93

Mutation Research, 36 (1976) 93--104 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE ISOLATION AND PRELIMINARY CHARACTERISATION OF 6-THIOGUANINE-RESISTANT MUTANTS OF HUMAN DIPLOID FIBROBLASTS

ROGER COX and W.K. MASSON

MRC Radiobiology Unit, Harwell, Didcot, Oxon. 0 X l l ORD (England)

(Received October 14th, 1975) (Accepted January 27th, 1976)

Summary

Mutant clones of human diploid fibroblasts deficient in the enzyme, hypo- xanthine-guanine phosphoribosyl transferase (HGPRT) were selected by their ability to grow in medium containing the cytotoxic purine analogue, 6-thio- guanine (6TG). The optimal conditions for mutant selection were 6TG concen- trations between 1 and 5 pg m1-1 and cell plating densities ~ 103 cells cm -z .

Nine spontaneous and four radiation-induced 6TG-resistant mutants had < 2% of the parental strain HGPRT activity and were unable to grow in medium containing azaserine. These mutants were phenotypically stable during >25 population doublings in non-selective medium and five mutants that were examined showed no gross change from the normal human karyotype.

Evidence is presented to show that 6TG is a better selective agent than 8-aza- guanine (8AG) for HGPRT-deficient mutants of human diploid fibroblasts.

Introduction

Recent experiments with cultured human diploid fibroblasts have shown that it is possible to select stable clones having phenotypic characteristics different from those of the normal parental cells [1,4,12]. Resistance to the cytotoxic purine analogue, 8-azaguanine (8AG) is a property which allows selection of mutan t clones having sub-normal activities of the purine salvage enzyme, hypo- xanthine-guanine phosphoribosyl transferase (HGPRT, E.C. 2.4.2.8) [7] and induction of 8AG-resistant mutants by physical and chemical agents has been used as a measure of mutagenicity [1,11]. However, 8AG is a poor selective agent for use in quantitative mutagenesis studies as judged by the growth of some HGPRT-proficient clones in 8AG medium [1,14,18], the effect of 8AG concentration on the biochemical phenotype of selected mutants [18] and the

94

antagonistic effect of some sera on the selective action of the analogue [1,19]. Human HGPRT has a much greater affinity for the purine analogue, 6-thio-

guanine (6TG) than for 8AG [10], suggesting that 6TG could be a more effi- cient selective agent for HGPRT-deficient cells. In this paper we describe the isolation and preliminary characterisation of 6TG-resistant mutants of human diploid fibroblasts and present some evidence supporting this view.

Materials and methods

Cells HF19 human diploid fibroblast cultures were initiated from ? foetal lung tis-

sue using the method previously described [5].

Media The normal growth medium was Eagle's MEM plus 10% foetal calf serum

(Gibco-Biocult). The medium for selection of 6TG-resistant mutants was nor- mal growth medium supplemented with 1--5 pg ml -~ 6TG (Sigma). Azaserine medium, for the counterselection of 6TG-resistant mutants, was normal growth medium plus hypoxanthine (6.8 pg ml -~ ), azaserine (1.7 or 3.5 pg m1-1 ) and thymidine (1.2 #g ml -~ ).

Culture and cloning techniques Cell cultures were maintained in glass roller-bottles using methods previously

described [5]. Early passage (4--15 population doublings) cultures of HF19 were used in mutant selection experiments and cells were harvested during the exponential phase of growth using 0.1% trypsin plus 0.4 mg ml-~ EDTA. Single cell suspensions were counted using a haemocytometer . Cells for feeder layers were irradiated with 3 krad doses of X-rays.

All cloning experiments were performed in 9 cm diameter tissue culture grade polystyrene Petri dishes (Sterilin) containing 10 ml medium. Unless otherwise stated the feeder cell technique was used to improve the cloning effi- ciency of cells [3]. Petri dishes were incubated at 37°C in 95% air plus 5% CO2 for 14--21 days and clones arising after this time were stained with 0.25% Azur A (Searle Diagnostic), dried and counted using a low power microscope.

Clones arising in 6TG medium were located using a low power dark field microscope, isolated using pasteur pipettes with silicone rubber tips and trans- ferred to normal growth medium. Cultures of mutants were maintained in nor- mal growth medium without 6TG; medium changes at 3 day intervals were nec- essary for adequate growth of some mutant cultures.

