Selection of aphidicolin-resistant CHO cells with altered levels of ribonucleotide reductase

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10-6. Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cellfree extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wildtype cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

Section: Selection Of Hydroxyurea-resistant Cell Linesupporting

confidence: 63%

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

Lewis¹,

Srinivasan²

1983

“…The initial pools of transfectants were then reselected for resistance to aphidicolin to isolate colonies overexpressing protein Ml. (Resistance to aphidicolin, an inhibitor of DNA synthesis, can be conferred by elevated levels of deoxynucleoside triphosphates resulting either from overproduction of ribonucleotide reductase or from desensitization of the enzyme to feedback regulation by dATP [1,25] recombinant protein Ml and to characterize the properties of their ribonucleotide reductase, we analyzed the following colonies further: WT-1, selected from the pER-WT population; dA-3 and dA-6, selected from the pER-dATPT population; and D-1, a control line containing only the DHFR gene.…”

Section: Resultsmentioning

confidence: 99%

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

Caras¹,

Martin²

1988

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit Ml. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit Ml. 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 Ml cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant Ml 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 Ml thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.

“…Overproduction of these "target" enzymes is often observed, which suggests that this may be a common mechanism of resistance (2,6,12,16,20,21,32,48,57). In the best-characterized systems, gene amplification has been demonstrated as the mechanism of overproduction (1,8,60) either in the form of DMs for unstable resistance or in the form of an expanded region, termed a homogeneously staining region (51), for stable resistance within (usually) one of the chromosomes.…”

Section: Discussionmentioning

confidence: 99%

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells.

Robertson¹,

Ling²,

Stanners³

1984

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of Pglycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.A genetic system of clinical relevance involving mammalian cell mutants resistant to a wide variety of cytocidal drugs has recently emerged (36). This system began with the isolation of a number of CHO cell mutants which were resistant to colchicine (38). These mutants were shown to be resistant to the drug as a result of a membrane permeability barrier, which reduced the cellular uptake of colchicine (38). They were also found to be cross-resistant to a number of structurally diverse and functionally unrelated drugs, some of which are used in chemotherapy of cancer, again, by virtue of reduced uptake of these compounds (10). Further analysis of the membranes of these cells, in conjunction with hybrid cell studies (37) and selection of revertants (35), clearly demonstrated the association of the multiple drug resistance phenotype and the reduced membrane permeability with the presence in the cell membrane of a glycoprotein of 160,000 to 180,000 daltons termed P-glycoprotein (27, 28). P-glycoprotein was undetectable in the membrane of the drug-sensitive parental CHO cells but present in drugresistant mutants in amounts directly correlated with the level of resistance. The overexpression of a 160,000-to 180,000-dalton protein has also been observed in several drug resistance systems where resistance has been selected for drugs other than colchicine (13,22,45), including a human leukemic cell line selected for vinblastine resistance (11). As it appears likely that the development of resistance to chemotherapy of human tumors after repeated treatment with these drugs involves, at least in part, the emergence of such mutants (10, 36, 52), it seems important to carry their genetic analysis further. As a first step in this direction, we * Correspondin...