genetic engineering of hybridoma glutamine metabolism

Genetic Engineering of Hybridoma Glutamine Metabolism Sarah L. Bell, Chris Bebbington, * Michelle F. Scott, J. Noel Wardell, Raymond E. Spier, Michael E. Bushel1 and Peter G. Sanders

School of Biological Sciences, University of Surrey, Guildford, England, and Telltech Limited, Slough, England

The murine hybridoma PQXBIIZ cannot be adapted to grow in culture media containing CO.5 mM glutamine. Transformants selected following electroporation of PQXBIR cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXBIIZ cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, suficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 kmol mint per 106 cells in transformants compared with parental levels of 0.25 pm01 mint per 106 cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabofic processes.

Keywords: Genetic engineering; glutamine synthetase; hybridoma physiology; ammonia detoxification

Introduction

Glutamine, the most versatile of all amino acids,’ is required for protein synthesis; it is an amino group donor for purine and pyrimidine biosynthesis and is a major energy source for mammalian cells. 2.3 Mammalian cell culture media normally contain glutamine at high levels (2-5 mu), and this can spontaneously decompose at up to 10% per day at 37°C to produce ammonia,4 which is also an end product of glutamine metabolism. Ammonia has been shown to have an inhibitory effect on the growth of mammalian cells,5.6 and ammonia production rates and cell growth rates are inversely related.7 Process analysis also suggests that intra- cellular ammonia produced as a result of metabolic processes is sufficient for normal hybridoma cell growth’ and

Address reprint requests to Dr. Peter G. Sanders, Molecular Microbiology Group, School of Biological Sciences, University of Surrey, Guildford GUZ 5XH, England. Dr. Bell’s present address is Kabi Phannacia, Bioscience Center, S-l 1287, Stockholm, Sweden. Dr. Scott’s present address is Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, England. Received 17 December, 1993; revised 28 April 1994; accepted 29 April 1994.

Enzyme and Microbial Technology 17:96-106, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

removal of excess exogenous ammonia would prevent the inhibition of growth caused by its presence, although inhibition of cell growth by ammonia may vary according to the cell line under study.8 Removal of exogenous ammonia would also reduce the expenditure of adenosine triphos- phate (ATP) by the Na+/K + -ATPase transporting NH,+ into the ce1L9 A number of protocols for reducing ammonia production, or removing ammonia from culture systems, have been established,“‘-r2 and recently, glutamine dipep- tide derivatives have been developed that are more stable in cell culture than free glutamine.13~14 Various cell types can be adapted to grow in glutamine-free media, which will in turn reduce the ammonia content of the environment, although supplementation of the media with increased levels of certain metabolites such as glutamic acid may be neces- sary . Is1 * Many rapidly proliferating lymphoid derived cell lines such as myelomas and hybridomas, however, are in- capable of growing in vitro in media lacking glutamine, although the YB2/0 and SP2/0 hybridomas generate glutamine-independent mutants.*O The mouse hybridoma, PQXB 112, which secretes an antibody against the herbicide paraquat, does not show this characteristic. Attempts to produce glutamine-independent derivatives of this cell line by adaptation and selection, including using media with increased glutamic acid levels, have been unsuccessful.

0141-0229/95/$10.00 SSDI 0141-0229(94)00056-W

PQXB1/2 hybridoma cells do not survive once glutamine levels in the culture media fall below 0.5 111~~’ although these cells possess the enzyme glutamine synthetase (EC 6.3.1.2).7~2’~22

Genetic engineering of glutamine metabolism: S, L. Bell et al.

a 500-bp, GS-specific fragment. The gpt gene probe was obtained from pEE6.gpt20 as a 2.0-kb BarnHI fragment. The p-actin gene probe. was obtained as a 2.0-kb PstI fragment from plasmid 41 ,30 a gift from M. Buckingham. All probes were purified from aga- rose gels by Geneclean (Stratech). DNA probes were labeled using the MultiPrime labeling kit (Amersham International). The cloning of genomic and cDNA sequences for Chi-

nese hamster glutamine synthetase (GS)18J3 and the development of vectors that use GS as a selectable markerz4 means that we have been able to investigate strategies involving genetic manipulation to transform hybridoma cells to glutamine independence, reducing the need for medium glutamine and the production of ammonia. We report the successful transformation to glutamine independence of the hybridoma cell line PQXB112 by transfection with a GS cDNA expressed from a viral promoter.

