adaptational origin of some purine-analogue resistant phenotypes in cultured mammalian cells

275

Mutation Research, 49 (1978) 275--296 © Elsevier/North-Holland Biomedical Press

ADAPTATIONAL ORIGIN OF SOME PURINE-ANALOGUE RESISTANT PHENOTYPES IN CULTURED MAMMALIAN CELLS

MARGARET FOX a and M. RADACIC b

a Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX. (Great Britain), and b Institute Riider Boskovie, Zagreb (Yugoslavia)

(Received 7 June 1977) (Revision received 23 August 1977) (Accepted 2 September 1977)

Summary

In an a t tempt to elucidate further the reasons for phenotypic diversity amongst purine-analogue resistant clones, Chinese hamster and mouse lymphoma cells were exposed to single and multistep selection procedures. The phenotypes of cells during exposure to, and on removal from, continuous low dose selection were determined and compared with those of cells surviving short- term exposure to high selective doses.

On exposure to low doses of 8-azaguanine (8AZ), of 6-thioguanine (6TG), HAT+ first step resistance developed within 2--3 days. First,step resistant lines showed no reduction in either HGPRT activity or [14C]hypoxanthine utilisation, consistent with their retained ability to grow in HAT. They were unstable when grown in the absence of selective pressure and gradually lost purine-analogue resistance over a period of 6--9 weeks.

Continued growth in the presence of low doses 8AZ resulted in progression to a higher level of resistance accompanied by a loss of ability to grow in HAT, a reduction in HGPRT activity, and in [14C]hypoxanthine utilisation.

Second step HAT- resistant lines gradually regained their ability to grow in HAT when cultured in the absence of selective pressure over 15--20 weeks. Re- covery of ability to grow in HAT was accompanied by a rise in [14C]hypoxan-

A b b r e v i a t i o n s : 8 A Z , 8 - a z a g u a n i n e ; A Z r , 8 -azaguan lne~res i s t an t l ine ; B U d R ° 5 - b r o m o - 2 ' - d e o x y u r i - d i n e ; 1 0 F , F i s c h e r ' s m e d i u m + 1 0 % horse serum; 20F, Fischer's m e d i u m + 2 0 % h o r s e s e r u m ; H A T , m e d i u m c o n t a i n i n g 4 X 1 0 -6 M a m e t h o p t e r i n , 5 X 1 0 -4 M t h y m i d i n e a n d 1 X 1 0 - 4 M h y p o x a n - t h i n e ; H G P R T + o r H G P R T - , indicates the possession of or lack of hypoxanthine--guanine phospho- riLosyl t r a n s f e r a s e ; LS , r ad iosens i t i ve s t r a i n o f L 5 1 7 8 Y ; L R , r a d i o r e s i s t a n t s t r a i n o f L 5 1 7 8 Y ; 1 0 M E M , Eag l e s ' m i n i m a l essen t ia l m e d i u m + 1 0 % f o e t a l c a l f s e r u m ; MS, multistep selection; P E , p l a t i n g e f f i c i e n c y ; R P E , relative efficiency; SS, s lng lemtep se l ec t i on ; 6 T G , 6 - t h i o g u a n i n e ; T G r , 6 - th io g u a n i n e - r e s i s t an t l ine .

276

thine incorporation and a reduction in level of resistance. The instability of these resistant lines, in contrast to those isolated after single high doses, suggests that they are non-mutants and are the result of drug-induced modifications in cellular biochemistry. The similarity of the phenotypes of these lines with those of many resistant lines described by others suggests that many other purine-analogue resistant lines may be of non-mutant origin. The implications of these observations are discussed.

Introduct ion

Purine-analogue resistant cell lines can be classified according to their stability and other characteristics as follows: (a) lines which are unstable, i.e. "revert" with high frequencies, have variable chromosome numbers, high HGPRT levels and continue to grow in HAT medium [10,30,37]; (b) lines which appear to be more stable with reversion frequencies within the range 1 X 10 -7 to 1 × 10 -4 [6,12,18,27,30]. Within this latter class however, hetero- geneity of phenotype exists. HAT + clones [8,20,23,35,39] with high HGPRT activity and HAT- clones [6,8,18,20,27,30,39] with little or no enzyme activity have both been described, as have lines which show cross-resistance to 8AZ and 6TG or are resistant to only one of the analogues [18,20,29].

Because of the apparent stability of variants in this latter class, they have generally been considered to be true mutants [32,36] and evidence that this is the case for some resistant lines is now available [6,16,29,33,36]. The phenotypic diversity and the progressive changes in enzyme levels and the biochemical properties of such enzymes during analogue exposure has been often interpreted as indicating the occurrence of multiple mutations conferring resistance [32--34,36]. However, several observations (in relation to purine-analogue resistance) led us to question the validity of this interpretation and to search for other factors which could account for the variability.

Firstly, in our earlier studies, stable HAT- high-level resistant clones were consistently isolated when doses of purine analogues well along the plateau region of the dose--response curve were used [18,25], and on extensive charac- terisation, little phenotypic diversity was encountered [18]. Estimation of the selective efficiency of different quoted external analogue concentrations used in different laboratories is difficult because of the .known modification of the dose--response curve by a variety of factors, e.g. hypoxanthine concentration, and presence of absence of degradative enzymes in serum [ 25,26,28,40]. Hence the "region of uncer ta in ty" , i.e. the dose range over which cells are killed less efficiently than in the initial steep part of the curve, but where the curve still has a positive slope before the true plateau region is reached, will vary from one laboratory to another. Thus, unless the complete dose--response curve is defined on each occasion, it is impossible to determine whether clones were isolated from the "resistant tail" or plateau region.

Thirdly, in a previous study [16] using IUdR as a selective agent for TK- variants of mouse lymphoma cells, we found that colonies isolated from the "steep region" and the "region of uncer ta in ty" of the dose--response curve showed transient low-level resistance to IUdR which could be stabilised and

277

increased on repeated exposure to the drug. Thompson and Baker [36] have suggested that surviving colonies from this steep region are generally small and are statistical anomalies resulting from incomplete inhibition of wild-type cells. They will, therefore, be non-mutant. The observations in the mouse- lymphoma system together with other reports suggesting the induction of transient resistance to purine analogues after exposure to low doses [10], and the difficulties of comparing quoted external concentrations of analogues discussed above, led us to examine the phenotypes of clones exposed to sub-toxic analogue concentrations in more detail. In addition, the often reported rapid and step-wise development of resistance to a number of antimetabolites [15,24,32,34] and several reports indicating an equally rapid alteration in target enzyme levels [11] or proportions of isoenzymes [22,24,32] in exposed cells suggests that first-step resistance may be of adaptive origin.

Thus, if low-level resistance is initially adaptive, or induced by the selective drug, and as suggested previously [18], continued growth of partially resistant cells under selective pressure results in a progression towards a higher level of resistance, then much of the observed phenotypic diversity could be accounted for. We therefore under took a detailed study of the relationship between cellular sensitivity, phenotypic stability, and duration of exposure to subtoxic analogue concentrations and compared the phenotypes of lines isolated by single and multistep selective procedures.

