Phenotypic lag and mutation to 6-thioguanine resistance in diploid human lymphoblasts

137

Mutation Research, 50 (1978) 137--144 © Elsevier/North-Holland Biomedical Press

PHENOTYPIC LAG AND MUTATION TO 6-THIOGUANINE RESISTANCE IN DIPLOID HUMAN LYMPHOBLASTS

W.G. THILLY, J.G. DELUCA, H. HOPPE IV and B.W. PENMAN

Toxicology Group, Department o f Nutrition and Food Science, Massachusetts Institute o f Technology, R o o m E18-666, Cambridge, Mass. 02139 (U.S.A.) (Received 4 August 1977) (Revision received 20 September 1977) (Accepted 26 September 1977)

Summary Mutants of a diploid human lymphoblast line resistant to 6-thioguanine (6TG) appear 6--16 generations after treatment with any of a diverse group of mutagens: methylnitrosourea (MNU), methylnitrosoguanidine (MNNG), ICR191, 5-bromodeoxyuridine (BUdR). A hypothesis is advanced that expression of the 6-thioguanine-resistant state may require the removal of essentially all pre-existing hypoxanthine--guanine phosphoribosyl transferase (HGPRT) molecules via division, dilution, and protein turnover. Design of protocols for quantitative mutation assays requires attention to this phenomenon.

Introduction

Several gene locus mutation assays have been developed using the X-linked enzyme HGPRT as the target in rodent cells [10,13,15] and human cells [1,17, 19]. Mutation at the presumptive hgprt locus results in resistance to the toxic effect of the purine base analogues 6TG or 8AG. A genetic lesion at the hgprt locus does not necessarily result' in an immediate change to the HGPRT-/6TG R state. The genetic change must first be fixed, i.e. the chemical lesion must become a chemically normal but genetically altered section of DNA in the strand from which mRNA is transcribed. Additionally, phenotypic change to 6TG a may require the loss of previously existing HGPRT molecules and the corresponding mRNA. In many reported mutation studies, selection for 6TG- or 8AG-resistant cells Abbreviations: 8AG, 8-azaguanine; BUdR, 5-bromodeoxyuridine; HGPRT, hypoxanthine---guanine phosphoribosyl transferase; MNNG, methylnitronitrosoguanidine; MNU, methylnitrosourea; 6TG, 6-thioguanine ; 6TG R, 6TG-resistant; PBS, phosphate-buffered saline.

138 has begun in the 3rd or 4th generation after treatment [1,4,10,11,13,17]. Early experiments with hamster fibroblast cultures showed that selection with 8AG 36--40 h after mutagen treatment yielded a mutant fraction significantly higher than at earlier or later times of selection [2,3,7]. The increase in mutant fraction as a function of time after treatment is consistent with the expected genotypic and then phenotypic expression time. The decline in mutant fraction cannot be explained in this fashion. However, fibroblasts in contact with each other can transfer phosphoribosylated 8AG or 6TG from H G P R T + to H G P R T cells. Thus, when cell concentration permits intercellular contacts, the growth of H G P R T - clones is suppressed [12,16]. The absence of a stable induced mutant fraction makes quantitative use of experimental results hazardous at best. The importance of phenotypic lag in interpreting quantitative mutation data at the H G P R T locus has been recognized in L5187Y mouse l y m p h o m a cells (a stable mutant fraction was not obtained) [23] and in V-79 Chinese hamster cells [24]. More recently, a stable mutant fraction after a prolonged expression has been reported in the mouse l y m p h o m a system [25]. We have developed a mutation assay at the H G P R T locus, expressed as 6TG R, using diploid human lymphoblasts as the target cells, the assay conditions do not permit the cross-feeding of phosphoribosylated 6TG and hence do n o t suppress the growth of H G P R T - clones. Mutants have been carefully studied for biases introduced during growth in nonselective media in reconstruction experiments, with results justifying a reasonable degree of confidence in the quantitative results [19]. Using this system, we have examined the length of time required to express the mutant p h e n o t y p e 6TG R, after treatment with the alkylating agents MNNG, MNU, the pyrimidine analogue BUdR, and the substituted, acridine half-mustard ICR-191. Materials and methods

