DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

af cultured cells derived ! omparison with human c

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Takashi Yagi Radiation Biology Center, Kyoto University, Yoshida-konoecho, Sakyo-ku, ', g

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(Received 23 November 1981) (Revision received 16 March 1982) (Accepted 25 March 1982)

aunary DNA repair in mouse cells derived from embryos ros of 3 i~ s were estigated in comparison with that in human cells. The levels of DNA Lthesis after UV irradiation appeared to change at different dif pa capaci; of host-cell reactivation of UV-irradiated herpes simplex always uced to the same levels as those in xeroderma pigm mentosum cells. This implied )lied I, l l V t tI, mouse cells Are reduced in excision-repair capaciti mcities and that the apparentlyY hig h levels of unscheduled DNA synthesis at certain pas passages Are not quantitatively ty related ~ted to high levels of cell survival. Essentially no diff~ differences in DNA repair were noted ed among 3 strains - - BALB/c, C 3 H / H e and C57E C57BL/10.

Th, t h e mouse is the most widely used test animal in can, cancer reseArch. To extrapolate )olate e x n e r i m e n t a l results re.~ult~ in tthe he m c m ~ tto a human I~lnm~n h~ino¢ the experimental mouse beings, it is indispensable to know the similarities and the differenq fferences between the two species. Involvement of D N A repair in carcinogenesis hass been suggested. Cancer proneness of patients with some hereditary diseases such as; xeroderma pigmentosum (XP), whose cells Are defective in DNA repair, supports the he idea of the importance of DNA repair in carcinogenesis. (For review, see refs. 20, 25.) In human cells, excisionI repair appeArs to be the predominant type of repair at least for UV damage, because , a u s e most XP cells, except those from variants, Are defective in excision repair with high UV sensitivity [3,21]. Although no mutant cells of mouse that were defectiv ve in any type of repair have been isolated so fAr, it has been suggested that mouse•. cells have a very low capacity for excision repair, and that post-replication repair'could be the predominant type of repair of UV damage in mouse cells [7,9,14]. Recentl, ~ n t l y , however, the presence of a considerable level of excision repair has been re~ported [2,17,18] and a gene presumably controlling the 002%5107/82/0000-0000/$02.75 © Elsevier Biomedical Press

:d on chromosome 4 [15]. s (UDS) has been used by many excision repair. Peleg et al. [18] ys 13-15 of gestation and from a lity to excise pyrimidine dimers th day embryo had no capacity fo: lowed different results with positi~ estation. Most mammalian cells h and the level usually becomes lc Tice [24]. Using I C R mouse, Sas incr level of U D S was low in cells from the day-17.5 embryo, emt ~ontaneous tran culturing the cells and went low again after spontam )ne aim of this study is to examine whether these thes appare nomena exist in mouse cells in culture. A comparison between s in D N A repair will be presented and discussed.

s as an at cells n adult UDS, 'pair of he cells Lt UDS he late nd that ally by dicting mouse

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ture media serum flemented wil Eagle's minimal essential medium (MEM) supplem~ um for E M with i bco) for successive transfers of mouse cells, and M ME M E M supplemented rated er experiments were used. For colony formation, a-modified a-mq 15% foetal bovine serum (Flow Laboratories) was iused.

Prelparation of mouse embryonic cells and successive trans.~fers Animal Experimental Anima Vlouse inbred strain B A L B / c was purchased from Shizuoka Shi M, by Drs. Yamazaki and nd kindly given and C 5 7 B L / 1 0 were Ltd., and C 3 H / H e rS Japan. Mouse embryos (day, gasawa, National Institute of Radiological Sciences, J Nag c u t internal organs removed were cul and 18 of gestation) and newborn mouse with intern 14 uffered times with phosphate-bufferec into) fragments by scissors and disaggregated several tirr n,= (PBS) (Pl:lq~ rcontaining nnt~inlno fl 25% 9~ tr'vr't~in aand n d 0.025 ('l I]9.~ M M E F]"D T A with rough shaking at trypsin al saline 0.25% w a s was centrifuged at 1500 r p m for 5 min. The pellet wa~ 37°C. The cell suspension than n, passed through 3 sheets of gauze, seeded at more that resuspended in the medium ge 2 × 107 cells per 150-cm 2 bottle and incubated for 3 days with a medium chang~ were riod, cells were too m a n y to proliferate. The cells wer~ every day. During this period, 6 detached with 0.1% trypsinLin 0.02% E D T A solution, counted and seeded at 3 × 10 ~ and passage 0 (P0). Every 3 days the cells were trypsinized ant cells per 150-cm 2 bottle as passag when fining 10% dimethyl sulphoxide at 80°C until use, whet stored in the medium containin the 'red into a new bottle. The medium was changed on th~ 3 X 106 cells were transferred next day after the transfer. ;thod The population doublingg number ( P D N ) was calculated as follows by the methoc aumber transfer, the growth rate (Gk), or increase in cell numbeJ of Ban et al. [1]. At every the ed as the number of cells in a bottle divided b y• th~ per passage, was calculated The cumulative growth rate at the N t h passage, inoculation size (3 × 106).