Methods

Cell lines Stock cultures of V79A Chinese hamster cells were maintained as mono-

layers as previously described [18,19,25] in 10MEM. Cells were maintained in exponential growth by subculture 2X weekly (1/1000 dilution) in 4 oz. medical flat bottles. Mouse-lymphoma cells LS and LR were derived from L5178Y [16]. Cells were routinely subcultured in suspension in 10F. 20F was used when cells were plated into soft agar (0.25%) for assay of colony-forming ability.

Growth curves and continuous culture in low purine-analogue concentrations For growth curves, V79 cells were trypsinised from exponentially growing

stock cultures, 1 X 106/4 oz. medical flat bott le, and plated into 9-cm plastic petri dishes (Nunc) at a density of 1 X 10S/dish. Purine analogues dissolved in 0.5% Na2CO3 were added to give the desired concentrations to groups of 10 dishes after allowing at tachment of cells (overnight). Pairs of dishes were subsequently trypsinised at appropriate times and cell counts made on an haemo- cytometer .

During the course of such experiments, control and drug-treated cultures were trypsinised and diluted 1/100 when cell densities reached 3.5 X 106/dish. When the interval between successive subcultures of drug-treated cells was greater than 60 h, due to slower growth in presence of analogues, drug contain- ing medium was renewed at 48-h intervals. Dilution factors on subculture of treated cells were adjusted to take account of slower growth. Procedures used for L5178Y cells were essentially similar except that cells were continually subcultured in suspension.

278

Cloning procedures Clones of V79 cells were isolated 7 days after plating 100 cells into replicate

60-mm petric dishes in 10MEM solidified with 0.25% agar. 8AZ 2 pg/ml was included or not as required. Colonies were subsequently visualised under a disecting microscope and one control size colony/dish was isolated using a ster- ile pasteur pipette. Clones were subsequently subcultured 1/500 dilution every 3--4 days in the presence of absence of selective drugs as required. Thus successive subcultures were initiated from low inocula 500 cells/bottle to ensure homogeneity.

L5178Y clones were isolated in a similar way except that 1 X 10 s cells were plated/dish for isolation of drug resistant lines.

Relationship between growth phase and 8AZ sensitivity V79 cells were initially plated into replicate dishes at a density of 5 × 104/

dish. Pairs of such plates were trypsinised at 24-h intervals and after determina- tion of total cells/dish, to establish the growth curve, aliquots of cells were replated into groups of 6 dishes at a density of 5 X 102. 8AZ 2 pg/ml was added to three of the dishes, the remaining three served as controls. When cultures reached stationary phase 1 X 10~/dish the medium was changed daily. 8AZ sensitivity was determined in the same way.

Selection versus induction in the origin o f drug-resistant phenotypes The frequency of spontaneously occurring HAT- purine-analogue resistant

clones isolated after exposure of V79 Chinese hamster cells to high doses of purine analogues has been established as 1--2 × 10 -s [18,25], therefore if only selection occurs when a sensitive population is exposed to such analogues, then: (a) survivors from lower concentrations should be predominantly of wild- type phenotype; (b) continued subculture of a sensitive population in sub-toxic drug concentrations should result in progressive inhibition of growth of sensitive cells followed by eventual outgrowth of a fully resistant fraction at control rate. Back-extrapolation of the outgrowth curve should thus indicate a surviving fraction of approx. 1 × 10 -s as for single-step selection; (c) the phenotypes of the resistant clones isolated under these two conditions should be identical.

Therefore, clones surviving various periods of exposure to low analogue concentrations, i.e. in the steep region of the dose-response curve, were isolated and characterized immediately, or after various periods of growth in the absence of the appropriate selective drug. The following conditions were used: (a) short exposure (up to 1 week) to a low concentration; (b) chronic exposure (2--4 weeks) to the same low concentration with continued sub-culture into fresh selective agent; (c) chronic exposure to gradually increasing drug concentrations. The concentration was doubled when cells grew at control rates in the original concentration. The phenotypes of these clones were compared with those of clones isolated after a single (7 day) exposure to high doses, with respect to level of resistance to 8AZ and/or 6TG and plating efficiency in HAT medium.

Measurement o f HGPR T activity and [~4C] hypoxanthine uptake Techniques used for such measurements have been described in detail previ-

280

109-

108

107-

106

105 o 0 48 96 144 192 240

(a) o/o// ///o

/./2 / / . - ' r

48 96 144 192

(b) 109 .

o

108

z o x

107-

106" o~_~/ /~ /~u

10 5

G

HRS IN 8-AZAGUANINE HRS IN 6-THIOGUANINE

/.;

Fig. l a . G r o w t h of V79 cells as m o n o l a y e r s in the c o n t i n u e d presence of increasing concen t r a t i ons of 8 A Z . Cul tures were in i t ia ted f r o m exponen t i a l l y g rowing s t o ck cu l tu res as descr ibed an d cont ro l s were d i lu ted to m a i n t a i n e x p o n e n t i a l every 2---3 days . Drug- t r ea t ed cu l tu res were d i lu ted and fresh drug ad d ed ~s ~lescribed in m e t h o d s , o, Cont ro l ; $, 1 p g / m l ; A 2 /~g/ml ; z~ 3 pg /ml ; B 5 pg /ml .

Fig. l b . G r o w t h of V79 cells in the c o n t i n u e d presence of increasing c o n c e n t r a t i o n s of 6 T G , o Cont ro l ; X, 0.1 pg]ml ; $, 0 .15 /~g]ml ; A, 0 .2 /~g]ml ; z~ 0.3 pg /ml .

the result of a progressive increase in cell cycle times (0.1 and 0.15 pg/ml) since little change in P.E. was observed at 0.15 pg/ml (Table 2).

(b) Growth of mouse lymphoma L51 78 Y cells in 6TG In common with other workers [1,14] we have found that mouse-lymphoma

cells are relatively insensitive to 8AZ but the dose--response for 6TG was similar to that in V79 cells. It is evident from Fig. 2 that L5178Y cell lines, when exposed to 6TG develop tolerance to low concentrations relatively rapidly, i.e. growth returns to control rates. After 10--12 days exposure, progressive increases in 6TG concentrations produced further growth inhibition, but both cell lines eventually grew at control rates in 20/lg/ml. These populations were cloned and their phenotypes were compared with those of clones isolated after a single (7 days) exposure to 20 pg/ml 6TG which reduced the surviving frac- t ion to approximately 1 X 10 -s.

(c) Phenotype o f cells after 0-28 days exposure to 8AZ or 6TG Cells were continually subcultured in 2/~g/ml 8AZ, and at intervals aliquots

were removed and their sensitivity to 8AZ determined relative to that of a control population. After 3 days, exposure to 2 pg/ml a decrease in PE of treated

281

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1° 9

io ~ >~

.J IO~

/ ! / y/j , 00:\

u.

