289

Mutation Research, 35 (1976) 289--310 © Elsevier Scientif ic Publishing Company , Ams*.erdam -- Pr inted in The Nether lands

BIOLOGICAL AND BIOCHEMICAL CHARACTERISATION OF PURINE ANALOGUE RESISTANT CLONES OF V79 CHINESE HAMSTER CELLS

M A R G A R E T FOX, J.M. BOYLE and B.W. FOX

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX United Kingdom)

Received August 8th, 1975) Revision received November 1 l t h , 1975) Accepted November 28th, 1975)

Summary

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous ori- gin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8- azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypo- xanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the pres- ence of amethopterin. The heat sensitivity and electrophoretic mobility of HGP RT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones repre- sent missense mutants.

Aboreviations: 8AZ, 8-azaguanine~ 8AZ r, 8-azaguanine resistant clone; EMS, e t h y l m e t h a n e sulphonate; HAT, medium containing hypoxanthine amethopterin and thymidine: HGPRT, hypoxanthine-guanine- phosphoribosyl transferase; IMP, inosinic acid; PRPP, phosphoribosyl-l-pyrophosphate; 6TG, 6-thio- guanine: 6TG r, 6-thioguanine resistant clone~ UV, ultraviolet light.

290

Introduction

Many variations in phenotype have been reported for purine analogue resis- tant clones isolated from a large number of cultured mammalian cell lines in vitro [1,6,7,24,25,35,37]. Genetic heterogeneity in cell lines isolated from Lesch--Nyhan patients has also been well documented [9,20,23]. In general the resistant clones can be subdivided into two major classes. Those which are apparently unstable, i.e. revert with high" frequencies [6,35,36], and those which appear to be stable, and have reversion frequencies within the range 1 in 10 -7 to 1 in 10 -4 [4,6,7,17,35]. Amongst the first class, chromosomal variation has been reported [36], and HGPRT levels are often high [35]. Some clones of the second class also have high in vitro HGPRT levels and are able to grow in HAT [17,25,37], whereas others had no detectable HGPRT activity [17,35]. These stable clones have been compared with cell lines isolated from patients with Lesch--Nyhan syndrome and have been classified by De Mars [9] into type I i.e. mutants with no measurable in vitro HGPRT activity and type II, those which show residual enzyme levels.

Amongst V79 Chinese hamster cell lines showing the extreme negative phenotype, Beaudet et al. [4] have demonstrated the existence of some which produce proteins that immunologically cross react with antibodies to wild type HGPRT, i.e. are CRM ÷ but are enzymologically HGPRT-, whilst others produce neither active enzyme nor immunologically recognisable protein. It has been suggested that the former represent nonsense mutations.

We have demonstrated the induction of 8-azaguanine resistant mutants in V79 cells with a number of chemical and physical mutagens [14]. In common with other authors, using other mutagens in similar assays we have observed a close correlation between lethal and mutagenic damage [10,31]. It has been suggested by Duncan and Brookes [11] and by Roberts et al. [32] that muta- tion to 8-azaguanine resistance in V79 cells may result from deletions and some evidence in support of this hypothesis has been presented [ 7 ,1!] .

Since, from a practical poi.nt of view, it is important to know the spectrum of mutations which can be detected in any particular cell line under standard assay conditions we have analysed the phenotypes of a number of purine ana- logue resistant mutants (spontaneous and induced), isolated from two different cell lines, to determine whether their behaviour was consistent with the idea that they are deletions. During the course of the investigation we also at tempted to answer some of the questions posed by various reports in the literature:

(1) Does the phenotype depend on whether 8AZ or 6TG is used as the pri- mary selective agent [35] ?

(2) Does the phenotype of the mutants depend on the dose of selective agent [37]?

(3) How is the phenotype, particularly the ability of cells to grow in HAT me- dium affected by variation in culture conditions, in particular serum [30,38,39] ?

(4) If HGPRT activity is implicated in purine analogue resistance, what alterations in activity can occur?

(5) What is the nature of the mutations affecting HGPRT activity?

291

Methods

Cell lines and isolation o f variants

The origin of the two V79 cell lines used, V79A and V79S, their sensitivity to 8AZ and 6TG, and the relationship of surviving fraction to cell density and degree of metabolic cooperation, have been described [14,26]. The technique used for exposure of V79 cells to various mutagens has also been described [14]. Resistant clones, spontaneous and EMS- or UV-induced, were isolated after plating untreated or treated cultures into soft agar containing various con- centrations of the two purine analogues. Concentrations of 8AZ and 6TG used were on the "pla teau" region of the dose response curve for both cell lines. Clones were isolated after 7--10 days culture in soft agar, subsequently grown as monolayers, and always maintained in the absence of the selective agent. They were designated A or S for their cell line of origin and were numbered on isolation. After various periods of culture in the absence of selective agents, clones were tested for their sensitivity to 8AZ and 6TG by plating 5 X 102 cells into increasing concentrations of the two purine analogues. Surviving colonies were scored after 7 days incubation.

Sensitivity o f isolated clones to HAT medium Each clone was tested for plating efficiency and ability to grow in HAT.

These tests were made on the same batch of cells as was used to test for 8AZ and 6TG resistance. Plating efficiency was determined by plating 5 X 102 cells into L HAT medium [21] containing 4 × 10 -7 M amethopterin, 1.6 × 10 -S M thymidine, and 1 × 10 -4 hypoxanthine. The growth rate of some mutant clones in HAT was measured by plating 5 × 102, 5 × 103 and 5 × 104 cells into replicate dishes. Duplicate dishes were subsequently trypsinised daily and cell numbers determined by counting in a haemocytometer . After initial testing, cells were stored in liquid nitrogen for various periods and subsequently retested for purine analogue resistance and HAT sensitivity in different batches of serum.

