Chromatid aberrations in a novobiocin-resistant subline of Chinese hamster V79 cells after exposure to novobiocin

Mutation Research, 174 (1986) 279-284

279

Elsevier

MRLett. 0877

Chromatid aberrations in a novobiocin-resistant subline o f Chinese hamster V79 cells after exposure to novobiocin K. T a k a h a s h i 1, I. K a n e k o 1'*, Y. Nishi 2 a n d N . I n u i 2 1Radiation Biology Laboratory, Institute of Physical and Chemical Research, Wako, Saitama 351-01, and 2Section of Cell Biology and Cytogenesis, Biological Research Center, Japan Tobacco Inc., Hatano, Kanagawa 257 (Japan) (Accepted 12 March 1986)

Summary We examined effects of novobiocin alone or in combination with ~,-irradiation, on the frequencies of chromatid-type aberrations in a novobiocin-resistant subline of Chinese hamster V79 cells (NOVOr-1). NOVOr-I cells were significantly resistant to novobiocin, as compared to wild-type V79 (WT) cells, with respect to cell survival and DNA synthesis. Survival responses of WT and NOVOr-1 cells to 7-rays in the range 2-10 Gy differed only slightly and the number of chromatid aberrations produced by irradiation at l Gy was fairly comparable in the two cell types. Post-irradiation treatment of cells with novobiocin at concentrations exceeding 200/~g/ml significantly increased the number of chromatid gaps plus breaks in WT cells as compared with NOVO~-I cells. With 200 #g/ml the increase was 1.2-fold (t-test, P < 0.05) and with 400 #g/ml, 2.3-fold (P<0.01) the number produced in NOVOr-1 cells.

Our previous study revealed that the frequencies of chromatid aberrations induced in Chinese hamster V79 cells irradiated by 7-rays in the G2 or plateau phase of the cell cycle were significantly increased by post-treatment with novobiocin (Takahashi et al., 1985a,b), a known inhibitor of eukaryotic type II DNA topoisomerase (Hsieh and Brutlag, 1980; Liu et al., 1980; Miller et al., 1981) or DNA polymerase (Edenberg, 1980; Meechan et al., 1984). Thus, the novobiocin-sensitive enzyme, most likely topoisomerase II, may be involved in the repair of 7-ray-induced lesions which can give rise to increased frequencies of chromosomal aberrations at mitosis, when their repair is incomplete.

To shed light on the possible involvement of topoisomerase II in repair processes after irradiation, we established a novobiocin-resistant subline from Chinese hamster V79 cells. In the present study, an attempt was made to examine the effects of novobiocin on the frequencies of chromatidtype aberrations induced by -r-irradiation in the novobiocin-resistant cells, and a comparison was made with the effects of novobiocin on those in wild-type V79 cells.

Materials and methods Cells

* To whom correspondence should be addressed.

Chinese hamster V79 cells maintained in our

0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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laboratory at 37°C in a humidified atmosphere of 5°70 CO2 in air in Eagle's minimum essential medium supplemented with 10070 fetal bovine serum, are referred to as wild-type (WT) cells in the present study. To obtain a novobiocin-resistant subline o f V79 cells, WT cells were treated for 3 h with ethyl methanesulfonate (0.8 mg/ml). After incubation for 1 week as the expression time, the cells were inoculated on 100 dishes at 1 x 106 cells/15 ml to form colonies on 0.33°/0 agar containing 200 tzg/ml novobiocin (Sigma). Cells in 1 out of 4 colonies which could survive on soft agar were selected and cultured in the presence of novobiocin. The novobiocin-resistant subline cells, named NOVO'-I, which exhibited stable proliferation with a population doubling time of 15.7 h were used. N o v o r - 1 cells were maintained in the presence of 200/~g/ml novobiocin.

Effects o f novobiocin on cell survival and D N A synthesis To examine the effect of novobiocin on cell survival, cells replated in appropriate numbers on dishes were cultured in the presence o f various concentrations of novobiocin. After incubation for 6-8 days, the cultures were fixed and stained with 0.1 07ocrystal violet. Colonies containing more than 50 cells were scored as survivors. For measurement of DNA synthesis in ceils when novobiocin was present, exponentially growing cells were labeled for 24 h with 0.05 ~Ci/ml [Me)H]thymidine (specific activity, 78.2 Ci/mmole) (New England Nuclear). Radioactivity incorporated into 1007o trichloroacetic acidprecipitable materials was measured in ACS II scintillation cocktails (Amersham).

tions were made according to a standard protocol and the slides were stained with 207o Giemsa (Merck) for analysis of aberrations.