N 10-2

10' 10 20 30 40 10"3 2 4 6 8 10 -// 3'0

TIME in DAYS 8AZ pg/ml

Fig . 2 . G r o w t h cu rve f o r s u s p e n s i o n c u l t u r e s o f m o u s e - l y m p h o m a cel ls ( L 5 1 7 8 Y LS) , subcul tured co nt in - u o u s l y in 6 T G . Cells w e r e d i luted to mainta in e x p o n e n t i a l g r o w t h in the co ntro l cul tures . The 6 T G wa s r e n e w e d at each sub-culture and the c o n c e n t r a t i o n d ou b led w h e n cel ls g~ew at a p p r o x i m a t e l y co ntro l rates in the presence o f the drug. C o n c e n t r a t i o n s (in ~ g / m l ) used are s tated o n the g r o w t h curve f o r d r u g - e x p o s e d cel ls , a , C o n t r o l , X , t r e a t e d . G r o w t h cu rves f o r L R cel ls w e r e essent ia l ly s imilar to tho se s h o w n f o r LS cel ls .

Fig. 3. Sens i t iv i ty o f V 7 9 cells to 8 A Z af ter c o n t i n u o u s subcul ture in 2 ~ g / m l 8 A Z f o r various per iods o f t i m e . D o s e - - ~ e s p o n s e curves obta ined b y plat ing 5 X 102 cel ls in to 9-era dishes and af ter 4 h i n c u b a t i o n t o a l low f o r cel l a t t a c h m e n t , 8 A Z was added at the appropriate c o n c e n t r a t i o n . Survival p lates w e r e incu- ba ted f o r 7 days be fore f ixat ion , o , c o n t r o l , n o e x p o s u r e t o 8 A Z ; e , dose -response curve af ter e x p o s u r e t o 2 ~ g / m l 8 A Z f o r 2 4 h ; A 4 8 h ; X, 3 d a y s ; ~, 1 4 d a y s ; i , 21 days .

population in 10MEM together with a decrease in 8AZ sensitivity was evident. The fail in PE is presumably due to killing of cells of wild-type phenotype. The surviving cells, although resistant to 10 pg/ml were not however, resistant to 30/ag/ml a concentration which consistently resulted in a surviving fraction of 1--2 X 10 -s when used as a single exposure {Fig. 3). Table 1 summarises the data on changes in relative plating efficiencies of 2/ag/ml grown cells over a 3-week period. After 20 days of continuous sub-culture in 8AZ, resistance to 30 #g/ml developed which was correlated with loss of ability of exposed populations to grow in HAT medium. Cultures resistant to only 10 pg/ml were consistently characterised by their ability to grow in HAT medium at the same rate and with the same PE as they did in 10MEM. Similar data have been obtained after exposure of cells to 1 pg/ml, a concentration which did not significantly affect PE in 10MEM. The results of a similar experiment in which cells were cultured in the presence of 0.15 #g/ml 6TG are shown in Table 2. After 2 days exposure, all cells became resistant to 1.0/~g/ml 6TG, by 7 days a few cells resistant to 5/~g/ml were evident but they still retained their ability to grow in HAT medium. Thus, adaptation to the presence of both 8AZ and 6TG occurs in these V79 cells.

282

T A B L E 1

D E V E L O P M E N T OF R E S I S T A N C E TO SAZ D U R I N G C O N T I N U E D G R O W T H IN P R E S E N C E OF 2

# g / m l 8 AZ

Days g r o w t h in 8AZ (2 ~tg/ml)

RPE in var ious tes t m e d i a a

MEM 2AZ 1 0 A Z 3 0 A Z H A T

0 1.0 0 .36 "~0.002 < 0 . 0 0 2 0.93 1 1.0 0 .65 < 0 . 0 0 2 < 0 . 0 0 2 0.69 2 1.0 0 .45 ~ 0 . 0 0 2 ~ 0 . 0 0 2 0.87 3 0 .16 0 .16 0 .16 ~ 0 . 0 0 2 0 .25 5 0 .17 0.2 0 .18 < 0 . 0 0 2 0 .22 7 0.21 0.21 0.21 < 0 . 0 0 2 0 .25 11 1 .05 1 .20 1.03 ~ 0 . 0 0 2 1.00 14 0.97 0.7 0 .77 0 .0 2 5 0 .76 21 0 .45 n t 0.4 0 .45 0 .0 7 5 28 0 .48 n t 0.6 0 .52 ~ 0 . 0 0 2

nt = n o t t e s t ed . The cell p o p u l a t i o n was con t inuous ly s ubc u l t u r e d in 8AZ, at the t i m e shown , a l iquots were r e m o v e d and p la t ed 5 × 102 cells/dish in to s u p p l e m e n t e d med ia . A f t e r 7 d a y s the n u m b e r s o f surviving colonies were scored . MEM, drug-f~ee m e d i u m . 2AZ; 10AZ; 30AZ, m e d i u m con ta in ing 2 p g / m l ; 10 p g / m l and 30

p g ] m l o f 8AZ. a PE of wi ld - type cells in 10MEM was 0.8, all o t h e r resul ts are expressed relat ive to this value.

Tests for stability of resistance (a) Low-level resistance in Chinese hamster cells To test whether individual cells in the V79 population behaved identically

with respect to resistance induction and to ensure that when stability of resistance was determined there could be no overgrowth by preexis t ing sensitive cells the previous experiment was repeated on several clones isolated 7 days after plating in 2 pg/ml 8AZ. Results identical to those described above were obtained, all clones isolated from 2 pg/ml showed resistance to 10 pg/ml i.e. when normal size surviving clones were tested, none had a wild-type phenotype. Resistance to 30 #g/ml developed in all clones after a further 14 days growth in 2 pg/ml 8AZ. Four further clones similarly isolated, were cultured in 2 pg/ml 8AZ for a further 0--1 weeks then subsequently in MEM. These clones

T A B L E 2

P R O G R E S S I V E D E V E L O P M E N T OF R E S I S T A N C E TO 6 TG ON C U L T U R E IN LOW DOSES OF T H E

A N A L O G U E

Per iod o f g r o w t h in 0 .15 ~ g / m l 6 T G (h)

Mean n u m b e r o f co lon ies (3 pla tes) in 6 TG a

in M E M 1 .0 ~ g / m l 2 .0 p g / m l 5 # g / m l

0 276 0 0 0 48 233 229 0 0 96 245 261 139 0 168 341 359 282 3 216 257 238 168 4 264 177 176 178 2

S.D. o n c o l o n y c o u n t s was less t h a n 10%. a 5 × 10 2 cells were p la t ed on each 9 -cm dish.

