REQUIREMENT FOR CELL DISPERSION PRIOR TO SELECTION OF INDUCED AZAGUANINE-RESISTANT COLONIES OF CHINESE HAMSTER CELLS1 B. C. MYHR

AND

J. A. DIPAOLO

Cytogenetics and Cytology Section, Biology Branch, Carcinogenesis Program, Division of Cancer Cause and Prevention, NatiomE Cancer Institute, Bethsda, Maryland 20014 Manuscript received September 16,1974 Revised copy received December 21,1974 ABSTRACT

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time M o r e adding the selection medium. V79-4 cells were exposed to 500 p M MMS, 7 pM AFAA, or 10 GM MNNG and allowed ta multiply before being reseeded at 4 x 104 cells/60 mm dish and selected with 10 p g / d AZG. Maximum frequencies of 4 x lW5, 4 x 10-4, and 2.4x 10-3 were obtained about 100,130,and 2 0 hrs after exposure to MMS, A I?& and M " G , reqmtively. The maximum frequencies folloiWing MMS or MNNG treatments were about 10.fold greater than those obtained when induction and selection of AZG-resistant colonies were perfolmed in the same culture dish. The reseeding of treated cells e m nated the possibility o'f metahlic cooperation within mosaic colonies of wildtype and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.-AZGresistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0-20 colonies aibsemed/dish. A mixed population d V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.

A NUMBER of reports have described the mutation of somatic mammalian

cells to 8-azaguanine (AZG) resistance after treatment with chemical or physical mutagens. The biochemical basis for this resistance is the lack of incorporation of AZG-nucleotide into DNA because of several possible deficiencies ROUFA which often include the functional loss of the enzyme, HGPRT (BEAUDET, and CASKEY 1973; SHARP,CAPECCHI and CAPECCHI 1973). The induction of and selection for AZG-resistant phenotypes have generally been carried out within the same dish of cultured cells, as described by CHU and MALLING(1968) who 1 Abbreviations: AZG, 8-azaguanine; HGPRT, hypoxanthine guanine phosphoribosyltransferase;HAT, hypoxanthine, aminopterin, and thymidine (see METHODS); MMS, methylmethane sulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; AFAA, N-acetoxy-2-fluorenylacetamide;BUdR, 5-bromodeoxyuridine; DMSO, dimethylsulfoxide; C. E.. cloning efficiency; PBS, DULBECCO'S phosphate buffered saline; FBS,fetal Lmvine serum.

Genetics 80: 157-169 May, 1975.

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demonstrated increased mutant frequencies in V79 Chinese hamster cell cultures exposed to MMS, EMS, and MNNG. The frequency of mutant colonies was influenced by the inoculum size, the concentration of selective agent (AZG) , and the time allowed for the mutagen-induced changes to be expressed (expression time) before exposing the cells to selection medium. As the expression time is increased, the number of presumptive mutant colonies characteristically rises to an optimum and then sharply falls. Thus, a maximum frequency of AZG-resistant colonies was observed at 42 hrs expression time M EMS treatment of V79 cells (CHUand MALLING 1968). More following recently, the optimum expression time has been found to increase with increasing and HARCOURT 1972a) and bromomethylbenz(a) andoses of UV light (ARLETT thracene (DUNCAN and BROOKES1973). The usual explanation for the sharp decline in mutant colony frequency is metabolic cooperation between wild-type and mutant colonies. As the colonies increase in size with time following treatment, contacts will be made between wild-type and mutant colonies, causing a shift to the HGPRT+ phenotype ( SUBAK-SHARPE, BURKand PITTS1969; Cox et al. 1972). This process limits the time available for expression of the HGPRT- phenotype and may, in fact, cause a technical reduction in the maximum observable mutation frequency. Therefore, the reliability of the assay procedure in studies of MMS, AFAA, and MNNG mutagenesis in V79 cell cultures was reexamined. The current study shwvs that the maximum frequency of AZG-resistant colonies cannot be reliably obtained when induction and selection are performed in the same dish of cultured cells. MATERIALS A N D METHODS