Results and discussion

Effects o f novobiocin on cell survival and DNA synthesis Survival responses of WT and NOVO'-I cells to novobiocin are shown in Fig. 1. Novobiocin at a level o f 50 #g/ml exhibited no significant influence on the colony-forming ability of WT cells, whereas novobiocin doses exceeding 100/~g/ml significantly reduced cell survival. The surviving fraction of NOVO'-I cells was considerably resistant to novobiocin, in terms of colony-forming ability. 1.0

0,1

> >

0.01

0.001

Analysis o f chromatid aberrations To score the chromatid-type aberrations, exponentially growing cells were irradiated with 6°Co y-rays (1 Gy) at room temperature. Immediately following irradiation, various concentrations o f novobiocin together with 0.1 ~g/ml demecolcine were added to the cells and the preparations were cultured at 37°C for 2 h. Chromosome prepara-

0

100

200

000

400

Novobiocin (~Jg/ml) Fig. 1. Survival response to novobiocin. WT (0) and NOVO~-I cells (e) were inoculated in appropriate numbers on dishes and cultured in the presence of various concentrations of novobiocin. Colonies containing more than 50 cells were scored as survivors. Each data point represents 3 experimental determinations and the error bars reflect the standard deviation of mean.

281 I

,.-g 100 v (a ~c c

0.1 < z o

5O

.c o 1

o

O 100

200

Novobiocin

300

400

(pglml)

Fig. 2. Effect of novobiocin on DNA synthesis. Exponentially growing WT (©) and NOVOr-I (O) were labeled with [3H]thymidine for 24 h in the presence of various concentrations of novobiocin. Radioactivity incorporated into acidprecipitable materials was measured. Each data point represents 3 experimental determinations and the error bars reflect the standard deviation of mean.

n

> > t/1

6.1

I

t

I

I

I

2

4

6

8

lO

Radiation

The surviving fraction of N O V O ' - I cells was 0.55 in the presence o f 300 #g/ml novobiocin. The concentration of novobiocin required to reduce the surviving fraction to 5007o of the control cultures not exposed to novobiocin was approximately 100 # g / m l for W T cells and 303 # g / m l for NOVOr-1 cells. Effects of novobiocin on D N A syntheses in W T and N O V O ' - I cells were examined by measuring the radioactivity of [3H]thymidine incorporated into acid-precipitable materials in the presence of novobiocin, at various concentrations. As can be seen in Fig. 2, the D N A synthesis of W T cells gradually decreased as the dose o f novobiocin was increased, and the concentration of novobiocin required for 50070 reduction of D N A synthesis in W T cells was approximately 113 #g/ml. In contrast to the W T cells, D N A synthesis o f NOVOL1 cells was strongly resistant to novobiocin. In concentrations up to 300 #g/ml, this drug exhibited no inhibitory effects on the D N A synthesis of NOVOr-1 cells. Even with 400 # g / m l o f novobiocin, the D N A synthesis o f N o v o r - 1 cells was only reduced to about 8007o o f the controls not exposed to the drug. These results suggest that the survival response of cells to

i

dose(Gy)

F i g . 3. S u r v i v a l r e s p o n s e

to y-irradiation.

Exponentially

grow-

cells were irradiated at room temperature by ~°Co y-rays. Immediately following irradiation, cells were replated in fresh medium. After 6-8 days, cultures were fixed and colonies containing more than 50 cells were scored as survivors. Each data point represents 3 experimental determinations and the error bars reflect the standard deviation of mean. (a) WT cells; (b) NOVOr-I cells. ing

novobiocin largely depends on the susceptibility o f D N A synthesis to novobiocin.

Survival response to ~-irradiation Fig. 3 shows a typical ~,-ray dose-survival relationship of W T and NOVOr-1 cells. Survival curves of W T and N O V O ' - I cells differed slightly after irradiation in the range 2-10 Gy and no shoulder r e g i o n was observed in the case of N O V O ' - I cells. The surviving curve is characterized by the following radiobiological parameters: n (extrapolation number) and Do mean lethal dose). These values were calculated by the least-squares method f r o m the linear part o f the curves constiting of at least 4 different doses of radiation. The values o f n for W T and N o v o r - 1 cells were 1.5 and 1.0, respectively, and the Do value for W T

282

was 358 rad and that for NOVOr-1 cells was 420 rad. Chromatid aberrations A 2-h treatment of unirradiated cells with novobiocin, even at the highest dose used here (400 #g/ml), induced few chromatid aberrations in NOVOr-1 cells, contrary to WT cells in which the incidence of abnormal metaphases (t-test, P<0.05) as well as the mean number of gaps plus breaks per metaphase (P<0.01) were significantly increased by 400 #g/ml novobiocin (Table 1). The frequency of exchanges, comprising chromatid intraexchanges and interexchanges, was not influenced in WT nor in N o v o r - I ceils by exposure to novobiocin at any concentration examined, as