rowth rate at every passage. PD

:sed as

OS) tcells v..~.lli~ O ¥O..lllJIJi~ l./O.~O,~ at. i , various passages were i ,ed and shed with fresh medium by centrifugation and plated into a fl ). Next ¢ the cells were trypsinized, counted and seeded at 5 X × 10 4 c e l l s Lameter cm) in which a coverslip had been placed. After 2~ 24 h incub ubator, the medium was removed and cells were wash washed with I aificant ference was found in the amount of UV-induced UDS UD', in the c 1 2 and flays or later after thawing. The ceils on the coverslip cove we1 to UV liation (30 J / m 2) at the dose rate of 1.5 j / m 2 / s e c . The Th, cells we t in the dium containing [3H]thymidine (10 # C i / m l , 24 Ci/l/mmole) f ~ed by ubation in the medium with non-radioactive thymid Lymidine (5 #l h [11]. e cells were fixed with methanol and then washed with v 5~ t Jc acid C) and water. Dried coverslips were dipped into 2-fold-dilut 2-J auclear ulsion (Sakura) and exposed for a week at 4°C. Th~ The coversli ,eloped ] fixed, then stained with 5% Giemsa solution for 5 To compare UDS in mouse use and human cells, normal human skin fibroblast cells c, H K [10] and xeroderma )derma pig~mentosum (XP) skin fibroblast cells, XP3OS [23], bel, belonging to the complementamentationa group A, were used with the same procedures. The numbers i of grains on lightly ghtly labelled elled nuclei were scored as the amount of UDS. Cells in S-phase that had heavily labelled elled nuclei (HLN) were clearly distinguished and eliminated from the grain rain scoring. JI. I V . a L . , / . , I I b , ' ~ i , K , / 1 K , IIJ ,ItJI.J.IIJUh~lF~

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UV survival :_ . 1" Appropriate numbers of[ cellss w~ w were inoculated into Lux Parmanox Contur dishes. After incubation for 18 h i na a 5%-CO 2 incubator at 37°C, the medium was removed, and the ceils were irradiated .ted with UV radiation through the bottom of the dish which had 45% transmittance nee of the UV radiation. After irradiation, fresh medium11 was added into the dish and aad cells were incubated in a 5%-CO 2 incubator without medium change for 10-12 days until colonies were formed. Colonies consisting of m o r e than 50 cells were fix[ated, stained and scored. A

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Host-cell reactivation (HC~~) of UV-irradiated virus Cell monolayers were pre •epared by inoculating 5 × l0 S cells into a dish (6 cm) and cultured for 3 days with a medium change at the second day. Herpes simplex virus (HSV) type 1 was used in this experiment. 1 ml of virus suspension (5 × 10 4 plaque-forming units per mi 11 in PBS) was irradiated with UV in a plastic dish. After appropriate dilution in PBS BS, 0.5 mi of virus suspension was added to the ceil

sed with PBS, and viruses were p CO 2 incubator. After the adsorp an y-globulin (Midori-Juji) was at zd the number of plaques was scol

~ociated with passage in vitro aouse embryos at gestation day 1 J ~ary phase at P4 or 5 with 4 or 5 PDN (Fig. 1). At P l l or ,arently transformed spontaneously and started proli ~roliferating 1 relation doubling was about 26 h in C 3 H / H e and an C57BI L B / c after the spontaneous transformation. Fhe fraction of cells undergoing semiconservative replicatioz r, thesis; SDS), which had heavily labelled nuclei (HLN), (HI was responding to the loss of proliferation between PO aJ and P10 a ,'r the transformation in all strains (Fig. 2). 9n the contrary, the levels of UDS induced by UV increase~ l reached a plateau, and then decreased in all strains str afte asformation. Representative histograms of UDS are shown ir shifts of UDS levels in each strain.

