BROMODEOXYURIDINE-INDUCED IN SYNCHRONOUS CHINESE HAMSTER CELLS: TEMPORAL INDUCTION OF 6-THIOGUANINE AND OUABAIN RESISTANCE DURING DNA REPLICATION

H. JOHN BURKP AND PAUL M. AEBERSOLD2 Group in Structural Biophysics, Biomedical Diuision, Lawrence Berkeley Laboratory', and Dept. of Molecular Biology2, Uniuersity of California, Berkeley, California 94720 Manuscript received November 14, 1977 Revised copy received April 25, 1978

ABSTRACT Mutations were induced in synchronous Chinese hamster cells by bromo- deoxyuridine (BUdR) incorporated into cells for one-hour periods in the . There was a very pronounced temporal dependence during the first half of the DNA synthesis period for th:! induction of damage leading to 6-thio- guanine (6TG) and ouabain resistance. No mutants above background were induced by exposure to BUdR in GI and G2 cells, and very few mutants were induced in the latter part of the DNA synthesis period. The peak for the induction of 6TG resistance occurs at about two hr in the DNA synthesis period; one hour later there is a peak for the induction of ouabain resistance. Both peaks occur before the time of maximum incorporation of BUdR into DNA. These results suggest that the mutagenesis by BUdR is associated with at least two nuclear genes, which replicate at two hr and three hr in the DNA synthesis period.

AMMALIAN cells replicate their several meters of DNA under culture Mconditions in about six to eight hr. Replicating units of the DNA replicate in two directions at a rate of about 1 micron per minute (HUBERMANand RIGGS 1968). The number of replicating units replicating during any one minute of the DNA synthesis period is on the average about lo4 (PAINTERand SCHAFER1969; PAINTER,JERMANY and RASMUSSEN1966). The time of replication of regions of chromosomes has been shown by many researchers to follow a temporal pattern in the DNA synthesis period (see, for example, STUBBLEFIELD1975; MUKHERJEE et al. 1968). Generally, euchromatic DNA replicates early, and heterochromatic and centromeric regions replicate later (COMINGS1972; UTAKOJIand Hsu 1965; STUBBLEFIELD1975). There are selected intervals in the DNA synthesis period when specialized DNA replicates, e.g., satellite DNA and nucleolus organizer DNA ( STAMBROOK19 74; AMALDI,GIACOMONI and ZITO-BIGNAMI 1969; BOSTOCKand PRESCOTT1971; FLAMM.BFRNHEIM and BRUBAKER1971). The time of replication of DNA appears to be related to organ differentiation (UTAKOJIand Hsu 1965), and there appears to be a specific time in the synthesis period when SV40 DNA is replicated (BALAZS,BROWN and SHILDKRAUT1973).

GeneliLs 90: 311-321 October, 1978. 312 H. J. BURKI AND P. M. AEEERSOLD The association of a particular cellular function with a region of the DNA can be accomplished by damaging a region in synchronous cells with the incorpora- tion of 5-bromodeoxyuridine (BUdR), or related agents such as iododeoxyuridine (IUdR), tritiated ( 3H-TdR), tritiated iodeoxyuridine (3H-IUdR), tri- tiated bromodeoxyuridine (6-?H-BUdR),and 1*51-iododeoxyuridine(IZ5I-IUdR) , into the DNA in short pulses during DNA synthesis. Inducing a change, using BUdR or other thymidine analogues, is usually a proof of function associated with damage to a particular DNA region, although the mechanism for the dam- age is not clear. Once BUdR is incorporated into DNA, damage can be induced by exposure to visible light (CHU.SUN and CHANG1972). These methods have been exploited to suggest that specific genes replicate at specific times during the cell cycle (SUZUKIand OKADA1975; AEBERSOLDand BURKI 1976; and KASUPSKIand MUKHERJEE1977). that “critical DNA” damage occurs in late replication DNA (BURKI 1974 and 1976), and that C virus and Epstein Barr viruses are activated at specified intervals within the S period (BESMERet (2.1. 1974; HAMPARet al. 1973). The work of SU~UKIand OKADA(1975) and AEBER- SOLD and BURKI(1976) suggests that the early DNA synthesis period is critical for mutagenesis. However, the results do not distinguish between a “hot time” in the cell cycle for mutagenesis and individual gene duplication, as suggested by the suppression of enzyme levels in mouse cells as a function of the time of incorporation of BUdR (KASUPSKIand MUKHERJEE1977). If the peak of BUdR-induced mutagenesis were due to a “hot time” for muta- genesis in the cell cycle, one would find that the induction of drug resistance related to two different enzymes occurs at the same time in the cell cycle. If, however, the peak of BUdR-induced mutagenesis were due to DNA damage within different DNA regions, “hot spots” during replication, one could find that different drug-resistant mutations occur at different times in the cell cycle. The drugs chosen to test these hypotheses were 6-thioguanine (6TG) and ouabain. The induction of different peaks for the different loci would also be evidence against other hypotheses to explain previous data, e.g.. thymidine pool effects, epigenetic mechanisms, and mitochondrial DNA (mDNA) replication. [Hamster cell mitochondria appear and disappear at different times in the cell cycle (ROSS and MEL 1972) ; in other species the mDNA replicates in S and G2, (KOCHand STOCHSTAD1967; BOSMAN1971 ; PICA-MATTOCCIAand ATTARDI1972), although no cyclic variation in mDNA replication has been seen after double thymidine block synchrony (RABINOWITZand SWIFT 197O)l.