ICR-191 was a generous gift from Dr. Hugh J. Creech, The Cancer Institute, Fox Chase, Penna. Fresh solutions of ICR-191 in dimethylsulfoxide (1 mg/ml) were prepared immediately before use. The alkylating agents MNNG and MNU were purchased from K&K Laboratories (Cleveland, Ohio). Desiccated, preweighed aliquots were stored at --80 ° C. Aliquots were dissolved in PBS(pH 7.2) and filter-sterilized immediately before use. BUdR was purchased from Sigma Chemical Company (St. Louis, Mo.) and stored as a filter-sterilized PBS solution. The human lymphoblast line (MIT-2) is a clonal derivative of the PGLC-33 line isolated from a young female mononucleosis patient b y Dr. Phillip R. Glade, University of Miami, Fla. Stock spinner cultures of the line grow in continuous exponential phase in antibiotic-free RPMI1640 (10% fetal-calf serum) with a doubling time of 16.8 h. Periodic chromosome counts have shown that more than 80% of the cells have 46 chromosomes. Lesch--Nyhan fibroblasts (Massachusetts General Hospital, Genetics Unit) were used as feeder layers in soft agar platings to determine cell survival and mutant fraction [17]. Stock fibroblast cultures were grown attached to 100-ml dishes in RPMI1640 (10% fetal-calf serum) and passaged weekly. Mycoplasma tests for all stock cultures (Microbiological Associates, Rockville, Md.) were

139 consistently negative. The mutant fraction was assayed by soft agar plating in the presence and absence of 10 pg 6TG/ml. All platings were done in at least quadruplicate. The details of our procedure and reconstruction experiments with this system have been previously described [19--21]. Mutagens were added to stationary suspension cultures (200 ml of 4 × 10 ~ cells/ml) and 24 h later the cultures were centrifuged, resuspended in fresh medium, and plated to determine cell survival. Dally thereafter, the cultures were electronically counted {Coulter Model B, Coulter Electronics, Hialeah, Florida) and diluted to 3.5 × l 0 s cells/ml with fresh medium. The mutant fraction was determined every other day for at least 20 days. Growth delay is examined by extrapolating the exponential growth curve to the end of the treatment period and comparing this number to the actual plating efficiency obtained at that time. If the plating efficiency is greater than the extrapolated number of survivors, we interpret this to be presumptive evidence of a growth delay among cells capable of division. The length of this growth delay is the time represented by the length of the horizontal line intercepting the surviving fraction following treatment and the back-extrapolated curve of exponential growth. This may represent a period of no growth or a somewhat longer period of slowed growth. Assuming reasonable homogeneity among the growing cells, it is possible to estimate the number of cell generations that have taken place between the time of treatment and each determination of the mutant fraction. The number of generations required for "full" expression of 6TG R was determined from a graph of induced mutant fraction as a function of generations after mutagen treatment. The value was taken as the intersection of

PHENOTYPIC LAG

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I I I I I I I I I I

> ==

0

0,0

SIMULTANEOUS INDEPEN DENT EXPERIMENTS

0

1

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I

0

5

I0

I 15

l

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L

20

25

50

35

GENERATIONS POST BUdR TREATMENT

Fig. 1. E x p r e s s i o n o f 6 T G R a f t e r t r e a t m e n t w i t h 50 ~ m o l a r B U d R . G e n e r a t i o n s are c a l c u l a t e d f r o m t h e b e g i n n i n g o f a 24-h m u t a g e n t r e a t m e n t . " O b s e r v e d m u t a n t f r a c t i o n " is t h e p l a t i n g e f f i c i e n c y i n t h e p r e s e n c e o f 1 0 / ~ g / m l 6 T G d i v i d e d b y p l a t i n g e f f i c i e n c y i n t h e a b s e n c e o f 6 T G . D a t a is t a k e n f r o m t w o s i m u l taneous independent experiments.

