mechanism of cytotoxic action of azaguanine and thioguanine in wild-type v79 cell lines and their...

165

Mutation Research, 80 (1981) 165--185 © Elsevier/North-Holland Biomedical Press

MECHANISM OF CYTOTOXIC ACTION OF AZAGUANINE AND THIOGUANINE IN WILD-TYPE V79 CELL LINES AND THEIR RELATIVE EFFICIENCY IN SELECTION OF STRUCTURAL GENE MUTANTS

MARGARET FOX and R.J. HODGKISS

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX (Great Britain)

(Received 11 April 1980) (Revision received 9 June 1980) (Accepted 28 July 1980)

Summary

The cyto toxic effects of azaguanine and thioguanine have been compared in two wild-type V79 cells. To achieve equitoxic effects in both cell lines a 10-- 20-fold higher concentrat ion of azaguanine than thioguanine was required. Affinity of HGPRT for azaguanine was 10-fold lower than for hypoxanthine in both cell lines and was similar to that for thioguanine in V79S cells. Affinity for thioguanine differed by a factor of 3 in the two cell lines. The rate of cell kill by azaguanine was markedly slower than by thioguanine in both cell lines. Reduct ion of whole cell uptake of [14C]hypoxanthine incorporation by unlabelled azaguanine was only demonstrable after prolonged incubation periods as was incorporation of [14C]azaguanine into acid-insoluble material. Experi- ments with cell-free extracts indicated that hypoxanthine acts as a non-competitive inhibitor of the enzyme. The slow rate of dissociation of the HGPRT-- azaguanine complex is reflected in the slow rate of killing of wild,type cells. Clones resistant to the cy to toxic effects of these analogues have been selected from both cell lines and have been shown to possess HGPRT with altered kinetic properties. Our data suggest that azaguanine and thioguanine may select for mutat ions at different sites on the HGPRT molecule in V79 cells and provide possiSle explanations for the differences in effectiveness of these two agents reported in other cell lines.

Abbrev ia t ions : A Z , a z a g u a n i n e ; A Z R a n d A Z S a z a g u a n i n e - r e s i s t a n t a n d -sensi t ive; H A T , m e d i u m c o n t a i n i n g h y p o x a n t h i n e , 1 X 1 0 -4 M0 a m e t h o p t e r i n , 4 X 1 0 - 7 M, a n d t h y m i d i n e , 5 X 1 0 --4 M; H A T R o r H A T S, res is tant or sens i t ive t o H A T m e d i u m ; H G P R T , h y p o x a n t h i n e g u a n i n e phos- p h o r i b o s y l t r a n s f e r a s e ; P R P P , 5 - p h o s p h o r i b o s y l p y r o p h o s p h a t e ; T G , t h i o g u a n i n e ; T G R a n d T G S, th ioguanine-res i s tant and -sensit ive.

166

Over the past few years there has been controversy as to the relative merits of azaguanine and thioguanine as agents for selecting HGPRT-deficient lines from mar~malian cells [1,5,18,20,24,27]. Azaguanine has been considered to be unsatisfactory for a variety of reasons. Firstly, some wild-type cell lines, e.g. rat liver [30] and mouse P388 and L1210 cells [2] are insensitive to the toxic effects of azaguanine. Secondly, the cy to toxic effects of azaguanine can be profoundly affected by presence of exogenous purines [1,28] and several authors have recommended the use of dialysed serum during selection [1,28]. Thirdly, many clones containing appreciable HGPRT levels can grow in azaguanine [8,11,15,17--19] . Fourthly, the degree of HGPRT deficiency in azaguanine-selected clones has in some cases been shown to be related to the concentration of azaguanine used for selection [29]. Lastly, some azaguanine- resistant clones have been shown to be thioguanine~sensitive [ 11,27]. Such data led Morrow [18] to suggest that azaguanine may preferentially select for a class of mutants arising as a result of gene inactivation, and Van Diggelen et al. [27] to postulate that many azaguanine-resistant clones arise as a result of non-mutational mechanisms e.g. guanase induction and inhibition of azaguanine incorporation by exogenous purines. Such conclusions were supported by the dem- onstration that purified mouse HGPRT has a 300-fold lower affinity for azaguanine than for thioguanine and hypoxanthine and thus competes very poorly with hypoxanthine both at the enzyme level and in whole cells.

In spite of these potential problems, we [9] and other workers [7,11] have used azaguanine satisfactorily to select clones with reduced HGPRT levels from one of our Chinese hamster V79 cell lines by single-step procedures. We have previously reported that the majori ty of such lines have a stable AZRTGRHAT s phenotype [9]. In addition, HGPRT from some of these lines had altered heat sensitivity and altered electrophoretic mobil i ty in polyacrylamide gels suggest- ing that they carry mutat ions in the structural gene for HGPRT [9]. Other workers have presented more extensive evidence to suggest that azaguanine can be used to select for HGPRT structural gene mutat ions in V79 Chinese hamster cells [3,8].

In an a t tempt to explain the effectiveness of azaguanine in one V79 cell line and to provide some explanations for the many conflicting observations in the literature we have analysed the biological and biochemical effects of the two purine analogues in whole cells and at the HGPRT level. In addition we have further characterized mutant lines and present evidence indicating that their phenotypes are dependent on the parental cell line from which they were isolated.

