Sequence requirements for the stimulation of gene amplification by a mammalian genomic element

HSAG-1 is a 3.4-kb genomic element from a human chronic lymphocytic leukemia--Chinese hamster ovary (CHO) hybrid cell line shown to stimulate the amplification of expression vectors in cis when transfected into a variety of cell lines [McArthur and Stanners, J. Biol. Chem. 266 (1991) 6000-6005]. Subfragments of HSAG-1 were tested for amplification activity by insertion into the vector, pSV2DHFR. The results suggest that multiple positive- and negative-acting elements were present that influenced amplification activity. The deletion of regions believed to contain positive-acting elements decreased or abolished the amplification stimulatory activity of the most active 1.45-kb fragment, supporting this hypothesis. The construction of composite sequences containing multiple positive elements and lacking negative elements, however, failed to enhance the activity; maximum activity was obtained only with the original intact configuration of elements. Two of two CHO HSAG-1-like elements tested had an activity equivalent to HSAG-1, while one of 24 random CHO genomic fragments tested had an activity as high as HSAG-1. The combination of sequence and structural features needed to affect the frequency of gene amplification may therefore be quite common in the mammalian genome.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.

Expression of human tissue-type plasminogen activator (t-PA) at high levels has been achieved in Chinese hamster ovary (CHO) cells by cotransfection and subsequent coamplification of the transfected sequences. Expression vectors containing the t-PA cDNA gene and dihydrofolate reductase (DHFR) cDNA gene were cotransfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in media lacking nucleosides and contained low numbers of t-PA genes and DHFR genes. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate generated cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA. To optimize both t-PA sequence amplification and t-PA expression, various modifications of the original procedure were used. These included alterations to the DHFR expression vector, optimization of the molar ratio of t-PA to DHFR sequences in the cotransfection, and modification of the methotrexate resistance selection procedure. The structure of the amplified DNA, its chromosomal location, and its stability during growth in the absence of methotrexate are reported.     

Selection for methotrexate resistance in mammalian cells bearing a <Emphasis Type="Italic">Drosophila</Emphasis> dihydrofolate reductase transgene

Antifolates, such as methotrexate (MTX), are the treatment of choice for numerous cancers. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms. In Drosophila, however, a resistant phenotype has only been observed to occur by mutation resulting in a MTX-resistant DHFR. It is unclear if differences in gene structure and/or genome organization between Drosophila and mammals contribute to the observed differences in acquired drug resistance. To investigate if gene structure is involved, Drosophila Dhfr cDNA was transfected into a line of CHO cells that do not express endogenous DHFR. These transgenic cells, together with wild-type CHO cells, were selected for 19 months for resistance to increasing concentrations of MTX, from 50- to 200-fold over the initial concentration. Since Drosophila Dhfr appears to have been amplified several fold in the selected transgenic mammalian cells, a difference in genome organization may contribute to the mechanism of MTX resistance.     

Expression of amplified cDNA and genomic sequences encoding human interleukin 2 in Chinese hamster ovary cells

Expression of human interleukin 2 (IL-2) at high levels has been achieved in Chinese hamster ovary (CHO) cells by amplification of transfected sequences. Plasmids containing the human IL-2 cDNA or genomic DNA and mouse dihydrofolate reductase (DHFR) cDNA were transfected into DHFR-negative CHO cells. Transformants expressing DHFR were selected in media lacking nucleosides, and cells which amplified both DHFR and IL-2 genes were obtained by exposure to increasing methotrexate (MTX) concentrations. These cell lines constitutively expressed elevated levels of IL-2 at a concentration of 2 mg/liter. These cell lines continued to produce IL-2 stably through at least 1 month, even in the absence of MTX.     

Amplified inverted duplications within and adjacent to heterologous selectable DNA.

Plasmids containing a dihydrofolate reductase (DHFR) expression unit were transfected into DHFR-deficient Chinese hamster ovary (CHO) cells. Methotrexate exposure was used to select cells with amplified DHFR sequences. Three cell lines were isolated containing amplified copies of transfected DNA that had integrated into the Chinese hamster genome. Plasmid DNA was found to co-amplify with flanking hamster sequences that were repetitive (2 cell lines) and unique (1 cell line). Fragments comprising the junctions of amplified plasmid and CHO DNA were found to exist as inverted duplications in all three cell lines. These observations provide evidence that inverted duplication occurred prior to DNA amplification, thus underscoring the importance of inverted duplication in the DNA amplification process.     

Dihydrofolate reductase activity in adriamycin and methotrexate sensitive and resistant P388 leukemia cells

P388 murine leukemia cells 18.4-fold more resistant to methotrexate (MTX) than the parent, drug susceptible line, were shown to possess a 1.5-fold higher dihydrofolate reductase (EC1.5.1.3) (DHFR) activity. This is in contrast to a MTX-resistant line, obtained from adriamycin-resistant cells, which is 27.9-fold more resistant to MTX and exhibits a 22.4-fold higher DHFR activity than that of the parent. The susceptibility of the enzyme to inhibition by MTX does not markedly change with the acquired drug resistance of the cell lines studied. Thus MTX-resistant cells obtained from an adriamycin-resistant line acquired resistance due to increased activity of the target enzyme, whereas other mechanisms are responsible for the resistance of cells derived from the adriamycin-sensitive parent.     

Construction of a dominant selectable marker using a novel dihydrofolate reductase.

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.     

Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.     

Chromosome instability associated with human alphoid DNA transfected into the Chinese hamster genome.

