411
Mutation Research, 250 (1991) 411-421
© 1991 Elsevier Science Publishers B.V. All rights reserved 002%5107/91/$03.50 ADONIS 0027510791001994 MUT 02545
Cell-cycle dependent mutation of human lymphoblasts: Bromodeoxyuridine and butyl methanesulfonate Henry Hoppe IV, Katherine M. Call, Phaik-Mooi Leong and William G. Thilly Massachusetts Institute of Technology, Centerfor Environmental Health Sciences, Cambridge, MA 02139 (U.S.A.)
Keywords: Cell-cycledependent mutation; Lymphoblasts,human; Bromodeoxy-uridine; Butyl methanesulphonate
Summary Cells of the human lymphoblast line WI-L2 and its derivative TK-6 were synchronized by centrifugal elutriation and cell-cycle dependent mutation to 6 T G R ( H P R T ) and O U A R (Na ÷, K + ATPase) measured. Bromodeoxyuridine induced 6TG R and O U A R mutations within S phase while butylmethylsulfonate induced mutation displayed no cell-cycle dependence. The data indicate that centrifugal elutriation is a facile means to obtain a useful degree of synchrony for these cell lines.
What are our reasons for studying the cell-cycle dependence of induced mutation? There is really only one reason. We wish to discover something of value in preventing diseases that begin with genetic change. T h e r e are two classes of disease which require genetic changes: those which can arise solely from inherited mutations and those which are combinations of inherited and somatic mutations. The former class include the thousands of dominant or recessive single gene mutations such as sickle cell anemia or cystic fibrosis. The latter include cancers, atherosclerosis and possibly the degenerative changes of age. The ceils in which heritable mutations occur comprise all linear descendants of a fertilized egg to the spermatocytes or the oocytes of an individual. Somatic mutations may arise in any cell division after fertilization in any organ. Each organ is
Correspondence: Dr. William G. Thilly, Massachusetts Institute of Technology, Center for Environmental Health Sciences, Cambridge, MA 02139 (U.S.A.).
a mosaic of differentiated cells operating cooperatively to achieve a physiologic function. Each of the differentiated cell types expresses an idiotypic set of genes and, in adults, maintains a constant cell number. This maintenance of a constant cell number is one of the wonders of physiology. Some cell types such as the crypt cells of the intestine are in a state of continuous cell division with losses by exfoliation matched for a lifetime by compensating cell division. Or is it that exfoliation exactly matches the products of cell division? A similar but slower process describes the squamous epithelium of the skin. Red blood cells have finite lifetimes after differentiation from the descendants of a pluripotent marrow stem cell. Some cell types in the nervous system may have zero capacity for cell replacement by cell division and were presumably distributed in early life in highly redundant physiologic arrays in which the deaths of a few individual cells would not inactivate the network. Because of these many division states of cell types in the body - - from continuous to zero - and because we really don't have a quantitative
412
picture of the kinetics of cell death and replacement in any human tissue, this area of research is bound to be speculative. Consider the rapid cell divisions of early embryonic life. The rate of gene duplication is prodigious. The fertilized egg appears to be packed with DNA-repair enzymes (Pfeiffer et al., 1988). But who has measured the rate or kinds of genetic change which occur in the first thirty divisions of life? During this period the single cell gives rise to 109 cells already differentiating into the rudiments of tissues and organs. Any mutation which does occur in the first precursor cells of an organ are subject to the occurrence of jackpot mutations as surely as Luria's bacterial populations and would be expected to be on the most probable pathway to diseases requiring more than one somatic mutation (Luria and Delbruck, 1943; Thiily, 1988). In organs maintaining a constant cell number with relatively infrequent cell division, the problem arises as to whether it is reasonable to regard the population of cells of the same histoiogic type as homogeneous. Aside from the variations in cell physiology from stem to a parenchymal cells, there is the fact that cells of identical physiology could differ enormously with regard to the time before their next cell divisions. Thus it becomes incumbent upon mutation researchers to think beyond the histologic separation of cell types to the dynamics of cell death and replacement and its consequences in mutagenesis: At the molecular level the availability of the D N A tbr chemical reactions may be affected by the replication or transcription state of the sequence in question. T r e a t m e n t s of cells by chemicals or irradiation can either increase or decrease the toxicity of subsequent treatments. (Thilly and Heidelberger, 1973; Kaden et al., 1987). These changes could come from expression of new enzymic activities or from structural changes in a D N A target or both. Certainly we must consider the possibility that the level of constitutive D N A (chromatin) repair systems will vary as a function of a cell's temporal position relative to its next division. The same may be said for the inducibility of repair functions and the well-known but little-understood p h e n o m e n a of cell-cycle progression delay (Plant and Roberts,
1971 a,b).
Generalizations have their place in describing these kinds of problems but an arithmetic example may be more compelling. Let us suppose that treatment of C H O cells with a mutagen at a subtoxic level has induced a mutant fraction of 10 4 by forward mutation of a gene like hprt. It the same experiment were carried out in a Chinese hamster ocary using the same number of DNA-reaction products per base pair one would expect ItXX) mutants in a 107 cell sample. However, if only 10 -" or 10 3 of the cells of the type chosen were within a day of their next cell division and repair of the potentially mutagcnic lesions proceeded with a half-life of 24 h then the outcome of the experiment would be 1(I or fewer mutants. Recognition of this kind of problem - - and extending it to treatments which change the number of cells proceeding to division - - is a fairly humbling experience for scientists who like to think that our common trick of using rapidly dividing microbes or mammalian cells in culture is the pathway to understanding. This paper, however, is about a preliminary study of induced mutation in continuously dividing cell human 13 cell lines. There is nothing preliminary about the depth or quality of the data, but it is preliminary in the sense that we recognize that the responses observed are only a beginning. We are working on a means to reversibly return B lymphoblasts to a non-dividing lymphocytic state in which we may be able to study the effect of mutagens on these ceils as a function of time between treatment and cell division over a period of weeks and months. This kind of experiment with UV-irradiated human fibroblasts has been performed (Maher et al., 1979) and extended to chemical mutagens (Yang et al., 1980) but has not been repeated by any other laboratory or extended to other cell types despite its apparent importance in building models of in vivo mutagenesis. As we move to the end of this century key mechanistic questions about mutation are being asked at the level of individual D N A sequences. What are the kinds, amounts and positions of D N A adducts as a function of D N A sequence within a gene and among similar sequence dispersed over transcribed and non-transcribed chromosomal regions'? What are the kinetics of
413
removal and subsequent repair of such reacted DNA bases? What is the resulting frequency of mutation or unrepairable block to DNA synthesis? What are the roles of the few known DNArepair enzymes and surely greater number of unknown chromatin/DNA-repair enzymes in this process? All of these questions have hidden within them the need to understand cell-cycle dependent changes in chromatin structure, amount and inducibility of repair enzymes, and the role and mechanism of progression delay in mutagenesis. The truth is that a relatively small number of mammalian cell lines have been used both in environmental mutagen assays and in studies of mutational mechanisms. Two of these cells lines, the hamster CHO line and the human TK-6 line are today frequently cited in both kinds of mutation studies. An area of general interest to mutation researchers but not frequently the subject of experimental reports however is the dependence of mutation on the cell cycle. That cell-cycle position was important in determining sensitivity to cell killing has been known since Terasima and Tolmach (1973a,b) reported that Hela cells synchronized by their new method of mitotic shakeoff were especially sensitive to killing by X-rays in G 2 phase and mitosis. After methods were developed to accurately measure induced point mutant fractions in mammalian cells similar experiments were possible with regard to mutation. The earliest of this genre explored the cycle dependence of bromodeoxyuridine (Aebersold and Burki, 1976; Carver et al., 1976; Burki and Aebersoid, 1978; Nakamura and Okada, 1979) induced mutations in rodent cell lines. Since that time only a few citations can be found for cell-cycle dependence of mutation despite an explosion in mammalian cell mutation studies. Among these are studies of UV light (Riddle and Hsie, 1978; Burki et al., 1980; Wood and Burki, 1982) ionizing radiation, (Burki, 1980) alkylating agents (Goth-Goldstein and Burki, 1980; McCormick and Bertram, 1982; Crespi et al., 1982; Smith et al., 1988), aflatoxin B~ (Kaden et al., 1987), nitroso acetyl amino fluorene, and benzopyrene diol epoxide (Smith et al., 1988; Lin and Kuo, 1990) and deoxyribonucleiotide pool imbalances (Mun and Mathews, 1991). Chief among the reasons for this low number