Methods

Cell lines. The origins of the two V79 lines used have been described previously [9]. Both wild-type lines V79A and V79S have been repeatedly recloned in our laboratories. The conditions under which single-step spontaneous and induced variants resistant to azaguanine and/or thioguanine were selected have been described previously and some mutants have previously been partially characterized [9].

All cell lines were routinely grown in 4-oz medical bott les as monolayers in

167

Eagle's MEM supplemented with 10% foetal calf serum (10 MEM). Cell lines were subcultured twice weekly, 1/000 dilution, to maintain exponential growth. Analogue-resistant lines were rountinely subcultured in the same way in the absence of selective agents.

Mycoplasma contamination of cell lines. Cell lines were periodically screened for mycoplasma infection by two methods. Scanning electron microscopy was used to examine the surface of cells grown as monolayers on coverslips and cells were examined by light microscopy after Hoechst staining [4]. All cell lines were free of mycoplasma contamination at the time of their characteriza- tion.

Preparation of cell-free extracts. Cells were grown to near confluence in 1-1 roller bott les containing 100 ml medium. Medium was changed 24 h before harvesting. Cells were harvested after removal of medium and rinsing the mono- layer with ice-cold PBS by scraping with a rubber policeman into a small vol- ume of PBS. Cell-free extracts were made either in PBS or in buffer containing 50 mM Tris, pH 7.7, 50 mM KC1, 6 mM MgC12, 0.1 mM EDTA and 2 mM dithiothreitol [22] but usually omitt ing PRPP.

The cell pellet was suspended in 3 ml and freeze-thawed 3 times. The lysate was centrifuged in an MSE superspeed centrifuge 1 h at 100 000 g. The resul- tant clear supernatant was dispensed into 1-ml aliquots in separate plastic vials and stored at --20 ° C. Protein concentrat ion in the extracts was determined by the method of Lowry et al. [16]. A standard protein curve made using 1 mg/ml solution of Bovine Serum Albumin was used to calibrate assays in each occa- sion they were performed.

Hypoxanthine guanine phosphoribosyl transferase assays. (a} Using thioguanine as a substrate: This assay was carried out using the method of Kong and Parks [14]. The reaction mixture contained 100 mM Tris, pH 7.5, 2 mM MgSO4, 1 mM PRPP and varying concentrations of thioguanine. The reaction was initiated by adding 20 pl of cell-free extract to 0.48 ml of the reaction mixture in a 1-ml microceU, Starna Ltd., and the rate bf increase in absorbance at 345 nm corresponding to the rate of conversion of 6-thioguanine to the 5 ' -mononucleotide was measured for several minutes in a Unicam SP1800 UV spect rophotometer linked to an AR25 linear recorder.

Control assays were carried out on each cell extract using a reaction mix containing 20 mM EDTA but omitting the MgSO4. Under these conditions HGPRT is inactive and any observed changes in absorbance are due to direct conversion of 6-thioguanine to the nucleoside by nucleoside phosphorylase (E.C.2.4.2.1).

Reaction rates were estimated b y drawing the best-f i t straight line through the initial port ion of the trace from the linear recorder.

(b) Using [14C]hypoxanthine and [14C]azaguanine and PRPP as substrates: The assay procedure using [ ~4C] hypoxanthine as a substrate has been described previously [9]. Concentrations of [14C]hypoxanthine were varied between 0.016 and 0.45 mM. The same procedure was used with PRPP as a substrate, in this case hypoxanthine concentrat ion was kept constant at 0.16 mM and PRPP concentrations were varied be tween 0.01 and 0.5 mM.

Under assay conditions in which [ ~4C]hypoxanthine is converted t6 IMP i:e. in the presence of PRPP and Mg 2÷, [~4C]azaguanine is also converted to-a~fast

168

migrating species with an R~ expected for the mononucleotide. Identification of the spot was confirmed by use of authentic reference 8-azaguanine mono- phosphate. [14C]Azaguanine concentrations were varied between 0.016 and 0.45 mM. All assays were incubated at 37°C for 10--15 min.

Polyacrylamide gel electrophoresis. Electrophoresis was carried out as previously described in 8% polyacrylamide gels at pH 8.9 using [14C]hypoxanthine as a substrate [9].

Assay for guanase activity. Cell-free extracts prepared as described above were incubated in the presence of [14C]azaguanine or ['4C]guanine in the absence of Mg 2÷ and in the presence of EDTA to suppress HGPRT activity. The products of the reaction were subjected to either thin layer chromatography [7] or high-voltage electrophoresis [27]. Azaxanthine and xanthine were identified by reference to authentic standards. Activity in cell-free extracts was compared with that of commercially available guanase (Sigma).

Purity of azaguanine, thioguanine and hypoxanthine. Samples of azaguanine obtained from Sigma and Koch-Light were subjected to two-dimensional chromatography together with [~4C]azaguanine (Fluorochem) in methanol : HCL : water, 65 : 17 : 18 vol/vol (1st dimension), and n-butanol : methanol : water : ammonia, 60 : 20 : 20 : 1 vol/vol (2nd dimension), on Polygram Cell 300 thin- layer plates. [14C]Hypoxanthine was chromatogrammed in the same solvent systems. Spots were visualized under UV light, cut out and counted individu- ally. The remainder of the thin-layer plate was cut into strips and also counted for radioactivity. At least 5 spots were detected in all azaguanine preparations. The major spot comprised approx. 60% of radioactivity. Unlabelled azaguanine was purified to chromatographic homogeneity by Dr. B.W. Fox.