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C/ ¥ "-induced -ln~ll4C~l UDS in the cells of primary cultures derived derit from mouse embryos at a~ different gestation periods Fable 1 shows UDS induced by UV exposure (30 J / m 2) in cells of primar ~nmary3 Table

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Fig. I. Growth curves o f the cellss derived from embryos of mouse at day 18 of gestation. B A L B / c • el C 3 H / H e II; and C 5 7 B L / 1 0 A. Population doubling numbers were calculated by the method of Ban et al. Ill.

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luled DNA synthesis (SDS) ( O ) and UV-induced UDS ( 0 ) at various Fig. 2. D N A replication or scheduled aore than 500 cells were scored for UDS and SDS respectively at each passages in vitro. 100 cells and more point. Normal human H K ( D ) md XP3OS (11) cells were used at the same time to compare UDS in mouse and human cells. rom culture (P0) derived from mouse embryos at days 14 and 18 of gestation and ffrom newborn mouse comparedrl with normal human H K and XP3OS cells. UDS iinn level., B A L B / c cells was higher than that in the other 2 strains. In all strains, UDS levels were not significantly different erent among the cells from different periods of gestatio~ ;station and newborn mouse.

U V survival

Although the primary pot ~opulation of mouse embryonic cells at P0 had a capacit3 ~acity

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Fig. 3. Histograms of UV-induced UDS at representative passages. A; Arrows and numbers indicate mean values of UDS.

of 4 or 5 doublings (Fig.• 1), a few of the cells in the population appeared tc to proliferate more to make colonies. olonies. But no colonies were formed at P5. The mouse embryonic cells zells at P0 of all strains showed the same sensitivity as normal human H K cells (Fil Fig. 4). After the spontaneous transformation (P19), the U V sensitivity of B A L B / c and C57BL/10 cells remained as resistant as the cells at P0 except for an appearance ~ce of small shoulders. However, C 3 H / H e cells were 2.4 times more sensitive than H?IK, K B A L B / c and C 5 7 B L / 1 0 cells at D O value.

HCR of UV-irradiated H S V The results of H C R assaiys of UV-irradiated HSV are shown in Fig. 5. All mouse embryonic cell strains at 3 different passages had similarly reduced H C R as XP30SS cells which had no ability to perform UDS. At P5 and 19, H C R in mouse cells appeared to be slightly m o rre e efficient than in X P 3 0 S cells, but the differences were

F PRIMARY CULTURES (P0) DERD STATION A N D F R O M NEWBORN (NB

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22.5 ± 1.18 23.7 ± 1.28 24.3 ±1.18 9.49 ± 0.44

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W 1 I .hin JUII experimental error. Although mouse cells had considerably c high ability to perform form UDS after UV exposure, particularly at P5 (Fi~g. 2), the H C R capacity was marke~ rkedly lower than that in normal human H K cells.

Discussion Th~ Fhe results on the change of UDS levels in mouse cells cell agreed with the results of Sasaki aki [19], but were inconsistent with the report by Meek Mee et al. [17]. The reason for i f f e r e n e a was w a ~ not n n t clear ~ l ~,~ar r hbecause ~ c - m ~ tthese h~ ~ n t h ~ r e ,,~,= suchh ddifference authors used B A L B / c cells which were also used in our experiments. Its. The only differences noted were the slightly different procedure for passage and1 the medium for the measurement of UDS. They used arginine-deficient Auto Pow w MEM with 5 m M hydroxyurea. The concentration of hydroxyurea used by them may have been too high and might have interfered with excision repair [8]. As shown in Fig. 5, mouse louse cells at 3 different passages had reduced H C R capacities, as low as that in in XP ceils belonging to the complementation group A, which were presumably defective ffective in excision repair. Host-cell reactivation of UVirradiated virus has been pro r proposed to be a good indication of relative capacity for excision repair [21,22]. However vever, even at passage 5, where UDS levels were 70-100% of normal human cells in all1 3 mouse-cell strains, H C R levels were as low as those of XP cells, suggesting that UD~ DS may not always represent the level of excision repair. Furthermore, neither reduced ed levels of HCR in different passages nor changes of the

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4. UV-survival curves of mouse and h u m a n cells. B A L B / c 0 ; C3 C3H/He • ; OS [3.