Measurement o f reversion frequencies L HAT medium did not however give opt imum growth of wild-type cells

under conditions of high cell density used for revertant selection and the recipe was subsequently modified to 5.0 X 10-4M thymidine, 1.0 X 10-4M hypo- xanthine, and 4 X 10 -7 M amethopterin and designated V HAT. These concen- trations gave opt imum growth of wild-type V79 cells when measured in terms of plating efficiency, colony size and saturation density (Fox and Boyle, un- publ. data). A more detailed analysis of the effects of variation of the constitu- ents of HAT medium and its effects on growth and plating efficiency in various cell lines will be presented elsewhere [15].

For detection of revertants, 8AZ resistant mutants were cultured in roller bottles to a cell density of 5 X 107/bottle. These cultures were then trypsinised, replated into 14 cm petri dishes at a density of 1 X 106 cells/dish in V HAT me- dium, and incubated for 10 days before scoring. EMS was used in an a t tempt to revert spontaneous and induced mutants. Cultures, also in roller bottles

292

(5 × 10 ~ cells) were exposed to 1 mg/ml EMS for 3 h then plated at a density of 1 × 106/14 cm dish in normal medium. After 48 h, normal medium was replaced with V HAT in one set of 5 dishes and a duplicate set of dishes were trypsinised and replated as previously described [14]. Although it should allow detection of revertants, this simple test will not necessarily yield optimum back mutation frequencies, since 1) 48 h may not be the optimal expression time for each mutant clone and 2) less than 100% recovery of mutants was achieved in sub- sequent back selection reconstruction experiments with cells plated at an equiv- alent density in 6 cm dishes [15].

Measurement of [t4C] hypoxanthine uptake into whole cells and nucleic acids Cultures of wild-type and mutant cells to be assayed for [14C] hypoxanthine

uptake were plated from stock cultures into 9ocm Falcon plastic petri dishes at a cell density of 1 × 10S/dish. After 18 h incubation 1.25 gCi [~4C]hypo- xanthine (62 mCi/mmol) were added to each dish. Dishes were removed at half hourly intervals up to 6 h after the addition of the label and immediately chilled on trays containing cracked ice. Growth medium was removed by aspira- tion, and each dish was washed twice with ice-cold phosphate buffered saline (PBS) + 0.1 mM cold hypoxanthine. After washing, 2.0 ml of 1 N NaOH was added to each dish to lyse the cells. The cell lysate was mixed with 2 ml ice cold 15% PCA together with 0.1 ml horse serum in a centrifuge tube then pro- cessed to determine radioactivity in cold and hot acid soluble fractions as previously described [ 29 ].

Growth of wild-type and mutant cultures for HGPRT assays V79 cells (wild-type and mutants) were grown in MEM + 10% foetal calf

serum in roller bottles to a density of 5 × 107 cells/bottle. (Confluence under such conditions was reached at 1 × l 0 s cells/bottle.) t~fter removal of the medium, the monolayer was washed twice with 20 ml PBS, and the attached cells scraped from the surface with a rubber policeman into 10 ml PBS. The cell suspension was washed twice by centrifugation and the cell pellet stored at --20°C for periods of up to 2--3 months.

Cell free extracts were prepared from such pellets as follows: after thawing, the pellet was resuspended in 0.5 ml PBS. The cells were then either sonicated by 3 × 5 sec bursts on a Mullard sonicator with cooling between each burst, or subjected to 3 cycles of freezing and thawing. Each lysate was then centrifuged at 50,000 g and the pellet discarded. Protein concentrations of the supernatants, determined by the method of Lowry [22] were between 1--2 mg/ml. Such ex- tracts could be stored without significant loss of HGPRT activity for 2 months.

Assay of HGPRT activity in wild-type cells and mutant clones The assay procedure for hypoxanthine-guanine phosphoribosyl transferase

(HGPRT E.C. 2.4.2.6.) was identical to that described by Duncan and Brookes [11]. Most assays of both wild-type and mutant extracts were performed at a hypoxanthine concentration of 1.6 × 10 -4 M and a protein concentration of 25--30 pg/50 #l of assay mix. Concentrations of phosphoribosyl-l-pyrophos- phate (PRPP), [14C]hypoxanthine, and Mg ~ were identical to those used by Duncan and Brookes [11] and were "s tandard" assay conditions in the other

293

experiments. Under these conditions 95% of added hypoxanthine was converted to IMP in 90 min.

In other experiments the duration of the assay, the substrate concentration, the protein concentration and the constituents of the standard assay mix were varied. These will be described where appropriate.

Heat inactivation o f enzyme in wild-type and mutant clones Cell free extracts were heated in sealed tubes in a Techne 'Dri-block' for

various periods of time, up to 90 m, at either 60°C or 80°C. 50 pl aliquots taken at 10 min intervals were subsequently cooled rapidly in ice and after adding [~4C] hypoxanthine and the other reaction components the products were as- sayed by chromatography in the usual way. A 50 pl aliquot was removed from the crude cell extract before heating, to serve as control.

Gel electrophoresis o f cell extracts Approx. 30--100 mg of protein was added to each polyacrylamide gel and

was subjected to electrophoresis as described by Bakay and Nyhan [2]. After electrophoresis, gels were incubated for 90 m with an enzyme reaction mix containing [ ~4C] hypoxanthine as label. After precipitation of labelled products with lanthanum chloride, the gels were washed 8--10 h in deionised water to remove unbound substrate then fractionated into 0.8 or 1.6 mm slices. Each slice was added to a scintillation vial with 0.5 ml of 30% hydrogen peroxide and the vials were capped and incubated overnight at 50°C. After solubilisation of the gel slices, 12 ml of phosphor containing 6 g PPO, 600 ml of ethoxy- ethanol/1 of toluene was added and samples counted in a Nuclear Chicago liquid scintillation spectrometer. Counting efficiency for ~4C under these conditions was 80%.