Uptake of [14C]hypoxanthine and [14C]azaguanine into whole cells. Uptake and incorporation of labelled purines into cell pools and acid-insoluble material was determined by methods previously described [9]. Whole cell incorporation of labelled precursors was determined by one of two methods described below, no real differences could be detected the two methods, l 0 s cells were plated into 9-cm petri dishes containing 9-cm coverslips or into sterile scintillation vials and incubated for various periods of time either in the presence of labelled purines, 0.1--0.35 pCi/ml, or with the labelled purine together and increasing molar ratios of unlabelled substrates. After removal of radioactive medium, cells were incubated for a further 1 h in the presence of unlabelled purine (10 -4 M), then washed 2X in ice-cold PBS. The labelled cultures were then processed in one of two ways:

Method 1. Labelled cells were washed overnight in running tap water and vials or plates were then allowed to dry. Cells were then solubilized by incu- bating the scintillation vials or vials containing broken coverslips for 20 min at 70°C for 0.3 ml NCS tissue solubilizer, Scintillant was then added to each vial and ~4C activity measured on a Packard 3375 Scintillation Counter.

Method 2. After washing with ice-cold saline, cells were extracted 3X with cold PCA, 1.5% v/v, added directly to the scintillation vials. Subsequently the samples were subjected to ethanol at room temperature to remove lipids and excess water. The ethanol was then decanted and after addition of 2 ml 5% PCA vials were incubated at 80°C for 40 min. After cooling 3 ml 3 : 1 Triton Phosphor was added and samples were counted.

169

Results

Sensitiuity of two V79 lines to cell killing by azaguanine and thioguanine. We have previously published dose-survival curves for V79S and A cells exposed to thioguanine or azaguanine indicating similar overall sensitivity of the two cell lines to the two drugs [20]. However, 20--30-fold higher concentrations of azaguanine are required to achieve the same level of kill in the two cell lines. At this time, we paid little attention to the cytotoxic effects of low concentrations of the analogues. A more detailed study of the cytotoxic effects of low concentrations is shown in Fig. 1. No difference was detectable to two cell lines with respect to their sensitivity to TG. Exposure to 1/~g/m] TG resulted in a surviving fraction of approx. 5 X 10 -s. However, in spite of some scatter of the data, V79S cells were consistently more sensitive to the cytotoxic effects of azaguanine than V79A cells. A concentration of 1--2 #g/ml reduced the surviving fraction of V79S cells to approx. 5 X 10 -4 whereas concentrations

o

10 -2- g

~ lO 3-

i0 -4-

10 -5

a

o o a i i i r i

0.2 0.6 1.0

concen!rat ion of

thioguanine b,g/ml

o o •

I I I 1.0 5.0 10.0

concentrat ion of azaguanioe

. g / m l

~ a

Fig. 1. D o s e - - r e s p o n s e re l a t i onsh i p for t h i o g u a n l n e and a z a g u a n i n e in V 7 9 A and V 7 9 S cel ls . Cells w e r e p l a t e d at 5 X 1 0 2 , 5 × 1 0 3 or 5 × 1 0 4 per d i sh d e p e n d i n g o n t h e l eve l o f ki l l e x p e c t e d . A f t e r 4 h i n c u b a - t i o n a z a g u a n i n e or t h i o g u a n i n e w a s a d d e d . A f t e r 7 days ' i n c u b a t i o n p la tes w e r e f i x e d and s ta ined a n d

c o l o n i e s c o u n t e d , o , V 7 9 A cel ls; o, V 7 9 S cel ls .

170

c~

x

y,

44~

40-

36-

32-

28-

24-

20

16

12-

8

4

2 4 6 8 I 0 3~0

Dose of azaguanine//thioguanine pg/rnl

Fig. 2. R a t e o f ce l l kil l ing o f V 7 9 A and S ce l l s b y d i f f e r e n t c o n c e n t r a t i o n s o f azaguanine and th ioguanine . Cells w e r e p lated at e i ther 5 X 102 or 5 × 104 pe r 9 -cm dish and azaguanine or th ioguan ine wa s added at the des ired c o n c e n t r a t i o n . A t approx . 5-h intervals up to 48 h drug-conta in ing m e d i u m wa s r e m o v e d and rep laced w i t h fresh 10 MEM and i n c u b a t i o n c o n t i n u e d for 7 days . C o l o n y survival at each dose was p l o t t e d against durat ion o f e x p o s u r e and f r o m these data the e x p o s u r e t i m e required for 90% lethal i ty was d e t e r m i n e d for each dose . e , V79S cells e x p o s e d to azaguanine; A V 7 9 A cel ls e x p o s e d to azaguanine; o, V79S cells; A, V 7 9 A cel ls e x p o s e d to th ioguanine .

in excess of 5 pg/ml were necessary before similar cytotoxicity was evident in V79A cells.