283

tO0

i

o_ c=

,0I

%. 5u gln~l ~.pg/ml

x\\

Weeks growth in MEM

Fig. 4. Changes in 8 A Z sensi t iv i ty w i t h durat ion of subcu l tu re in MEM of c l o n e s i so la ted after e x p o s u r e t o 2 ~zg/ml 8 A Z for e i ther 1 or 2 w e e k s . Co lon ie s w e r e i so la ted 1 w e e k a f ter plat ing in agar conta in ing 2 /~g /ml 8 A Z . Several c lones w e r e cu l tured in 1 0 M E M f r o m the t i m e o f i so la t ion o n w a r d s , o thers in 8 A Z conta in ing m e d i u m for a further w e e k b e f o r e culturing in 1 0 M E M . 8 A Z sensi t iv i ty w a s d e t e r m i n e d b y plating a l iquots f r o m such cul tures 5 X 10 2 / 9 - c m dish to w h i c h approp1~late c o n c e n t r a t i o n s o f 8 A Z w e r e added . The n u m b e r o f surviving c lones was d e t e r m i n e d 7 days later , Resul ts s h o w n are typ ica l o f r e sponse o f several c l o n e s t e s t ed in each case o and e ; sens i t iv i ty of a c lone i so la ted after 1 w e e k o f expost tre to 8 A Z t o subsequent chal lenge w i t h 5 # g / m l e ; a nd 10 # g / m l o; ~ an d • as a bo v e for c lones e x p o s e d for 2 w e e k s . Plating e f f i c i e n c y in 8 A Z is expres sed relat ive to that o f the s a m e c lo ne in 10MEM (RPE) ,

were tested for stability of resistance to higher 8AZ concentrations at weekly intervals. Data for two such clones which were resistant to 10 ~ug/ml 8AZ are shown in Fig. 4. Resistance to both test doses of 8AZ was gradually lost at a similar rate in both clones so that after 9 weeks growth in 10MEM the sensitivity of the clones was approaching that of the wild-type population.

(b) High-level resistance in Chinese hamster cells Clones of Chinese hamster cells which had developed resistance to 30 #g/ml

as a result o f exposure to 2 pg/ml for 3 weeks or longer, were subsequently cultured in 10MEM and tested weekly for stability of resistance and HAT sensitivity (Table 3). No detectable loss of resistance to 30AZ occurred initially but after 45 days a significant frequency of HAT+ colonies was detected. The frequency of HAT+ colonies progressively increased so that by between 16--18 weeks it approached 100%. This increase in HAT+ colonies was associated with a progressive loss in 8AZ resistance. To test whether the observed loss of resistance was due to progressive overgrowth of wild-type HAT* cells, 6 clones were isolated from the 8AZ-resistant line after 11 weeks growth in 10MEM. Such clones were independantly maintained in 10MEM and changes in their phenotype monitored by weekly plating of each clone into media supplemented with 8AZ or HAT. The results of this experiment (Table 4) demonstrate that, with a l lc lones 8AZ resistance was lost at a rate similar to that in the uncloned population.

Variation in 8AZ sensitivity with growth phase Since it is possible that the observed resistance to 8AZ is associated with

284

T A B L E 3

W e e k s i n C l o n e M e a n c n u m b e r o f c o l o n i e s

M E M a n u m - (3 p l a t e s ) / 5 × 102 ce l l s p l a t e d in b e t b

1 0 M E M 3 0 A Z H A T

M e a n n u m b e r

c o l o n i e s / 5 × 104 H A T

S . F . i n

3 0 A Z H A T

0 1 1 2 7 1 3 7 0

2 2 2 0 1 6 6 0 3 1 3 5 1 3 9 0 4 1 7 6 1 5 3 0

1 1 4 6 1 5 1 0 0

2 1 8 6 2 1 6 0

2 1 4 1 9 4 6 3 0

2 3 8 1 3 9 4 0

3 1 8 2 1 8 5 0

4 1 4 6 1 7 7 0

6 1 1 4 4 1 5 8 0 2 1 9 0 2 2 8 0

3 1 3 5 1 3 8 0

8 1 4 0 5 5 2 1 n d

2 3 4 6 5 0 4 n d 3 2 1 1 2 3 2 n d 4 2 1 9 2 3 7 n d

9 1 3 0 9 2 9 2 n d 3 2 8 4 3 1 0 n d

4 2 6 2 2 7 0 n d

11 1 3 2 6 3 8 1 3 7 . 0 ± 3 . 2

2 3 4 6 3 9 0 4 1 . 0 ± 2 .6

12 1 4 5 1 3 9 9 86 ± 1 2 . 0

13 2 3 8 2 3 0 5 9 7 ± 9 . 0

14 1 2 1 6 1 1 1 1 0 0 ± 7 . 0

2 2 2 0 1 1 2 1 0 1 ± 6 . 9

1 5 1 2 5 7 79 1 7 7

16 1 4 3 8 1 2 3 4 2 7 ± 8 . 5

0

0

0 0

0

0

0 0

0 3 . 0 ±

0 . 7 5 -+

1 3 . 0 +

5 1 . 0

1 9 8 . 0

8 . 0 1 2 . 6

1 6 8 . 8

t m

t m

n d

n d

n d

n d

n d

n d

1 .9

0 . 8 2

4 .6

-+ 6 . 4

± 1 4 . 2

± 3 . 5 -+ 2 . 1 4

± 3 1 . 0

1 . 0 7

0 . 7 5

1 . 0 2 0 . 8 6

1 . 1 0

1 . 1 6

1 . 1 0

1 . 0 3

1 .01

1 .21

1 . 0 9 1 .2 1 . 0 2

1 . 2 8

1 . 4 5

1 . 0 9 1 . 0 8

0 . 9 4 1 . 0 9

1 . 0 3

1 . 1 6

1 . 1 2

0 . 8 8

0 . 7 9

0 . 5 1

0 . 5 1

0 . 3 1

0 . 2 8

m

0 . 0 0 0 1 5

0 . 0 0 0 1 5

0 . 0 0 1 3

0 . 0 0 1 4

0 . 0 0 9 0

0 . 0 0 0 2 0 . 0 0 0 4

0 . 0 0 6

0 . 1 1

0 . 1 2

0 . 1 9

0 . 2 5

0 . 4 6

0 . 4 6

0 . 6 8

0 . 9 7

a O r i g i n a l c l o n e s w e r e g r o w n f o r 4 w e e k s i n 2 # g / m l o f 8 A Z .

b C l o n e s w e r e t e s t e d i n w e e k s 3 - - 5 , p h e n o t y p e as f o r w e e k 2. c S D o n c o l o n y c o u n t s w a s less t h a n 1 0 % . n d , n o t d o n e .

t i n , t o o m a n y c o l o n i e s t o c o u n t .

T A B L E 4

C H A N G E S I N R P E s O F T H E O R I G I N A L P O P U L A T I O N A N D 6 S E P A R A T E L Y I S O L A T E D S U B - C L O N E S W I T H T I M E O F G R O W T H I N M E M

W e e k s R a t i o o f n u m b e r o f c o l o n i e s i n 3 0 p g / m l 8 A Z / n u m b e r o f c o l o n i e s in H A T in M E M

O r i g i n a l S e p a r a t e l y i s o l a t e d c l o n e s p o p u l a t i o n a

1 2 3 4 5 6

1 4 1 . 1 0 1 . 1 0 1 . 2 1 1 . 1 0 1 . 0 5 1 5 0 . 4 4 0 . 6 6 0 . 5 7 0 . 8 8 0 . 7 8 16 0 . 2 9 0 . 1 6 0 . 4 1 0 . 2 8 0 . 2 3 18 0 . 1 3 0 . 1 5 0 . 3 0 0 . 4 0 0 . 1 6 20 0 . 0 3 0 . 0 6 0 . 0 9 0 . 1 0 0 . 0 3

1 . 5 1 .5

0 . 0 6

1 . 0 5 0 . 6 8 0 . 4 2 0 . 2 4 0 . 0 8

a Original population was clone I of Table 3.

