68, 200-214

VIROLOGY

Isolation

(1975)

of Simian

Virus 40-Transformed

Lines Heterogeneous

for Virus Induction

Inbred

Hamster

Cell

by Chemicals

or Radiation JOAN C. KAPLAN,’ SHARON M. WILBERT.2 JEFFREY J. COLLINS,3 TAMARA RAKUSANOVA, GLES B. ZAMANSKY, AND PAUL H. BLACK Department

of Medicine,

Massachusetts General Hospital. and Departments o/ Microbioiogv and Mole< Genetics and Medicine, Harcard Medical School. Boston. Massachusett.\ 021 II Accepted June 16. 197.5

Cloned cell lines have been isolated after simian virus 40 (SV40) transformation 01 kidney cells of an inbred hamster strain. Considerable heterogeneity for the induction of’ infectious virus was observed between the lines, ranging from the spontaneous production of infectious virus to nonproducer characteristics. In spite of their differences in virus inducibility, all the clones were found to contain equivalent small numbers of SF’40 genomes (l-2) per cell as measured by Cot analysis. The induction of infectious virus was studied after treating these clones with four agents that cause strand hreakage either directly or indirectly in DNA: mitomycin C. Brdli and visible light. uv irradiation or 60Co y-irradiation. A direct dose-response relationship was established hetween virus yield and dose of inducing agent used. Yields of infectious virus in induced cells were increased hl factors of 1WlO” compared to untreated cultures. In producer cell lines the percentage ot induced cells was estimated to he Z-6”; as determined by V antipen production or infectious center formation after treatment with the most effective inducing agent. mitomycin C. Our studies suggest that DNA damage and single-strand break formation may be early events in the induction of infectious SV40 from transformed cells hy chemical and physical agents.

was not possible to study the mechanism of virus induction because of the low levels of infectious virus produced by these clones. We have subsequently isolated a number of SV40-transformed hamster kidney cell lines resulting from the infection of cells of the inbred hamster strain LSH. A group of clones has been obtained which are heterogeneous for their capacity to produce virus spontaneously or to be induced by chemical agents or radiation. Whereas some clones yield considerably more virus after induction than those previously described, others are noninducible. These lines will be used to examine the molecular basis of virus induction. Also, comparative studies of virus activation in these inbred, transformed cell clones will be carried out to determine the level of control of virus genome expression which may be responsi-

INTRODUCTION

In previous studies it was demonstrated that the synthesis of infectious simian virus 40 (SV40) could be induced in SV40-transformed hamster kidney cell clones by chemical and physical agents, including mitomycin C (Burns and Black. 1969), 5bromodeoxyuridine (BrdU). irradiation (Rothschild and Black, 1970). or by growth of the cells in amino acid deficient medium (Kaplan et al., 1972). However, it ‘Send reprint requests to cJ. C. K.. Infectious Disease Unit, Massachusetts General Hospital, Boston, Mass. 02114. ‘Present address: Department of Biochemistry and Biophysics. University of California. School of Medicine, San Francisco, Calif. 94143. 3Present address: Surgical Virology Laboratory, Department of Surgery, Duke University Medical Center, Durham, N. C. 27710. 200 Copyright All rights

0 1975 by Academic Press, Inc of reproductmn in any form reserved.

SV.40 INDUCTION

BY CHEMICALS

ble for the observed differences in virus inducibility between the clones. In this paper we describe the isolation and properties of three representative clonal lines that differ considerably in their capacity to produce infectious virus. Since the basis for this heterogeneity could be related to different numbers of SV40 genome equivalents integrated per transformed cell, these values were determined for each of the clones. It is likely that DNA damage and strand breakage are early events in the induction of virogenic transformed cells by chemical and physical agents. In order to test this hypothesis we utilized four different inducing agents that produce DNA strand breakage either directly or indirectly by cellular DNA repair processes: mitomycin C, BrdU and visible light, uv irradiation or 6oCo y-irradiation. Our results showed that under optimum conditions of virus induction, virus yields were directly proportional to the dose of inducing agent used.

OR RADIATION

201

plasma contamination was determined by two procedures. One which measures the incorporation of [methyl-3H]dT into confluent cultures has been described (Culp and Black, 1972). The other method, a cultural technique, was performed in the laboratory of Dr. Leonard Hayflick. Assay of SV40 infectivity. Cell-free extracts were tested for SV40 infectivity on TC-7 monkey kidney cells by a modification of a plaque assay which has been described (Black et al., 1964). Aliquots (0.2 ml) of cell-free extracts were added to confluent TC-7 monkey cell cultures in 60-mm plastic petri dishes. After a 2-hr adsorption period, the virus inoculum was removed from the plates which were then overlaid with 5 ml of maintenance medium containing 0.9% agar and 5% FCS. After 7 days, an additional 3 ml of overlay medium were added to the plates. On day 11 the plates were stained with 0.01%) neutral red, and t.he plaques were counted on days 12 and 14. Immunofluorescent staining. Cells were MATERIALS AND METHODS stained for the SV40 nuclear tumor (T) Cell cultures. Secondary tissue cultures antigen as described (Pope and Rowe, prepared from weanling hamster kidneys 1964; Collins and Black, 1973a). SV40 (inbred Syrian hamster, strain LSH virion (V) antigen was detected by the ssLAK, Lakeview) were infected with puriindirect immunofluorescence technique by using goat antiserum made against purified SV40 as described (Collins and Black, 1973b). Transformed lines were established fied SV40 virions (Flow Labs; neut.ralizafrom separate cultures. Each established tion titer, 1: 160). Coverslips were subsecell line was cloned once in the absence and quently treated with fluorescein-conjuonce in the presence of 1% rabbit antigated rabbit anti-goat immunoglobulin SV40 antiserum (neutralization titer, serum (Sylvana Co.; final dilution, 1:lO) in 1:2560) as described previously (Collins rhodamine counterstain (1:20 dilution). and Black, 1973b). Cloned lines were grown Percentages of fluorescent-positive cells in Eagle’s minimum essential medium were calculated from the results of count(Gibco) with fourfold the usual concentraing approximately 2 x lo3 cells per covertion of vitamins and essential amino acids slip. Infectious center assay. The infectious (MEM x 4), 10% fetal calf serum (FCS; Gibco), glutamine (2 mM), penicillin and center assay performed was similar to the streptomycin sulfate in concentrations ot procedure described by Kit et al. (1966). Two-day-old cultures were treated with 250 units/ml and 250 pg/ml, respectively (maintenance medium). Twice cloned lines mitomycin C for various lengths of time, were subjected to in viva passage as de- washed with phosphate-buffered saline Viable cells (estiscribed previously (Burns and Black, 1968) (PBS) and trypsinized. mated by trypan blue exclusion) were pelto insure that those cells used for experileted, resuspended at a concentration of mental purposes were free of mycoplasma contamination. Cells were tested every 10 lo6 viable cells/ml in Dulbecco’s medium 1% rabbit anti-SV40 serum passages and no cultures were found to be containing contaminated. The presence of myco- (Gibco; neutralization titer, 1:2560) and

