Human cell clones, RSa and UVr-1, differing in their capability for UV-induced virus reactivation and phenotypic mutation

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER Mutation Research 306 (1994) 211-222

Human cell clones, RSa and u v r - 1 , differing in their capability for UV-induced virus reactivation and phenotypic mutation

Nobuo Suzuki a,,, Hiroshi Kimoto b, Haruhiko Koseki c, Nobuyuki Miura d, Takashi Watanabe e, Noriyuki Inaba f, Hiroyoshi Takamizawa f, So Hashizume g

Department of a Biochemistry, c Center for Neurobiology and Molecular Immunology, a Pediatrics and f Obstetrics and Gynecology, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba-shi, Chiba 260, Japan, b Department of Cellular and Molecular

Immunology, National Institute of Health, Ohsaki 2-10-35, Shinagawa-ku, Tokyo 141, Japan, e Department of Chemotherapy, Tokyo Metropolitan Komagome Hospital, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan,

g Japan Poliomyelitis Research Institute, Kumegawa 34-4-5, Higashimurayama-shi, Tokyo 189, Japan

(Received 18 August 1993; revision received 23 November 1993; accepted 24 November 1993)

Abstract

u v r - 1 is a human cell clone established as a variant with increased resistance to cell killing by ultraviolet light (UV, principally 254 nm wavelength) from a UV-sensitive cell clone, RSa. Both cells have been characterized to have much the same capacity of UV-induced DNA repair synthesis in whole cells, and the parent RSa cells were recently found to be hypermutable. In the present study uv r -1 cells were characterized in comparison with RSa cells with respect to UV-induced virus reactivation and phenotypic mutation. Survival levels of UV-irradiated vaccinia virus and herpes simplex virus type 1 (HSV-1) were much the same in logarithmically proliferating UVr-1 and RSa cells. Correlated with these host cell reactivation levels, the same extent of UV-induced DNA repair replication synthesis was observed in isolated nuclei of the two cell clones. Enhancement of survival levels of UV-irradiated HSV-1 was detected when proliferating RSa cells were irradiated with UV prior to the virus infection. In contrast, this enhanced virus reactivation (EVR) was not detected in similarly irradiated and infected UVr-1 cells. As for phenotypic mutation frequencies assessed by the cloning efficiency of ceils with increased resistance to ouabain cell killing (OuaR), Oua R mutants were not obtained from UV~-I cells either with or without UV irradiation. When the proliferation of cells was synchronized, both EVR and Oua R mutations were detected in RSa cells irradiated with UV at any cell cycle phase, being greatest in the later half of the G1 phase. However, there was no detectable EVR or mutation in any phase of synchronous UV~-I cells. The hypomutability of UV'- I cells and hypermutability of RSa cells in a G1 cell cycle phase was also found even if 4-nitroquinoline 1-oxide was used as a mutagen or mutant cells with increased resistance to 6-thioguanine cell killing were estimated.

Key words: Human cell clones; UV; Virus reactivation; Phenotypic mutation; G1 cell cycle phase

* Corresponding author. Tel. (+ 81) 43-222-7171 (Ext. 2091); Fax: ( + 81) 43-222-7853.

Abbreviations: EVR, enhanced virus reactivation; 4NQO, 4- nitroquinoline 1-oxide; HCR, host cell reactivation; HuIFN, human interferon; HSV-1, herpes simplex virus type 1; Oua R, resistance to ouabain cell killing; 6-TG a, resistance to 6- thioguanine cell killing; UV, ultraviolet light.

0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0027-5107(93)E0217-E

212 N. Suzuki et al. / Mutation Research 306 (1994) 211-222

1. Introduction

Far-ultraviolet light (UV, principally 254 nm) is an intriguing tool for investigating biological functions of human cells. In fact, various cellular responses such as DNA repair and phenotypic mutation are detectably induced in UV-irradiated cells. However, the molecular mechanism under- lying the responses has not been unequivocally clarified (Ronai et al., 1990; Lehmann et al., 1992). In particular, understanding of the process inducing mutational events is poor, and has de- pended largely on the molecular mechanism operating in Escherichia coli (Hanawalt, 1989), cate- gorized as a so-called SOS response (Radman, 1975; Witkin, 1976).

The SOS theory developed as a result of re- cent advances in genetic engineering and protein chemistry, but is based on the establishment and characterization of isogenous cell strains which have distinct UV susceptibility. Ingenious usage of infectious viruses such as a A bacterial phage also contributed to the construction of the theory (Radman, 1975; Witkin, 1976; Sobels, 1986; Ha- nawalt, 1989). Isogenous human cell clones with different sensitivities to UV effects are thus de- sirable to define the mechanisms which govern the UV response. Only a few revertant cells with increased resistance to UV cell killing were reported from UV-sensitive xeroderma pigmentosum (Royer-Pokora and Haseltine, 1984; Leh- mann, 1985; Schultz et al., 1985; Cleaver et al., 1987). However, in order to investigate the cellular functions responding to UV mutagenicity pairs of hypermutable and hypomutable human cell clones are required. Up to now there have been no such cell clones available.

In 1979 we succeeded in establishing cells with increased resistance to UV cell killing from human RS cell clones which are unusually highly sensitive to this killing (Suzuki and Kuwata, 1979). Among the cells established the following two cell clones were intriguing: RSa, which was established as a fibroblastic cell clone from human embryo-derived cells by double infection with simian virus 40 and Rous sarcoma virus and later characterized as sensitive to UV cell killing (Suzuki and Fuse, 1981); and UVr-1, established

as a variant with increased resistance to UV cell killing from RSa cells mutagenized with ethyl methanesulfonate and then irradiated with UV (Suzuki, 1984).

