Gene amplification in a single cell cycle in Chinese hamster ovary cells

We have employed Chinese hamster ovary cells synchronized by mitotic selection to study the replication and amplification of the dihydrofolate reductase gene. Using bromodeoxyuridine to differentially label newly replicated DNA, we show that the dihydrofolate reductase gene is replicated during the first 2 h of S phase, a time when, at most, 10% of the total genome has been replicated. We find that a 6-h inhibition of DNA synthesis by hydroxyurea beginning 2 h after the initiation of S phase markedly increases the frequency with which cells become resistant to a 100-fold increment in methotrexate. When DNA synthesis resumes following removal of the hydroxyurea, virtually all of the DNA replicated prior to inhibition, including the dihydrofolate reductase gene, is rereplicated. Analysis of the dihydrofolate reductase enzyme content of cells 24 h after treatment with hydroxyurea using the fluorescence-activated cell sorter reveals a subset of cells with elevated dihydrofolate reductase. It is this subset that contains additional copies of the dihydrofolate reductase gene and from which emerge highly methotrexate-resistant cells. We propose that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome. As cells are subsequently placed under selection, a number of processes, including recombination events and loss of nonselected DNA sequences occur, resulting in what appears as differential gene amplification.     

Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate

We have investigated the role of dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo purine synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Previous studies have shown that cytotoxic concentrations of MTX that inhibit dihydrofolate reductase produce only minimal depletion of the reduced folate cofactor, 10-formyltetrahydrofolate, required for purine synthesis. At the same time, de novo purine synthesis is totally inhibited. In these studies, we show that 10 microM MTX causes inhibition of purine synthesis at the step of phosphoribosylaminoimidazolecarboxamide (AICAR) transformylase, as reflected in a 2-3-fold expansion of the intracellular AICAR pool. The inhibition of purine synthesis coincides with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of AICAR transformylase. When the generation of H2PteGlu is blocked by pretreatment with 50 microM 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase, MTX no longer causes inhibition of purine synthesis. Intermediate levels of H2PteGlu produced in the presence of lower (0.1-10 microM) concentrations of FdUrd led to proportional inhibition of purine biosynthesis, and the exogenous addition of H2PteGlu to breast cells in culture re-established the block in purine synthesis in the presence of FdUrd and MTX. The early phases of inhibition of purine biosynthesis could be ascribed only to H2PteGlu accumulation. MTX polyglutamates, also known to inhibit AICAR transformylase, were present in breast cells only after 6 h of incubation with the parent compounds and were not formed in cells preincubated with FdUrd. The lipid-soluble antifolate trimetrexate, which does not form polyglutamates, produced modest 10-formyltetrahydrofolate depletion, but caused marked H2PteGlu accumulation and a parallel inhibition of purine biosynthesis. This evidence leads to the conclusion that MTX and the lipid-soluble analog trimetrexate cause inhibition of purine biosynthesis through the accumulation of H2PteGlu behind the blocked dihydrofolate reductase reaction.     

High level expression of human proteins in murine hybridoma cells: induction by methotrexate in the absence of gene amplification.

We have developed an inducible system for high level expression of heterologous genes in murine hybridoma cells. The rapid induction by methotrexate (MTX) does not involve gene amplification and is controlled at the level of mRNA accumulation. Transfection was achieved by protoplast fusion with an expression vector containing the cDNA of interest and a marker gene encoding dihydrofolate reductase. The initial clones, selected at 100 nM MTX, produced high levels of the protein of interest and contained about 100-400 copies of the integrated plasmid DNA. They could adapt to a 100- to 1000-fold stepwise increase in MTX concentration in a few weeks, during which the expression of the gene of interest but not its copy number, increased several-fold. Furthermore, the induction is freely reversible. If cells were propagated in MTX-free media, the expression level decreased, but the cells could be reinduced to their original high level of expression by adding MTX back to the media. A several-fold increase in the mRNA levels of the dihydrofolate reductase and the gene of interest could be detected after induction for 18 h.     

Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea.

We investigated various parameters associated with the initial selection of mouse 3T6 cells for resistance to single concentrations of methotrexate and characterized resistant colonies for the presence of additional (amplified) copies of the dihydrofolate reductase gene. Our results indicate that the frequency of occurrence of dihydrofolate reductase gene amplification varies with the selecting concentration of methotrexate and is highly variable between clonally derived sublines of mouse 3T6 cells. Second, we increased the frequency of occurrence of cells with amplified dihydrofolate reductase genes by transiently inhibiting DNA synthesis with hydroxyurea before the selection of cells in single concentrations of methotrexate. This effect was dependent on the concentration of hydroxyurea, the time of exposure to the drug, and the time interval between the removal of hydroxyurea and the selection of cells in methotrexate.     

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.     

Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification

Recombinant Chinese hamster ovary (CHO) parental clones expressing a humanized antibody against S surface antigen of hepatitis B virus were obtained by cotransfection of heavy chain (HC) and light chain (LC) cDNA expression vectors into dihydrofolate reductase (DHFR)-deficient CHO cells. When 23 representative parental clones were subjected to stepwise selection for increasing methotrexate (MTX) resistance, such as 0.02, 0.08, 0.32, and 1.0 microM, their clonal variations in regard to antibody expression were found to be significant. Among 23 parental clones, only one clone (hu17) showed the significant increment of specific antibody productivity (q(Ab)) with increasing MTX concentration up to 0.32 microM. Compared with the parental clone (hu17), the q(Ab) of hu17 resistant at 0.32 microM MTX (hu17-0.32) was enhanced approximately 12.5-fold. To clarify the reason for the occurrence of clonal variations, Southern blot analyses of chromosomal DNAs derived from each amplified clone at 0.32 microM MTX were performed. Only the hu17-0.32 clone did not experience severe genetic rearrangement during gene amplification, and it had only one 49-kb amplification unit including the LC and HC cDNAs. A fluorescent MTX competition assay showed that the resistance against MTX toxicity of the other clones without enhanced q(Ab) at 0.32 microM MTX was obtained by mechanisms such as an impaired MTX transport system. Taken together, the data obtained here show that clonal variations in regard to antibody expression are found to be significant because clones can acquire MTX resistance by mechanisms other than DHFR-mediated gene amplification despite the stepwise selection.     

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.     

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.     

Comprehensive characterization of dihydrofolate reductase-mediated gene amplification for the establishment of recombinant human embryonic kidney 293 cells producing monoclonal antibodies

Human embryonic kidney 293 (HEK293) cells with glycosylation machinery have emerged as an alternative host cell line for stable expression of therapeutic glycoproteins. To characterize dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification in HEK293 cells, an expression vector containing dhfr and monoclonal antibody (mAb) gene was transfected into dhfr-deficient HEK293 cells generated by knocking out dhfr and dhfrl1 in HEK293E cells. Due to the improved selection stringency, mAb-producing parental cell pools could be generated in the absence of MTX. When subjected to stepwise selection for increasing MTX concentrations such as 1, 10, and 100 nM, there was an increase in the specific mAb productivity (q_mAb) of the parental cell pool upon DHFR/MTX-mediated gene amplification. High producing (HP) clones with a q_mAb of more than 2-fold of the corresponding cell pool could be obtained using the limiting dilution method. The q_mAb of most HP clones obtained from cell pools at elevated MTX concentrations significantly decreased during long-term culture (3 months) in the absence of selection pressure. However, some HP clones could maintain high q_mAb during long-term culture. Taken together, a stable HP recombinant HEK293 cell line can be established using DHFR/MTX-mediated gene amplification together with dhfr⁻ HEK293 host cells.     

Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells

Stable expression of human insulin-like growth factor of binding protein-1 (hIGFBP-1)at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of expression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reductase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR⁺ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX). Expression of hIGFBP-1 in mixed clones was found to increase with increasing copy number and an apparent correlation between intra- and extracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIGFBP-1 25 times. The productivity did not increase further after five more months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplification in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44

We have developed a systematic and generic way to improve recombinant protein productivities in stable transfections by applying mRNA and protein destabilizing elements to reduce selection marker expression strength. Interferon-gamma (IFNgamma) expression vectors containing different combinations of AU-rich elements (ARE) and mouse ornithine decarboxylase (MODC) PEST region on the amplifiable dihydrofolate reductase (dhfr) selection marker were stably transfected into CHO-DG44 cells. Improvements in specific IFNgamma productivities were 1.7-, 6.6- and 13.3-fold with the application of ARE, MODC PEST, and both ARE and MODC PEST, respectively. To further enhance productivities, compatibility of the destabilizing sequences with methotrexate (MTX) amplification was validated by amplifying the transfected cells to 50nM MTX. A 14- to 27-fold increase in specific IFNgamma productivities were observed after amplification, indicating the compatibility of the two systems. A high specific IFNgamma productivity of 1.05pg/cell/day was also attained by the amplified cell pool with both ARE and MODC PEST.     

Inefficient growth arrest in response to dNTP starvation stimulates gene amplification through bridge-breakage-fusion cycles.