MATERIALS AND METHODS

Cell culture The CHO cell line was received from L. KAPP and R. KLEVECZ,City of Hope Medical Center, Duarte, California, and had been selected for iise on the cell-cycle analyzer (Talandic Research Corporation). Stocks of this CHO cell line, which we call “CHO-kk”, have been kept frozen in our laboratory since 1976. Fresh cultures are started about four times a year to insure that the genetic changes in the cell lines during growth at 37” are minimal and reproducible from year to year. MUTATIONS IN CHINESE HAMSTER CELLS 313

This cell line has been shown to be free of PPLO, and has a modal chromosome number of 21, as others have reported. The G-banded karyotype has been determined, and shows four abnormal autosomes and only one complete X chromosome (BURKI,unpublished). The popula- tion doubling time of the cells is about 12 hr under optimum conditions with a four-hour G1 period, six-hour S period, and a G2 plus M period of two hours. The cells are grown at 37" in a CO, incubator or in close tissue culture flasks at 37" in McCoy's 5A media, supplemented with 15% fetal calf serum and 100 units per ml of penicillin and 100 micrograms per ml of streptomycin, and 1 miv HEPES buffer to slow pH changes during experiments. Care must be taken to avoid exposure of the cells or the growth medium to normal white laboratory fluorescent light (JOSTES,DEWEY and HOPWOOD1977; BRADLEYand SHARKEY1977), since, with induction of mutations, the population doubling time of the cells may be extended due in part to the killing of cells after fluorescent light exposure. Gold fluorescent light exposure does not cause these effects (BURKIand LAM1978). Synchronization methods Cells were grown for several days in logarithmic growth phase in roller bottles turned at 0.5 rev,"in. They were subcultured into one or more roller bottles at 2 to 3 x IO7 cells per bottle. After 48 hr, the cells were synchronized in a 37" room using a "Cell Cycle Analyzer." (Talandic Research Corporation, Pasadena, California). This apparatus is patterned after the instrument developed by KLEVECZ(1972). The mitotic cells were usually shaken off at one-hour intervals at the following speeds: 54 min at 0.5 RPM, three min at 180 RPM, two min for cell collection, and one min for media replacement in the roller battle. This apparatus thus gives continuous samples of cells spaced in these experiments one hour apart in cycle time. The typical yield per roller bottle is 2 x 106 cells in very early G1. More than 90% of the cells at harvest appear as small doublets. These cells are permitted to attach to 75 cm' plastic tissue culture flasks, and are labelled with BUdR or thymidine according to each experimental format. Cell life cycle progress analysis afier mitotic detachment (a) Cell volume spectrosccpy: The modal size and the volume distribution of the trypsinized synchronous cells at different positions in the cell cycle were determined, using a Coulter Counter Model ZBI matched to a Coulter Channelyzer. This system was calibrated, using 10 micron spheres and ragweed pollen provided by the Coulter Company. An X-Y plotter was used to determine graphically the coefficient of variation cf the distribution of pulses created by the cell populations as a function of the cell-cycle time. The volume of the cells appeared to increase almost linearly until the cells divided after approximately 12 hr. (b) Flow cytometry (flowmicrofluorimetry): Cells at different times after mitotic detach- ment were collected by gentle centrifugation and fixed at OD, using a fixitive containing three parts 200 mM MgCl, + one part absolute alcohol, and kept at 4". They were then stained with chromomycin A3 (100 pgm/ml), (Cal Biochemical), for at least one hr (TOBYand CRISSMAN 1975). Analysis of the fluorescence was made, using an instrument according to the design of STEINKAMPet al. (1973), and described by HAWKESand BARTHOLOMEW(1977). In these experi- ments, the wavelength used for excitation was 457nm at a laser power of 0.6 watts, with approximately 500 cells per second intersecting the beam from the Argon-Ion laser. The coeffi- cient of variation was calculated hy standard methods. Although the coefficient of variation has been as small as 2.5%, it is usually 4% in non3 cells (see text). (c) Uptake of tritiated BUdR: Usually 106 cells were washed three times with cold 10% TCA, and then kept at 90" €or one hr to hydrolyze the cell DNA for a determination of the amount of precursor incorporated into DNA at different times. The radioactive mixture was added to PCS solubilizer for liquid scintillation counting (Amersham and Searle Co.). The absolute counting efficiency for the samples was determined by using tritium quench standards and the channels ratio method. 