Somatic Cell and Molecular Genetics, Vol. I0, No. 3, 1984, pp. 275-281

Variants Inducible for Glutamine Synthetase in V79-56 Cells

Morgan Harris

Department of Zoology, University of California, Berkeley, California 94720

Received 22 November 1983--Final 27 December 1983

Abstract--V79-56 cells have an absolute requirement for exogenous glutamine and are not inducible for glutamine synthetase. Prototrophs arise spontaneously at approximately 1.0 x 10 -5 per cell per generation as measured by fluctuation tests. Higher frequencies of glutamine-independent variants may be obtained by treatment with the mutagen ethyl methane sulfonate, as well as by exposure to 5-azacytidine and sodium butyrate, which act primarily by affecting gene expression. Variants of all types show marked elevation of glutamine synthetase activity. Although this activity declines toward constitutive levels in the presence of glutamine, it is still inducible in variant cells. These populations, after a lag, regain the ability for progressive growth in glutamine-free medium. Thus, the stable variation seen here is in the potential for induction, rather than steady-state expression of glutamine synthetase at a higher level.

INTRODUCTION

The early studies of Eagle and his collab- orators (1) showed that while mammalian cells can derive glutamine from glutamic acid, synthesis at the constitutive level is inade- quate for growth and proliferation except in dense populations. However, glutamine may be formed at higher levels when some lines are maintained without glutamine in nutrients containing glutamic acid and NHaC1. De- Mars (2) showed that glutamyltransferase or glutamine synthetase activity is induced in HeLa cells by this means, and similar increases have been reported with mouse L cells (3) and Chinese hamster cells (4).

Several investigators have isolated glu- tamine auxotrophs as sublines from inducible wild-type cells. Chu et al. (5) isolated gluta- mine-requiring variants from V79 Chinese hamsters cells by exposure to aminopterin in the absence of thymidine. Ctonal populations

of these cells reverted to glutamine prototro- phy at a rate of 1.4 • 10 -7 as measured by fluctuation tests. In a similar study, Var- shaver et al. (6) obtained glutamine-depen- dent variants by exposing V79 cells to BrdU- black light treatment (7). Most of the clones obtained were leaky auxotrophs, but one chosen for further study showed a stable glu- tamine dependence of 40~ In this population prototrophs arose spontaneously at a rate of a p p r o x i m a t e l y 10 -6 , and the frequency was enhanced 10x by MNNG treatment.

In the present experiments we have used an auxotroph derived without selection to examine transitions between glutamine de- pendence and independence in V79 cells. This subline, V79-56, has an absolute requirement for glutamine and is not inducible for gluta- mine synthetase even with elevated levels of glutamic acid and NH4C1 (4). However, pro- totrophs appear spontaneously in these popu- lations and can be produced in greater num-

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276 Harris

bers by exposure to EMS, a mutagen, as well as by treatment with 5-azacytidine and sodium butyrate, which appear to act pri- marily in eukaryotic cells by causing changes in gene expression (8, 9). Glutamine synthe- tase is inducible in all of these variants, and provides the basis for progressive growth in glutamine-free media.

MATERIALS AND METHODS

Culture Procedures. All experiments were performed with V79-56, a glutamine auxotroph obtained by random cloning with- out selection from wild-type populations. Stock cultures were maintained in 10% fetal calf serum, plus 90% Dulbecco's modification of Eagle's medium containing 4.5 mg/ml glu- cose with pyruvate omitted (10FCSDB). For selection of glutamine-independent variants, cells were plated into 10% dialyzed fetal calf serum plus 90% a-MEM medium with glu- tamine omitted (G-c~-MEM). Fluid changes were made every 5-7 days over a 3 to 4-week culture interval. When colonies were well- formed, experiments were terminated by staining the cultures for 30 rain in a saturated solution of crystal violet in 0.85% NaC1, after which the dishes were washed and air dried.

Fluctuation Tests. Each assay was ini- tiated with a single colony of V79-56 cells which, after 4 days of growth in 10FCSDB, contained about 200 cells. The colony was dissociated and plated in 10FCSDB to give subclone colonies, 15 of which were trans- ferred to prescription bottles and grown to mass populations. Aliquots from the 15 sub- lines were then assayed for glutamine-inde- pendent variants in G - a - M E M (six petri dish cutures at 100,000 cells per subline). Colony counts were evaluated for significance by a chi-square test, and reversion rates to gluta- mine independence were determined by the methods of Lea and Coulson (10) and Capizzi and Johnson (11).