Interestingly, the frequency of phenotypic mutation in RSa cells, when estimated by the cloning efficiency of cells with increased resistance to ouabain cell killing (Ouar~), was found to be induced to an unusually high extent (more than one Oua R mutant per 103 cell survivors) after either UV irradiation (Suzuki et al., 1985) or treatment with various chemical mutagens (Suzuki et al., 1985; Suzuki and Suzuki, 1988b). UVr-1 cells unexpectedly showed much the same capacity of so-called excision repair after UV as did RSa cells (Suzuki, 1984). The interrelationship between UV-induced DNA repair and phenotypic mutation has been speculated to arise as follows: low excision repair capacity induces error-prone repair, thereby resulting in phenotypic mutation (Friedberg, 1985; Suzuki et al., 1985; Hanawalt and Sarasin, 1986; Tatsumi et al., 1987). On the contrary, an increase of excision-repair capacity seems to lead to the reduction of phenotypic mutation frequency (Cleaver et al., 1987; Suzuki et al., 1988; Suzuki and Suzuki, 1988a). If so, it is intriguing to learn whether or not u v r - 1 cells also remain hypermutable.

In the present study we first compared DNA virus reactivation activities between UV-irradiated UVr-1 and RSa cells, as the reactivation is considered to be a sensitive parameter reflecting the ability of host cells for DNA repair a n d / o r its related functions (Day, 1981). We next examined the mutability of u v r - 1 cells in comparison with the parent RSa cells. As UV mutagenicity is suppressed in cells pretreated with human interferon-a (HulFN-a) prior to irradiation with UV (Suzuki and Suzuki, 1988a), it was further dis- cussed whether phenotypes of u v r - 1 are affected by HulFN-c~.

2. Materials and methods

2.1. Agents

Natural HulFN-a (5.0 × 108 I U / m g protein), produced by treating Namalwa cells with Sendai

N. Suzuki et al. / Mutation Research 306 (1994) 211-222 213

virus (Suzuki et al., 1984), was provided by Dr. S. Yonehara (The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Anti-HulFN-a polyclonal antibodies (1.0 x 106 neutralizing uni ts /mg protein) were described elsewhere (Sugita et al., 1992). Eagle's minimal essential medium (EMEM) and fetal bovine serum were both supplied by Gibco (Grand Island, NY), and antibiotics (Meijiseika, Tokyo, Japan) were purchased from Sanko-junyaku (Osaka, Japan). [methyl-3H]Thymidine ([3H]dThd) (20 /zCi/ mmole; New England Nuclear, Boston, MA), [32p]phosphate (Japan Atomic Energy Research), and [methyl-3H]thymidine 5'-triphosphate ([3n]- dTI'P) (19 Ci/mmole; New England Nuclear) were purchased from Japan Radioisotope Associ- ation (Tokyo, Japan). Other agents were purchased from Nacalai Tesque (Kyoto, Japan) unless specifically noted.

2.2. Cells and culture conditions

High UV sensitivity and hypermutability of RSa cells was described elsewhere (Suzuki and Fuse, 1981; Suzuki et al., 1985; Suzuki and Suzuki, 1988a,b). Establishment and partial characterization of UV resistance of uvr -1 cells were described in previous papers (Suzuki, 1984; Suzuki et al., 1986). Normal UV sensitivity of YO human fibroblast cells was also described elsewhere (Sugita et al., 1987), as were HeLa cells (Suzuki and Fuse, 1981).

The medium for cell culture was EMEM containing 10% (v/v) fetal bovine serum and antibiotics (100/xg streptomycin/100 ml and 100 units penicillin G/ml) . Cells were cultured with the medium in plastic dishes (Iwaki Glass, Tokyo, Japan) at 37°C in a humidified atmosphere containing 5% CO 2.

All of the cell clones used here were free of mycoplasma infection, as determined by MY- COTRIM-TC (Hana Biologics Inc., Berkeley, CA).

2.3. U V irradiation conditions

A germicidal lamp (principally 254 nm wavelength) described elsewhere (Suzuki and Kuwata,

1979) was used to irradiate cells and viruses. Conditions for the irradiation of cultured cells with UV were as described (Suzuki and Fuse, 1981). For irradiation of virus, stocked viruses were diluted 102-fold with cold phosphate- buffered saline. An aliquot of virus suspension no greater than 5 ml was irradiated in an uncovered 100 mm diameter plastic dish with constant agita- tion during the irradiation. Other irradiation techniques and the dosimetry were as described (Suzuki and Fuse, 1981).

2.4. Survival test for UV-irradiated viruses

Assays of virus reactivation, so-called host cell reactivation (HCR) and enhanced virus reactivation (EVR), were done using DNA viruses. The strain of vaccinia virus was described elsewhere (Hashizume et al., 1985). The macro-plaque strain of HSV-1 (Takebe et al., 1974) was provided by Dr. O. Nikaido (Kanazawa University, Kanazawa, Japan). The plaque-purified vaccinia virus and HSV-1 were grown in YO human fibroblast ceils with usual HCR capacity (Kojima et al., 1985), and stocks of each virus (2 x 107 plaque forming units/ml) were obtained as the supernatant from the virus-infected YO ceils 2 days after the infection.

Virus plaque assays were performed on freshly confluent monolayers of cells, basically according to the method described by Kojima et al. (1985). Briefly, the assay of HCR using UV-irradiated vaccinia virus was done by the agar overlay proce- dure (Lytle et al., 1972), while that using irradiated HSV-1 was done by the method described by Takebe et al. (1974), using human y-globulin (Midori Juji, Osaka, Japan). For the assay of EVR, monolayered cells were irradiated with UV and then inoculated with appropriate dilutions of irradiated HSV-1. Three days after the virus inoc- ulation cells were stained with 0.01% neutral red or hematoxylin, and plaques were scored.

The ratio of the titer of UV-irradiated viruses to that of unirradiated viruses on similarly treated ceils gave the virus surviving fraction, HCR value. The D37 value, which is the UV dosage required to reduce virus survival to 37%, was calculated from the virus survival curve. The EVR value was

214 N. Suzuki et al. /Muta t ion Research 306 (1994) 211-222

determined by the ratio of the surviving fraction of irradiated HSV-1 on UV-irradiated cells to that on similarly treated but unirradiated cells. Values were the mean of three independent experiments in which more than five replicate dishes were used at each UV dose tested.