Vector construction

Materials and methods

Cells

Insertion of GS cDNA and gpt gene into pKSVl0. The eukary- otic expression vector pKSVl0 (Pharmacia) was modified to express both GS and gpt genes. Two synthetic oligonucleotides that would anneal to provide an adaptor with BgnI and Hi&III ends were synthesized (Applied Biosystems DNA synthesizer), phosphorylated with y3*P-ATP, and ligated to Hi&III-digested linear pSV2.GS. The adaptor structure was:

The murine hybridoma PQXB1/2 (ECACC 8905) was produced by fusion of X63.Ag8.653 myeloma cells with splenocytes from a mouse immunized with the herbicide paraquat. The cell line was obtained from ICI (Alderley Edge, UK) and secretes an immuno- globulin G (IgG) antibody against paraquat.25 COS-1 cells26 were obtained from ATCC.

BglII Not1 Oligo 1 S’GATCTGCGGCCGCA 3’

Oligo 2 3’ A CGCCGGCGTTCGA 5’

Hind111

Culture media and cell growth

Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mu glutamine, 18 mu sodium bicarbonate, and 10% (voYvo1) heat-inactivated fetal calf serum (FCS), (Life Technologies Inc.), or glutamine-free (G-DMEM) supplemented with heat-inactivated dialysed FCS (dFCS), nucleosides, nones- sential amino acids, glutamate, and asparagine.18*20 For selection involving xanthine-guanine-phosphorybosyltransferase (gpt) expression, the medium used was either DMEM or G-DMEM (minus guanosine), 10% FCS, or dFCS, containing 3 pg ml - 1 mycophenolic acid, 250 pg ml-’ xanthine, and 15 pg ml-’ hypo- xanthine, to give DMEM/gpt or G-DMEMlgpt media, respectively. Cells were grown at 37°C in 5% CO, in air.

Digestion with BgAI released the GS gene on a 1.2-kb BgAI fragment that was purified by Gene Clean (Stratech) and ligated into BgfII-digested, de-phosphorylated pKSV10. The ligated DNA was used to transform Escherichiu coli DHSa cells (Life Technolo- gies), and recombinant plasmids were analyzed by standard protocols. A plasmid with the GS gene inserted in the correct orien- tation was renamed pKSVlO.lGS.

Subsequently, a 2.0-kb EarnHI fragment containing the gpt gene with its own SV40 promoter was purified from pEE6.gptzo and cloned into the unique BumHI site of pKSVlO.lGS to form pKSVlO.lGS.gpt (pKSVlO.GS.gpt in Figure I).

pCMGS and pCMGS.gpt have been described previously.2o

Cell viability

Exclusion of Erythrosin-B (Sigma), (0.04% wt/vol in 0.85% wt/ vol NaCl) was taken as an indication of cell viability.’

Mycoplasma testing

pEEB.gpt s S

pSVP.GS

P P

pKSVlO.GS.gpt

Cell stocks were tested for the presence of mycoplasma by ECACC (Porton Down, Salisbury, UK). All of the cells were found to be negative.

Molecular biology techniques

Molecular biology techniques were performed according to Ma- niatis et ~1.~~ Extraction of cytoplasmic RNA from eucaryotic cells was done according to Favarolo et a1.29 Hybond membranes were used for nucleic acid blotting (Amersham International.)

pCMGS.gpt

DNA probes

A GS-specific DNA probe was prepared from pSV2.GSZo (Figure I) by digestion of plasmid DNA with Bg/II and EcoRI, releasing

Figure 1 Schematic diagrams of vectors used. Vectors are shown linearized for electroporation. WE, SV, early promoter; hCMV, human cytomegalovirus promoter.