of papers must be death of H.J. Burki whose laboratory accounted for half of the published literature in this area. Another reason appears to be that while the importance of cycle-dependent cell killing is generally appreciated in optimizing protocols for cancer radio- and chemo-therapy, the role of cycle dependent mutation in causation of mutation leading to cancer and heritable mutations is not widely appreciated, A final reason for experimental inactivity appears to be that cell cycle mutation experiments are perceived as technically difficult to perform. The technical barrier to obtaining sufficient cell numbers (108 cells, z.b.) for facile cell cycle studies in CHO cells was essentially eliminated by the combination of microcarrier technology with of Terasima and Tolmach's (1963) mitotic shakeoff technique (Crespi et al., 1982). Mass synchronization of suspension cell lines such as HeLa was possible with careful matching of DNA inhibition to cell growth parameters (Thilly, 1976) however, repeated efforts to apply this approach to human lymphoblast lines have not succeeded. An alternative synchronization method based on Lindahl's 1948 design of a counter streaming centrifuge was introduced in 1977 (Meistrich et al., 1977; Mitchell and Tupper, 1977; Sanderson and Bird, 1977). The necessary equipment, though not inexpensive, is commercially available. This method, centrifugal elutriation, separates cells on the basis of differential sedimentation velocity and should yield a degree of synchrony and cell number sufficient for a wide variety of quantitative mutation studies. This method provides the synchronous human B cell populations which we believe will be necessary for key future experiments with human lymphoblastoid lines. Materials and methods
Cells The WI-L2 line (Levy et al., 1968), was provided by Dr. Arthur Bloom Columbia College of Physicians and Surgeons, New York. A subpopulation (HHIV) capable of forming colonies in the absence of feeder layers was used in these experiments. TK-6 (aka H2BT) was isolated as a thymidine kinase heterozygote from this population (Skopek et al., 1979). Cells were grown in RPM1
414 1640 media supplemented with 10% horse serum. Average doubling times for both lines were 18 h.
Chemicals 5-Bromodeoxyuridine (BUdR) and [3H]thymidine were obtained from New England Nuclear (Boston, MA), butyl methylsulfonate (BMS) from Pfaltz and Bauer (Stanford, CT), DNAase I, Worthington, (Feerhold, N J).
The sample to be separated (4 x 108 cells) was suspended in I(X) ml complete medium and placed in the sample reservoir. It was pumped into the separation chamber in the rotor at 10 m l / m i n . After the loading of the sample medium from the buffer reservoir was pumped at incremental pump speeds of 4 m l / m i n and samples collected. The total sample collection amounted to a volume of 250-300 ml. The last p u m p speed at which samples were collected was 38 m l / m i n .
Synchronization by centrifugal elutriation The basic components for centrifugal elutriation were a Model J-21B Centrifuge (Beckman Instruments) and a Cole-Palmer Masterflex Pump (Cole-Palmer Instrument Co., Chicago, IL). The controller for the p u m p was modified by the replacement of the supplied speed regulator with a 10-turn 2-ohm millipot (M.I.T. Electronics Shop). This modification allowed small, reproducible adjustments of the p u m p speed. The rotor was assembled as instructed in the manual. The partially assembled rotor was autoclaved at 275 o C, 15 p.s.i, for 45 min. The gasket for the separation chamber was not able to be autoclaved and was therefore sterilized by immersion in 70% ethanol for several hours prior to assembly. The assembly of the rotor was completed in an aseptic manner in a still air hood. The rotor was flushed with 70% ethanol and warmed to 37 ° C for several hours before use. The centrifuge was set up next to a still air hood and 37 ° C water bath. Sufficient tubing was present to allow sterile manipulations within the hood. The buffer reservoir, sample reservoir and collection flask were kept in the water bath. Just prior to use, the assembled rotor was placed in the centrifuge and the appropriate connections was made. Air bubbles were flushed from the system and the p u m p speed was recalibrated. Separations were done at 19 600 rpm. The speed was checked stroboscopically. N.B. Use of horse serum was found to be associated with precipitation during operation of the elutriator. These problems were overcome by heat treating the horse serum (57 ° C x 2 h) followed by a 1-h incubation at 3 7 ° C with filter sterilized DNAase I, 300 units/ml. The use of DNAase treatment was essential to clean separations.
Cell counts and t,olume determinations The number of cells and their volume distributions were determined using differential threshold settings on a Coulter electronic particle counter. Latex spheres of known diameter were used to calibrate the process.
DNA synthesis The amount of [3H[thymidine incorporated into acid-precipitable materials was measured as an estimate of D N A synthetic rate. Cells were incubated for one hour in the presence of 1 /xM ~H-TdR (500 C i / m o l e ) . The culture was resuspended in fresh medium with treated serum, 2 x 107 cells were removed for the asynchronous control, and the remainder was separated according to volume by centrifugal elutriation. The asynchronous control and 2 x 107 cells from each of the samples post-elutriation were resuspended in 5% trichloroaretic acid at 4 ° C and allowed to sit on ice for at least 20 rain. An aliquot from each sample was filtered onto glass filter which were held in a standard filter-holding apparatus. The filters were washed with 20 ml T C A at 4 ° C . followed by 20 ml of 95% ethanol. The filters were then placed in glass scintillation vials and allowed to dry overnight and scintillation counting performed the next day.
Assays for suruieal and mutation Assay for survival and mutation to resistance to 6-thioguanine (6TG), ouabain ( O U A ) or trifluorothymidine (F3-TdR) were performed and analyzed according to the methods reported in Furth et al. (1981). This approach involves distributing cells directly into microwells in the presence or absence of selective conditions and using the Poisson distribution to calculate the mean and
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Fig. 1. Size distribution of WI-L2 cells separated by centrifugal elutriation. Modal volumes increased from 310 ~3 to 810 /z3 in approximately 100-.. 3 increments with each fraction. The TK-6 clone separation parameters and results were identical to WI-L2. Fraction 0 contained many dead cells and cell debris. Fractions 5 and 6 contained cell doublets.
standard deviation of resulting estimates of surviving and mutant fractions. Estimates of mutant fractions and their dispersion were analyzed according to Leong et al. (1985) and found to lie within the expected range of outcomes based on numerical variation. Treatment of asynchronous populations with mutagens for 1 hour immediately preceeded elutriation. Results
Tritiated thymidine uptake, Fig. 3, revealed that all fractions contained some cells in S phase but fractions 2 and 3 were significantly enriched. Microscopic examination of all fractions revealed that fractions 4, 5 and 6 with the greatest V
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Synchronization The separation of these cells bases on sedimentation velocity may be regarded as moderately successful in that fractions of successively greater modal volume were eluted (Fig. 1) and found to have successively longer periods between elution and cell division as indicated in Fig. 2. That it required 6 h of a 18-h doubling time for each cohort to pass through mitosis is consistent with the large overlap among fractions with regard to size distribution, i.e. cell size is not an exact indicator of cell-cycle position.