Neither of the two following examinations re- vealed the presence of endogenous HSV-1 in the cells used: assay of HSV-1 antigens in culture medium using fluorescein isothiocynate-labeled monoclonal antibodies against HSV-1 (Gleaves et al., 1985), or assay of DNA homogenous to HSV-1 genomes by the DNA hybridization method after amplification of DNA from cells with the polymerase chain reaction (Cao et al., 1989).

2.5. Repair replication synthesis in isolated nuclei

al., 1985; Suzuki and Suzuki, 1988a). Briefly, at the indicated expression time after UV irradiation or treatment with 4-nitroquinoline 1-oxide (4NQO) for 1 h as described (Suzuki, 1987), cells were replated at a density of 5 x 10 4 cells/100- mm dish and supplemented with medium containing 0.1 /xM ouabain or 1 /zM 6TG. Calcula- tion of mutation frequency was done by dividing the total number of the agent-resistant mutant colonies by the total number of cells plated, and was corrected by the cloning efficiency of the cells during mutant selection. Frequencies are expressed as mutants per 10 4 cells. Values are the mean of those obtained from two independent experiments in which more than 10 replicate dishes were used to count the number of mutants and calculate cloning efficiency.

Cellular DNA was prelabeled by culture of cells with [32p]phosphate, followed by UV irradiation or mock-irradiation of the cells as described (Suzuki and Fuse, 1981). Immediately after the irradiation dishes containing the cells were placed on ice, cell nuclei were isolated using a modifica- tion of the method originally developed by Her- shey et al., as described in our previous report (Suzuki et al., 1984).

The isolated nuclei (about 2 X 106 nuclei/ assay) were incubated with [3H]dTTP (100 /~C/ ml) at 37°C, according to the method developed by Smith and Hanawalt (1978). The incubated nuclei were lysed, and then unreplicated parental DNA was isolated in neutral CsC1 and analyzed in alkaline CsC1 gradients, as described elsewhere (Suzuki and Fuse, 1981). Values of repair activity were expressed in the unreplicated DNA as the ratios of the total amount of tritium radioactivity to the total amount of 32p radioactivity or 260 nm absorbance (an A260 of 1.0 corresponds to 50 ~g DNA/ml ) as described (Suzuki and Fuse, 1981). The repair activity increased linearly during the 3-h incubation of the nuclei.

2. 6. Analyses of phenotypic mutation

Frequencies of Oua R and resistance to 6- thioguanine cell killing (6TG R) mutations were determined principally as described (Suzuki et

2. 7. Assay of anti-viral actiuity

The anti-viral activity of the culture medium was measured by the virus yield reduction test described elsewhere (Suzuki et al., 1982).

2.8. Detection of HuIFN antigens

Culture medium was collected from dishes containing cells irradiated with UV and mock- irradiated, and clarified by centrifugation at 1000 x g for 5 min, followed by freezing at -70°C until shortly before assay. The medium was analyzed for the level of HuIFN-a by monoclonal-antibody-based immunoassays, as described elsewhere (Suzuki et al., 1992). The minimum detectable dose was 2 IU/ml .

2.9. Analyses of mutability and EVR in synchronized cells

Synchronization of cells at the G1 /S boundary phase was carried out by two cycles of thymidine block, basically according to the method described (Suzuki, 1984). Briefly, the first block of logarithmically growing cells with 2.6 mM thymidine for 24 h was followed by culture of the cells in thymidine-free medium for 14 h. The second block was then performed with 2.6 mM thymidine for 14 h. After being released from the second


thymidine block, the cells progressed through S and into the following phases.

Viable ceils were determined by the trypan blue exclusion test and counted with a hemocy- tometer. The rate of DNA synthesis was moni- tored by pulse-labeling of cellular DNA with medium containing 1/xCi/ml of [3H]dThd for 10 min and subsequently measuring the incorporation of radioactivity into 10% (w/v) trichloroace- tic acid-insoluble materials. For analyses of phenotypic mutation synchronized cells were irradiated or treated with 4NQO and then incubated during the expression time (2 days for the Oua R test and 8 days for the 6TG R test), followed by exposure to ouabain or to 6TG as described above. The EVR assay was done by infection of 50 J / m 2 UV-irradiated HSV-1 24 h after UV (2 J / m 2) irradiation to synchronized cells.

lO -t

0 IIL

Q

O" g [ Q

~ 10 -~ 0

10-~ ! !

o 50

1.0

B

i i i i

1110 0 50

UV Influence on Virus (Jim 2)

! !

108

Fig. 1. Survival curves for UV-irradiated vaccinia virus (A) and HSV-1 (B). 13, HeLa cells; II, u v r - 1 cells; o , RSa cells. The data are the mean d:SE of three independent experi- men ts.

2.10. Other conditions

Experiments were done under a yellow lamp (National, FL20S-Y-F). Results are expressed as the mean of values obtained from two independent experiments unless otherwise noted.

3. Results

3.1. HCR test

Seeking a clue to the difference in UV-affected cellular functions between UVr-1 and RSa cells, estimates of survival were made for UV- irradiated DNA viruses. For vaccinia virus, the survival curves for the two cell clones were similar at all UV doses tested, and also much the same as the curve for HeLa cells examined comparatively (Fig. 1A). Survival levels of HSV-1 on HeLa cells, however, were higher than those on uvr-1 and RSa cells, although the survival curves for the latter two cell clones were nearly the same (Fig. 1B). The D37 values on HeLa cells were calculated as about 75 J / m 2 and 50 J / m 2 from the first and second components of the survival curve, respectively, each of which was more than threefold larger than the corresponding values on UVr-1 and RSa cells, 25 J / m 2 and 9 J / m 2 (Fig. 1B).

3.2. DNA repair replication in cell nuclei

In addition to HCR DNA repair replication levels in nuclei of uvr-1 and RSa cells were estimated in comparison with those in HeLa cell nuclei.