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le cells. the UV sen fls of UDS during the passages accurately reflected th of sites prehminary results of the other excision-repair assay assa - - dis~ sion in ~eptible to bacteriophage T4 endonuclease V and accumula bation [A in the medium containing hydroxyurea and ara-C during pc evel of indicated that mouse cells had a very low but si~;nificantl is•on repair at P5 (Yagi, in preparation). i the levels of UDS durin[g IFhere nere may be 3 possibilities to explain the changes m ,me activity, as reported by som~ s u c ccessive e s s i v e passages; changes in thymidine kinase acti changes in the endogenou., ~ n o u s inv,estigators [5,16], changes in repair patch size, and ress in our laboratory on thes~ these midine nucleotide pool. Experiments are in progress thymidine est sibilities, and the results will be presented elsewhere. elsewher Preliminary data sugges possibilities, the pool, might be the nu thatt the third possibility, changes in the thymidine nucleotide

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SV in mouse and h u m a n cells. B A L B / c l ; C 3 H / H e • ; C 5 7 B L / 1 0 •A ; Fig. 5. H C R of UV-irradiated HSV H K O ; XP3OS El.

DS in certain passages of mouse c~ hty of C R levels were related to high Lg.4 in was not so with mouse cells as ! ',eption lad been shown by Takebe et al. [,' ¢ than 19. They were 2.4 times more sen which cells, but were not as sensitive as ,' ruse of ive than normal human cells. Alth aown, it could be due to the c ted to '-nitroalkylating agents such as N-methy human cells, the change of sensit agents loss of tted to SV40 transformation has been reported [4,6] [4,( as beil airing ability of O6-alkylguanine. However, no corn parable 'e ever ~e cells n presented in reference to UV damage. Chromosome numbers re fore, ~19 were 73.7 for B A L B / c , 76.7 for C 3 H / H e and 68.4 68.. for C57 amount of D N A could not have caused the difference differenc in UV a the 3 tins. They were 40 at P0 and the number increased after aft sponta formaL D N A reorganization associated with the mcrease increas of chr Lumber ,,ht change the UV sensitivity. Finally, data by Peleg et al. [18] were most contrad contradictory to • They erved positive U D S in the cells from an embryo at days d 13-1 JDS at rs 17--19 of gestation. One possible cause could be that th the st~ ed was identical with that in this work. Lee and Suzuki [1 [13] showe )ng 14 use strains they used, B A L B / c J was the lowest in the th~ level of U D S after methyl memanesur Lhanesulphonate treatment. Klein stated in his book boo~ [12] that B A L B / c had a different !erent origin from other inbred mouse strains. This m maa y suggest that B A L B / c is a rather aer difficult strain to work with. But essentially no difference difl among the 3 strains, whiqkch were representatives of the most widely used strain strail~ in cancer studies in Japan, were •e found in this study. Mouse cells were definitely different dit from human cells in D N A repair characteristics. -

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Acknowledgements I am grateful to Dr. Hirakl aku Takebe for his advice and encouragement throughout the work. I thank Drs. Masao Iasao S. Sasaki, Osamu Nikaido and Kanji Ishizaki for advice and discussion, and Dr. Junzo Yamada for advice and information on mouse breeding. This work was su supported by Grants-in-Aid from the Ministry of Education, Science and Culturee and by the Princess Takamatsu Cancer Research Fund.

References 1 Ban, S., O. Nikaido and T. Sulgahara, Acute and late effects of a single exposure of ionizing radiation radiation on cultured human diploid cell ~ll populations. Radiat. Res., 81 (1980) 120-130. 2 Ben-Ishai, R., and L. Peleg, Excision-re xcision-repair in primary culture of mouse embryo cells and its decline m i~ progressive passage and established cel lines, in: P.C. Hanawalt and R.B. Setlow (Eds.), Moleculai ~lishedt cell lolecular Mechanisms for Repair of DNA hlA, Part B, Plenum, New York, 1975, pp. 607-610.