Induction of Variants. In experiments with ethyl methane sulfonate (EMS), cells were exposed to graded concentrations of

mutagen in 10FCSDB for 24 h, and after a recovery period of 3 days, were plated out in G-a -MEM. In other studies, V79-56 cells were treated for 24 h with graded levels of 5-azacytidine (5-aza-CR) in 10 FCSDB. Two days later each population was assayed for colony formation in G a-MEM. In a third series of experiments, cells were grown for 4 days in 10 FCSDB containing graded concen- trations of sodium butyrate, after which the cultures were trypsinized and plated out directly in G-a -MEM.

Assays for Glutamine Synthetase. Glu- tamine synthetase activity in cell extracts was determined by measuring the conversion of labeled glutamate to glutamine (4, 12). Log- phase cultures containing about 30 x 1 0 6 cells were trypsinized, rinsed 1 • in Hanks' saline, and sedimented for 5 min at 500g. The pellet was resuspended in 1.6 ml glass-distilled water and frozen in a CO2-methanol bath. Immediately upon thawing, 0.12 ml of 75 mM dithiotreitol was added, together with 0.2 ml of a buffer containing 0.5 M Tricine, pH 7.5, 0.5 M KC1, 0.2 M MgCI2, and 1.0 mM EDTA. After centrifugation for 10 min at 20,000g, the supernatant was frozen for use within a few days. Protein determinations were made by the procedure of Bradford (13), using bovine plasma gamma globulin as a standard, and reagents obtained from BioRad Laboratories. All assays for glutamine synthe- tase were performed with [~4C]glutamate (Amersham, 280 mCi/mmol), using 1.0 uCi/ sample. A standard assay mixture of 100 ~zl contained 50 mM Tricine, pH 7.5, 20 mM MgClz, 15 mM ATP, 5 mM NH4C1, 2.0 mM dithiothreitol, and about 100 ug extract pro- tein. After incubation for 20 rain at 37~ the reaction was stopped by adding 250 #1 ice-cold 1.0 mM imidazole HC1, pH 7.0. To separate out [~4C]glutamine, 250 ul of the final reac- tion mix was added to a Pasteur pipet column (5 x 0.5 cm) filled with Dowex ! (BioRad AG1 x 8, 200-400 mesh), previously equili- brated with 2.5 ml 0.1 mM imidazole HC1, pH 7.0. The columns were then washed with 1.5 ml 0.1 mM imidazole-HCl and effluents

Variants Inducible for Glutamine Synthetase 277

collected in scinti l lat ion vials. Aquasol (15 ml /v ia l ) was added and the vials counted in a Beckman LS7000 liquid scinti l lat ion system.

R E S U L T S

Spontaneous Reversion Frequen- cies. Since g lu tamine- independent colonies appeared at low incidence in unt rea ted popu- lations of V79-56 cells, f luctuation tests were performed to measure the spontaneous ra te of reversion to g lu tamine prototrophy. Our cells, like other g lu tamine-dependen t lines (5, 6) are leaky auxotrophs. However, re l iable selec- tion was obta ined by avoiding high populat ion densi ty in petri dish cultures, and careful adherence to a schedule of fluid changes every 5-7 days over the 3 to 4-week assay period. Table 1 gives the results obta ined in three independent experiments . The t ransi t ion from g lu tamine dependence to independence is c lear ly spontaneous and random, with an overall ra te of approx imate ly 1 x 10 5 per cell per generat ion.

Production of Variants with Ethyl Methane Sulfonate. The frequency of glu- t amine- independent var iants in V79-56 popu- lations can be increased somewhat with the mutagen, EMS. Figure 1 shows a representa- tive exper iment in which stock populat ions