Rate of killing of the two V79 lines by azaguanine and thioguanine. Previous data suggested that V79A cells were killed at the same rate by 1 pg/ml TG and 20 #g/ml AZ [20]. A more detailed study of the rates of kill of the two cell lines by the two analogues is shown in Fig. 2. The rate of cell killing by thioguanine is dose<lependent in both cell lines for concentrations below 1.0 pg/ ml. At higher concentrations kill is independent of dose and differs only slightly in S and A cells. In S cells concentrations of azaguanine in excess of 10 pg/ml kill almost as rapidly as 10-fold lower thioguanine concentrations. How- ever, in A cells the rate of cell killing even by high concentrations of azaguanine (30 pg/ml) was considerably slower than in S cells and much slower than the rate of kill by thioguanine.

Inhibition of [14C]hypoxanthine incorporation into intact cells by purine analogues. The differential rates o f kill by the two analogues in the two cell lines suggest differences in their rates of incorporation, either due to differences in endogenous purine pools or to differences in affinity of HGPRT for

171

la)

I00-

x

100-

x

50- c~

¢

~: loo-

50

Ibl

° ox~,x x o , . . . . ~ ~ ~_

(el

?,.

"o-o. :---T i 2 4 6 8 10 12

Molar ratio

Fig. 3. I nh ib i t i on , by pur ine analogues , of the i n c o r p o r a t i o n o f [ 1 4 C ] h y p o x a n t h i n e (spec. act . 56 m C i /

m m o l e ) in V79S cells a f t e r d i f f e r en t pe r iods o f i nc uba t i on . (a) 4 h , (b) 8 h, (c) 24 h. Th e d o t t e d line in (a) r ep resen t s the t heo re t i ca l i so tope d i lu t ion curve for [ 14 C ] h y p o x a n t h i n e . Th e c o n c e n t r a t i o n of [ 14 C]- h y p o x a n t h i n e fo r 100% i n c o r p o r a t i o n was 0 ,008 mM . Resul ts r ep r e sen t m e a n s of repl ica te e s t ima t ions at each m o l a r ra t io in 3- -4 sc int i l la t ion vials, e , azaguan ine ; o, t h ioguan ine ; X, h y p o x a n t h i n e .

the different analogue substrates, or both. We therefore determined the relative effectiveness with which increasing molar concentrat ions of pur.ine analogues reduced the incorporation of labelled [ 14C]hypoxanthine in the two lines. The results in Figs. 3 and 4 show that addition of unlabelled hypoxanthine decreased the incorporation of [14C]hypoxanthine as would be expected as a result of isotopes dilution in both cell lines. The effect was demonstrable after

T A B L E 1

I N C O R P O R A T I O N OF [ 1 4 C ] H Y P O X A N T H I N E IN V79 C E L L L I N E S

Cell l ine I n c u b a t i o n Cell c o u n t d . p . m . / 1 0 5 cells × 10 -3 period (X 10 -4 ) (h) Acid-soluble Acid-insoluble

fraction a fraction a

V 7 9 A 4 4 .4 -+ 0 .78 130.7 + 40 .1 66 .9 + 14 .23 7 4.9 + 0 .64 113 .7 + 48 .0 168 .8 ± 53 .30

24 15.1 + 2 .82 72 .8 ± 16.2 252.1 ± 9 .35

V79S 4 1 .26 + 0 .14 320 .2 ± 28.6 167 .0 ± 21 .3 7 1.7 ± 0 .26 300 .1 + 16 .8 .302 .6 ± 26 .8

24 8.0 ± 1 .96 280 .0 ± 28 .0 606 .1 + 179.6

a D a t a are con t ro l s i.e. w i t h o u t a dde d nuc leos ides f r o m whole cell inh ib i t ion Expts . s h o w n in Figs. 3 and 4. Resul t s axe m e a n -+ S.E. o f 6 - - 1 0 separa te d e t e r m i n a t i o n s .

172

I00=

50-

~ 100- ==

== o* 5o-

o lO0- g

-~ 5o-

100-

50-

ta) • • - - r

2~'~.~. -_ - - -

[b)o " ~ ' " o ~ o ~ o - ~x x

o \

0 X

- - 0 -

c)

~x • o \

0 X

~ o ~ ,

O-

~ 0 " 0

o

(d)

O X X

~°~ o.. o o ? // 2 4 8 1 12 14 20

M01ar ratio

Fig. 4. Inhibi t ion by purine analogues of the incorporat ion of [ 14C]hypoxanth ine into V79A cells after different periods of incubat ion: (a) 1 h, (b) 4 h, (e) 7 h, (d) 24 h. o, azaguanine; o, thioguanine; ~, guanine; X, hypoxanth ine . Dotted line in (a) represents theoretical isotope di lut ion curve for hypoxanthine .

a 1-h incubation and was similar in both cell lines, and indicates that [14C]- hypoxanthine equilibrates rapidly with endogenous purine pools.