Cell lines: The male Chinese hamster lung cell line, V79-4, and an AZG-resistant derivative, designated 38@23, were provided by DR.E. H. Y. CHU. Line 380-23 was MMS-induced and selected in medium containing 15% whole FBS.Another AZG-resistant clone, MSI, was isolated from V 7 9 4 cells exposed to MMS and selected in medium containing 10 pg/ml AZG and 5% dialyzed FBS. Both resistant lines are HAT-sensitive. Materials: DULBECCO’S modification of EAGLE’Sminimal essential medium was purchased from Schwarz/Mann; fetal bovine m m and IOOX penicillin G-streptomycin sulfate, from GIBCO. Hypoxanthine and 8-azaguanine.i/zH20were purchased from Calbiochem; aminopterin, from Lederle; thymidine, from Schwarz/Rlann; MMS, from Eastman; AFAA, from Starks Associates; MNNG, from Aldrich. AFAA and MNNG were stored at -W. Falcon plastic tissue culture dishes were used for d l cultures. Serum dialysis was performed with the D m c/HFD-15 Mini Plant Dialyzer with Bio-Fiber 50 fibers (5,CMN molec. wt. cutoff) or with cellulose tubing (12,000 molec. wt. cutoff) from Arthur H. Thomas. Cell culture: Cultures were maintained in DuLBEXX”s medium supplemented with 5% FBS and penicillin G-streptomycin sulfate (100 U and 100 pg/ml, respectively) ; this medium is referred to as CM. At 3-4-day intervals, cultures were subcultured using 1015 cells/100 mm dish in 20 ml CM and incubated at 37” in a humidified atmosphere that contained 10% CO,. The doubling time was consistently 11-12 hrs. Cultures were discarded after 3 4 months and fresh ones were started from cell stocks held in liquid nitrogen. Tests by Flow Laboratories, Rockville, Md., for Mycoplasma cmtaminatim were negative. The AZG-resistant lines were grown in the appropriate selection medium for two weeks before use in reconstruction experiments. Prior to expelimental use, V79 cells were routinely subcultured in HAT medium (CM containing 1 x l e 5 M hypoxanthine, 3.2 x 10-6 M aminopterin, and 5 x 1 0 ; 6 M thymidine) for at

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least 7 days to ensure a low background frequency of AZG-resistant cells. The cells were removed from HAT by washing once with HT medium (HAT minus aminopterin) and cultured in HT medium for one day, followed by one day in CM,before being subcultured for ex*mental use. The doubling time of the cultures returned to 12 hrs. Dialyzed serum for selection use was initially prepared in 100-ml batches using cellulose tubing and 3 5 0 ml s a h e dialysates at 4" for 24 hrs; the dialysate was replaced at 12 hrs. In later experiments the hollow fiber dialysis technique was used. Cold serum (1200 d)was of a D m Miniplant at 300 ml/min while 14 liters of saline was recirculated through the fih pumped in the opposite direction through the jacket at 1500 ml/min. The saline was recirculated for 1 hr and replaced with fresh saline for a second h m of dialysis. After sterilization by filtration, the dialyzed sewas heated to 56" for 30 min. Mutagenesis and selection: The procedure for induction and selection of AZG-resistant colonies is shown in Figure 1. Cells (104 to 106) were seeded into replicate 150 mm dishes and, 1 6 2 0 hrs later, treated with a chemical mutagen for one or two hours. After m w a l of the mutagen, the cultures were incubated for a variable number of days for expression of the induced changes; t h i s period included subculturing if the cells became confluent. The cells were seeded at 4 X 1W cells/60 mm dish for the mutation assay and at 1oy) cells/dish for C.E. determination. At different intervals (referred to as the colony selection time) the CM in the mutation assay dishes was changed to medium containing 10 p g / d AZG and 5% dialyzed F"BS (IO pg/ml AZG/dCM) . This selection medium was replaced 2 or 3 times at 2-day intervals. Thirteen days after d i n g , the resistant colonies were washed with PBS, fixed in methanol, stained with Giemsa, and counted. The dishes prepared for the C.E. determination were refed 24-48 hrs later with medium containing 5% dialyzed serum (dCM); colonies were fixed and stained after 9 days of g r d . The background mutation frequency and C.E. of untreated V79-4 cells were similarly determined for each experiment. Azaguanine stock solutions were prepared in 0.05 M NaOH and stared at 4" for a maximum of 4 weeks. Further sterilization was unnecessary and not done to avoid adsorption losses on bacteriological filters. The mutagen stock solutions were prepared prior to use and kept on ice: 0.E M MMS in HANK'Ssalt solution, 1.4 x 16-4 M AFAA in DMSO, and 0.01 M M " G in acetone. The solutions were diluted with serum-free medium at ropoyyIIL tempwature to WC the treatment concentration. The CM in the 150 mm dishes was replaced with 20 ml CM containing 10% FBS, and the mutagen in U) ml serum-free medium was quickly added and mixed by swirling. The pH was initially adjusted to remain near 7 or below during the 1-2 hr chemical incubation period at 37" in 10% CO,. Mutagen treatmats were terminated by removing the medium and replacing it with 41)ml CM. Cultures were refed two days later. Mutant frequency calculatiom The average number of colonies/dish in &12 C.E. dishes containing dCM was used as an estimation of the plating efficiency of cells in the mutagenesis assay dishes. Thus, the mutant frequency per unit number d s u r v i v m was obtained by dividing the observed mutant; colony frequency by the C.E. of the cells. The C.E. was determined every time a treated or control culture was reseeded for mutant selection. The mutant colony frequency at of 24 dishes (106 total cells each selection time after reseeding was determined from a t-1 plated). Colony splitting, which occurs when Chinese hamster colonies are refed, can be minimized by gentle pipetting during the selection medium changes. The daughter colonies are usually