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Fig. 2. Growth curves of four fractions of TK-6 cells separated as in Fig. 1. The initial modal volume of the fraction marked by hexagons was 800/.L3, by triangles 650 p3, by squares 500 p.3 and by circles 400 p3. Growth of the parent line WI-L2 after separation was identical to TK-6.
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Butyl methanesulfonate The toxicity and mutagenicity of BMS to the WI-L2 line and its TK-6 thymidine kinasc hctcrozygote subclone arc shown in Figs. 4 and 5 respectivcly. Survival may reasonably be described as log linear and mutagenicity linear for both lines and genes studied. No significant difference in the concentration-dependent toxicity between WI-L2 and TK-6 cells was found. Treatment with 5 mM BMS for 1 h resulted in relative survivals of about 0.65 or close to 0.5 lethal hits per ccll. Mutation responses were however significantly different for the two cell lines. The slope of the 6TGR mutant fractions a function of initial BMS concentration was 4.7 x 10 -~' cell m u t a t i o n s / m M
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model cell volume contained many doublets which were converted to single cells upon resuspension in the spinner cultures. Formation of these doublets in the elutriator is a technical limitation to be considered in interpretation. The proportion of cells which actually underwent cell division after elutriation varied among fractions. 75% of cells a p p e a r e d to double synchronously in the fraction containing the largest ceils (4, 5, 6) while a somewhat smaller proportion were involved in cell division in the fractions containing smaller ceils (Fig. 2). This proportion of cells actually undergoing cell division varied among experiments and in some runs a fraction less than 50% divided despite the fact that the cells were in exponential growth and had high ( ~ 50%) cloning efficiency both before and after elutriation. Since no cell division occurred in the first hour after separation for any fraction, it is probable that the elutriation process occasioned some cell-cycle delay.
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BMS for W I - L 2 cells but was 9 . 0 × 10 -6 for TK-6. Similarly the slope of induced O U A ~ mutations was 5 x 10 -7 for W I - L 2 and 1.8 x 10 -~ for TK-6. T h e ratio of slopes O U A R / 6 T G R for WIL2 was thus 5 x 1 0 - 7 / 4 . 7 x 10 -6 = 0.11 while this ratio was 1.8 x 1 0 - 7 / 9 x 10 -6 = 0 . 0 2 for T K 6 cells. Because it was possible to measure mutations at the thymidine kinase locus in T K 6 ceils this assay was also performed. It was found that the slope for tk mutation (F 3 T d R R) was identical to that seen for hprt ( 6 T G a ) . That W I - L 2 and TK-6 lines show differential sensitivity at both the hprt and N a +, K ÷ A T P a s e genes is statistically significant but s o m e w h a t disconcerting for the assumption that they are isogenic with regard to the genes involved in mutagenesis induced by BMS (Liber et al., 1982). W h e n the elutriated fractions were examined for BMS killing and mutation to 6TG R in two i n d e p e n d e n t experiments a greater sensitivity was reproducibly found in fractions with smaller cells than with larger. No significant difference in in-
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Fig. 5. Concentration dependence of BMS toxicity and mutagenicity to TK-6 cells in a l-h exposure of asynchronous populations. Triangles, 6TG R, squares, F3 TdR R, and circles, OUAR mutant fractions.
W I - L 2 and TK-6 cells differ markedly in their response to B U d R in the medium. Since TK-6 cells have only 50% o f the thymidine kinase activity of W I - L 2 cells (Skopek et al., 1978) D N A synthetic pathway this may not be surprising. I n d e e d t r e a t m e n t o f W I - L 2 cells with 200 tzM B U d R for 1 h p r o d u c e d a 6 T G R m u t a n t fraction of 4 × 10 -3 but no increase in 6 T G R m u t a n t fraction was seen in TK-6 cells even when expo-
418
sure was extended to 2 h. Independent study (H.L. Liber, Ph.D. Thesis) has shown that under these conditions BUdR uptake into the D N A of both cell lines is saturated above 5 0 / z M and that at 200 izM for 2 h WI-L2 cells have twice was much BUdR in their D N A as TK-6. Since TK-6 cells were not mutated at all by BUdR we were limited in its study to the WI-L2 parent line. As shown in Fig. 7 the concentration dependence of B U d R toxicity to asynchronous WI-L2 cells was complex but monotonically decreasing. For induction of mutation to 6TG R or O U A a no increase was observed at 50 IzM but then mutant fraction rose linearly with B U d R concentration from 50 to 200 /xM. The ratio of slopes in this range between the two gene loci was 0.15 similar to the ratio 0.11 observed for the two loci when mutated by BMS.
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Cell-cycle dependence of BUdR toxicity and mutagenicity was performed using the elutriated fractions as in Fig. 1 with a l-h exposure to 200 /xM BUdR. As seen in Fig. 9 smaller cell fractions were more susceptible to killing than larger ones. A clear but curious non-correspondence between a fraction's ability to incorporate tritiated thymidine (Fig. 3) and its susceptibility to B U d R killing (Fig. 8) is evident. With regard to mutagenicity two independent experiments measuring 6TG R gave comparable results. Fraction 3 which displayed the highest tritiated thymidine uptake also had the maximum B U d R induced 6TG R mutant fraction in both experiments. A displacement of O U A R mutation
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to a larger cell fraction seems evident and is confirmed by the ratios of ouabain R to 6TG R in fractions 2 through 6. Fig. 9 compares tritiated thymidine uptake (Fig. 3) to BUdR induced mutation (Fig. 8) directly. It seems clear that the BUdR mutational effect is restricted to a narrower size range of WI-L2 cells than thymidine uptake. Discussion
In dealing with data purporting to describe cell-cycle dependence of killing or mutation, one should first check the degree of synchrony achieved and investigate the effects of the synchronization procedure used on untreated ceils. In the use of centrifugal elutriation we found that background mutant fraction was not affected by the procedure but that some interruption in cellcycle progression occurred (Fig. 2) as well as some loss of cells' ability to grow as isolated colonies. The degree of synchronization may be formally calculated by the method of Engelberg (1964) or
its graphic equivalent (Bakke and Petterson, 1976). Here it suffices to point out that the smallest cell fraction representing Gt phase cells required some 8 h to complete a 'synchronous' wave of division in an 18-h doubling time. This may be compared to some 5-h duration for the division wave 18 h after mitotic selection of CHO cells by shake-off from microcarriers (Crespi et al., 1982). Centrifugal elutriation does not achieve the degree of synchrony of mitotic shake-off but does yield a significant degree of synchrony sufficient for many purposes. Tritiated thymidine labelling shows a clear differentiations among fractions (Fig. 3) although the difficulty of cell doublets contaminating the largest cell fraction must be noted. Interpretation of survival curves must account for the observation that cells in the larger cell fractions had a somewhat higher probability of subsequent cell division than those in smaller cell fractions (Fig. 2). The apparent lower relative survival of smaller cell fractions after BUdR or BMS treatment may therefore arise from actual cycle-dependent sensitivity to treatment or a synergistic interaction between the toxicity of the synchronization procedure and that of the treatment. With regard to mutagenicity these problems are reduced since all determinations of mutant fraction are made after the treated populations have returned to exponential growth phase and have a colony-forming efficiency equal to that of untreated control populations. That BMS had no significant cell-cycle dependent effect on mutation to 6TG R is evident in Fig. 6. The alkylating agent EMS showed a modest (_< 2 x ) cell-cycle dependence for mutation in mitotically synchronized CHO cells with a minimum sensitivity in S phase (Crespi et al., 1982). The lower degree of synchrony obtained by centrifugal elutriation would have presented observations of so small a differential in the experiments reported here. Because TK-6 cells do not remove or repair O6-methyl guanine or any other methyl base product except 3-methyl adenine (Goldmacher et al., 1986) a lack of cell-cycle dependent mutation may be regarded as consistent with a model in which the time between adduct formation and DNA replication is inconsequential. This same
420
cell line showed no cell cycle dependence on aflatoxin B~ induced mutation (Kaden et al., 1987). BUdR did show clear cell-cycle dependence for mutation as would bc expected for a pyrimidine analogue but, of course, the actual mechanism of BUdR mutagcncsis - - replacement of thymidinc or deoxycytidinc or both - - is not yet settled and awaits future mutational spectra studies. Sensitivity to 6TG R or OUA ~ mutation appear to have different cell-cycle positions in BUdR mutagenesis (Fig. 8) a result also seen in Chinese hamster ceils (Crespi et al., 1982). That the cycle dependence of H P R T vs. Na +, K* ATPase gene mutation can in fact be observed in elutriation-synchronizcd B cell culturcs indicatcs that the degree of synchrony achieved will be useful in future work. It is also clear that in the experiment shown in Fig. 9 that the period of sensitivity to BUdR mutation is shorter than the entire S phase consistent with the synthesis of the hprt genc at a reproducible point in S phase. This demonstration gives the experimenter an estimate of the resolution to be expected using these cells and centrifugal elutriation in future experiments. Our understanding of why different replicon clusters are synthesized at specific points S phase is still rudimentary. Do the rules of mutation differ among genes duplicated at different S phase points? Only by doing the challenging experiments with synchronized populations of sufficient size for quantitative mutation assays can we expect to find out. Failure to account for cell-cycle dependence puts off, perhaps for another generation, understanding of key issues in mutation mechanisms. The decision not to do cell-cycle studies does avoid some technically difficult experiments. The method of Ng ct al, (1980) provides a facile means to obtain the necessary numbers of synchronized cells for Chinese hamstcr cells. We hope that this paper demonstrates that a facile technology is available and can bc readily applied to the human B cell lymphoblastoid lines.