Similar inhibition of [I4C]hypoxanthine utilization was evident in both cell lines at longer incubation times. The exception was 24 h in V79A cells when the competit ive effectiveness of unlabelled hypoxanthine was apparently reduced. The data in Table 1 indicate that at least two cell doublings occurred inboth cell lines during this period, therefore the effect is not due to toxicity or cell loss. Also shown in Table 1 are data supporting the conclusion that equili- bration of cell pools withexogenous [ I4C]hypoxanthine is rapid as there was no changes in d.pzn, recovered in the acid-soluble fraction of either cell line between 4 and 24 h. Uptake of [ I4C]hypoxanthine into cell pools (acid-soluble fraction) and incorporation into nucleic acids was 2--3-fold higher in V79S cells than in V79A cells indicating a smaller endogenous hypoxanthine pool in V79S cells. I n V79S cells the rate of incorporation into nucleic acids was approx, linear over the 24-h incubation period but decreased between 7 and 24 h in V79A cells. This reduced rate of incorporation is probable the result of a switch from salvage to de novo nucleic acid synthesis as evidenced by an increase in the relative utilization of [14C]formate at this time (data not

173

_

?

3-

• . ~ 2-

1 .

,m 7 , , , , ?

1 2 3 4 5 6 7 8

Hrs incubation in 14Cazaguanine

Fig. 5. Incorporat ion of [ 14C]azaguanine, spec. act. 22.0 mCi/mmole , into hot and cold acid-soluble hot acid fract ions of V79A and S cells (see Methods). o, V79S cells, 0.35 ~tCi/ml; s, V79S cells, 0.1 #Ci/ml; o, V79A cells, 0.35/~Ci/ml; o, V79A cells, 0.1 DCi/ml; X, V79A cells, 0.1 ~Ci/ml, cold acid-soluble fraction.

shown) which was more marked in V79A cells than in V79S cells. This switch will presumably alter the size of the endogenous hypoxanthine pools and may be related to the observed alteration in competit ive effectiviness of unlabelled hypoxanthine at 24 h. Without further detailed experimentation, however, no firm conclusions can be drawn.

Addition of increasing molar ratios of guanine or thioguanine also resulted in rapid inhibition of [14C]hypoxanthine incorporation. Azaguanine however behaved differently. After 1 h incubation with molar ratios of up to 30 : 1 excess azaguanine, no inhibition was evident. Prolongation of the incubation per iod to 4 h also resulted in little inhibition. However, by 8 h significant inhibition was evident at high molar ratios and this effect was more marked by 24 h in both cell lines.

Similar Expts. in mouse cells were described by Van Diggelen et al. [27] which also indicated that azaguanine was a highly inefficient inhibitor of [ ~4C]- hypoxanthine uptake, whereas thioguanine and guanine reduced hypoxanthine incorporation at molar ratios similar to those used in hamster cells. However, only 1 h incubation periods were used. It is therefore not clear whether effects similar to those observed in hamster cells also occur in mouse cells.

Azaguanine uptake in wild-type V79A and S cell lines. Incorporation of [~4C]azaguanine into acid-insoluble material of V79S cells increased linearly with time (0.1 pCi/ml) bu t incorporation was undetectable in V79A cells incubated with the same azaguanine concentrations over the same time period. An increase in azaguanine concentrat ion to 0.35 pCi/ml resulted in a .5-fold increase in incorporation in S cells over the first 3 h, thereafter incorporation

O "~ 0.3 | o / /

"!0. x

0 20 40 60 1/S hypoxanthine mM

0.8- b

x

¢ 0 ' 2'0 ' 4'0 ' 6'0

1/S azaguanine mM

• = o . e

E"

,= E

0.4

E

0.,;

.~, 25-

E ~ 2.0 20-

1.5 15-

1.0 10-

~0.5 5-

o

20 ' 4'o s'o 8'o 16o 1/S PRPP mM

Fig. 6. Lineweaver--Burke plots for (a) hypoxanthine, (b) azaguanine, and (c) PRPP in different wild-type V79 lines. For assay of hypoxanthine and azaguanine the PRPP concentration in the reaction mix was 12.5 mM. For PRPP assay hyPoxanthine concentration was 0.16 raM. ¢, X, V79A wild-type; o, V79S wild-type at 0.008 mM hypoxanthine (inner scale for V0).

1 7 5

plateaued or even decreased. Incubation of V79A cells with 0.35 pCi/ml resulted in a detectable incorporation after 1 h incubation bu t little increase in incorporation occurred between 1 and 6 h (Fig. 5). The lack of incorporation in A cells is no t due to lack of uptake of azaguanine, as 14C-labelled material accumulated in acid-soluble fractions over the 8-h incubation period (Fig. 5). The plateau in incorporation of 14C-azaguanine in S cells incubated with 0.35 pCi/ml (approx. 3 pg/ml) seems likely to be due to effects of this concentration on cell growth. Proliferation of S cells under these conditions ceased at 8 h whereas in A cells incubated under the same conditions growth continued exponentially at control rates (data not shown).

Guanase activity in cell extracts. When [14C]guanine was used as substrate for a commercial guanase preparation rapid conversion to xanthine was observed, i.e. by 20 min 98.5% of radioactivity appeared as xanthine. [14C]Aza- guanine was a poor substrate for commercially available guanase, only 14% conversion to azaxanthine was observed over a 90-min incubation period. Cell- free extracts of wild-type V79A or S cells had undetectable activity using either [ 14C]guanine or [ 14C] azaguanine as substrate (Table ~).

