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BROMODEOXYURIDINE-INDUCED MUTATIONS 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 cell cycle. 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 thymidine ( 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.

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