202

KAPLAN ET AL.

incubated at 37” for 30 min. Confluent cultures of BSC-B monkey kidney cells were inoculated with 0.1 ml of sample and overlaid with 0.5 ml of agar-overlay (0.9’7, ) medium. After it solidified, another 5 ml of agar-overlay medium were added. Plates were incubated in 5% CO, for 12 days, stained with 0.01% neutral red and the plaques counted on days 13 and 15. Induction by mitomycin C. Confluent cultures in 60-mm petri dishes (Falcon) were exposed to different concentrations of mitomycin C (Nutritional Biochemicals Corp.) for various lengths of time in the dark. Then the mitomycin C was removed, the cells washed with PBS, and fresh maintenance medium was added. The cells were incubated at 37” and harvested at the indicated times. Cell-free extracts were prepared by three cycles of freeze-thawing followed by sonication in a Raytheon Sonifier (3 A for 3 min). Induction by BrdU and visible light. Two-day-old subconfluent cultures in 60-mm petri dishes were treated with BrdU (Calbiochem Corp.) in the dark for 24 hr and then illuminated for various lengths of time with G.E. cool-white 40 W fluorescent bulbs. Cells were exposed through plastic petri dish covers at a distance of 10 cm from the light source, as described by Fogel (1973). Before irradiation, cell counts were determined on replicate plates so that final virus titers could be expressed as plaque forming units (PFU) per lo6 cells at the time of irradiation. After irradiation, the BrdU-containing medium was removed and the cultures were washed twice with maintenance medium. The cells were reincubated with fresh maintenance medium in the dark and harvested at the indicated times. Cell-free extracts were prepared as described above. Induction by uu irradiation. The uv source was a calibrated 5-bulb mercury vapor G.E. germicidal lamp providing a 90% output at 2539 A with a constant fluence of 4.5 ergs/mm ‘/sec. Confluent cultures in 60-mm petri dishes were exposed to various doses of irradiation (25-150 ergs/ mm’; exposure times, 5.6-33.3 set) at room temperature (24”) in the absence of medium after one wash with PBS. Following irradiation, the cells were incubated in

maintenance medium and harvested at the indicated times. Cell-free extracts were made as described above. Induction by @‘Coy-irradiation. Confluent cultures in maintenance medium were exposed to various doses of ‘%o y-irradiation at room temperature (24”). The doserate was 250 radlsec. Following irradiation the cultures were incubated in fresh maintenance medium and harvested at the indicated times. Cell-free extracts were made as described above. Induction by cell fusion. Cell fusion procedures for induction of infectious SV40 were carried out using ,8-propriolactoneinactivated Sendai virus as described (Burns and Black, 1968). Chromosome analyses. Chromosome analyses were performed on trypsinized cells by a modification of the method of Moorhead et al. (1960). At least 30 mitotic cells were examined for each ceil line. DNA content per cell. The amount of DNA per cell was determined using the method described by Kraemer et al. (1971). Fixed cells stained with a fluorescent dye were passed through a laser beam of light, and the amount of DNA per cell was calculated from the height of the pulse of light emitted. Results are presented as relative numbers comparing the DNA content of the G, phase of a given cell line with that of a control hamster cell line in the diploid range (BHK-21) as established by Dr. D. Peterson (Los Alamos Scientific Lab., Los Alamos, N. M.). Extraction of cellular DNA. Cellular DNA extraction was performed according to the method of Marmur (1961) with minor modifications. The RNase (Calbiothem Corp.) treatment was followed by Pronase digestion (Calbiochem Corp.; 50 pg/ml for 1 hr at 37”) and additional deproteinization steps with chloroformisoamyl alcohol. In some experiments the Hirt extraction procedure was used to separate high molecular weight from low molecular weight DNA (Hirt, 1967). The Hirt pellet was solubilized prior to DNA extraction by resuspension in 0.02 M EDTA, pH 7.4, and by treatment with Pronase: 50 pg/ml for 224 hr at 37” followed by 16 hr at room temperature. Preparation of 32P-labeled SV40 DNA.

SV40 INDUCTION

BY CHEMICALS

Confluent cultures of MA-134 or TC-7 monkey cells were infected with plaquepurified SV40 virus at a multiplicity of infection equal to 0.1-l PFU per cell. Approximately 20-24 hr after infection, carrier-free [32P]orthophosphate (New England Nuclear Corp.) was added to the cultures in phosphate-free maintenance medium (50-100 pCi/ml) containing 10% dialyzed FCS. The cells were harvested 7-8 days after infection. SV40 was purified according to Black et al. (1964). After equilibrium density centrifugation in C&l, Form I viral DNA was isolated and purified as described by Gelb et al. (1971). SV40 and cell DNA’s were fragmented by sonication in a Raytheon Sonifier (3 A, 7 min) to a 6-8 S size as confirmed by alkaline sucrose gradient sedimentation. C,t analysis. This was performed according to the method of Gelb et al. (1971) with the following minor modifications. Fragmented, denatured 32P-labeled SV40 DNA was allowed to reanneal at 68” in 0.4 M phosphate buffer, pH 6.8, in the presence of unlabeled salmon sperm DNA, normal hamster or monkey cell DNA, or DNA from SV40-transformed hamster cells. The degree of reassociation of 32P-labeled SV40 DNA was determined at various times during the incubation. The proportion of single- and double-stranded DNA was assayed by chromatography on hydroxyapatite columns. Duplexes containing 32Plabeled SV40 DNA were eluted from the columns and counted directly in 2.5 volumes of Aquasol (New England Nuclear Corp.). Results were plotted as a function of C,t (the product of moles of nucleotides x seconds per liter) and the number of copies of SV40 DNA was calculated from these values using a figure of 3.9 x 10” daltons of DNA per diploid cell. RESULTS