Cells often acquire resistance to the antiproliferative agents methotrexate (MTX) or N-phosphonacetyl-L-aspartate (PALA) through amplification of genes encoding the target enzymes dihydrofolate reductase or carbamylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), respectively. We showed previously that Syrian hamster BHK cells resistant to selective concentrations of PALA (approximately 3 x ID50) arise at a rate of approximately 10(-4) per cell per generation and contain amplifications of the CAD gene as ladder-like structures on one of the two B9 chromosomes, where CAD is normally located. We now find that BHK cells resistant to high concentrations of PALA (approximately 15 x ID50) appear only after prior exposure to selective concentrations of PALA for approximately 72 h. Furthermore, in contrast to untreated cells, BHK cells pretreated with selective concentrations of MTX give colonies in high concentrations of PALA, and cells pretreated with selective concentrations of PALA give colonies in high concentrations of MTX or 5-fluorouracil. As judged by measuring numbers of cells and metaphase cell pairs, BHK cells do not arrest completely when starved for pyrimidine nucleotides by treatment with selective concentrations of PALA for up to 72 h. We propose that DNA damage, caused when cells fail to stop DNA synthesis promptly under conditions of dNTP starvation, stimulates amplification throughout the genome by mechanisms--such as bridge-breakage-fusion cycles--that are triggered by broken DNA. Amplified CAD genes were analyzed by fluorescence in situ hybridization both in cells where amplification was induced by PALA pretreatment and in cells in which the amplification occurred spontaneously, before selection with PALA. The ladder-like structures that result from bridge-breakage-fusion cycles were observed in both cases.     

The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.     

Limitations to the amplification and stability of human tissue-type plasminogen activator expression by Chinese hamster ovary cells

Chinese hamster ovary cell production of recombinant tissue-type plasminogen activator (t-PA) was increased by amplification of cotransfected dihydrofolate reductase cDNA using stepwise adaptation to increasing methotrexate (MTX) concentrations. The highest producing clones were isolated at 5 microM MTX and yielded 26,000 U/10(6) cells/day t-PA (43 microgram/10(6) cells/day). Above 25 microM MTX, cell specific t-PA production rates became increasingly variable and the cDNA copynumbers decreased. No apparent correlation between the cell specific t-PA production rate and the growth rate was observed upon subcloning of the amplified cells. When MTX selection was removed, the t-PA production rate decreased up to tenfold within 40 days; this was accompanied by an up to 60% drop in cDNA copynumber. Subclones isolated after 108 days of culture in the absence of MTX were, on average, sixfold more stable than their parental cells. In culture without MTX, the maximum stable t-PA production rate obtained (over 250 days) was 7000 +/- 750 U/10(6) cells/day (approximately 12 microgram/10(6) cells/day), approximately threefold lower than the maximum unstable levels of production reached under selective pressure. Taken together, these results define a wide range of the highest t-PA expression rates obtained under MTX selection, for which stable expression without selection has not been reported.     

Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate

We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic antifolates, respectively, were established. Increased antifolate resistance was associated with a prominent overexpression of dihydrofolate reductase as determined from the elevated folate reductase activity, cellular labeling with fluorescein-methotrexate, and steady-state mRNA levels as a result of a consistent dihydrofolate reductase gene amplification. However, upon subsequent incremental increases in trimetrexate, further resistance was also associated with amplification of the multidrug resistance gene. This resulted in overexpression of P-glycoprotein and a subsequent 20-50-fold collateral resistance to pleiotropic drugs such as adriamycin, actinomycin D, vinca alkaloids, etoposide, and colchicine. In contrast, initial resistance following selection with low piritrexim concentrations resulted from an unknown mechanism(s) not involving overproduction of either dihydrofolate reductase or P-glycoprotein. This piritrexim resistance was shared with trimetrexate but not with methotrexate. Upon further selection with piritrexim, resistant variants emerge with amplified dihydrofolate reductase but not with multidrug resistance genes. These variants were subsequently resistant to both hydrophilic and lipophilic folate antagonists but retained sensitivity to pleiotropic drugs. The pattern of resistance with methotrexate, trimetrexate, and piritrexim shared a common mechanism, dihydrofolate reductase gene amplification, but differed regarding the additional amplification of the multidrug resistance gene in trimetrexate-resistant cells as well as the emergence of an additional unknown mechanism(s) of resistance to lipid-soluble antifolates upon initial selection in piritrexim.     

Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase

Methotrexate-resistant forms of human dihydrofolate reductase have the potential to protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained by combining two active-site mutations that independently confer weak MTX-resistance. In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR) variants for this application, we used a semi-rational approach to obtain combinatorial active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial mutant library encoding various amino acids at residues Phe31, Phe34 and Gln35. In vivo library selection was achieved in a bacterial system on medium containing high concentrations of MTX. We characterized ten novel MTX-resistant mutants with different amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters of the purified mutants revealed that higher MTX-resistance roughly correlated with a greater number of mutations, the most highly-resistant mutants containing three active site mutations (Ki(MTX)=59-180 nM; wild-type Ki(MTX)<0.03 nM). An inverse correlation was observed between resistance and catalytic efficiency, which decreased mostly as a result of increased KM toward the substrate dihydrofolate. We verified that the MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting the most resistant mutants into DHFR-knock-out CHO cells. The transfected variants conferred survival at concentrations of MTX between 100-fold and >4000-fold higher than the wild-type enzyme, the most resistant triple mutant offering protection beyond the maximal concentration of MTX that could be included in the medium. These highly resistant variants of hDHFR offer potential for myeloprotection during administration of MTX in cancer treatment.