Enzyme Microb. Technol., 1995, vol. 17, February 99

Papers

Cell transfections

Electroporation protocol for PQXB1/2 cells. PQXB l/2 hybridoma cells were transformed by electroporation3i using a Gene Pulser (Biorad). Exponentially growing cells with a minimum viability of 92% were washed in phosphate-buffered saline (PBS) (177 mM NaCl, 4 mu KCI, 10 rrt~ Na,HPG,, 1 mu KH,PO,, pH 7.2) pelleted by centrifugation at 80 g for 5 min, and resuspended at a density of 10’ cells ml-’ in ice-cold PBS. Then, 40 pg of linearized plasmid DNA was added to the cells to give a total volume of 0.8 ml. Transformation was optimized using &Zl linearized pEE6.gpt, and maximal when the cells were subjected to two pulses of 2 kV and 3 pF, with a 10-s interval between pulses. Changes in pulse duration, capacitance, and incubation temperature from these values did not improve the transformation frequency (Table I). After pulsing, the cells were left on ice for 10 mitt, resuspended in nonselective growth medium, and distributed in OS-ml volumes into the wells of 24-well tissue culture plates. Selective media were added after 24 h as required according to previously published procedures.20 The number of colonies surviving were determined after 10 to 14 days. Transformation frequencies in the range of 30 to 50 colonies per lo6 cells were regularly achieved (Table I).

DEAE-dextran transfection protocol. The DEAE-dextran transfection protoco13* was used with COS-1 cells.

Antibody production

Antibody concentration in the medium was determined by a sand- wich enzyme-linked immunosorbent assay (ELISA). Medium samples for the assay were centrifuged at 200 g for 5 min to remove cells and debris. Immunoplates (Nunc) were coated with 100 pl sheep anti-mouse IgG (Sigma) diluted l/l000 in coupling buffer (0.1 M sodium carbonate, pH 9.6) and left in the dark overnight at 4°C. The plates were washed four times with wash buffer [PBS, pH 7.4, containing 0.1% Tween 20 (vol/vol)]. Ap- propriate dilutions of the samples in PBS were applied to the plates in duplicate at 100 l~l per well. Affinity purified IgG (Sigma) antibody standard was used as a control. The plates were incubated in the dark at 37°C for 1 h, then washed four times with wash buffer. Next, 100 p,l of a l/l000 dilution of sheep anti-mouse IgG-alkaline phosphatase (Sigma) in PBS was applied to each well and incubated for 1 h at 37°C in the dark. Plates were washed four times with wash buffer. Then, 100 Pl enzyme substrate (Sigma

Table 1 Optimization of electroporation conditions for PQXBl/Z cells: Effect of altering electroporation pulse conditions

Transformation frequency

Voltage Capacitance (colonies/

Sample Pulse no. NJ) (+I lo6 cells)

A 1 2.0 3 30

: 2 2.0 3 52 1 2.0 25 18

E” 2 2.0 25 20 1 1.5 3 21

L 2 1.5 3 49 1 1.5 25 11

t-i 2 1.5 25 8

104-O) at 1 mg ml-’ in 0.5 M glycine, pH 10.4, 5 mu zinc chloride, 5 mM magnesium chloride was applied to all wells. The plates were incubated in the dark at 37°C for 30 to 60 mitt before reading the absorbance at 410 nm in a Dynatech MR600 plate reader.

Protein determinations

Protein concentrations in samples were determined according to Lowry et ~21.~~

Glutamine synthetase assay

A i4C-radiometric assay based on the method described by Tiemeier and Milman34 was used to measure GS activity. Cell pellets were washed twice in PBS and resuspended in 1 ml extraction buffer [50 mM Tris-HCl and 2 ITIM EDTA (pH 7.9)] at 10’ cells ml- I. Cells were lysed by sonication and the lysate was incubated in ice for 10 min. Next, 40 pl of sample was added to 360 pl of reaction mix containing 50 mu imidazole, 20 mu magnesium chloride, 20 mu ammonium chloride, 10 mM creatine phosphokinase, 15 mu Na-ATP, 1.2 U creatine kinase, 20 mu L-glutamic acid, 0.25 pCi L-(1-r4C)-glutamic acid, and incubated at 37°C for 30 min. All samples were assayed in duplicate. Rat liver and E. co/i grade V glutamine synthetases (Sigma), both native and heat inactivated, were used as controls. The reaction was stopped with 100 pl of 12% perchloric acid (vol/vol) and placed in ice. Samples were neutralized to pH 7.0 by adding 10 pl pH indicator solution 678 (BDH Ltd.), 20 Pl 5 M potassium hydroxide, and 70 p,l TRA [0.5 M triethanolamine hydrochloride and 2 M potassium hydroxide (BDH Ltd)], and centrifuged at 10,000 g for 2 min to remove any precipitate formed. Then, 350 p,l of sample supematant was passed through a Dowex-Cl (8% cross- linked) column (Sigma) pre-equilibrated with 50 mu Tricine buffer, pH 7. The column was then washed with 5 ml Tricine buffer and the effluent collected. We then counted 0.5-ml samples of effluent using OptiScint Hi-Safe Scintillation fluid (LKB). Compensation for quenching was made by using standard “‘C discs containing a known amount of radioactivity (104,900 dpm) per disc (Amersham International).