Passage History

of

the Transformed

OR RADIATION

tumors. During the isolation and cloning procedures, virus production was measured by exposing cultures to mitomycin C (2 pg/ml for 8 hr). Patterns of virus release, either spontaneous or following mitomycin C treatment, were relatively constant for each clonal line at the various stages of their passage history. However, considerable heterogeneity in virus inducibility was observed between the lines, ranging from spontaneous production of infectious virus to nonproducer characteristics. Three representative clones were chosen for further studv and the yields of infectious virus obtained spontaneouslv or subsequent to their induction with mitomycin C at various stages of cell passage are presented in Table 1. Ten in vitro passages elapsed between the first cloning and passage of the cell lines in animals. THK 22A Cl l-l, AP, is an example of a spontaneous producer and will be referred to as clone A. THK 22E Cl l-l, AP, (clone E) occasionally releases small amounts of virus spontaneously and can be stimulated further by inducing agents. THK 22G Cl l-1, AP,, (clone G) is a nonproducing, noninducible clonal line. Subsequent experiments demonstrated that clones A and E produced maximum yields of infectious virus when exposed to mitomycin C at a concentration of 0.5 pg/ml for 24 hr (see Fig. 2). Therefore, clone G was subjected to these optimal inducing conditions but was still found to be completely noninducible for infectious virus. Attempts were made to rescue SV40 from this line by fusing it to permissive monkey cells with inactivated Sendai virus. Although heterokaryons were formed, no infectious virus was released. This technique has been used previously to rescue infectious SV40 from other transformed hamster cells which were not inducible with mitomycin C (Burns and Black, 1968). Characteristics

Lines

Each of the six transformed lines isolated was cloned, passaged twice and recloned in the presence of SV40 antiserum. Subsequently each line was carried for two passages in the presence of SV40 antiserum and transplanted to inbred hamsters. Cell lines were established from 16 different

203

of

the Clonal Lines

Cells from the three different clones are morphologically similar. In general they are epithelioid and resemble SVBO-transformed hamster kidney cells previously described (Black et al., 1963). There is some variation in size between the clones, with cells of clone A being smaller than those of clones E or G. Each line contains

204

KAPLAN

ET AL.

TABLE INDUCIBII.ITY

CHARACTERISTICS

First cloning THK 22 Cl 1

Cell line

Clone A Clone E Clone G

1

OF CLONAL

LINES

AT VARIOIX

Second cloning THK 22 Cl 1-l

Control”

Induced

Control

Induced

7.0 x 10’ 0 0

1.8 x 105 1.6 x 10’ 0

2.1 X 102 1.3 0

1.7 X 10’ 1.0 X 105 0

STAGES

OF PASSAGE

Following animal passage THK 22 Cl 1-1, AP, Control 6.4 x 10’ 5.0 0

Induced 7.3 x 105 6.2 x 10’ 0

a Virus yield expressed as plaque forming units (PFU) per milliliter; 1 ml of extract contained approximately lo6 cells. Cultures were exposed to mitomycin C (2 pg/ml) for 8 hr and harvested after 96 hr.

A ; P “6

=J 2 B

4

k

2

% a

O

CLONE

CLONE

E ; P tt7

I

II,

80

90

I

G ; P 87

I

30

40

50

60

CHROMOSOME

70

100

NUMBER

FIG. 1. Chromosome analysis of clones A, E and G. Chromosome number was determined in approximately 30 mitotic cells. Cell passage numbers are indicated.

a small percentage of multinucleated, syncytial giant cells. The SV40 T antigen was present in the nuclei of nearly all the cells of the three clones, but SV40 V antigen was never detected in uninduced cells of any clone. Cell clones were analyzed for their chromosome content, and the modal number of chromosomes was determined (Fig. 1). Repeated analyses showed that the distribution of chromosomes for each cell line was fairly constant over several passages. Clone A had a relatively sharp peak in the diploid range with a mean modal number of 39

chromosomes per cell. Most clone G cells contained between 49 and 55 chromosomes, while the cells of clone E were more aneuploid with chromosome numbers ranging between 56 and 80 per cell. In clone E cells there was evidence of cytogenetic aberrations, including rearrangements and translocations. DNA content per cell was estimated in each of the three cloned lines and reported as a ratio of the DNA content of the G, phase of a given cell line with that of a control cell line (BHK-21). Clones E and G contained more DNA than untransformed BHK-21 cells (Table 2). The relative amounts of DNA per G, cell in clones A, E and G were in the proportion of 1.0:2.0:1.5, respectively, which was in general agreement with results from the chromosome analyses described above. Total Amount

of SV40 DNA per Cell

In order to determine whether differences in virus inducibility between the lines were related to the number of SV40 viral genomes they contained, we estimated the amount of SV40 DNA per cell by C,t analysis (Gelb et al., 1971) (Fig. 2). The acceleration of reassociation of 32P-labeled SV40 DNA in the presence of unlabeled DNA from SV 40-transformed cells is proportional to the number of SV40 genome copies present in these cells. The results presented in the first column of Table 2 are calculated on the basis of the diploid quantity of host cell DNA. However, as described above, clones A, E and G contain different quantities of host DNA per cell. Therefore, the data were recalculated

SV40 INDUCTION

BY CHEMICALS TABLE

205

OR RADIATION

2

AMOUNT o~SV40 DNA IN THESV~@TRANSFORMEDHAMSTERCELLS Cell line

Number of copies of SV40 DNA per diploid amount of cell DNA

Relative amount of DNA per G, cell compared to BHK 21 cells

Number of copies of SV40 DNA per G, cell”

Clone A Clone E Clone G

2.1 0.85 0.90

1.0 2.0 1.5

2.1 1.7 1.4

a The values presented represent an average of 8-11 determinations. Methods of DNA determination of number of SV40 copies per cell are described in Materials and Methods.

