Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.     

Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry

The cell cycle-dependent regulation of the cellular dihydrofolate reductase content (DHFR) and tissue plasminogen activator (t-PA) production and secretion in plasmid-amplified cells was investigated in the DHFR-negative CHO cells transfected with the plasmid pSV-tPA.dhfr. This plasmid, carrying the dhfr and t-PA gene under control of different promotors, was amplified by serial passages in 5 microM methotrexate (MTX) for dhfr gene amplification. The intracellular amount of DHFR was quantitated in viable cells by MTX-FITC labeling and flow cytometric analysis of the FITC fluorescence. In comparison with the original CHO cells, the pSVtPA.dhfr-amplified cells showed a greater than 230-fold increase in MTX-FITC fluorescence. Using dual laser flow cytometry (uv: vital cell cycle with Hoechst 33342; 488 nm: DHFR with MTX-FITC), we show a maximum increase in the intracellular DHFR content during G1 and/or at G1/S transition (100 to 157%), followed by a continuous increase to 200% during S and G2/M. To determine t-PA production CHO cells were sorted from G1-, early/late S-, and G2/M-phase. After 1-, 2-, and 4-h incubation periods, t-PA production was quantitated using a sensitive t-PA ELISA technique. We found that t-PA production and secretion (2-h assay), unlike the expression of DHFR, increased continuously from relatively 100% in G1 to 127% in early S and reached its maximum of 159% in late S, whereas in G2/M-phase it decreased to 118%. Our results show that in pSVtPA.dhfr-coamplified CHO cells gene products DHFR and t-PA both exhibit different cell cycle-correlated accumulation and secretion, respectively, indicating that the brightest MTX-FITC-positive cells (G2/M) do not display the highest t-PA secretion rate.     

Effect of amplification ofdhfr andlac Z genes on growth and β-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 β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-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 β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-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 ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

Evolution of genomes, host shifts and the geographic spread of SARS-CoV and related coronaviruses

Severe acute respiratory syndrome (SARS) is a novel human illness caused by a previously unrecognized coronavirus (CoV) termed SARS‐CoV. There are conflicting reports on the animal reservoir of SARS‐CoV. Many of the groups that argue carnivores are the original reservoir of SARS‐CoV use a phylogeny to support their argument. However, the phylogenies in these studies often lack outgroup and rooting criteria necessary to determine the origins of SARS‐CoV. Recently, SARS‐CoV has been isolated from various species of Chiroptera from China (e.g., Rhinolophus sinicus) thus leading to reconsideration of the original reservoir of SARS‐CoV. We evaluated the hypothesis that SARS‐CoV isolated from Chiroptera are the original zoonotic source for SARS‐CoV by sampling SARS‐CoV and non‐SARS‐CoV from diverse hosts including Chiroptera, as well as carnivores, artiodactyls, rodents, birds and humans. Regardless of alignment parameters, optimality criteria, or isolate sampling, the resulting phylogenies clearly show that the SARS‐CoV was transmitted to small carnivores well after the epidemic of SARS in humans that began in late 2002. The SARS‐CoV isolates from small carnivores in Shenzhen markets form a terminal clade that emerged recently from within the radiation of human SARS‐CoV. There is evidence of subsequent exchange of SARS‐CoV between humans and carnivores. In addition SARS‐CoV was transmitted independently from humans to farmed pigs (Sus scrofa). The position of SARS‐CoV isolates from Chiroptera are basal to the SARS‐CoV clade isolated from humans and carnivores. Although sequence data indicate that Chiroptera are a good candidate for the original reservoir of SARS‐CoV, the structural biology of the spike protein of SARS‐CoV isolated from Chiroptera suggests that these viruses are not able to interact with the human variant of the receptor of SARS‐CoV, angiotensin‐converting enzyme 2 (ACE2). In SARS‐CoV we study, both visually and statistically, labile genomic fragments and, putative key mutations of the spike protein that may be associated with host shifts. We display host shifts and candidate mutations on trees projected in virtual globes depicting the spread of SARS‐CoV. These results suggest that more sampling of coronaviruses from diverse hosts, especially Chiroptera, carnivores and primates, will be required to understand the genomic and biochemical evolution of coronaviruses, including SARS‐CoV. © The Willi Hennig Society 2008.     

Influence of hydrophobic and electrostatic residues on SARS‐coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS‐coronavirus (SARS‐CoV). SARS‐CoV entry is facilitated by the spike protein (S), which consists of an N‐terminal domain (S1) responsible for cellular attachment and a C‐terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS‐CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site‐directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH‐independent retroviral fusion proteins, SARS‐CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS‐CoV S2 analysis showed that specific hydrophobic positions at the C‐terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C‐terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C‐terminal hydrophobic residues led us to identify a 42‐residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections.     

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.     

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.     

Chemically defined medium for the production of biologically active substances of CHO cells

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.Abbreviations CHO Chinese hamster ovary - hGH human growth hormone - dFCS dialyzed fetal calf serum - dhfr dihydroforate reductase - MTX methotrexate     

Expression of recombinant classical swine fever virus E2 glycoprotein by endogenous Txnip promoter in stable transgenic CHO cells

As the main immunogen that could stimulate neutralized antibody in pigs, recombinant E2 protein of CSFV was expressed in CHO‐dhfr^?cells driven by endogenous Txnip promoter from Chinese hamster. Different fragments of Txnip promoter were amplified by PCR from isolated genomic DNA of CHO cells and cloned into different expression vectors. Compared with CMV promoter, CHO‐pTxnip‐4‐rE2 (F12) cell clone with the highest yield of rE2 protein was established by random insertion of the expression cassette driven by 860 bp sequences of Txnip promoter. In combination with treatment of 800 nM MTX for copy amplification of inserted expression cassette, the dynamic expression profile of rE2 protein was observed. Then inducible expression strategy of balance between viable cell density and product yield was conducted by mixed addition of 0.1 mM NADH and 0.1 mM ATP in culture medium at day 3 of batch‐wise culture. It could be concluded that Txnip promoter would be a promising alternative promoter for recombinant antigen protein expression in transgenic cells.     