285

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.,: I0~

E z

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/o //o / [ o

~ o

<

"0 I0 -r

co

N

m

10-2

10-3 I I ~'0 8'0 i~o i~o 20o 2~0 2~0 Hours after Plating IO 5 Celts/Dish

Fig. 5. Sensit ivity to 8 A Z relative to gro~vth phase o f popu la t ion . Cells were initial ly plated at a dens i ty o f 1 X 1 0 S / m e d i c a l fiat bot t le in 10MEM and at intervals bot t l e s were trypsinised, their cel l c o u n t recorded and al lquots plated into 2 ~zg/ml 8 A Z a t a d e n ~ t y o f 5 X 1 0 2 [ 9 - c m p l a t e . A l i q u o t s were also plated at a dens i ty o f 5 X 102 [plate in MEM to determine P E . 8 A Z sensit ivity is expressed relative to control PE and results f rom several such exper iments are s h o w n , e , cell number/d ish; o , R P E i n 2 / ~ g / m l 8 A Z .

changes in endogenous purine pools related to a particular growth phase, the sensitivity of cells to 8AZ at different phases of growth was determined. The results are shown in Fig. 5. In early exponential growth, sensitivity to 8AZ was high, but showed a considerable decrease as cells progressed through exponential and approach stationary phase. In stationary phase, the PE of the population fell to 50% but the surviving viable cells were all resistant to 2 #g/ml 8AZ. Therefore, careful control of cell density of cultures from which cells are har- vested is necessary if reproducible dose--response data is to be obtained, e.g. in 10 separate previous determinations of cell sensitivity to 2/~g/ml 8AZ survival values ranged from 0.1--75% when the cell density of the parent culture was not precisely controlled.

Many other factors have been reported to affect the survival of cells plated into 8AZ, including variation in serum batch with respect to endogenous pu- fine concentration [28], breakdown of analogues by serum enzymes [40], and plating density [8,25,35]. We, however, have been unable to show significant effects of variation in serum batch, or breakdown of the purine analogues during preincubation in medium [25]. The magnitude of the change in sensitivity with growth phase suggests that this also may be a source of uncontrolled variability in cellular response.

Biological phenotype and stability of TG r clones of mouse-lymphoma lines Both single and multistep TG r clones from the mouse-lymphoma lines were

cultured in 10F for approximately 3 weeks between isolation and characterisa-

286

T A B L E 5

C O M P A R I S O N OF T H E S E N S I T I V I T Y OF W I L D - T Y P E S I N G L E AND M U L T I S T E P TG r MOUSE- L Y M P H O M A L I N E S A N D T H E I R R E V E R T A N T S TO S U B S E Q U E N T E X P O S U R E TO H A T AND 6 TG

Cell line Surviving fraction in 6TG HAT

5 ~ g / m l 10 /~g/ml 15 # g ] m l 20 # g / m l

LSWt 1.1 X 10 -5 2.0 × 10 - s 1.0 × 10 -5 1.3 × 10 -5 1.0 LRWt 5.0 × 10 -S 3.0 _+ 10 -5 1.3 × 10 -5 1.0 + 10 -S 0 .88 LSTG r (MS) 0 .96 0 .98 0 .96 0 .96 0 LSTGr(SS) 0 .92 1.0 0 .97 0 .97 0 L R T G r ( M S ) 1.0 0 .96 0 .56 0 .40 2.32 X 10 -5 L R T G r ( S S ) 0.99 0 .99 0 .93 0 .93 0 L R T G r ( R 1 ) 0 nd nd 0 0 .90 L R T G r ( R 2 ) 0 .88 nd nd 0.69 0 .93 L R T G r ( R 3 ) 0 .57 nd nd 0 .26 0.99

Wt, wild t y p e ; MS and SS, mul t i - and single-step res is tance select ion, r espec t ive ly ; nd, n o t d o n e .

t ion of their biological and biochemical phenotypes. A summary of their biological properties is presented in Table 5. Both single and multistep TG r mutants of the LS line were fully resistant to 20 pg/ml 6TG and did not grow in HAT. The single-step clone isolated from the LR line was also fully resistant, but the multistep " m u t a n t " from this line had a consistently " l eaky" phenotype in that only 40% of the population was resistant to 20 pg/ml 6TG and a low frequency of clones was consistently observed on plating in HAT.

3 such HAT + clones were isolated from the LR TG r multistep line, and designated R~--R3. Only R~ behaved as a true revertant i.e. was 6TG-sensitive, R2 survived in HAT probably because it was also amethopterin resistant (Fox and Dale, unpublished} whilst R3 remained partially 6TG-resistant.

Biochemical pheno type The biochemical properties of these lymphoma lines, their TG ~ and HAT +

derivatives are summarised in Table 6. HGPRT activities were residual in both the parental resistant lines, one rever-

tant (R1) had wild-type enzyme activity and high [~4C]hypoxanthine uptake consistent with its ability to grow in HAT. R2 had very low HGPRT activity and was TG r whilst R3 had approximately 25% of normal HGPRT activity and showed partial resistance to 6TG. [~4C] Hypoxanthine uptake in this clone was markedly increased in the presence of amethopterin.

HGPRT activity and [14C]hypoxanthine uptake in V79 cells resistant to 8AZ The HGPRT activities in wtV79 cells grown in 2 pg/ml 8AZ for up to 56

days were compared with those of cultures grown in MEM (Table 7). At 7 days 8AZ-treated cells were already resistant to 10 pg/ml but possessed as much HGPRT activity as the control population. On further subculture a progressive fall in enzyme activity of the treated population was observed consistent with the progression towards higher levels of 8AZ resistance and loss of ability to grow in HAT (Table 1). [14C]Hypoxanthine uptake, starting with cells grown in 2 pg/ml for 4 weeks, was initially ~2% of control. After 11 weeks in MEM up-

287

T A B L E 6

U P T A K E O F [ 1 4 C ] H Y P O X A N T H I N E A N D H G P R T A C T I V I T I E S IN W I L D - T Y P E M O U S E - L Y M P H O M A L I N E S , T G r V A R I A N T S A N D T H E I R " R E V E R T A N T S "