140 a horizontal line drawn through the plateau of the induced m u t a n t fraction and a straight line through the rising values of the m u t a n t fraction (see Fig. 1). Results Fig. 1 shows the m u t a n t fraction observed for the mutagen BUdR as a function of generations after treatment. As can be seen from the graph, approximately 15 generations were required before the m a x i m u m observed m u t a n t fraction was reached. Once the m u t a n t fraction reached a m axi m um , it remained constant (within the limits of the sensitivity of the assay). Similar curves were obtained with MNU, MNNG and ICR-191 [19--21]. The n u m b e r of generations required to achieve full expression of the 6TG R state ranged from a low of 6 for an experiment with MNNG to a high of 16 for an e x p e r i m e n t using ICR-191. For MNU and MNNG, the length of this p h e n o t y p i c lag is n o t inconsistent with an interpretation of concentration dependence. The minimum detectable m u t a n t fraction is 2 X 10 -6 , well below the normal background of 5--40 X 10 .6 . This limit arisis because two million cells (as determined by electronic particle counting) were plated in the presence of 6TG in a quadruplicate determination of the m u t a n t fraction. Plating efficiencies of control cultures (and treated cultures that had resumed exponential growth) were ab o u t 25%. However, since the mutagen treatments were toxic (see Table 1), early platings contained a substantial portion of dead cells and the minim u m detectable m u t a n t fraction was above the background level (i.e., no mutant clones were found) or the standard deviation of the observed m u t a n t fraction was very large (only a few m u t a n t clones were found). These points have been omitted from Fig. 1. In experiments with very high toxicities or low induced m u t a n t fractions, accurate determination of the length of the phenotypic lag is n o t possible; i.e., whenever the plating efficiency X m u t a n t fraction X 2 X 106 <~ 16 at a time before the end of p h e n o t y p i c lag. TABLE

1

Agent

Concentration (pmolar)

MMU

37.5 50

MNNG

BUdR ICR-191

0.075 0.150 0.225 50 0.625 1.25 2.50

Percent survival

Growth delay (days)

Days before full expression of 6TG R

Generations before full expression of 6TG R

Induced mutant fraction X 104

7 0.10

2--3 2--3

8 14

7 13

1.1 4.2

10 1 0.06

2--3 2--3 2--3

12 10 10

6 7 9

1.0 2.4 2.5

9

0--1

12--14

15

3.7

42 18 11

3--4 3--4 3--4

16 16 20

16 16 15

4.8 5.3 8.4

Shown are concentration of mutagen, relative survival of treated cultures measured immediately upon resuspension after treatment, the apparent growth delay after treatment (see Materials and methods) numb e r o f d a y s o f g r o w t h b e f o r e f u l l e x p r e s s i o n o f 6 T G R s t a t e , n u m b e r o f g e n e r a t i o n s o f g r o w t h b e f o r e full expression of 6TG R state, stable induced mutant fraction after phenotypic expression. The control m u t a n t f r a c t i o n fell w i t h i n t h e r a n g e 0 . 0 5 - - O . 4 0 X 1 0 - 4 .

141

Table 1 summarizes the specific data regarding treatments for several experiments in which the length of the phenotypic lag could be reliably ascertained: survival (cloning efficiency relative to controls immediately following treatment), the period of growth delay after treatment before cellular multiplication apparently began, the number of generations and days of growth before full expression of 6TG R, and the stable induced mutant fraction observed. However, experiments performed under similar conditions of mutagen concentration, duration of treatment, and resulting toxicity have yielded disparate results when performed on different days. For instance, in a series of subsequent experiments with ICR-191, all cultures expressed full 6TG R by 10--12 generations (12 days) after treatment. There is, however, spectral evidence that the ICR-191 used in the second experiment may have undergone some decomposition. Discussion

What could reasonably account for the marked lag between mutagen treatment and the expression of the 6TG R phenotype? 6TG presumably kills via incorporation into cellular DNA [30]. If there were no H G P R T activity in a cell, no 6TG would be phosphoribosylated, none would be incorporated into the DNA, and the cell would survive. A complete lack of cellular H G P R T activity would therefore be the simplest explanation for 6TG a. We have in fact found no detectable HGPRT activity in 6TG R mutants isolated from this system [19]. One molecule of H G P R T in a cell could conceivably generate enough phosphoribosylated 6TG in a generation to cause cell death. This conclusion is based on the following reasoning: (1) The kinetics of 6TG phosphoribosylation are comparable to those for hypoxanthine [ 14]. (2) The lowest concentration of 6TG that we have found to be toxic to our wild-type cells is about 5.5 X 10 -7 M [19]. (3) The concentration of 6TG in the selective medium (10 pg/ml) is several times higher than the Km of the enzyme [14]. {4) The internal aqueous volume of a lymphoblast is no greater than 1000 p3. The phosphoribosylation of 6TG at Vmax can be calculated by multiplying the specific activity of H G P R T by the molecular weight of the protein. This calculation yields 180 molecules phosphoribosylated hypoxanthine/molecule H G P R T X min. The presence of one molecule of HGPRT in a volume of 1000 tt 3 (10 -12 liter) operating at Yma x would bring that volume to 5.5 X 10-TM phosphoribosylated 6TG in about 1800 min (30 h). Since 5.5 X 10 -7 M is the concentration of 6TG that is toxic to the wild-type cells, these calculations indicate that one remaining active H G P R T molecule could conceivably produce enough phosphoribosylated 6TG to cause cell death. The molecular weight of HGPRT is 68 000 [5]. Its specific activity at saturating substrate concentration is 2.7 X 10 -6 moles phosphorylated hypoxanthine/mg protein X min [6]. The cellular activity of H G P R T in our lymphoblast line at saturating substrate concentration is approximately 7.5 X 10 -16 moles phosphoribosylated hypoxanthine/cell X min [19]. The amount of H G P R T per lymphoblast can therefore be calculated as about 2.5 X 106 or 221 molecules/

