Studies of mutagenicity on quiescent and proliferating hamster cell populations

Cell Biology

International STUDIES

Reports,

OF MUTAGENICITY

PROLIFERATING

R. Laboratorio

Vol. 4, No. 3, March

1980

303

ON QUIESCENT

HAMSTER

CELL

AND

POPULATIONS

VERGARA and M. TERZI di Mutagenesi e Differenziamento deZ CNR, Pisa, Italy ABSTRACT

A few known mutagens were tested on proliferating hamster cells and on cells made quiescent by L-histidino1 block. No differences were found in the effectiveness of two alkylating agents and a base analogue; whereas the carcinogen 4-nitroquinoline-l-oxide was more effective on quiescent than on proliferating cells. The implications of these data on the mechanism of action of mutagens are discussed. INTRODUCTION Mammalian cell populations have become in recent years one of the systems of choice for studies of mutaThis because it is commonly thought that the genesis. extrapolation to man is somewhat more reliable starting from mammalian cells that from, say, Enterobacteriaceae. Virtually all those studies however, are done on proliferating cell populations and this, particularly if we want to extrapolate to the situation in viva - where most cells at any time are in a state of quiescence might be incorrect. Therefore we thought it interesting to compare the mutagenic action of a few known mutagens on cell popula tions, proliferating or kept quiescent by some type ofinduced block. MATERIALS

AND

The Chinese hamster cell line V79 cultivated in Dulbecco's modification of mented with 5% foetal calf serum (Flow). bling time of V79 was 11-12 hrs and its

METHODS was used throughout. It was Eagle's medium (DMBM)supple In our conditions the dou-plating efficiency 90-100%.

Chemicals were purchased from Sigma, except Ethylmethanesulfonate (EMS) from Eastman, Hydroxyurea (HU) from BDH, 4-Nitroquinoline-l-oxide (4NQO) and N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) from K. and K. A reversible shortly after cell at 4.103/cm2, were tion of the drug. 0309-16511801030303-1om.m/0

block in cell growth was achieved by adding, attachment, 1mM L-histidinol. The cells, seeded considered fully quiescent 2-3 days after addi0 1980

Academic

Press

Inc. (London)

Ltd.

304 Thymidine pitable material fit radioactivity

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Vol. 4, No. 3, March

1980

incorporation was measured by counting the TCA preci after 30' pulses of tritiated thymidine at a spec: of 225 mC/mM, 5 UC/ml.

For mutagenesis the cell were seeded in Corning flasks at about lo4 cells/cmL and treated next day; in the case of quiescent cells, they were seeded at about 2.104 cells/cm2, then L-histidinol was ad ded and mutagenesis performed two days later. The chemical mu.agenwas added to the flasks and left for two hours. After its removal, the cells were washed twice with buffer (PBS) and seeded at different cell densities: - a series of plates with 7 cells/cm2 was used to measure survival; the colonies were stained and counted after one2week; - a series of plates was seeded at a cell density of 6*103 /cm when the subculture was to be used two days later (if instead three days of subculture were necessary, the density of the inoculum was four times lower in order to reach the same final density). lutant colonies were scored by plating the mutagenized cell cuiture or its 2-3 days subcultures at a density of 2.5~10~ cells/ cm . 2.5 FM 6-thioguanine (6TG) was added soon after cell attachment. Resistant colonies were stained and counted after 9 days of incubation without medium change. For additional expression times, subculturing was repeated with the same procedure. This way curves of the expression time of the mutation were determined as suggested by van Zeeland and Simons (1976). Mutation frequency was determined from the average number of colonies/dish corrected for survival and plating efficiency. The background of spontaneous mutants (about 0.5*10-6 in our conditions) was neglected. RESULTS 1.