roderma pigmentosum, Biochemical and

16 17

18

19 20 21

22

23

24 25

teristics,

D.A. Scudiero, S.A. Meyer, A.S. Lubinie~ di, S.M. ~0-transive repair of alkylated DNA by human tr London), 288 (1980) 724-727. ase and thymine deoxyribonucleotide pho, g growth em., 240 (1965) 2607-2611. arkey and K.W. Korn, DNA cross-linking ct repair cells, Nature (London), 288 (1980) 727-7 eplication after UV-irradiation in rodent different 1973) 359-376. 3arrier. D.P. Smith and J.D. Regan, Inhibi repair in 92. ltraviolet-irradiated human cells by hydroxyurea, Biochim. Bioph 3~s. Acta, 56 mouse L :ujiwara, Y., and T. Kondo, Caffeine-sensitive repair dr of ultraviolet ultraviole~ light-dam~ ells, Biochem. Biophys. Res. Commun., 47 (1972) 557-564. I. High ,tease inhibitors o shizaki, K., T. Yagi and H. Takebe, Cytotoxic effects of protease J ensitivity of xeroderma pigrnentosum cells to antipain, Cancer Lett., Let 10 (198(] repair ¢ndrome shizaki, K., T. Yagi, M. lnoue, O. Nikaido and H. Takebe, DNA D H3-219. R, ibroblasts after UV irradiation or treatment with mitomycin C, Mutation lX ~pringer, ;.lein, J., The mouse and its forms, in: Biology of the Mouse Histocompatibilit Histoc ~ew York, 1975, pp. 16-39. ts mouse ,ee, I.P., and K. Suzuki, Differential DNA-repair activity m in prespermiogenic presp trains, Mutation Res., 80 (1981) 201-211. lanawalt .ehmann, A.R., Postreplication repair of DNA in UV-irradiated mammalian m rk, 1975, nd R.B. Setlow (Eds.), Molecular Mechanisms for Repair of DNA,, Part B, Pk p. 617-623. [on), 289 m chr, chromosome 44, Nature (London), .in, P., and F.H. Ruddle, Murine DNA repair gene located on 1981) 191-194. Biochim. Biophys. Acta, Attlefield, J.W., Studies on thymidine kinase in cultured mouse fibroblasts, fil Littlefield 95 (1965) 727-729. deek, R.L., T. Rebeiro and C.W. Daniel, Patterns of unscheduled DNA synthesis in mouse embryo Meek formation to a continuous cell line, Exp. ells associated with in vitro aging and with spontaneous transforma cells Cell Res., 129 (1980) 265-271. repair in mouse embryonic 'eleg, L., E. Raz and R. Ben-Ishai, Changing capacity for DNA excision eJ Pel¢ ells in vitro, Exp. Cell Res., 104 (1976) 301-307. cells Changes during serial in vitro ',asaki, M.S., Repair of damage to DNA and chromosome mutation, mutati Sasaki. (1975) 492 (Abstract, in Japanese). ransfer of embryo cells of mouse and man, Jpn. J. Genet., 50 (197 transfer ;etlow. R.B., R.B.. Reoair deficient human disorders and cancer,', Nature (London), 271 (1978) 713-717. Setlow, ;~ ntosum: DNA repair defects and skin cancer, in: T. Sugimura, H. Endo, Takebe, H., Xeroderma pigmentosum: T. Ono and H. Sugano (Eds.), Progress in Cancer Biochemistry, Jpn. Sci. Soc. Press, Tokyo, 1979, pp. 103-117. lyTakebe, H., S. Nil, M.I. Ishii and H. Utsumi, Comparative studies of host-cell reactivation, colony'epair after UV irradiation of xeroderma pigmentosum, normal human forming ability and excision re ells, Mutation Res., 25 (1974) 383-390. and some other mammalian cells tka, J. Furuyama, K. Tanaka, M.S. Sasaki, Y. Fujiwara and H. Akiba, Takebe, H., Y. Miki, T. Kozuka ad skin cancers of xeroderma pigmentosum patients in Japan, Cancer DNA repair characteristics and Res., 37 (1977) 490-495. ,#ng, -repair capability, in: E.L. Schneider (Ed.), The Genetics of Aging, Tice, R.R., Aging and DNA-re Plenum, New York, 1978, pp. 53-89. he role of mutagenesis in carcinogenesis, Photochem. Photobiol. Rev., 3 Trosko, J.E., and C. Chang, The (1978) 135-162.