COMPARISON OF THE FREQUENCIES OF SPONTANEOUS AND CHEMICALLY-INDUCED 5-BROMODEOXYURIDINE-

RESISTANCE MUTATIONS IN WILD-TYPE AND REVERTANT BHK-2 1 /13 CELLS

MICHEL CABOCHE

Laboratoire de Genetique CelEulaire, INRA, 31320 Castanet-Tolosan, B.P. No. 12, France

Manuscript received December 14, 1973

ABSTRACT

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations vaned with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxy- uridine and azaguanine resistance markers. Ten independent bromodeoxy- uridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mu- tation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 1G-6 / cell / generation) and low in the wild- type cells (< 3.5 x 10-8 / cell / generation). A comparison of induced muta- tion frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these re- sults are discussed.

MOST genetic studies of a specific locus in cultured mammalian cells have involved the X-linked hypoxanthine-guanine phosphoribosyl transferase

locus (SZYBALSKI and SMITH 1959; LITTLEFIELD 1963; CHU and MALLING 1968, CHU et al. 1969; ALBERTINI and DE MARS 1970; MORROW 1970; GILLIN et al. 1972). The study of an autosomal locus using the methods developed for 8-aza- guanine resistance analysis should provide further information concerning the effect of gene multiplicity on genetic stability (HARRIS 1971 ; MEZGER-FREED 1971 ) . Also, by this method the effect of gene multiplicity on chemically-induced mutation rates can be studied by comparison of heterozygous revertant clones to the homozygous wild-type cells. However, many established cell lines are aneuployd and the presence of the two homologous chromosomes bearing the marker under study can rarely be assumed. Genetics 77 : 309-322 June. 1974.

310 M. CABOCHE

Chromosome banding studies of the BHK 21/13 strain (MARSHALL 1972) in- dicate that its karyotype is very close to that established from the Syrian hamster. For this reason the BHK 21/13 strain, isolated by STOKER and MAC PHERSON (1964), was chosen. Moreover, LITTLEFIELD and BASILICO (1966) have shown that the thymidine kinase locus of BHK 21/13 cells is a suitable marker for genetic studies because forward and back mutations can easily be selected. Mu- tations affecting this locus are responsible for 5-bromodeoxyuridine resistance (KIT et al. 1963). This locus is autosomal in man (MILLER et al. 1971) and is not linked to the hypoxanthine-guanine phosphoribosyltransferase locus in any mammalian strains where this linkage was studied.

The present report describes genetic and biochemical studies of this presumably autosomal locus in BHK 21/13. Spontaneous mutations and mutagen-induced mutations for 5-bromodeoxyuridine resistance are compared in the wild-type cells and revertant cells. The processes involved in the resistance mechanism are discussed.

MATERIALS AND METHODS

Cells: BHK 21/13, a male baby Syrian hamster kidney cell strain, was supplied by the American Type Culture Collection. A derived line C3 was cloned twice by plating single cells and by picking developing colonies with Pasteur capillary pipettes. The absence of mycoplasmas was established according to the test of TODARO, AARONSON and RANDS (1971). The C3 line karyotype has a modal number of 43 chromosomes with a very low, but significant, level of tetra- ploids (1-3%). The variants isolated from this strain were preserved by freezing.

Culture methods: All media were buffered at pH 7.2 with HANKS basal saline solution con- taining tris hydroxy methyl amino methane (10-2 M), and penicillin (100 UI/ml) and strepto- mycin (100 pg/ml), (H.B.S.S.). Cells, were grown at 36.5" in stoppered glass bottles containing Eagle's minimum essential medium (MEM) supplemented with non-essential amino acids (IOW M each), 4% fetal calf serum and 6% calf serum (MEMS). Nutritional requirements were tested with medium containing dialyzed serum (D). Serum was dialyzed 24 hrs against cold running tap water and 6 hrs against 20 volumes of 0.15 M sodium chloride in cold distilled water. In MEM supplemented only with dialyzed serum, clonal growth did not occur. Repro- ducible clonal growth in this medium was obtainec! by adding serine, asparagine and sodium pyruvate at the concentration of 5 x IO-4 M (MEM.CL.D), (CABOCHE 1973). Nutritional re- quirements and drug sensitivity were measured by plating 20 cells/cm2 in MEMS, in replicate bottles. Six hours later cells were washed in H.B.S.S. and the drug or metabolite was added with ME1LI.CL.D. Seven to ten days later colonies were fixed in 10% trichloroacetic acid and stained. C3 plating efficiency was 25% in MEM.CL.D and 32% in MEM.S. The standard error of the mean was usually less than 15% in a given experiment. Cell growth was measured by plating IO3 cells/cmz in MEM.S in replicate bottles. Each day duplicate bottles were sampled and separated cells counted in a solution (EDTA.B) composed of sodium ethylene diamine tetra- acetate 0.2 g, sodium chloride 8 g, potassium chloride 0.2 g, disodium phosphate 1.1 g, mono- potassium phosphate 0.2 g and glucose 0.2 g to one liter pH 7.4, as described by PAUL (1961).

