scholarly journals Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines.

1981 ◽  
Vol 1 (12) ◽  
pp. 1084-1093 ◽  
Author(s):  
R J Kaufman ◽  
P C Brown ◽  
R T Schimke
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Stable State ◽  
Unequal Distribution ◽  
Continuous Growth ◽  
Daughter Cells ◽  
Reductase Gene ◽  
Double Minute ◽  
Multiple Copies ◽  
Double Minute Chromosomes

We studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. We also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.

Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines

1981 ◽  
Vol 1 (12) ◽  
pp. 1084-1093
Author(s):  
R J Kaufman ◽  
P C Brown ◽  
R T Schimke
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Stable State ◽  
Unequal Distribution ◽  
Continuous Growth ◽  
Daughter Cells ◽  
Reductase Gene ◽  
Double Minute ◽  
Multiple Copies ◽  
Double Minute Chromosomes

We studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. We also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.

Mathematical models of gene amplification with applications to cellular drug resistance and tumorigenicity.

Genetics ◽  
1990 ◽  
Vol 125 (3) ◽  
pp. 633-644
Author(s):  
M Kimmel ◽  
D E Axelrod
Keyword(s):  
Mathematical Models ◽  
Cell Lines ◽  
Gene Amplification ◽  
Dihydrofolate Reductase ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Published Data ◽  
Reductase Gene ◽  
Double Minute ◽  
Double Minute Chromosomes

Abstract An increased number of copies of specific genes may offer an advantage to cells when they grow in restrictive conditions such as in the presence of toxic drugs, or in a tumor. Three mathematical models of gene amplification and deamplification are proposed to describe the kinetics of unstable phenotypes of cells with amplified genes. The models differ in details but all assume probabilistic mechanisms of increase and decrease in gene copy number per cell (gene amplification/deamplification). Analysis of the models indicates that a stable distribution of numbers of copies of genes per cell, observed experimentally, exists only if the probability of deamplification exceeds the probability of amplification. The models are fitted to published data on the loss of methotrexate resistance in cultured cell lines, due to the loss of amplified dihydrofolate reductase gene. For two mouse cell lines unstably resistant to methotrexate the probabilities of amplification and deamplification of the dihydrofolate reductase gene on double minute chromosomes are estimated to be approximately 2% and 10%, respectively. These probabilities are much higher than widely presumed. The models explain the gradual disappearance of the resistant phenotype when selective pressure is withdrawn, by postulating that the rate of deamplification exceeds the rate of amplification. Thus it is not necessary to invoke a growth advantage of nonresistant cells which has been the standard explanation. For another analogous process, the loss of double minute chromosomes containing the myc oncogene from SEWA tumor cells, the growth advantage model does seem to be superior to the amplification and deamplification model. In a more theoretical section of the paper, it is demonstrated that gene amplification/deamplification can result in reduction to homozygosity, such as is observed in some tumors. Other applications are discussed.

scholarly journals Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

1981 ◽  
Vol 1 (12) ◽  
pp. 1077-1083 ◽  
Author(s):  
P C Brown ◽  
S M Beverley ◽  
R T Schimke
Keyword(s):  
Dihydrofolate Reductase ◽  
Deoxyribonucleic Acid ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Gene Sequences ◽  
Resistant Mouse ◽  
Methotrexate Resistance ◽  
Double Minute ◽  
Relationship Of ◽  
Double Minute Chromosomes

Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.

scholarly journals Increased UV resistance of a xeroderma pigmentosum revertant cell line is correlated with selective repair of the transcribed strand of an expressed gene.

1993 ◽  
Vol 13 (2) ◽  
pp. 970-976 ◽  
Author(s):  
L Lommel ◽  
P C Hanawalt
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Xeroderma Pigmentosum ◽  
Uv Resistance ◽  
Cyclobutane Pyrimidine Dimers ◽  
Pyrimidine Dimers ◽  
Reductase Gene ◽  
Group A ◽  
A Cell ◽  
Xeroderma Pigmentosum Group A

A UV-resistant revertant (XP129) of a xeroderma pigmentosum group A cell line has been reported to be totally deficient in repair of cyclobutane pyrimidine dimers (CPDs) but proficient in repair of 6-4 photoproducts. This finding has been interpreted to mean that CPDs play no role in cell killing by UV. We have analyzed the fine structure of repair of CPDs in the dihydrofolate reductase gene in the revertant. In this essential, active gene, we observe that repair of the transcribed strand is as efficient as that in normal, repair-proficient human cells, but repair of the nontranscribed strand is not. Within 4 h after UV at 7.5 J/m2, over 50% of the CPDs were removed, and by 8 h, 80% of the CPDs were removed. In contrast, there was essentially no removal from the nontranscribed strand even by 24 h. Our results demonstrate that overall repair measurements can be misleading, and they support the hypothesis that removal of CPDs from the transcribed strands of expressed genes is essential for UV resistance.

Amplification of a cloned Chinese hamster dihydrofolate reductase gene after transfer into a dihydrofolate reductase-deficient cell line

1983 ◽  
Vol 3 (7) ◽  
pp. 1274-1282
Author(s):  
J D Milbrandt ◽  
J C Azizkhan ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Genomic Dna ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Ovary Cell ◽  
Dhfr Gene ◽  
Reductase Gene ◽  
Ovary Cell Line

We have transformed a dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary cell line to the DHFR+ phenotype with a recombinant cosmid (cH1) containing a functional Chinese hamster DHFR gene (J.D. Milbrandt et al., Mol. Cell. Biol. 3:1266-1273, 1983). After exposure of cells to successive increases in methotrexate, we have isolated a resistant cell line (JSH-1) that grows in 1 microM methotrexate. We show here that JSH-1 contains 300 to 500 copies of the integrated cosmid and that these copies are located predominantly at one position on a chromosome identified as Z5a. Hybridization analysis of restriction digests of genomic DNA indicates that the cosmid has been integrated intact into the genome and that upon amplification, the original cosmid/genomic junction fragments are also amplified in JSH-1. Furthermore, the pattern of amplified bands observed in ethidium bromide-stained gels indicates that the unit amplified sequence (amplicon) may be as large as 120 to 135 kilobases and therefore includes considerable amounts of flanking DNA in addition to the 45 kilobases of integrated cosmid. We also show that the protein overproduced by the amplified cosmid in JSH-1 comigrates with the 21,000-dalton polypeptide characteristic of the methotrexate-resistant cell line (CHOC 400) from which cH1 was cloned. However, the DHFR mRNA species overproduced in JSH-1 appear to be larger than those detected in CHOC 400, indicating that not all of the normal transcription and processing signals are preserved in the integrated recombinant cosmid.

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