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The isolation and characterization of asparagine-requiring mutants of Chinese hamster cells
Primed in Sweden Copyright @ 1977 by Academic Press, Inc. All rights of reproduction in anyform resewed
ISSN00144827
Experimental
THE ISOLATION
Cell Research 104 (1977) 357-367
AND CHARACTERIZATION
ASPARAGINE-REQUIRING
MUTANTS
CHINESE
CELLS
HAMSTER
P. S. G. GOLDFARB, B. CARRITT,
OF OF
M. L. HOOPER and C. SLACK
Cancer Research Campaign Somatic Cell Genetics Group, Institute of Genetics, University of Glasgow, Glasgow, Scotland
SUMMARY Nine asparagine-requiring mutants were isolated in culture from the Don line of Chinese hamster cells. Investigation of the asparagine requirements of the mutants, the effect of asparagine deprivation on macromolecular synthesis, and the rates of reversion to asparagine independence indicated that there were differences between the mutant clones. Biochemical analysis revealed that the defect in the mutants was due to a deficiency of the enzyme asparagine synthetase, and that the enzyme activity in the mutants and Asn+ revertants obtained from them was not influenced by the concentration of asparagine in the growth medium. Complementation analysis by S&&i virusmediated cell fusion indicated that the lesion behaved as a recessive trait, and was probably located in the same gene in all the mutant clones.
The nutritional requirement of certain tu- contain increased levels of asparagine synmour-derived malignant cell lines for L- thetase [6,7,8, 161appear at low frequency asparagine has been well documented [ 141. in the population and are derived from The inability of such cell lines to synthesise mutational events, rather than as a response asparagine [5] has in all cases been shown to the absence of asparagine [17-211. In to be due to the absence of, or low level of, some instances [14,22] the level of enzyme asparagine synthetase (EC6.3.1.1) in cells activity found in the Asn+ cell lines and passaged both in vitro and as tumours in tumours can be increased by removal of vivo [6-8]. The sensitivity of these aspara- asparagine, suggesting that a regulatory gine-requiring (Asn-) cell lines and tumours system may be present. To date there has been no report of the to L-asparaginase (EC3.5.1.1) from either E. coli or guinea pig serum [9, 10, 111has isolation in cell culture of Asn- cells from formed the basis of chemotherapeutic ap- an Asn+ cell line. The possession of such proaches using asparaginase [12-U]. One cell lines would be of interest not only for Gndrance to these approaches has been the use in the biochemical and genetical anaappearance, when both Asn- tumours and lysis of the regulatory processes involved in 4sn- cell lines are treated with aspara- the expression of asparagine synthetase cinase, of cells which no longer require activity, but also to investigate the sugrsparagine (Asn+ cells). These cells which gested [18, 191 correlation between maExp
Cell
Res
104 (1977)
358
Goldfarb
et al.
lignancy and the appearance of an asparagine requirement in cell lines. In this paper we describe the isolation of Asn- cell lines from an Asn+ line of diploid Chinese hamster cells (Don), and the subsequent derivation from the Asn- lines of Asn+ revertants. Biochemical analysis of the Asnlines and their Asn+ revertants suggests that the observed defect in the Asn- lines is due to a deficiency of asparagine synthetase activity. Complementation analysis suggests that the lesion is probably located in the same gene in all the Asn- clones. MATERIALS
AND METHODS
Chemicals and radiochemicals Ethylmethanesulphonate, 5fluorodeoxyuridine (FUdR), Na*ATP, and L-asparaginase were obtained from Sigma. Aminopterin was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Colcemid was supplied by Ciba Laboratories, Horsham, Sussex. UV-inactivated Sendai virus was obtained from Searle Diagnostic Ltd, High Wycombe, Bucks. [6-3H]thymidine 20 Cilmmole, [5 ,6-3H]uridine 40 Ci/ mmole, Lj4,5-3H]leucine 40 Cilmmole, and L#J-“‘Claspartic acid 200 mCi/mmole were obtained from the Radiochemical Centre, Amersham, Bucks.
Cells and cell culture The Don line of Chinese hamster lung fibroblasts was obtained from the American Type Culture Collection, Rockville, Md, as was the Jensen Sarcoma (JS) line of rat cells. The Don TK- derivative a23 was isolated by Dr A. Westerveld and kindly supplied by him. Cells were normally grown in 90 cm* plastic tissue culture flasks (A/S Nunc) in Eagle’s medium (Glasgow modifica~on) contaiining 10% v/v foetal calf serum (GJBCO-Biocult) under an atmosphere of 5% CO, in air, at 37°C. For growth at low cell density (<50 cells/ cm%)and for the routine growth of Asn- cell lines, the medium was supplemented with non-essential amino acids (alanine, aspartic acid, asparagine, glutamic acid, glycine each 0.1 mM; serine, proline each 0.2 mM), nucleosides (adenosine, guanosine, cytidine, uridine, each 30 PM; thymidine, 10 fiM), and 1 r&l sodium pyruvate. Cloning efficiencies of 30-50% were achieved in this medium. For asparagine-free medium the non-essential amino acid supplement was omitted, and replaced by L-aspamginase 0.02 U/ml. Cells were passaged twice weekly by trypsinization using 0.05 % trypsin in Ca-Mg-free isotonic phosphatebuffered saline (PBS) containing 0.6 mM EDTA. After neutralization with serum-containing medium, washing and resuspension in growth medium, 10% of the cells were reseeded into plastic flasks. Exp Cell Res 104 (1977)
Regular examination of cells for mvcoolasma contamination was performed by the method of Fogh & Fogh [23]. No evidence of such contamination was found during this study.
Recloning
of cell lines
Log-phase cells were trypsinized, washed and resuspended in complete supplemented medium at a density of 5 cells/ml. 0.2 ml aliquots were dispensed into each of 96 plastic micro-wells (Linbro) and the wells incubated overnight at 37°C. On inspection, wells containing more than one cell were discarded, and incubation continued for a further 7-10 days. At the end of this period the wells were inspected and cells removed by trypsinization from those containing a single large colony. Each clone was then oassaged to a population size of 5 x 10’ cells prior to storage in liquid N2 in supplemented medium containing 10% dimethylsulphoxide.
Selection of Asn- cells A confluent monolayer of 2X 10’ Don cells was mutagenized with 4 mM ethylmethanesulphonate in complete supplemented medium for 4 h at 37°C (10% survival). After mutagenesis the cells were trypsinized, resuspended in complete supplemented medium, seeded into four replicate 90 cm* plastic flasks and allowed to grow for 2 days. At the end of this period the medium was changed to asparagine-free medium and incubation continued for 24 h. The medium was removed and replaced by asparagine-free medium lacking thymidine which contained FUdR 25 pg/ml and uridine 125pg/ml. After a further 48 h incubation, the medium was replaced by complete supplemented medium. This was renewed at 3 day intervals. Cell death and detachment was apparent after 2-3 days and surviving colonies were visible after 14 days. The colonies were trypsinized and taken en masse for further rounds of selection, care being taken to ensure that survivors from the four parallel selections were kept separate. In all, three rounds of selection were employed. At the end of the 3rd round of selection 2-3 morphologically distinct colonies were picked from each of the 4 bottles, grown to a population size of 5x 10’ cells, and stored in liquid Nr.
Incorporation of radioactive precursors into macromolecules of Asn- cells In experiments to measure the rate of shutdown of macromolecular synthesis in Asn- cells deprived of asparagine, cells were seeded on to sterile Deconwashed 13‘mm dia. coverslips in plastic wells (Linbro) at 1X 10’ cells/well in complete supplemented medium. After 24 h at 37°C the medium was replaced with asparagine-free medium lacking nucleosides, and carrier-free [3H]thymidine (TdR), [3Hjmidine, or [3H]leucine added to 5 &i/ml. At appropriate intervals coverslips were removed for extraction which trichloroacetic acid (TCA) and scintillation counting as
Asparagine described previously [24]. In experiments to measure the rate of switch-on of macromolecular synthesis in starved Asti cells to which asparagine had been restored, cells were seeded as above. After 24 h the medium was changed to asparagine-free medium and incubation continued for a further 48 h. The medium was replaced with complete medium lacking nucleosides, and [3H]TdR, [3H]uridine or [3H]leucine added as above. Coverslips were removed for TCA extraction and scintillation counting [24]. In both ‘switch-off and ‘switch-on’ experiments a parallel series of coverslips was taken for estimation of total protein.
Karyology Metaphase spreads were prepared from colcemidtreated cells, and stained with aceto-orcein or Gbanded as described previously [24].