Selection of drug-resistant cells: 106 cells or more were seeded in MEM.S. The medium con- taining the drug was substituted 6 hrs later and replenished 24, 72 and 144 hrs after plating. The following drug concentrations were used: 5-bromodeoxyuridine (BrdU) 25-50 pg/ml in MEM.S, 5-fluorodeoxyuridine (FrdU) 0.01 pg/ml in MEM.CL.D, and 8-azaguanine (AZG) 15 pg/ml in MEM.S. The seeding of cells was carried out at a concentration of 4 x IO3 cells/cm2 except for the selection of AZG-resistant cells where a lower concentration was used ( 2 x lo3 cells/ cm2). The resulting experiments demonstrated that the plating efficiency of mutants was not affected by the presence of the wild-type cells under these selection conditions (CABOCHE 1974).

BROMODEOXYURIDINE RESISTANCE 31 1

Selection of revertants: Clones reverted to BrdU, or FrdU sensitivity were selected according to LITTLEFIELD and BASILICO (1966). Cells were plated at 4 x IO3 cells/ml in MEM.CL.D con- taining 5 x 10-4 M alanine, glycine, proline, aspartic acid, glutamic acid, 10-4 M hypoxanthine, 10-5 M uridine and thymidine and 2 x 10-6 M aminopterin (HAT medium). The medium was replenished 2.E, 72 and 144 hrs later. The plating efficiency of C3 and revertant cells was unaffected in these selection conditions.

Standard conditions for mutagenesis: 2 x 10" cells/cmZ were plated in MEMS. Six hours later they were treated with mutagen for 16 hrs in the same medium, then washed twice with H.B.S.S. and reincubated for 4 days in MEM.S. Mutagenized cells were plated for isolation of mutants after a minimum of three division cycles. The toxic effect of each mutagen was tested by measuring the plating efficiency of single cells and the growth rate of cultures subjected to the same mutagenesis conditions in MEM.S. For mutagenesis the following standard conditions were used: ethyl methanesulfonate (EMS) 300 pg/ml (survival 65%), N-methyl-N-nitro-N- nitrosoguanidine (MNNG) 0.5 pgjml (s = IO%), methyl methanesulfonate (MMS) 20 pg/ml (s=22%), BrdU 10 pg/ml (s=25%). Induced mutation frequency was estimated as the number of selected mutant clones divided by the product of the mutant clone's plating efficiency under the selection conditions and the number of cells subjected to selection. The frequencies of spontaneous appearance of mutant cells were determined by the same procedure in non-treated cultures. Values for this control were routinely subtracted from the mutation frequency observed in the corresponding mutagen-treated culture.

Cellular uptake of labelled metabolites: 2 x IO5 cells were incubated for 24 hrs in MEM.S. Then M E M S was replaced by MEM CLD containing 10-5 M 14C-8 hypoxanthine 0.1 pCi/ml or M 3H methyl thymidine 1 pCi/ml and cells were incubated a further 15 to 120 min at 37". The incorporation was stopped by washing with cold H.B.S.S. Cells were stored frozen in EDTA.B. After thawing and release from glass, 400 pg bovine serum albumin were added in each flask and cell extracts were precipited with 10% trichloroacetic acid and collected on What- man GFC fiber filters. The filters were successively washed in beakers containing 10% trichloro- acetic acid, absolute ethanol and ether, then dried and incubated for 30 min at room temperature in Packard Soluene according to the procedure of BIRNBOIM (1970). The radioactivity of filters was counted in a Packard Tri-carb apparatus.

