DRUG RESISTANCE AND MEMBRANE ALTERATION IN MUTANTS OF MAMMALIAN CELLS

1975 ◽  
Vol 17 (4) ◽  
pp. 503-515 ◽  
Author(s):  
Victor Ling
Keyword(s):  
Drug Resistance ◽  
Mammalian Cells ◽  
Molecular Conformation ◽  
Membrane Lipids ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Cross Resistance ◽  
Permeability Barrier ◽  
Colchicine Resistance

Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicates that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.

Cross-resistance and biochemical characteristics of second-step mutants of HeLa cells resistant to cardiac glycosides

1990 ◽  
Vol 68 (5) ◽  
pp. 852-857
Author(s):  
Arvind Chopra ◽  
Anil K. Dudani ◽  
Radhey S. Gupta
Keyword(s):  
Atpase Activity ◽  
Hela Cells ◽  
Cardiac Glycosides ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Cell Extract ◽  
Cross Resistance ◽  
Second Step ◽  
Ovary Cells

From ouabain-resistant (OuaR) mutants of HeLa cells which do not show any cross resistance to the digoxin analog SC4453, stable second-step mutants resistant to either SC4453 or those exhibiting increased resistance to digoxin have been isolated. The mutants obtained exhibited highly specific cross resistance towards different cardiac glycosides (CGs) and, based on their cross-resistance patterns, contained more than one type of genetic lesion. Biochemical studies with these mutants showed that cellular uptake of 86Rb was inhibited by specific CGs to which they showed increased resistance. The mutants showed reduced binding of [3H]ouabain and [3H]digoxin in comparison with the parental OuaR cells and about 50–60% of the Na+,K+-ATPase activity in the mutant cell extract was highly resistant to inhibition by ouabain and digoxin. In contrast to the above changes, these mutants showed no evidence of amplification, enhanced transcription, or gross alterations in the genes for the α or β subunits of Na+,K+-ATPase. These observations indicated that these mutants involved a second-specific alteration in Na+,K+-ATPase. In contrast to these mutants, Chinese hamster ovary cells, which naturally exhibit comparable levels of resistance to CGs, showed no significant binding of either [3H]ouabain or [3H]digoxin and all of their Na+,K+-ATPase activity was resistant to inhibition by CGs.Key words: cardiac glycosides, mammalian cell mutants, mutants resistant to cardiac glycosides, Na+,Ka+-ATPase.

scholarly journals Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

1988 ◽  
Vol 8 (7) ◽  
pp. 2698-2704 ◽  
Author(s):  
I W Caras ◽  
D W Martin
Keyword(s):  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
Mutant Mouse ◽  
Chinese Hamster Ovary Cells ◽  
Spontaneous Mutation ◽  
Single Point ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Single Point Mutation ◽  
Mutator Phenotype

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

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 A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

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.

Further Evidence That the Majority of Primary Nuclear RNA Transcripts in Mammalian Cells Do Not Contribute to mRNA

1982 ◽  
Vol 2 (6) ◽  
pp. 701-707
Author(s):  
M. Salditt-Georgieff ◽  
J. E. Darnell
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cdna Clones ◽  
Ovary Cells ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Polyadenylic Acid ◽  
Nuclear Molecules ◽  
Nuclear Rna

Nuclear RNA from Chinese hamster ovary cells was effectively separated into polyadenylic acid [poly(A)]-containing [poly (A) + ] and non-poly(A)-containing [poly(A) − ] fractions so that ∼90% of the poly(A) was present in the (A) + fraction. Only 25% of the 5′-terminal caps of the large nuclear molecules were present in the (A) + class, but about 70% of the specific mRNA sequences (assayed with cDNA clones) were in the (A) + class. It appears that many long capped heterogeneous nuclear RNA molecules are of a different sequence category from those molecules that are successfully processed into mRNA.

scholarly journals Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

1987 ◽  
Vol 105 (6) ◽  
pp. 2713-2721 ◽  
Author(s):  
D J Yamashiro ◽  
F R Maxfield
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Ph Value ◽  
Ph Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Cell Biol ◽  
Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

scholarly journals Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

2009 ◽  
Vol 186 (4) ◽  
pp. 615-628 ◽  
Author(s):  
Pinkesh Bhagatji ◽  
Rania Leventis ◽  
Jonathan Comeau ◽  
Mohammad Refaei ◽  
John R. Silvius
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Folate Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fundamental Property ◽  
Membrane Lipid ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Lipid Markers

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

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