Cytotoxicity o f 6TG and selection of 6TG-resistant mutants The cytotoxic i ty of 6TG at concentrations up to 10 pg m1-1 was measured

in terms of the fraction of HF19 cells capable of clonal growth in normal growth medium containing the analogue. The cell plating density on all Petri dishes was ~103 cells cm -2 . At surviving fractions >10 -4 (6TG at 0---0.5 pg ml-I ) feeder cells and experimental cells were plated together so that the total cell density was 103 cells cm -: . At surviving fractions <10 -4 (6TG at 0.5--10 pg m1-1 ) no feeder cells were used. Spontaneous 6TG-resistant mutants were

95

selected in normal growth medium containing 1--5 pg ml-I 6TG. Radiation-in- duced 6TG-resistant mutants were selected at 2 pg m1-1 6TG after a 7 day post-irradiation growth period in normal growth medium to allow expression of induced mutants.

The effect of cell plating density on the frequency of spontaneous mutants was examined by varying plating density from 3 • 102--3 • 103 cells cm -2 and selecting mutants resistant to 2 pg ml -~ 6TG. The efficiency of recovery of iso- lated 6TG-resistant mutants under selective conditions was determined by adding a mixture of 250 mutant cells from isolated mutants and 5 • 104 paren- tal strain cells to Petri dishes containing 6TG (2 pg m1-1 ) medium. Cloning effi- ciencies of four mutants under these selective conditions were compared with the cloning efficiencies of the same mutants in normal (non-selective) medium using the feeder cell technique.

The incorporation o f radioactive hypoxanthine and adenine it2to mutant and wild type cells

Hypoxanthine guanine phosphoribosyl transferase (HGPRT, E.C. 2.4.2.8) and adenine phosphoribosyl transferase (APRT, EC 2.4.2.7) were assayed indi- rectly by the cellular incorporation of radioactive hypoxanthine and adenine into trichloracetic acid (TCA) insoluble cell fractions [9]. Assays were per- formed in 3 cm diameter plastic petri dishes each containing a monolayer of l 0 s cells and 1 ml Eagle's MEM (without serum) plus 0.2 pCi ml -~ [14C]hypo- xanthine ( 3 . 1 0 -° M) and 0.4 pCi [3H]adenine ( 1 . 5 - 1 0 -s M). The labelled substrates were added at zero time and the cell monolayers incubated at 37°C in 95% air plus 5% CO2. The reactions were terminated at hourly intervals up to 4 h by two washes with 2 ml ice-cold phosphate buffered saline (pH 7.3}. Cells were immediately lysed with 1 ml of 1% sodium dodecyl sulphate (SDS) and the SDS lysates then washed into test tubes with 2 × 2 ml of buffer solution. Nucleic acids were precipitated by addition of 5 ml 10% TCA at 4°C and the precipitates collected on Whatman GF filters. Filters were washed with 5 ml 5% TCA at 4 ° C, air-dried and 14C and 3H activities determined by liquid scintilla- tion counting using toluene-butyl PBD (Koch--Light) scintillation fluid. All samples were assayed in duplicate and enzyme activities expressed as the mean hourly increase in ~4C and 3H incorporation into nucleic acids during the incu- bation period.

Growth in azaserine medium The growth of 6TG resistant mutants and HF19 parental strain cells in aza-

serine medium was examined by plating 5 . 1 0 4 cells into each of 35 petri dishes containing 10 ml azaserine medium. Incubation conditions were the same as those described above.

Ka ry o typing Mitotic cells were accumulated by exposing exponentially growing cultures

of HF19 parental strain and mutants to 0.1 pg ml -~ colcemid for 4 h. Air dried preparations of cells on glass slides were treated with hypotonic citrate solu- tion, fixed with methanol--acetic acid (3 : 1) mixture and stained with Giemsa [13]. The karyotypes of 100 randomly chosen mitotic cells from each culture were recorded.

