Amplified DHFR Gene Research Articles

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells.

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

DNA Repair of Benzo[A]Pyrene Diol Epoxide-DNA Adducts in the DHFR Gene of a Human Embryonic Kidney Cell Line

The repair of polycyclic aromatic hydrocarbon (PAH)-DNA adducts by nucleotide excision repair is a critical factor in the human response to exposure to these environmental carcinogens. In this study, we utilized a laser cleavage technique and Southern Blotting to measure the repair of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts in the transcribed and nontranscribed strands of a human cell line containing an amplified DHFR gene. Treatment of the cultures with 1.5 μM BPDE resulted in little adduct removal for 24 hours, whereas 1 μM BPDE-treated cells removed most of the adducts over a 24 hour period. Southern analyses of repair in individual strands of the human cell line revealed that adducts on the transcribed strand are repaired more rapidly than those on the non-transcribed strand during the first 8 hours after 1 μM BPDE treatment. These results suggest that human cells can repair BPDE-DNA adducts through a transcription-coupled repair process.

Unbalanced growth in mouse cells with amplified dhfr genes

When grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase (dhfr) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G1/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G1/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub-cytotoxic concentrations, also inhibits the cell cycle at the G1/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.

Unbalanced growth in mouse cells with amplified dhfr genes

AbstractWhen grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase (dhfr) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G1/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G1/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub‐cytotoxic concentrations, also inhibits the cell cycle at the G1/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.

A new myeloblastic leukemia cell line with double minute chromosomes. Induction of methotrexate resistance and dihydrofolate reductase gene amplification

To test the relationship between DMs and drug resistance in newly established AML cell lines, KY821, and its clone KY821A3, the latter had lost DMs during cloning, were cultured in increasing concentrations of MTX, KY821 became resistant against 2 × 10 −4 M MTX, whereas KY821A3 did against 2 × 10 −5 M MTX in a same period. Enhanced enzyme activities of DHFR were correspondent to the increased DMs numbers and DHFR gene amplification in both resistant clones. The amplified DHFR gene was located on DMs by in situ hybridization. These data indicated that the presence of DMs in KY821 would facilitate the acquisition of drug resistance.

Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells.

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which codes for beta-galactosidase (beta-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10(-7), and 10(-6) M methotrexate (MTX), and the beta-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplified dhfr gene content and foreign beta-gal gene expression in the cell populations. A biochemical assay for beta-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10(-7) M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10(-6) M MTX was 20% lower than that of recombinant CHO cells at 10(-7) M MTX. There was no effect of 10(-5) M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect of dhfr and foreign gene amplification and increased beta-galactosidase expression.

An amplified insect dihydrofolate reductase gene contains a single intron

We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.

Novel submicroscopic extrachromosomal elements containing amplified genes in human cells

In previous work, several methotrexate (MTX)-resistant variants were isolated frm the human cell line HeLa BU25, which exhibited a high degree of dihydrofolate (DHFR) gene amplification (estimated to be 250- to 300-fold). These variants did not contain any chromosome with a homogeneously staining region (HSR) and exhibited only a small average number of minute chromosomes per cell: these two types of karyotypic abnormalities generally accompany selective gene amplification. We now report that structures containing amplified DHFR genes in one of these variants (HeLa BU25-10B3) can be isolated by pulsed-field gradient or field-inversion gel electrophoresis as homogeneous DNA molecules of approximately 650 kilobases (kb). Electron microscopy of metaphase spreads from these cells reveals chromatin fibres with a similar DNA content, which are probably related to the above elements. These represent a novel type of extrachromosomal structures in mammalian cells.

Gene amplification: an example of accelerated evolution in tumorigenic cells.