In nuclei from mock-irradiated cells of the three clones there was no discrete peak of [3H]dTTP incorporation at the parental DNA density region detected by either 32p radioactivity or absorbance at 260 nm; but peaks were detected in all of the nuclei from cells irradiated with UV (Fig. 2). The specific activities of UV-induced [3H]dTTP incorporation into DNA in HeLa cell nuclei (3H/32P radioactivity ratios) were calculated to be 2.4 at 16 J / m 2 and 2.3 at 32 J / m 2 (Fig. 2A), more than twice as high as

2.5

5 10 15

2.0

# o 1.5

L

1.0

8

0.5

0


B

5 10 15

Fraction Number

c

× g

~ 1 0 . 4 o e

(1,1 ~ 5 10 15

Fig. 2. Repair replication analyzed by alkaline CsCI gradients of the parental density DNA from nuclei of HeLa (A), uwr-1 (B) and RSa (C) cells. Centrifugations are from right to left. ©, ~ ; and e, [3H]dTTP incorporation after mock-irradiation, irradiation with 16 J / m 2 UV and irradiation with 32 J / m 2, respectively; t3, 32p radioactivity in mock-irradiated cells; A, absorbance in mock-irradiated cells.

those in u v r - 1 cell nuclei with values of 1.2 and 0.91 (Fig. 2B), respectively. In addition, values of 3H cpm per /zg DNA in the parental region of HeLa cells were calculated to be 44 cpm//xg DNA at 16 J / m 2 and 42 cpm/ /zg DNA at 32 J / m 2 (Fig. 2A), each of which was also larger than that of u v r - 1 cells, 22 cpm//~g and 16 cpm//~g (Fig. 2B), respectively.

Levels of the repair activities in RSa cell nuclei, however, were almost the same as those in UVr-1 cell nuclei, because the comparative 3 H / 32p and 3H//xg DNA ratios in the former were 1.2 and 23 at 16 J / m 2 and 0.96 and 18 at 32 J / m 2 (Fig. 2C).

3.3. E V R test

Instead of HCR, HSV-1 EVR levels were compared between u v r - 1 and RSa cells. In EVR- positive cells survival levels of UV-irradiated HSV-1 are expected to increase when the cells are lightly UV-irradiated prior to HSV-1 infection. More than twice the number of 50 J / m 2 UV-irradiated HSV-1 survived on 2 J / m 2 UV- irradiated RSa cells than on non-irradiated RSa cells (Fig. 3A, B). The enhanced HSV-1 survival was detected by infection at all times following

irradiation to the host RSa cells, and a maximal fourfold extent was obtained at 24 h (Fig. 3A). Among various UV doses to RSa cells 2 J / m e UV induced the greatest increase in HSV-1 survival at any dose (10-100 J / m e) of UV to the virus (Fig. 3B).

g

,,o

g

>~

24 48

Time after UV Application to Cells (h)

C

i i i i I i

2 4 6 2 4 6

OV Inf luence on Cells (d /m ~)

Fig. 3. Examination of HSV-1 EVR on RSa and u v r - I cells. Infection of HSV-1 irradiated with 50 J / m 2 at various times after UV (2 J / m 2) irradiation to cells (A), that of HSV-1 irradiated with various UV doses 24 h after UV irradiation to RSa cells (B) and to wvr-1 cells (C), was done as described in Materials and methods. (A) e, RSa cells; II, UVr-1 cells. (B and C) Doses to HSV-1 were 10 J / m 2 (0), 50 J / m 2 (e) and 100 J / m 2 (zx). The data are the m e a n + S E of three independent experiments.


6O

.£

b -g *~ 30

g

I . . _ - - - . .

T I I I I l

1 2 3 4 0

Expression Time (days)

B

. . . . , . .

! I I 2 • 4 8 1

UV (J / m 2)

Fig. 4. UV mutagenicity in exponentially proliferating UVr-I RSa cells. (A) Expression time for the induction of Oua R mutation after 12 J / m 2 UV irradiation. (B) UV mutagenicity after 2 days of expression time. I , uvr -1 cells; o , RSa cells. The data are the mean±SE of three independent experiments.

However, after irradiation of UVr-1 cells with 2 J / m 2 UV there was no increase at any time, and rather a slight decrease in survival levels of 50 J / m 2 UV-irradiated HSV-1, in comparison

with survival levels of non-irradiated cells (Fig. 3A). No UV dose (up to 6 J / m 2) to UV~-I ceils nor any dose (up to 100 J / m 2) to HSV-1 enhanced HSV-1 survival (Fig. 3C).

3.4. UV mutagenicity test

We next examined the susceptibility of UW-1 cells to UV mutagenicity in comparison with that of RSa cells. Frequencies of Oua R mutation in UV-irradiated RSa cells increased steadily with time up to 2 days (Fig. 4A) and with UV dose to 12 J / m 2 (Fig. 4B), while there was no appear- ance of the mutation fraction in uvr-1 cells at any expression time (Fig. 4A) or UV dose (Fig. 4B).

3.5. Estimation of HulFN-a antigens in culture medium

To examine the probability that the refractori- ness of UVr-1 cells to UV mutagenicity was due to extracellular HuIFN molecules produced in

X

I 32

z

--t

A

- -- \

' 'o ' ' g 0 ' 1 20 30 50

B-3

B - 4

i | i | i

10 20 30 40 50

Time alter Block Reversal (h)

Fig. 5. UV-induced Oua R mutation and HSV-1 EVR in synchronized RSa (A) and uvr -1 (B) cells. At indicated times after release from thymidine block cells were subjected to each assay as described in Materials and methods. The UV dose for UV mutagenicity testing was 8 J / m 2 and those for EVR were 2 J / m 2 to cells and 50 J / m 2 to HSV-1. (A-1 and B-l), Rate of DNA synthesis; (A-2 and B-2), number of cells proliferated; (A-3 and B-3), Oua R mutation frequencies; (A-4 and B-4), EVR levels.