Acknowledgments We gratefully acknowledge the support of the office of Health and Environmental Research
U.S. Department of Energy (DE-FG02-86ER 60448) and the U.S. National Institute of Environmental Health Sciences, (P30-ES021(}9, P01ES3926) HHIV and KMC were NIEHS Predoctoral Trainees.
References Aebersold. P.M. (1976) Mutagenic mechanism ot 5bromodeoxyuridine in Chinese hamster cells, Mutation Res.. 36, 357-362. Acbersold, P.M.. and H.J. Burki (1976) 5-Bromodeoxyuridine mutagenesis in synchronous hamster cells. Mutation Res,. 40, 63-66. Bakke, O., and E.O. Petlersen (19761 A fasl and accurate method R~r calculating Engelberg's synchronization index. ('ell Tissue Kinet., 9, 389393. Burki, H.J. (19811) Ionizing radiation-induced 6-lhioguanineresistant clones in synchronous CI-tO cells, Radiat. Res.. 81(1), 76-84. Burki, I I.J., and P.M. Aehcrsold (19761)Bromodet~x,'yuridineinduced mutations in synchronous Chinese hamster cells: temporal induction of 6-thioguanine and ouabain resistance during DNA replication. Genetics. 9t1, 311-321. Hurki, lI.J.. C . K . I . a m and RD. Wood (1980) [.;V-Light induced mutations in synchronous (711(.) cells, Mutation Res., 6cR2). 347-356. Carver. J.H., W.C. Dewey and L.E. tlopwood (1976) X-Rayinduced mutants resistant to b~-azaguanine. 11. Cell cycle dose response, Mutation Res., 34, 465-480 Crespi, ('.1,., and W.G. Thilly (1982) Selection of mitotic Chinese hamster ovary cells from microcarriers: ('ell cycle-dependent induction of mutation by 5-bromo-2'-deoxyuridine and ethyl methanesulfonate. Mutation Rcs., 106, 123-135. Engelberg, J. (1964) Measurement of degrees of synchronization in cell p~pulations, in: E. Zeuthen (Ed.), Synchmny in ('ell Division and Growth. lnterscience, New York. Furth, E.E.. W.G. Thilly, B.W. Penman, 11.L. I,iber and W.M. Rand (1981) Quantitative assay for mutation in diploid human [ymphoblasts using microtiter plates, Anal Biochem., 110. 1-8. Goldmacher, V.S., R.A. Cuzick Jr. and W.G lhiHy (lqgt~) Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, J. Biol. ('hem., 261(27), 12462-12471. Goth-Goldstein, R., and H.J. Burki (19801 Ethylnitrosoureainduced mutagenesis in asynchronous and synchronous Chinese hamster ovary cells. Mutation Res., 69, 127--137. Kaden, D.A., K.M. (.'all, P.-M. Leong, E.A. Komives and W.G. Thilly (19871 Killing and mutation of human lymphoblasts cells by aflatoxin B~: Evidence for an inducible repair response. Cancer Res., 47, 1993--2001. l,eong, P.-M., W.G. Thilly and S. Morgenthaler (19851 Variance estimation in single-cell mutation assays: Comparison
421 to experimimtal observations in human lymphoblasts at 4 gene loci, Mutation Res., 150, 403-410. Levy, J.A., M. Virolainer and V. Defendi (1968) Human lymphoblastoid lines from lymph node and spleen, Cancer, 22, 517-524. Liber, H.L., and W.G. Thilly (1982) Mutation assay at the thymidine kinase locus in diploid human lymphoblasts, Mutation Res., 94, 467-485. Lin, J.K., and M.L. Kuo (1990) Induction of ouabain-resistance mutation and cycle-dependent transformation of C 3 H / 1 0 T 1 / 2 cells by N-nitroso-2-acetylaminofluorene, Mutation Res., 230(1), 35-43. Lindahl, P.E. (1948) Principle of a counter-streaming centrifuge for the separation of particles of different sizes, Nature (London), 161,648-655. Luria, S.E., and M. Delbruck (1943) Of bacteria from virus sensitivity to virus resistance, Genetics, 28, 491-511. Maher, V.M., D.J. Dorney, A.L. Mendrala, B. Konze-Thomas and J.J. McCormick (1979) DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation, Mutation Res., 62, 311-323. McCormick, J.J., and J.S. Bertram (1982) Differential cell cycle phase specifically for neoplastic transformation and mutation to ouabain resistance induced by N-methyI-N'nitro-N-nitrosoguanidine in synchronized C3H 10T1/2 CI 8 cells, Proc. Natl. Acad. Sci. (U.S.A.), 79, 4342-4346. Meistrich, M.L., R.E. Meyn and B. Barlogie (1977) Synchronization of mouse L-P59 cells by centrifugal elutriation separation, Exp. Cell Res., 105, 169-177. Mitchell, B.F., and J.T. Tupper (1977) Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation, Exp. Cell Res., 106, 351-355. Mun, B.J., and C.K. Mathews (1991) Cell cycle-dependent variations in deoxyribonucleotide metabolism among Chinese hamster cell lines bearing the Thy-mutator phenotype, Mol. Cell. Biol., 11(1), 20-26. Nakamura, N., and S. Okada (1979) Cell-cycle dependency of BUdR-induced mutation for six genetics markers in cultured mammalian cells, Mutation Res., 60, 83-89. Ng, J.J.Y., C.L. Crespi and W.G. Thilly (1980) Selection of mitotic Chinese hamster ovary cells from microcarriers, Anal. Biochem., 109, 231-238. Pfeiffer, P., and W. Vielmetter (1988) Joining of nonhomologous DNA double-strand breaks in vitro, Nucl. Acid. Res., 16, 907-924. Plant, J.E., and J.J. Roberts (1971a) A novel mechanism for the inhibition of DNA synthesis following methylation: The effect of N-methyI-N-nitrosourea on HeLa cells, Chem.-Biol. Interact., 3, 337-342.