TABLE 1 F R E Q U E N C I E S OF C H R O M A T I D A B E R R A T I O N S IN W T AND Novor-I CELLS Cell

Treatment y-Ray Novo(Gy) biocin

Abnor- N u m b e r o f aberrations/ mal me- 100 metaphases (mean taphase

~g/ml) WT

0

NOVOr- ! 0

± S.E.) Gaps and breaks

0 100 200 400

4 5 7 14'

0 100 200 400

61 65 78** 94**

0 100 200 400 0 100 200 400

Exchanges

4 5 8 16

± ± ± _+

2.0 2.2 2.7 3.9**

0 0 0 0

125 134 203 349

_+ ± ± ±

13.7 14.2 16.2"* 25.6**

4 5 9 4

6 3 9 8

6 3 9 10

+_ _+ _+ ±

2.4 1.7 2.9 3.6

0 0 0 0

65 75 83** 80*

127 148 168 153

+ ± ± ±

13.2 12.4 12.8" 14.2

4 i 3 5

_+ ± ± ±

1.9 2.2 3.2 2.4

± ± + ±

2.0 1.0 1.7 2.2

100 metaphases were scored per point and the m e a n ± S.E. per 100 metaphases was given. Significantly different from the value for control not exposed to novobiocin: * P < 0 . 0 5 , * * P < 0 . 0 1 .

measured at this sample time. Since our previous results revealed that 400 #M (254 #g/ml) novobiocin produced few chromatid aberrations in unirradiated V79 cells (Takahashi et al., 1985a), at doses exceeding 254 #g/ml, novobiocin itself may produce chromatid aberrations, except for exchanges, in unirradiated cells, by interfering with normal cellular processes such as chromosome condensation. This assumption was also suggested by previous data indicating that novobiocin treatment resulted in a significant mitotic delay and a reduction of mitotic frequency in unirradiated cells (Takahashi et al., 1985b). The present results also suggest that the normal cellular processes of unirradiated Novor-1 cells seem to be more resistant to novobiocin than the WT cells. Irradiation of exponentially growing cells by 3,rays at 1 Gy produced nearly comparable numbers of chromatid aberrations, comprising gaps, breaks, and exchanges, in WT and NOVOr-1 cells, although these increases are not statistically significant (P<0.05). Novobiocin at 200/zg/ml or 400 /zg/ml significantly increased the frequencies of chromatid gaps plus breaks as well as the incidence of abnormal metaphases in WT cells which had been irradiated for 2 h before harvesting. Novobiocin at 200 #g/ml could significantly increase the number of gaps plus breaks in NOVOr-1 cells (P< 0.05), whereas 400 #g/ml novobiocin did not result in a statistically significant increase in gaps or breaks (P<0.05). Irradiation alone or in combination with novobiocin treatment (up to 200 #g/ml) produced comparable amount of gaps plus breaks in WT and NOVO~-I cells (P<0.05); however, post-irradiation treatment with 400 /~g/ml novobiocin produced 2.3-fold (P<0.01) as many gaps or breaks in WT cells compared with those in NOVO~-I cells. Contrary to gaps plus breaks, the frequency of exchanges induced in WT and Novor-1 cells by irradiation was not influenced by novobiocin, even at the highest dose given. Chromatid-type aberrations have been shown to be produced directly from double-strand breaks in the DNA (Natarajan and Obe, 1978; Bryant, 1984), and DNA lesions other than double-strand breaks induced by ionizing radiations were sug-

283

gested to be converted into chromatid aberrations when repair of these lesions was inhibited by chemical agents (Preston, 1980; Bender and Preston, 1982). Our present results suggest that 7ray-induced repair is more resistant to novobiocin in N o v o r - 1 cells than in WT cells, so that higher concentrations of novobiocin are required for increases in the frequencies of gaps or breaks, except for exchanges, in the N o v o r - 1 cells. It was suggested that production of exchanges requires DNA synthesis (Bender and Preston, 1982) and that the DNA synthetic activity exists in the G2 phase of the cell cycle (Schvartzman et al., 1981). A known inhibitor of DNA synthesis, such as hydroxyurea (Kihlman et al., 1982) or aphidicolin (Natarajan et al., 1982), also failed to increase the frequency of exchanges after Xirradiation, and this was suggested to be due to the possible inhibition of the DNA synthesis by these drugs (Natarajan et al., 1982). However, it seems unlikely that novobiocin fails to increase the frequency of exchanges by interfering with DNA synthesis in G2, because up to 300 #g/ml novobiocin, that fails to influence significantly the DNA synthesis in NOVOr-1 cells (Fig. 2), does not increase the frequency of exchanges in N o v o r - 1 cells previously induced by irradiation (Table 1). Alternatively, it seems most likely that novobiocin may inhibit chromosome condensation or restoration of higher-order DNA structures after irradiation (Mattern et al., 1983; Takahashi and Kaneko, 1985) by interfering with topoisomerase II, other than DNA polymerase, thereby leading to increased frequencies of chromatid gaps or breaks, without influencing significantly the frequency of exchanges.