Table 1. Rate of Reversion to Glutamine Independence in V79-56 Cells

Fluctuation tests I II Ill

No. of sublines 15 15 15 Initial cell no. 1 1 1 Mean final cell no. • 106 1.6 41.4 36.8 Total cells tested per subline

x 106 0.6 0.6 0.6 Mean no. of glutamine-in-

dependent colonies per subline sample 27.2 88.1 64.0

Variance 1242 19,600 1101 Ratio of variance to mean 45.7 222.5 17.2

X z 685 3337 258 P <0.001 <0.001 <0.001

Reversion rate per cell per generation • 106

Lea and Coulson (10) 5.8 10.5 12.2 Capizzi and Johnson ( 11 ) 8.5 15.9 12.0 Average 7.2 13.2 12.1

were exposed for 24 h to graded concentra t ion of E M S in 10FCSDB. The cul tures were r insed and cont inued in 10FCSDB alone for a 6-day recovery period, with one subcul ture . Assays for g lu tamine- independent var iants were then set up in G - ~ - M E M , and plat ing efficiency for each populat ion was de te rmined in the same medium supplemented with glu- tamine. Survival ranged from 84.2% in control cul tures to 42.5% for those exposed to E M S at 500 # g / m l . Under these condtions, the inci- dence of g lu tamine- independent var iants per surviving cell rises 10-20• over the range of E M S concentrat ions used. The magn i tude observed is s imilar to increases previously repor ted for induction of g lu tamine proto- t rophs from V79 cells with another chemical mutagen, M N N G (6).

Treatment with 5-Azacytidine. In ear- l ier studies we showed that 5 - aza -CR can induce high-frequency change in several well- defined marke r systems. These include reap- pearance of thymid ine kinase act ivi ty in

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~ I0

~ ,;o 2~o 3~o ,;o 5;~ EMS,~g/ml

Fig. 1. Induction of glutamine-independent variants in V79-56 cells by ethyl methane sulfonate. Cultures were exposed to graded levels of EMS for 24 h, with a 6-day expression time in 10FCSDB. Assays are shown as colo- nies per surviving cell in G-a-MEM (6 petri dishes at 0.1 • 106 cells at each concentration shown).

278 Harris

enzyme-deficient Chinese hamster cells (14), reexpression of asparagine synthetase in Jensen rat sarcoma cells (15), and simulta- neous reactivation of pyrroline-5-carboxylate synthetase and ornithine aminotransferase activity in CHO-K1 cells (16). In each case, brief exposure to 5-aza-CR at optimal levels leads to increases of variant frequency of 100,000- to 1,000,000-fold in the treated pop- ulations, and variants isolated from these cul- tures are stable phenotypically in the absence of further selection. Similarly, treatment with 5-aza-CR raises the incidence of glutamine- independent variants in cultures of V79-56 cells. Figure 2 illustrates one of several experi- ments performed. The inductive effect is quantitatively less than observed after 5-aza- CR treatment in the systems mentioned above, but is well above the level of increase obtained with EMS in V79-56 cells (see pre- ceding section). Figure 2 also shows that population density can influence colony for- mation in G-~-MEM, with or without induc-

I000

>

~o 100

<

a~ 10.0

c l,( o 8

+ (D

0.1

200,000 cells

/ ~ 50,000 ce Is per culture

i

" o , o13 llo ~Jo ' ' I0.0 50.0

Azacy'ddine,)Jg/ml

Fig. 2. Production of glutamine-independent variants by 5-azacytidine in V79-56 cells. Cultures were treated with graded concentrations of 5-aza-CR for 24 h and con- tinued for a 2-day expression time in 10FCSDB. Assays were performed at two population density levels (6 petri dishes each at 50,000 and 200,000 cells in G c~-MEM, for each 5-aza-CR concentration shown).

tive treatment. Similar observations have been reported by others working with gluta- mine auxotrophs (5, 6). But while cell density may modulate variant frequency, it does not explain the inductive effects obtained with 5-aza-CR and other agents, since dose- response curves are in parallel at different population levels.

Exposure to Sodium Butyrate. Induc- tion of variants with sodium butyrate has a special significance, since butyrate is a normal constituent of animals cells and lacks muta- genic potential. On the other hand, gene expression can be altered by exposure to buty- rate (9, 17), and we have found it effective in reactivation of thymidine kinase in Chinese hamster cells (14). Our present experiments extend this to the production of glutamine prototrophs in V79-56 cells. Induction by butyrate is less rapid than with 5-aza-CR and shows a time-graded progression. Figure 3 gives the results of a typical experiment in which V79-56 cells were grown for 10 days in

I000

10(

o~

> >

I 0 0 %

o_

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o / / o15 ,Io 21o 4!o No butyrate, mM

Fig. 3. Effect of sodium butyrate on frequency of gluta- mine-independent variants in cultures of V79-56 cells. Populations were grown in graded levels of butyrate for 10 days, with one transfer, and were assayed directly by subculture to G-c~-MEM (6 petri dishes at 0.1 x 10 6 cells at each concentration shown).