Thymidine kinase assays: Thymidine kinase assays were performed according to BOLLUM end POTTER (1959) and BREITMAN (1963): 2 x 107 exponentially growing cells were pelleted 24 hrs after the last medium replenishment, then washed in H.B.S.S. and resuspended in 0.5 ml of an extraction buffer containing 10 mM Tris HC1 pH 8, 1 mM Zmercaptoethanol, 0.1 M KC1 and 0.1 mM thymidine. Cells were sonicated at 4" with a Braun Sonic 300 sonifier (setting 60, two times 15 sec sonications). The lysate was centrifuged 30 min at 4000 g and the pellet was discarded. The incubation mixture, for a total volume of 200 81, contained 50 mM Tris HC1 p H 8, 2.5 mM MgCl,, 2.5 mM ATP, 20 pM 3H methyl thymidine (0.1 pCi) and 20 p1 of enzyme extract. The mixture was incubated at 37" and 20 pl aliquots were spotted on Whatman DE 81 paper discs after variable incubation times (5 to 40 min). Discs were immediately washed in 2 X le3 M ammonium formate for 30 min, in absolute ethanol and finally in ether. They were then dried and counted. ATP was omitted in controls. Protein concentration was estimated in cell extracts by the Folin method. Incorporations were converted to pp moles of thymidine phos- phorylated per minute and per mg of protein in the extract. Thermal stability was estimated by incubating at 65" the cell extract diluted 5 times (2 x 10-5 M final thymidine concentration). For the determination of the Km of the enzyme in cell extracts the extraction buffer without thymidine was used. The Km of thymidine kinase was graphically determined by reciprocal plot of enzyme activity versus thymidine concentration. Cytoplasmic, mitochondrial and nuclear fractions were separated by centrifugation after Dounce homogenization according to DE SAINT VINCENT and BUTTIN (1 973).

Karyotypes: 106 exponentially growing cells were treated 4 hrs with colchicine (4 pg/ml) . They were then trypsinized and resuspended for 45 min at 37" in an hypotonic medium made of 16% calf serum, 250 UI/ml hyaluronidase and distilled water. The cells were fixed in glacial

312 M. CABOCHE

acetic acid-absolute ethanol (lv:3v) for 10 min at 4". Drops of the fixed material were air-dried on slides which were then washed and stained for 7 min in standard buffered Giemsa stain. Chromosome number was estimated under the microscope (ECHARD 1973).

RESULTS

Spontaneous 5-bromodeoxyuridine-resistant cells: Under defined conditions corresponding to BrdU concentrations varying between 25 and 50 pg/ml and cell density of less than lo4 cells/cmz, plating efficiency of resistant cells is unaffected by the presence of the wild-type cells (CABOCHE 1974). In an attempt to perform a fluctuation test, ten independent cultures of C3 were grown for about 30 gen- erations and then tested for spontaneous resistant cells. No resistant clones were isolated among 3 X IO6 cells of each culture submitted to selection. Therefore spontaneous mutation rate in this strain is significantly lower than 3.5 X per cell per generation (P < 0.02). BrdU-resistant strains can be stepwise se- lected by first growing cultures for one week in medium containing 10 pg/ml BrdU and then selecting for resistance to 25 pg/ml BrdU. Since BrdU has been shown to be a mutagenic agent for mammalian cells (HUBERMAN and HEILDEL- BERGER 1972, Table 4) mutagenesis occurs during stepwise selection. Conse- quently the isolated resistant clones are not necessarily of spontaneous origin.

Mutation expression time after mutagenesis by alkylating agents: Single-step selection of drug-resistant clones after mutagenesis of V79 hamster cells was first described by CHU and MALLING ( 1968). In the same manner ORKIN and LITTLE- FIELD (1971) isolated BrdU-resistant clones from BHK 21/13 cells. This latter

10 0

8 0

6 0

FIGURE 1.-Toxic effects of ethyl methane sulfonate on C3 plating efficiency and doubling time. Closed circles: relative plating efficiency of cells treated for 16 hours with variable EMS concentrations ( P / P , ) ; open circles: ratio T / T , of the doubling time of control cultures to mutagenized cultures.