Kinetics o f wild-type HGPRT with respect to various substrates. Van Digge- len et al. [27] have suggested that the kinetics of inhibition of hypoxanthine uptake by purine analogues directly reflects the relative affinity of HGPRT for the various substrates. We therefore measured the Kms of HGPRT of the two cell lines for hypoxanthine and the two purine analogues. Typical data for hypoxanthine and azaguanine are shown in Fig. 6. The Km of HGPRT for hypoxanthine and azaguanine was similar in the two cell lines. The affinity of the enzyme for azaguanine was approx. 10-fold lower than for hypoxanthine in both cell lines. The Km and Vmax data for these substrates. PRPP, and the thioguanine are summarized in Table 3.No differences in K~s were detected except in the case of thioguanine. The affinity of HGPRT for this substrate was 3-fold higher in V79A cells than in V79S cells.

Inhibition by purine analogues o f conversion o f [14C]hypoxanthine to IMP in cell-free extracts. Incubation of cell-free extracts in the presence of [~4C]hypoxanthine and increasing molar ratios of unlabelled hypoxanthine or thioguanine resulted in a marked reduction in the conversion of labelled substrate to IMP (Fig. 7). These data indicate that thioguanine is acting as a competit ive inhibitor, and from these data and those in Fig. 6 a K i of 5 #M can be calculated for thioguanine by the method of Dixon [5]. Azaguanine, however, at a 20- fold excess did not inhibit the conversion of hypoxanthine to IMP. Molar ratios

T A B L E 2

G U A N A S E A C T I V I T Y a IN C E L L - F R E E E X T R A C T S OF W I L D - T Y P E V79 CELLS

Cell e x t r a c t f r o m

X a n t h i n e or a z a x a n t h i n e p r o d u c e d ( n m o l e s / m i n / m g ) f r o m

[ 14 C ] Gua n ine [ 14 C ] Azaguan ine

V 7 9 A 0 .001 0 .003 V79S 0 . 003 0 . 002 G u a n a s e 56.0 0 . 208

a A s s a y r o u t i n e l y p e r f o r m e d in the p r e s e n c e o f E D T A to i n h i b i t H G P R T act iv i ty .

176

TABLE 3

KINETICS OF HGPRT FOR VARIOUS SUBSTRATES IN WILD-TYPE V79 CELL LINES

Cell line V79A V79S

K m (M) Vma x n.m./min/mg K m (M) Vma x n.m./min/mg

H y p o x a n t h i n e 1.0 × 10 -5 14.2 1.0 × 10 -5 7.6 PRPP 5.6 × 10 -5 8.0 5.6 × 10 -5 2.5

Thioguanine 3.0 × 10 -6 11.0 1.2 × 10 -5 14.2 Azaguanine 1.0 X 10 -4 25.0 1.0 X 10-4 12.5

of up to 50 : 1 also did not inhibit. Reduction of the hypoxanthine concentration in the assay and dialysis of the cell-free extracts had no effect (data not shown).

When [14C]azaguanine was used as a substrate and different hypoxanthine concentrations were added to the assay mix, inhibition of AZ conversion to AZGMP by hypoxanth ino was non-competit ive (Fig. 8a). Similar experiments in which [14C]hypoxanthine was used as substrate and 0 .10 mM thioguanine added, are shown in Fig. 8b and provide further evidence for the largely competitive nature of thioguanine inhibition.

Frequency of spontaneous mutants and phenotypes of resistant clones. The frequency of colonies recovered when wild-type V79A cells were plated in AZ, 30 pg/ml, was 4.3 + 1.01 × 10 -4 (mean 10 determinations i.e. plating 1 × l 0 s cells into 10 plates on 10 separate occasions) whilst that recovered after plating

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the same population in 10 pg/ml TG was 4.5 ± 1.8 × 10 -6. The frequency of V79 colonies recovered from 10 pg/ml TG was 5.0 ± 0.5 × 10 -6 (mean 10 determinations). The frequency: of colonies recovered from the same population plated in 30/zg/ml azaguanine was similar but clones were only evident after 10 days ' growth as apposed to 7 days for other selective conditions. A summary of the phenotypes of isolated clones is presented in Table 4.

All but two clones selected in either analogue from V79A cells were resistant to both AZ and TG and HAT s. The two clones which were AZ • and TG s grew in HAT when plated at high density.

In contrast all clones selected from V79S cells in either analogue were TGaHAT s but showed partial AZ sensitivity.

Azaguanine sensitivity o f TG R V79S lines. The partial AZ sensitivity of V79STG a cells was mainly evident when cells were plated at low density. The effects of 30/~g/ml on growth of TG R clones plated at a density of 5 × 104 per dish are shown in Fig. 9. At this density a cytostatic effect only is evident. The rate of kill by azaguanine of TG R clones plated at low density was slow, 40%

179

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f r o m t h e m . o, V79S wi ld- type ; ~, ~, +, c on t ro l g r o w t h curves for 3 d i f f e r en t 6 T G R m u t a n t s . A e f fec t of 30 ~ g / m l azaguan ine on g r o w t h ra te of c on t ro l cells; I , e M , e f f e c t o f 30 p g / m l azaguanine on g r o w t h of three d i f f eren t 6 T G R l ines der ived f r o m V79S cells.

kill with a 96-h exposure (data not shown). The toxicity of azaguanine was not the result of impurities present in the commercially available samples, since purification of azaguanine to chromatographic homogenei ty did not eliminate this secondary toxicity.