218 N. SuzuM et al. / Mutation Research 306 (1994) 211-222

Table 1

Es t ima t ion of ant i -vi ra l act ivi ty and a m o u n t of H u l F N - a ant i -

gen in cu l tu re m e d i u m a

Cell I r r ad i a t i on H u l F N - a Ant i -v i ra l H u l F N clone wi th U V b a d d e d c activi ty an t igen

( I U / m l ) ( I U / m l )

RSa - - 0 0

- + 100 99 + - 0 0

+ + 100 100

u v r - 1 - - 0 0

- + 100 100

+ - 0 0 + + 100 99

a Cu l t u r e m e d i u m was t aken f rom dishes con ta in ing cel ls 24 h af ter U V (8 J / m 2) i r r ad ia t ion or mock- i r rad ia t ion , and then

H u I F N - a was or was not a d d e d to each cu l tu re m e d i u m in a

f inal concen t r a t ion of 10 2 I U / m l . Ant i -v i ra l act ivi t ies and

levels of H u I F N - a an t igens were e s t i m a t e d as desc r ibed in

Ma te r i a l s and methods . Va lues a re m e a n of two de t e rmina -

tions. b --, mock- i r rad ia t ion ; + , UV- i r rad ia t ion .

c + , addi t ion ; - , non-addi t ion .

the cell and released into the culture medium, anti-viral activities and HulFN-a antigen concen- trations were estimated in the medium obtained from culture dishes containing cells with and without UV irradiation. However, there were no detectable levels of anti-viral activity or HulFN-a antigens, as shown in Table 1. As amounts of HulFN-a added to medium samples were recov- ered (Table 1), there appeared no substances inhibiting the detection of HulFN molecules.

On the other hand, even if UVr-1 cells were cultured with the medium containing antibodies against HulFN-a , their susceptibility to UV mutagenicity was similar to that without the antibody treatment (data not shown).

3.6. UV mutagenicity and EVR in synchronized cells

It was further of interest to investigate whether UV-induced Oua l~ mutation and HSV-1 EVR were detected after synchronization of the proliferation of u v r - 1 cells.

As shown in Fig. 5, tritiated thymidine uptake levels (Figs. 5A-I and 5B-l) and cell numbers

(Figs. 5A-2 and 5B-2) were clearly differentiated among cell cycle fractions. The increase of [3H]thymidine into DNA of RSa and u v r - 1 cells increased after release from the thymidine block as the cells progressed through the S phase, reaching a maximum value at 6 h. As the cells proceeded to the G2, M and G1 phases, the cell number per culture at 14 h was twice the initial value (Figs. 5A-2 and 5B-2). The ceils then en- tered the next cycle. After release from the thymidine block cells at 0 -9 h and 24-33 h, 9-10 h and 33-34 h, 10-14 h and 34-38 h, and 14-24 h and 38-48 h appeared to be in the S, G2, M and G1 phase of the cell cycle, respectively.

In RSa cells both Oua R mutation (Fig. 5A-3) and HSV-1 EVR (Fig. 5A-4) occurred after irradiation with 8 J / m 2 UV and with 2 J / m 2 UV, respectively, at any cell cycle phase. In the latter half of the G1 phase under both the first and second cell cycles the highest peaks of the mutation and EVR were identified (Figs. 5A-3 and 5A-4). In UV~-I cells, in contrast, neither Oua R mutation (Fig. 5B-3) nor EVR (Fig. 5B-4) was observed at any phase during the two cell cycle rotations examined.

There was no great difference in cloning efficiency between synchronous RSa and u v r - 1 cells at any cell cycle phase, being 5 -10% and 8-14%, respectively. When repair replication activities were estimated in nuclei of RSa and UV~-I cells irradiated with 16 J / m 2 UV at the late G1 phase (20 h after release from thymidine block), the two cells had much the same activity, 20 cpm//zg DNA.

3.7. 6TG n mutation and 4-NQO mutagenicity at a late G1 cell cycle phase

In order to confirm the discrepancy of mutability between UVr-1 and RSa cells when mutagenized at a late G1 cell cycle phase, 6TG was used instead of ouabain as an agent for selection of phenotypic mutation. In UVr-1 cells 6TG R mutants were not identified, but in RSa cells they were identified, although less frequently than Oua R mutants (Fig. 6A). On the other hand, instead of UV another mutagen, 4NQO, was ap- plied for the Oua R mutation test at the late G1


A B

10 30

2

_ _ _

I I L ~ b I I I I 4 8 12 0 9 8 7 6

UV (J / m 2) 4 N Q O ( - log M )

Fig. 6. UV mutagenicity for 6TG R (A) and 4NQO mutagenicity for Oua R (B) in synchronous uvr-1 and RSa cells at the late G1 cell cycle phase. 20 h after release from thymidine block synchronous uvr-1 and RSa cells were irradiated with UV (A) or treated with 4NQO (B), then subjected to 6TG R (A) and Oua R (B) selection as described in Materials and methods. II, uvr-1 cells; o, RSa cells.

cell cycle phase. Again, Oua R mutants were not identified in 4NQO-treated UVr-1 cells (Fig. 6B). In contrast, the mutants were identified in 4NQO-treated RSa cells at high frequency (Fig. 6B).

4. Discussion

A human cell clone, UVr-1, showed much the same levels of HCR and repair replication synthesis in nuclei as did the parent RSa cell clone (Figs. 1 and 2). However, uvR-1 cells had no EVR response (Fig. 3) and displayed hypo-

Table 2 Comparative characteristics of UV response between UVr-1 and RSa cells

Assay Characteristics of cell clones

UVr-1 RSa

HCR of vaccinia virus high high HCR of HSV-1 low low DNA repair in cell nuclei low low EVR of HSV-1 undetectable detectable Frequency of

Oua R mutation undetectable high Frequency of

6TG R mutation at a late G1 phase undetectable high

mutability (Fig. 4) after UV irradiation, in contrast to the definite EVR and hypermutability in RSa cells. The similarities and differences of UV response are comparatively summarized in Table 2.