Repetitive DNA sequences have been implicated in the mediation of DNA rearrangement in mammalian cells. We have tested this hypothesis by using a dihydrofolate reductase (DHFR) expression vector into which candidate sequences were inserted. DHFR- Chinese hamster ovary (CHO) cells were transfected with this vector, the amplification of which was then selected for by methotrexate (MTX) exposure. Cells transfected with the vector alone (and resistant to 0.02 or 1.0 microM MTX) or with a poly(dG-dT) insert (and resistant to 0.05 or 1.0 microM MTX) showed little change in chromosome aberrations or sister chromatid exchange frequencies. In contrast, transfection of DHFR- CHO cells with a vector containing either of two distinct 0.34-kilobase human alphoid DNA segments (and selection to 0.05 to 10.0 microM MTX) showed an approximately 50% increase in chromosome number and marked changes in chromosome structure, including one or two dicentric or ring forms per cell. The sister chromatid exchange frequency also increased, to more than double the frequency of that in cells transfected without insert or those containing poly(dG-dT). In situ hybridization of one 0.34-kilobase insert in some cells suggested clustering of homologous sequences in structurally abnormal recipient CHO cell chromosomes. The approach described provides an introduction to a unique means for a coordinate molecular and cytological study of dynamic changes in chromosome structure.     

Antifolate resistance and its circumvention by new analogues.

We have established human leukemia cell lines made resistant to various antifolate drugs and analyzed resistance mechanisms developed in these cells at the cellular and molecular levels. The cells acquired resistance to antifolate drug(s) through: (1) impaired drug uptake via the reduced folate carrier, (2) increased activity of the target enzymes[dihydrofolate reductase(DHFR) or thymidylate synthase(TS)] resulted from a concomitant amplification and overexpression of their gene, (3) induction of a variant DHFR with a low affinity for antifolate drug(s) used for the selection of resistance, and (4) defective polyglutamation. Each resistance mechanism was not necessarily induced at random, but appeared to relate to the biochemical and pharmacological properties of the drug exposed, biological dispositions of the cells, drug-exposure manners to, or culture conditions of the cells. Since it has been shown that a minor modification at the specified position of the folate structure resulted in a drastic change in its pharmacological properties, many new compounds have been rationally designed on the basis of the knowledge of relationships between structure modifications and pharmacological properties. The step-by-step approach to the development of new analogues led to the discoveries of several promising antifolate drugs such as trimetrexate and raltitrexed, which can overcome the acquired and natural resistance to methotrexate, a classical antifolate, and clinical trials of these newer classes of antifolate compounds are currently underway.     

Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.     

Dihydrofolate reductase gene as a versatile expression marker.

The Escherichia coli dihydrofolate reductase (DHFR) gene has been used as a genetic marker specifying trimethoprim resistance (TmpR). In order to use the DHFR gene as a versatile expression marker, we have constructed three types of plasmids: promoter cloning vector, terminator cloning vector, and the plasmid containing the DHFR gene cassette. In these systems, the selection of recombinant plasmids was carried out just by examining the TmpR phenotype of the transformed cells. Then, levels of the enzymatic activity of DHFR were measured to evaluate the efficiency of promoters and terminators in the fused DNA fragment. An expression plasmid which resulted in the E. coli host cells being able to produce DHFR up to 20% of total cellular proteins was also constructed by changing the promoter and Shine-Dalgarno sequences of the DHFR gene.     

The organization of the dihydrofolate reductase gene of baby hamster kidney fibroblasts.

Using cloned DNA complementary to mouse dihydrofolate reductase (DHFR) mRNA, the organization of the hamster DHFR gene has been determined in two baby hamster kidney (BHK) cell lines, A5 and B1. A5 cells are highly methotrexate-resistant, containing 200-fold more copies of the DHFR gene than do the parental B1 cells. The DHFR gene has the same organization in A5 and B1 cells, suggesting that it has not been altered by the amplification process. The BHK DHFR gene spans a maximum of 10.7 kb and contains at least three introns. Thus the BHK DHFR gene is much smaller than the mouse DHFR gene, which has a minimum size of 42 kb and at least five introns. This striking size difference is probably due to much smaller introns in the BHK DHFR gene.     

Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.     

A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes.

The retroviral gene transfer system is a powerful tool for somatic gene therapy. A retroviral stock with a high viral titer and lacking replication-competent virus (RCV) is desirable for this type of gene transfer. To fulfill these requirements, we made a new packaging cell line, designated ampli-GPE. To reduce the homology between proviral DNA in the packaging cell and retroviral vector, the gag-pol and env genes of Moloney murine leukemia virus were separated onto two different plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human beta-globin gene containing the polyadenylation site as control units for the gag-pol and env genes. In addition, these plasmids contained 69% of the bovine papillomavirus gene for gene amplification to obtain production of virus at a high titer. NIH 3T3 clones containing approximately 20 to 50 copies of the gag-pol and env genes were selected and designated ampli-GPE. When ampli-GPE was transfected with the N2 vector or pZipNeoSV(DHFR) derived from pZipNeoSV(X)1, we established clones producing titers of 5 x 10(6) and 1 x 10(6) CFU/ml, respectively. There was no sign of RCV generation in any virus-producing cells from ampli-GPE. However, virus-producing cells derived from psi 2 cells transfected with N2 did generate RCV. Thus, we showed that ampli-GPE, possessing the minimum complement of proviral genes, has potential for the development of a gene transfer system.