BROMODEOXYURIDINE RESISTANCE 31 3

TABLE 1

Average expression time for half muxima1 expression of induced muiations (Te) ~~ ~~~~~~

Number of resistant clones per 4 X 108 mutagenized cells

Mutagenesis AT: 8 hrs 32 hrs 56hrs 80 hrs 128 hrs Te Td Ne=Te/Td

EMS 250 pg/ml 0 3 6 7 9 44 hrs 21 hrs 2.1 EMS 4.00 pg/ml 0 0 9 16 19 58 hrs 26 hrs 2.2 MNNG 0.5 pg/ml 0 0 2 12 10 63 hrs 31 hrs 2.0

At various intervals (AT) after the first half of mutagenesis 4 x 10s cells were submitted to selection. The number of resistant clones was plotted as a functions of AT and Te was graphically estimated. Td is the doubling time of cultures after mutagenesis.

strain was used to study the action of mutagens on cellular proliferation. AS shown on Figure 1 and Table 1, the growth rate of mutagenized cells is reduced. However. it remains exponential for more than three days after mutagenesis, until confluency is reached. The doubling time of EMS-treated cultures increases exponentially with mutagen concentration (Figure 1) . MNNG acts in a similar way. When cells are plated for selection at various intervals after mutagenesis, resistant clones gradually appear with increasing expression time until a plateau is reached. Then their number decreases, probably by elimination of slowly growing mutants as suggested by MORROW (1972). The half expression time for these mutations is defined as the period between the middle of the mutagenesis treatment and the time at which half of the final number of resistant clones ap- pears. This half expression time increases with the concentration of EMS em- ployed for inducing mutations (Table 1). The ratio between the half expression time and the doubling time for a given mutagen concentration is a measure of the number of cell division cycles necessary to express induced mutations. Table 1 shows that a constant number of two cell cycles is necessary to express half of induced mutations after EMS or MNNG mutagenesis. Induced BrdU-resistance mutation frequencies for different mutagens under optimal conditions of muta- tion expression are given in Table 4.

Reversion to 5-bromodeoxyuridine sensitivity: When they are grown in the absence of drug for several months, all the clones selected for their BrdU re- sistance remain resistant. BrdU-sensitive clones have been selected from those resistant clones. 2 X lo6 cells from ten independently isolated resistant clones were incubated in HAT medium as described in the methods section, with or without previous EMS mutagenesis. C3.E8BU3 clone reverted spontaneously at a rate of approximately 1 O-T/cell/generation, when estimated by a fluctuation test. The induced reversion frequency of another clone, C3.E2BU1 was 1.8 X 10-5 after standard EMS treatment. This clone did not revert spontaneously. All the isolated clones were BrdU-sensitive like the wild type cells.

Spontarreously occurring BrdU-resistant mutants from revertants: Seven re- \-ertant clones were isolated from C3.E8BU3 and C3.E2BU1. The number of BrdU-resistant cells increases progressively in cultures of revertant cells grown in MEM.S. Forward mutation rates €or two revertant clones were estimated by

314 M. CABOCHE

a fluctuation test. The two clones were tested by cloning cells in HAT medium and then picking clones and allowing them to multiply independently in MEM.S. The C3.E8BU3.SREV2 clone was also tested by incubating cells in HAT medium for five days and then by plating these cells in HAT medium with aminopterin omitted (1 00 cells per flask) . When large colonies appeared they were trypsinized and plated without dilution. The resulting cultures were trypsinized once mo,re and plated without dilution in larger flasks and samples of confluent cultures were tested for BrdU-resistant cells. In each experiment a minimum of 20 cell cycles occurred between the initial plating and the test for resistant clones.

The resistant clones arising in these tests were recorded and the mutation rates were estimated according to LURIA and DELBRUCK (1943) by calculating the average number of resistant cells per culture (Table 2). When resistant clones were selected at a BrdU concentration of 50 ,pg/ml, a mutation rate of 1.4 X IO-' per cell per generation was observed for the clone C3.E2BUI.E21REVl.

In each experiment the ratio of variance to average for sampling distribution was significantly greater than 1. Measurements of the fluctuation of the number of BrdU-resistant clones in a reconstruction experiment gave a value of 1.14 fo r this ratio.