Results

Optimization of electroporation conditions

To optimize the electroporation protocol, the effect of different incubation conditions and electroporation pulsing re- gimes on the transformation frequency of PQXB1/2 cells was examined. The most efficient pulse conditions were 2.0 kV and 3 PF (Table I, sample B). The most effective incubation conditions were found to be 0°C before pulsing and 22°C afterward. However, as only slightly fewer colonies were obtained by using 0°C before and after pulsing, this temperature was used in subsequent experiments (Table 2, sample A).

Transfection of PQXB l/2 cells with pEE6.gpt under the chosen conditions (two pulses of 2.0 kV, 3 pF, and incubation at O’C) resulted in 5 1 colonies surviving in selective media per lo6 cells. Selection for transformants containing vectors expressing gpt was more efficient than using vectors expressing the neomycin resistance gene (Table 3), and therefore gpt-based vectors were used in subsequent studies .

100 Enzyme Microb. Technol., 1995, vol. 17, February

Genetic engineering of glutamine metabolism: S. L. Bell et al.

Table 2 Optimization of electroporation conditions for PQXBllP cells: Effect of altering electroporation incubation conditions

pEE6.gpt. Two related strategies were therefore pursued involving (a) cotransfection of pEE6.gpt with pSV2.GS and (b) incorporation of the GS and GPT expression cassettes in one vector prior to transfection.

Transformation frequency

Prepulse Postpulse Postpulse (coloniesi

Sample temp (“Cl temp 1°C) time (min) lo6 cells)

0 22 37

0 0 0 0 0

0 0 0

22 37

0 22 37

10 49 IO 41 10 12 IO 54 IO 31 30 48 30 52 30 33

Transfection of PQXB1I2 cells with SV#O promoter-based expression vectors

Initial attempts to develop cell lines that would grow in the absence of exogenous glutamine involved transfection of PQXB1/2 cells with vectors expressing glutamine synthetase from the SV40 early promoter, with transformants being selected by their ability to survive in glutamine-free medium (G-DMEM).

Transfections with pSV2.GS. Following transfection of the PQXB1/2 hybridoma with the vector PSV2.GS (Figure 1), the numbers of surviving colonies were assessed after 10 to 14 days. Putative transformed clones were found to be initially present on all of the GS transformation plates; however, after a period of 14 to 17 days, the colonies stopped proliferating, irrespective of whether G-DMEM was used alone for selection or with supplements (200 PM glutamine from 24 to 120 h post-transfection, or 4 mM glutamic acid and 1 mM ammonium chloride from 24 h post-transfection onward) added to help the cells overcome the initial glutamine deficiency and to provide nutrients for glutamine synthesis. In control transformations using pEE6.gpt a mean frequency of 43 colonies per lo6 cells was obtained. No colonies were observed on the “mock” transformation plates.

Cotransfection with SV40 based vectors expressing GS and gpt genes. In experiments using pSV2.GS transformed clones were routinely obtained using the control vector

Table 3 Optimization of electroporation conditions for PQXBl/Z cells: Results obtained from transforming PQXBIR hybridoma cells with pEEG.gpt and pSV2neo under optimal conditions

Transformation frequency (colonies/106 cells)

pEEG.gpt 51 51 pSV2.neo 35 46

(a) Vectors pSV2.GS and pEE6.gpt were linearized using PvuI and SalI, respectively, and cotransfected into the cells by electroporation. The total plasmid DNA concentration was maintained at 40 pg per transfection, but the ratio of pSV2.GS to pEE6.gpt was varied with a five- to IO-fold excess of pSV2.GS being used.