t--t

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Colony selection

t

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Add

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FIGURE 1.-Experimental flow diagram for mutagenesis assays.

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smaller than the large, well-defined original colonies. Each selection condition was therefore examined for colony size distribution and the smaller colonies disregarded or the dishes were discarded. Some small colonies may be bona fide mutants with impaired growth rates, but elidnation of small colonies minimizes the possibility of inflated mutation frequencies, as proven in reconstruction experiments. RESULTS

Assay Linearity and eficimcy: The efficiency of detection of mutants over the range of colony numbers expected per dish in mutagenesis experiments was determined by a reconstruction assay. The C.E. of 100 cells of line 380-23 in CM in the absence of V79-4 cells was 97%. A small, variable number of AZG-resistant cells (line 380-23) and 4 x IO4 V79-4 cells were seeded in CM in 60-mm dishes (20 cellsJmmZ). The cultures were refed 24 hrs later with CM containing 30 pu&/mlAZG. This selection medium was replaced twice at 2-day intervals and colonies were counted after 9 days’ incubation. The recovery of resistant colonies was linear from 0 to 20 expected colonies per dish (Figure 2 ) . Each point, corrected for 97% C.E., was slightly above the 100% recovery line, probably because of the incomplete killing of wild-type cells. In the absence of any added mutants, an average of 0.5 mlony/dish was found. Under the more stringent selection conditions of 10 pg/ml AZG/dCM used in mutagenesis experiments, however, line 380-23 grew slowly and cloned at less than 2%. In dCM the C.E. was 94% of that obtained in CM; consequently, loss of serum nutrients after dialysis was not responsible for the poor recovery in the selection medium. In contrast to line 380-23, the MSI line has a population doubling time of 15 hrs and 90% C.E. when grown in dCM or dCM containing 10 pg/ml AZG. A reconstruction experiment with this selection medium resulted in a linear, 100% recovery of mutants but with complete killing of wild-type cells. Furthermore, if MSI cells were selected in medium containing an inferior dialyzed FBS, a “feeder effect” was observed. The C.E. of MSl cells plated without V79-4 cells in the inferior dCM was reduced to 40%, and the colonies were less than one-fourth -

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FIGURE 2.-Recovery of AZG-resistant colonies. See text for conditions. Each pint is the average count d 24 dishes.