This high variance among independent cultures, which does not appear to be

TABLE 2

Fluctuation test on the revertant clone C3.E8BU3.SREVZ

Number of cultures: 1 7 20 Number of observed

resistant clones per sample Number of samples

0 1 2 3 4 5

6-1 0 11-20 21-30 31-40 41-50

Average per sample Variance (corrected for sampling) Variance/average BrdU concentration during selection Plating efficiency of BrdU-resistant cells

Final number of cells per culture (Nt) Number of plated cells for selection in each sample Average resistant cells per culture* Mutation rate per cell per generation

in the selection conditions

10 1 2 2 0 0 1 0 1 0 0 2.28

30 13.1 50 ag/ml

23 % 3 x 106 6x 105

20 2.3 x 10-6

4 3 3 0 1 1 3 2 1 1 1 8.50

127 14.9 25 P g / d

30% 25 x IO6 6 x ID5 1180

e.1 x 10-6

* The average number of resistant cells per culture is the average number of observed resistant clones in the tested samples divided by the plating efficiency of resistant cells in the selection conditions and multiplied by the dilution factor of the sampling.

BROMODEOXYURIDINE RESISTANCE 315

TABLE 3 Induced mutation frequencies (I.M.F.) for BrdU-resistance mutations with

increasing EMS concentrations

c3 C3.E8BU3.SREV2 EMS concentration Resistant clones per I.M.F. Resistant clones per I.M.F.

( w d d 4 X 108 plated cells P.E. ( X 10-8) 3.2X 106 plated cells P.E. ( X lo-')

Control 0 32% (<.8) 5 30% ( 5.2) 100 2 30% 1.6 14 30% 9.4

4'00 14 26% 13.5 20 16% 34.0 500 18 18% 25.0 - - -

200 4 30% 3.3 w 30% 18.8 300 7 26% 6.7 31 28% 29.6

Cells were incubated for 16 hrs with the mutagen and allowed to multiply during five days until a minimum of three division cycles had occurred in each culture submitted to mutagenesis. Cells were then plated for selection of BrdU 25 pg/ml resistant clones and their plating efficiencies (P.E.) were simultaneously estimated in MEM.S. I.M.F. was calculated as described in MATERIAL A N D METHODS.

due to sampling error or inefficiency of selection method, indicates that the re- sistant cells are pre-existent in each culture and not induced by selection.

Induced resistance mutation frequency in C3 and revertant clones: All the tested revertant clones yield spontaneous as well as EMS-induced resistant cells. Under EMS or MNNG standard mutagenesis Conditions, resistance mutation frequencies are five to ten times higher for the revertant clones than for the wild- type cells (Table 3 ) . Induced resistance mutation frequency was estimated for \7arying concentrations of EMS. Care was taken to ensure a sufficient expression time at high mutagen concentrations. Figure 2 shows a striking difference be- tween wild-type and revertant cell response: the wild-type induced-mutation irequency is very low at low EMS concentration and increases sharply at high concentration in a non-linear manner; on the contrary, induced resistance muta- tion frequency of the revertant C3.E8BU3.SREV2 increases linearly with EMS concentration. This latter result is in agreement with the observations of CLIVE et al. (1972).

Induced mutation frequencies for different mutagens and different loci: The optimal conditions for mutagenesis and expression of mutations, studied for the induction of BrdU resistance mutations, were used to compare the action of dif- ferent mutagens at different loci. The effects of four chemical mutagens were compared: EMS, MNNG, MMS and BrdU (Table 4). Spontaneous mutations were observed only in the experiments concerned with AZG resistance. The in- duced mutation rates for three different markers were compared using the stan- dard mutagenesis conditions described above. In all cases, screening for mutants was carried out on-populations in the range of IO6 to 5 x I O 6 cells. Drug-resistant clones were isolated. A comparison of the wild type and standard variants pheno- type in the two last columns of Table 4 shows a clear-cut distinction between them. The isolated resistant clones keep their phenotype in the absence of drug.

Growth characteristics of BrdU-resistant and -sensitive lines: Growth rates o€

316

0 6

M. CABOCHE

* 1 0 0 Z O O 3 0 0 4 0 0 5 0 0

5

4

h Y)

I o - 2 . 3

L r - 1

1

EMS (pg /ml )

FIGURE 2.-Induced mutation frequency (I.M.F.) for BrdU-resistance mutations. Mutagenesis conditions at variable EMS concentrations are discussed in MATERIAL AND METHODS. Open circles: wild-type C3 cells; closed circles: revertant clone C3.E8BU3.SREV2. Spontaneous resistance mu- tations are not deduced. Three experiments were performed on C3 cells and one experiment on two revertant clones. A multihit curve was always observed for C3 cells and a single-hit curve was observed for the revertant clones.