Results

8 A Z and 6 TG sensitivity o f spontaneous and induced clones The selective levels of 8AZ and 6TG used for isolation of spontaneous and

UV- or EMS-induced clones together with the subsequent sensitivity of the isolated clones to 30 pg/ml of 8AZ and 10 pg/ml 6TG are shown (Table I). Also summarised are the sensitivities of the various clones to HAT medium. Wild type clones of both V79A and V79S cells showed sensitivity to 8AZ and 6TG.

All the clones (spontaneous or induced) isolated after growth in 8AZ at 7.5, 30.0 or 100.0 #g/ml showed resistance when rechallenged with 30 pg/ml 8AZ. These tests were performed after the clones had been cultured for various periods of time in medium without inhibitors and in most cases were repeated at intervals to test for stability of resistance. None of the clones showed loss of resistance when cultured in the absence of the selective agent. The majority of the clones also showed stable cross-resistance to 6TG (10 pg/ml). There were however two exceptions, A10 and A17, which on successive testing consistently showed resistance to 8AZ but partial sensitivity to 6TG.

After selection in 6TG all the V79 A clones tested showed stable resistance to both 6TG and 8AZ. In contrast, selection of V79S cells in 6TG consistently

294

T A B L E I

B I O L O G I C A L B E H A V I O U R O F 8 A Z R E S I S T A N T C L O N E S O F V 7 9 A C E L L S

S e l e c t i v e Dose O r i g i n C l o n e % w i l d t y p e P.E. in

a g e n t ( p g / m l ) )

8 A Z 6 T G

( 3 0 p g / m l ) ( 10 U g / m l )

H A T

(1) (2) (3)

8AZ

S A Z 7 .5 AS a 1 1 1 2 101 0 2 1 0 2 1 0 0 1 0 6 b

3 1 0 2 91 0

4 111 - - - -

5 96 -- - -

7 .5 A U 6 1 0 6 1 0 4 106 b

50 e r g s / m m 2 7 1 0 1 - - - -

8 9 8 - - - -

9 1 1 4 - - - -

1 0 1 1 5 0 . 2 5 0 . 0 7

AU I i 105 95 85 b

200 ergs/mm 2 12 120 107 0

30.0 AS 13 98 98 0 --

1 4 108 1 1 5 0 --

15 1 1 5 108 0 --

16 I01 I 0 0 0 - -

AE 17 102 8 94 97

1 .0 m g / m l 1 8 89 9 2 4 0 0

1 0 0 AS 19 1 0 4 101 0 - -

20 1 0 2 1 0 0 0 - -

21 102 106 0 --

22 1 0 0 1 0 3 0 - -

A E 23 1 0 2 97 0 - -

1 .0 m g ] m l 24 1 1 4 87 0 0

a S s p o n t a n e o u s l y a r i s i ng c l o n e . U i n d u c e d b y u l t r a - v i o l e t i r r a d i a t i o n a t d o s e l eve l s h o w n . E i n d u c e d b y

e t h y l m e t h a n e s u l p h o n a t e a t t h e d o s e l eve l s h o w n . Wi ld t y p e p l a t i n g e f f i c i e n c y was c o n s i s t e n t l y c lose to

1 0 0 % . A d e n o t e s i s o l a t i o n f r o m V 7 9 A cel ls see M e t h o d s . T h e t h r e e c o l u m n s H A T ( 1 ) ( 2 ) ( 3 ) i n d i c a t e t h e

r e s p o n s e o f o r i g i n a l l y H A T p o s i t i v e c l o n e s i n t h r e e d i f f e r e n t s e r u m b a t c h e s .

b I n d i c a t e s t h a t t h e s e c l o n e s w e r e a l l t e s t e d a t t h e s a m e t i m e i n t h e s a m e s e r u m b a t c h .

0

0 0

0 _

_

0 0 _

_

_

0 . 0 5 0.08

0 0

0

98 0

produced clones which were resistant only to 6TG and sensitive to 8AZ {Table II).

Sensitivity o f clones to H A T medium All clones selected for resistance to 6TG were sensitive to growth in HAT

medium {Table II), as were most clones selected for resistance to 8AZ (Table I). Of 16 mutants of the latter class tested for growth in HAT, six initially produced clones in HAT. Subsequently these six were all retested using HAT medium supplemented with different batches of foetal calf serum. Four mutants A2, A6, A l l , A18, that initially showed high plating efficiency in HAT, failed to grow with HAT supplemented with two alternative batches of serum {Table I). One mutant, A17, however, consistently showed high plating efficiency in HAT which was independent of serum batch. Another, A10, also consistently showed some growth in HAT, bu t produced only 1--5 colonies/5 × 10 ~ cells.

295

T A B L E II

B I O L O G I C A L C H A R A C T E R I S T I C S O F V 7 9 C L O N E S S E L E C T E D IN 6 - T H I O G U A N I N E

A d e n o t e s d e r i v a t i o n f r o m V 7 9 A cel ls ; S f r o m V 7 9 S cel ls ; AS d e n o t e s a s p o n t a n e o u s l y r e s i s t a n t A c l o n e ;

SS d e n o t e s a s p o . n t a n e o u s l y r e s i s t a n t S c l o n e ; E is E M S i n d u c e d c l o n e .

S e l e c t i v e D o s e Orig in C l o n e No. % w i l d - t y p e P.E. in

a g e n t ( p g / m l ) 8 A Z 6 T G

( 3 0 ~ g / m l ) ( 10 ,~Zg/ml)

H A T

6TG

6 T G

1 .0 AS 25 107 101 0

1 0 . 0 AS 26 101 9 0 0 27 1 0 3 9 4 0

1 0 . 0 A E 28 101 9 2 0

1 .0 m g / m l 29 9 4 4 8 0

30 1 0 9 99 0

31 1 0 4 9 4 0

0 . 5 SS 1 - - 1 0 4 - -

2 - - 1 1 0 - -

3 - - 101 - -

4 0 99 0

1 . 0 S $ 5 - - 8 9 - -

6 - - 9 6 - -

7 0 9 8 0

10.0 SS 8 2.8 98 0 9 4.4 96 0

Both these clones showed partial sensitivity to 6TG which was not observed in any of the other 8AZ selected clones.