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the size usually obtained in dCM. However, when MS1 cells were seeded with 4 x l o 4 wild-type cells in the same dCM containing lOpg/ml AZG, the C.E. increased t o 53% and the colonies were dense and normal in size. Selection conditions: Fetal bovine serum is known to contain substances (probably hypoxanthine and other purines) that lower the effective concentration of and DEMARS 1971 ;THOMPSON and BAKER1973). These substances AZG (FELIX influence the number of colonies surviving selection with 10 pgJml AZG control cultures (the background frequency). Large numbers of colonies were obtained with selection medium containing 10% FBS, and this background was reduced to about 200 colonies/106 plated cells when 5% FBS was used; the surviving colonies varied considerably in size. After dialysis of the serum, however, only well-defined colonies remained and the background frequencies were Similar results were obtained with seven different lots of on the order of le6. FBS. Mutagenesis assays with consistently low backgrounds and few problems in colony sizing thus become possible. The effective concentration of AZG is also increased by periodic replacement of the selection medium. To avoid problems with colony splitting or culture contamination, it is desirable to use as few replacements as necessary for low background frequencies. Alternate-day refeeding reduces the number of surviving colonies in V79 control cell cultures exposed to selection medium 48 hrs or 72 hrs after seeding 4 X lo4 cells/dish (Table 1). Since small colonies occurred with insufficient refeeding, only those with more than 100 cells were recorded. Two changes of selection medium for 48 hrs growth and three changes for 72 hrs growth were necessary to reduce the background colony frequency to about 1 O-6. Assay reconstruction: The assay scheme outlined in Figure 1, with the selection conditions discussed, was used to determine whether the measured frequency of resistant colonies would agree with the known frequency of mutants in a constructed cell population. V79-4 and MS1 cells were mixed at an MSl frequency of 5.3 X and the mixed population (5.7 x IO5 cells) inoculated into a 150-mm dish. Three days later (corresponding to an expression period after mutagen TABLE 1 T h effect of refeeding and cell growth on the background frequency of resistant colonies

Number of changes of selection medium

0

1 2

3

Colony frequency per 106 cells at two selection times 48 hr

85 6.5 1.1 0

7a hr

75 51 6.7 2.6

V79 cells previously exposed to HAT for two weeks were seeded at 4 x 104 cells/60 mm dish in a total of 24 dishes (0.96x 106 cells) for each data point. At the indicated times, the medium was replaced with 6 ml selection medium containing 10 p g / d AZG and 5% dialyzed FBS (8 ml in the caw of no changes). Each subsequat change was performed at two-day intervals using

6 ml selection mediua, except the last change used 8 ml medium. Incubation was continued for 13 days following seeding, and the colony frequency was based on the n u m k of seeded cells.

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treatment) the culture was confluent, and the cells were reseeded into a series of 60-mm dishes. Colony selection was initiated at different times by the addition of 10 pg/ml AZG/dCM (Figure 1) . The frequency of surviving colonies remained constant for selection times up to 43 hrs but decreased sharply to one-half this level by 67 hrs (Figure 3 ) . This was the predicted result for a cell population in which AZG resistance is fully expressed (not acquired as a result of treatment) and in which metabolic coeperation occurs at sufficiently high cell densities (Cox et al. 1972). The average plateau frequency of 5.6 colonies/l O4 survivors agrees closely with the original This result shows that colony splitting is not a serious mixture at 5.3 x problem when the medium is changed carefully and undersized colonies are not scored. Thus, this assay scheme with the selection procedures used is appropriate for the quantitation of mutants in treated V79 cell populations. C.E. recovery following treatment: After mutagen treatment, the C.E. of the cells may be severely depressed for several days, even though the survival of untrypsinized cells may be no lower than 30-40%. Cell survival is normally determined with cells cloned immediately after treatment or, as in these experiments, by treating a freshly cloned population. If, in contrast, a day or more elapses between treatment and cloning, the C.E. measures the ability of damaged cells to survive both chemical treatment and trypsinization and to form definitive colonies. The increase in C.E. as a function of time after exposure of V79 cells to MMS, AFAA, or MNNG is shown in Figure 4, and the respective survivals of untrypsinized cells are given in Table 2. The C.E. of control cells varied between SO-SO% for cultures cloned in parallel with treated cells. A 1-hr exposure to 500 pM MMS caused a small reduction in C.E. and a 2-hr,1-mM treatment produced an 84% reduction. More than 100 hrs would probably be required for the mass population to recover and clone between SO-SO%. For 7 J.M AFAA (1 hr), the C.E. fell to l o % , although survival of untrypsinized cells was 30%; about 140

i7?-,--

10 20 COLONY

30 40 50 60 SELECTION TIME ( H R S )

70

FIGURE 3.--Selection c m e fm MSI fquency in a h w n mixture with V79 cells. Cells from a full V79 culture containing MSI wlLs added 3 days earlier at a frequency d 5.3 x 10-4 were reseeded at 4 x 104 cells/60 mm dish. Selection was performed at the indicated times with 10 pg/ml AZG/aCM. Each point carresponds to the colonies found in 2.4 dishes (0.96x IDS plated cells) after 13 days and is c ~ t ~ e ~for t eadC.E. of 90%.