Preparation
of cell extracts
Extracts were prepared from late log phase cells grown in complete supplemented medium in 180 cm2 plastic flasks (A/S Nunc). Typically 2x10’ cells were trypsinized into 10 ml of medium and pelleted at 200 g for 5 min. After resuspension and gentle washing in 10 ml ice-cold PBS, cells were recentrifuged at 200 g for 5 mitt, and the pellet resuspended in 1 ml of 10 mM Tris-HC1+0.5 mM /3-mercaptoethanol pH 7.4 at 2°C. The suspension was sonicated using an MSE-Mullard Sonicator for two bursts of 10 set with 1 min cooling between. The sonicate was either assayed directly for enzyme activity, or divided into 100 ~1 aliquots for storage at -70°C. No reduction in enzyme activity was found after one cycle of freeze thawing, even in extracts which had been stored for up to 3 months.
Determination concentration
of protein
Determinations were performed on duplicate samples containing 10-100 pg protein using the Miller [25] modification of the method of Lowry et al. [26]. In the case of cells attached to coverslips, three washes with isotonic saline at 4°C were employed to remove the serum component of the growth medium, and reactions performed directly on the attached cells using l/S vol.
auxotrophs
359
of each in 10 ~1 HzO) added to tubes 1 and 2. Tube 3 was heated to 100°C for 2 min to inactivate the enzyme, cooled to 37°C and 0.01 U of aspamginasein 5 ~1 H,O added. After 15 min incubation at 37°C the reaction was terminated by heating to 100°Cfor 2 mitt, the tube placed in ice, and carrier aspartic acid and asparagine added as above. Separation of [Wlasparagine from [Ylaspartic acid was performed by the electrophoretic method of Horovitz et al. [6]. The amount of radioactivity co-electrophoresing with cartier asparagine was determined and the value obtained for tube 3 subtracted from the mean of the values for tubes 1 and 2. The resulting difference was used to calculate the enzyme activity. The asparaginase-resistant counts represented approx. 0.2% of the input radioactivity.
Complementation cell fusion
analysis by
2x iOBcells of each parental cell type were seeded into a 20 cm* plastic Petri dish (Falcon) in complete supplemented medium to form a confluent monolayer and incubated for 18 h at 37°C. The medium was removed, the cells washed with medium lacking serum, and 1 ml Hanks’ balanced salts solution containing 500 haemagglutinating units of UV-inactivated Sendai virus added. After 30 min adsorption at 4”C, 1 ml of medium conmining serum was added and incubation continued for a further 14 h at 37°C. The cells were washed twice with asparagine-free medium and trypsinized into 10 ml of the same medium. After centrifugation at 200 g for 5 min the cells were resuspended and seeded into a 150cm* plastic flask (Corning). In the case of fusions between Asti parents the medium for resuspension and selection of complementing hybrids was asparagine-free medium, whereas for fusions between Don a23 and Asn- lines the resuspension and selection medium was asparagine-free medium (lacking nucleosides) to which 0.1 mM hypoxanthine, 0.8 PM aminopterin, 3 I.LM glycine, and 20 PM TdR (HAT) had been added. Incubation was continued for 14days with medium changes every 3 days and surviving colonies picked or stained with Leishman stain (1.5 g/I in absolute methanol). Controls to which no Sendai virus was added were performed in parallel.
RESULTS Assay of asparagine synthetase activity Asparagine synthetase assays were performed in triplicate in plastic reaction tubes (Eppendorf) at 37°C in a final volume of 50 ~1. Each reaction contained 5 pmoles Tris-HCl (pH 8 at 37°C). 0.4 pmoles MgClp, 0.4 pmoles Na,ATP, 1 pmole L-glutamine, 0.1 Pmole L-[U-W]aspartic acid (spec. act. 2 &i//Lmole), and 100-500 pg protein cell extract. After 5 min equilibration at 37°C reactions were started by the addition of cell extract (usually 25 ~1). After 15 min incubation, reactions were terminated by placing the tubes in ice, and carrier aspartic acid and asparagine (0.1 pmoles 24-761813
Isolation
of Asn- clones
The frequency with which surviving colonies appeared following one round of selection was 1x 10e4.However, analysis of a representative sample of these colonies indicated that
360
Goldfarb
et al.
Don
,x,3
lull
b
- - - - - - - - 13L/lO
b A___-13u30
13L/3e13LIJ
lL----12
a
A
1 1W3eR,
t
3
L
104hL
time (days); ordinate: cell no. O-O, No asparagine; O-0, 1O-5 M; A-A, 2x1O-5 M; Cm, 5x lo+ M. (a) 134/3; (b) 134/7. Effect of asparagine on the growth of Asn- clones 134/3 and 134/7 in mass culture. IX 105 cells were seeded on to replicate 20 cm* plastic Petri dishes in asparagine-free medium. After attachment and spreading, the medium was changed to that containing the indicated concentrations of asparagine. Each day cells from one dish at each concentration were removed by trypsinisation, diluted, and counted using a Coulter Counter (Coulter Electronics Ltd, St Albans).
Fig. 2. Abscissa:
13u3efI,
Fig. 1. Genealogy of (a) Asn+ revertants and (b) Asn-
subclones of Asn- clone 13413.
After a total of three rounds of selection the survival rate had increased to 1X 10d3,indicating that although the selection had enriched for Asn- colonies, there was either a continued leak through of Asn+ cells or a low survival of Asn- cells in the selection system. Reconstruction experiments in grew vigorously in mass culture and could which a small number of Asn- cells were be passaged using 10% splits. mixed with Don cells and put through the selection indicated that the survival of Asn- Requirement of Asn- clones for asparagine cells in the system is about 1%. A total of ten colonies were picked fol- The Asn- clones showed an absolute relowing three rounds of selection and on quirement for asparagine both for maximum initial screening were found to require as- growth rate at high cell density (5 X 103cells/ paragine for growth. One clone 134/2 had a cmz) and for colony formation at low cell density (60 cells/cm2). This requirement low cloning efficiency, and was eventually lost. The nine remaining Asn- clones (fig. 1) could not be satisfied by any other nonTable 1. Fluctuation
analysis
of the Asn- to Asn+ reversion
in asparagine
auxotroph
clones
Clone 134/l 3 4 5 6 7 8 9 10
Total cells/ No. of subclones subclone 8 9 7 8 6 7 8 6
2x 108 4x 10’ 2x106 2x 106 4x 10’ 4x 10’ 2x 106 2Xlog 2x 106
Cells seeded/ replicate dish 1x10”
5x105 1x10s 1x105 1x1@ 1x106 1x105 1x10s 1x1@
Average no. of reversions/ subcloneD
Reversions/ cell/ generation”
1.3f0.7 115. +20 1.3kO.8 co.3 3.3f 1.2 3.9+ 1.6 0.8f0.5 co.3 ll.Of3.4
4.5+2.3x 2.0*0.3x 4.6*2.6x <1x10-’ 5.7+2.1x 6.7f2.7~ 2.7f 1.8x <1x10-’ 3.8f I .2x
a Calculated by the median method of Lea & Coulson [28], and expressed as estimate +S.E. Exp CeNRes 104 (1977)
lo-’ 1O-6 lo-’ 1O-8 1OV lo-’ lo-@
Asparagine L.lOS
0
b I
2.105
L
/
361
auxotrophs
the response to asparagine-free medium varied from clone to clone, in some instances cell detachment occurring after 2-3 days, whilst in others the cells remained viable for up to 5 days.
x---'
Reversion of Asn- cells to Am+
04 / 0
12
2L
Fig. 3. Abscissa: time (hours); ordinate: (u-c) cpm; (d) Kg protein/well. O-O, Don; O-O, 134/7a.
Effect of removal of asparagine on incorporation of (a) rH]TdR; (b) rH]uridine; (c) [3Hjleucine into TCAprecipitable material; and on (d) total cell protein in Asn- subclone 134/7a.
essential amino acid either alone or in combination. However, at high cell density some growth (at a rate of about 20 % of that in the presence of asparagine) was observed in some instances if all the non-essential amino acids other than asparagine were supplied. It is not known whether this was a real sparing effect, or if the non-essential amino acids maintained the cells in a viable condition long enough for asparagine to be formed by serum proteolysis. Because of this effect, all the non-essential amino acids were routinely omitted from asparagine-free medium. In the absence of other non-essential amino acids a relationship between the amount of growth at high cell density and the concentration of added asparagine could be demonstrated. As illustrated in fig. 2 for clones 134/3 and 134/7, the growth response to added asparagine differed reproducibly from clone to clone. It was also found that
When Asn- cells at a density of 5~ 103cells/ cm2 were maintained in asparagine-free medium for 14 days, colonies which did not require asparagine for growth appeared at low frequency in the cultures. Since the appearance of these Asn+ colonies could have been due either to reversion of the Asncells at low frequency, or to the persistence of wild-type Don cells in the culture, the Asn- clones were recloned by dilution and 6-9 subclones derived from each. Cells from each subclone were then seeded at early passage into five replicate 20 cm2 plastic Petri dishes in asparagine-free medium, and the appearance of Asn+ colonies 2.105 I
a
L:
0
0
lo5
7
/*
/
k
.-
1
2.105
L
b
pi. 0' i 0' ,*
2.103
.