Plant, J.E., and J.J. Roberts (1971b) Extension of the pre-DNA synthetic phase of the cell cycle as a consequence of DNA alkylation in Chinese hamster cells: a possible mechanism of DNA repair, Chem.-Biol. Interact., 3, 343-351. Riddle, J.C., and A.W. Hsie (1978) An effect of cell-cycle position on ultraviolet-light-induced mutagenesis in Chinese hamster ovary cells, Mutation Res., 52, 409-420. Sanderson, R.J., and K.E. Bird (1977) Cell separations by counterflow centrifugation, in: Methods in Cell Biology, Vol. 15, Part 1, Academic Press, New York. Skopek, T.R., H.L. Liber, B.W. Penman and W.G. Thilly (1978) Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: Possibility for a rapid human cell mutation assay, Biochem. Biophys. Res. Commun., 84, 411-416. Smith, G.J., J.W. Grisham and K.S. Bentley (1988) Mutagenic potency at the N a ~ / K + ATPase locus correlates with cycle-dependent killing of 10TI/2 cells, Environ. Mol. Mutagen., 12(3), 299-309. Terasima, T., and L.J. Tolmach (1963a) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa $3 cells, Esp. Cell Res., 30, 344-362. Terasima, T., and L.J. Tolmach (1963b) Variations in several responses of HeLa cells to X-irradiation during the division cycle, Biophys. J., 3, 11-33. Thilly, W.G. (1976) Maintenance of perpetual synchrony in HeLa $3 cultures: Theoretical and empirical approaches, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. 14, Academic Press, New York, pp. 273-285. Thilly, W.G. (1988) Looking ahead: Algebraic thinking about genetics, cell kinetics and cancer, in: H. Bartsch, K. Hemminiki and I.K. O'Neill (Eds.), Methods for Detecting DNA Damaging Agents in Humans: Applications in Cancer Epidemiology and Prevention, IARC Monograph No. 89, 1ARC Scientif. Publ., Lyon, pp. 486-492. Thilly, W.G., and C. Heidelberger (1973) Cytotoxicity and mutagenicity of ultraviolet irradiation as a function of the interval between split doses in cultured Chinese hamster cells, Mutation Res., 17, 287-290. Wood. R.D., and H.J. Burki (1982) Repair capability and the cellular age response for killing and mutation induction after UV, Mutation Res., 95(2-3), 505-514. Yang, L.L., V.M. Maher and J.J. McCormick (1980) Error-free excision of the cytotoxic, mutagenic N2-deoxyguanosine DNA adduct formed in human fibroblasts by (+)-7beta,8-alpha-dihydroxy-9-alpha,10-alpha-epoxy-7,8,9,10-tetrahydro-benzo{a]pyrene, Proc. Natl. Acad. Sci. (U.S.A.), 77, 5933-5937.
Mutation Research, 250 (1991) 411-421
© 1991 Elsevier Science Publishers B.V. All rights reserved 002%5107/91/$03.50 ADONIS 0027510791001994 MUT 02545
Cell-cycle dependent mutation of human lymphoblasts: Bromodeoxyuridine and butyl methanesulfonate Henry Hoppe IV, Katherine M. Call, Phaik-Mooi Leong and William G. Thilly Massachusetts Institute of Technology, Centerfor Environmental Health Sciences, Cambridge, MA 02139 (U.S.A.)
Keywords: Cell-cycledependent mutation; Lymphoblasts,human; Bromodeoxy-uridine; Butyl methanesulphonate
Summary Cells of the human lymphoblast line WI-L2 and its derivative TK-6 were synchronized by centrifugal elutriation and cell-cycle dependent mutation to 6 T G R ( H P R T ) and O U A R (Na ÷, K + ATPase) measured. Bromodeoxyuridine induced 6TG R and O U A R mutations within S phase while butylmethylsulfonate induced mutation displayed no cell-cycle dependence. The data indicate that centrifugal elutriation is a facile means to obtain a useful degree of synchrony for these cell lines.
What are our reasons for studying the cell-cycle dependence of induced mutation? There is really only one reason. We wish to discover something of value in preventing diseases that begin with genetic change. T h e r e are two classes of disease which require genetic changes: those which can arise solely from inherited mutations and those which are combinations of inherited and somatic mutations. The former class include the thousands of dominant or recessive single gene mutations such as sickle cell anemia or cystic fibrosis. The latter include cancers, atherosclerosis and possibly the degenerative changes of age. The ceils in which heritable mutations occur comprise all linear descendants of a fertilized egg to the spermatocytes or the oocytes of an individual. Somatic mutations may arise in any cell division after fertilization in any organ. Each organ is
Correspondence: Dr. William G. Thilly, Massachusetts Institute of Technology, Center for Environmental Health Sciences, Cambridge, MA 02139 (U.S.A.).
a mosaic of differentiated cells operating cooperatively to achieve a physiologic function. Each of the differentiated cell types expresses an idiotypic set of genes and, in adults, maintains a constant cell number. This maintenance of a constant cell number is one of the wonders of physiology. Some cell types such as the crypt cells of the intestine are in a state of continuous cell division with losses by exfoliation matched for a lifetime by compensating cell division. Or is it that exfoliation exactly matches the products of cell division? A similar but slower process describes the squamous epithelium of the skin. Red blood cells have finite lifetimes after differentiation from the descendants of a pluripotent marrow stem cell. Some cell types in the nervous system may have zero capacity for cell replacement by cell division and were presumably distributed in early life in highly redundant physiologic arrays in which the deaths of a few individual cells would not inactivate the network. Because of these many division states of cell types in the body - - from continuous to zero - and because we really don't have a quantitative
412
picture of the kinetics of cell death and replacement in any human tissue, this area of research is bound to be speculative. Consider the rapid cell divisions of early embryonic life. The rate of gene duplication is prodigious. The fertilized egg appears to be packed with DNA-repair enzymes (Pfeiffer et al., 1988). But who has measured the rate or kinds of genetic change which occur in the first thirty divisions of life? During this period the single cell gives rise to 109 cells already differentiating into the rudiments of tissues and organs. Any mutation which does occur in the first precursor cells of an organ are subject to the occurrence of jackpot mutations as surely as Luria's bacterial populations and would be expected to be on the most probable pathway to diseases requiring more than one somatic mutation (Luria and Delbruck, 1943; Thiily, 1988). In organs maintaining a constant cell number with relatively infrequent cell division, the problem arises as to whether it is reasonable to regard the population of cells of the same histoiogic type as homogeneous. Aside from the variations in cell physiology from stem to a parenchymal cells, there is the fact that cells of identical physiology could differ enormously with regard to the time before their next cell divisions. Thus it becomes incumbent upon mutation researchers to think beyond the histologic separation of cell types to the dynamics of cell death and replacement and its consequences in mutagenesis: At the molecular level the availability of the D N A tbr chemical reactions may be affected by the replication or transcription state of the sequence in question. T r e a t m e n t s of cells by chemicals or irradiation can either increase or decrease the toxicity of subsequent treatments. (Thilly and Heidelberger, 1973; Kaden et al., 1987). These changes could come from expression of new enzymic activities or from structural changes in a D N A target or both. Certainly we must consider the possibility that the level of constitutive D N A (chromatin) repair systems will vary as a function of a cell's temporal position relative to its next division. The same may be said for the inducibility of repair functions and the well-known but little-understood p h e n o m e n a of cell-cycle progression delay (Plant and Roberts,
1971 a,b).