Acknowledgements This work was supported in part by a Grant-inAid from the Ministry of Education, Science and Culture of Japan. We thank M. Ohara (Kyushu Univ.), for comments on the manuscript.

References Bender, M.A. and R.J. Preston (1982) Role of base damage in aberration formation: Interaction of aphidicolin and X-rays, in: A.T. Natarajan, G. Obe and H. Altmann (Eds.), DNA Repair, Chromosome Alterations and Chromatin Structure, Elsevier, Amsterdam, pp. 37-46. Bryant, P.E. (1984) Enzymatic restriction of mammalian cell DNA using Pvu II and Barn HI: evidence for the doublestrand break origin of chromosomal aberrations, Int. J. Radiat. Biol., 46, 57-65. Edenberg, H.J. (1980) Novobiocin inhibition of simian virus 40 DNA replication, Nature (London), 286, 529-531. Hsieh, T., and D. Brutlag (1980) ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings, Ceil, 21, 115-125. Kihlman, B.A., K. Hansson and F. Palitti (1982) Potentiation of induced chromatid-type aberrations by hydroxyurea and caffeine in G2, in: A.T. Natarajan, G. Obe and H. Altman (Eds.), DNA Repair, Chromosome Alterations and Chromatin Structure, Elsevier, Amsterdam, pp. 11-24. Liu, L.F., C.-C. Liu and B.M. Alberts (1980) Type II DNA topoisomerase: Enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break, Cell, 19, 697-707. Mattern, M.R., L.A. Zwelling, D. Kerrigan and K.W. Kohn (1983) The reconstitution of higher-order DNA structure after X-irradiation of mammalian cells, Biochem. Biophys. Res. Commun., 112, 1077-1084. Mcechan, P.J., S. Killpack and J.E. Cleaver (1984) Novobiocin-mediated inhibition of polymerization and ligation of DNA in vitro, Mutation Res., 141, 69-73. Miller, K.G., L.F. Liu and P.T. Englund (1981) A homogenous type II DNA topoisomerase from HeLa cell nuclei, J. Biol. Chem., 256, 9334-9339. Natarajan, A.T., and G. Obe (1978) Molecular mechanisms involved in the production of chromosomal aberrations, I. Utilization of Neurospora endonuclease for the study of aberration production in G2 stage of the cell cycle, Mutation Res., 52, 137-149. Natarajan, A.T., I. Csuk/ls, F. Degrassi, A.A. Zeeland, F. Palitti, C. Tanzarella, R. de Salvia and M. Fiore (1982) Influence of inhibition of repair enzymes on the induction of chromosomal aberrations by physical and chemical agents, in: A.T. Natarajan, G. Obe and H. Altmann (Eds.), DNA Repair, Chromosome Alterations and Chromatin Structure, Elsevier, Amsterdam, pp. 47-59. Preston, R.J. (1980) The effect of cytosine arabinoside on the frequency of X-ray-induced chromosome aberrations in normal human leukocytes, Mutation Res., 69, 71-79. Schvartzman, J.B., B. Chenet, C. Bierknes and J. van 't Hof (1981) Nascent replicons are synchronously joined at the end of S phase or during G2 phase in peas, Biochim. Biophys. Acta, 653, 185-192.

284 Takahashi, K., and I. Kaneko (1985) Changes in nuclease sensitivity of mammalian cells after irradiation with 6°Co 7-rays, Int. J. Radiat. Biol., 48, 389-395. Takahashi, K., I. Kaneko, C. Nishiyama and K. Nakano (1985a) Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following 7-irradiation, Mutation Res., 144, 265-270.

Takahashi, K., C. Nishiyama and I. Kaneko (1985b) Increase in the frequency of ~,-ray induced chromosomal aberrations in mammalian cells by post-treatment with novobiocin, Int. J. Radiat. Biol., in press. Communicated by F.H. Sobeis