Hypoxanthine and azaguanine incorporation in TGR-resist.ant clones o f V79S cells. We have previously reported very low hypoxanthine incorporation in analogue resistant V79S cells in spite of measureable in vitro enzyme activity [9]. Under similar conditions V79S TG R clones all failed to incorporate either hypoxanthine or azaguanine into acid-insoluble material.

Kinetics o f HGPRT of resistant clones with respect to various substrates. The affinity of HGPRT of a number of TGRAZRHATSV79A clones for hypoxanthine and azaguanine was measured and found to be identical with that of the wild-type enzyme. Similarly, no alteration in affinity for guanine was detectable. Affinity for PRPP (Fig. 10) was however reduced in mutant cell lines.

In V79S TG R mutants, affinity for thioguanine was much reduced [13] and no conversion of hypoxanthine or azaguanine to the mononucleot ide could be detected in cell-free extracts. A similar reduction in affinity for TG was found in V79A mutants, typical data is shown in Fig. 10. HGPRT of mutant lines consistently showed substrate inhibition at higher thioguanine concentrations. The concentrat ion at which this effect was evident, however, varies with different cell extracts. Substrate inhibition at similar TG concentra t ion was also seen in extracts of V79S wild-type cells [13] but no t in V79A cell extracts. The effect thus appears to be a characteristic o f the cell line and not specifically of the mutants.

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No labelled peaks could be detected when cell-free extracts of several V79S TG R mutants were run on polyacrylamide gels using either hypoxanthine or guanine as a substrate. In contrast V79A mutants had detectable enzyrhe activity with altered electrophoretic mobili ty (Fig. 11) as previously described for mutants of this cell line [9].

Discussion

The affinity of HGPRT for hypoxanthine was identical in extracts of the two wild-type cell lines. The affinity for azaguanine was also identical but was 10-fold lower than for the natural substrate. An approx. 3-fold difference in Km for thioguanine was evident between extracts of the two cell lines. These differences in relative Kms are probably sufficient to account for the 20--30- fold difference in dose of the two analogues required to produce equitoxic effects but do not account for the slow rate of kill by azaguanine or its slow rate of incorporation into acid-insoluble material in both cell lines.

Thioguanine acts as a competitive inhibitor of [ 14C]hypoxanthine conversion to IMP with a Ki of 2/~M in CHO cells [23]. We have made similar observations in V79 cells and have obtained a similar Ki value for thioguanine, 5/~M. These results are consistent with the observed biological effectiveness of thioguanine

182

in hamster cells, and indicate that thioguanine and hypoxanthine bind at similar if not identical sites on the HGPRT molecule.

In similar experiments, azaguanine, even at high molar ratios of 80 : 1, did not affect '4C-hypoxanthine conversion to IMP. In contrast, when ['4C]azaguanine was used as a substrate, its conversion to AzaGMP could be inhibited profoundly by the addition of low concentrations of hypoxanthine (0.008 mM). The inhibition in this case was of the non-competit ive type which indicates that azaguanine and hypoxanthine bind to different sites on the HGPRT molecule.

These data provide a biochemical basis for our observations and for some of the other observations reported in the literature. Firstly, the effectiveness of hypoxanthine as an inhibitor of azaguanine phosphorylat ion together with the approx. 10-fold difference in Km of HGPRT for the two substrates accounts for the ineffectiveness of low concentrations of azaguanine and the frequently reported serum effects [1,28]. The greater sensitivity of S cells to azaguanine toxicity does not seem to be related to a difference in affinity of HGPRT for this substrate. It is therefore likely to be due either to a more rapid breakdown of the enzyme--inhibitor--substrate (EIS) complex in S cells or to differences in affinity of other enzymes on the purine catabolic pathway for AzaGMP [211.

The ability of clones with appreciable HGPRT activity [10,11,15,19] to grow in azaguanine, and the apparent relationship between level of enzyme activity and dose of azaguanine used for selection [10,29] are also probably explicable. In the first case, any endogenous hypoxanthine will significantly reduce azaguanine incorporation in vivo, in spite of differences or similarities in the Km of HGPRT for the two substrates, allowing HGPRT + cells to survive. Measurement of HGPRT activity in vitro using '4C-hypoxanthine as a substrate will indicate near normal levels. In lines selected from different doses of azaguanine and subsequently maintained in azaguanine presumably because of their unstable phenotypes, the slow dissociation of the EIS complex will result in different amounts of azaguanine and hypoxanthine remaining bound to HGPRT. A reduced rate of protein synthesis is known to occur as a result of azaguanine incorporation [21] and hence a reduced rate of synthesis of new enzyme. Together these phenomena will result in progressive lowering of HGPRT levels and azaguanine resistance in the absence of mutation in the structural gene [10].

It has been suggested previously, that purine analogues behave as alternative substrates for HGPRT rather than inhibitors [14]. In the absence of exogenous hypoxanthine this appears to be the case, however in competi t ion experiments thioguanine clearly acts largely as a compet i t ive inhibitor of hypoxanthine and hypoxanthine as a noncompet i t ive inhibitor of azaguanine phosphorylat ion at the HGPRT level.