96

Results

Dose-response to inactivation by 6TG The dose-response curve of human diploid fibroblasts to 6TG (Fig. 1) was

similar in shape to that published for 8AG with an established line of human cells [15] . The fraction of cells capable of clone format ion fell rapidly with in- creasing 6TG concentrat ions up to 0.5 pg m1-1 . A plateau region of cell survival (at surviving fractions between 10 -s and 10 -6 ) was reached at a 6TG concen- tration of 1.0 pg m1-1 and was maintained up to at least 5 t~g m1-1 6TG. 6TG at 10 pg ml -~ fur ther reduced cell survival in HF19 cultures but this was not al- ways the case for other human fibroblast cultures examined. It was assumed that the decrease in cell survival up to 0.5 pg ml -~ 6TG represented the cyto- toxic effect of 6TG on normal analogue-sensitive cells and the plateau region of survival between 1.0 and 5.0 pg ml -~ 6TG represented the survival of a small fraction of spontaneous 6TG resistant mutant cells in the populat ion.

Selection of mutants was usually complete after 14 days incubation and few additional clones arose in 6TG medium during the third week of incubation. However, 21-day incubation period resulted in larger mutant clones which fa- cilitated their isolation. Medium changes at 3 day intervals during the incuba- tion period did not affect the yield of mutants although mutant clones that had been re-fed were larger in size and more densely packed than clones grown wi thout medium changes.

The data points of Fig. 1 were obtained using four different batches of se- rum. There were no significant differences in the response of HF19 to 6TG in normal medium containing these sera and of twelve other batches of serum tested at 2 and 3 gg ml -~ 6TG only three caused changes greater than a factor of 2 in the cy to tox ic i ty of the analogue.

c ii 0 1 2 3 4 5

6 Thioguo nine (/Jg/ml)

Fig. 1. D o s e - r e s p o n s e o f h u m a n d i p l o i d f ibroblas t s to i n a c t i v a t i o n b y 6 - t h i o g u a n i n e . Baxs represent ranges o f surviving f rac t ions o b t a i n e d us ing f o u r d i f f e r e n t sera.

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98

Effects o f cell density on recovery of mutants The yield o f spon taneous m u t a n t clones resistant to 2 pg ml-~ 6 TG was af-

fec ted by the cell plat ing dens i ty (Table I). At cell densit ies be tween 0.3 and 1.0 • 103 cells cm -2 m u t a n t c lones were ob ta ined at a f r eq u en cy of ~ 4 . 5 • 10 -6. At higher cell densit ies m u t a n t yields decreased unti l at a dens i ty of 3 • 10 :* cells cm h2 the yield was reduced by a fac to r o f 10.

R e c o n s t r u c t i o n expe r imen t s with artificial mix tures o f wild type and m u t a n t cells (Table II) showed tha t at a to ta l cell plat ing dens i ty o f 7.9 • 102 cells cm -2 (5 • 104 cells in a 9 cm diam. dish) the c loning e f f ic iency of 6TG-res is tant mu- tants in 6TG m e d ium was ~ 50% of tha t observed for the same mutan t s c loned in normal med ium.

The in vitro growth of mutants The major p rob lem e n c o u n t e r e d in the charac ter isa t ion of m u t a n t clones was

tha t the f inite in vi t ro l i fe t ime o f h u m a n f ibroblasts l imited the g rowth poten- tial o f m a n y of the clones to very few generat ions. Approx . 50% of c loned mu- tants c o m p l e t e d less than 20 popu l a t i on doubl ings dur ing and a f te r select ion and fo r these the scope for f u r t he r expe r imen t s was very l imited. Exper imen t s tha t involved a second c loning o f the mu tan t s were par t icular ly d i f f icul t and m a n y mutan t s had insuff ic ient g rowth po ten t i a l fo r this t ype of expe r imen t . However , the m u tan t s descr ibed here initially exh ib i ted g rowth character is t ics and c loning eff iciencies in no rma l m e d i u m comparab le with the parenta l strain cells and were resis tant to 6TG at 2--5 pg m1-1 af te r m o re than 25 popu la t i on doubl ings in non-select ive med ium.

The g rowth o f H F 1 9 paren ta l strain cells in azaserine m ed iu m conta in ing 1.7 or 3.5 pg m1-1 azaserine was comparab le with tha t observed in normal medium. At cell plat ing densi t ies o f ~ 103 cells cm -2 no m u t a n t cells p r o d u c e d clones in azaserine me d ium with azaserine at e i ther co n cen t r a t i o n ; c loning eff iciencies o f mu tan t s in azaserine med ium were es t imated to be < 1 0 -6 (Table III).