0
MO
/
MO
0 _(-
L.105
p:,‘ "
/.
0
0
12 0
0
‘
0
1
0
12
c 0
- 10'
2.10' / */O L0
/"
L103
(..-J. 0 L
0
Fig. 4. Abscissa:
\~\;/protein/well.
12
0
L
time (hours); ordinare: (a-c) cpm; (L&f) O-O, Don; (right) O-0,
Effek of restoring asparagine to starved cells on incorporation of (a) rH]TdR; (b) rH]uridine; (c) [3H]leucine into TCA-precipitable material; and on (d) total cell protein. Exp Cell Res 104 (1977)
362
Goldfarb et al.
2513u3e
‘3‘k
lYi6b 23
scored after 7-10 days. The results were subjected to fluctuation analysis [27, 281 and in all cases the between-subclone variance was greater than the within-subclone variance, indicating that the Asn- to Asn+ reversion was a process randomly distributed in time generating a stably inherited phenotype, rather than a response to medium lacking asparagine. As shown in table 1, a low but finite reversion rate was observed in most of the clones. This rate could have accounted for the presence of Asn+ cells in stocks of the Asn- clones, although the possibility that they were surviving parental Don cells is not ruled out. Table 1 also shows that the reversion rates differed between Asn- clones over a 102-fold range. In order that further characterisation of the Asn- clones would not be confused by the presence of Asn+ cells, all subsequent work was performed on recloned material at early passage, one subclone from each clone being used for this. For comparative purposes revertant Asn+ clones were isolated from certain Asn- subclones by seeding 5 x 106cells in duplicate into asparaginefree medium in 180 cm2 plastic bottles. After 14 days one revertant colony was picked from each bottle and grown to a population size of 5X 10’ cells prior to storage in liquid NZ. Exp Cell Res IO4 (1977)
13*r ‘Ci 22
Effect of asparagine on incorporation of radioactive precursors
As shown in fig. 3 the withdrawal of asparagine from the growth medium of Asnclones, as demonstrated by subclone 134/ 7a, resulted in a decrease in the incorporation of precursors into DNA, RNA and protein. Although there appeared to be a rapid decrease in the rate of incorporation of all three precursors there were marked differences in the length of time which elapsed prior to complete inhibition of incorporation. Whereas incorporation of rH}TdR into DNA ceased after 8 h, incorporation of r3H]uridine into RNA was not inhibited until after 12 h. Incorporation of rH]leucine into TCA-precipitable material also did not appear to be shut off until more than 12 h had elapsed. The results obtained with other Asn- subclones were in agreement with those for subclone 134/7a except that variations of 2-3 h were observed in the time at which [3H]TdR incorporation ceased. It is apparent therefore that asparagine deprivation in the Asn- cells leads directly or indirectly to a rapid inhibition of DNA synthesis, which precedes any fall in the rates of uridine or leucine incorporation or total protein synthesis. This decrease in the rate of DNA synthesis cannot therefore be a result of an effect on protein synthesis
Asparagine
Fig. 6. Trypsin-Giemsa banded chromosomes from
Asn- subclones and Asn+ revertants derived from
unless asparagine deprivation in Asn- cells leads initially to the depletion of a specific sub-set of cellular proteins. The effect of restoring asparagine to starved Asn- cells is demonstrated for subclone 134/7a in fig. 4. As can be seen there is a lag period before rH]TdR incorporation commences. This lag is not seen in the case of [3H]uridine or rH]leucine incorporation. It is also of note that there is a large increase in the total protein of cultures of Asn- cells following restoration of as-
auxotrophs
363
them. (a) 134/1Oband 134/10bR,; (b) 134/7a and 134/ 7aR,.
paragine, suggesting that not only is there increase in cell number, but also in the amount of protein per cell. An increase in cell size is also observed during this period. Karyotypic
analysis
Twenty-five aceto-orcein or Giemsastained metaphases of each subclone were examined and the chromosome numbers determined. In all cases the modal number was diploid or close to diploid, no gross structural rearrangements being observed. Exp Cell Res 104 (1977)
364
Goldfarb et al.
Table 2. Asparagine synthetase activities of the Asn- subclones and their revertants
Clone Asn134/lc 3e 3f 4b
5a 6b 7a 8a 9a lob Asn+ 134/3eRl 6bRl 7aRl 1ObRl Don LMTK-
Asparagine synthetase activity (nmoles/mg/h)
2.8kO.3
(5)
1.4f0.5 (4) 1.7kO.3 (6) co.2 3.4k1.2
(4) (3)
5.1kO.9 (4) 2.3kO.3 2.4kO.7
(3) (4) 1.9kO.8 (3j 3.2f0.4 (2)
13.ork1.3 13.4f1.8
(4) (2)
13.9k2.0 11.5f0.4 18.1kl.l 36.2f2.1
(3) (2) (11) (3)
% Don 15.5 7.7 9.4
Cl.0 18.8 28.2 12.7 13.3
10.5 17.7 71.8 74.0 76.7 63.5
-
Activity is expressed as nmoles asparagine produced/ mg protein/hfS.E.M. Figures in parentheses indicate the number of determinations made using at least two extracts from cells of different passage numbers, each determination being the mean of two assays.
Asparagine synthetase activity of the Asn- subclones and their revertants
As shown in table 2 the asparagine synthetase activity in extracts of the Asn- subclones was markedly reduced compared with the parent Don cells. In one instance (subclone 134/4b) enzyme activity was never detected. It was not possible, however, to determine whether this was due to a complete lack of functional enzyme protein, or whether the residual activity was more unstable than in other subclones. Experiments in which extracts from Don cells and subclone 134/7a were mixed prior to assay showed that the deficiency of enzyme in the Asn- subclones was not due to the presence of a diffusible inhibitor. In order to investigate whether the levels of asparagine synthetase were regulated by the external asparagine concentration, as in the case of Asn+ revertants of JS cells [14, 221, extracts were made from cells which had been starved of asparagine. It was found that neither in Don cells nor in any Asn- subclone was enzyme activity increased by pretreatment for 48 h with asparagine-free medium. Also shown in table 2 is the enzyme activity found in extracts of four revertant clones. As can be seen these have signiticantly higher enzyme activity than their Asn- parents, although in no case does the activity equal that of Don cells. This activity would appear to be the maximum level expressed in the revertant clones, as prior growth for one passage in asparaginefree medium did not lead to any significant increase in enzyme activity.
As shown in fig. 5 the modal chromosome numbers of the Am+ revertant clones were also close to diploid and resembled that of their Asn- parent subclone. Examination of trypsin-Giemsa banded preparations indicated that there was no evidence of translocation having occurred within the karyotype. Fig. 6a shows a G-banded preparation of subclone 134/10b and a revertant 134/ 10bRl derived from it. No differences were observed between the Asn- and Asn+ lines, both showing a banding pattern identical to that of the Don line [24, 291. In the case of subclone 134/7a (fig. 6b), the deviation from diploidy is due to an extra small metacentric chromosome. The origin of this extra chromosome, which is also present in Complementation analysis a derived Asn+ revertant 134/7aR,, is not In order to determine whether the defect in the Asn- clones behaved as a dominant or known. EXP Cell RPS 104 (1977)
Asparagine
Table 3. Complementation
analysis
Fusion of Don a23x 134/7a and selection of complementing hybrids in asparagine-free medium+HAT
occur in intracistronic
auxotrophs
365
complementation
1301.
Cells of subclone 134/7a were also fused with a recloned Asn- line derived from rat Culture No. of JS cells, and colonies surviving in asparano. a23 cells gine-free medium scored. The results indi1 1x10s 1x10s + 340,243, 272 cated that intercistronic complementation 2 1x106 1x106 16, 47, 30 had not occurred, and that the defect in 3 1x1@ + 0, 0, 0 134/7a was in the same gene as that in the 4 1x106 0, 0, 0 5 ix106 + 1, 0, 0 JS cells. This conclusion is drawn on the as6 1 1XlW 0, 0, 0 sumption that the fusion index between Data shown are the results of three replicate fusion Don Asn- and JS cells is similar to that beexperiments. tween Don Asn- and Don a23, and that there is sufficient retention of rat chromorecessive trait, cells of subclone 134/7a somes under selective conditions to allow were fused with those of the Don TK- line the detection of complementation. a23, and the number of colonies surviving a selective system toxic to both parents DISCUSSION scored. The frequency of hybrid formation using this method has not been established, In this paper we have demonstrated that by but by comparison with other fusions using using a modification of the method of Kao Don a23 (our unpublished results) the ex- & Puck [31] it is possible to isolate asparapected frequency would be < 1x 10T3. As gine auxotrophs from a population of wildshown in table 3 the frequency of survivors type cells. The apparently high selectivity from the fusion was significantly greater of the system for Asn- mutants is probably than that in controls from which Sendai due to two factors. (1) The FUdR treatment virus or one of the parents had been used was highly toxic to growing cells. (2) omitted. This indicates that complementa- The selection was performed in undialysed tion of the Asn- defect by the Asn+ parent serum, the concentrations of non-essential had occurred, and that Asri behaves as a amino acids in this probably being suffirecessive trait. cient to allow continued growth of any When cells from the various Asn- sub- other auxotrophic mutants. Presumably, in clones were fused with each other in two- the case of asparagine, the concentration parent crosses and colonies surviving in was low enough (due to the presence of asparagine-free medium scored, no increase added asparaginase) to cause complete inabove controls was observed. Assuming hibition of DNA synthesis in a proportion that the fusion index was the same in these of the Asn- cells, thus allowing them to crosses as in 134/7aXDon a23, this would escape the effects of FUdR. The toxicity of indicate that in all cases there was an ab- the selection system to Asn- cells combined with the leak through of a proportion of sence of intercistronic complementation and would suggest that the defect in all the Asn+ parent cells makes it difficult to deterAsn- clones lies in the same gene. This mine a mutation frequency for the Asn+ to technique would not, however, detect low Asn- event. However, the results of anallevels of complementation such as might ysis of a representative sample of colonies No. of 134/7a cells
No. of Sendai surviving virus colonies
Exp Cell Res 104 (1977)
366
Goldfarb et al.