3144 H. J. BURKI AND P. M. AEBERSOLD Selection for drug resistance Clones resistant to 5 pgm/ml of 6TG or 3 miv ouabain were selected after an expression time of five days had passed. Previous experiments had shown that this was an adequate expression time for 6TG resistance (AEBERSOLD1975). Although more rapid expression times are found for ouabain resistance, it was convenient to use the same expression time as for 6TG. It is important to note that in the usual experiment the cells were kept in log growth for the entire expression Feriod. The usual procedure was initially to use an aliquot of cells of about 2 x IO5 cells, permit growth in a large tissue culture flask for 2.5 to 3 0 days, and then sub- culture to 2 x 105 to 106 cells again in order to grow sufficient cells for challenge on the fifth day. Growth curves of cells after these treatments shoved that at least seven to ten population doubling times occurred under the experimental conditions. The plating efficiency of surviving cells five days after synchiony and BUdR exposure was indistinguishable from stock CHO cells, although initially BUdR is more toxic to early cells than to late s phase cells (KAJIWARA and MUELLER1964.; AEBERSOLD1976). After eight days in the selective drug, the number of clones was determined by counting the number of colonies stained with 1% methylene blue. Experiments were designed to insure that at least 100 clones above background were obtained after treatment at peak times for BUdR mutagenesis to insure 10% statistical reliability at the peak time of mutagenesis. Thus, five plates were inoculated at 2 x 106 and at 2 x IO5 cells per plate for ouabain and 6TG selection, respectively.

RESULTS CHO-kk cells were detached during mitosis from glass roller bottles. using the cell-cycle analyzer. The peak of the size distribution was determined, and the data from a typical experiment are given in Figure 1. In other experiments not reported here, the synchronous change in cell volume as a function of time con- tinued for at least the next generetion, with a very slow rate of decay of the degree of synchrony in the cell population. The progress of the synchronous CHO-kk cells was also followed through the cell cycle, using the flow cytometric method (flow microfluorimetry) ; the data are shown in Figure 2. The first panel shows the initial distribution of fluores- cence of the population one hr after mitotic detachment. The amount of fluorescence corresponds to (he amount of dye bound by the DNA in the cell. If this dye binding is stochiometric for the cellular DNA content under these conditions, then this first panel shows a population that is 99% in the G1 stage, with one unit of DNA per cell. The coefficient of variation of this group is about 4%. The cells proceed through the S period, as seen in the panels corresponding to 4, 5.5, and 7 hr, respectively. The coefficient of variation characteristically increases through the S period to 15% during mid S in these cells. Most of the cells are in G2 or mitosis after 10.0 hr. and half the population has divided at 11.5 hr. After 13 hr, the cells are again in the G1 state of the cycle, although the coefficient of variation has increased slightly to about 6% on the main peak, and a small portion (5%) of the cells appear to be moving through the cycle more slowly than most of the population. This data may be summarized in terms of the peak channel corresponding to the DNA content and the time in the cell cycle since mitotic detachment. In the experiment shown in Figure 3, the cell-life cycle corresponds to a G1 period of 4 hr, a S period of about G hr, and a G2 plus M period of about 1.5 hr. MUTATIONS IN CHINESE HAMSTER CELLS 315