The incorporation of radioactive hypoxanthine and adenine into cells H G P R T and A P R T activit ies in whole cells, as measured by the incorpora-

t ion o f radio-label led substrates , were linear wi th incuba t ion t ime up to at least 7 h .

T A B L E I I

E F F I C I E N C Y O F R E C O V E R Y O F 6 T G - R E S I S T A N T M U T A N T S F R O M A R T I F I C I A L M I X T U R E S W I T H P A R E N T A L S T R A I N C E L L S A T C E L L P L A T I N G D E N S I T I E S O F 7 .9 • 1 0 2 C E L L S C M -2

L

TG2 TG9 T G 2 3 TG24

Cel l strain C l o n i n g e f f i c i e n c y (%) i n C l o n i n g e f f i c i e n c y (%) in % R e c o v e r y o f m u t a n t s in n o r m a l g r o w t h m e d i u m a se lect ive ( 6 T G ) g r o w t h se lec t ive ( 6 T G ) g r o w t h

m e d i u m b m e d i u m

5 0 . 4 2 7 . 2 5 4 . 0 5 5 . 2 2 5 . 8 4 6 . 7 5 9 . 5 4 4 . 5 5 6 . 3 4 7 . 7 2 0 . 4 4 2 . 8

a 2 5 0 m u t a n t cei ls p l a t e d w i t h 5 " 104 rad i a t i on - i nac t i va ted parenta l s t r a i n cel ls . b 2 5 0 m u t a n t cel ls p la ted w i t h 5 " 104 v i a b l e p a r e n t a l s t r a i n cel ls .

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Chromosome complement

Fig. 2. C h r o m o s o m e c o m p l e m e n t s obse rved in cu l tu res of H F 1 9 pa ren t a l s t ra in and five der iva t ive 6TG- res is tant m u t a n t s .

Nine spontaneous and four radiation induced 6TG-resistant mutants had less than 2% of the parental strain activity of HGPRT and there was no correlation between enzyme activity and the concentra t ion of 6TG used for selection (Ta- ble III).

APRT activities were generally similar to that of the parental strain (Table III). Two mutants , TG21 and TG23, had activities of ~25% that of the paren- tal strain but none showed the extreme enzyme deficiency observed for HGPRT.

Karyotypes of mutants Karyotypic analyses of mitot ic preparations of late passage HF19 parental

strain cells and six spontaneous 6TG-resistant mutants axe shown in Fig. 2. All mutants exhibited a modal chromosome number of 46. The propor t ions of sub- diploid, tetraploid and sub-tetraploid cells in mutant populat ions were similar to those of the parental strain and were, in our experience, typical of late pas- sage human fibroblasts. Few chromosome aberrations were detected in the preparations and none of the mutants showed stable chromosome alterations that might be associated with the phenotypic change to 6TG-resistance.

Discussion

The dose response for inactivation of human diploid fibroblasts by 6TG showed a plateau of cell survival between 1.0 and 5.0 #g m1-1 6TG and all clones isolated as resistant to these concentrat ions showed properties consistent with a mutat ion affecting the activity of the purine salvage enzyme, HGPRT. Spontaneous 6TG-resistant mutants were detected in HF19 cell populat ions at frequencies of approximately 5 • 10 -6 mutants per viable cell but, because cell density effects during selection may reduce mutant yields by 50%, the true spontaneous mutan t f requency was probably ~ 1 0 -s . The cy to tox ic i ty of 6TG was no t significantly altered when different sera were used in the growth me- dium. This observation contrasts with our experience of 8AG as a selective agent where differences in the 8AG sensitivity of cells as great as 10-fold were

101

frequently observed amongst different sera or with one serum after different storage times at --20 ° C. The selective actions of 8AG and 6TG may also be dif- ferent. Selection of resistant clones by 8AG requires frequent medium changes and many resistant clones only appear after 3 weeks incubation of cells under selective growth conditions [18]. In contrast, the selection of clones resistant to 6TG did not require a medium change and almost all clones could be de- tected after two weeks incubation.