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene‐of‐interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long‐term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non‐UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one‐step amplification procedure. However, non‐UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long‐term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non‐UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1014–1025, 2015     

Structural isomers of the S2 state in photosystem II: do they exist at room temperature and are they important for function?

In nature, an oxo‐bridged Mn₄CaO₅ cluster embedded in photosystem II (PSII), a membrane‐bound multi‐subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light‐induced charge separations in the reaction center of PSII. The Mn₄CaO₅ cluster accumulates four oxidizing equivalents to enable the four‐electron four‐proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S‐states, S₀ – S₄ in the Kok cycle. One important question related to the catalytic mechanism of the oxygen‐evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S‐states and if such equilibria are essential for the mechanism of the O‐O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X‐ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S₂ state of PSII with structural data collected of the S₁, S₂ and S₃ states by serial crystallography at neutral pH (～6.5) using an X‐ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S₂ state, the room temperature crystallography data can be well‐described by just one S₂ structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.     

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings.     

利用谷氨酰胺合成酶基因(GS)作扩增标记在CHO细胞中高效表达乙型肝炎表面抗原基因

利用谷氨酰胺合成酶基因（ＧＳ）［１］作扩增选择标记,结合ＣＭＶ－ＩＥ启动子,在ＣＨＯ细胞中高效表达乙型肝炎表面抗原基因。初筛克隆表达水平ＲＰＨＡ检测为１：６４,经过谷氨酰胺合成酶基因的抑制剂ＭＳＸ的两轮基因扩增,ＨＢｓＡｇ的表达水平ＲＰＨＡ在１：２５６以上。方瓶静置培养收液,ＲＩＡ检测ＨＢｓＡｇ最高产量为９．５μｇ／毫升。表达水平较以前利用ｄｈｆｒ基因扩增选择系统所得到的高表达细胞系Ｂ４３高一倍以上。利用ＧＳ基因扩增选择系统可以在哺乳动物细胞中高水平表达外源基因。     

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.     

Comparing the binding properties of peptides mimicking the Envelope protein of SARS‐CoV and SARS‐CoV‐2 to the PDZ domain of the tight junction‐associated PALS1 protein

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS‐CoV and SARS‐CoV‐2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ‐binding motif at its C‐terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS‐CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS‐CoV and SARS‐CoV‐2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS‐CoV‐2 compared to SARS‐CoV may rely on the increased affinity of its Envelope protein for PALS1.     

Shortlisting SARS‐CoV‐2 Peptides for Targeted Studies from Experimental Data‐Dependent Acquisition Tandem Mass Spectrometry Data

Detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a crucial tool for fighting the COVID‐19 pandemic. This dataset brief presents the exploration of a shotgun proteomics dataset acquired on SARS‐CoV‐2 infected Vero cells. Proteins from inactivated virus samples were extracted, digested with trypsin, and the resulting peptides were identified by data‐dependent acquisition tandem mass spectrometry. The 101 peptides reporting for six viral proteins were specifically analyzed in terms of their analytical characteristics, species specificity and conservation, and their proneness to structural modifications. Based on these results, a shortlist of 14 peptides from the N, S, and M main structural proteins that could be used for targeted mass‐spectrometry method development and diagnostic of the new SARS‐CoV‐2 is proposed and the best candidates are commented.     

Scientific research progress of COVID‐19/SARS‐CoV‐2 in the first five months

A cluster of pneumonia (COVID‐19) cases have been found in Wuhan China in late December, 2019, and subsequently, a novel coronavirus with a positive stranded RNA was identified to be the aetiological virus (severe acute respiratory syndrome coronavirus 2, SARS‐CoV‐2), which has a phylogenetic similarity to severe acute respiratory syndrome coronavirus (SARS‐CoV). SARS‐CoV‐2 transmits mainly through droplets and close contact and the elder or people with chronic diseases are high‐risk population. People affected by SARS‐CoV‐2 can be asymptomatic, which brings about more difficulties to control the transmission. COVID‐19 has become pandemic rapidly after onset, and so far the infected people have been above 2 000 000 and more than 130 000 died worldwide according to COVID‐19 situation dashboard of World Health Organization ( https://covid19.who.int ). Here, we summarized the current known knowledge regarding epidemiological, pathogenesis, pathology, clinical features, comorbidities and treatment of COVID‐19/ SARS‐CoV‐2 as reference for the prevention and control COVID‐19.     

Effects of palindrome structure on Dhfr amplification in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are widely used in producing therapeutic proteins. Gene amplification techniques are frequently used in improving protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used for the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and found one BAC clone (Cg0031N14) containing a CHO genomic DNA sequence adjacent to Dhfr. The BAC clone contained a large palindrome structure with a small inverted repeat in the junction region. To investigate the effect of the palindrome structure derived from the BAC clone Cg0031N14 on Dhfr amplification in CHO cells, we constructed plasmids that contain part or the whole junction region of the palindrome structure. The transfected CHO DG44 cells containing part or the whole junction region of the palindrome structure could adapt quickly to high methotrexate (MTX) concentrations. Moreover, the cells containing the whole junction region of the palindrome structure showed a high ratio of GFP-positive cells during gene amplification. On the basis of these results, we estimated that the junction region plays an important role in gene amplification in CHO cells.     

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.     

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.