Reversion of resistant clones to wild type phenotype Eleven clones were tested for spontaneous reversion using V HAT medium

for selection (Methods). All had measurable in vitro enzyme levels, 7 were spontaneous, 1 EMS induced and 3 UV induced. Screening of up to 5 × 106 cells from each of these clones i.e. A1, 2, 6, 10, 14, 20, 24 and 25 produced no revertant colonies. Five mutants (A1 and 13 spontaneous, A l l , 12, 24, EMS in- duced) were scored for revertants after EMS treatment. In each case revertant colonies were obtained at apparent frequencies ranging from 8.0 × 10 -7 to 1.4 × 10 -6. The revertant colonies continued to grow in HAT after isolation, but to date have not been characterised further.

Utilisation of exogenous hypoxan thine The uptake of [14C]hypoxanthine into whole cells in a number of mutants

is shown in Table III. All clones tested showed significantly reduced levels of ['4C]hypoxanthine incorporation into whole cells, mainly due to a reduction of incorporation of radioactivity into the acid soluble fraction. The uptake of label by either wild type or mutant cells was unaffected by the presence of amethopterin in the six clones tested. Thus very poor utilisation of exogenous hypoxanthine occurred in clones which show positive enzyme levels in vitro.

296

T A B L E II I

ENZYME L E V E L S A N D H Y P O X A N T H I N E U P T A K E IN V 7 9 A CLONES

Clone No. IMP produced a H G P R T activity n m o l / h / m g % control

[ 14C] h y p o x a n t h i n e uptake into whole cells % cont ro l (4 h)

- -Amethopter in +Amethopter in

A1 5.1 A2 16.2 A3 5.4 A4 13.6 A5 12.5 A6 10.6 A7 15.9 A8 10.5 A9 13.8 A10 19.9 A l l 17 .5 A12 20.9 A13 3.6 A14 51.5 A15 47.8 A16 14.9 A17 23.0 A l S 55.7 A19 17.7 A20 20.7 A21 25.3 A22 28.5 A23 35.2 A24 34.3 A25 56.0 A26 53.7 A27 48.3 A28 23.5 A29 57.5 A31 28.9 AWt 230 .0 -+ 25.1

2.2 7.0 2.3 5.9 5.4 4.6 6.9 4 .5 6.0 8.6 20.4 23.9 7.6 1.4 9.0 1.2 1.5 1.0 4.2

22.3 2.0 20.7

6.4 10.0 6.9 24.2

7.6 3.6 5.7 9.0 0.S

11.0 12.3 15.3 14.9 1.7 24.3 3.5 23.3 5.3 11.7 21 .0 10 .2 25.0 11.0 19.1 12 .5

4.4 2.7 0.4

0.6

a Assay performed at a substrate concentrat ion of 1.6 X 10 -4 M.

Measurement of hypoxanthine guanine phosphoribosyl transferase activity

Dependance of HGPRT activity on incubation time Conversion of [ ~4C] hypoxanthine to IMP, assayed under standard conditions

at a hypoxanthine concentration of 1.6 × 10 -4 M (8 nmoles per 50 pl reaction mix), was linear for 60 or 90 m depending on the method of extraction. The data are shown together with that for two 8AZ resistant clones in Fig. 1. Much less IMP was produced during the assay period in both mutant clones. Activity in extracts prepared by freezing and thawing was in general higher than that in sonicated extracts.

Relationship between protein concentration and IMP production Protein concentration varied in different assays but was in general kept be-

tween 20--30 pg//50 pl of reaction mix. The precise relationship between

297

o ~ 10(~

~o

P ~ ~

30 60 90 120 Minutes of incubation

Fig. 1. Kine t ics o f IMP f o r m a t i o n in wild t y p e and m u t a n t cell e x t r a c t s ; assays were p e r f o r m e d at a sub- s t ra te c o n c e n t r a t i o n of 1.6 × 10 -4 M h y p o x a n t h i n e : (~",) wild t y p e e x t r a c t p r e p a r e d by f r eeze - thaw m e t h -

od. M a x i m u m c o n v e r s i o n , 362 .1 n m ] m g p ro te in ; (©) wi ld - type e x t r a c t p r e p a r e d by son i ca t i on . M a x i m u m

c o n v e r s i o n , 179.6 n m / m g p ro t e in ; (A) m u t a n t A17 e x t r a c t p repaxed by f r eeze - thaw m e t h o d . M a x i m u m

conve r s ion , 97 .4 n m ] m g p ro te in ; (o) m u t a n t A2 s o n i c a t e d e x t r a c t e d . M a x i m u m c onve r s ion 50.3 n m / m g

p ro te in .

9O

~ 8o o -~70 ,~

~SC

~ S~ ._ .l~

,~ 4C g ~3~

_'2" ~ l~

o/ /

~ z'o 3'0 4~ ~g protein/assay

60

50

4C

/ / 7 °'-° I O / S J ' ,

IO 20 30 4'0 ~g protein/assay

Fig. 2. a) Re la t i onsh ip b e t w e e n p r o t e i n c o n c e n t r a t i o n / 5 0 #1 assay and H G P R T ac t i v i t y in cell free e x t r a c t s o f V79 Acel ls . Assay was p e r f o r m e d u n d e r s t a n d a r d c o n d i t i o n s at a s u b s t r a t e c o n c e n t r a t i o n o f 1.6 X 10 -4 M

h y p o x a n t h i n e : (o) 90 ra in i n c u b a t i o n ; (o) 60 m i n i n c u b a t i o n , b ) Re l a t i onsh ip b e t w e e n p r o t e i n concen t r a - t i on and H G P R T ac t i v i t y in cell f ree e x t r a c t s f r o m var ious 8 AZ r m u t a n t s . C o n d i t i o n s o f assay were as for Fig. 2a. I n c u b a t i o n was for 90 m i n : (o) A13; (A) A2; (A) A25.