r 1 I I 1 I I I 1 I I I I I I

- su+ Q) c c 0 0 0 40-

Y 0 0 z30- il 1I I I I I I I I I I I Hours after shake off XBL771-3015 FIGURE1.-Individual sample cultures at various times from the time of shake-off were trypsinized, and aliquots of the cells were counted on a Model ZBI Coulter counter equipped with a 100 x 120 micron orifice. The distribution of pulses, proportional to cell volume, was analyzed, using a 100-channel analyzer (Coulter Channelyzer), and the peak channel was recorded for each sample of cells.

For mutagenesis of synchronized cells, some of the various cell population samples were exposed to one-hour pulses of 0.5 mM BUdR or tritiated BUdR, 1 pC/ml at 0.5 mM. After labelling and washing, the cell number, cell volume, DNA content, and uptake of tritiated BUdR were determined. The results for controls exposed to one-hour pulses of 0.5 mM thymidine are shown in Figure 4. The average frequency for the whole cycle given by the dotted line was 0.33 x 2 0.3 x for 6TG, and 0.5 X 0.5 x for ouabain. The individual means for drug resistance in the cycle were not significantly dif- ferent from the cycle mean. The induction of mutations by exposure to BUdR for one-hour pulses is given in Figure 5. There is a peak for the induction of mutations to 6TG resistance at six hr after mitotic detachment; the peak is approximately 50 times the back- ground frequency. Ouabin resistance is induced maximally at seven hr after mitotic detachment at a rate 25 times background. The peaks are separated by about one hour in the DNA synthesis period. These peaks do not correspond to the peak time for incorporation of tritiated BUdR into DNA, which occurs at 316 H. J. BURKI AND P. M. AEBERSOLD - -FMF DATA I .o 2.5

4 5.5 7.0 i 13.0

d I

XBL771-3013 FIGURE2.-Individual sample cultures at various times after shake-off were trysinized, and aliquots of fixed cells stained with chromomycinL A3 were analyzed, using the FCM method. The data from a typical experiment for the distribution of fluorescence (proportional to DNA content) are given on the nine panels. The maximum number of cells in each channel analyzed was 4,000. The numbers in each panel are the times, in hours, after detachment. The abscissa is the amount of fluorescence recorded. - E 648 - b- ..- - r lifll- T -I L IIW LL c c r0 0 Y 0 70 Q) a, 1.1 T 1 I I I I 0 2 4 6 8 IO 12 14 Hours after shake off XBL771-3014 FIGURE3.-The data of Figure 2 can be used to determine the average DNA in the samples (proportional to fluorescence) as a function of time after selective detachment of the cells. The mean and the coefficient of variation of the mean is given in the graph. These data, combined with data on the incorporation of tritiated BUdR (Figure 6, left panel) enable the life-cycle parameters of the cells to be determined. about nine hr, as seen in Figure 6. For convenience, the S period is taken as the time when there is significant uptake of 6-’H-BUdR (5% of peak value) , since some cells begin synthesis slightly earlier than others. Induction of mutation with the peaks normalized to 1.0 is also shown in Figure 6. The peaks within the DNA synthesis period are at two hr for 6TG and at three hr for ouabain. These results are very similar to previous results with CHO cells after mitotic selection and hydroxyurea resynchrony at the G1/S boundary (AEBERSOLD 1975). There are also some smaller peaks above background in the S period, which appear to be significant and which may be related to 5% of the population that transverses the cycle at a slower rate. There do not appear to be any signifi- cant peaks for BUdR mutagenesis in the G1 period.