Cell l ine H G P R T n m o l / h / m g prote in

[ 1 4 ] H y p o x a n t h i n e uptake d . p . m . / l O 5 cel ls X 10 --4

- - A m e t h o p t e r i n + A m e t h o p t e r i n

Hot acid Cold acid Hot acid Cold acid soluble a soluble b soluble soluble

L S W t 3 6 3 . 6 5 .0 1 .5 7 .0 2.6 L R W t 3 2 9 . 6 5 .0 3 .5 2.9 1.9 L S T G r ( M S ) 0 .91 0 . 0 0 1 0 . 0 9 0 . 0 0 1 0 .1 L S T G r ( S S ) 0 .47 0 . 0 0 1 0 . 1 5 0 . 0 0 1 0 .1 L R T G r ( M S ) 0 . 6 7 0 . 0 0 1 0 . 1 2 0 .09 0 . 1 4 L R T G r ( S S ) 0 . 8 3 0 . 0 0 1 0 . 1 0 0 . 0 0 1 0 . 1 2 L R T G r ( R 1 ) 4 1 1 . 3 0 2 .3 0 .6 2 .0 1 .5 L R T G r ( R 2 ) 2 . 3 0 0 . 0 0 1 0 .3 0 . 0 0 1 0 .3 L R T G r ( R 3 ) 1 2 1 . 2 0 0 . 0 0 7 0 . 2 0 .8 0 . 5

Abbreviat ions as for Table 5. a Hot acid soluble radioactivity represents that incorporated into nucleic acids, and extractable wi th h o t 5% perchloric acid. b Cold acid soluble , that incorporated into cell poo l s , and extractable wi th co ld 7.5% perchloric acid.

take was 6% of control and by 13 weeks was 90% of control values (Table 8). No effect of amethopterin was detectable. The rise in hypoxanthine uptake parallels the rise in HAT+ colonies (Tables 3 and 4).

Fluctuation tests at high and low doses of 8AZ Conventional fluctuation tests were performed as described in Methods and

results are shown in Table 9. When 5 × 102 cells/dish were plated into 2 pg/ml the variance/mean ratio exceeded unity in both independently maintained cultures and in replicate samples from the same culture. Plating of cells (5 X 104/ dish) from independently maintained cultures into 30 pg/ml 8AZ gave a consid- erably higher variance however, than did replicate plating from the same culture. In this latter case, the variance/mean ratio was close to unity.

Clones surviving exposure to 2 pg/ml also survived exposure to HAT medium whereas those surviving 30/~g/ml were HAT-sensitive. We have already presented data suggesting that clones surviving 2 pg/ml show induced transient

T A B L E 7

H G P R T A C T I V I T Y IN V 7 9 C E L L S IN 2 ~ g l m l 8 A Z F O R I N C R E A S I N G P E R I O D S IN TIME

Time o f HGPRT acitivity sampling n m o l / h / m g (days)

% Control

0 1 5 4 . 6 6 133 .9 86 .6 14 1 6 2 . 2 1 0 4 . 9 28 6 1 . 3 3 9 . 6 56 1 5 . 5 1 0 . 2

b~

00

00

TA

BL

E 8

[14

C]H

YP

OX

AN

TH

INE

U

TIL

ISA

TIO

N

WIT

H

TIM

E

OF

G

RO

WT

H

IN M

EM

IN

V7

9

CE

LL

S

INIT

IAL

LY

E

XP

OS

ED

T

O

2 p

g/m

l 8

AZ

F

OR

4

CH

AN

GE

S

IN

WE

EK

S

Co

ntr

ol

Tim

e o

f [1

4C

]Hy

po

xa

nth

ine

u

pta

ke

d.p

.m./

10

5

cell

s X

10

-3

aft

er

4 h

inc

ub

ati

on

8A

Z-H

AT

+

gro

wth

in

M

EM

wee

ks

Ho

t ac

id s

olu

ble

a

Co

ld a

cid

so

lub

le b

T

ota

l %

Co

ntr

ol

--A

me

th

+A

me

th

--A

me

th

+A

me

th

--A

me

th

+A

me

th

11

.2

11

.5

11

.9

14

.3

23

.1

25

.9

--A

me

th

+A

me

th

Res

ista

nt

1 0

.01

4

0.0

26

0

.3

0.5

0

.31

4

0.5

26

1

.3

2.0

v

aria

nt

11

0

.6

0.7

0

.8

1.0

1

.4

1.7

6

.0

6.5

8

AZ

+H

AT

- 1

2

3.9

4

.2

6.2

6

.2

10

.1

10

.4

43

.7

90

.3

13

1

2.6

1

1.6

1

1.0

1

1.8

2

2.6

2

3.4

9

7.8

9

0.3

a H

ot

acid

so

lub

le r

ad

ioa

cti

vit

y i

s th

at

ex

tra

cta

ble

wit

h h

ot

5% p

erc

hlo

ric

aci

d.

b C

old

aci

d s

olu

ble

ra

dio

ac

tiv

ity

is

tha

t so

lub

le i

n c

old

7.5

% p

erc

hlo

ric

aci

d.

TA

BL

E 9

LU

RIA

--D

EL

BR

UC

K F

LU

CT

UA

TIO

N

TE

ST

IN

V7

9 C

EL

LS

AT

DIF

FE

RE

NT

D

OS

ES

OF

8

AZ

Ind

ep

en

de

nt

cu

ltu

res

Re

pli

ca

te c

on

tro

ls

Ex

ptl

A.

Ex

ptl

.l.

Ex

ptl

.1.

Ex

ptl

.1

Nu

mb

er

of

cult

ure

s 1

5

15

1

Init

ial

nu

mb

er

cell

s/cu

ltu

re

10

1

0

10

F

inal

nu

mb

er

cell

s/cu

ltu

re X

10

S

4.2

-+

0.4

3

5.0

4 +

0.6

1

16

.0

Nu

mb

er

of

pla

tes/

cu

ltu

re

5 5

10

C

orr

ec

tio

n r

atio

(F

inal

nu

mb

er

cell

s/ce

lls/

pla

ted

) 1

.66

2

.0

3.3

1 1

0

20

.0

15

2.2

Cu

ltu

re

nu

mb

er

Me

an

(5

pla

tes)

co

lon

ies/

dis

h

Nu

mb

er

of

co

lon

ies/

dis

h

Ex

ptl

. 1

Ex

p tl

. 2

Ex

ptl

. 1

Ex

ptl

. 2

2 /~

g/m

l 3

0

#g

/ml

2 Iz

g/m

l 3

0 p

g/m

l 2

#g

/ml

30

#

g/m

l 2

~zg

/ml

30

pg

/ml

1 6

6

2 4

3

3 1

9

4 3

8

5 5

7

6 4

1

7 3

6

8 7

2

9 1

01

10

6

0

11

2

7

12

2

2

13

3

8

14

7

9

15

4

0

Me

an

/sa

mp

le

49

.2

Va

ria

nc

e/s

am

ple

4

87

.4

Va

ria

nc

e/m

ea

n

9.9

4

Me

an

mu

t/c

ult

ure

8

1.6

X 1

02

M

uta

tio

n r

ate

0 2

9

0 4

1

5 1

2

37

0

.2

12

1

1 4

1

0 3

3

6

0.2

1

5

0 1

5

2 0

.4

10

0

16

2

0 3

7

0 3

9

1

0 4

6

0 4

8

1 0

27

0

24

1

0 3

1

0 1

1

2

0 2

2

0 1

8

4 0

53

0

0 3

5

0.2

0 1

2

0 1

44

--

0

16

0

.2

0.9

7

30

.2

0.0

4

25

.7

2.5

8

.9

12

8.4

0

.00

14

1

63

.6

2.9

6

9.1

7

4.2

5

28

.5

6.3

1

.18

1

.61

6

0.6

× 1

02

0

.08

4

82

.2 X

10

2

8.2

1

.7 X

10

-6

2.5

X 1

0 -7

41

3

20

5

22

2

27

1

23

5

29

1

41

2

37

1

27

1

30

1

39

3

48

1

24

2

11

1

18

4

29

.1

2.2

9

7.1

2

.02

3

.3

0.9

18

1

18

X 1

02

5

.7

t~

(30

¢.0

290

resistance and the data from the fluctuation tests indicate that such colonies apparently arise at random in both replicate control and independently maintained cultures.

