scholarly journals Isolation of Chinese hamster ovary cell mutants requiring the continuous presence of taxol for cell division.

1983 ◽  
Vol 97 (1) ◽  
pp. 22-29 ◽  
Author(s):  
F R Cabral
Keyword(s):  
Cell Division ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Normal Growth ◽  
Time Lapse ◽  
Single Step ◽  
Ovary Cell ◽  
M Phase ◽  
Continuous Presence

Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two-dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.

Chinese hamster ovary cell mutants defective in the internalization of ricin

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

Revertants of a Chinese Hamster Ovary Cell Mutant with an Altered β-Tubulin: Evidence that the Altered Tubulin Confers Both Colcemid Resistance and Temperature Sensitivity on the Cell

1982 ◽  
Vol 2 (6) ◽  
pp. 720-729
Author(s):  
Fernando Cabral ◽  
Irene Abraham ◽  
Michael M. Gottesman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Temperature Sensitivity ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Β Tubulin

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell 20 :29-36, 1980). This mutant carries an altered β-tubulin but still grows normally at 37°C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3°C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered β-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant β-tubulin polypeptide. This second alteration appears to make the mutant β-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in β-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered β-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells

1983 ◽  
Vol 3 (3) ◽  
pp. 391-398
Author(s):  
I L Andrulis ◽  
C Duff ◽  
S Evans-Blackler ◽  
R Worton ◽  
L Siminovitch
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Asparagine Synthetase ◽  
Chromosome 1 ◽  
Ovary Cell ◽  
Synthetase Activity

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

scholarly journals Isolation and Characterization of a Mutant Chinese Hamster Ovary Cell Line That Is Resistant to Chlamydia trachomatis Infection at a Novel Step in the Attachment Process

2001 ◽  
Vol 69 (9) ◽  
pp. 5899-5904 ◽  
Author(s):  
Reynaldo A. Carabeo ◽  
Ted Hackstadt
Keyword(s):  
Chlamydia Trachomatis ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chemical Mutagenesis ◽  
Chlamydia Trachomatis Infection ◽  
Isolation And Characterization ◽  
Ovary Cell Line

ABSTRACT Host factors involved in Chlamydia trachomatispathogenesis were investigated by random chemical mutagenesis of Chinese hamster ovary (CHO-K1) cells followed by selection for clones resistant to chlamydial infection. A clonal mutant cell line, D4.1–3, refractory to infection by the C. trachomatis L2 serovar was isolated. The D4.1–3 cell line appears to be lacking in a previously undescribed temperature-dependent and heparin-resistant binding step that occurs subsequent to engagement of cell surface heparan sulfate by L2 elementary bodies. This novel binding step differentiates the lymphogranuloma venereum (LGV) serovar from other serovars and may contribute the different pathologies associated with LGV and non-LGV strains.

scholarly journals Chinese hamster ovary cell mutants defective in the internalization of ricin.

1982 ◽  
Vol 2 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Electron Microscopic ◽  
Ovary Cells ◽  
Internalization Process ◽  
In The Wild ◽  
Mutant Cells

Chinese hamster ovary mutants simultaneously resistant to ricin and Pseudomonas toxin have been isolated. Two mutant cell lines (4-10 and 11-2) were found to retain normal levels of binding of both ricin and Pseudomonas toxin. They were defective in the internalization of [125I]ricin into the mutant cells, as measured by both a biochemical assay for ricin internalization and electron microscopic autoradiographic studies. Although pretreatment of Chinese hamster ovary cells with a Na+/K+ ionophore, nigericin, resulted in an enhancement of the cytotoxicities of ricin and Pseudomonas toxin in the wild-type Chinese hamster ovary cells, preculture of the mutant cells did not alter the susceptibility of the mutant cells to either toxin. These results provide further evidence that there is a common step in the internalization process for ricin and Pseudomonas toxin.

scholarly journals Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

1983 ◽  
Vol 3 (3) ◽  
pp. 391-398 ◽  
Author(s):  
I L Andrulis ◽  
C Duff ◽  
S Evans-Blackler ◽  
R Worton ◽  
L Siminovitch
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Single Step ◽  
Asparagine Synthetase ◽  
Chromosome 1 ◽  
Ovary Cell ◽  
Synthetase Activity

The amino acid analog albizziin was used to isolate Chinese hamster ovary cell lines which overproduce asparagine synthetase. Mutants selected in a single step after ethyl methane sulfonate mutagenesis were approximately 10-fold more resistant to the drug than the parental lines and expressed 8- to 17-fold elevations in enzyme activity. The karyotypes of these lines show alterations such as breaks and translocations affecting the long arm of chromosome 1. Cell lines isolated in several steps by growth in progressively increasing concentrations of albizziin were more resistant to the drug and exhibited up to 300-fold enhancement of asparagine synthetase activity. The multistep albizziin-resistant cell lines usually had expanded chromosomal regions which stained somewhat homogeneously, often on the long arm of chromosome 1. These results suggest that resistance to albizziin in the multistep lines may be due to gene amplification.

scholarly journals Synthesis and biochemical properties of a new photoactivatable cholesterol analog 7,7-azocholestanol and its linoleate ester in Chinese hamster ovary cell lines

2002 ◽  
Vol 43 (8) ◽  
pp. 1341-1347 ◽  
Author(s):  
Jonathan C. Cruz ◽  
Matthew Thomas ◽  
Edmund Wong ◽  
Nobutaka Ohgami ◽  
Shigeki Sugii ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cholesterol Metabolism ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Biochemical Properties ◽  
Ovary Cell ◽  
Cyclodextrin Complex ◽  
Mutant Cell Line

We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1.These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.

scholarly journals Revertants of a Chinese Hamster Ovary Cell Mutant with an Altered β-Tubulin: Evidence that the Altered Tubulin Confers Both Colcemid Resistance and Temperature Sensitivity on the Cell

1982 ◽  
Vol 2 (6) ◽  
pp. 720-729 ◽  
Author(s):  
Fernando Cabral ◽  
Irene Abraham ◽  
Michael M. Gottesman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Temperature Sensitivity ◽  
Chinese Hamster Ovary ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Β Tubulin

We recently described the isolation of a mutant Chinese hamster ovary cell (Cmd 4) resistant to the cytotoxic effects of colcemid (Cabral et al., Cell20:29-36, 1980). This mutant carries an altered β-tubulin but still grows normally at 37°C. In the present study we found that Cmd 4 is temperature sensitive for growth at 40.3°C. A class of revertants selected for temperature resistance had simultaneously lost colcemid resistance and the altered β-tubulin. In addition, we isolated a temperature-resistant revertant which carries a further alteration in the mutant β-tubulin polypeptide. This second alteration appears to make the mutant β-tubulin incompetent to assemble into microtubules, resulting in a strain which is again colcemid sensitive. These revertant cell lines provide strong evidence that a mutation in β-tubulin can confer both colcemid resistance and temperature sensitivity on a mammalian cell line. Cellular microtubules studied by indirect immunofluorescence in both mutant and revertant cell lines had an apparently normal distribution at permissive and nonpermissive temperatures, yet mitosis appears to be abnormal in the mutant cell line. We conclude from these studies that incorporation of the altered β-tubulin into microtubules does not affect their distribution but may affect their function during mitosis.

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