p*P] DNA

extraction

and

Cot IMx set/l)

FIG. 2. 3ZP-labeled SV40 DNA reassociation in the presence of preparations of unlabeled DNA from SV40-transformed hamster cells. Each incubation mixture contained sheared 32P-labeled SV40 DNA, sheared, unlabeled DNA (in concentrations indicated below) in 0.4 A4 phosphate buffer, pH 6.8, O.Oo:! M EDTA. DNA preparations were heat denatured and allowed to reassociate at 68”. At appropriate time intervals, aliquots were withdrawn, diluted to a final concentration of 0.14 M phosphate and subjected to hydroxyapatite column chromatography as described in Materials and Methods. (O----O), 32P-labeled SV40 DNA (8 x lOme-9.6 x 10e5 OD units/ml) incubated with salmon sperm DNA (20-50 OD units/ml); (.A), 32P-labeled SV40 DNA (2.2 x lo-’ OD units/ml) incubated with normal monkey cell DNA (line MA-134) (47 OD units/ml); (A---A), 32P-labeled SV40 DNA (2.2 x 10m5 OD units/ml) incubated with clone A DNA (16 OD units/ml); (A---A). 82P-labeled SV40 DNA (2.2 x IO-’ OD units/ml) incubated with clone E DNA (56 OD units/ml); (O--U), 32P-labeled SV40 DNA (2.2 x lo-” OD units/ml) incubated with clone G DNA (58 OD units/ml). Each final point represents 200-400 cpm.

206

KAPLAN

E?‘ilL ‘lr

based on the total amount of DNA actually present in each of the transformed cell lines in order to estimate the true number of SV40 genome equivalents in each line. Results are presented in Table 2, column 3, and range from 1.4 for clone G to 2.1 for clone A. Therefore, the three lines contain essentially the same relatively small numbers of SV40 genomes per cell in spite of’ their observed differences in vrirus inducibility.

Virus Induction by Mitomycin

EXPCSbRE

C

Experiments were performed to determine the optimal conditions of induction of infectious SV40 from clones A and E. Exposure of clone E to mitomycin C (0.5 pug/ml) for 24 hr resulted in maximum production of infectious SV40 when cultures were harvested 120 hr after initiating mitomycin C treatment (Fig. 3). A marked rise in virus production was observed between 24 and 72 hr which slowlv leveled off by 120 hr. The length of time of exposure of cells to this inducing agent is an important factor in determining the efficiency of virus rescue. A 1-hr exposure resulted in approximately 200-fold less virus produced per lo6 cells of the culture than seen with a 24-hr treatment. Clone A consistently produced lo-loo-fold more infectious virus at various times after induction than was observed with clone E. Uninduced clone A cells demonstrated a baseline level of approximately lo*-lo3 PFU of infectious virus per lo6 cells (data not shown). As illustrated in Fig. 4, the amount of virus induced from clone E was dependent on the dose of mitomycin C used at the shorter exposure times. A direct relationship between dose and virus yield was observed in the range of O.j-2.0 pg/ml of mitomycin C. However, exposure to concentrations of mitomycin C greater than 0.5 pg/ml for 16- and 24.hr periods resulted in decreased virus yield, presumably because of toxicity to the cells. Since higher yields of virus were obtained after mitomycin C treatment of these clones than had been rescued previously (Burns and Black, 1969) or with the other inducing agents used in this study, experiments were done to determine the

48

24 TIME

OF

72

HARVEST,

96

120

hours

FIG. 3. Kinetics of virus release from clone E as a function of exposure time to mitomycin C. Mitomycin C (0.5 kg/ml) in maintenance medium was added to confluent cultures of clone E at time zero. Replicate cultures were exposed to mitomycin C for various times between 1 and 24 hr, and then the medium was removed. Cultures were washed once and incubated in maintenance medium at 37” until they were harvested at daily intervals during a 120-hr period. Contents of two petri dishes were pooled and assayed for infectious virus at each time point as described. Virus titer is expressed as PFU/lO’ cells counted at time zero. Exposure times to mitomycin C were: (04) 1 hr; (O-----O) 2 hr; (A---A) 4 hr: (A-A) 8 hr; (m-e) 16 hr; (U----O) 24 hr.

number of cells activated treatment.

by mitomycin

C

Percentage of Cells Induced by Mitomycin C Treatment The proportion of induced cells in clones A and E was determined in two ways: (i) Estimation of SV40 virion (V) antigen production and (ii) infectious center assay. When confluent cultures of clones A and E were induced with mitomycin C, V antigen production was first observed by immuno-

SV40 INDUCTION

BY CHEMICALS

fluorescence after 48 hr and continued to increase up to 96 hr after initiating treatment (Table 3). Many nuclei became enlarged and a certain percentage of these showed extensive fragmentation. All Vpositive nuclei were of the enlarged type. This effect has also been observed in mouse fibroblast cells treated with mitomycin C (Shatkin et al., 1962). In both clones A and E only 223% of the nuclei were positive for V antigen after mitomycin C treatment. Positive cells appeared in clusters. No V antigen was ever detected in uninduced cultures of either clone. Although cells containing V antigen represent induced cells, they may not necessarily contain or release complete infec-

[MITOMYCIN

C’,,ug/ml

FIG. 4. Dose-response relationship of mitomycin C concentration and virus release from clone E. Mitomycin C (0.5-2.0 pg/ml) in maintenance medium was applied to confluent cultures of clone E at time zero. Cultures were exposed to mitomycin C for various times between 1 and 24 hr, and then the medium was removed. Cultures were washed, incubated in maintenance medium at 37” and harvested after a total incubation time of96 hr. Contents of two petri dishes were pooled for each point and assayed for infectious virus as described. Virus titer is expressed as PFU/106 cells counted at time zero. Exposure times to mitomycin C are indicated; symbols are defined in the legend to Fig. :i.