Induction of quiescence Quiescence was induced by treatment with L-histidinol, a histidine analogue known to block the cells in Gl phase (Warrington and Hechtman, 1977). In our hands this type of treatment was more effective than serum limitation (Todaro et al.,, 1965) and it produced a complete inhibition of growth as can be seen from figure 1. The block has also been checked by measuring the incorporation of tritiated thymidine in normally proliferating and inhibited cells. Quiescent cells incorporated less than 1% of the control (1.2~10~~ counts per min. per cell). Fig. 2 shows that after removal of the drug, growth resumes its normal rate within 24-48 hrs. In the same figure it is shown that lmll HU - an S-phase specific cytotoxic drug - (Rozengurt and PO, 1976) kills the reversed cells approx. at the same rate of the control, whereas during the L-histidinol block it had no effect (data not shown). L-histidinol inhibition is therefore reversible,

Cell Biology

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Reports,

Vol. 4, No. 3, March

1980

although a decrease in plating efficiency is apparent plating is done immediately after drug removal. The fect on cell proliferation in mass cultures however much smaller.

time

(days)

if efis

Fig.1

Fig.1

EFFECT OF 1 mM L-HISTIDINOL ON THE GROWTH OF V79 CELLS o Cells grown in medium containing L-histidinol 0 Proliferating cells

Fig.2

EFFECT OF 1 mM HYDROXYUREA ON V79 CELLS AFTER REVERSAL OF L-HISTIDINOL BLOCK o untreated control; o Cells treated with L-histidinol; n cells reversed from growth block;A cells reversed and treated with hydroxyurea; A proliferating cells treated with hydroxyurea; + time of L-histidinol removal; + time of hydroxyurea addition.

2.

Mutagenesis on proliferating and resting populations The spontaneous background of mutants resistant to 2.51~M 6TG was determined several times. There was no difference in the two cases of proliferating and quiescent populations. i) Ethylmethanesulfonate (EMS). This mutagen was used at a final concentration of 0.1% (v/v> for two hrs. The results, presented in Table 1, show that mutant induction, at different expression times, is not very different in the two growth conditions.

Cell Biology

306 Table

1

e.t. days

International

MUTAGENIC

ACTION

eopX prol.

Reports,

Vol. 4, No. 3, March

OF EMS Mut./lO' prol.

quiesc.

1980

surv. quiesc.

0

120

18

2

63

43

3.3

11.5

4

93

91

71.5

68.7

6

75

74

84.4

92.7

e.t. : expression time of the mutation; eop : ef ficiency of plating; Mut./105surv. : number of induced mutants per 105 survivors, that is revised for eop.; prol. : proliferative phase; quiesc. : quiescent phase.

Table

2

e.t. days

iii)

prol.

ACTION

OF MNNG Mut./lO' prol.

cop% quiesc.

surv. quiesc.

0

37

17

2

15

20

34

22

4

18

14

260

235

6

76

58

107

189

For ii)

MUTAGENIC

the

explanation

of

abbreviations

see

Table

1.

N-Methyl-N' -nitro-N-nitrosoguanidine (MNNG). This alk lating agent was used at a concentration of 7x10' ): M for 2 hrs. Table 2 shows that, again, the difference between proliferating and quiescent cells is irrelevant. 5-Bromodeoxyuridine (BUdR). The mutagenesis protocol used for this base analogue was modified in order to accommodate a few spe the efficiency of the muta cial needs that improve genie treatment : a) lower cell density b) longer exposure to the drug

Cell Biology

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Vol. 4, No. 3, March

1980

c)

incubation in thymidine at the end of the BUdR treatment (Davidson and Kaufman, 1978). Therefore the protocol we used was the following (modified from Huberman and Ueidelberger 1972) : cells were seeded at a density of 2.5x10 3 xcmB2, then L-histidinol was added. BUdR (3x10e5M) was ad ded two days later and left for 51 hrs. In the pro liferating control 6x103cells/cm2 were inoculatedand BUdR was added next day. In both cases at the end of the BUdR treatment, the cells were trypsini zed, their efficiency of plating (eop) was measured, and they were reseeded for 3 days in a medium containing (2 mM) thymidine. Then subcultures for the expression times were done as in Materials and Methods. Table 3 shows that even with this base analogue the cells in the two growth phases behaved in the same way. Similar results were obtained when the same con centration of BUdR was added soon after seeding, left for 3 days and repleced with a thymidine-contai ning (10~M) medium for 2 days (protocol modified from Davidson and Kaufman, 1978). The mutant induction was somewhat lower in this last case, but no differences were apparent in the two cell populations

Table

3

e.t. days

prol.

