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.

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.

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.

Cytogenetic Effects Induced by Cytochalasin B in Chinese Hamster Ovary Cells

1995 ◽  
Vol 2 (1) ◽  
pp. 30-35
Author(s):  
M. Gabriele ◽  
C. Cantemir ◽  
F. Majone
Keyword(s):  
Chinese Hamster Ovary ◽  
Cytochalasin B ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Cytogenetic Effects

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 EVIDENCE FOR PLEIOTROPIC CHANGES IN LINES OF CHINESE HAMSTER OVARY CELLS RESISTANT TO CONCANAVALIN A AND PHYTOHEMAGGLUTININ-P

1973 ◽  
Vol 56 (3) ◽  
pp. 666-675 ◽  
Author(s):  
J. A. Wright
Keyword(s):  
Concanavalin A ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Step ◽  
Cross Resistance ◽  
Wild Type ◽  
Con A ◽  
Intact Cells ◽  
Ovary Cells ◽  
Resistant Lines

Lines of Chinese hamster ovary cells resistant to the lectins concanavalin A (Con A) and phytohemagglutinin-P (PHA-P) have been isolated and characterized. Lines were isolated by a stepwise, a single-step, or a cycling single-step procedure, from both mutagen-treated and untreated cultures. The resistant lines showed a higher efficiency of colony formation in the presence of the appropriate lectin than did the wild-type parental line. The cell lines resistant to Con A did not exhibit any detectable cross resistance to PHA-P, nor did the PHA-resistant cells exhibit cross resistance to Con A. The toxicity of Con A from the wild-type and Con A-resistant lines was reduced in the presence of methyl α-D-glucopyranoside; this effect was not seen with the PHA-resistant line. Using 125I-labeled Con A, it was found that Con A was bound preferentially to the surface of intact cells, and that the amount of labeled Con A bound to intact cells was similar for the wild-type and lectin-resistant lines. The Con A-resistant lines were found to be more susceptible to the toxic effects of a number of different compounds, including cyclic AMP and its dibutyryl derivative, sodium butyrate, high concentrations of glucose, phenethyl alcohol, phenol, ouabain, and testosterone. It appears that, in these lines, acquisition of resistance to Con A gave rise to pleiotropic effects which were detected by changes in the sensitivity of the cells to a variety of agents.

Regulation of Na+/Ca2+exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

1998 ◽  
Vol 275 (1) ◽  
pp. C50-C55 ◽  
Author(s):  
Yu Fang ◽  
Madalina Condrescu ◽  
John P. Reeves
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Hill Coefficient ◽  
Acetoxymethyl Ester ◽  
Ovary Cells ◽  
Whole Cells ◽  
Order Of Magnitude ◽  
The Difference ◽  
Exchange Activity

Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+concentration was clamped with gramicidin at ∼20 mM. [Ca2+]iwas varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0–1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+regulation between whole cells and membrane patches is unknown.

scholarly journals Microtubule-nucleating activity of centrosomes in Chinese hamster ovary cells is independent of the centriole cycle but coupled to the mitotic cycle.

1981 ◽  
Vol 91 (3) ◽  
pp. 822-826 ◽  
Author(s):  
R Kuriyama ◽  
G G Borisy
Keyword(s):  
Chinese Hamster Ovary ◽  
Mitotic Cycle ◽  
Cytochalasin B ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Whole Mount ◽  
Microtubule Formation ◽  
Brain Tubulin

The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes. However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity. Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome. Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared. In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle. Nucleating activity did not change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle.

scholarly journals Immunocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane-depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells.

1983 ◽  
Vol 31 (12) ◽  
pp. 1385-1393 ◽  
Author(s):  
B L Armbruster ◽  
H Wunderli ◽  
B M Turner ◽  
I Raska ◽  
E Kellenberger
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Protein A ◽  
Chinese Hamster ◽  
Cytoskeletal Proteins ◽  
Metaphase Chromosomes ◽  
Thin Sections ◽  
Ovary Cells ◽  
Whole Cells ◽  
Immunocytochemical Techniques

Immunocytochemical techniques employing protein A-gold labeling were used to locate actin, tubulin, and histone 2B in thin sections of embedded, isolated membrane-depleted nuclei, metaphase chromosomes, and whole Chinese hamster ovary (CHO) cells. Actin and tubulin were detected in significant amounts in the cytoplasm and the nucleus of whole cells. The isolated membrane-depleted nuclei and metaphase chromosomes showed levels of actin and tubulin labeling either comparable to or sometimes lower than the levels of labeling in whole interphase cells. The results suggest that actin and tubulin are normal components of nuclei throughout the cell cycle. A mouse monoclonal antibody to histone 2B gave relatively weak labeling of nuclei and chromosomes in whole cells but intense labeling of isolated nucleoids and chromosomes. An increased concentration of antibody at the periphery of interphase nucleoids was noted.

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