207

OR RADIATION TABLE

3

PERCENTAGEOF (V) ANTIGEN-POSITIVE CELLS AFTER MITOMYCIN C TREATMENF’ Cell line

Time after mitomycin &

Clone A CloneE

0 0

C treatment

(hr)

(?,

(E,

(f ,

(?,

$,

0 0

0 0

0.01 0.03

1.5 1.0

2-3 1.8

a Cells on coverslips were induced with mitomycin C (0.5 fig/ml for 24 hr) and then washed and incubated in maintenance medium. Coverslips were fixed at the indicated times and assayed as described in Materials and Methods. Approximately 2000 nuclei were counted per coverslip. Fragmented nuclei contained within a cell were counted as one nucleus.

tious virus. To estimate the number of cells yielding infectious virus in an induced population it was first necessary to determine the distribution of infectious virus at various times after initiating mitomycin C treatment. Therefore, we measured the proportion of infectious SV40 which remained cell associated or was released into the culture medium as a function of time after mitomycin C treatment of clone E (Table 4). At 24 hr after initiating mitomytin C treatment, all of the infectious virus was still cell associated, but, by 48 hr, 41% of the virus had been released into the medium. and after 96 hr only 23% remained associated with the cells. It is probable that, once activated, cells are lysed rapidly. Therefore, cultures for the infectious center assay were harvested 24 hr after initiating mitomycin C treatment, and the percentage of virus-producing cells was determined to be 6% for clone A and 1.2% for clone E (data not shown). These data demonstrate that the infectious center assay is more sensitive than the determination of nuclear V antigen. Induction by RrdU and Visible Light When cells are grown in BrdU, the BrUra-substituted DNA becomes sensitive to breakage by visible-light irradiation (Puck and Kao, 1967) and single-strand breaks are produced at the substituted sites (Ben Hur and Elkind, 1972). It has been shown that BrdU is capable of inducing virus only if it has been incorporated into the cellular

208

KAPLAN

DNA (Teich et al., 1973). Therefore clones A and E were exposed to BrdU and visible light to determine whether any correlation existed between irradiation dose and the induction of infectious virus. Results of a representative experiment are shown in Table 5, in which clone A cells were exposed t,o different concentrations of BrdU (O-80 pg/ml) as described in Materials and Methods. The data demonstrated a direct relationship between the length of exposure time to visible light and virus yield at each concentration of BrdU used. The optimal combination tested was BrdU at a concentration of 40 pg/ml with a 40-min exposure to light. Cellular toxicity was apparent at a BrdU concentration of 80 pg/ml. When clone E was treated by a similar procedure, the same type of virus activation pattern was observed, including a dose-response relationship with the amount of visible light used. Approxi-

E?’ AL.

mately lo-fold less virus was activated in clone E than in clone A. Although these data suggest a relationship between DNA breakage and virus activation, BrdU is known to have a variety of effects on mammalian cell metabolism other than the induction of DNA strand breaks (Rutter et al., 1973). Therefore, this possible mechanism of virus induction requires additional more direct evidence. Induction

It is known that various forms of irradiation cause strand breakage in DNA. With irradiation, short exposure times can be used, and the physiological complications inherent in the addition of drugs to cell cultures are minimized. Subconfluent cultures of clone E were irradiated with different doses of uv light (25-150 ergs) at time zero (Fig. 5) and

TABLE PAKTWIONING

OF INDUCED VIRCS

4

BETWEEN CELL FRACTION

AND C~LTITRE

MEDIL-M”

Fraction

Time of harvest (hr)

0 1.7 x 5.7 x 0.9 x 3.0 x

Total yield PFU

Medium

Cell PFU

0 24 48 72 96

by uv Irradiation

Percentage of total yield

PFU

Percentage of total yield

0 100 59 31 23

0 0 4.0 x 10’ 2.0 x 10’ 1.0 x 105

-

100 10’ 10’ 10’

1.7 9.7 2.9 1.3

41 69 77

0 X 100 Y 10’ x 10’ Y 105

DConfluent cultures of clone E were exposed to mitomycin C (0.5 pg/ml for 24 hr) at which time they were washed once and fresh maintenance medium was added. Cell and supernatant fractions were harvested separately at various times after initiation of treatment and assayed for infectivity. TABLE EFFECT OF BrdU Time

AND VISIBLE

LIGHT

ON INDIJCTION

of

0

OF INFECTIOIJS

VIRIJS

FROM CLONE

A”

BrdU k/ml)

irradiation (min)

10 20 40

5

(PFUi1°06 cells)

10 (PFU/lOE cells)

40 (PFU/lO” cells)

106 125 260 135

110 125 565 3010

240 475 1800 5770

300 380 925 2800

“Subconfluent monolayer cultures of clone A were pretreated with BrdU for 24 hr and then exposed to visible light. The BrdU-containing medium was replaced with maintenance medium after two washes and the cultures harvested 72 hr later and assayed for infectivity.

SV40 INDUCTION

BY CHEMICALS

uv, ergs

the dose of uv irradiation used (Fig. 6). Increasing amounts of virus were produced with doses up to 75-100 ergs, at which point the curves leveled off. At the highest dose used (150 ergs) virus yield began to decrease slightly in both clones. Induction

0

24 HOURS

I

I

48

72

AFTER

I 96

(20

209

OR RADIATION

by 6oCo y-Irradiation

The kinetics of virus induction by yirradiation were followed in clone E (Fig. 7) over a 5day period. There was a very rapid increase in virus production between 24 and 72 hr which plateaued beyond that time. At doses greater than 10 krad, almost all the virus was produced by 72 hr. A direct relationship was observed between virus yield and dosages in the range of 5-25 krad for both clones A and E (Fig. 8). Virus yields leveled off after 30 krad and

IRRADIATION

Fro. 5. Kinetics of virus release after uv irradiation of clone E. Cultures of clone E were exposed to uv irradiation at time zero. After irradiation, the cultures were incubated at 37” and harvested at daily intervals during a 120.hr period. Contents of two petri dishes were pooled for each time point and assayed for infectious virus as described. Virus titer is expressed as PFU/106 cells counted at time zero. Ultraviolet doses were: (04) 0 ergs; (O--O) 25 ergs: (A-A) 50 ergs; (A-A) 100 ergs; (W---m) 150 ergs.

cultures were harvested at daily intervals for 120 hr. At each uv dose there was an initial rise of virus production between 24 and 72 hr which leveled off between 72 and I20 hr. Optimum induction occurred with a uv dose of 100 ergs/mm* and resulted in a 200-fold stimulation of virus production which was about lo-fold lower than the amounts induced under optimal conditions of mitomycin C treatment. In control cultures an increase in virus production was observed as a function of time spent in unchanged culture medium. This value varied from one experiment to another within a lo-fold range. When the kinetics of virus induction by uv irradiation were followed in clone A, patterns were similar and at each irradiation dose virus yields were IO-loo-fold higher than seen with clone E (data not shown). The total amount of virus produced from clones A and E was directly dependent on

10% 1 0

25

50 UV,

I

I

I

I

75

100

125

150

ERGS

FIG. 6. Dose-response relationship for uv irradiation and virus release in clones A and E. Cultures of clone A (O--Q) and clone E (04) were exposed to uv irradiation (25-150 ergs) at time zero. Then the cells were incubated at 37” and harvested after a total incubation time of 96 hr. Contents of two petri dishes were pooled for each point and assayed for infectious virus as described. Virus titer is expressed as PFU/lO’ cells counted at time zero.