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[ A 20

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100

110

140

160

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FIGURE 4.-The C.E. d V79 cells at different times following mutagenic treatment. Untreated controls ( V ) , 5 0 pM MMS for 1 hr (O), 1 mM MMS for 2 hrs (e),7 pM AFAA for 1 hr (W), 10 pM MNNG for 2 hrs (A).Data were collected from several experiments over a 4-month period. Each point is the average of 6 dishes cloned at 1001cells:each.

hrs were required for the C.E. to return to the control value. Ten pM MNNG treatment for 2 hrs (38% survival) had an even more drastic effect on C.E. and growth rates. The C.E. increased from 1% to only 40% in 7 days and the colonies (1971) reported simiwere much smaller than normal. ORKINand LITTLEFIELD lar results for BHK cells treated with MNNG. Therefore, to avoid C.E. corrections greater than 2- or %fold to the observed mutation frequency, V79-4 cells must be allowed at least 4 days for recovery after 7 pM AFAA treatment and more than 7 days after 10 pM MNNG. When the time course of mutant expression is being studied, larger corrections to the observed frequency must be made. Chemical mutagenesis: Assays for the frequency of AZG-resistant colonies induced by 500 p M MMS for 1 hr are shown in Figures 5 and 7. The selection curve for cells reseeded at 43 hrs after treatment rose linearly from a near-zero value, indicating that the expression of resistant colonies was increasing (Figure TABLE 2 A comparison of induced mufant frequencies obtained by the reseeding method and the standard procedure introduced by CHU and MALUNG(1968)

Treatment

500 pM MMS 1 hr 1 mM MMS 2 hr 7 pM AFAA 1hr 10 pM MNNG 2 hr

Survival*

Expression time (hrs)+

Analysis C.E. (%)$

95 6

100

75 30 60 42

(%I

32 38

110 130 200

Mutants/iOs survivors Reseeding Standard method methods

4 23

-

40

-

240

37

0.8

* Determined by treatment of a cloned population and expressed as a percentage of the control population C.E. -f Time after treatment when a colony expression curve yielded the maximum frequency of resistant colonies. $ The C E. of treated cells at the time of reseeding when a maximum frequency was obtained. The number of definitive colonies selected at 42 hrs expression with 30 pg/ml AZG in the (1968). presence of 15% whole FBS,as shown in Figure 4 of CHU and MALLING

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FIGURE 5.-Colony selection curves for V79 cultures (2 x 10s cells) exposed to 500 p M MMS for 1 hr. Analysis dishes seeded at hrs and 74 hrs after treatment. C.E.'s were 73% and SI%, respectively. Only one change of selection medium.The background frequency varied from less than 1 x 10-6 at 24 hrs to 6 x 10-6 at 4 8 hrs selection time.

5). The last two points were high because of only one change of selection medium, The second selection curve at 74 hrs post MMS was obtained after one subculture of the treated cells. If there had been no loss in the resistant cell frequency with subculturing, the curve should have begun at the non-zero value indicated by the arrow (74 hrs = 43 hrs 4-31 hrs selection time). The small apparent drop in frequency is indicative of a lower average plating efficiency for the new mutants than for the wild-type cells. The slope of the second curve was less than the first curve and indicates that a maximum of nearly 4 mutants/106 survivors would have been attained. Three selection curves for 500 pM MMS were obtained in experiments performed 6 months later (Figure 7). Each curve corresponds to an independently treated culture and shows the variability between experiments for this relatively mild mutagenic treatment. The mutant frequency varied from 2.2 - 5.5 x with an average value near 4 X At 60 hrs selection time, a large number of colony-to-colony contacts was seen. This result is consistent with a metabolic cooperation explanation for the drop in mutant frequency. The complete expression of AF'AA-induced mutants was more delayed in time than for MMS. An analysis at 95 hrs after AFAA treatment showed that the frequency of AZG-resistant colonies was still increasing (Figure 6). At 121 hrs The treated the selection curve appeared to reach a maximum near 4.2 x cells were not subcultured before reseeding for analysis at 121 hrs and the selection curve was continuous with the curve begun at 95 hrs. Other replicate, treated cultures were subcultured 2 days before the third analysis was begun at 147 hrs; there was a drop in the expected position of the curve and the maximum mutation frequency fell to 3.5 x Additional experiments with AFAA confirmed the repeatability of the maximum frequency. Three independently treated cultures gave virtually the same (Figure 7). There may be some selection curve with a maximum level at 4 x variability in the time necessary for f u l l expression, however, since a maximum