During selection for methotrexate resistance, tumorigenic CHEF/16 cells and derivatives from CHEF/16 tumors underwent amplification of the dihydrofolate reductase gene (DHFR) at accelerated rates compared with closely related nontumorigenic CHEF/18 cells. "Dot blot" analysis showed that the CHEF/16 cells contained many more copies of the DHFR gene than did the CHEF/18 cells, when assayed at similar elevated levels of methotrexate resistance. Chromosome analysis of cell samples taken at several time points during amplification revealed large differences between the nontumorigenic CHEF/18 cells and the two tumorigenic cell lines. The tumorigenic cells developed few chromosome rearrangements over a 4-log increase in methotrexate resistance, other than increased length of a single chromosome, which was shown by in situ hybridization to contain most or all of the amplified DHFR gene copies. In contrast, the CHEF/18 cells underwent complex, progressive changes in almost every chromosome, and in situ hybridization suggested a dispersed pattern of gene amplification. The data support the hypothesis that unregulated amplification is a pathological process, occurring readily in neoplastic but rarely in normal cells, that together with other chromosomal disturbances contributes to the rapid evolution and progression of cancer.

Genetic demonstration of mitotic recombination in cultured chinese hamster cell hybrids

Chinese hamster ovary cell hybrids were constructed that are heterozygous for two markers, leuS and emtB, linked to the long arm of chromosome 2. In addition, the chromosome 2 carrying the wild-type leuS and emtB alleles contains, on its short arm, a homogeneously staining region (hsr) in which the gene encoding dihydrofolate reductase (dhfr) is amplified ∼50-fold. This provides a convenient cytogenetic and biochemical means to distinguish the chromosome 2s from the different parents. Analysis of emetine-resistant segregants isolated from such hybrids identified three distinct classes of segregants. One rare class of segregants loses the wild-type leuS and emtB gene functions on the long arm of the hsr chromosome 2 (H-2) but retains the amplified dhfr genes on the opposite arm. Detailed genetic analysis of two such segregants that did not arise by chromosome loss or deletion revealed that new gene linkage relationships had been established on the H-2 chromosome in each, demonstrating that the segregation events in these cell lines involved mitotic recombination.

Moderate-Level Gene Amplification in Methotrexate-Resistant Chinese Hamster Ovary Cells is Accompanied by Chromosomal Translocations At or Near the Site of the Amplified DHFR Gene

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Moderate-level gene amplification in methotrexate-resistant Chinese hamster ovary cells is accompanied by chromosomal translocations at or near the site of the amplified DHFR gene.

In previous studies, we have described several classes of methotrexate-resistant Chinese hamster ovary cell lines. Although the RI class is resistant because of an altered target enzyme, dihydrofolate reductase, the RIII class derived from RI cells is somewhat more resistant because of a moderate amplification of the altered dhfr structural gene (Flintoff et al., Mol. Cell. Biol. 2:275-285, 1982). In one RIII line, a translocation between the short arm (p) of chromosome 2 and the long arm (q) of chromosome 5 was observed, and the amplified RIII gene complex was mapped to the p arm of the 2p-marker chromosome derived from the translocation (Worton et al., Mol. Cell. Biol. 1:330-335, 1981). We tested the hypothesis that chromosomal translocation is a general feature of RIII cells and that such translocation involves a site at or near the dhfr structural gene. Thus, we examined four independently derived RIII-type mutants and found that each had a moderate amplification of the dhfr gene sequences, and karyotype analysis revealed that each carried a translocation involving the 2p arm at or near band 2p25. That this chromosomal rearrangement involves a site near the dhfr locus was demonstrated by mapping the altered but unamplified structural gene coding for the RI phenotype to the short arm of an unaltered chromosome 2. This suggests that a highly specific rearrangement involving an exchange at or near the site of the unamplified gene is a necessary prerequisite for the amplification process. A model for gene amplification involving chromosomal rearrangements and sister chromatid exchange is described.

Selective amplification of polymorphic dihydrofolate reductase gene loci in Chinese hamster lung cells.

A series of antifolate-resistant Chinese hamster lung sublines that overproduce either a Mr 20,000 or a Mr 21,000 class of dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3), known to contain amplified DHFR genes, has been analyzed by DNA and RNA transfer techniques. The results suggest that the Mr 20,000 and Mr 21,000 DHFRs are encoded by at least two polymorphic DHFR genes, both of which are expressed in drug-sensitive parental cells. In drug-resistant sublines only one of the two DHFR gene types is amplified, thus accounting for the overproduction of one or the other molecular weight class of DHFR. In addition to the known differences between the DHFRs whose overproduction they direct, these allelic genes differ in restriction endonuclease profiles and in the relative abundances of their multiple mRNA transcripts.