HCR is considered to be the result of the ability of host cells to reactivate UV-irradiated DNA viruses. The ability is considered to be governed by the excision repair capacity of the host cells (Intine and Rainbow, 1990). HSV-1 replicates in cell nuclei (Spear and Roizman, 1981; Wagner, 1985), although an episomal form of the HSV-1 genome was suggested within latently infected nerve cells (Rock and Fraser, 1983). By contrast, vaccinia virus replicates in cytoplasma (Traktman, 1990), although it has been suggested that a selective few host nuclear pro- teins are recruited (Morrison and Moyer, 1986; Bloom et al., 1989). Thus, increased HCR levels in HSV-1 but no increase in vaccinia virus on HeLa cells (Fig. 1) imply a high capacity of DNA repair in the cell nuclei. In fact, HeLa cells had more increased levels of DNA repair replication synthesis in cell nuclei than did uvr -1 and RSa cells (Fig. 2). The similarity of the repair replication capacity in nuclei of the latter two cells was in agreement with previous findings that they had much the same DNA repair capacity in cellular DNA prepared from whole cells (Suzuki, 1984).

Nevertheless, HSV-1 EVR was detected in exponentially proliferating RSa cells but not in the proliferating uwr-1 cells (Fig. 3). EVR is the enhanced survival of UV-inactivated DNA viruses in host cells that have been irradiated prior to infection of the viruses. The EVR is thought to be analogous to Weigle reactivation in bacteria (Radman, 1975; Witkin, 1976; Sobels, 1986; Hanawalt, 1989), although the analogy has been disputed (Rossman and Klein, 1985). An increase in viral survival is thought to be due to inducible cellular DNA repair and /o r replication activities operating on the genome of an infecting virus. The inducible activities have been suggested to be error-prone (DasGupta and Summers, 1978; Abrahams et al., 1984, 1988; Shwartz et al., 1988). In agreement with this suggestion EVR was asso- ciated with a high frequency of Oua R mutation in RSa cells, while neither EVR nor highly frequent


mutation occurred in u v r - I cells (Figs. 3 and 4). In the culture media from RSa and uvr -1

cells either with or without UV irradiation there was no detectable anti-viral activity or HuIFN-a antigens (Table l). Thus, neither HCR nor EVR seems to be modulated by extracellular HuIFN molecules. Anti-HuIFN-o~ antibodies added to the culture medium did not affect the mutability of UVr-1 cells. Therefore, it seems unlikely that the hypomutability of UVr-1 cells is due to suppres- sive activities of extracellular HuIFN-a molecules against UV mutagenicity.

The association between EVR and Oua R mutation was distinctively observed at a late G1 phase of synchronously proliferated RSa cells (Fig. 5). By contrast, even if UV'- I cells were synchronously proliferated EVR and Oua ~ mutations were not detected (Fig. 5). The hypermutability of RSa cells and hypomutability of uv r -1 cells at a late G1 phase were also found in the analysis of UV-induced 6TGR mutation (Fig. 6A). In RSa cells the mutation frequency to Oua R was greater than that to 6TGR. However, Fortini et al. (1993) reported that the former frequency was smaller than the latter in Chinese hamster ovary cells treated with alkylating agents. Thus, it seems likely that the frequency of the respective phenotypic mutation varies depending on the cells and mutagens tested.

Furthermore, synchronous RSa cells showed high sensitivity to 4NQO mutagenicity at the G1 phase, but u g r - 1 cells did not show any sensitivity (Fig. 6B). It was found earlier that cellular DNA synthesis in both exponentially and synchronously proliferating uvr -1 cells is inhibited by 4NQO to the same extent as that in RSa cells (Suzuki, 1984). In addition, the capacity of 4NQO-induced DNA repair replication synthesis is much the same in the two cell types (Suzuki, 1984). Thus, in the two cell clones 4NQO response patterns appear to be similar to UV response patterns.

Despite the importance of cell cycle position in determining radiation sensitivity (Kastan et al., 1992), only a few citations can be found regarding the mutability of synchronous human cells (Hoppe et al., 1991). The cell cycle phase at which phenotypic mutation is strongly influenced in human

cells, has been reported to be S a n d / o r G1 and S boundary phases (Enninga et al., 1985). Irradia- tion with UV at a G1 phase has been reported to result in a much lower mutation frequency than irradiation at the S phase (Enninga et al., 1985). In this investigation, however, hypermutability of RSa cells and hypomutability of UVr-1 cells were identified at a late G1 phase, irrespective of the kind of mutagen (UV or 4NQO) or selected phe- notype (Oua R or 6TG R) (Figs. 5 and 6). The pair of UVr-1 and RSa cells will act as good biological models to aid us in understanding the mechanisms of cell cycle-specific mutability of human cells.

Cells derived from xeroderma pigmentosum and Cockayne's syndrome patients resemble RSa cells in their high sensitivity to UV cell killing and hypermutability (Suzuki et al., 1985; Hanawalt and Sarasin, 1986; Norris et al., 1991). Thus, the mutability of the patient-derived cell clones has been compared with that of our cell clones. For instance, XP20S and XP2YO, simian virus 40- transformed xeroderma pigmentosum cell clones (Takebe et al., 1974; Yagi and Takebe, 1983), have been found to have one or fewer Oua R mutants per 105 survivors after UV irradiation (unpublished results). Therefore, under our assay conditions XP20S and XP2YO cells appear to be mutable, contrary to the non-detectable mutability of UVr-1 cells, but to be less mutable than RSa cells. On the other hand, the characteristics of our cell clones are not at all analogous to those of bacterial mutants. Escherichia coli mutants lacking the SOS response, though UV-immuta- ble, are highly sensitive to UV cell killing (Rad- man, 1975; Witkin, 1976). However, UVr-1 cells, having lost the ability to be mutated by UV, have gained an increased resistance to the killing. Fur- ther investigation of the analogy is now in progress between the phenotypes of a pair of RSa and uvr-1 cells and pairs of UV-sensitive prokaryote or eukaryote mutants and their parent a n d / o r normal cells.

5. Acknowledgements

We thank Dr. Masamiti Tatibana for his gen- eral support. This work was funded in part by a


grant-in-aid from the Ministry of Education, Sci- ence and Culture of Japan (No. 03680179).