298

protein concentration and enzyme activity in wild type V79A cells and three mutan t clones is shown in Fig. 2a, b. Again the assay was performed at a stan- dard substrate concentration of 1.6 × 10 -4 M for both wild type and mutant clones. The amount of IMP produced by wild type and mutant extracts in- creased with increasing protein concentration. For wild type extracts a maxi- mum of 90% conversion of added labelled [~4C]hypoxanthine to IMP was reached when between 20 and 30 pg protein were present/assay. Significantly lower conversion was observed for two mutants over the same range of protein concentration.

Characteristics o f wild-type enzyme The specific activity of extracts of wild type V79A cells was 230.0 -+ 25.1

nmoles IMP produced/mg protein/h (mean 24 determinations). That of V79S cells, under comparable conditions, was lower, 95.93 ± 14.88 nmoles IMP/mg protein/h (mean 4 determinations) and similar to that previously reported for these cells [11]. The percentage of added [14C]hypoxanthine converted to IMP in the two cell lines at comparable protein, different substrate concentra- tions and under different assay conditions for V79A and V79S cells is shown in Table IV. The V79A extracts showed the expected response to omission of Mg 2÷ and PRPP (tetrasodium salt) i.e. HGPRT activity was much reduced, as previously reported for S cell extracts [ 11 ].

Under standard assay conditions inosine was also produced by extracts from both cell lines. The proportion of added [14Cl hypoxanthine converted to ino- sine was similar in different extracts of V79A cells. Consistently less inosine production was observed in extracts of V79A cells than in extracts of V79S cells. (Table IV). Specific activity for inosine production in V79S cells was comparable to that reported previously for this cell line [ 11]. In the absence of added enzyme after 90 m incubation 0.86% of ~4C activity was recovered as

T A B L E IV

C H A R A C T E R I S T I C S OF WILD-TYPE E N Z Y M E S

Cell l ine Substrate I n c u b a t i o n % a d d e d [ 14 C]- Specif ic ac t iv i ty c o n c e n t r a t i o n c o n d i t i o n s h y p o x a n t h i n e c o n - n m o l / h / m g

ver ted to

IMP Inos ine IMP Inos ine

V 7 9 A 1.6 X 10 -S S tanda rd a 85.6 1.7 41 .4 1.4 1.6 × 10 -4 S tanda rd 96 .6 1.7 207 .7 3.6 3.2 X 10-4 S tanda rd 70.1 4.3 301 .6 18 .5 1.6 X 10 -5 Dialysed 91.7 0.7 42 .6 1.4 1.6 X 10-4 Dialysed 95 .5 0.9 308.1 2.8 3.2 X 10 -4 Dialysed 59.6 0.6 384 .6 3.6

V79S 1.6 X 10 -5 S t anda rd 47 .5 5.8 34 .8 14 .75 1.6 X 10-4 S t anda rd 18.1 3.6 140 .3 66.8

V 7 9 A 1.6 X 10 -4 --Mg 2~ 4.9 9.6 36.1 69 .7 1.6 X 10-4 - -PRPP 0 .8 1.6 5.2 10.6

a As de f ined in Methods i.e. in p r e s e n c e o f Mg 2 . and PRPP.

299

IMP and 0.16% as inosine. This background has been subtracted from all values given for wild type and mutant clones.

Enzyme activity in mutant clones The spontaneous and induced clones selected after growth in 7.5 pg/ml 8AZ

(Clones A1--12, Table I) all showed positive enzyme levels ranging from 2.2--9.0% of wild type values (Table III). It is possible that at these low levels of selective agent, although resistance is stable, mainly " leaky" mutants are recovered. Consequently mutants were isolated after growth in high 8AZ con- centrations i.e. 30 pg/ml and 100 pg/ml (Clones A13--24, Table I). The levels of enzyme activity in these clones were, if anything, higher than those in the previous group, ranging from 1.5--25.2% (Table III).

It has been inferred by a number of authors [35,38], that 6TG is a better agent for selecting HGPRT negative mutants. Mutants were therefore selected from V79A cells after growth in a range of 6TG concentrations (Clones A25-- 31, Table II) and their enzyme levels assayed. Again a range of enzyme activi- ties was seen in both spontaneous and induced mutants (10.2--24.3% of wild type activity) (Table III).

Very low enzyme levels were reported by Duncan and Brookes [11] in V79S--SAZ r mutants, both spontaneous and carcinogen induced. To test whether the recovery of zero enzyme mutants was characteristic of this cell line, we isolated 6TG resistant mutants. Results are summarised in Table V. Again a range of activities was observed, but in general, the levels were lower than those of V79A mutants and two mutants with near zero levels were observed. The en- zyme levels must be viewed with some reservation however, because of some dependence of mutan t enzyme activity on substrate concentration (Table VI). It is evident, particularly in clones with enzyme levels above 50%, that at lower substrate concentrations the HGPRT shows activity in vitro similar to that in wild type cells. It also seems possible that in some cases where zero enzyme activities have been reported by other workers [11,17,35] assays could have been carried out at inhibitory substrate concentrations.