Generalizations have their place in describing these kinds of problems but an arithmetic example may be more compelling. Let us suppose that treatment of C H O cells with a mutagen at a subtoxic level has induced a mutant fraction of 10 4 by forward mutation of a gene like hprt. It the same experiment were carried out in a Chinese hamster ocary using the same number of DNA-reaction products per base pair one would expect ItXX) mutants in a 107 cell sample. However, if only 10 -" or 10 3 of the cells of the type chosen were within a day of their next cell division and repair of the potentially mutagcnic lesions proceeded with a half-life of 24 h then the outcome of the experiment would be 1(I or fewer mutants. Recognition of this kind of problem - - and extending it to treatments which change the number of cells proceeding to division - - is a fairly humbling experience for scientists who like to think that our common trick of using rapidly dividing microbes or mammalian cells in culture is the pathway to understanding. This paper, however, is about a preliminary study of induced mutation in continuously dividing cell human 13 cell lines. There is nothing preliminary about the depth or quality of the data, but it is preliminary in the sense that we recognize that the responses observed are only a beginning. We are working on a means to reversibly return B lymphoblasts to a non-dividing lymphocytic state in which we may be able to study the effect of mutagens on these ceils as a function of time between treatment and cell division over a period of weeks and months. This kind of experiment with UV-irradiated human fibroblasts has been performed (Maher et al., 1979) and extended to chemical mutagens (Yang et al., 1980) but has not been repeated by any other laboratory or extended to other cell types despite its apparent importance in building models of in vivo mutagenesis. As we move to the end of this century key mechanistic questions about mutation are being asked at the level of individual D N A sequences. What are the kinds, amounts and positions of D N A adducts as a function of D N A sequence within a gene and among similar sequence dispersed over transcribed and non-transcribed chromosomal regions'? What are the kinetics of
413
removal and subsequent repair of such reacted DNA bases? What is the resulting frequency of mutation or unrepairable block to DNA synthesis? What are the roles of the few known DNArepair enzymes and surely greater number of unknown chromatin/DNA-repair enzymes in this process? All of these questions have hidden within them the need to understand cell-cycle dependent changes in chromatin structure, amount and inducibility of repair enzymes, and the role and mechanism of progression delay in mutagenesis. The truth is that a relatively small number of mammalian cell lines have been used both in environmental mutagen assays and in studies of mutational mechanisms. Two of these cells lines, the hamster CHO line and the human TK-6 line are today frequently cited in both kinds of mutation studies. An area of general interest to mutation researchers but not frequently the subject of experimental reports however is the dependence of mutation on the cell cycle. That cell-cycle position was important in determining sensitivity to cell killing has been known since Terasima and Tolmach (1973a,b) reported that Hela cells synchronized by their new method of mitotic shakeoff were especially sensitive to killing by X-rays in G 2 phase and mitosis. After methods were developed to accurately measure induced point mutant fractions in mammalian cells similar experiments were possible with regard to mutation. The earliest of this genre explored the cycle dependence of bromodeoxyuridine (Aebersold and Burki, 1976; Carver et al., 1976; Burki and Aebersoid, 1978; Nakamura and Okada, 1979) induced mutations in rodent cell lines. Since that time only a few citations can be found for cell-cycle dependence of mutation despite an explosion in mammalian cell mutation studies. Among these are studies of UV light (Riddle and Hsie, 1978; Burki et al., 1980; Wood and Burki, 1982) ionizing radiation, (Burki, 1980) alkylating agents (Goth-Goldstein and Burki, 1980; McCormick and Bertram, 1982; Crespi et al., 1982; Smith et al., 1988), aflatoxin B~ (Kaden et al., 1987), nitroso acetyl amino fluorene, and benzopyrene diol epoxide (Smith et al., 1988; Lin and Kuo, 1990) and deoxyribonucleiotide pool imbalances (Mun and Mathews, 1991). Chief among the reasons for this low number
of papers must be death of H.J. Burki whose laboratory accounted for half of the published literature in this area. Another reason appears to be that while the importance of cycle-dependent cell killing is generally appreciated in optimizing protocols for cancer radio- and chemo-therapy, the role of cycle dependent mutation in causation of mutation leading to cancer and heritable mutations is not widely appreciated, A final reason for experimental inactivity appears to be that cell cycle mutation experiments are perceived as technically difficult to perform. The technical barrier to obtaining sufficient cell numbers (108 cells, z.b.) for facile cell cycle studies in CHO cells was essentially eliminated by the combination of microcarrier technology with of Terasima and Tolmach's (1963) mitotic shakeoff technique (Crespi et al., 1982). Mass synchronization of suspension cell lines such as HeLa was possible with careful matching of DNA inhibition to cell growth parameters (Thilly, 1976) however, repeated efforts to apply this approach to human lymphoblast lines have not succeeded. An alternative synchronization method based on Lindahl's 1948 design of a counter streaming centrifuge was introduced in 1977 (Meistrich et al., 1977; Mitchell and Tupper, 1977; Sanderson and Bird, 1977). The necessary equipment, though not inexpensive, is commercially available. This method, centrifugal elutriation, separates cells on the basis of differential sedimentation velocity and should yield a degree of synchrony and cell number sufficient for a wide variety of quantitative mutation studies. This method provides the synchronous human B cell populations which we believe will be necessary for key future experiments with human lymphoblastoid lines. Materials and methods
Cells The WI-L2 line (Levy et al., 1968), was provided by Dr. Arthur Bloom Columbia College of Physicians and Surgeons, New York. A subpopulation (HHIV) capable of forming colonies in the absence of feeder layers was used in these experiments. TK-6 (aka H2BT) was isolated as a thymidine kinase heterozygote from this population (Skopek et al., 1979). Cells were grown in RPM1
414 1640 media supplemented with 10% horse serum. Average doubling times for both lines were 18 h.
Chemicals 5-Bromodeoxyuridine (BUdR) and [3H]thymidine were obtained from New England Nuclear (Boston, MA), butyl methylsulfonate (BMS) from Pfaltz and Bauer (Stanford, CT), DNAase I, Worthington, (Feerhold, N J).
The sample to be separated (4 x 108 cells) was suspended in I(X) ml complete medium and placed in the sample reservoir. It was pumped into the separation chamber in the rotor at 10 m l / m i n . After the loading of the sample medium from the buffer reservoir was pumped at incremental pump speeds of 4 m l / m i n and samples collected. The total sample collection amounted to a volume of 250-300 ml. The last p u m p speed at which samples were collected was 38 m l / m i n .
Synchronization by centrifugal elutriation The basic components for centrifugal elutriation were a Model J-21B Centrifuge (Beckman Instruments) and a Cole-Palmer Masterflex Pump (Cole-Palmer Instrument Co., Chicago, IL). The controller for the p u m p was modified by the replacement of the supplied speed regulator with a 10-turn 2-ohm millipot (M.I.T. Electronics Shop). This modification allowed small, reproducible adjustments of the p u m p speed. The rotor was assembled as instructed in the manual. The partially assembled rotor was autoclaved at 275 o C, 15 p.s.i, for 45 min. The gasket for the separation chamber was not able to be autoclaved and was therefore sterilized by immersion in 70% ethanol for several hours prior to assembly. The assembly of the rotor was completed in an aseptic manner in a still air hood. The rotor was flushed with 70% ethanol and warmed to 37 ° C for several hours before use. The centrifuge was set up next to a still air hood and 37 ° C water bath. Sufficient tubing was present to allow sterile manipulations within the hood. The buffer reservoir, sample reservoir and collection flask were kept in the water bath. Just prior to use, the assembled rotor was placed in the centrifuge and the appropriate connections was made. Air bubbles were flushed from the system and the p u m p speed was recalibrated. Separations were done at 19 600 rpm. The speed was checked stroboscopically. N.B. Use of horse serum was found to be associated with precipitation during operation of the elutriator. These problems were overcome by heat treating the horse serum (57 ° C x 2 h) followed by a 1-h incubation at 3 7 ° C with filter sterilized DNAase I, 300 units/ml. The use of DNAase treatment was essential to clean separations.