6. References

Abrahams, P.J., B,A. Huitema and A.J. Van der Eb (1984) Enhanced reactivation and enhanced mutagenesis of herpes simplex virus in normal human and xeroderma pigmentosum cells, Mol. Cell. Biol., 4, 2341-2346.

Abrahams, P.J., A.A.M. Van der Kleij, R. Schouten and A.J. Van der Eb (1988) Absence of induction of enhanced reactivation of herpes simplex virus in cells from xeroderma pigmentosum patients without skin cancer, Cancer Res., 48, 6054-6057.

Bloom, D.C., R. Massung, L. Savage, D.K. Morrison and R.W. Moyer (1989) Recruitment to the cytoplasm of a cellular laminin-like protein from the nucleus during a poxvirus infection, Virology, 169, 115-126.

Cao, M., X. Xiao, B. Egbert, T.M. Darragh and T.S. Benedict Yen (1989) Rapid detection of cutaneous herpes simplex virus infection with the polymerase chain reaction, J. In- vest. Dermatol., 82, 391-392.

Cleaver, J.E., F. Cortes, L.H. Lutze, W.F. Morgan, A.N. Player and D.L. Mitchell (1987) Unique DNA repair properties of a xeroderma pigmentosum revertant, Mol. Cell. Biol., 7, 3353-3357.

DasGupta, U.B., and W.C. Summers (1978) Ultraviolet reactivation of herpes simplex virus is mutagenic and inducible in mammalian cells, Proc. Natl. Acad. Sci. USA, 75, 2378- 2381.

Day, R.S. III (1981) Use of human adenoviruses 2 and 5: purification, plaque assay, and inactivation, in: E.C. Fried- berg and P.C. Hanawalt (Eds.), DNA Repair: A Labora- tory Manual of Research Procedures, Vol. 1, part B, Dekker, New York, pp. 587-604.

Enninga, I.C., R.T.L. Groenendijk, A.A. Van Zeeland and J.W.I.M. Simons (1985) Differential response of human fibrobiasts to the cytotoxic and mutagenic effects of UV radiation in different phases of the cell cycle, Mutation Res., 148, 119-128.

Fortini, P., A. Calcagnile, A. Di Muccio, M. Bignami and E. Dogliotti (1993) Quantitative relationship between eth- ylated DNA bases and gene mutation at two loci in CHO cells, Environ. Mol. Mutagen., 21, 154-159.

Friedberg, E.C. (1985) Error-prone DNA synthesis, in: DNA Repair, Freeman, New York, pp. 435-445.

Gleaves, C.A., D.J. Wilson, A.D. Wold and T.F. Smith (1985) Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation, J. Clin. Microbiol., 21, 29-32.

Hanawalt, P.C. (1989) Concepts and models for DNA repair: from Escherichia coli to mammalian cells, Environ. Mol. Mutagen., 14, 90-98.

Hanawalt, P.C., and A. Sarasin (1986) Cancer-prone heredi- tary diseases with DNA processing abnormalities, Trends Genet., 2, 124-129.

Hashizume, S., H. Yoshizawa, M. Morita and K. Suzuki (1985) Properties of attenuated mutant of vaccinia virus, LC16mS, derived from Lister strain, in: G.V. Quinnan (Ed.), Vaccinia Viruses as Vectors for Vaccinia Antigen, Elsevier, Amsterdam, pp. 87-99.

Hoppe IV, H., K.M. Call, P.M. Leong and W.G. Thilly (1991) Cell-cycle dependent mutation of human lymphoblasts: Bromodeoxyuridine and butyl methanesulfonate, Mutation Res., 250, 411-421.

Intine, R.V.A., and A.J. Rainbow (1990) Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells, Environ. Mol. Mutagen., 15, 19-23.

Kastan, M.B., Q. Zhan, W.S. EI-Deiry, F. Carrier, T. Jacks, W.V. Walsh, B.S. Plunkett, B. Vogelstein and A.J. For- nace (1992) A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia- telangiectasia, Cell, 71,587-597.

Kojima, T., M. Fujita, S. Okamoto and N. Suzuki (1985) Xeroderma pigmentosum variant, Nishi Nippon Dermatol., 47, 1047-1052 (in Japanese with English abstract).

Lehmann, A.R., J.H.J. Hoeijmakers, A.A. Van Zeeland, C.M.P. Backendorf, B.A. Bridges, A. Collins, R.P.D. Fuchs, G.P. Margison, R. Montesano, E. Moustacchi, A.T. Natarajan, M. Radman, A. Sarasin, E. Seeberg, C.A. Smith, M. Stefanini, L.H. Thompson, G.P. Van der Schans, C.A. Weber and M.Z. Zdzienicka (1992) Workshop on DNA repair, Mutation Res., 273, 1-28.

Lehmann, A.R. (1985) Use of recombinant DNA techniques in cloning DNA repair genes and in the study of mutagenesis in mammalian cells, Mutation Res., 150, 61-67.

Lytle, C.D., S.A. Aaronson and E. Harvey (1972) Host-cell reactivation in mammalian cells: II. Survival of herpes simplex virus and vaccinia virus in normal human and xeroderma pigmentosum cells, Int. J. Radiat. Biol., 22, 159-165.

Morrison, D.K., and R.W. Moyer (1986) Detection of a sub- unit of cellular pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions, Cell, 44, 587-596.

Norris, P.G., C.F. Arlett, J. Cole, A.R. Lehmann and J.L.M. Hawk (1991) Abnormal erythemal response and elevated T lymphocyte HRPT mutant frequency in Cockayne's syndrome, Br. J. Dermatol., 124, 453-460.

Radman, M. (1975) SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis, in: P.C. Hanawalt and R.B. Setlow (Eds.), Molec- ular Mechanisms for Repair of DNA, Part A, Plenum, New York, pp. 355-367.

Rock, D.L., and N.W. Fraser (1983) Detection of HSV-1 genome in central nervous system of latently infected mice, Nature, 302, 523-525.