DISCUSSION The induction of mutations due to exposure of cells to BUdR during DNA synthesis occurs at specific times in the first half of the DNA synthesis period. BUdR-induced mutation to 6TG resistance is maximum about two hr after the initiation of DNA synthesis; one hour later there is a peak in induced ouabain 318 H. J. BURKI AND P. M. AEBERSOLD ..

Lu Hours after mitotic detachment XBL778-3665 FIGURE4.-Synchronized cells were exposed to one-hour pulses of 0.5 mM thymidine and grown in a dark 37" walk-in incubator. After five days, the cells were challenged to 6TG or ouabain (OUA). The dotted lines are the average number of induced drug-resistant clones throughout the complete cell cycle.

U 4 0 IL U L) 0 IL Hours after mitotic detachment XBL778-3666

FIGURE5.-Synchronized cells were exposed to one hr pulses of 0.5 mM BUdR. After an expression time of five days, cells were challenged to either 6TG or ouabain (OUA) . The values are the mean and standard deviation from a typical experiment. Background levels are not sub- tracted in this graph. Note that the vertical scale is different than that of Figure 4. MUTATIONS IN CHINESE HAMSTER CELLS 319 to Hours after commencement of DNA synthesis 0 X 02468 0246 .-c 7 E 2.0 - -1 al 0 \ 1.5 .-0 t g 1.0 t .-c .-v) U E 0.5 .-3 .-t $0 4 8 12 0 4 8 12 Hours after mitotic detachment

XBL77S-3664 FIGURE6.-Left: Samples of cells were permitted to incorporate 3H-BUdR at 0.5 miv and 1 pCi/ml, under the same conditions in which mutations were induced. Right: Induction of 6TG resistant clones (dotted line, closed circles) or ouabain resistant clones (solid line, open circles) are plotted for comparison with the peaks arbitrarily set to 1.0. resistance. There does not appear to be any induction of drug resistance for cells that are exposed to BUdR in other phases of the cell cycle or in the late S period. These data constitute strong evidence that the induction of drug resistance is due to BUdR-induced mutations in the gene or genes associated with the production of the HPRT enzyme and the Na+ K+ ATPase enzyme. They do not support the hypothesis that there are “hot times” for mutagenesis in the cell cycle in the early S period. It is also difficult to explain these data on the basis of epigenetic events, mitochondrial DNA mutation: or thymidine pool effects. The simplest explanation is that mutations or premutational lesions are introduced to specific genes during their replication. It is not surprising that these results and those of others (SUZUKIand OKADA1975) show that nuclear genes coding for various endpoints are replicated in the first half of the DNA synthesis period. It has been long known that euchromatic DNA, which codes for messenger RNA, replicates earlier in the DNA synthesis period than does heterochromatic, centromeric, or pericentromeric DNA. Although the mechanism for the induction of mutations by BUdR in mam- malian cells is not yet known, the induction rate as a function of BUdR concen- tration is not linear (AEBERSOLD1976). This nonlinearity is similar to results seen in bacteria (RYDBERG1977). Bacterial studies with repair-deficient stains suggest that “mismatch repair” occurs, leading to base transitions in the DNA after the incorporation of BUdR. This may be a good preliminary hypothesis to test for the mechanism of BUdR mutagenesis in mammalian cells. 320 H. J. BURKI AND P. M. AEBERSOLD We originally proposed to study the functional organization of the nucleus, using incorporated radioisotopes and related agents with the hope of being able to study intranuclear regions associated with specific cell functions. By damaging these regions, we hypothesized that we could study such phenomena as intra- nuclear repair, mutagenesis, and intranuclear toxicity. The results reported here, together with previous reports of “critical DNA” within the nucleus, suggest that intranuclear damage studies are feasible. The data in this paper suggest that temporal replication of genes can be demonstrated in Chinese hamster cells. The competent technical assistance of MILDREDHUGHES, JAN CURTIS, and VELMAMCNEAL is greatly appreciated. I also thank D. LEACHand G. WALPOLEfor the preparation of the manu- script. This work was supported by Public Health Service Grant #14310, and by the Division of Biomedical and Environmental Research of the U.S. Department of Energy.

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