T A B L E V

F, N Z Y M E L E V E L S IN 6 - T H I O G U A N I N E R E S I S T A N T V 7 9 S C L O N E S

C l o n e No. IMP p r o d u c e d n r n o l / h / m g a p r o t e i n

$1 6.6 $2 3.6 $3 13 .4 $4 18 .0 $5 6 .8 $6 2.8 $7 2.0 $8 1.51 $9 0 .76 SWt 95 .93 -+ 14 .88

a A s s a y p e r f o r m e d at a s u b s t r a t e c o n c e n t r a t i o n o f 1.6 × 10 -4 M.

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301

Determination o f Michaelis constants The Km for the wild type HGPRT as determined by a Lineweaver--Burke

plot was 6.2 × 10 -5 M and the Yma x 250 nmoles/h/mg protein. Fig. 3 shows plots of Vmax/V for wild type enzyme and enzyme from four

8AZ r mutants so that data for all extracts can be plott.ed on the same scale as suggested by Olsen and Milman [27]. HGPRT in crude extracts from these four mutants had much lower Vmax values than the wild type enzyme, but the ap- parent Km was the same as that for wild type enzyme, i.e. 6.2 × 10 -5 M. The mutant enzyme in many cases appeared to be susceptible to substrate inhibi- tion as typified by the data for two clones A10 and A12 in Fig. 3. Inosine production by mutan t extracts was very similar to that in wild type extracts, and double reciprocal plots gave mean Km and Vm~ values for wild type ex- tracts of 1.3 × 10-4M and 110 nmoles/h/mg protein respectively (data not shown).

Sensitivity o[ wild type and mutant enzymes to heat inactivation Initial experiments at 80°C indicated that the wild type enzyme incubated

under standard conditions lost 90% of its activity after 90 min incubation. The extracts from mutan t clones lost activity too rapidly at this temperature for satisfactory measurement to be made. After 90 min at 60°C two wild type ex- tracts lost 30% and 5% of the activities respectively. A comparison of the heat sensitivity of wild type and mutant cell extracts is shown in Fig. 4. Extracts

20 ~

18

16

i ~ ~ • ~

~2 V m~x

V I0

i';'~ ~ ~ .~.o~O

l .s ~.o o.s o'.s ~2o ~'.s ~'.o ~'.s ~'.o Hypo~anthine "I mM'1

~g. 3. Dete~ination ol ~chae~s const~, HGPRT acuity in c~de cell ex~ac~ o[ wild type ~d

mut~t ce~ w~ me~u~d ~ desc~bed using hypox~t~ne ~ subs~za~e ~d ~0 ~g p~oteinlS0 ~l ~say.

~e velocity V is the amoun~ o[ IMP ~o~ed in nmoles~Img pzote~ ~d Vmax w~ de~e~ined fzom lhe

o~dinate of a double zecipzocal plot of (velocity) -I venus (substrate) -I. ~e Vmax v~ues weze lot wild-

t~pe (~) 250; ~0 (o) A2.5; ~2 (~) 3~.5; ~ (~) 130 ~d ~4 (m) 30.0. The common K m [oz hypox- ~hine w~ 6.2 X 10 -s M.

302

IO0

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,

,~ ~'o ;o 4'o s'o ~'o / 0 % ~'o ~im~tes 3t 60'-C

blg. 4. Rate of inac t iva t ion of I-IGPRT act ivi ty in wild t y p e and m u t a n t e n z y m e ex t rac t s hea t ed at 60°C. At 10 rain intervals a l iquots were r e m o v e d and the hea ted cell ex t rac t s were assayed u n d e r s t anda rd con- di t ions in the presence of 1.6 X 10 -4 M h y p o x a n t h i n e and 1 m M PRPP, as desc r ibed in Methods : (m) A17: (A) A13; (&) A12: (X) A24; (~) A2: (©) Wt e x t r a c t e d by f reeze - thaw m e t h o d ; (m) Wt ex t r ac t p r epa red by sonica t ion .

from four different mutant clones showed considerably enhanced heat sensitivi- ty compared with either of the two wild type extracts. The heat sensitivity of HGPRT activity from clone A17 however was considerably less than that from other mutants and was only slightly greater than that of the more sensitive wild type extract.

Electrophoretic mobility of mutant and wild type enzymes Polyacrylamide gel electrophoresis profiles of crude cell extracts from wild

type cells and three mutant clones are shown in Fig. 5. The profiles of wild type extracts show at least three peaks, whilst those of mutants showed only a single peak. The electrophoretic mobility of HGPRT activity from mutant clones was consistently 20--28% less than that of the wild-type enzyme. These results contrast with the alteration in the relative mobility of residual HGPRT activity in extracts from erythrocytes of homozygous and heterozygous Lesch- Hyhan patients [3]. In these studies however, the activity in mutant extracts showed 15% greater mobility than that of wild-type. The mobility of wild type HGPRT from V79 cells used in the present study was similar to that of human enzyme using the same technique [2,3].

To rule out possible artefacts, extracts from wild type and mutant cells were run separately and as a mixture in separate gels at the same time. The results

303

210 Hinu~es 3mA/tube ~"

, ,

w. ~ , ~.~ 2 ~33 6fig) ~

A24 ~ 0.! (41 6~g) ~

I

2 (80~ g)

I

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32 48 64 80 96 Distance Migrated (mini

Fig. 5. M i g r a t i o n o f H G P R T a c t i v i t y f r o m w i l d - t y p e a n d m u t a n t e x t r a c t s i n . p o l y a c r y l a m i d e ge ls . Ge l s

w e r e f r a c t i o n a t e d i n t o 1 . 6 - n m s l i ce s w h i c h w e r e c o u n t e d as d e s c r i b e d i n M e t h o d s . M u t a n t s i d e n t i f i e d o n p a n e l t o g e t h e r w i t h p r o t e i n c o n c e n t r a t i o n i n t h e gel .

are shown in Fig. 6. Mutant enzyme activity appeared in the same place on the gel and showed similar activity whether a mixture or separate extracts were applied. Thus there was no evidence for activation of mutan t enzyme when mixtures of wild type and mutan t extracts were run together, in contrast to the activation of mutan t enzyme observed by Bakay and Nyhan [3] .