Using DMEM/gpt-selective media, a transformation frequency of 30 to 50 transformants per 10h cells was obtained. A number of these clones were expanded, and found to be stable in gpt-selective medium, but they failed to grow in the absence of glutamine. Additional selection regimens involving glutamine-free G-DMEMlgpr medium to provide dual selection for both the gpf- and GS-genes, also failed to select cell lines that could grow in glutamine-free medium. Supplementation of G-DMEMlgpt medium with 200 pM glutamine between 24 and 72 h, or the addition of 4 mM glutamic acid and 1 mM ammonium chloride from 24-h post-transfection was similarly unsuccessful.

(b) The dual expression vector pKSVlO.lGS.gpt was subsequently constructed (see Materials and methods) and initially expressed in COS-1 cells to confirm the functioning of the GS and gpt expression cassettes. Radiolabeled GS and gpr-specific probes hybridized to mRNAs of 2 kb (GS) and 2.2 kb (gpt) on Northern blots of RNA extracted from COS-1 cells 48 h after transfection with pKSV10. lGS.gpt vectors, the bands corresponding to the expected sizes of the transcripts (Figure 2). GS enzyme activity was also elevated in COS cells transfected with pKSVl0. lGS.gpt compared with untransfected controls (Table 4). The vector clone pKSVlO.lGS.gpt2. which gave the highest levels of GS enzyme activity in COS-1 cells, was subsequent used to transform PQXB1/2 cell-lines.

Plasmid pKSV 10. IGS .gpt2 was linearized with TthIII and then introduced into PQXB112 cells using electroporation. pEE6.gpt and DNA minus control transfections were also performed.

After 10 to 14 days, transformants selected with gpr-media gave 64 colonies per lo6 cells with pKSVlO.lGS.gpt2 and a similar frequency of 55 transformants per lo6 cells, with pEE6.gpt. However, no transformed colonies were obtained subsequent to selection in either G-DMEM or G-DMEM/gpt media. In these transformations, colony growth was initially observed in the wells, but proliferation ceased after 10 days. These results are similar to those found during attempts to transform PQXB1/2 hybridomas with pSV2.GS.

Transfection of PQXBlI2 cells with hCMV promoter-based vectors

15 days

It was concluded that the failure to obtain transformed hybridoma cells capable of growing in glutamine-free media was probably a consequence of the levels of GS expression attained, rather than the transformation and selection protocols; evidence for this came from our consistent success in obtaining transformed hybridomas when selecting for expression of the gpt gene. Vectors pCMGS and pCMGS.gpt,


Plasmid 10 days

Papers

a. M

123456 b. 1 2 3 4 5 6 c. 12 3456

Figure 2 Autoradiographs of Northern blots of COS-1 cell RNA following transfections with the stated vectors. (a) Probed with GS cDNA. (b) Probed with a gpt gene probe. (c) Probed with a @actin gene probe. All tracks contain 10 hg of RNA. Positions of RNA size markers, in kilobases, are in the M column. Track 1, COS-1 cell RNA. Track 2, COS-1 cells transformed with pKSV10. Track 3, CDS-1 cells transformed with pKSVlO.lGS.gpt. Track 4, COS-1 cell RNA incubated with DNAse. Track 5, COS-1 cells transformed with pKSV10, incubated with DNase. Track 6, COS-1 cells transformed with pKSVlO.lGS.gpt, incubated with DNase.

Table 4 Specific activities of glutamine synthetase in transformed COS-1 cells

Cell line

Mean GS activity (~mol/min/106 cells)

COS-1 (heat inactivated) 0.03 cos-1 0.09 COS-1 + pKSV10 0.08 COS-1 + pKSVlO.lGS.gptl 0.68 COS-1 + pKSVlO.lGS.gptP 0.74 COS-1 + pKSVlO.lGS.gpt3 0.39 COS-1 + pKSVlO.lGS.gpt4 0.44

which express GS from the stronger human cytomegalovirus immediate early promoter were therefore used20 (Figure 1). The plasmids were linearized with BumHI and PvuI, respectively, and then independently transformed into PQXB l/2 cells by electroporation. Control transfections were performed using pKSVlO.lGS.gpt2, pEE6.gpt, and no DNA.