CHINESE HAMSTER CELL MUTAGEh'ESIS

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FIGURE 6.-Cdony selection curves for V79 cultures (2 x 106 cells) exposed to 7 pM AFAA for 1 hr. C.E.'s far each assay were as follorws: 5&% at 95 hrs, 61% at 121 hrs, 77% at 147 hrs. Background same as in Figure 6.

was achieved by 120 hrs in experiments shown in Figure 7, while 160 hrs were required for those described in Figure 6. Preliminary experiments with 10 pM MNNG have yielded a mutation frequency at least 6 times that of 7 JLMAFAA at approximately 180 hrs after treatment (Table 2). Subculturing caused a drop in the observed mutation frequency (66% of the original culture) when the cells were subcultured 4 days after treatment and 3 days before reseeding for analysis.

FIGURE 7.--Replicate selection curves for V79 cu1tm-e~exposed to 7 pM AFAA or 500 fiM MMS for 1 hr. Assays were performed at 95 hrs and 70 hrs past treatment, respectively, for three independent cultures (0,Q A ) . The C.E. range for the AFAA assays was 59-65%; MMS assays, 72-78%. The background was less than 1 x 10-6 at 30 hrs, 6 x 10-6 at 48 hm, and 3 x 10-8 at 64 hrs. The background was subtracted from the MMS data points.

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These results show that maximum induced mutation frequencies are obtained when mutagen-treated V79 cells are dispersed after a suitable expression period and prior to selection with AZG. The time necessary for the complete expression of an induced phenotype in mammalian cells must be determined for each mutagen. A correlation between the optimum expression time and colony size after treatment has been suggested to indicate that 3-4 cell divisions are required (CHU and MALLING1968; DUNCAN and BROOKES1973). This requirement may be correct, but the correlation is not statistically valid, since only wild-type colonies are being observed; the frequency of colonies destined to be AZG-resistant is generally less than one in ten thousand. Moreover, the present study indicates the frequency optimum is an artifact caused by metabolic cooperation within colonies of mixed genotype. This situation would arise if the progeny of a treated cell included both HGPRT+ and HGPRT- genotypes and the HGPRT- genotype appeared after several cell divisions. Upon application of the selection conditions, the survival or loss of the colony would depend upon the makeup of it’s population. Many colonies might barely survive cooperation and exist in such a damaged condition that the resultant colonies would be too small to be scored. Thus, the frequency of induced AZG-resistant cells will be considerably underestimated. To achieve longer expression times and to avoid the possibility of selecting mosaic colonies, treated populations were reseeded for subsequent selection. Wild-type and HGPRT- cells in each original colony will therefore be mixed into the total population and differences in growth rates will lead to three general results as time elapses after treatment: (1) If HGPRT- cells divide mare rapidly than HGPRT+ cells, the frequency of AZG-resistant colonies will steadily increase; (2) If the two cell types have equal division times, the frequency of colonies will stay constant; (3) If HGPRT- cells generally divide more slowly than wild-type, the codony frequency will decline with time after treatment. In the case of V79 cell cultures, the first possibility is highly unlikely, since V79 cells have a doubling time of 11-12 hrs. Thus, the mutant colony frequency could be expected to stay constant or decline with time after treatment, and the true mutation frequency might be underestimated but never overestimated. Experiments showed that the frequencies of AZG-resistant colonies increased with the time intervals between treatments with AFAA or MMS and the reseeding of cells for colony selection (Figures 5 and 6). The frequencies did not increase indefinitely but reached maximum values (Figure 7). The frequency increases were interpreted as the continued expression of new mutants. The attainment of a maximum frequency was regarded as strong evidence for the validity of the reseeding procedure in V79 cell cultures. The maximum induced mutation frequencies obtained with several treatments (Table 2) are compared to values reported by CHU and MALLING(1968). The striking increase in mutant frequencies by the reseeding method is apparently the result of allowing full expression and the selection of colonies of a single