142 cell. Since these calcultations are based on in vitro determinations, this value may be viewed as an upper limit. Depending on the precise molecular mechanism (e.g., positive or negative strand lesion) of a mutation, one to two generations should be required for fixation. After fixation mutants will lose HGPRT activity via division, dilution, and normal protein degradation. HGPRT is degraded with a half life of 24-48 h [8,19]. Under the stationary suspension culture conditions of our experiments, cell division takes place approximately once every 24 h. After fixation, the cellular HGPRT concentration should therefore be diluted by a factor of 2 t'5 to 25 every 24 h. At this rate, it would take 10--14 days after fixation of the mutation to dilute the pre-existing HGPRT to one molecule per cell. We would also expect that the time (generations and degradation) required should be reasonably constant and independent of mutagen and dose. Our observations do not entirely conform to this latter expectation. The phenotypic lag seen in BUdR and ICR-191 treated cultures is consistent with the hypothesis that the 6TG R state is reached by the complete loss of pre-existing HGPRT via division, dilution, and degradation. However, less time is required for expression of 6TG R in cultures treated with the alkylating agents MNU or MNNG and the expression time may even be dependent on the dose. As can be seen in Fig. 1, the estimate of the number of generations required for full expression of the m u t a n t phenotype is somewhat imprecise. Furthermore, in one ICR-191 experiment, full expression of 6TG R t o o k 15--16 generations, while in a later experiment expression was complete by 10--12 generations. In spite of these variations, the differences w i t h i n an e x p e r i m e n t of expression time as a function of dose with the alkylating agents MNU and MNNG seems real. A dose
143 to affect phenotypic lag for 6TG resistance: (1) The number of HGPRT molecules in the cell at the time of treatment; (2) the doubling time of a particular cell population; (3) the rate of degradation of HGPRT; (4) the activity of a particular HGPRT to 6TG; (5) the sensitivity of a particular cell type toward the 6TG phosphoribosyl derivative. Thus, each potential mammalian cell mutation assay should be carefully characterized with regard to the length and possible variability of phenotypic lag. The data comprising such characterization should be offered simultaneously with a demonstration of stability of mutant fraction if quantitative use of results is intended. Acknowledgements Dr. Lee Jacoby of the Genetics Unit of the Massachusetts General Hospital kindly provided starter cultures of both lymphoblasts and Lesch--Nyhan fibroblasts. This work was partially supported by Grant No. 5-RO1-CA151010-02ET from the National Cancer Institute, by Grant No. 5-PO1-ES-00597 from the National Institute of Environmental Health Sciences, and by a grant from the Massachusetts Institute of Technology Environmental Laboratory. B.W. Penman, H. Hoppe IV and J.G. DeLuca are predoctoral trainess of the National Institute of Environmental Health Sciences. References 1 A l b e r t i n i , R~I., a n d R . De M a r s , D e t e c t i o n a n d q u a n t i f i c a t i o n o f X - r a y i n d u c e d m u t a t i o n in c u l t u r e d , diploid human fibrobiast, Mutation Res., 18 (1973) 199--224. 2 Arlett,C.F., and S.A. Harcourt, The induction of 8-azaguanine-resistant mutants in cultured Chinese h a m s t e r cells b y u l t r a v i o l e t light: T h e e f f e c t o f c h a n g e s in p o s t i r r a d i a t i o n c o n d i t i o n s , M u t a t i o n Res., 14 (1972) 431--437. 3 A r l e t t , C . F . , a n d S . A . 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