The. difference in apparent K,, for thioguanine in the two wild-type lines may relate to the presence in V79A cells of higher endogenous hypoxanthine levels as suggested by the data in Table 1 and Fig. 5 since no difference is electrophoretic mobil i ty of the two wild-type enzymes could be detected ([9,13] and Fig. 11). The observation illustrates the difficulty of accepting kinetic changes alone as evidence for structural gene mutation.

183

The majori ty of resistant lines isolated from V79A cells had the same phenotype i.e. AZRTGRHAT s which was associated with the following: (1) Reduced HGPRT activity in vitro measured in the presence of saturating PRPP concentrations [9]. {2) HGPRT with unaltered affinity for azaguanine and hypoxanthine, bu t reduced affinity for PRPP and thioguanine (Fig. 10). (3) HGPRT with altered electrophoretic mobil i ty ([9] and Fig. 11).

The reduction in affinity of HGPRT for PRPP results in reduced availability of PRPP to the salvage enzyme in vivo, provides an explanation for the reduced invivo 14C-hypoxanthine incorporation [9], and even in the absence of a change in affinity of HGPRT for azaguanine can account for AZ R because of the low affinity of this analogue for the enzyme. The cross resistance to thioguanine does however appear to be associated with a change in affinity of the enzyme for this substrate.

AZ R lines of V79 cells in which affinity of HGPRT for PRPP is reduced have been described previously [3,8]. The enzyme also had reduced electrophoretic mobil i ty and altered subunit molecular weight [8]. These particular AZ R lines however, in contrast to the majority of ours, grew in HAT and were TG s sug- gesting a mutat ion specifically affecting the binding site for PRPP. Rarely, clones with AZRTG s phenotype were recovered from our V79A cells (Table 4). They had the following characteristics: (1) Ability to grow in HAT when plated at high density. (2) Near normal in vitro HGPRT activity. (3) HGPRT with reduced affinity for PRPP bu t unaltered affinity for thioguanine, azaguanine and hypoxanthine (data not shown). (4) HGPRT with minimal alteration in electrophoretic mobil i ty.

Again, azaguanine resistance was associated with altered kinetics with respect to PRPP. The absence of a change in electrophoretic mobil i ty in these clones, in contrast to those which are cross-resistant to both analogues, suggests that the altered mobil i ty of clones of the latter class may be associated with an altered TG binding site.

Lines selected in either TG or AZ from V79S cells were invariably TGRAZSHAT s. HGPRT had demonstrably altered affinity for TG but other at tempts to characterize the enzyme failed because of the very low enzyme activity. Evidence from reversion analysis [12,13] has indicated that many of these lines carry deletions in the structural gene. However, several lines, includ- ing the one most extensively studied revert on exposure to alkylating agents and therefore probably carry missense or nonsense mutations.

The persistent association of AZ sensitivity with TG R and HAT s in V79S clones is in marked contrast to the situation in the majority of V79A isolates. The effect was however cytostat ic, rather than cytotoxic , and was evident mainly in cells plated at low density. Since no incorporation of azaguanine could be demonstrated either in whole cells or at the enzyme level, this toxicity may reflect a second site of action of AZ which is masked in cells which possess HGPRT activity. Cytostatic effects manifested as reduced colony size have been observed in V79A cells exposed to high AZ concentrations (100 pg/ml; our unpublished observations). The second site of action of azaguanine has not been identified.

It has frequently been implied that azaguanine and thioguanine can be used interchangeably to select for mutants at the HGPRT locus in V79 cells bu t that

184

thioguanine selects more stringently for an enzyme-negative phenotype. Many of our results are consistent with this conclusion. However, although the two analogues may possibly be considered interchangeable in V79A cells several features of our data suggest that they may be selecting for mutations at different sites on the HGPRT molecule. Firstly, spontaneous mutant frequencies are consistently higher in AZ. Secondly, AZ R lines are frequently but n o t invariably cross-resistant to TG [8,11] (Table 3). Thirdly, the two analogues obvi- ously bind to different sites on the HGPRT molecule (Fig. 8). In V79S cells they cannot be used interchangeably as all TG R mutants were AZ s.

In conclusion, therefore, although the Km of HGPRT for azaguanine was 10- fold lower than for thioguanine and hypoxanthine in our hamster cell lines, this did not interfere significantly with the cytotoxic effectiveness of this analogue or preclude its use as a selective agent for structural gene mutants in V79A cells providing that high concentrations were used. Other complications reported in other cell lines e.g. breakdown of AZA-GMP intracellularly, postulated to occur in rat cells on the basis of observed breakdown of IMP to inosine [30] and induction of guanase [27], could not be demonstrated. Thus for hamster cell mutants studied here, the hypothesis that azaguanine selects for a unique class of mutants arising as a result of gene inactivation [18] or that AZ R lines are of non-mutational origin [ 27], appear untenable.

The reported ineffectiveness of azaguanine in some other hamster [25,26] and human [5] cells may result from greater differences in affinity of HGPRT for the purine analogues and natural substrates, differences in endogenous hypoxanthine pools or differences in rates of breakdown of the EIS complex, all of which will result in lowered azaguanine incorporation.

Acknowledgements

The excellent technical assictance of Mrs. M. Bloomfield and Mr. M. Greaves is gratefully acknowledged. The work was supported by grants from the Medi- cal Research Council and the Cancer Research Campaign.

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mechanism of cytotoxic action of azaguanine and thioguanine in wild-type v79 cell lines and their...

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