KAPLAN

YIRRAD, Krod

24 HOURS

48 AFTER

72

96

I2C

ET AL

interest in that they represent the products of distinct individual transformation events occurring after the infection of genetically identical hamster kidney cells. Nevertheless. despite their similar origin, the clones examined are markedly heterogeneous with respect to their virogenic properties. SV40-specific DNA was measured in these clones to see if quantitative differences in the number of SV40 genomes per cell could account for the observed differences in virus inducibility. However, our data indicate that all three clones contain equivalent amounts of SV40-specific DNA (l-2 genome equivalents per cell) at a level which agrees with recent reports on the quantity detectable in other SV-40 transformed hamster cells (Levine et al., 1969). Previous attempts to relate the amount of SV40 DNA per cell to virus rescuability from transformed cells cocultivated with 406:

IRRAD!PT/ON

FIG. 7. Kinetics of virus release after -r-irradiation of clone E. Cultures of clone E were exposed to -r-irradiation at zero time. After irradiation they were incubated at 37” and harvested at daily intervals during a 120.hr period. Contents of two petri dishes were pooled for each time point aml assayed for infectious virus as described. Virus titer is expressed as PFU/106 cells counted at time zero. Doses of y-irradiation were: (04) 0 krad; (04) 5 krad: (A---A) 10 krad; (A--A) 20 krad; (W---W) 30 krad: (04) 50 krad.

in clone A actually decreased beyond this point so that with 70 krad it had diminished by 30-fold. This decrease could be a reflection of cessation of macromolecular synthesis and is being investigated. With clone E there was only a five-fold decrease in virus production in the dose range of 50-70 krad of irradiation. It is of interest that doses ofy-irradiation that induced the highest yields of virus are extremely lethal to the cell. Preliminary observations suggest that only 0.01% of the cells are capable of division once they have been y-irradiated with doses greater than 2 krad. DISCUSSION

The SV40-transformed kidney cell clones described in this report are of particular

:

I’ P/’

‘b --“‘9

FIG. 8. Dose-response relationship for y-irradiation and virus release from clones A and E. Cultures of clone A (04) and E (O---O) were exposed to y-irradiation (5-70 krad) at time zero. Then the cells were incubated at 37” and harvested after a total incubation time of 96 hr. Contents of two petri dishes were combined for each point and assayed for infectious virus as described. Virus titer is expressed as PFU/lOG cells counted at time zero.

SV40 INDUCTION

BY CHEMICALS

permissive cells also demonstrated no correlation between virus yield and the number of viral genome equivalents per cell (Ozanne et al., 1973). Differences in the probability of infectious virus induction may therefore be related to the nature or sites of integration or to mechanisms controlling genome excision, viral DNA replication, transcription, or posttranscriptional events. We therefore investigated an early event in the induction process. Since DNA strand breakage is thought to facilitate viral genome excision, a number of different inducing agents were studied, all of which make breaks either directly or indirectly in DNA. These agents, mitomytin C, BrdU and visible light, uv irradiation or y-irradiation were all found to induce SV40 from clones A and E. Although the types of DNA damage caused by these agents may differ, the end result is single-strand break formation. Mitomycin C is an alkylating agent that induces crosslinks in DNA (Iyer and Szybalski, 1963) which are removed by cellular repair enzymes (Cole, 1973). BrdU incorporation into DNA sensitizes it to radiation so that direct single-strand breakage occurs at the BrUra-substituted sites (Puck and Kao, 1967; Ben-Hur and Elkind, 1972). The DNA damage produced by uv or ionizing radiation and the repair of such lesions have been quite well defined in molecular terms. The type of repair that is initiated depends on the kind of radiation used. DNA lesions produced by uv irradiation cause localized distortions in the DNA helix. It is believed that this damage is removed from mammalian cells by DNA repair enzymes in the same manner as in bacteria (Regan and Setlow, 1974). In bacteria, a specific endonuclease makes singlestrand breaks in the DNA near the lesions and the damage is removed by exonuclease action (Kaplan et al., 1969; Grossman, 1974; Regan et al., 1968). In mammalian cells, ionizing radiation either directly or indirectly produces many DNA singlestrand breaks which are rapidly resealed (Lohman, 1968; Ormerod and Stevens, 1971) with the accompanying excision of a few bases (Cerutti, 1974). When optimal conditions of induction

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were established in clones A and E for each of the inducing agents, we observed that the amount of infectious SV40 produced from induced cultures was directly proportional to the dose of inducing agent used. Under these conditions, approximately lo’-lo3 more infectious virus could be induced from producer clones A and E than had been induced previously from transformed hamster clones by chemical and physical agents (Burns and Black, 1969; Rothschild and Black, 1970). Using these conditions it has been shown that SV40 viral DNA is excised from its integrated state after treatment of these cells with mitomycin C or y-irradiation (Rakusanova et al., manuscript in preparation). Maximum virus induction occurred in clones A and E with doses of uv light and y-irradiation which inhibited multiplication and promoted cell death. With uv irradiation, the optimum virus-inducing dose (100 ergs/mm2) was still within the range of about 54 cell survival. However, y-irradiation doses which produced barely detectable amounts of virus were already lethal and completely inhibited cell division (lethality is defined here as the cell’s inability to divide and form colonies despite the fact that the original irradiated cell continues to undergo macromolecular synthesis). Doses of y-radiation which induced maximum yields of virus were extremely high (30-50 krad). One explanation could be that strand rejoining occurs much more rapidly following y-irradiation than after uv irradiation and, if the probability of virus activation is dependent on the accumulation of single-strand breaks, it may be necessary to induce many more breaks with y-irradiation than with uv. The accumulation of DNA strand breaks may be an important factor in the control of viral genome excision and virus activation. In fact, in the dose range of y-radiation that elicits a linear response of virus production (5-30 krad), we have measured increasing DNA strand breakage in clones A and E by alkaline sucrose gradient analysis (unpublished observations). We plan to compare the rates of DNA break formation and strand rejoining in these clones to determine whether differences in cellular