after the first round of selection would indicate that following mutagenesis the frequency could be as high as 1X 10V5. In the case of the Asn- to Asn+ reversion event the results of the fluctuation tests showed that the spontaneous reversion rate was low in all of the clones, the range of values being 3.8+1.2x low6 to 5.7+2.1x lo-*. Although these values are in general lower than those reported for reversion events in Asn- tumour lines [18, 19, 201 they are in the range accepted as being indicative of single step mutation [32]. It is of note that the different Asn- clones demonstrated different reversion rates. This difference in reversion rates when considered in conjunction with the observed differences between clones in response to asparagine, behaviour on asparagine deprivation, and asparagine synthetase activity is consistent with the hypothesis that they are of independent origin. However, we cannot exclude the possibility that they are derived from a single spontaneous mutation event, differences having arisen during the course of their subsequent multiplication. The results of the karyotypic analysis indicated that chromosome rearrangement had not occurred during the forward or back mutation events. This would suggest that these events were not the result of major deletions of genetic material. In fact both the retention of some residual enzyme activity in the majority of the Asn- clones and the occurrence of Asn+ revertants makes it unlikely that any of the Asn+ to Asn- mutations were due to deletion of the structural gene. The presence in some of the Asnsubclones and their Asn+ revertants of a small extra chromosome is unlikely to be associated with the Asn- mutation as a proportion of the parental Don cells also have 23 chromosomes. Differences between the Asn- subclones Exp CeNRes IO4 (1977)
were revealed by the assays for asparagine synthetase activity in the cells, there being a difference in residual activity from subclone to subclone. There was, however, little difference between two subclones derived from the same clone. In general the residual asparagine synthetase activities were higher than those found in extracts of Asn- tumour cell lines [6, 7, 81, although it is difficult to decide with what “wild-type” level of activity the latter should be compared. The level of activity found in the Asn+ revertants was 64-77 % of that in Don cells, although in no case could the activity be increased to that of Don cells. Since we have been unable to demonstrate the presence of a control system by removal of asparagine from the medium the simplest hypothesis is that the deficiency of asparagine synthetase activity in the Asn- subclones is due to mutation in the structural gene for the enzyme. The absence of such a control system in our cells would not be unexpected if one considers that they are all fibroblast derived. It is possible that the retention of a control system in previously described Asn- lines [ 14,221 may be related to their tumour derivation. Certainly the retention of control systems and other differentiated functions in cell culture has only been demonstrated in tumour derived cell lines, e.g. inducibility of glutamine synthetase and tyrosine aminotransferase in HTC hepatoma cells [33, 341, and inducibility of glycerol-3-phosphate dehydrogenase in RG6A glioma cells [35], although in these cases the tissue specificity of the enzymes and the tissue of origin of the cell line may be of primary importance. Although complementation analysis indicated that the lesion was situated in the same gene in our Asn- clones and the JS cells and behaved as a recessive trait in hybrids with Don a23, these results do not
Asparagine
auxotrophs
367
provide evidence that the structural gene for asparagine synthetase is the one involved. Further investigations to detect a low level of complementation (as might be seen in intracistronic complementation) would require the introduction of forcing markers into the Asn- subclones and selection of all hybrids produced following Sendai mediated fusion. The method used here, in which complementation was tested directly by using asparagine-free medium as the selective system may have been too stringent to allow the survival of cells with intermediate levels of enzyme activity. Confirmation that our Asn- clones represent structural gene mutants will depend on biochemical and immunological study of the enzyme protein present in both the Asn- mutants and their revertants. Since the cell of origin is defined and can be grown under controlled conditions in culture, such an approach is readily feasible.
8. Patterson, M K Jr & Orr, G R, Cancer res 29 (1969) 1179. 9. Broome, J D, J exp med 127 (1968) 1055. 10. Dolowv. W C. Shrek. R. Henson. D. Comet. J & Brown; E, Nature 219 (1%8) 1028. 11. Patterson, M K Jr, Conway, E, Whittle, W & McCoy, T A, Proc sot exp biol 119(1%5) 5. 12. Mashbum, L T & Wriston, J D Jr, Arch biochem biophys 105 (1964) 450. 13. Roberts, J, Prager, M D & Bachynski, N, Cancer res 26 (1966) 2213. 14. Hill, J M, Roberts, J, Loeb, E, Khan, A, McLelIan, A & Hill, R, J Am med ass 202 (1967) 882. 15. Oettgen, H F, Old, L J, Boyse, E A, Campbell, H A, Philips, F S, Clarkson, B D, Tullal, L, Leper, R D, Schwartz, M K & Krim, J H, Cancer res 27 (1967) 2619. 16. Broome, J D, Trans NY acad sci (2) 30 (1968) 690. 17. Patterson, M K Jr, Maxwell, M D & Conway, E, Cancer res 29 (1969) 2%. 18. Morrow, J, J cell physio177 (1971) 423. 19. Summers, W P & Handschumacher, R E, Biochem pharmacol20 (1971) 2213. 20. Capizzi, R L, Smith, W J & Papirmeister, B, Proc Am assoc cancer res 13 (1972) 58. 21. Colofiore, J, Morrow, J & Patterson, M K Jr, Genetics 75 (1973) 503. 22. Patterson, M K Jr, Recent results in cancer res 33 (1970) 22. 23. Fogh, J & Fogh, H, Proc sot exp biol med 117 (1964) 899. 24. Slack, C, Morgan, R H M, Car&t, B, Goldfarb, P S G & Hooper, M L, Exp cell res 98 (1976) 1. 25. Miller, G L, Anal them 31 (1959) 964. We wish to thank Professors J. A. Pateman and J. H. 26. Lowry, 0 H, Rosebrough, N J, Farr, A L & Subak-Sharpe for their advice and interest in this Randall, R J, J biol them 193 (1951) 265. work. Expert technical assistance was provided by 27. Luria, SE & Delbriick, M, Genetics 28 (1943)491. Mrs Jeanette Daniel, Mr Liam Meagher and Mrs Rose28. Lea, D E & Coulson, C A, J genet 49 (1947) 264. mary Morgan. The investigation was supported by a 29. Hsu, T C & Zenzes, M T, J natl cancer inst 32 grant from the Cancer Research Campaign. (1964) 857. 30. Fincham, J R S, ‘Genetic complementation’. Microbial and molecular biology series no. 3 (ed. B D Davis). Benjamin, New York (1966). REFERENCES 31. Kao, F T & Puck, T T, Proc natl acad sci US 60 1. Newman, R E & McCoy, T A, Science 124 (1956) 32 (1967) 1275. Chu, E H Y, Brimer, P, Jacobson, K B & Mer124. 2. McCoy, T A, Maxwell, M & Kmse, P F, Cancer 33. riam, E V, Genetics 62 (1969) 359. Kulka, R G, Tomkins, G M & Crook, R B, J cell res 19 (1959) 591. 3. Haley, E E, Fischer, G A & Welch, A D, Cancer 34 biol54 (1972) 175. Tomkins, G M, Geleheter, T D, Granner, D, res 21 (1961) 532. Martin, D W Jr, Samuel, H H & Thompson, E B, 4. Broome, J D, Nature 191 (1961) 1114. 5. Broome, J D & Schwartz, J H, Biochim biophys 35 Science 166(1969) 1474. Davidson, R L & Benda, P, Proc natl acad sci US acta 138 (1967) 637. 67 (1970) 1870. 6. Horowitz, B, Madras, B K, Meister, A, Old, L J, Boyse, E & Stockert, E, Science 160 (1968) 533. 1976 7. Prager, M D & Bachynski, N, Biochem biophys Received Accepted May Ju,y 31, l6 1976 res commun 127 (1968) 645.