Variants Inducible for Glutamine Synthetase 279

graded concentrat ions of butyrate . The cells were then t rypsinized and assayed di rec t ly in G-c~-MEM, without addi t ional expression t ime. The dose- response curve obta ined shows a concent ra t ion-dependent induction of glu- t amine- independent colonies, rising to about 200 • basal levels. Since the plat ing efficiency even af ter growth for 10 days in 4 m M buty- ra te was 64% tha t of populat ions in 10FCSDB alone, it is clear tha t differential survival does not account for the results obtained.

Phenotypic Stability of Variants. W e compared the propert ies of var iants derived from V79-56 af ter main tenance in the pres- ence or absence of L-glutamine, respectively. Table 2 summar izes da ta on plat ing eff• and levels of g lu tamine synthetase activity. Colony formation by V79-56 stock cells is high in G o~-MEM with 4 m M L-glutamine added, but falls to about 10 5 in unsupple- mented medium. Dependence of V79-56 cells on exogenous g lu tamine is associated with a

low, but measurable , const i tut ive level of glu- t amine synthetase activity.

G lu tamine- independen t var iants of all types show high p la t ing e f f ic iency and enhanced levels of g lu tamine synthetase activ- i ty when test populat ions are precul t iva ted in G c~-MEM. But when cells are grown for 2 days in the same nutr ient with added gluta- mine, levels of g lu tamine synthetase act ivi ty dropped sharply, along with the potent ia l for colony format ion in G oL-MEM alone. These observations show that g lu tamine indepen- dence is unstable phenotypica l ly unless cells a re main ta ined in a g lu tamine- f ree medium.

Al though g lu tamine synthetase act ivi ty in var iant cells is subject to end-produc t repression, it remains inducible. This allows repressed cells to resume growth, af ter a lag, in g lu tamine- f ree media , while the precursor V79-56 cells are unable to do so. This differ- ence can be i l lus t ra ted by 7-day growth tests in G-c~-MEM (Table 3). The da ta also show

Table 2. Phenotypic Stability of Glutamine-Independent Variants Derived from V79-56 Cells

Plating efficiencyb Glutamine synthetase

Inductive Maintenance activity c Cells agent medium" + G - G (cpm/~g protein)

V79-56 +G 101.0 2.3 • 10 -5 7.3 91.3 4.0 • 10 5 7.4

1832-71 Spontaneous - G 47.0 38.3 770 +G 46.3 36.7 50.6

1832-72 - G 47.0 40.7 795 +G 52.0 38.3 23.0

1838-5 EMS - G 87.0 75.0 468 +G 102.7 15.0 11.6

1838-6 G 58.7 34.3 695 +G 52.7 12.0 28.0

1755-1 5-Azacytidine - G 93.0 39.5 509 +G 104.0 3.2 9.3

1756-3 - G 88.7 91.3 777 +G 96.5 8.7 16.4

1759-2 Sodium - G 53.7 30.3 233 butyrate +G 66.0 8.5 11.3

1759-3 - G 58.3 36.3 381 +G 62.0 5.8 20.0

aPopulations of variants growing in G-a-MEM were trypsinized and sublines maintained for 2 days in the same nutrient with or without the addition of 4 mM L-glutamine (+G and - G media). Extracts were then prepared for assays of glutaminc synthetase activity, and plating efficiency tests were performed.

bPlating efficiencies were determined by inoculating cells from each subline into + G and - G media (6 petri dishes at 100 cells per series). Values shown represent average numbers of colonies formed per 100 cells plated.

CFigures shown represent average values for four determinations.

280 Harris

tha t var iants arising from t r ea tmen t with EMS, 5 -aza -CR, or bu ty ra te do not in genera l possess a growth advan tage as compared to those appear ing spontaneously. In all var iants , the charac ter i s t ic conferred as a s table change is induc ib i l i ty for g l u t a m i n e syn the tase , ra ther than s teady-s ta te expression of enzy- mat ic act ivi ty at a higher level.