The mobil i ty of the mutant enzyme was similar in all clones tested except that of clone A17 which differed from that of all o ther mutants in that it showed almost the s a m e mobili ty as that of the wild type enzyme (Fig. 7).

210 M i n u t e s ~ - ÷ _ _ ,

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0

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~ ~~~~ ~ . . . . , , ,

16 ~2 ~8 64 80 96 D ~ s t a n c e ~ i g r a t e d (ram)

~ . G. ~ g r a t i o n of mi~;ed extracts f r o ~ wild ~y~e ~ d ~ u ~ eels in polyae[ylamide gels. ~ top three p~e ls , gels were fza¢Uonate~ into 0 . 8 - ~ slices in ~ at tempt to i ~ r o v e resolution. ~ot~o~ ~ e l , gel w ~ fracUona~ed i~to ] . 6 - ~ slices.

wt I] ~ ~ . . . . Wt2 A2 ,,3 ~N .~ ~

~ 5 ~ ~

~ ~l ~ ~

~ ~'0 3'0 4; s; ~0 7'0 i0 ~'0 ~;0 - Di stance migrated (%) +

Fig. 7. Relative mobi l i ty o f I-IGPP~T acUvity (normaJised to 100 fractions/gel and expressed as %) f rom di f ferent wi ld type and mutant cell ex~ae~. Blocl~ represent the width of the enzyme peak in each case; the bar indicates the fraction in which maximum enzyme activity was detected.

304

305

Discussion

In the introduction we posed certain questions concerning the selection and characterisation of purine analogue resistant mutants. The results we have pre- sented allow some qualified answers to be given.

The mutant phenotype is somewhat dependent on the selective agent, but the phenotypic pattern may be influenced by the parental subline from which the mutants are derived

All of the mutants isolated in the present study showed stable resistance to the purine analogue used for their isolation. Of those selected in 8AZ only two failed to show complete cross-resistance to 6TG. Those selected in 6TG from the V79A line showed cross resistance to 8AZ, but a group of 4 spontaneous mutants selected in 6TG from the V79S line showed no cross resistance when tested in 8 AZ.

Clones which were 8AZ r but not cross resistant to 6TG have been reported [17]. Hence the phenotype of mutants isolated from a single cell line (V79A) does not seem to be determined by whether the primary selective agent is 6TG or 8AZ [35,38], but 6TG selected mutants from V79A and V79S cells showed different phenotypes. Therefore, it is likely that the spectrum of phenotypes isolated from different cell lines from the same and different species will also differ. In this respect it is interesting to note that although genetic complemen- tation was observed between groups of mouse cell mutants selected in 8AZ and 6TG, no complementat ion occurred within a group of mutants selected in either analogue [33]. This suggests that a similar mutat ion had occurred in all clones selected with one particular purine analogue.

The biochemical basis for the 6TG r 8AZ s phenotype has yet to be established in detail; however, similar mutants have been described in Lactobacillus casei [12]. One possible explanation for the differential sensitivity is that 8AZ could be deaminated by guanase to azaxanthine which can then be incorporated by xanthosine 5'-phosphate pyrophosphorylase and produce toxic effects. 6TG would not be incorporated because of mutant hypoxanthine guanine phos- phoribosyl transferase. Clones would also be HAT sensitive. Enzymes capable of deaminating guanine and 8AZ have been demonstrated in several mouse tumour tissues [ 13,18,34], but using the same assay procedures no breakdown of 6TG could be detected [5,13].

The pheno type is largely independent o f the dose o f the initial selective agent The doses of both purine analogues used in the present study, were on the

plateau region of their respective dose response curves [26] and isolated clones were fully resistant after growth in the absence of selective agents (Table I}. Different results were obtained in human [37] and mouse cells [35]. In the former case the phenotype of the clones was apparently strongly dependent on the dose of selective agent, however, both degree of resistance and HGPRT levels were highly variable and no positive correlation between the two proper- ties was detected. One group of clones studied by Sharp et al. [35] also had high and variable HGPRT levels. In both these cases, clones were routinely maintained in the presence of selective media suggesting some instability, which

306

was manifest in the case of the mouse clones, as a high reversion frequency. Chromosome instability has been correlated with instability of resistance in some cases [36].

Growth of partially resistant cells in the presence of the selective agent will gradually select a more resistant population. In addition, the continued presence of purine analogues may modulate enzyme levels. These two factors could significantly affect enzyme levels in partial.ly resistant cells but may have less effect in fully resistant lines.

The phenotype can be affected by cultural conditions, particularly serum The effect of dialysable serum factors on the use of HAT media has been

discussed by Peterson et al. [30]. We have observed that four of our mutants (Clones A2, 6, 11, 18; Table I) were able to grow in HAT in one batch of foetal calf serum, but not in two other batches. Hence all except two mutants isolated from V79A cells had a phenotype 8AZ r, 6TG r, HAT- and, in spite of significant in vitro enzyme levels, utilised exogenous hypoxanthine very poorly.

This distribution of mutant phenotype appears to differ from that reported by Gillen et al. [17] where 18 of 35 of their mutants were HAT ÷.

However, in both studies the ability of 8AZ r clones to grow in HAT was as- sociated with sensitivity to 6TG.

The mutants described here also apparently differ from those described by Gillen et al. [17] and Sharp et al. [35] in that we were unable to isolate mutants with the extreme negative phenotype reported by them. The set of 6TG r mutants isolated from the V79S line showed the lowest levels of HGPRT activity, but it was detectable in all cases (Table III) and, at low substrate con- centrations, was similar to those derived from V79A cells. Thus it may not be possible to conclude that clones are truly enzyme negative unless assays are performed over a range of substrate concentrations.