Exp Cell Res IO4 (1977)
ISSN00144827
Experimental
THE ISOLATION
Cell Research 104 (1977) 357-367
AND CHARACTERIZATION
ASPARAGINE-REQUIRING
MUTANTS
CHINESE
CELLS
HAMSTER
P. S. G. GOLDFARB, B. CARRITT,
OF OF
M. L. HOOPER and C. SLACK
Cancer Research Campaign Somatic Cell Genetics Group, Institute of Genetics, University of Glasgow, Glasgow, Scotland
SUMMARY Nine asparagine-requiring mutants were isolated in culture from the Don line of Chinese hamster cells. Investigation of the asparagine requirements of the mutants, the effect of asparagine deprivation on macromolecular synthesis, and the rates of reversion to asparagine independence indicated that there were differences between the mutant clones. Biochemical analysis revealed that the defect in the mutants was due to a deficiency of the enzyme asparagine synthetase, and that the enzyme activity in the mutants and Asn+ revertants obtained from them was not influenced by the concentration of asparagine in the growth medium. Complementation analysis by S&&i virusmediated cell fusion indicated that the lesion behaved as a recessive trait, and was probably located in the same gene in all the mutant clones.
The nutritional requirement of certain tu- contain increased levels of asparagine synmour-derived malignant cell lines for L- thetase [6,7,8, 161appear at low frequency asparagine has been well documented [ 141. in the population and are derived from The inability of such cell lines to synthesise mutational events, rather than as a response asparagine [5] has in all cases been shown to the absence of asparagine [17-211. In to be due to the absence of, or low level of, some instances [14,22] the level of enzyme asparagine synthetase (EC6.3.1.1) in cells activity found in the Asn+ cell lines and passaged both in vitro and as tumours in tumours can be increased by removal of vivo [6-8]. The sensitivity of these aspara- asparagine, suggesting that a regulatory gine-requiring (Asn-) cell lines and tumours system may be present. To date there has been no report of the to L-asparaginase (EC3.5.1.1) from either E. coli or guinea pig serum [9, 10, 111has isolation in cell culture of Asn- cells from formed the basis of chemotherapeutic ap- an Asn+ cell line. The possession of such proaches using asparaginase [12-U]. One cell lines would be of interest not only for Gndrance to these approaches has been the use in the biochemical and genetical anaappearance, when both Asn- tumours and lysis of the regulatory processes involved in 4sn- cell lines are treated with aspara- the expression of asparagine synthetase cinase, of cells which no longer require activity, but also to investigate the sugrsparagine (Asn+ cells). These cells which gested [18, 191 correlation between maExp
Cell
Res
104 (1977)
358
Goldfarb
et al.
lignancy and the appearance of an asparagine requirement in cell lines. In this paper we describe the isolation of Asn- cell lines from an Asn+ line of diploid Chinese hamster cells (Don), and the subsequent derivation from the Asn- lines of Asn+ revertants. Biochemical analysis of the Asnlines and their Asn+ revertants suggests that the observed defect in the Asn- lines is due to a deficiency of asparagine synthetase activity. Complementation analysis suggests that the lesion is probably located in the same gene in all the Asn- clones. MATERIALS
AND METHODS
Chemicals and radiochemicals Ethylmethanesulphonate, 5fluorodeoxyuridine (FUdR), Na*ATP, and L-asparaginase were obtained from Sigma. Aminopterin was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Colcemid was supplied by Ciba Laboratories, Horsham, Sussex. UV-inactivated Sendai virus was obtained from Searle Diagnostic Ltd, High Wycombe, Bucks. [6-3H]thymidine 20 Cilmmole, [5 ,6-3H]uridine 40 Ci/ mmole, Lj4,5-3H]leucine 40 Cilmmole, and L#J-“‘Claspartic acid 200 mCi/mmole were obtained from the Radiochemical Centre, Amersham, Bucks.
Cells and cell culture The Don line of Chinese hamster lung fibroblasts was obtained from the American Type Culture Collection, Rockville, Md, as was the Jensen Sarcoma (JS) line of rat cells. The Don TK- derivative a23 was isolated by Dr A. Westerveld and kindly supplied by him. Cells were normally grown in 90 cm* plastic tissue culture flasks (A/S Nunc) in Eagle’s medium (Glasgow modifica~on) contaiining 10% v/v foetal calf serum (GJBCO-Biocult) under an atmosphere of 5% CO, in air, at 37°C. For growth at low cell density (<50 cells/ cm%)and for the routine growth of Asn- cell lines, the medium was supplemented with non-essential amino acids (alanine, aspartic acid, asparagine, glutamic acid, glycine each 0.1 mM; serine, proline each 0.2 mM), nucleosides (adenosine, guanosine, cytidine, uridine, each 30 PM; thymidine, 10 fiM), and 1 r&l sodium pyruvate. Cloning efficiencies of 30-50% were achieved in this medium. For asparagine-free medium the non-essential amino acid supplement was omitted, and replaced by L-aspamginase 0.02 U/ml. Cells were passaged twice weekly by trypsinization using 0.05 % trypsin in Ca-Mg-free isotonic phosphatebuffered saline (PBS) containing 0.6 mM EDTA. After neutralization with serum-containing medium, washing and resuspension in growth medium, 10% of the cells were reseeded into plastic flasks. Exp Cell Res 104 (1977)
Regular examination of cells for mvcoolasma contamination was performed by the method of Fogh & Fogh [23]. No evidence of such contamination was found during this study.
Recloning
of cell lines
Log-phase cells were trypsinized, washed and resuspended in complete supplemented medium at a density of 5 cells/ml. 0.2 ml aliquots were dispensed into each of 96 plastic micro-wells (Linbro) and the wells incubated overnight at 37°C. On inspection, wells containing more than one cell were discarded, and incubation continued for a further 7-10 days. At the end of this period the wells were inspected and cells removed by trypsinization from those containing a single large colony. Each clone was then oassaged to a population size of 5 x 10’ cells prior to storage in liquid N2 in supplemented medium containing 10% dimethylsulphoxide.
Selection of Asn- cells A confluent monolayer of 2X 10’ Don cells was mutagenized with 4 mM ethylmethanesulphonate in complete supplemented medium for 4 h at 37°C (10% survival). After mutagenesis the cells were trypsinized, resuspended in complete supplemented medium, seeded into four replicate 90 cm* plastic flasks and allowed to grow for 2 days. At the end of this period the medium was changed to asparagine-free medium and incubation continued for 24 h. The medium was removed and replaced by asparagine-free medium lacking thymidine which contained FUdR 25 pg/ml and uridine 125pg/ml. After a further 48 h incubation, the medium was replaced by complete supplemented medium. This was renewed at 3 day intervals. Cell death and detachment was apparent after 2-3 days and surviving colonies were visible after 14 days. The colonies were trypsinized and taken en masse for further rounds of selection, care being taken to ensure that survivors from the four parallel selections were kept separate. In all, three rounds of selection were employed. At the end of the 3rd round of selection 2-3 morphologically distinct colonies were picked from each of the 4 bottles, grown to a population size of 5x 10’ cells, and stored in liquid Nr.
Incorporation of radioactive precursors into macromolecules of Asn- cells In experiments to measure the rate of shutdown of macromolecular synthesis in Asn- cells deprived of asparagine, cells were seeded on to sterile Deconwashed 13‘mm dia. coverslips in plastic wells (Linbro) at 1X 10’ cells/well in complete supplemented medium. After 24 h at 37°C the medium was replaced with asparagine-free medium lacking nucleosides, and carrier-free [3H]thymidine (TdR), [3Hjmidine, or [3H]leucine added to 5 &i/ml. At appropriate intervals coverslips were removed for extraction which trichloroacetic acid (TCA) and scintillation counting as
Asparagine described previously [24]. In experiments to measure the rate of switch-on of macromolecular synthesis in starved Asti cells to which asparagine had been restored, cells were seeded as above. After 24 h the medium was changed to asparagine-free medium and incubation continued for a further 48 h. The medium was replaced with complete medium lacking nucleosides, and [3H]TdR, [3H]uridine or [3H]leucine added as above. Coverslips were removed for TCA extraction and scintillation counting [24]. In both ‘switch-off and ‘switch-on’ experiments a parallel series of coverslips was taken for estimation of total protein.
Karyology Metaphase spreads were prepared from colcemidtreated cells, and stained with aceto-orcein or Gbanded as described previously [24].
Preparation
of cell extracts
Extracts were prepared from late log phase cells grown in complete supplemented medium in 180 cm2 plastic flasks (A/S Nunc). Typically 2x10’ cells were trypsinized into 10 ml of medium and pelleted at 200 g for 5 min. After resuspension and gentle washing in 10 ml ice-cold PBS, cells were recentrifuged at 200 g for 5 mitt, and the pellet resuspended in 1 ml of 10 mM Tris-HC1+0.5 mM /3-mercaptoethanol pH 7.4 at 2°C. The suspension was sonicated using an MSE-Mullard Sonicator for two bursts of 10 set with 1 min cooling between. The sonicate was either assayed directly for enzyme activity, or divided into 100 ~1 aliquots for storage at -70°C. No reduction in enzyme activity was found after one cycle of freeze thawing, even in extracts which had been stored for up to 3 months.
