Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.

Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3H]deoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heat-labile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene.     

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.     

An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments.

We have developed a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) containing a 500-fold amplification of a 135-kilobase chromosomal DNA sequence. This sequence includes the gene for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3). The high copy number of the amplified sequence permits it to be visualized as a distinct series of restriction fragments in genomic digests separated on ethidium bromide-stained agarose gels. Initiation of DNA replication in the amplified sequence was studied by radiolabeling DNA synthesized during the onset of S phase in synchronized CHOC 400 cells. Autoradiography of Southern blots of labeled genomic digests shows that DNA synthesis initiates in a small subset of the EcoRI fragments derived from the amplified units. These early labeled fragments are not synthesized at later times during S phase, when different subsets of fragments are synthesized. Regardless of the drug used to collect cells at the beginning of S phase, the replication pattern observed remains the same. These data suggest that replication of the amplified sequence initiates at specific sites within each repeated unit and proceeds in nonrandom order throughout the remainder of the sequence--i.e., that initiation of DNA synthesis in the chromosomes of mammalian cells is sequence specific.     

S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells

We investigated the cell cycle modulation of dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase, 7,8-dihydroxyfolate:NADP+ oxidoreductase, EC 1.5.1.3) levels in methotrexate-resistant Chinese hamster ovary cells synchronized by mitotic selection. DNA content and DHFR concentration were analyzed throughout the cell cycle by standard biochemical techniques and by double fluorescence staining utilizing the fluorescence-activated cell sorter. We found an S phase-specific period of DHFR biosynthetic activity. Commencing within hour 2 of S phase and continuing throughout the duration of S phase, there is a 90% increase in DHFR specific activity. This results from an approximately 2.5-fold increase in the level of DHFR, while total soluble protein increases 50% during the same period. This increase is the result of new synthesis of DHFR molecules initiated after the cell is physiologically committed to DNA replication. This increase in DHFR activity through S phage parallels the increasing rate of [3H]thymidine incorporation during the same interval. The maximum peak of DHFR activity is coincident with the maximum rate of DNA synthesis, both activities occurring during the bulk of DNA replication within the last stages of the 6.5-hr S phase.     

Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells.

Exposure of Chinese hamster cells to reduced oxygen partial pressure results in a marked enhancement in the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. The frequency of enhanced resistance is a function of the length of exposure to hypoxic conditions and the time after recovery from hypoxia when cells are plated into methotrexate-containing medium. Hypoxia results in an inhibition of DNA synthesis; upon return to normal oxygen atmosphere, greater than 60% of cells in S phase at the time hypoxia was started subsequently undergo overreplication of DNA within a single cell cycle. The cells with the increased frequency of gene amplification are derived from this subset of overreplicated cells. These results are discussed within the context of the hypoxic state of many solid tumors and the high frequency of aneuploidy, chromosomal aberrations, and spontaneously occurring resistances to a number of cancer chemotherapeutic agents.     

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinii DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinii DHFR and, in fact, is a more potent inhibitor of human DHFR.     

Expression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells.

We have constructed a number of abbreviated dihydrofolate reductase (DHFR) genes by using cloned mouse genomic and cDNA sequences. These genes contain 1.0 kilobase of 5' flanking genomic sequence and varying portions of the 3' non-coding region. Two of the genes contain the first two introns of the DHFR gene; the other three lack introns. Transfection of DHFR-deficient Chinese hamster ovary cells with any of these constructed genes results in cells with the DHFR+ phenotype. Treatment of the transfectants with methotrexate, a folate antagonist, leads to the emergence of methotrexate-resistant colonies which have amplified the transfected genes.     

Isolation and expression of an altered mouse dihydrofolate reductase cDNA.

We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate. From this library we have isolated a cDNA clone similar to, but distinguishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding. To confirm that this substitution was responsible for the altered properties of the enzyme, we genetically localized the region of the cDNA that specified resistance to methotrexate by in vitro recombination. These results reveal that a single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.     

Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea.