After 14 days, PQXB l/2 cells electroporated with either pCMGS or pCMGS . gpt produced large numbers of clones that would grow in G-DMEM (Table 5). Using G-DMEM as the selective medium with pCMGS and pCMGS.gpt vectors in fact produced six to 20 times more transformants than were obtained using DMEM/gpt selective media on cells transfected with pCMGS.gpt and other vectors expressing gpt from the SV40 promoter. No glutamine- independent transformants were obtained using selection in G-DMEM on cells transfected with vectors expressing the GS-gene from the SV40 early promoter or from cells transfected with no DNA, which indicated that introduction of the CMV-GS expression cassette was required for growth without glutamine (Table 5).

These results provide further evidence that the SV40 early promoter is unable to transcribe the GS-gene in GS- transformed PQXB1/2 hybridomas at sufficient levels to enable glutamine-independent growth. The hCMV promoter though, was capable of expressing sufficient GS to enable growth in glutamine-free media. The transformation frequency when selecting for clones transformed with pCMGS . gpt was 1 O-fold higher if glutamine-free selection was used than when using gpt selection. The transformed glutamine-independent cell lines were recloned by limiting dilution, employing mouse spleen feeder cells. The pCMGS clones were expanded in G-DMEM, and the pCMGS.gpt clones in both G-DMEM and gpt-selective media. The pCMGS transformants were subsequently referred to as CMGS-PQXB cell-lines.

Analysis of CMGS-PQXB hybridornu cells

Molecular studies of CMGS-PQXB hybridoma cell lines were performed to confirm that their ability to grow in

Table 5 PQXBl/Z transformation results

Vector Selective

media Colonies/ lo6 cells

pCMGS pCMGS.gpt pCMGS.gpt pEEG.gpt pKSVl0.1 GS.gpt pKSVlO.lGS.gpt Mock Mock

G-DMEM 250 G-DMEM 250

gPt 20

gPt 42 G-DMEM 0

gPt 12

!JPt 0 G-DMEM 0



glutamine-free media was a consequence of transformation with the hCMV-GS transcription unit, and not a result of an alteration in the levels of expression of the endogenous gene.

Antibody secretion by glutamine-independent PQXBl/Z lines. To determine whether the pCMGS transformed hybridomas still secreted antibody, 20 pCMGS-transformed clones were screened for antibody production by ELISA. The clones were found to secrete varying levels of antibody, ranging from 2.5 to 25 p,g ml - * with six of the transformants not secreting detectable levels of antibody. Two CMGS-PQXB-transformed cell lines, C and E, producing parental levels of antibody (23.6 and 25.5 p.g ml- I, respectively) were chosen for assay of GS enzyme activity and nucleic acid hybridization studies,

Nucleic acid hybridization. DNA was extracted from both CMGS-PQXB C and E and untransformed parental PQXB1/2 hybridomas. Then, 10 p.g samples were digested with BgA and B&II and used for Southern hybridization. Using a GS-specific probe, DNA from cells transfected with pCMGS contained a 2.1-Kb band (Figure 3), which is the size predicted for the exogenous GS-gene in pCMGS after BgnlBgflI digestion. The GS probe also cross hybridized with the mouse endogenous GS-gene in PQXB1/2

123 456 7 M.

tG iS+

23.1 9.6 6.4

4.6

2.3 2.0

1.3

1.1

0.8

0.6

9.3

Figure 3 Autoradiograph of a Southern blot of DNA from transfected PQXBl/P cells probed with GS cDNA sequences. Track 1, pCMGS equivalent to 100 copies per cell. Track 2, pCMGS equivalent to 50 copies per cell. Track 3, pCMGS equivalent to 10 copies per cell. Track 4, CMGS-PQXBlR clone E. Track 5, CMGS- PQXBlI2 clone C. Track 6, PQXBlR. Track 7, Lambda HindIll and PhiX174 Haelll molecular weight markers. M, sizes of the molecular weight markers in kilobase pairs. Tracks 2,3, and 4 contain 10 r.r,g of DNA each. tGS, II, position of GS sequences from transfected DNA. DNAs in tracks 1 to 6 were digested with Bg/ll and Bg/l, which releases the GS cDNA.

cells, which was seen as a doublet of 6 and 5.5 Kb, and a lower doublet of 3.5 and 3 Kb. These bands served as an internal control for loading of the same amount of DNA on each track of the gel. By comparison with known amounts of vector DNA loaded in adjacent tracks, the average vector copy number in the CMGS-PQXB1/2 hybridomas was es- timated to be approximately one GS-gene copy per cell.