Determination concentration
of protein
Determinations were performed on duplicate samples containing 10-100 pg protein using the Miller [25] modification of the method of Lowry et al. [26]. In the case of cells attached to coverslips, three washes with isotonic saline at 4°C were employed to remove the serum component of the growth medium, and reactions performed directly on the attached cells using l/S vol.
auxotrophs
359
of each in 10 ~1 HzO) added to tubes 1 and 2. Tube 3 was heated to 100°C for 2 min to inactivate the enzyme, cooled to 37°C and 0.01 U of aspamginasein 5 ~1 H,O added. After 15 min incubation at 37°C the reaction was terminated by heating to 100°Cfor 2 mitt, the tube placed in ice, and carrier aspartic acid and asparagine added as above. Separation of [Wlasparagine from [Ylaspartic acid was performed by the electrophoretic method of Horovitz et al. [6]. The amount of radioactivity co-electrophoresing with cartier asparagine was determined and the value obtained for tube 3 subtracted from the mean of the values for tubes 1 and 2. The resulting difference was used to calculate the enzyme activity. The asparaginase-resistant counts represented approx. 0.2% of the input radioactivity.
Complementation cell fusion
analysis by
2x iOBcells of each parental cell type were seeded into a 20 cm* plastic Petri dish (Falcon) in complete supplemented medium to form a confluent monolayer and incubated for 18 h at 37°C. The medium was removed, the cells washed with medium lacking serum, and 1 ml Hanks’ balanced salts solution containing 500 haemagglutinating units of UV-inactivated Sendai virus added. After 30 min adsorption at 4”C, 1 ml of medium conmining serum was added and incubation continued for a further 14 h at 37°C. The cells were washed twice with asparagine-free medium and trypsinized into 10 ml of the same medium. After centrifugation at 200 g for 5 min the cells were resuspended and seeded into a 150cm* plastic flask (Corning). In the case of fusions between Asti parents the medium for resuspension and selection of complementing hybrids was asparagine-free medium, whereas for fusions between Don a23 and Asn- lines the resuspension and selection medium was asparagine-free medium (lacking nucleosides) to which 0.1 mM hypoxanthine, 0.8 PM aminopterin, 3 I.LM glycine, and 20 PM TdR (HAT) had been added. Incubation was continued for 14days with medium changes every 3 days and surviving colonies picked or stained with Leishman stain (1.5 g/I in absolute methanol). Controls to which no Sendai virus was added were performed in parallel.
RESULTS Assay of asparagine synthetase activity Asparagine synthetase assays were performed in triplicate in plastic reaction tubes (Eppendorf) at 37°C in a final volume of 50 ~1. Each reaction contained 5 pmoles Tris-HCl (pH 8 at 37°C). 0.4 pmoles MgClp, 0.4 pmoles Na,ATP, 1 pmole L-glutamine, 0.1 Pmole L-[U-W]aspartic acid (spec. act. 2 &i//Lmole), and 100-500 pg protein cell extract. After 5 min equilibration at 37°C reactions were started by the addition of cell extract (usually 25 ~1). After 15 min incubation, reactions were terminated by placing the tubes in ice, and carrier aspartic acid and asparagine (0.1 pmoles 24-761813
Isolation
of Asn- clones
The frequency with which surviving colonies appeared following one round of selection was 1x 10e4.However, analysis of a representative sample of these colonies indicated that
360
Goldfarb
et al.
Don
,x,3
lull
b
- - - - - - - - 13L/lO
b A___-13u30
13L/3e13LIJ
lL----12
a
A
1 1W3eR,
t
3
L
104hL
time (days); ordinate: cell no. O-O, No asparagine; O-0, 1O-5 M; A-A, 2x1O-5 M; Cm, 5x lo+ M. (a) 134/3; (b) 134/7. Effect of asparagine on the growth of Asn- clones 134/3 and 134/7 in mass culture. IX 105 cells were seeded on to replicate 20 cm* plastic Petri dishes in asparagine-free medium. After attachment and spreading, the medium was changed to that containing the indicated concentrations of asparagine. Each day cells from one dish at each concentration were removed by trypsinisation, diluted, and counted using a Coulter Counter (Coulter Electronics Ltd, St Albans).
Fig. 2. Abscissa:
13u3efI,
Fig. 1. Genealogy of (a) Asn+ revertants and (b) Asn-
subclones of Asn- clone 13413.
After a total of three rounds of selection the survival rate had increased to 1X 10d3,indicating that although the selection had enriched for Asn- colonies, there was either a continued leak through of Asn+ cells or a low survival of Asn- cells in the selection system. Reconstruction experiments in grew vigorously in mass culture and could which a small number of Asn- cells were be passaged using 10% splits. mixed with Don cells and put through the selection indicated that the survival of Asn- Requirement of Asn- clones for asparagine cells in the system is about 1%. A total of ten colonies were picked fol- The Asn- clones showed an absolute relowing three rounds of selection and on quirement for asparagine both for maximum initial screening were found to require as- growth rate at high cell density (5 X 103cells/ paragine for growth. One clone 134/2 had a cmz) and for colony formation at low cell density (60 cells/cm2). This requirement low cloning efficiency, and was eventually lost. The nine remaining Asn- clones (fig. 1) could not be satisfied by any other nonTable 1. Fluctuation
analysis
of the Asn- to Asn+ reversion
in asparagine
auxotroph
clones
Clone 134/l 3 4 5 6 7 8 9 10
Total cells/ No. of subclones subclone 8 9 7 8 6 7 8 6
2x 108 4x 10’ 2x106 2x 106 4x 10’ 4x 10’ 2x 106 2Xlog 2x 106
Cells seeded/ replicate dish 1x10”
5x105 1x10s 1x105 1x1@ 1x106 1x105 1x10s 1x1@
Average no. of reversions/ subcloneD
Reversions/ cell/ generation”
1.3f0.7 115. +20 1.3kO.8 co.3 3.3f 1.2 3.9+ 1.6 0.8f0.5 co.3 ll.Of3.4
4.5+2.3x 2.0*0.3x 4.6*2.6x <1x10-’ 5.7+2.1x 6.7f2.7~ 2.7f 1.8x <1x10-’ 3.8f I .2x
a Calculated by the median method of Lea & Coulson [28], and expressed as estimate +S.E. Exp CeNRes 104 (1977)
lo-’ 1O-6 lo-’ 1O-8 1OV lo-’ lo-@
Asparagine L.lOS
0
b I
2.105
L
/
361
auxotrophs
the response to asparagine-free medium varied from clone to clone, in some instances cell detachment occurring after 2-3 days, whilst in others the cells remained viable for up to 5 days.
x---'
Reversion of Asn- cells to Am+
04 / 0
12
2L
Fig. 3. Abscissa: time (hours); ordinate: (u-c) cpm; (d) Kg protein/well. O-O, Don; O-O, 134/7a.
Effect of removal of asparagine on incorporation of (a) rH]TdR; (b) rH]uridine; (c) [3Hjleucine into TCAprecipitable material; and on (d) total cell protein in Asn- subclone 134/7a.
essential amino acid either alone or in combination. However, at high cell density some growth (at a rate of about 20 % of that in the presence of asparagine) was observed in some instances if all the non-essential amino acids other than asparagine were supplied. It is not known whether this was a real sparing effect, or if the non-essential amino acids maintained the cells in a viable condition long enough for asparagine to be formed by serum proteolysis. Because of this effect, all the non-essential amino acids were routinely omitted from asparagine-free medium. In the absence of other non-essential amino acids a relationship between the amount of growth at high cell density and the concentration of added asparagine could be demonstrated. As illustrated in fig. 2 for clones 134/3 and 134/7, the growth response to added asparagine differed reproducibly from clone to clone. It was also found that
When Asn- cells at a density of 5~ 103cells/ cm2 were maintained in asparagine-free medium for 14 days, colonies which did not require asparagine for growth appeared at low frequency in the cultures. Since the appearance of these Asn+ colonies could have been due either to reversion of the Asncells at low frequency, or to the persistence of wild-type Don cells in the culture, the Asn- clones were recloned by dilution and 6-9 subclones derived from each. Cells from each subclone were then seeded at early passage into five replicate 20 cm2 plastic Petri dishes in asparagine-free medium, and the appearance of Asn+ colonies 2.105 I
a
L:
0
0
lo5
7
/*
/
k
.-
1
2.105
L
b
pi. 0' i 0' ,*
2.103
.
0
MO
/
MO
0 _(-
L.105
p:,‘ "
/.