Previous work has shown that mammalian cells that carry unstably amplified genes for dihydrofolate reductase (DHFR) gradually lose the amplified DHFR genes when grown in the absence of the DHFR inhibitor methotrexate (MTX). Unstably amplified genes occur on small acentric chromosomes called double minutes (DMs) or even smaller chromatin fragments, in contrast to stably amplified genes, which reside in centromere-containing chromosomes. We have found that the rate of loss of the unstably amplified DHFR genes can be greatly oncreased by growing the cells in the presence of a nonlethal concentration of hydroxyurea. For example, in one MTX-resistant subline studied, approximately equal to 90% of the original DHFR gene dosage is lost in 25-30 cell doublings in the absence of MTX. The same degree of loss is achieved, however, in less than 4 doublings if cells are grown in the presence of 50 microM hydroxyurea. This new effect of hydroxyurea does not appear to be due to changes in plating efficiency or selective cytotoxicity. In particular, no increase in cell death occurs at 50 microM hydroxyurea, and cells continue to multiply, albeit 1/2 to 2/3 as fast as in the absence of hydroxyurea. The ability to selectively accelerate the loss of amplified genes from mammalian cells as shown in the present work may have important implications both for the problem of drug resistance in cancer chemotherapy and for curing mammalian cells of extrachromosomally maintained DNA genomes of pathogenic viruses.     

Isolation and characterization of DNA sequences amplified in multidrug-resistant hamster cells.

The mechanism by which mammalian cells acquire resistance to chemotherapeutic agents has been investigated by using molecular genetic techniques. LZ and C5, two independently derived multidrug-resistant Chinese hamster cell lines, share specific amplified DNA sequences. We demonstrate that commonly amplified DNA sequences reside in a contiguous domain of approximately equal to 120 kilobases (kb). We report the isolation of this DNA domain in cosmid clones and show that the level of amplification of the domain is correlated with the level of resistance in multidrug-resistant cell lines. The organization of the amplified domain was deduced by a unique approach utilizing in-gel hybridization of cloned DNA with amplified genomic DNA. We show that the entire cloned region is amplified in adriamycin-resistant LZ cells and independently derived, colchicine-resistant C5 cells. A mRNA species of approximately equal to 5 kb is encoded by a gene located within the boundaries of this region. Genomic sequences homologous to the 5-kb mRNA span over 75 kb of the amplified DNA segment. The level of expression of this mRNA in multidrug-resistant cells is correlated with the degree of gene amplification and the degree of drug resistance. Our results strongly suggest that the 5-kb mRNA species plays a role in the mechanism of multidrug resistance common to the LZ and C5 cell lines.     

Mitogenic hormones and tumor promoters greatly increase the incidence of colony-forming cells bearing amplified dihydrofolate reductase genes.

Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12,13-butyrate, at their optimal concentrations (approximately equal to 0.1 microM) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbol 12,13,20-triacetate, have no effect on the incidence of MTX-resistant 3T6 colonies. Anthralin, a nonphorbol tumor promoter, is approximately equal to 40% as active as PMA in the MTX selection assay. Remarkably, the hormones insulin, arginine vasopressin, and epidermal growth factor, all of which are mitogenic for 3T6 cells, also exert a strong PMA-like effect on the incidence of MTX-resistant 3T6 colonies under conditions of MTX selection. The effect of insulin at its optimal concentration (approximately equal to 1 microgram/ml) is approximately equal to 70% that of PMA. Although the effect of PMA on the incidence of MTX-resistant 3T6 colonies does not significantly depend on the initial density of seeded cells or volume of the medium added, the analogous effect of insulin is strongly influenced by these parameters. Mevalonic acid, arachidonic acid, thymidine, caffeine, and nicotine, all of which are known to influence patterns of DNA synthesis in mammalian cells, were tested at their highest noncytotoxic concentrations and failed to produce any significant effect on the incidence of MTX-resistant 3T6 colonies. We discuss possible mechanisms of hormone- and tumor promoter-facilitated gene amplification in mammalian cells, relationship of mitogenic hormones to tumor promoters, and also implications of our findings for the problem of drug resistance in cancer chemotherapy.     

Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes.

Selection of mammalian cells in progressively increasing concentrations of methotrexate results in selective amplification of DNA sequences coding for dihydrofolate reductase (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). In some cell variants the amplified genes are stable with growth in the absence of methotrexate, whereas in other variants the amplified genes are lost from the population. We have previously reported that in a stably amplified variant of Chinese hamster ovary cells, the genes are localized to a single chromosome. Herein we report that in mouse S-180 and L5178Y cell lines unstably amplified dihydrofolate reductase DNA sequences are associated with small, paired chromosomal elements denoted "double minute chromosomes," whereas in stably amplified cells of the same origin, the genes are associated with large chromosomes.     