212

KAPLAX

repair processes could account for observed differences in virus inducibility. The production of single-strand breaks could be a common step leading to generalized derepression in cells transformed by DNA viruses. Following derepression, viral genome excision might be specifically controlled by viral or host excision enzymes. It is also possible that viral genome excision and infectious virus production results from random cleavage of cellular DNA following treatment with chemicals or radiation. Factors determining cellular permissiveness for viral replication are not completely understood. In these studies it is apparent that the degree of permissiveness for viral genome expression varies both within a population of cloned cells and between different syngeneic clonal lines of the same species. Interspecies variation has also been observed with respect to inducibility of infectious SV40 from transformed cells (Dubbs and Kit. 1971; Watkins, 1973). We found that the probability of virus activation was quite low in these hamster cells. As measured by V antigen production or infectious center formation, only a small percentage (2-6%) of cells was induced after mitomycin C treatment. However, more cells may actually be induced than is evident by these detection methods as was observed for the induction of EB virus from Raji lymphoblastoid cells (Hampar et al., 1972). Our data show that a few cells are spontaneously permissive in an inducible clone and allow virus replication to occur even in the absence of inducing agents. At present, we are unable to determine whether virus activation is increased in such cells or if other cells are activated de nouo when cultures are exposed to inducing agents like mitomycin C or radiation. The magnitude of stimulation of virus production by all the inducing agents was approximately the same for both clones A and E even though their spontaneous yields of infectious virus were different. Clone G could not be induced by any of the agents utilized or even after fusion with permissive monkey cells so it may contain functionally defective SV40 genomes. Hamster cells have previously been con-

ET AL

sidered totally nonpermissive for SV30 replication. Clone A is unusual among other SV40-transformed hamster lines in that it continuously produces small amounts of infectious SV40 even in the absence of inducing agents. This could be a unique feature of inbred LSH strain cells. However, it is clear that the production of infectious virus from uninduced cells does not reflect the establishment of a carrier culture, since this property is maintained after cloning and repeated passage of the cells in high titered SV40-neutralizing antiserum. Thus, it seems probable that a small proportion of cells are spontaneously induced to synthesize and release infectious virus. A polyoma virus-transformed rat cell line with similar properties has also been described (Fogel and Sachs. 1969, 1970; Fogel. 1973). The hamster cell is relatively inefficient for SV40 synthesis. As demonstrated by the infectious center assay, under optimum conditions of mitomytin C induction, clone A has a burst size of at most lo-20 PFU per cell, approximately lo-100 times (l-2 logs) less than the burst size in a lytic infection of monkey cells (Black et al., 1964; Manteuil et al., 1973). In the accompanying article, further studies that examine the effects of cell synchronization on virus induction will be reported. ACKNOWLEDGMENTS This investigation was supported by I!.S.P.H.S. Grant No. CA-11126-07 from the National Cancer Institute and Training Grants No. GM 00 177 to Jeffrey J. Collins and No. ST01 GM 00 175. 16 to Glen B. Zamansky while in the Department of Microbiology and Molecular Genetics, Harvard Medical School. Joan C. Kaplan is the recipient of a Cancer Research Scholar Award from the American Cancer Society (Massachusetts Division). We thank Lawrence F. Kleinman for his excellent technical assistance, Dr. dohn B. Little for providing radiation facilities and advice for these experiments, Dr. Leonard Atkins for performing chromosome analyses, and Dr. Donald Petersen for estimating cell DNA content. REFERENCES BEN-HUH, E., and ELKIXI, M. M. t19i’2). Damage and repair of DNA in 5-bromodeoxyuridine-labeled Chinese hamster cells exposed to fluorescent light. Ehphys. J. 12, 636-647.

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BY CHEMICALS

BLACK, P. H., CRAWFORD, E., and CRAWFORD. L. (1964). The purification of simian virus 40. Virolog) 24, 381-387. BLACK, P. H., ROWE, W. P., and COOPER,W. L. (1963). An analysis of SV40-induced transformation of hamster kidney tissue in vitro. II. Studies of three clones derived from a continuous line of transformed cells. Proc. Nat. Acad. Sci. USA 50, 847-854. BLJHNS,W. H., and BLACK, P. H. (1968). Analysis of simian virus 40.induced transformation of hamster kidney tissue in uitro. V. Variability of virus recovery from cell clones inducible with mitomycin C and cell fusion. J. Viral. 2, 606-609. BURNS, W. H., and BI.ACK, P. H. (1969). Analysis of SV40-induced transformation of hamster kidney tissue in vitro. VI. Characteristics of mitomycin C induction. Virologv 39, 626-634. CEHLITTI, P. A. (1974). Effects of ionizing radiation on mammalian cells. Naturu,issenschaften 61, 51-59. COIX, R. S. (1973). Repair of DNA containing interstrand cross links in E. coli; sequential excision and recombination. Proc. Nat. Acad. Sci. IJSA 70, 10641068. COLI.INS, J. J., and BLACK, P. H. (1973a). Analysis of surface antigens on simian virus 40.transformed cells. I. IJnique antigenicity of simian virus 40. transformed outbred hamster kidney cell lines. J. Nat. Cancer Inst. 51, 95-114. COLLINS, J. .I., and BI.ACK, P. H. (1973b). Analysis of surface antigens on simian virus 40.transformed cells. II. Exposure of simian virus 40.induced antigens on transformed rabbit kidney and inbred hamster kidney cells by phospholipase C. J. Nat. Cancer Inst. 51, 115-135. CCLP, L. A., and BLANK, P. H. (1972). Contact-inhibited revertant cell lines isolated from simian virus 40.transformed cells. III. Concanavalin A-selected revertant cells. J. Viral. 9, 611-620. DUBBS, D. R., and KIT, S. (1971). Spontaneous virus production by clonal lines of simian virus 40.transformed cells and effects of superinfection by deoxyribonucleic acid from mutant simian virus 40 strains. J. Viral. 8, 430-436. Fo~EI., M., and SACHS, L. (1969). The activation of virus synthesis in polyoma transformed cells. Virol0g.v 37, 327-334. FOGEI., M., and SACHS, L. (1970). Induction of virus synthesis in polyoma transformed cells by uv light and mitomycin C. Virology 40, 174-177. FOGEI., M. (1973). Induction of polyoma virus synthesis by fluorescent (visible) light in polyoma-transformed cells pretreated with 5bromodeoxyuridine. Nature New Rio/. 241, 1822184. GELB, L. D., KOHNE, D. E., and MARTIN, M. A. (1971). Quantitation of simian virus 40 sequences in African green monkey, mouse and virus-transformed genomes. J. Mol. Biol. 57, 129-145.