BrdU-resistant cells are not significantly affected by 25 pg/ml BrdU. BrdU- resistant clones are cross-resistant to FrdU and IrdU. The clones selected for FrdU resistance are also resistant to BrdU and IrdU (Table 5 ) . As first observed by KIT et al. (1963), exogeneous thymidine utilization is greatly reduced in re- sistant cells, as shown by the measurement of the incorporation of tritiated thymidine (Table 6) . These cells are unable to grow in HAT medium even if

TABLE 4

Induced mutation frequencies by different mutagens at different loci ~~~~~ ~ ~

Induced mutation frequency ( X io-'') EMS MMNG BrdU MMS

Cell phenotype*

Selection condition 250 pg/ml 0.5 pg/ml 10 pg/ml 20 pg/ml c3 V " T

AZG 15 pg/ml 120 70 34 23 3.5 pg/ml 60 pg/ml BrdU 30 pg/ml 8.3t 20.1 1.8 7.4 0.5 pg/ml 150 fig/ml FrdU 0.1 pg/ml 5.2 - 0.6 0.6 3mpg/ml 2pg/ml

* Phenotypes of wild-type C3 cells and variant clones are compared by measuring the drug concentration inducing a 20% plating efficiency when compared to plating efficiency in optimal growth conditions.

$ The 95% confidence limits for the measurement of I.M.F. after EMS mutagenesis (250 ag/ml, 16 hrs) are I.M.F. = 8.7 x 10-6 i 3.7 x 10-6, when estimated by t-test on five independent measurements.

BROMODEOXYURIDINE RESISTANCE

TABLE 5

The effect of drugs on the plating efficiency of BrdU-resistant or -sensitiue cells

31 7

Drug added

C3.ESBU3.SREV2. C3.ESBU3 C3.ESBU3.SREVZ E30 B,U1

c3 EMS-induced spontaneous resistant isolated wild type resistant revertant from revertant

MEM.S

BrdU Ipg/ml BrdU 10 pg/ml BrdU 100 pg/ml FrdU 0.1 pg/ml IrdU 20 pg/ml TdR 250 pg/ml AZG 15 pg/ml HAT medium

31’% 29 % 30% 28%

8 0 Q 0 0 0 0

96

97 95 74 83 85

100 0 0

12 0 01 0 0 0 0

84

98 99 89 90 73 84 0 0

Results are expressed in percent plating efficiency of controls, except for the first line, where absolute plating efficiency in MEM.S is given.

1 Ow3M thymidine and deoxycytidine are added. Another characteristic of BrdU- resistant clones is their ability to grow in the presence of 2 X thymidine, con- trary to the wild type and revertant clones which are killed by 5 X lo4 M thymidine. No other modification of metabolic utilization is observed: BrdU- resistant cells are sensitive to AZG and ARA-C; they incorporate labelled hy- poxanthine, uridine, deoxycytidine, glucose and galactose at the same rate as the wild type. Therefore, the mechanism of resistance is probably not an aspecific modification of permeation. Growth rates, drug sensitivities and exogeneous thymidine metabolism of revertant and wild-type cells are identical.

Karyotypes: LITTLEFIELD and BASILICO (1 966) observed modifications of karyotypes in BrdU-resistant cells isolated by stepwise selection. However, we were not able to observe such karyotype modifications in resistant and revertant cells isolated from C3 (Table 7). Therefore, the resistance mechanism is not cor- related with the loss of chromosomes bearing the studied markers.

Thymidine kinase activities of cell extracts: KIT et al. (1963) first discovered the correlation between the resistance mechanism and the loss of thymidine

TABLE 6

Incorporation of 3H-methyl thymidine and 14C hypoxanthine in 5-bromodeoxyuridine- resistant or -sensitiue cells

Wmeth 1 thymidine 14C8-hypoxanthine Cells 1 fici/y,110-j M 0.1 fiCl/ml lo-‘ M

- ~ _ . _ _ _

c 3 14700 2190 C3.E8BU3 980 261 0 C3 .E8BU3 .SREV2 15400 241 0 C3.E8BU3.SREV2.E30BUl 6990 2040

Incorporations are linear during the first two hours. Results are expressed in CPM incorpor- ated/hour/lOS cells.