Isolation of a replication origin complex from Escherichia coli.

A complex consisting of replicative origin DNA and several proteins was isolated from Escherichia coli. Cells of temperature-sensitive mutants were labeled at the origin and fractionated by sucrose gradient centrifugation. A complex highly purified in origin DNA sedimented as a unique band. This complex dissociated at high concentration, above 0.2 M KCl. Upon dialysis, the complex reformed, allowing further purification of its constituents. Three major protein bands were found, corresponding to proteins of the outer membrane. The complex did not sediment with membrane fractions, but adhered to the outer membrane in the presence of magnesium.     

Lipid-soluble diaminopyrimidine inhibitors of dihydrofolate reductase.

On the basis of activity against experimental tumors and potency as inhibitors of human dihydrofolate reductase, two compounds were selected for pharmacokinetic evaluation: metoprine ((2,4-diamino-5-(3',4'-dichlorophenyl)-6-methyl pyrimidine, DDMP, B.W. 197U) and etoprine, the corresponding 6-ethyl analog (DDEP, B.W. 276U). These lipid-soluble compounds readily cross the blood-brain barrier and penetrate rapidly into brain and brain tumors induced in rats by ethylnitrosourea. Both compounds are extensively bound to human plasma protein and their slow elimination from plasma and tissues contrasts with the kinetics of methotrexate. Cerebrospinal fluid levels of "folate" were elevated following oral administration of citrovorum factor to rats but not following equivalent doses of folic acid. The balance between selective action of the drug and selective protection by the vitamin is discussed with regard to differential distribution into separate compartments.     

Dynamics of trimethoprim bound to dihydrofolate reductase.

The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 1H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase. 13C relaxation measurements reveal dihedral angle changes of +/- 25 degrees to +/- 35 degrees on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least +/- 65 degrees on the millisecond time scale. 1H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.     

Single-molecule and transient kinetics investigation of the interaction of dihydrofolate reductase with NADPH and dihydrofolate

The interaction of dihydrofolate (H(2)F) and NADPH with a fluorescent derivative of H(2)F reductase (DHFR) was studied by using transient and single-molecule techniques. The fluorescent moiety Alexa 488 was attached to the structural loop that closes over the substrates after they are bound. Fluorescence quenching was found to accompany the binding of both substrates and the hydride transfer reaction. For the binding of H(2)F to DHFR, the simplest mechanism consistent with the data postulates that the enzyme exists as slowly interconverting conformers, with the substrate binding preferentially to one of the conformers. At pH 7.0, the binding reaction has a bimolecular rate constant of 1.8 x 10(7) M(-1).s(-1), and the formation of the initial complex is followed by a conformational change. The binding of NADPH to DHFR is more complex and suggests multiple conformers of the enzyme exist. NADPH binds to a different conformer than H(2)F with a bimolecular rate constant of 2.6-5.7 x 10(6) M(-1).s(-1), with the former value obtained from single-molecule kinetics and the latter from stopped-flow kinetics. Single-molecule studies of DHFR in equilibrium with substrates and products revealed a reaction with ensemble average rate constants of 170 and 470 s(-1) at pH 8.5. The former rate constant has an isotope effect of >2 when NADPD is substituted for NADPH and probably is associated with hydride transfer. The results from stopped-flow and single-molecule methods are complementary and demonstrate that multiple conformations of both the enzyme and enzyme-substrate complexes exist.     

Isolation and mapping of plasmids containing the Salmonella typhimurium origin of DNA replication.

A purified EcoRI restriction endonuclease fragment that determines resistance to kanamycin and is incapable of self-replication was used to select autonomously replicating fragments from an EcoRI digest of a Salmonella typhimurium F' plasmid containing the chromosomal region believed to include the S. typhimurium origin of DNA replication. Both the F factor and S. typhimurium chromosome replication origins were cloned by this procedure. The EcoRI fragmentment containing the S. typhimurium origin of replication is 19.4 kilobase pairs long and includes functional asp+ and uncB+ genes. Restriction endonuclease analysis of deletions obtained from the S. typhimurium origin plasmid indicated that the replication origin (ori region) is contained within a 3.3-kilobase pair region. Comparison with Escherichia coli origin plasmids shows colinearity of gene arrangement on the chromosomes in this region and suggests that some, but not all, regions of the nucleotide sequence in the origin region may be conserved (identical) in these two bacterial species.