The antagonism of 8AG by serum components may in some cases be reduced by dialysis of the serum used in selective media. However, in our experience, se- rum dialysis had little effect on the selective action of 6TG and only served to depress the overall cloning efficiency of cultured human fibroblasts. With 6TG as the selective agent serum dialysis may not be an important factor in mutant selection and, provided that a suitable serum batch is chosen, dialysis may be omitted as a routine procedure.

Cell density effects on the efficiency of 6TG-resistant mutant selection were similar to those reported for 8AG-resistant mutants [1,17] with significant loss of mutan t clones above a cell plating density of 103 cells cm -z . ReconsLruction experiments showed that, with four different 6TG-resistant mutants in artificial mixtures with parental strain cells, the efficiency of recovery of mutants in 6TG medium was ~50%. Similar experiments with mixtures of 8AG-resistant Lesch Nyhan cells and normal human fibroblasts have shown recoveries of be- tween 39% and 100% [1,14]. Metabolic cooperation between normal and mu- tant cells, causing the mutants to incorporate the analogue, has been suggested as an explanation for this density-related loss of mutants in selective medium [6,17].

HGPRT activities in 6TG-resistant mutants were all ~ 2% that of the parental strain. This result contrasts with HGPRT activities previously found in 8AG-re- sistant mutants which were often comparable with that of their respective parental strains [1,14,18]. There was also no difference in HGPRT activity of 6TG-resistant mutants selected at different 6TG concentrations. This was not found for the 8AG-resistant mutants examined by Van Zeeland et al. [18] which had enzyme activities that were dependent to some degree upon the 8AG concentration used for their selection. However, only a small sample of mutants has been examined in the present study and enzyme activity was as- sayed by an indirect method. Consequently the effect of the selective agent on mutant phenotype described by Van Zeeland et al. [18] cannot yet be ex- cluded from 6TG selection of HGPRT-deficient mutants.

Azaserine is known to inhibit de novo synthesis of purines in cultured hu- man cells and cells dependent for growth on de novo purine synthesis, as a re- sult of defective purine salvage enzymes, might be expected to exhibit impaired growth in azaserine medium [8]. All 6TG-resistant mutants had cloning effi- ciencies of ~10 -6 in azaserine medium, confirming the absence of a fully func- tional purine salvage pathway predicted by the low HGPRT activities in these mutants. The largely unaffected APRT activities in 6TG-resistant mutants further suggests that the phenotype of those mutants examined was specifically associated with reduced activity of HGPRT and resistance to 6TG did not arise by an overall reduction in the activities of the enzymes in the salvage pathway. The extremely low cloning efficiency of 6TG-resistant mutants in azaserine me-

1 0 2

dium confirms their phenotypic stability and suggests that spontaneous rever- sion of the phenotype did not occur at a high frequency. Unpublished experi- ments (Cox and Masson) also showed that t reatment with MNNG at 3 #g ml- did not increase the reversion frequency of the 6TG-resistant phenotype above 10 -6 per viable cell. However, the opt imum conditions for selection of 'wild type revertants' from mutants have not yet been fully examined.

It has been suggested that phenotypic variation in cultured mammalian cells may arise by either true gene mutat ion or as a consequence of chromosomal variation [ 16]. The karyotypes of five mutants, examined within the first few generations following isolation, showed a modal chromosome number of 46 with no more variation than observed in the parental strain. Preliminary ASG chromosome banding experiments [2] with two spontaneous mutants, TG24 and TG38, showed no obvious differences from the normal human banding pat- tern. Thus, the 6TG-resistant phenotype was not associated with gross chromo- somal changes but it should be considered that even ASG banding can only re- solve relatively major changes in chromosome structure. The absence of detec- table chromosome variation in 6TG-resistant mutants is therefore not direct evidence of gene mutation.

In summary, 6TG was an effective agent for the selection of stable HGPRT- deficient mutants of human diploid fibroblasts and although further genetic and biochemical characterisation of mutants is required, 6TG appears to be a more suitable selective agent than 8AG for use in quantitative mutagenesis studies with cultured human cells.

Acknowledgements

We wish to thank the John Radcliffe Hospital, Oxford for samples of foetal tissue and also Drs. R.J. Munson and R.H. Mole for their advice on "the prepara- tion of this manuscript.

References

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