318 M. CABOCHE

TABLE 7

Auerage karyotype chromosome number for BrdU-resistant or -sem&e cells

C3.E8BU3.SRF,V2 Cells c3 C3, E8BU3 C3. E8BU3. SREVZ E30BU1 ____ ~~ ____

Average chromosome number 42.42 0.7 42.0k 1.1 42.3 +- 0.5 42.8 i 0.6 ~ ~

Twenty diploid metaphases were counted fo r each of the four cell lines.

kinase activity of cell extracts. Because this enzyme activity widely varies with growth phase and medium composition as shown by WEISSMAN, SMELLIE and PAUL (1960) and by EKER (1966), optimal conditions for test have been care- fully investigated. Reproducible results are obtained when thymidine kinase activity is measured with cells obtained from exponentially growing cultures, in 24-hour-old medium, containing 10-5 M thymidine. The cell extracts were assayed immediately after preparation. All BrdU- or FrdU-resistant clones have a low but significant residual thymidine kinase activity (Table 8) ATTARDI and ATTARDI (1972) have shown that this residual activity might be best ex- plained by the presence of the mitochondrial enzyme in resistant cells. The BrdU- resistance mechanism might only be associated with the loss of the cytoplasmic enzyme. Intracellular localization of thymidine kinase activities in the wild type and revertant cells have been compared (Table 9) . The enzyme is mainly 10- cated in the cytoplasmic fraction. Thus an over-production of the mitochondrial enzyme in revertant clones is not the mechanism responsible for reversion. How- ever, because mitochondrial and cytoplasmic enzyme are not enzymologically distinGished in this experiment, the release of mitochondrial enzyme into the cytoplasm during preparation cannot be excluded. Thymidine kinase activities OE extracts of the revertant clones are generally lower than in the wild-type ex- tracts. Thymidine kinase activities of C3 and the revertant clone C3.E8BU3. SREV2, are further analyzed in Table 9. These activities are, to the same extent,

TABLE 8

Thymidine kinase actiuities of different clones

Cells Specific activity

Selection (fipM TMP/mg/min)

c3 C3.E2BU1 C3.NG2BU1 C3.E8BU3 C3.E2FU1 C3.E2BU1 .E21REVl C3.E2BU1 .E21REV4 C3.E8BU3.SREV2 C3.E2BUl.E21REVI.NG24BU2 C3.E2BUI.E21REV4.SBUll C3.E8BU3.SREV2.E30BUI

wild type resistant resistant resistant FrdU resistant revertant revertant revertant resistant resistant resistant

380 12 4 2 6

125 1 63 270

8 2 3

Percent wild type

100 3 1 0.5 1.5

33 43 71 2 0.5 0.8

BROMODEOXYURIDINE RESISTANCE 31 9

TABLE 9

Thymidine kinase activities of the wild-type cells and a revertant done

Test Conditions C3 C3.E8BU3.SREVe

Thymidine kinase activity exponential growth 380 C _ 35 contact-inhibited cells 903-10

(wM/mg/min) 2 x 10-6 aminopterin and 1 W M hypuxanthine in MEM.S for 16 hrs

Thermal stability Time of 50% enzyme inactivation at 65"

Cellular distribution of activity cytoplasm 61 % mitochondria and membranes 9%

(percent homogenate activity) nucleus 9% Km* (X 10-6M) determined for thymidine 3.7

570 & 50 37 2 5 sec

3.0

270 * 35 30210

420 k 45 4o)+lOSec

84% 11% 4% 5.9 5.0

* The probability that this Km discrepancy has occurred by chance i s PzO.07 (tz3.69).

increased by aminopterin treatment or decreased by contact inhibition of growth and medium depletion. Thermal stabilities have been compared according to the method of MIGEON, SMITH and LEDDY (1969). An exponential decrease of thymidine kinase activity as a function of incubation time at 65" is observed. The 50% average inactivation times of extracts of the two strains are not significantly different. The affinity of thymidine kinase for thymidine has been also measured in the two strains: the Km of the wild type enzyme for thymidine is significantly lower than that of the revertant type.

DISCUSSION

The BrdU-resistance phenotype is specific and hereditary. Resistant cells are only affected in their ability to use exogeneous thymidine and toxic analogs. Cul- tures are resistant to BrdU even after growth for months without the addition of drug into the medium to prevent the accumulation of revertant cells. This herit- able phenotype is not a long-term adaptation to the drug during selection, as shown by fluctuation tests on revertant clones. The resistant phenotype is in- ducible by classical chemical mutagens, strongly suggesting a genetic basis to this modification of phenotype ( CHU and MAILING 1968).