RNA was also extracted from both pCMGS-transformed hybridomas and from untransformed control PQXB l/2 cells, and 25-p.g samples were used to prepare Northern blots. RNA from SPG2 cells, a variant of SP2/0 selected for glutamine-independent growth that has an increased endogenous GS-gene copy number (Bell and Bebbington, unpub- lished) were included on the gel as “positive” controls. The GS-gene probe detected a band of approximately 2 Kb in the cells transformed with pCMGS and cross reacted with the endogenous GS-mRNA in the SPG2 control, but did not hybridize with RNA from the PQXB1/2 cell lines (Fig- ure 4).

The GS probe was subsequently removed from the filter, and the filter rehybridized with @actin, demonstrating that similar amounts of RNA had been loaded in each track. The additional band present in the RNA extracted from transformed cells, and absent from the RNA extracted from untransformed cells, was therefore a consequence of the transcription of the exogenous GS-gene from pCMGS.

Glutamine synthetase enzyme activity. GS enzyme activity was measured using a radiometric assay that demon- strated (Table 6) that GS-specific activity was greater in the two hybridomas transformed with the GS-gene construct than in untransformed parental PQXB l/2 cells.

a. M. b.

-2.8 -

_ 1.4,

Figure 4 Autoradiographs of Northern blots of RNA from the stated cells hybridized with (a) B-actin and fb) GS probes. All tracks contain 25 pg RNA unless otherwise stated. Track 1, 0.5 Pg pCMGS. Track 2, PQXBl/Z cells grown in RPM1 1540 + 2 mM glutamine. Track 3, PQXBl/Z cells grown in G-DMEM + 2 mM glutamine. Track 4, SPG2 cells grown in G-DMEM. Track 5, CMGS-PQXBl/P clone E grown in G-DMEM. GS + 0: the position of GS mRNA in CMGS-PQXBl/P clone E cell RNA. n : GS mRNA in SPG2 cells.


Papers

Table 6 Specific activity of glutamine synthetase in CMGS- PQXB clones C and E

Cell line

Mean GS activity (~mollmin/106 cells)

PQXBl/P (heat inactivated) 0.02 PQXBl/S 0.25 CMGS-PQXB clone C 0.57 CMGS-PQXB clone E 0.63

Discussion

Electroporation was found to be a simple, rapid, and repro- ducible means of effectively transforming the PQXB l/2 hybridoma with exogenous DNA. Transformation frequencies in the range lop5 to 10e4 were regularly achieved, depend- ing on the vector construction employed. These results are comparable with those obtained by others using lymphocytes, myeloma, and lymphoma ce11s.27,31.35,36

Attempts to transform the PQXBU2 hybridoma to glutamine independence by using the SV40 early promoter to drive GS expression were unsuccessful. The vector pSV2.GS, which expresses the GS cDNA from the SV40 early promoter, has been used to transform, at a low frequency, NSO myeloma cells to glutamine independence.*O However, similar selection regimens used with PQXB112 cells in the present study failed to produce clones that could survive in the absence of media glutamine. The hCMV promoter has been shown to be stronger than the SV40 early promoter in lymphoid cells3’ and expression of GS from this promoter enables transfected PQXB l/2 cells to produce sufficient GS to overcome the absence of this amino acid from the culture medium. The earlier failure of SV40 early promoter-driven GS expression to produce glutamine- independent PQXB l/2 cells is presumed to be due to insuf- ficient production of GS. However, the ability of the SV40 promoter to function in the PQXBU2 cells is shown by the successful introduction and expression of the gpt-gene under its control using the same electroporation conditions. This led to the selection of transformants using DMEMlgpt media containing mycophenolic acid and xanthine and dem- onstrated the effectiveness of both the transformation protocol and SV40-driven gpt expression in PQXB1/2 cells.