D I S C U S S I O N

Fluctua t ion tests show tha t V79-56 cells, which are auxotrophic for g lutamine, can convert by a random and spontaneous process to the protot rophic state. However , the na ture of the stochast ic events which take place is not clear. Still higher frequencies of reversion are seen af ter t r ea tment with EMS, a known mutagen, as well as by exposure to 5 - aza -CR or sodium bu ty ra te which act p r imar i ly if not exclusively by al ter ing gene expression. These var iants appear to be s imilar phenotypical ly , and thus the question is whether conversion

proceeds by para l le l events or by a common mechanism. Gene muta t ion has been proposed as the basis for reversion of g lu tamine auxo- trophs in ear l ier studies (5, 6) and could account for the effects of E M S in the present exper iments as well. But it is more difficult to extend this concept to other inductive agents, especial ly butyra te , for which any indicat ion of mutagenic potent ial is lacking.

Al ternat ively , reversions to g lu tamine independence may be t r iggered by D N A demethyla t ion at sites regula t ing the induc- ibil i ty of g lu tamine synthetase, a model which accords well with the act ion of 5 -aza -CR in the present invest igat ion. The abi l i ty of 5 -aza -CR to cause hypomethyla t ion in newly formed D N A is well known (18), and reduc- tion in methyla t ion at specific sites following exposure to 5 -aza -CR has been correla ted, for example, with reappearance of herpes thymi- dine kinase act ivi ty after repression in mouse cells (19). Exper iments on the metal lothi- onein-1 (MT-1) gene by Compere and Pal-

Table 3. Growth of V79-56 and Variant Sublines in G a-MEM after Maintenance in Presence and Absence of L-Glutamine

Terminal cell counts • 106 Maintenance per culture after 7-day growth Relative

Cells Origin medium a in G a-MEM b increase

V79-56 +G 0.040 0.0 0.037 0.0

1832-71 Spontaneous G 7.6 76X +G 7.7 77X

1832-72 - G 7.4 74X +G 5.4 54X

1838-5 EMS - G 10.0 100X +G 3.6 36X

1838-6 - G 6.1 61X + G 2.0 20X

1755-1 5-Azacytidine - G 5.1 51X +G 1.0 10X

1756-3 - G 7.2 72X +G 4.6 46x

1759-2 Sodium - G 3.7 37X butyrate + G 1.8 18X

1759-3 G 4.7 47X +G 2.3 23X

~ preparation for growth tests, variants growing in G-a-MEM were trypsinized and mass populations maintained for 4 days in the same nutrient with or without the addition of 4 mM L-glutamine (+G and - G media).

bGrowth tests were performed by inoculating cells from each subline into 3-oz prescription bottles at 0.1 • 1 0 6 in G a-MEM (no added glutamine). After incubation for 7 days, each culture was trypsinized and cell number determined with a Coulter counter. All tests were performed in duplicate.

Variants Inducible for Glutamine Synthetase 281

miter (20) in W7 mouse thymoma cells are of special significance for our current study. The W7 cells do not express the MT-I gene i n the presence of cadmium or glucocorticords, unlike most other lines. But on treatment of W7 cells for a few hours with 5-aza-CR, the MT-1 gene becomes inducible with these agents. Coincidentally, the Hpa H sites within the MT-1 gene and flanking sequences were found to be unmethylated, in contrast to the methylated condition of these sites in parent cells. In this context, the action of 5-aza-CR on V79-56 cells is most reasonably explained by assuming that methylation changes occur which permit the induction of glutamine synthetase to take place.

While the relevance of DNA methyl- ation changes to induction of glutamine proto- trophs by butyrate and EMS is not obvious, a possible association cannot be excluded. In Friend erythroleukemic mouse cells, the effects of butyrate on gene expression are correlated with shifts in acetylation of nuclear histones (21, 22), but DNA demethylation by butyrate occurs in the same cells as well (23). Similarly, while EMS is a well-known muta- gen, it also causes generalized damage to DNA which conceivably may result in hypomethylation at specific sites in recovery cells. Evidence for this concept can be found in a recent study (24) in which methylation of deoxycytidine incorporated by DNA excision repair was studied in human fibroblasts, fol- lowing damage by UV and other agents. Methylation in repair patches was slow and incomplete, suggesting that DNA damage and repair may lead to a heritable loss of methylation at some sites in the surviving cells. In other experiments, Wilson and Jones (25) have shown directly that EMS and a variety of alkylating agents can inhibit DNA methylation reactions in vitro. This adds fur- ther support to the hypothesis that EMS, 5-aza-CR, and butyrate are acting here by a similar mechanism and suggests new possibili- ties for future study.

ACKNOWLEDGMENTS

I thank Patrick Link and Janet Conley for excellent technical assistance. This work was supported by Public Health Service grant CA 12130 from the National Cancer Insti- tute.

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