Cell counts and t,olume determinations The number of cells and their volume distributions were determined using differential threshold settings on a Coulter electronic particle counter. Latex spheres of known diameter were used to calibrate the process.
DNA synthesis The amount of [3H[thymidine incorporated into acid-precipitable materials was measured as an estimate of D N A synthetic rate. Cells were incubated for one hour in the presence of 1 /xM ~H-TdR (500 C i / m o l e ) . The culture was resuspended in fresh medium with treated serum, 2 x 107 cells were removed for the asynchronous control, and the remainder was separated according to volume by centrifugal elutriation. The asynchronous control and 2 x 107 cells from each of the samples post-elutriation were resuspended in 5% trichloroaretic acid at 4 ° C and allowed to sit on ice for at least 20 rain. An aliquot from each sample was filtered onto glass filter which were held in a standard filter-holding apparatus. The filters were washed with 20 ml T C A at 4 ° C . followed by 20 ml of 95% ethanol. The filters were then placed in glass scintillation vials and allowed to dry overnight and scintillation counting performed the next day.
Assays for suruieal and mutation Assay for survival and mutation to resistance to 6-thioguanine (6TG), ouabain ( O U A ) or trifluorothymidine (F3-TdR) were performed and analyzed according to the methods reported in Furth et al. (1981). This approach involves distributing cells directly into microwells in the presence or absence of selective conditions and using the Poisson distribution to calculate the mean and
415 I
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Fig. 1. Size distribution of WI-L2 cells separated by centrifugal elutriation. Modal volumes increased from 310 ~3 to 810 /z3 in approximately 100-.. 3 increments with each fraction. The TK-6 clone separation parameters and results were identical to WI-L2. Fraction 0 contained many dead cells and cell debris. Fractions 5 and 6 contained cell doublets.
standard deviation of resulting estimates of surviving and mutant fractions. Estimates of mutant fractions and their dispersion were analyzed according to Leong et al. (1985) and found to lie within the expected range of outcomes based on numerical variation. Treatment of asynchronous populations with mutagens for 1 hour immediately preceeded elutriation. Results
Tritiated thymidine uptake, Fig. 3, revealed that all fractions contained some cells in S phase but fractions 2 and 3 were significantly enriched. Microscopic examination of all fractions revealed that fractions 4, 5 and 6 with the greatest V
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Synchronization The separation of these cells bases on sedimentation velocity may be regarded as moderately successful in that fractions of successively greater modal volume were eluted (Fig. 1) and found to have successively longer periods between elution and cell division as indicated in Fig. 2. That it required 6 h of a 18-h doubling time for each cohort to pass through mitosis is consistent with the large overlap among fractions with regard to size distribution, i.e. cell size is not an exact indicator of cell-cycle position.
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Fig. 2. Growth curves of four fractions of TK-6 cells separated as in Fig. 1. The initial modal volume of the fraction marked by hexagons was 800/.L3, by triangles 650 p3, by squares 500 p.3 and by circles 400 p3. Growth of the parent line WI-L2 after separation was identical to TK-6.
416 i
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Butyl methanesulfonate The toxicity and mutagenicity of BMS to the WI-L2 line and its TK-6 thymidine kinasc hctcrozygote subclone arc shown in Figs. 4 and 5 respectivcly. Survival may reasonably be described as log linear and mutagenicity linear for both lines and genes studied. No significant difference in the concentration-dependent toxicity between WI-L2 and TK-6 cells was found. Treatment with 5 mM BMS for 1 h resulted in relative survivals of about 0.65 or close to 0.5 lethal hits per ccll. Mutation responses were however significantly different for the two cell lines. The slope of the 6TGR mutant fractions a function of initial BMS concentration was 4.7 x 10 -~' cell m u t a t i o n s / m M
270
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Fig. 3. D N A Synthesis in fractions of WI-L2 cells of Fig. 1. 3H-TdR into acid precipitable cell material was measured after 1 h incubation and was used to calculate the D N A synthetic rate. The solid symbol indicates the value for the asynchronous exponentially growing parent culture.
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model cell volume contained many doublets which were converted to single cells upon resuspension in the spinner cultures. Formation of these doublets in the elutriator is a technical limitation to be considered in interpretation. The proportion of cells which actually underwent cell division after elutriation varied among fractions. 75% of cells a p p e a r e d to double synchronously in the fraction containing the largest ceils (4, 5, 6) while a somewhat smaller proportion were involved in cell division in the fractions containing smaller ceils (Fig. 2). This proportion of cells actually undergoing cell division varied among experiments and in some runs a fraction less than 50% divided despite the fact that the cells were in exponential growth and had high ( ~ 50%) cloning efficiency both before and after elutriation. Since no cell division occurred in the first hour after separation for any fraction, it is probable that the elutriation process occasioned some cell-cycle delay.
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Fig. 4. Concentration dependence of BMS toxicity and mutagenicity to WI-L2 cells in a l-h exit)sure of asynchronous populations. Open symbols, 6TG a mutant fraction, closed symbols O U A R mutant fraction.
417 i
BMS for W I - L 2 cells but was 9 . 0 × 10 -6 for TK-6. Similarly the slope of induced O U A ~ mutations was 5 x 10 -7 for W I - L 2 and 1.8 x 10 -~ for TK-6. T h e ratio of slopes O U A R / 6 T G R for WIL2 was thus 5 x 1 0 - 7 / 4 . 7 x 10 -6 = 0.11 while this ratio was 1.8 x 1 0 - 7 / 9 x 10 -6 = 0 . 0 2 for T K 6 cells. Because it was possible to measure mutations at the thymidine kinase locus in T K 6 ceils this assay was also performed. It was found that the slope for tk mutation (F 3 T d R R) was identical to that seen for hprt ( 6 T G a ) . That W I - L 2 and TK-6 lines show differential sensitivity at both the hprt and N a +, K ÷ A T P a s e genes is statistically significant but s o m e w h a t disconcerting for the assumption that they are isogenic with regard to the genes involved in mutagenesis induced by BMS (Liber et al., 1982). W h e n the elutriated fractions were examined for BMS killing and mutation to 6TG R in two i n d e p e n d e n t experiments a greater sensitivity was reproducibly found in fractions with smaller cells than with larger. No significant difference in in-
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Fig. 6. Cell-cycle dependence of BMS toxicity and mutagenicity to WI-L2 cells. Two independent experiments are shown (0. o) with the mean value (e) for survival indicated. Indicated as (A) and (B) are the untreated control values and the values for simultaneously treated asynchronous WI-L2 population. Large variations in one experiment (o) arose from the uncertainty introduced from low cloning efficiency of the elutriated BMS treated cultures. The straight line represents the interpretation of no cell-cycle dependent effect on mutation.
x
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duction of 6 T G R mutations was found a m o n g the 6 fractions, however (Fig. 6).
Bromodeoxyuridine oI~J~Ia::
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.o
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1 4
I 5
Fig. 5. Concentration dependence of BMS toxicity and mutagenicity to TK-6 cells in a l-h exposure of asynchronous populations. Triangles, 6TG R, squares, F3 TdR R, and circles, OUAR mutant fractions.