Genes involved in resistance mechanism: biochemical study: The cytoplasmic thymidine kinase activity is specifically affected in the resistant and revertant clones. This enzyme has a variable intracellular level, probably repressed by thymidine nucleotides as shown by EKER (1966). Structural and regulatory genes can be involved in the mutation processes. However, thymidine kinase ac- tivity is identically regulated, or protected, in wild-type cells and revertant clones (Table 9). If a repression mechanism occurs in the control of thymidine kinase level, revertant clones are not constitutive mutants, Moreover, BrdU-resistance phenotype is recessive (this property of BrdU-resistance mutations has been

320 M. CABOCHE

widely used in hybridization experiments). Therefore, the involved mutations in resistance or reversion mechanisms probably do not affect a repressor gene. However, an operator mutation, if this exists in mammalian cells, or a mutation affecting a regulator gene that is necessary for the expression of the thymidine kinase structural gene, would behave in the same fashion as a mutation affecting the structural gene itself.

BEAUDET, ROUFA and CASKEY (1973) have shown modifications of structural gene in hypoxanthine guanine phosphoribosyltransferase-deficient mutants of mammalian cells. We have only observed a small but significant thymidine kinase K m discrepancy between the wild type and a revertant clone. There is no decisive biochemical demonstration for the involvement of the thymidine kinase structural gene in resistance and reversion mechanisms; nevertheless. all the above results agree with this hypothesis.

Genes inuolued in resistance mechanisms: genetic study: The thymidine kinase locus is not X-linked in several mammalian cell strains and is autosomal in man (MILLER et al. 1971). As BHK 21/13 cells have an almost diploid karyotype, two functional structural genes for thymidine kinase are likely to be present in this strain, although a heterozygous wild-type clone cannot be excluded. Assuming that two functional structural genes for thymidine kinase are present in the wild-type cells and must be inactivated to get the BrdU-resistant phenotype, then, according to this hypothesis, resistant clones are homozygous, where as revertant clones are heterozygous for the structural thymidine kinase genes. The experi- mental results for the measurements of spontaneous resistance mutation rate agree with this hypothesis: CLIVE et al. (1972) have estimated the spontaneous mutation rate from the wild type to resistant phenotype to be 5 x 10-ll/cell gen- eration; for the C3 clone this mutation rate is lower than 3 X 10-8/cell/genera- tion. The spontaneous mutation rate from revertant to resistant phenotype is about 4 X 10-6/cell/generation. Curves of induced resistance mutation frequen- cies as a function of mutagen concentration also show a clear discrepancy be- tween C3 and revertant clones. Curves for revertant clones are linear and suggest a single-hit mutation process. On the contrary, the wild-type curves are typical of a multihit process (two or three hits). Therefore under the above hypothesis, mutation rates for spontaneous BrdU-resistance and mutation frequencies for chemically induced resistance are affected by the number of genes coding for active thymidine kinase. These results are not in agreement with observations of HARRIS (1971) on the effect of ploidy on spontaneous mutation rates.

If the two genes in the wild type and one in revertant clones have to be af- fected to induce the resistant phenotype, then the. induced resistance mutation frequency for the wild type should be the square of the induced mutation fre- quency for revertant clones. This is not experimentally observed. An explanation for this would be the existence of a correlation between the inactivations of the two thymidine kinase genes in the wild type. For instance, the mutagen could induce in the same cell a point mutation of one of the two structural genes for thymidine kinase, and a crossing over on the involved homologous chromosomes after replication of the altered gene. Such an EMS-induced crossing over has

BROMODEOXYURIDINE RESISTANCE 32 1

been described by YOST, CHALEFF and FINERTY (1967) in yeast. Another expla- nation for this would be the occurrence of two modes by which resistance to BrdU could come about: one involving the inactivation of the two genes for thymidine kinase, the other involving some completely different mechanism. The apparent homogeneity of resistant clones argues against this second hy- pothesis: Among 17 individually tested clones, resistant to BrdU 25 pg/ml, only one was able to grow in HAT medium and showed a consistant thymidine kinase activity.

The author wishes to thank MISS GINETPE GIFFARD and MR. FRANCIS BENNE for their skillful and conscientious technical assistance; DRS. GERARD BUTTIN, BARRY FAULKNER, and GERARD TIRABI for helpful criticism and for reading the manuscript.

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Corresponding editor: E. H. Y. CHU