0
0
12 0
0
‘
0
1
0
12
c 0
- 10'
2.10' / */O L0
/"
L103
(..-J. 0 L
0
Fig. 4. Abscissa:
\~\;/protein/well.
12
0
L
time (hours); ordinare: (a-c) cpm; (L&f) O-O, Don; (right) O-0,
Effek of restoring asparagine to starved cells on incorporation of (a) rH]TdR; (b) rH]uridine; (c) [3H]leucine into TCA-precipitable material; and on (d) total cell protein. Exp Cell Res 104 (1977)
362
Goldfarb et al.
2513u3e
‘3‘k
lYi6b 23
scored after 7-10 days. The results were subjected to fluctuation analysis [27, 281 and in all cases the between-subclone variance was greater than the within-subclone variance, indicating that the Asn- to Asn+ reversion was a process randomly distributed in time generating a stably inherited phenotype, rather than a response to medium lacking asparagine. As shown in table 1, a low but finite reversion rate was observed in most of the clones. This rate could have accounted for the presence of Asn+ cells in stocks of the Asn- clones, although the possibility that they were surviving parental Don cells is not ruled out. Table 1 also shows that the reversion rates differed between Asn- clones over a 102-fold range. In order that further characterisation of the Asn- clones would not be confused by the presence of Asn+ cells, all subsequent work was performed on recloned material at early passage, one subclone from each clone being used for this. For comparative purposes revertant Asn+ clones were isolated from certain Asn- subclones by seeding 5 x 106cells in duplicate into asparaginefree medium in 180 cm2 plastic bottles. After 14 days one revertant colony was picked from each bottle and grown to a population size of 5X 10’ cells prior to storage in liquid NZ. Exp Cell Res IO4 (1977)
13*r ‘Ci 22
Effect of asparagine on incorporation of radioactive precursors
As shown in fig. 3 the withdrawal of asparagine from the growth medium of Asnclones, as demonstrated by subclone 134/ 7a, resulted in a decrease in the incorporation of precursors into DNA, RNA and protein. Although there appeared to be a rapid decrease in the rate of incorporation of all three precursors there were marked differences in the length of time which elapsed prior to complete inhibition of incorporation. Whereas incorporation of rH}TdR into DNA ceased after 8 h, incorporation of r3H]uridine into RNA was not inhibited until after 12 h. Incorporation of rH]leucine into TCA-precipitable material also did not appear to be shut off until more than 12 h had elapsed. The results obtained with other Asn- subclones were in agreement with those for subclone 134/7a except that variations of 2-3 h were observed in the time at which [3H]TdR incorporation ceased. It is apparent therefore that asparagine deprivation in the Asn- cells leads directly or indirectly to a rapid inhibition of DNA synthesis, which precedes any fall in the rates of uridine or leucine incorporation or total protein synthesis. This decrease in the rate of DNA synthesis cannot therefore be a result of an effect on protein synthesis
Asparagine
Fig. 6. Trypsin-Giemsa banded chromosomes from
Asn- subclones and Asn+ revertants derived from
unless asparagine deprivation in Asn- cells leads initially to the depletion of a specific sub-set of cellular proteins. The effect of restoring asparagine to starved Asn- cells is demonstrated for subclone 134/7a in fig. 4. As can be seen there is a lag period before rH]TdR incorporation commences. This lag is not seen in the case of [3H]uridine or rH]leucine incorporation. It is also of note that there is a large increase in the total protein of cultures of Asn- cells following restoration of as-
auxotrophs
363
them. (a) 134/1Oband 134/10bR,; (b) 134/7a and 134/ 7aR,.
paragine, suggesting that not only is there increase in cell number, but also in the amount of protein per cell. An increase in cell size is also observed during this period. Karyotypic
analysis
Twenty-five aceto-orcein or Giemsastained metaphases of each subclone were examined and the chromosome numbers determined. In all cases the modal number was diploid or close to diploid, no gross structural rearrangements being observed. Exp Cell Res 104 (1977)
364
Goldfarb et al.
Table 2. Asparagine synthetase activities of the Asn- subclones and their revertants
Clone Asn134/lc 3e 3f 4b
5a 6b 7a 8a 9a lob Asn+ 134/3eRl 6bRl 7aRl 1ObRl Don LMTK-
Asparagine synthetase activity (nmoles/mg/h)
2.8kO.3
(5)
1.4f0.5 (4) 1.7kO.3 (6) co.2 3.4k1.2
(4) (3)
5.1kO.9 (4) 2.3kO.3 2.4kO.7
(3) (4) 1.9kO.8 (3j 3.2f0.4 (2)
13.ork1.3 13.4f1.8
(4) (2)
13.9k2.0 11.5f0.4 18.1kl.l 36.2f2.1
(3) (2) (11) (3)
% Don 15.5 7.7 9.4
Cl.0 18.8 28.2 12.7 13.3
10.5 17.7 71.8 74.0 76.7 63.5
-
Activity is expressed as nmoles asparagine produced/ mg protein/hfS.E.M. Figures in parentheses indicate the number of determinations made using at least two extracts from cells of different passage numbers, each determination being the mean of two assays.
Asparagine synthetase activity of the Asn- subclones and their revertants
As shown in table 2 the asparagine synthetase activity in extracts of the Asn- subclones was markedly reduced compared with the parent Don cells. In one instance (subclone 134/4b) enzyme activity was never detected. It was not possible, however, to determine whether this was due to a complete lack of functional enzyme protein, or whether the residual activity was more unstable than in other subclones. Experiments in which extracts from Don cells and subclone 134/7a were mixed prior to assay showed that the deficiency of enzyme in the Asn- subclones was not due to the presence of a diffusible inhibitor. In order to investigate whether the levels of asparagine synthetase were regulated by the external asparagine concentration, as in the case of Asn+ revertants of JS cells [14, 221, extracts were made from cells which had been starved of asparagine. It was found that neither in Don cells nor in any Asn- subclone was enzyme activity increased by pretreatment for 48 h with asparagine-free medium. Also shown in table 2 is the enzyme activity found in extracts of four revertant clones. As can be seen these have signiticantly higher enzyme activity than their Asn- parents, although in no case does the activity equal that of Don cells. This activity would appear to be the maximum level expressed in the revertant clones, as prior growth for one passage in asparaginefree medium did not lead to any significant increase in enzyme activity.
As shown in fig. 5 the modal chromosome numbers of the Am+ revertant clones were also close to diploid and resembled that of their Asn- parent subclone. Examination of trypsin-Giemsa banded preparations indicated that there was no evidence of translocation having occurred within the karyotype. Fig. 6a shows a G-banded preparation of subclone 134/10b and a revertant 134/ 10bRl derived from it. No differences were observed between the Asn- and Asn+ lines, both showing a banding pattern identical to that of the Don line [24, 291. In the case of subclone 134/7a (fig. 6b), the deviation from diploidy is due to an extra small metacentric chromosome. The origin of this extra chromosome, which is also present in Complementation analysis a derived Asn+ revertant 134/7aR,, is not In order to determine whether the defect in the Asn- clones behaved as a dominant or known. EXP Cell RPS 104 (1977)
Asparagine
Table 3. Complementation
analysis
Fusion of Don a23x 134/7a and selection of complementing hybrids in asparagine-free medium+HAT
occur in intracistronic
auxotrophs
365
complementation
1301.
Cells of subclone 134/7a were also fused with a recloned Asn- line derived from rat Culture No. of JS cells, and colonies surviving in asparano. a23 cells gine-free medium scored. The results indi1 1x10s 1x10s + 340,243, 272 cated that intercistronic complementation 2 1x106 1x106 16, 47, 30 had not occurred, and that the defect in 3 1x1@ + 0, 0, 0 134/7a was in the same gene as that in the 4 1x106 0, 0, 0 5 ix106 + 1, 0, 0 JS cells. This conclusion is drawn on the as6 1 1XlW 0, 0, 0 sumption that the fusion index between Data shown are the results of three replicate fusion Don Asn- and JS cells is similar to that beexperiments. tween Don Asn- and Don a23, and that there is sufficient retention of rat chromorecessive trait, cells of subclone 134/7a somes under selective conditions to allow were fused with those of the Don TK- line the detection of complementation. a23, and the number of colonies surviving a selective system toxic to both parents DISCUSSION scored. The frequency of hybrid formation using this method has not been established, In this paper we have demonstrated that by but by comparison with other fusions using using a modification of the method of Kao Don a23 (our unpublished results) the ex- & Puck [31] it is possible to isolate asparapected frequency would be < 1x 10T3. As gine auxotrophs from a population of wildshown in table 3 the frequency of survivors type cells. The apparently high selectivity from the fusion was significantly greater of the system for Asn- mutants is probably than that in controls from which Sendai due to two factors. (1) The FUdR treatment virus or one of the parents had been used was highly toxic to growing cells. (2) omitted. This indicates that complementa- The selection was performed in undialysed tion of the Asn- defect by the Asn+ parent serum, the concentrations of non-essential had occurred, and that Asri behaves as a amino acids in this probably being suffirecessive trait. cient to allow continued growth of any When cells from the various Asn- sub- other auxotrophic mutants. Presumably, in clones were fused with each other in two- the case of asparagine, the concentration parent crosses and colonies surviving in was low enough (due to the presence of asparagine-free medium scored, no increase added asparaginase) to cause complete inabove controls was observed. Assuming hibition of DNA synthesis in a proportion that the fusion index was the same in these of the Asn- cells, thus allowing them to crosses as in 134/7aXDon a23, this would escape the effects of FUdR. The toxicity of indicate that in all cases there was an ab- the selection system to Asn- cells combined with the leak through of a proportion of sence of intercistronic complementation and would suggest that the defect in all the Asn+ parent cells makes it difficult to deterAsn- clones lies in the same gene. This mine a mutation frequency for the Asn+ to technique would not, however, detect low Asn- event. However, the results of anallevels of complementation such as might ysis of a representative sample of colonies No. of 134/7a cells
No. of Sendai surviving virus colonies
Exp Cell Res 104 (1977)
366
Goldfarb et al.