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GROSSMAN,I,. (1974). Enzymes involved in the repair of DNA. Aduan. Radiat. Biol. 4, 77-129. HAMPAH, B., DERGE, J. G., MARTOS, L. M., and WALKER, J. L. (1972) Synthesis of Epstein-Barr virus after activation of the viral genome in a “virus negative” human lymphoblastoid cell (Raji) made resistant to 5.bromodeoxyuridine. Proc. Nat. Acad. Sci. USA 69, 78-82. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. IYER, V. N., and SZYBALSKI, W. (1963). A molecular mechanism of mitomycin action. Linking of complementary DNA strands. Proc. Nat. Acad. Sci. USA 50, 355-362. KAPI.AN, J. C., KUSHNER, S. R., and GROSSMAN, L. (1969). Enzymatic repair of DNA. I. Purification of two enzymes involved in the excision of thymine dimers from uv-irradiated DNA. Proc. Nat. Acad. Sci. USA 63, 144-151. KAPLAN, J. C., WILBEHT, S. M., and BLACK, P. H. (1972). Analysis of simian virus 40-induced transformation of hamster kidney tissue in vitro. VIII. Induction of infectious simian virus 40 from virogenie transformed hamster cells by amino acid deprivation of cycloheximide treatment. J. Viral. 9, 448-453. KIT, S., DUBBS, D. R., FREARSON,P. M., and MELNICK, J. L. (1966). Enzyme induction in SV40-infected green monkey kidney cultures. Virology 29, 69-83. KRAEMEK, P., PETEKSEN, D., and VAN DILLA, M. (1971). DNA constancy in heteroploidy and the stem line theory of tumors. Science 174, 714-717. LEVINE, M. J., OXMAN, M. N. DIAMANDOPOCLOS,G. T., LEVINE, A. S., HENRY, P. H., and ENDERS, J. F. (1969). Virus-specific nucleic acids in SV40-exposed hamster embryo cell lines: Correlation with S and T antigens. Proc. Nat. Acad. Sci. USA 62, 589-596. LOH~AN, P. H. M. (1968) Induction and rejoining of breaks in the DNA of human cells irradiated at various phases of the cell cycle. Mutat. Res. 6, 449-458. MANTEIIIL, S., PAGES, J., STEHELIS, D., and GIRARD, M. (1973). Replication of simian virus 40 deoxyribonucleic acid: Analysis of the one-step growth cycle. J. Viral. 11, 98-106. MAHM~K, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3, 208-218. MOOHHEAD, P. S., NOHVELL, P. C., MELLMAS, W. J., and BALTIPS, D. M. (1960). Chromosome preparations of leucocytes cultured from peripheral blood. Ewp. Cell Res. 20, 612-616. ORMEHOI), M. G., and STEVENS, V. (1971). The rejoining of X-ray induced strand breaks in the DNA of a murine lymphoma cell (L5178Y). Biochim. Biophys. Acta. 272, 457-462. OZANKE, B., SHAKP, P. A., and SAMBKOOK, J. 11973).

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Transcription of SV40. II. Hybridization of RNA extracted from different lines of transformed cells to the separated strands of simian virus 40 Dh’A. J. Viral. 12, 90-98. POPE, J. H., and ROWE, W. P. (1964). Detection of specific antigen in SV40-transformed cells by immunofluorescence. J. Erp. Med. 120, 121-128. PUCK, T. T., and KAO, F. (1967). Genetics of somatic mammalian cells. V. Treatment with &bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc. Nat. Acad. Sci. USA 58, 1227-1234. REGAL, J. D., TROSKO, J. E., and CARRIER, W. L. (1968). Evidence for excision of uv-induced pyrimidine dimers from the DNA of human ceils in citro. Biophys. J. 8, 319-325. REGAN, J. D., and SETLOW, R. B. (1974). Two forms of repair in the DNA of human cells damaged by chemical carcinogens and mutagens. Cancer Res. 34, 3318-3325. ROTHSCHILD, H., and BI.ACK, P. H. (1970). Analysis of

ET AL SV40 induced transformation of hamster kidney tissue in vitro. VII. Induction of SV40 from trans. formed hamster cell clones by various agents. Viro/ogy 42, 251-256. RI’TTEK, W. J., PICTE.I., R. L.. and MOKHI~. P. W. (1973). Toward molecular mechanisms of developmental processes. Annu. Rev. Hiochem. 42, 601-646. SHATKIN, A. J., REICH, E., FHANKLIN, R. M.. and TAT~M, E. L. (1962). Effect of mitomycin C on mammalian cells in culture. Hiochim. Biophys. Acta 55, 277-289. TEICH, N., Lowv. D. R., HAHTI.E\, J. R.. and ROWE, W. P. (1973). Studies of the mechanism of induction of infectious murine leukemia virus from AKR mouse embryo cells by +5-iododeoxyuridine and 5-bromodeoxyuridine. Virology 51, 163-173. WATKISS, d. F. (1973). Studies on a virogenic clone of SV40-transformed rabbit cells using cell fusion and in situ hybridization. J. Gen. Viroi. 21, 69-81.