ACTION

cop% quiesc.

OF BUdR Mut./105surv. prol. quiesc.

0

72

25

3

77

76

2.6

2.4

5

72

100

2.3

2.8

7

75

79

2.3

2.4

For iv)

MUTAGENIC

the

explanation

of

abbreviations

see

4-nitroquinoline-loxide (4NQO) This mutagen was used for 2 hrs according rials and Methods at a final concentration 10m7M. Table 4 shows that mutant induction ter in the quiescent cell populations at sion times tested.

Table

1.

to Mateof is greaall expres

308

Cell Biology Table

4

e.t. days

International

MUTAGENIC

prol.

ACTION

Reports,

Vol. 4, No. 3, March

OF 4NQ0 Mut./105 prol.

cop% quiesc.

surv. quiesc.

0

108.5

71.2

4

67.0

81.7

0.6

2.3

6

87.2

97.5

0.2

2.7

9

72.7

115.5

0.1

2.6

11

89.7

67.25

0.2

2.7

13 -

82.5

70.5

0.8

3.5

For

the

explanation

of

1980

abbreviations

see

Table

1.

Growth inhibition after mutagenic treatment. In order to understand at what time the L-histidino1 block interferes with mutation induction (in case it does, and whatever the mechanism), quiescence was in duced immediately after mutagenic treatment and the results compared with those obtained by blocking before mutagenesis. The protocol was as described for the proliferating cells, except that after mutagenic treatment the cells were subcultured for 96 hrs in the presence of L-histidinol. The results for 4NQ0 are reported in table 5. They show that mutant induction is maximum if the mutagenic treatment is done during L-histidinol block. If the cells remain proliferating or if the block is indumutant induction is low. ced after mutagenesis, The same experiment was performed with EMS adding L-histidinol after treatment for 48 and 96 hrs. The results presented in fig. 3 indicate that the various time are almost superimcurves of mutant induction vs. posible if they are corrected for the time of induced quiescence during which, apparently, the process of mutant formation was as blocked. 3.

Cell Biology Table

5

International

Reports,

Vol. 4, No. 3, March

309

1980

MUTAGENESIS BY 4NQ0 IN POPULATIONS BLOCKED BEFORE AND AFTER MUTAGENIC TREATMENT

e.t. days

4NQO

cop% hol+4NQO

4NQ0

Mut./lODsurv. hol+4NQO

4NQWhol

0

54

10

4

76

76

47

0.6

2.7

0.6

6

71

63

49

0.3

4.3

0.2

8

87

98

91

0.5

3.1

0.1

11

74

67

98

0.9

2.7

0.2

13

64

70

58

1.1

3.6

0.2

4NQO+hol

4NQO: proliferative phase; hol+4NQO: quiescent and during the treatment; 4NQCHhol: quiescent after the treatment and lasted 96 hrs. The other abbreviations are as in Table 1.

Fig.3

MUTANT INDUCTION BY EMS vs TIME Mut/l05surv.: number of induced mutants per vors; e.t.: expression time of the mutation; proliferative phase; l hisol+EMS: quiescent fore and during the treatment; 0 EMS+hisol48: phase induced after the treatment and lasted AEMSthisol96:quiescent phase induced after ment and lasted 96 hrs.