The mutat ion rate to resistance to 30 pg/ml is similar to that quoted by other workers [9,36] i.e. in the range 2.5 X 10 -~ to 1.7 X 10-6/cell/generation.

Discussion

Phenotypic resistance to purine analogues was apparent after 72 h exposure to low doses of both analogues. These observations indicate that resistance is induced for the following reasons. (1) The frequency of spontaneously occurring HAT-AZ r clones is 1--4 X 10 -S therefore, exposure of the sensitive population to the drug levels which show little effect on the PE of the population would not allow selection of this resistant fraction within 3 days (Fig. 6 illus- trates the number of population doublings which would be required to select such pre-existing mutants if no induction of resistance occurred). (2) The phenotype of these more frequent resistant clones differs markedly from that of the apparently pre-existing spontaneous mutants in that they initially show a lower level of resistance, are HAT + and HGPRT ÷. (3) Cell lines isolated after low dose exposure (1--2 weeks) are phenotypicaUy unstable and a progressive loss of resistance is detectable from 4 weeks onwards.

-~ IO l

u~

10-2

o ~. 10-3

10-4

I0"~

Populatlon doublings

2 4 6 8 I0 12 14 16 18 20 22 24

/ / / /"

/ • Le,,dese e o High dose

o/.

?' 2'0 6'0 ' ' ~ I00 140 180 2 0

Hrs growth in 8AZ

Fig. 6. R e l a t i o n s h i p b e t w e e n n u m b e r of p o p u l a t i o n d o u b l i n g s and t i m e requ ixed to se lec t ou t p re -ex i s t ing m u t a n t s in i t ia l ly p r e sen t a t a f r e q u e n c y of 1 X 10 -5 . o , ca lcu la ted on the a s s u m p t i o n t h a t if a h igh S A Z dose is g iven , w t cells are c leared f r o m the p o p u l a t i o n w i t h a ha l f t i m e of 20 h ; o, ca lcu la ted on the a s s u m p t i o n a 6ofold increase in p .d . t , of w i ld - type cells in t h e p r e s e n c e of 8 A Z . I f on ly a 3-fold increase in p .d . t , o c c u r r e d t h e n it w o u l d t a k e 270 h b e f o r e 50% o f the p o p u l a t i o n c o m p r i s e d o f p re -ex i s t ing 30 Mg/ml

res i s tant cells.

291

Many other workers have described purine-analogue resistant clones with a HAT* phenotype which (in some cases} appeared to be stable [8,20,23] (and reviews [33,36]). However, in the present study more than 100 population doublings in the absence of selection were necessary before a significant decline of this first-step resistance was detected. Moreover, since these clones are resistant only to a limited range of analogue concentrations, it seems likely that many clones isolated from regions of the dose--response curve which show a markedly reduced, but still positive slope will be of this type, and although resistant, may not be mutant [14,35]. These observations could affect the interpretation of other data: (a) it is possible that the complex dose-response curves which have been obtained with purine analogues and other antimetabolites reflect an inducible fraction in the population [1,14,34,35]; (b) we have reported that clones isolated after growth (1 week) in HAT medium show an enhanced resistance to amethopterin [19]. This resistance is probably the result of a rise in dihydrofolate reductase activity, since the activity of this enzyme has been shown to increase approximately 3-fold within 48 h of exposure to folic acid analogues [11]; (c) the reported lack of effectiveness of 8AZ compared with 6TG in mouse-lymphoma cells [1,13,14] suggests that these cell lines are particularly susceptible to induction of resistance by the former analogue. The reports from several laboratories that 6TG is the more effective selective agent for several other cell lines [35,40] (including V79) may also be related to the relative effectiveness of the two analogues in inducing resistance in cells of different origins which may in turn be related to the preferential incorporation of 8AZ into RNA [3].

Thus, survivors of purine-analogue exposure can be of several distinct types depending on dose used.

(1) Non-mutant, non-resistant clones. The small clones described by Thacker et al. [35], apparently survive because they incorporated insufficient purine analogue for the following reasons: (a) the external analogue concentration may be too low to overcome intrinsic purine pools, (b) cells are in an intrinsi- cally resistant growth phase, e.g. sensitivity to 8AZ and also to methotrexate [11] decreases as cells progress from early to late exponential growth, (c) there may be competition between the applied concentration of analogue and extrinsic purine sources, e.g. purines present in serum or released by killed cells, (d} temporarily non~ycling of slowly cycling cells, e.g. those suffering mutagen induced division delay may show transient resistance as a result of reduction of DNA and RNA synthetic rates and consequently the size of purine pools.

(2) Non-mutant clones showing quasi-stable first step "induced" drug resistance. These clones are HAT* and their resistant phenotype is probably the result of drug-induced modifications in cell biochemistry. Their frequency decreases with increasing concentration of analogue since this first-step resistance permits survival only over a limited concentration range (Fig. 3). We have already argued that the acquisition of resistance is probably the result of drug- induced changes, because of the difference in phenotypes between the first-step resistant clones and "pre-existing" mutants and the rapidity of onset of resistance. The rate of loss of resistance on the other hand is slow and appears to be related to the level of resistance acquired and the duration of exposure to selective pressure. It seems unlikely in view of the data in Table 4 that loss of resis-

292

tance is due to selective overgrowth of resistant cells by those with a wild-type phenotype.

Thus we suggest that the rapid acquisition of purine-analogue resistance in V79 cells and its subsequent progressive loss on growth under non-selective conditions may be related to reversible changes in the relative affinity of HGPRT for hypoxanthine and the two purine analogues in exposed cells.

Changes in relative proportions of multiple molecular forms of several enzymes during the development of antimetabolite resistance have been reported [22,24], e.g. in Novikoff haematoma cells exposed to azacytidine, resistance was evident by the fourth transplant generation and was associated with a relative increase in uridine kinase enzyme with a lower Km for uridine. These early changes were essentially reversible on removal of cells from selective pressure.

(3) Non-mutant clones showing second-step resistance which is unstable. In populations exposed to low<lose selective pressure over long periods, there was a progression towards a higher level of resistance accompanied by a fall in the proportion of cells able to grow in HAT and in the average HGPRT activity. The resultant phenotype was thus indistinguishable from that of clones isolated after a single short exposure to a high selective dose.