Changes in HGPRT protein result in purine analogue resistance The subunit structure of HGPRT has now been convincingly demonstrated

in partially purified enzyme from a number of different sources [19,27,28]. The profile observed for the wild type HGPRT in the present study is consistent with the interpretation that the enzyme has multiple subunits. The altered heat sensitivity and electrophoretic mobility of HGPRT in extracts of purine ana- logue resistant clones reported here suggests that mutations in the structural gene(s) for HGPRT are the basis of the observed phenotypes. The possibility that distortion of the enzyme protein caused by these mutations results in defective binding of HGPRT to hypoxanthine has been ruled out in at least four mutants by the observed similarities in Km for this substrate. It has been suggested that mutants showing relatively high in vitro HGPRT levels but poor utilisation of exogenous hypoxanthine, as we have observed, may have transport defects [17]. However thiopurines have been shown to enter cells by passive diffusion [5] therefore this explanation seems unlikely. Although Km values for PRPP have not yet been estimated for these cell lines it seems more likely that resistance in our mutants is associated either with defective binding of HGPRT to PRPP in vivo or that PRPP concentrations in vivo are limiting. Preliminary indirect evidence obtained from a comparison of the activity of

307

adenine phosphoribosyl transferase in wild type and one of the mutant clones (Fox, unpubl, obs.) suggested that both lines contained similar a m o u n t s of PRPP. A human HGPRT mutant has been described [23] which showed altered Km values for both purine substrates and PRPP.

It seems unlikely that regulatory genes are involved in the majority of cases since this would be more likely to produce a change in quant i ty but not quality (mobili ty) of the enzyme [9]. Clone A17 may fall into this class. This clone produced only 10% of the HGPRT of V79A but the enzyme had a Vmax closer to that of the wild type enzyme, 130 nmol/h/mg protein compared with 250 nmol/h/mg protein, than any of the other clones. It also showed wild type characteristics with respect to heat sensitivity and electrophoretic mobility. Utilisation of exogenous hypoxanthine was however comparable to that in other clones.

The mu ta t i on even t in the H G P R T s tructural gene m a y be a missense m u t a t i o n One possible interpretation of the altered mobili ty of HGPRT in mutant ex-

tracts is that it is the result of deletion of DNA coding for one or more of the enzyme sub-units. However, since both spontaneous and induced mutants appear to have the same range of phenotypes and both classes are revertible with EMS, this possibility seems unlikely and hence we would favour the inter- pretation that they result from point mutations.

Our thermosensitivity and electrophoretic data are compatible with the demonstrat ion by Beaudet et al. [4] of immunologically active bu t enzymati- cally inactive proteins in extreme negative mutants. This latter observation was interpreted to be the result of a nonsense mutat ion in the structural gene for HGPRT. Our mutants clearly differ from those of Beaudet et al. in possessing enzyme activity and since nonsense mutat ions generally result in loss of en- zyme activity and missense mutat ions frequently permit residual activity [17] , the most likely interpretation is that our mutants belong to the latter class. However, in view of the problems discussed elsewhere [15] , a more detailed analysis of the specificity of reversion and the biochemistry of the apparent revertants is obviously necessary before further conclusions can be drawn.

Acknowledgements

The authors are grateful for the technical assistance of Mrs. M. Bloomfield and Mr. S. McMillan.

The work was supported by grants from the Medical Research Council and the Cancer Research Campaign.

References

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2 Bakay, B. and W.L. Nyhan, The sepaxation of adenine and hypoxanthine-guanine phosphoribosyl transferase isoenzymes by disc gel electxophoresis, Biochem. Genet., 5 (1971) 81--90.

3 Bakay, B. and W.L. Nyhan, Activation of variants of hypoxanthine guanine phosphoribosyl transferase by the "normal enzyme", Proc. Nat. Acad. Sci. U.S.A., 59 (1972) 2523--2527.

4 Beaudet, A.L., D.A. Roufa and C.T. Caskey, Mutations affecting the structure of hypoxanthine-

3 0 8

guanine phosphor ibosy l t ransferase in cu l tu red Chinese h a m s t e r cells Proc. Natl. Acad. Sci. U.S.A., 70 ( 1 9 7 3 ) 320 - -324 ,

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guanine p h o s p h o r i b o s y l t ransferase , J. Biol. Chem. , 250 (1975) 120 - -127 . 20 Kelley, W.N. and J.C. Meade, Studies on h y p o x a n t h i n e guanine phosphor ibosy l t r ans fe ra se in fibro-

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b inants , Science, 145 (1964) 709. 22 L o w r y , O.H., N.J. Ros e b rough , A.L. Farr and R.J. Randal l , Prote in m e a s u r e m e n t wi th folin reac t ion

reagen t , J. Biol. Chem. , 193 (1951) 2 6 5 - - 2 7 5 . 23 McDonald , J .A. and W.N. Kelly, L e s c h - - N y h a n s y n d r o m e : a l tered kinet ic p roper t i e s of m u t a n t en-

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(1970) 2 7 9 - - 2 8 7 . 25 Mor row, J., J. Colof iore and D. Rintoul , Azaguan ine res is tant h a m s t e r cell lines n o t de f ic ien t in h y p o -

xan th ine guanine p h o s p h o r i b o s y l t ransferase , J. Cell Physiol . , 81 (1973) 9 7 - - 1 0 0 . 26 Nikaido, O. and M., Fox, The relative e f fec t iveness o f 6 - th ioguan ine and 8-azaguanine in select ing

res is tant m u t a n t s f rom V79 Chinese h a m s t e r cells, Muta t ion Res., s u b m i t t e d . 27 Olsen, A.S. and Milman, G., Chinese h a m s t e r h y p o x a n t h i n e - g u a n i n e phosphor ibosy l t ransferase :

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