after the first round of selection would indicate that following mutagenesis the frequency could be as high as 1X 10V5. In the case of the Asn- to Asn+ reversion event the results of the fluctuation tests showed that the spontaneous reversion rate was low in all of the clones, the range of values being 3.8+1.2x low6 to 5.7+2.1x lo-*. Although these values are in general lower than those reported for reversion events in Asn- tumour lines [18, 19, 201 they are in the range accepted as being indicative of single step mutation [32]. It is of note that the different Asn- clones demonstrated different reversion rates. This difference in reversion rates when considered in conjunction with the observed differences between clones in response to asparagine, behaviour on asparagine deprivation, and asparagine synthetase activity is consistent with the hypothesis that they are of independent origin. However, we cannot exclude the possibility that they are derived from a single spontaneous mutation event, differences having arisen during the course of their subsequent multiplication. The results of the karyotypic analysis indicated that chromosome rearrangement had not occurred during the forward or back mutation events. This would suggest that these events were not the result of major deletions of genetic material. In fact both the retention of some residual enzyme activity in the majority of the Asn- clones and the occurrence of Asn+ revertants makes it unlikely that any of the Asn+ to Asn- mutations were due to deletion of the structural gene. The presence in some of the Asnsubclones and their Asn+ revertants of a small extra chromosome is unlikely to be associated with the Asn- mutation as a proportion of the parental Don cells also have 23 chromosomes. Differences between the Asn- subclones Exp CeNRes IO4 (1977)
were revealed by the assays for asparagine synthetase activity in the cells, there being a difference in residual activity from subclone to subclone. There was, however, little difference between two subclones derived from the same clone. In general the residual asparagine synthetase activities were higher than those found in extracts of Asn- tumour cell lines [6, 7, 81, although it is difficult to decide with what “wild-type” level of activity the latter should be compared. The level of activity found in the Asn+ revertants was 64-77 % of that in Don cells, although in no case could the activity be increased to that of Don cells. Since we have been unable to demonstrate the presence of a control system by removal of asparagine from the medium the simplest hypothesis is that the deficiency of asparagine synthetase activity in the Asn- subclones is due to mutation in the structural gene for the enzyme. The absence of such a control system in our cells would not be unexpected if one considers that they are all fibroblast derived. It is possible that the retention of a control system in previously described Asn- lines [ 14,221 may be related to their tumour derivation. Certainly the retention of control systems and other differentiated functions in cell culture has only been demonstrated in tumour derived cell lines, e.g. inducibility of glutamine synthetase and tyrosine aminotransferase in HTC hepatoma cells [33, 341, and inducibility of glycerol-3-phosphate dehydrogenase in RG6A glioma cells [35], although in these cases the tissue specificity of the enzymes and the tissue of origin of the cell line may be of primary importance. Although complementation analysis indicated that the lesion was situated in the same gene in our Asn- clones and the JS cells and behaved as a recessive trait in hybrids with Don a23, these results do not
Asparagine
auxotrophs
367
provide evidence that the structural gene for asparagine synthetase is the one involved. Further investigations to detect a low level of complementation (as might be seen in intracistronic complementation) would require the introduction of forcing markers into the Asn- subclones and selection of all hybrids produced following Sendai mediated fusion. The method used here, in which complementation was tested directly by using asparagine-free medium as the selective system may have been too stringent to allow the survival of cells with intermediate levels of enzyme activity. Confirmation that our Asn- clones represent structural gene mutants will depend on biochemical and immunological study of the enzyme protein present in both the Asn- mutants and their revertants. Since the cell of origin is defined and can be grown under controlled conditions in culture, such an approach is readily feasible.
8. Patterson, M K Jr & Orr, G R, Cancer res 29 (1969) 1179. 9. Broome, J D, J exp med 127 (1968) 1055. 10. Dolowv. W C. Shrek. R. Henson. D. Comet. J & Brown; E, Nature 219 (1%8) 1028. 11. Patterson, M K Jr, Conway, E, Whittle, W & McCoy, T A, Proc sot exp biol 119(1%5) 5. 12. Mashbum, L T & Wriston, J D Jr, Arch biochem biophys 105 (1964) 450. 13. Roberts, J, Prager, M D & Bachynski, N, Cancer res 26 (1966) 2213. 14. Hill, J M, Roberts, J, Loeb, E, Khan, A, McLelIan, A & Hill, R, J Am med ass 202 (1967) 882. 15. Oettgen, H F, Old, L J, Boyse, E A, Campbell, H A, Philips, F S, Clarkson, B D, Tullal, L, Leper, R D, Schwartz, M K & Krim, J H, Cancer res 27 (1967) 2619. 16. Broome, J D, Trans NY acad sci (2) 30 (1968) 690. 17. Patterson, M K Jr, Maxwell, M D & Conway, E, Cancer res 29 (1969) 2%. 18. Morrow, J, J cell physio177 (1971) 423. 19. Summers, W P & Handschumacher, R E, Biochem pharmacol20 (1971) 2213. 20. Capizzi, R L, Smith, W J & Papirmeister, B, Proc Am assoc cancer res 13 (1972) 58. 21. Colofiore, J, Morrow, J & Patterson, M K Jr, Genetics 75 (1973) 503. 22. Patterson, M K Jr, Recent results in cancer res 33 (1970) 22. 23. Fogh, J & Fogh, H, Proc sot exp biol med 117 (1964) 899. 24. Slack, C, Morgan, R H M, Car&t, B, Goldfarb, P S G & Hooper, M L, Exp cell res 98 (1976) 1. 25. Miller, G L, Anal them 31 (1959) 964. We wish to thank Professors J. A. Pateman and J. H. 26. Lowry, 0 H, Rosebrough, N J, Farr, A L & Subak-Sharpe for their advice and interest in this Randall, R J, J biol them 193 (1951) 265. work. Expert technical assistance was provided by 27. Luria, SE & Delbriick, M, Genetics 28 (1943)491. Mrs Jeanette Daniel, Mr Liam Meagher and Mrs Rose28. Lea, D E & Coulson, C A, J genet 49 (1947) 264. mary Morgan. The investigation was supported by a 29. Hsu, T C & Zenzes, M T, J natl cancer inst 32 grant from the Cancer Research Campaign. (1964) 857. 30. Fincham, J R S, ‘Genetic complementation’. Microbial and molecular biology series no. 3 (ed. B D Davis). Benjamin, New York (1966). REFERENCES 31. Kao, F T & Puck, T T, Proc natl acad sci US 60 1. Newman, R E & McCoy, T A, Science 124 (1956) 32 (1967) 1275. Chu, E H Y, Brimer, P, Jacobson, K B & Mer124. 2. McCoy, T A, Maxwell, M & Kmse, P F, Cancer 33. riam, E V, Genetics 62 (1969) 359. Kulka, R G, Tomkins, G M & Crook, R B, J cell res 19 (1959) 591. 3. Haley, E E, Fischer, G A & Welch, A D, Cancer 34 biol54 (1972) 175. Tomkins, G M, Geleheter, T D, Granner, D, res 21 (1961) 532. Martin, D W Jr, Samuel, H H & Thompson, E B, 4. Broome, J D, Nature 191 (1961) 1114. 5. Broome, J D & Schwartz, J H, Biochim biophys 35 Science 166(1969) 1474. Davidson, R L & Benda, P, Proc natl acad sci US acta 138 (1967) 637. 67 (1970) 1870. 6. Horowitz, B, Madras, B K, Meister, A, Old, L J, Boyse, E & Stockert, E, Science 160 (1968) 533. 1976 7. Prager, M D & Bachynski, N, Biochem biophys Received Accepted May Ju,y 31, l6 1976 res commun 127 (1968) 645.
Exp Cell Res IO4 (1977)