If, once the lines have become HAT- they are removed from selective media and grown in 10MEM, no detectable loss of 8AZ resistance occurs initially. However, the cells gradually regain their ability to grow in HAT so that by 16 weeks the "reversion" frequency was near 100%. Thus, although HAT ÷ clones were occurring at an undetectable frequency for the first 5--6 weeks, this frequency progressively increased with duration of culture in MEM. This behav- iour is typical of chronically selected V79 clones and also of LR clones isolated by multistep selection although the latter were not studied in such detail.

A similar progression, i.e. a fall in PEs in HAT paralleled by a reduction in TK activity was reported by Harris [21] in segregants from a hybrid clone when continually cultured in BUdR. These changes were interpreted as being induced by the selective agent, however, the possibility that resistance was induced in the two parental lines and in the original early segregants was not considered.

The level of methotrexate resistance in L5178Y cells selected by multistep procedures also shows a progressive decline on removal from selective conditions [15], and over a similar time scale. Similar slow declines in levels of resistance on cultivation in the absence of selective pressure have been reported for Chinese hamster cell lines selected by multistep procedures, for resistance to actinomycin D, amethopterin in BUdR [7]. Thus, the wide range of revertant frequencies which have been reported in the literature (see Introduction) may well be purely a reflection of the time interval which has elapsed between removal from the selective purine analogue and plating in HAT medium.

The second problem in interpretation of reversion frequencies is the possible multiplicity of phenotypes associated with growth in HAT medium [19]. The data for L5178Y indicate that this phenotypic diversity is no t restricted to V79 since at least 3 different phenotypes were associated with HAT + clones derived from a multistep TG ~ L5178Y mutant .

These observations indicate that even reports of very low reversion frequen-

293

cies, in lines isolated by multistep selection, e.g. 1R and A9 [4,5,31,42] should be viewed with some caution as many such lines are routinely maintained under selective pressure and reversion frequencies may have been measured only after a very limited period of growth in the absence of selection [27]. Our observations suggest that these lines are not true mutants in agreement with Watson et al. [42]. Preliminary chromosome analysis of our unstable resistant lines has detected no associated karyotypic instability, however, our observations can be reconciled with those of Terzi [37,38] (and others, see [38]) as follows:

The initial event on exposure of wild-type cells to antimetabolites is the induction of resistance in cells of normal karyotype. However, if the frequency of chromosomal re-arrangements and non<lisjunction in cultured cells is increased in the presence of selective agents, then when resistant lines are maintained under selective conditions, cells with altered gene dosage may well have a selective advantage, and continue to proliferate. They will therefore, increase in proport ion relative to the rest o f the population. Hence the observations of an association between cells with abnormal karyotype and resistance. On removal from selection, cells with abnormal karyotypes may disappear from the population as there may no longer be a selective advantage and the karyotype of these populations will tend towards that of wild-type cells.

(4) Mutant clones with stable resistance, and no measurable spontaneous reversion frequency. We have no evidence that lines isolated after a single exposure to high doses of selective agents are unstable. In fact, we have observed no change in phenotype and no spontaneous revertants after more than 2 years continuous cultivation in the absence of selection. These clones are HAT-, show residual HGPRT levels and do not incorporate hypoxanthine even in the presence of amethopterin. The majority of evidence available suggests that such clones are true mutants [6,18,19,30,33,36,41].

That such drug-resistant clones preexis t in the wild-type population has frequently been inferred from results of fluctuation tests [8,23,38]. However, many published fluctuation tests have been performed at sub-toxic analogue concentrations which allow the survival of a variety of phenotypes, e.g. HGPRT ÷ HAT* colonies [8,23] and in many cases, the phenotypes of the clones scored has not been checked. The interpretation of fluctuation data as indicating both random and non-adaptive origin of resistant clones should now therefore be questioned [8,38]. Such tests may well indicate random events (Table 9) bu t since the only way of detecting~resistance of antimetabolites is to plate cells into the selective drug, one cannot exclude the adaptive origin of some resistant clones.

The multiplicity of phenotypes reported for purine-analogue resistant clones therefore appear to be associated with multiple mechanisms of origin both genetic and adaptational. The various possible classes of resistant clones which can occur are illustrated in Fig. 7.

In conclusion, the present data suggest: (1) That many, though not all, drug- resistant lines, which arise both in vitro and probably in vivo, during tumour chemotherapy are of non-mutational origin. The frequently reported step-wise development of resistance [ 7,15,22,24] probably therefore reflects the progressive adaptation of cells to the presence of the selective drug, and is not the result of multiple mutations conferring resistance. (2) Adaptat ion or induction

294

Induction selection and chromosome variation in the origin of resistant

variants in cultured mammalian cells

~ ' ) Near diploid initial sensitive cell

I ~ Growth in 8AZ 2/~g/ml ~ ~3-7 daysl

o k ~ ~ 6~- Induced resisr.ance I st step ~ 8AZ ~ HAT~

°c" ~ / $ 1 I Continued growth

Induced resistance 2nd step Possible 8AZ ~ HAT- Spontaneous HGPRT: ~.. HGPRT-

HGPRT 8 AZ ++ HAT-

® Loss of resistance on growth in r'dEM

i

II - 18 weeks | 8AZ + 8 A Z - ~,,,,,,,,,...~ HAT ~

No loss of resistance 8 A Z ++ HAT- HGPRT- " Non revertible"

Nixed origin population maintained phenotypically stable in the presence of selective agent HGPRT" HAT- 8AZ ~

Fig. 7. Diagramatic representat ion of poss ible origins of drug-resistant p h e n o t y p e s in cells exposed to purine analogues . H G P R T - represents residual e n z y m e concentrat ion; H G P R T -+, b e t w e e n 20 and 60% of wt levels; H G P R T ÷, >60% wi ld- type level; 8 A Z - , cells sensit ive to 8AZ; 8 A Z +, cells resistant to 10 #g/m1; 8 A Z ++, resistant to 30 ~g/ml; H A T +, cells able or unable to grow in H A T medium; 2n , ceil w i th diploid c h r o m o s o m e number; 2n+, indicates a cell w i th an increased c h r o m o s o m e number .

occurs rapidly, after only 6 population doublings, in the present study and may well appear stable, even after considerable (>15 weeks in some cases) periods of growth in the absence of selection. Initially adapted clones are HAT+ but when maintained under selective pressure, progression to a more stable higher level of resistance occurs which is accompanied by a loss of ability to grow in HAT. Such lines can be distinguished from those selected by single high doses only by repeated measurements of reversion frequencies. The most important criterion for the mutational origin of a resistant line would therefore appear to be the long-term stability of the phenotype in the absence of selective pressure. (3) Several workers have used antimetabolites as selective agents in the study of dose--response relationships in induced mutagenesis in cultured mammalian cells [2 ,8 ,9 ,13,14,35] . It seems premature, however, at this stage to draw detailed conclusions from such data as only a small proportion of colonies

295

scored may be of true mutational origin. Even using the HGPRT- system which so far is the best characterised, great care must be taken.

Acknowledgements

The work was supported by Grants from the Medical Research Council and the Cancer Research Campaign. The authors are grateful for the technical assis- tance of Mrs. M. Bloomfield and Mr. M. Greaves.

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adaptational origin of some purine-analogue resistant phenotypes in cultured mammalian cells

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