CHINESE HAMSTER CELL MUTAGENESIS

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genotype. A dose-response relationship is also evident for MMS treatments; and an the basis of mutagen concentrations at equal survival levels, the order of mutagenic potency is MNNG> >AFAA> >MMS. The large increases in mutation frequencies were not caused by the survival of wild-type color-ies. Background frequencies as high as to 3 x le3 have been reported for supposedly equally selective concentrations of AZG in hamster cell 1972b; BRIDGESand HUCKLE 1970; HUBERMAN cultures (ARLETTand HARCOURT and SACHS1974). With 10 pg/ml azaguanine as the selection agent, dialyzed FBS and two replacements of the selection medium were found to be both necessary and sufficient to consistently yield backgroulld frequencies near (Table 1 ) . These two operations apparently serve to maintain the effective concentration of AZG near the added level (LITTLEFIELD 1963; FELIXand DEMARS1971; THOMPSON and BAKER1973). PETERSON, PETERSON and HEIDELBERGER (1974) have also shown that dialysis of FBS reduces the frequency of phenotypic reversion of HGPRT- V79 colonies in HAT medium, apparently by the removal of purine nucleosides. Serum dialysis may not be generally required, however, if calf serum is used in place of FBS during selection (FELIXand DEMARS1971). The “stringency” of selection with 10 pg/ml AZG/dCM was further indicated by the low recovery (2%) of line 380-23 cells. This MMS-induced line was isolated in AZG selection medium contair-ing undialyzed FBS. Since line 380-23 is probably representative of at least some of the AZG-resistant colonies counted in the standard assay (Table 2), the differences between the frequencies yielded by the two analysis methods might be even greater than indicated. The maximum mutation frequencies can be expected to be increased still further by improved recovery of metabolic-deficient mutants with enriched selection medium. Subculturing of treated populations tends to reduce the mutant frequencies (Figures 5 and 6), suggesting that newly arisen mutants generally have a lower C.E. than wild type. The C.E. determined at the time of reseeding for mutant analysis is statistically that of surviving wild-type cells, so the mutant frequency calculated per unit number of survivors will always be underestimated. This underestimation may become more serious when the C.E. of the total population is strongly depressed, such as soon after mutagen treatment. Such considerations indicate that the initial number of cells to be treated must be adjusted to allow time for complete expression before the culture becomes too dense. To determine the maximum induced mutation frequency, it is esselltial to use replicate-treated cultures and to reseed at various times following treatment for selection. The procedure of reseeding cells before selection has also been used in studies of EMS- and MNNG-induced resistance to 6-thioguanine by ORKINand LITTLEFIELD (1971) for BHK 21/13 cells and by SATO,SLESINSKI and LITTLEFIELD (1972) for human lymphoblasts. None o€ the frequencies were corrected for depressed plating efficiencies, but the apparent maximal frequency of EMS-induced mutant BHK 21/13 cells was reported to occur after 6 days’ expression during which the treated population doubled 3 or more times. Recently, SLESINSKI ( 1974) has extended the lymphoblast studies to show maximal expression between 3.7 and 4.7 population doublings following EMS treatment. An extensive

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study by CABOCHE(1974) of 3 different loci in BHK 21/13 cells showed that a constant number of two population doublings was necessary to express half of the BUdR-resistant mutants induced by EMS or MNNG. The actual expression time increased with the dose. In the current study, maximum expression of AFAA-induced mutants was achieved after approximately 4 population doublings, but no attempt has been made to correlate mutant expression with total population growth. The attainment of maximum induced mutation frequencies is essential for quantitative studies, such as the determination of dose-response relationships and comparisons of mutagenic efficiencies with different mutagens and different genetic loci. Also, the effects of DNA repair-modifying agents or mutagen treatments separated in time should be determined relative to the maximum attainable frequencies. The procedures described in this report are appropriate for such studies. LITERATURE CITED

ARLETT, C. F. and S . A. HARCOURT, 1972a The induction of 8-azaguanineresistant mutants h cultured Chinese hamsterr cells by ultraviolet light. The effect of changes in post-irradiation conditions. Mutation Res. 14: 431-487. -, 1972b Expression time and spontaneous mutability in the estimation of induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light. Mutation Res. 16: 301-306. BFAUDET, A. L., D. J. ROUFAand C. T. CASKEY, 1973 Mutations affecting the stsucture of hypoxanthine : guanine phosphoribmyltransferase in cultured Chinese hamster cells. Proc. Natl. Acad. Sci. U.S. 70: 320-324. BRIDGES, B. A. and J. HUCKLE, 1970 Mutagenesis of cultured mammalian cells by X-radiation and ultraviolet light. Mutation Res. 10: 141-151.

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Mutation

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