals Correlation of unstable multidrug cross resistance in Chinese hamster ovary cells with a homogeneously staining region on chromosome 1.

1983 ◽  
Vol 3 (9) ◽  
pp. 1634-1647 ◽  
Author(s):  
S H Grund ◽  
S R Patil ◽  
H O Shah ◽  
P G Pauw ◽  
J K Stadler
Keyword(s):  
Cytochalasin B ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Proteins ◽  
Chromosome 1 ◽  
Cross Resistance ◽  
Metabolic Labeling ◽  
Ovary Cells ◽  
Whole Cells ◽  
Multiple Phenotype

An enrichment selection method using repeated pulses of low drug concentration (1 microgram/ml) was used to isolate CHO (AK412) variants that are 20-fold more resistant to cytochalasin D (CD). CD-resistant (CydR) variants possess a unique unstable phenotype, including a longer doubling time in nonselective medium, a higher frequency of multinucleate cells in the population (probably due to a defect in cytokinesis), an altered morphology, and increased resistance or sensitivity to a number of unrelated drugs. In each of two variant lines examined cytologically, this multiple phenotype is associated with a small homogeneously staining region on chromosome 1. The homogeneously staining region is present in the CydR variants, but absent both in the CD-sensitive parent and in a CD-sensitive revertant subpopulation. Studies of CD-displaceable binding of [3H]cytochalasin B show a fourfold reduction in CD binding or uptake when whole cells of the variant line were examined. Lactoperoxidase-catalyzed iodination and metabolic labeling with [H3]fucose of cell surface proteins of the CydR variants showed multiple differences in electrophoretic band migration when compared with parental proteins.

Maytansine-resistant mutants of Chinese hamster ovary cells with an alteration in α-tubulin

1985 ◽  
Vol 63 (6) ◽  
pp. 503-510 ◽  
Author(s):  
Matthew J. Schibler ◽  
Fernando R. Cabral
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Vinca Alkaloid ◽  
Chinese Hamster ◽  
Single Step ◽  
Cross Resistance ◽  
Two Dimensional ◽  
Wild Type ◽  
Electrophoretic Variants ◽  
Ovary Cells

Mutant clones of Chinese hamster ovary cells resistant to killing by the Vinca alkaloid maytansine have been isolated using a single-step procedure. These mutants are threefold more resistant to killing by the drug than the wild-type parent. The majority of the clones (30 of 34) probably contain alterations in membrane permeability based on their cross-resistance to an unrelated drug, puromycin. Two of the four puromycin-sensitive clones were found to contain "extra" spots which migrated close to α-tubulin on two-dimensional gels. The "extra" spots were shown to be electrophoretic variants of α-tubulin with an identical two-dimensional tryptic peptide map to that of the wild-type α-tubulin. The α-tubulin mutants were cross-resistant to other microtubule disrupting drugs such as griseofulvin, vinblastine, and colcemid, but were more sensitive to the microtubule-stabilizing agent taxol than the wild-type parental cells. Mutant – wild-type hybrids were found to be resistant to levels of maytansine intermediate between the lethal doses for mutant and wild-type cells. A possible explanation for the drug resistance of these mutants is discussed.

Chinese hamster ovary cell mutants specifically affected in the phosphorylation of C-purine nucleosides

1985 ◽  
Vol 63 (9) ◽  
pp. 1044-1048 ◽  
Author(s):  
Kamal D. Mehta ◽  
Radhey S. Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Nucleoside Analogs ◽  
Adenosine Kinase ◽  
Ovary Cell ◽  
Cross Resistance ◽  
Ovary Cells ◽  
C And N ◽  
High Degree

In Chinese hamster ovary cells, two different types of mutants resistant to purine nucleoside analogs have been isolated. One type of mutants selected in presence of C-nucleosides formycin A and formycin B (FomR mutants) exhibited a high degree of cross resistance to different C-nucleosides but showed very slight to no cross resistance to various N-nucleosides. In contrast, mutants selected in presence of toyocamycin (Toyr mutants) exhibited a high degree of cross resistance to all C- and N-adenosine analogs. Studies on the cellular uptake and phosphorylation of [3H]adenosine, [3H]tubercidin, and [3H]formycin A show that, unlike the Toyr mutants which show reduced phosphorylation of all three compounds, the FomR mutants show reduced cellular phosphorylation of only [3H]formycin A. It is suggested that the genetic lesion in the FomR mutants affects adenosine kinase in a novel manner that specifically affects the phosphorylation of C-purine nucleosides.

Development of PCL-PEG nanofibrous mats as alternative carriers for recombinant Chinese hamster ovary cells

2012 ◽  
Vol 32 (6-7) ◽  
pp. 453-461
Author(s):  
Mehdi Shakibaie ◽  
Marieh Ghafari Rahbar ◽  
Fatemeh Tabandeh ◽  
Hossein Tavanai ◽  
Alireza Zomorodipour ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chemical Structure ◽  
Protein Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Animal Cells ◽  
Ovary Cells ◽  
Mass Cultivation ◽  
Cell Densities ◽  
Nanofiber Mats

Abstract In this study, six polyblended nanofiber mats, composed of poly ε-caprolactone (PCL)-polyethylene glycol (PEG) mixed at different proportions in an electrospinning solution were fabricated, and then utilized alongside commercial polyester microcarriers for the mass cultivation of recombinant Chinese hamster ovary (rCHO) cells. The SEM micrographs showed that the rCHO cells were attached to the nanofiber mats with significant differences in their affinities, which can be related to the chemical structure and hydrophilicity of the nanofiber mats. The cell densities of the rCHO cells grown on the polyblended PCL-PEG nanofiber mat (80:20)14% were much higher when compared with the commercial polyester microcarrier. Moreover, recombinant protein production increased by approximately 20% when cells were grown on the PCL-PEG nanofiber mat (80:20)14%. The results of this research indicated a potential for the replacement of the commercial polyester microcarrier with the PCL-PEG polyblended nanofiber mats, in order to achieve mass cultivation of recombinant animal cells in industrial bioreactors.

Correlation of unstable multidrug cross resistance in Chinese hamster ovary cells with a homogeneously staining region on chromosome 1

1983 ◽  
Vol 3 (9) ◽  
pp. 1634-1647
Author(s):  
S H Grund ◽  
S R Patil ◽  
H O Shah ◽  
P G Pauw ◽  
J K Stadler
Keyword(s):  
Cytochalasin B ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Proteins ◽  
Chromosome 1 ◽  
Cross Resistance ◽  
Metabolic Labeling ◽  
Ovary Cells ◽  
Whole Cells ◽  
Multiple Phenotype

An enrichment selection method using repeated pulses of low drug concentration (1 microgram/ml) was used to isolate CHO (AK412) variants that are 20-fold more resistant to cytochalasin D (CD). CD-resistant (CydR) variants possess a unique unstable phenotype, including a longer doubling time in nonselective medium, a higher frequency of multinucleate cells in the population (probably due to a defect in cytokinesis), an altered morphology, and increased resistance or sensitivity to a number of unrelated drugs. In each of two variant lines examined cytologically, this multiple phenotype is associated with a small homogeneously staining region on chromosome 1. The homogeneously staining region is present in the CydR variants, but absent both in the CD-sensitive parent and in a CD-sensitive revertant subpopulation. Studies of CD-displaceable binding of [3H]cytochalasin B show a fourfold reduction in CD binding or uptake when whole cells of the variant line were examined. Lactoperoxidase-catalyzed iodination and metabolic labeling with [H3]fucose of cell surface proteins of the CydR variants showed multiple differences in electrophoretic band migration when compared with parental proteins.

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