phase before phase induced

10 5 survio EMS: phase bequiescent 48 hrs; the treat-

310

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1980

DISCUSSION The comparative analysis of mutagenic effectiveness of known mutagens on proliferating and quiescent cell populations has shown few differences. These differences concern either the final level of mutants or the ti me of expression of the mutation. Our results are not easily usable for extrapolating from proliferating systems in vitro to quiescent systems in vivo except in introducing a new word of caution in that each mutagen could be different from the rest. Instead more immediate implications of the present work are on the definition of the mechanism of action of the mutagens on eukaryotic cells. The alkylating agents EMS and MNNG acted with the same effectiveness on quiescent and proliferating cells. This might have come as a surprise, at least in the case of MNNG,which in procaryotes is known to act at the replication fork, hence mostly in the replicative phase (Cerda-Olmedo et al., 1968). It should be mentioned however, that doubts have already been raised on this type of mutagenic mechanism by Orkin and Littlefield who failed to obtain greater induction of muta(19711, nts during the S-phase in syncronized Syrian hamster fi broblasts. Another action mechanism which our data do not sup one proposed for BUdR whose muport is the "classical" tagenic action has been interpreted as due to its incorporation in lieu of thymidine with consequent pairing If this were the case in eukamistakes (Freese, 1959). ryotes, one would expect a big difference between proli ferating and quiescent cells. Our results are more in line with the finding by Kaufman and Davidson (1978) of lack of linear relationship between incorporation of The same Authors (DavidBUdR and induction of mutants. son and Kaufman, 1978).discovered that the mutagenic action of BUdR can be inhibited by deoxy-cytidine and is increased by thymidine. We also found that a concentration of 3x10B5M BUdR induced fewer mutants if the thymidine concentration was lowered from 2 mM to 10pM and this even if the mutagen was left to act 72 hrs instead of 51 of our protocol. Therefore our results support the interpretation of Kauf man and Davidson (1978) who postulated an important role of the nucleotide pools for the mutagenic action of BUdR Our results on the mutagenicity of 4NQ0 before and after L-histidinol block could be interpreted as if 4NQ0 mutagen and its action became amplified were a very poor in the presence of L-histidinol. However, in the light of what is known from the li-

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1980

311

other interpretations are more plausible. In terature, particular, we know that 4NQ0 induces DNA-repair synthe sis throughout the cell cycle (Stich and San, 1970) and that most of this repair occurs within 8 hrs, long befo re the cells enter the S-phase. However, when the cellreplicates its DNA, the damage becomes fixed, i.e. the level of mutations depends on the level of unrepaired lesions (Stich et al., 1971). Since L-histidinol not only prevents DNA replication but also repair synthesis (Abbondandolo, pers. comm.), the cells cannot repair du ring the block and when L-histidinol is removed there is a race between replication and repair. A good proportion of lesions becomes fixed and the level of mutations is high. Instead, if the block is induced after mutagene sis, L-histidinol cannot stop the DNA repair which has already started and hence the level of mutations is the same as in proliferating cells. If we now turn to differences, not on the final level, but on the expression time of mutations, we think that they can be explained on a simple physiological ba sis: cells resistant to purine analogues are devoid (or show reduced levels of activity) of the enzyme hypoxanthine-phosphoribosyl-transferase. In order for the muta tion to be expressed, in a cell where the relevant DNAchange has occurred, the enzyme has to be diluted: if cell division is prevented by L-histidinol, this dilution cannot occur or is retarded. In conclusion : i ) Alkylating agents such as EMS and MNNG behave as direct mutagens and there is no difference in their ef fectiveness on quiescent and proliferating cell populations. ii) The mutagenic action of BUdR is not to be interpreted only as mismatch but more likely it has to do with disturbances in nucleotide pools. iii)The action of 4NQ0 is strongly influenced by an error-free repair system: if this system is allowed to act it repairs the majority of premutational lesions. If instead the cell enters division, the unrepaired lesions may become expressed mutations. ACKNOWLEDGEMENTS R. Vergara was supported by a fellowship from Scuola Normale Superiore, Pisa. The Authors express their thanks to Drs. A. Abbondan dolo and G. Rainaldi for many helpful discussions. -

Received:

14th November

1979

Accepted:

10th December

1979

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1980

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