W I - L 2 and TK-6 cells differ markedly in their response to B U d R in the medium. Since TK-6 cells have only 50% o f the thymidine kinase activity of W I - L 2 cells (Skopek et al., 1978) D N A synthetic pathway this may not be surprising. I n d e e d t r e a t m e n t o f W I - L 2 cells with 200 tzM B U d R for 1 h p r o d u c e d a 6 T G R m u t a n t fraction of 4 × 10 -3 but no increase in 6 T G R m u t a n t fraction was seen in TK-6 cells even when expo-
418
sure was extended to 2 h. Independent study (H.L. Liber, Ph.D. Thesis) has shown that under these conditions BUdR uptake into the D N A of both cell lines is saturated above 5 0 / z M and that at 200 izM for 2 h WI-L2 cells have twice was much BUdR in their D N A as TK-6. Since TK-6 cells were not mutated at all by BUdR we were limited in its study to the WI-L2 parent line. As shown in Fig. 7 the concentration dependence of B U d R toxicity to asynchronous WI-L2 cells was complex but monotonically decreasing. For induction of mutation to 6TG R or O U A a no increase was observed at 50 IzM but then mutant fraction rose linearly with B U d R concentration from 50 to 200 /xM. The ratio of slopes in this range between the two gene loci was 0.15 similar to the ratio 0.11 observed for the two loci when mutated by BMS.
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Fig. 8. Cell-cycle dependence of BUdR toxicity and mutagenicity to WI-L2 cells. Two independent experiments are shown for survival (c,, o) and the mean values indicated by (×). Two independent experiments are shown for 6TG R (0. ,) and one for OUAR (©). Indicated as (A) and (B) are the values for untreated cultures and simultaneously treated asynchronous cultures respectively.
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Fig. 7. Concentration dependence o f B U d R toxicity and mutagenicity to W l - L 2 cells in a l-h exposure o f an asynchronous population. N.B. T K - 6 cells were refractory to m u t a t i o n to 6 T O R or O U A R under these conditions.
Cell-cycle dependence of BUdR toxicity and mutagenicity was performed using the elutriated fractions as in Fig. 1 with a l-h exposure to 200 /xM BUdR. As seen in Fig. 9 smaller cell fractions were more susceptible to killing than larger ones. A clear but curious non-correspondence between a fraction's ability to incorporate tritiated thymidine (Fig. 3) and its susceptibility to B U d R killing (Fig. 8) is evident. With regard to mutagenicity two independent experiments measuring 6TG R gave comparable results. Fraction 3 which displayed the highest tritiated thymidine uptake also had the maximum B U d R induced 6TG R mutant fraction in both experiments. A displacement of O U A R mutation
419
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Fig. 9. Comparison of ~H-Td R uptake rate to BUd R induced 6TG R mutation in each elutriated WI-L2 fraction.
to a larger cell fraction seems evident and is confirmed by the ratios of ouabain R to 6TG R in fractions 2 through 6. Fig. 9 compares tritiated thymidine uptake (Fig. 3) to BUdR induced mutation (Fig. 8) directly. It seems clear that the BUdR mutational effect is restricted to a narrower size range of WI-L2 cells than thymidine uptake. Discussion
In dealing with data purporting to describe cell-cycle dependence of killing or mutation, one should first check the degree of synchrony achieved and investigate the effects of the synchronization procedure used on untreated ceils. In the use of centrifugal elutriation we found that background mutant fraction was not affected by the procedure but that some interruption in cellcycle progression occurred (Fig. 2) as well as some loss of cells' ability to grow as isolated colonies. The degree of synchronization may be formally calculated by the method of Engelberg (1964) or
its graphic equivalent (Bakke and Petterson, 1976). Here it suffices to point out that the smallest cell fraction representing Gt phase cells required some 8 h to complete a 'synchronous' wave of division in an 18-h doubling time. This may be compared to some 5-h duration for the division wave 18 h after mitotic selection of CHO cells by shake-off from microcarriers (Crespi et al., 1982). Centrifugal elutriation does not achieve the degree of synchrony of mitotic shake-off but does yield a significant degree of synchrony sufficient for many purposes. Tritiated thymidine labelling shows a clear differentiations among fractions (Fig. 3) although the difficulty of cell doublets contaminating the largest cell fraction must be noted. Interpretation of survival curves must account for the observation that cells in the larger cell fractions had a somewhat higher probability of subsequent cell division than those in smaller cell fractions (Fig. 2). The apparent lower relative survival of smaller cell fractions after BUdR or BMS treatment may therefore arise from actual cycle-dependent sensitivity to treatment or a synergistic interaction between the toxicity of the synchronization procedure and that of the treatment. With regard to mutagenicity these problems are reduced since all determinations of mutant fraction are made after the treated populations have returned to exponential growth phase and have a colony-forming efficiency equal to that of untreated control populations. That BMS had no significant cell-cycle dependent effect on mutation to 6TG R is evident in Fig. 6. The alkylating agent EMS showed a modest (_< 2 x ) cell-cycle dependence for mutation in mitotically synchronized CHO cells with a minimum sensitivity in S phase (Crespi et al., 1982). The lower degree of synchrony obtained by centrifugal elutriation would have presented observations of so small a differential in the experiments reported here. Because TK-6 cells do not remove or repair O6-methyl guanine or any other methyl base product except 3-methyl adenine (Goldmacher et al., 1986) a lack of cell-cycle dependent mutation may be regarded as consistent with a model in which the time between adduct formation and DNA replication is inconsequential. This same
420
cell line showed no cell cycle dependence on aflatoxin B~ induced mutation (Kaden et al., 1987). BUdR did show clear cell-cycle dependence for mutation as would bc expected for a pyrimidine analogue but, of course, the actual mechanism of BUdR mutagcncsis - - replacement of thymidinc or deoxycytidinc or both - - is not yet settled and awaits future mutational spectra studies. Sensitivity to 6TG R or OUA ~ mutation appear to have different cell-cycle positions in BUdR mutagenesis (Fig. 8) a result also seen in Chinese hamster ceils (Crespi et al., 1982). That the cycle dependence of H P R T vs. Na +, K* ATPase gene mutation can in fact be observed in elutriation-synchronizcd B cell culturcs indicatcs that the degree of synchrony achieved will be useful in future work. It is also clear that in the experiment shown in Fig. 9 that the period of sensitivity to BUdR mutation is shorter than the entire S phase consistent with the synthesis of the hprt genc at a reproducible point in S phase. This demonstration gives the experimenter an estimate of the resolution to be expected using these cells and centrifugal elutriation in future experiments. Our understanding of why different replicon clusters are synthesized at specific points S phase is still rudimentary. Do the rules of mutation differ among genes duplicated at different S phase points? Only by doing the challenging experiments with synchronized populations of sufficient size for quantitative mutation assays can we expect to find out. Failure to account for cell-cycle dependence puts off, perhaps for another generation, understanding of key issues in mutation mechanisms. The decision not to do cell-cycle studies does avoid some technically difficult experiments. The method of Ng ct al, (1980) provides a facile means to obtain the necessary numbers of synchronized cells for Chinese hamstcr cells. We hope that this paper demonstrates that a facile technology is available and can bc readily applied to the human B cell lymphoblastoid lines.
Acknowledgments We gratefully acknowledge the support of the office of Health and Environmental Research
U.S. Department of Energy (DE-FG02-86ER 60448) and the U.S. National Institute of Environmental Health Sciences, (P30-ES021(}9, P01ES3926) HHIV and KMC were NIEHS Predoctoral Trainees.
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