Glutamine-independent transfotmants were obtained using two vectors (pCMGS and pCMGS.gpt) that expressed the GS-gene from the hCMV promoter. Analysis of cells transformed by pCMGS showed that they probably contained a single copy of the transfected GS-gene (Figure 3). This copy number is consistent with other studies27.36,3x involving the transfection of heterologous genes into mammalian cells. GS mRNA of the size expected for transcripts from the transfected GS-gene was also detected (Figure #), and GS assays showed that levels of active GS enzyme were elevated in the transformed CMGS-PQXB cell lines (Tu- ble 6).

GS vector-based amplification of transfected genes has been performed in myeloma and nonlymphoid cells with the aim of producing large amounts of recombinant pro-

teins.*Os2’ In contrast, the work presented here with hybridoma cells involves making cells with a minimal change in GS-gene copy number (one additional constitutively expressed gene per cell) and ultimately with different levels of GS expression, so that the physiologic effects of small changes in GS expression on glutamine-related physiologic processes can be evaluated. We have not attempted to quan- tify the levels of mRNA production in cells transfected with the SV40 promoter-driven GS cDNA, which would enable us to determine the critical amount of transcription and translation needed to get glutamine independence. How- ever, the levels of GS mRNA and enzyme produced by hCMV-driven expression may well be near the limit for the production of sufficient glutamine for growth in the absence of this as a medium supplement, as shown by the slower growth of CMGSE-PQXB cells compared with the parents in l-l stirred flasks*’ (Bell et al., manuscript in preparation).

The levels of heterologous GS enzyme constitutively expressed in the two transformed PQXB l/2 cell lines producing parental levels of antibody were found to be equal to the highest levels seen only transiently during batch culture of the parental cells, and are increased by 128 and 152% compared with the PQXB1/2 parents grown in glutamine- containing medium. ‘,*I There is obviously scope for a further increase in GS expression by elevation of the GS-gene copy number via methionine sulphoximine amplification,r8 or the use of an even stronger promoter to see whether cells with parental growth rates can be restored. Further media supplementation may also be of value.

The ability of certain animal cell lines to grow in glutamine-free media has traditionally been correlated with the cells’ capacity for increasing GS expression when glutamine is removed from the culture medium. Kitoh ef ~1.~~ have shown that there is a good inverse correlation between glutamine dependence and activity of GS, with GS activity being induced in glutamine-independent cells in response to the removal of glutamine.

Jenkins et al,,22 however, have recently shown that in two cell types with similar GS synthetic capabilities, Mc- Coy and MDCK cells, glutamate transport may be a deci- sive factor in determining whether the cells can adapt to glutamine-free culture media. In our study, the elevation of GS levels alone produced cells which could grow in the absence of glutamine. Future strategies for manipulating cells to grow in glutamine-free media may therefore involve manipulation of either (or both) GS expression and glutamate transport systems. In addition to removing the need for glutamine, another aim of genetically manipulating glutamine synthetase expression was to see how this would affect the accumulation of ammonia in the culture media. Ammonia cannot be detected in glutamine-free culture medium containing the transformed cells when grown in batch culture2’ (Bell et al., manuscript in preparation).

Another important consideration when altering cellular metabolism by genetic engineering is whether the manipu- lated cells will be so stressed by the selection process or new growth conditions that product generation is affected. Although antibody levels of transfected glutamine- independent PQXB1/2 cells did vary, two clones with pa-



rental levels of antibody were isolated that could survive in glutamine-free medium, showing that the cells can cope with glutamine depletion when GS enzyme levels are modified within the cell.

In conclusion, we have investigated protocols for the successful transformation of the PQXB1/2 hybridoma to glutamine independence and shown the feasibility of at- tempting to manipulate mammalian cell physiology by genetic engineering. The physiologic manifestations and con- sequences of this interference in the control of GS expression, and its ramifications with regard to nitrogen metabolism in these cells, are now being analyzed in more detail.

Acknowledgments

This work was supported by an SERC cooperative grant with Celltech, Glaxo, ICI, SmithKline-Beecham, Porton International, and The Wellcome Foundation. The authors are grateful for the skilled technical assistance of N.K. Hig- bee and T. O’Shaughnessy.

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genetic engineering of hybridoma glutamine metabolism

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