CHINESE HAMSTER CELL LINES RESISTANT TO THE CYTOTOXIC ACTION OF FLUORIDE

1978 ◽  
Vol 20 (1) ◽  
pp. 71-84 ◽  
Author(s):  
Ralph Mankovitz ◽  
R. Kisilevsky ◽  
Marie Florian
Keyword(s):  
Molecular Basis ◽  
Culture Medium ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Cytotoxic Action ◽  
Wild Type ◽  
Resistant Lines ◽  
Ovary Cell Line ◽  
Resistant Cells

The proliferation and efficiency of colony formation of a Chinese hamster ovary cell line, CHO, was found to be inhibited by concentrations of fluoride ≥ 10−3 M. From mutagenized populations of CHO cells, clones were isolated that were from 1.6 to 13 times more resistant than the wild-type to the cytotoxic action of fluoride. The resistant clones were found to be stable in the absence of selection. The fluoride sensitivity of wild-type and fluoride resistant clones was not altered by changes in the pyruvate concentration in the culture medium, indicating that the cytotoxic effect of fluoride is not due to the action of fluoride on the glycolytic pathway. On the other hand, both the incorporation of 3H-leucine into acid precipitable material and the distribution of polyribosomes were sensitive only to fluoride concentrations that were cytotoxic, suggesting that the molecular basis of fluoride induced cytotoxicity in both wild-type and fluoride resistant cells is the sensitivity of protein synthesis to fluoride. At concentrations of fluoride at which the wild-type cells are inhibited but fluoride resistant cells are not, the intracellular concentration of fluoride in the fluoride resistant cells was found to be 1/5 to 1/10 that of the wild-type, suggesting that fluoride exclusion is the basis for resistance in the resistant lines.

Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line

1990 ◽  
Vol 10 (4) ◽  
pp. 1338-1346
Author(s):  
C Ma ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Multiple Origins ◽  
Chromosomal Walking ◽  
Chinese Hamster Cell Line ◽  
S Period ◽  
Ovary Cell Line

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

Overexpression and amplification of five genes in a multidrug-resistant Chinese hamster ovary cell line

1986 ◽  
Vol 6 (5) ◽  
pp. 1671-1678
Author(s):  
A M Van der Bliek ◽  
T Van der Velde-Koerts ◽  
V Ling ◽  
P Borst
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Multidrug Resistant ◽  
Ovary Cell ◽  
Wide Range ◽  
Gradient Gel Electrophoresis ◽  
Ovary Cell Line ◽  
Resistant Cells

Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.

scholarly journals Overexpression and amplification of five genes in a multidrug-resistant Chinese hamster ovary cell line.

1986 ◽  
Vol 6 (5) ◽  
pp. 1671-1678 ◽  
Author(s):  
A M Van der Bliek ◽  
T Van der Velde-Koerts ◽  
V Ling ◽  
P Borst
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Multidrug Resistant ◽  
Ovary Cell ◽  
Wide Range ◽  
Gradient Gel Electrophoresis ◽  
Ovary Cell Line ◽  
Resistant Cells

Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells

1983 ◽  
Vol 3 (2) ◽  
pp. 198-202
Author(s):  
K Nielsen-Smith ◽  
S McGill ◽  
M Frahm ◽  
D J Roufa
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Molecular Product ◽  
Translation Apparatus ◽  
Hybrid Clones

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

scholarly journals Multiple origins of replication in the dihydrofolate reductase amplicons of a methotrexate-resistant chinese hamster cell line.

1990 ◽  
Vol 10 (4) ◽  
pp. 1338-1346 ◽  
Author(s):  
C Ma ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Multiple Origins ◽  
Chromosomal Walking ◽  
Chinese Hamster Cell Line ◽  
S Period ◽  
Ovary Cell Line

We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.

Relief of temperature sensitivity in a concanavalin A resistant Chinese hamster ovary cell line auxotrophic for cholesterol

1987 ◽  
Vol 65 (7) ◽  
pp. 635-641
Author(s):  
Robert A. R. Hurta ◽  
David N. Burton
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Temperature Sensitivity ◽  
Concanavalin A ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cell ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ovary Cell Line

The concanavalin A resistant, glycosylation-deficient, Chinese hamster ovary cell variant CR-7 is auxotrophic for cholesterol owing to an inability to adequately convert lanosterol to cholesterol. It is also temperature sensitive for growth, being unable to proliferate at 39 °C. Temperature sensitivity was relieved by addition of mevalonolactone, dolichol, or dolichyl-P to the growth medium, provided that cholesterol was also present in amounts sufficient to overcome cholesterol auxotrophy at 34 °C. Other metabolites of mevalonolactone (squalene, ubiquinone, lanosterol, and isopentenyladenine) were inactive in this regard. Measurement of dolichol levels in CR-7 and wild-type cells at 34 °C and after exposure to 39 °C showed that dolichol increased at 39 °C to an approximately equal extent in both cell types. Dolichol, dolichyl-P, ubiquinone, and isopentenyladenine had no effect on the sensitivity of either wild-type or CR-7 cells to the cytotoxic effects of concanavalin A. Mevalonolactone or lanosterol markedly increased the resistance of CR-7 to the lectin, but had no effect on wild-type cells. This raises the possibility that the presence of unusually large amounts of lanosterol, coupled with low amounts of cholesterol, in the membranes of CR-7 may be related to its concanavalin A resistance and other characteristic phenotypic abnormalities.

scholarly journals Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

1983 ◽  
Vol 3 (2) ◽  
pp. 198-202 ◽  
Author(s):  
K Nielsen-Smith ◽  
S McGill ◽  
M Frahm ◽  
D J Roufa
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Molecular Product ◽  
Translation Apparatus ◽  
Hybrid Clones

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

scholarly journals Complementation of a methotrexate uptake defect in Chinese hamster ovary cells by DNA-mediated gene transfer.

1989 ◽  
Vol 9 (4) ◽  
pp. 1754-1758 ◽  
Author(s):  
T M Underhill ◽  
W F Flintoff
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Ovary Cells ◽  
Ovary Cell Line ◽  
Human Specific

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from either wild-type Chinese hamster ovary or human G2 cells. Primary and secondary transfectants regained the ability to take up methotrexate in a manner similar to that of wild-type cells, and in the case of those transfected with human DNA, to contain human-specific DNA sequences. The complementation by DNA-mediated gene transfer of this methotrexate-resistant phenotype provides a basis for the cloning of a gene involved in methotrexate uptake.

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