Ronai, Z.A., M.E. Lambert and I.B. Weinstein (1990) In- ducible cellular responses to ultraviolet light irradiation and other mediators of DNA damage in mammalian cells, Cell Biol. Toxicol., 6, 105-126.

Rossman, T.G., and C.B. Klein (1985) Mammalian SOS sys-

222 N. Suzuki et al. /Mutation Research 306 (1994) 211-222

tem: A case of misplaced analogies, Cancer Invest., 3, 175-187.

Royer-Pokora, B., and W.A. Haseltine (1984) Isolation of UV-resistants from a xeroderma pigmentosum complementation group A cell line, Nature, 311, 390-394.

Schultz, R.A., D.P. Barbis and E.C. Friedberg (1985) Studies on gene transfer and reversion to UV resistance in xeroderma pigmentosum cells, Somat. Cell Genet., 11,617-624.

Shwartz, H., O. Shavitt, D. Hervoni, Y. Tadmor, O. Cohen and Z. Livneh (1988) In vitro replication of damaged DNA: A model for SOS-mutagenesis, in: E.C. Friedberg and P.C. Hanawalt (Eds.), Mechanisms and Consequences of DNA Damage Processing, Liss, New York, pp. 471-475.

Smith, C.A., and P.C. Hanawalt (1978) Phage T4 endonucle- ase V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts, Proc. Natl. Acad. Sci. USA, 75, 2598-2602.

Sobels, F.H. (1986) Changing concepts in mutation research, in: C. Ramel, B. Lambert and J. Magnusson (Eds.), Ge- netic Toxicology of Environmental Chemicals, Part A, Liss, New York, pp. 129-146.

Spear, P.G., and B. Roizman (1981) Herpes simplex viruses, in: J. Tooze (Ed.), DNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 615-746.

Sugita, K., N. Suzuki, T. Kojima, Y. Tanabe, H. Nakajima, A. Hayashi and M. Arima (1987) Cockayne syndrome with delayed recovery of RNA synthesis after ultraviolet light irradiation but normal ultraviolet survival, Pediatr. Res., 21, 34-37.

Sugita, K., N. Suzuki, E. Isogai and H. Niimi (1992) Serum factors responsible for unusual induction of plasminogen activator activity in tuberous sclerosis, J. Neurol. Sci., 108, 202-206.

Suzuki, H., and N. Suzuki (1988b) Mutagenicity of saccharin in a human cell strain, Mutation Res., 209, 13-16.

Suzuki, H., N. Suzuki, M. Sasaki and K. Hiraga (1985) Or- thophenylphenol mutagenicity in a human cell strain, Mu- tation Res., 156, 123-127.

Suzuki, N. (1984) A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line, Mutation Res., 125, 55-63.

Suzuki, N. (1987) 4NQO- or MNNG-resistant variants established from a human cell line, RSb, with high sensitivity to both agents, Mutation Res., 178, 135-141.

Suzuki, N., and A. Fuse (1981) A UV-sensitive human clonal cell line, RSa, which has low repair activity, Mutation Res., 84, 133-145.

Suzuki, N., and T. Kuwata (1979) Establishment of

ultraviolet-resistant cells from the highly sensitive human clonal cell line, RSb, Mutation Res., 60, 215-219.

Suzuki, N., and H. Suzuki (1988a) Suppression of UV mutagenicity by human interferon, Mutation Res., 202, 179-183.

Suzuki, N., J. Nishimaki and T. Kuwata (1982) Characteriza- tion of a UV-resistant strain, uvr-10, established from a human clonal cell line, RSb, with high sensitivity to UV, 4-NQO, MNNG and interferon, Mutation Res., 106, 357- 376.

Suzuki, N., T. Kojima, T. Kuwata, J. Nishimaki, Y. Takakubo and T. Miki (1984) Cross-sensitivity between interferon and UV in human cell strains: IF r, HEC-1 and CRL1200, Virology, 135, 20-29, 1984.

Suzuki, N., I. Watanabe, J. Nishimaki, A. Fuse, K. Sugita, S. Sekiya, Y. Takakubo and K. Terao (1986) Increased resistance to the anticellular effect of interferon in an ultraviolet light-resistant human cell line, UVr-1, J. Gen. Virol., 67, 651-661.

Suzuki, N., H. Suzuki, T. Kojima, K. Sugita, N. Takakubo and S. Okamoto (1988) Effects of human interferon on cellular response to UV in UV-sensitive human cell strains, Muta- tion Res., 198, 207-214.

Suzuki, N., Y. Oiwa, I. Sugano, N. Iuaba, S. Sekiya, I. Fukazawa, J. Yoshida, Y. Takakubo, E. Isogai and M. Saito-Ebihara (1992) Dipyridamole enhances an antiprolif- erative effect of interferon in various types of human tumor cells, Int. J. Cancer, 51, 1-7.

Takebe, H., H. Nii, M. Iida-Ishii and H. Utsumi (1974) Comparative studies of host-cell reactivation, colony forming ability and excision repair after UV irradiation of xeroderma pigmentosum, normal human and some other mammalian cells, Mutation Res., 25, 383-390.

Tatsumi, K., M. Toyoda, T. Hashimoto, J. Furuyama, T. Kurihara, M. Inoue and H. Takebe (1987) Differential hypersensitivity of xeroderma pigmentosum lymphoblas- told cell lines to ultraviolet light mutagenesis, Carcinogen- esis, 8, 53-57.

Traktman, P. (1990) The enzymology of poxvirus DNA replication, Curr. Topics Microbiol. Immunol., 163, 93-123.

Wagner, E.K. (1985) Individual HSV transcripts: Characteri- zation of specific genes, in: B. Roizman (Ed.), The Herpes Viruses, Vol. 3, Plenum, New York, pp. 45-104.

Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869- 907.

Yagi, T., and H. Takebe (1983) Establishment by SV40 trans- formation and characteristics of a cell line of xeroderma pigmentosum belonging to complementation group F, Mu- tation Res., 112, 59-66.

Documents

Human cell clones, RSa and UVr-1, differing in their capability for UV-induced virus reactivation and phenotypic mutation