A case of caffeine-mediated cancellation of mitotic delay without enhanced breakage in V79 cells

Mutation Research, 304 (1994) 203-209 © 1994 Elsevier Science B.V. All rights reserved 0027-5107/94/$07.00

203

MUT 05340

A case of caffeine-mediated cancellation of mitotic delay without enhanced breakage in V79 cells Alison N. Harvey * and John R.K. Savage MRC Radiobiology Unit, Chilton, Didcot, OXll ORD, UK (Received 23 February 1993) (Revision received 25 May 1993) (Accepted 19 August 1993)

Keywords: Caffeine; Chromosomal aberrations; Mitotic delay

Summary Chinese hamster cells (V79 379A)were grown for 17 h in the presence of 10/~g/ml bromodeoxyuridine (BrdU) to obtain cells with potential sister-chromatid differentiation. At this time batches were irradiated (1.5 Gy, 250 kVp X-rays) and the medium of all flasks replaced with one containing 10/~g/ml thymidine with or without 400 ~ g / m l caffeine. Metaphases from irradiated and unirradiated batches were sampled every hour for 7 h and FPG stained. All categories of chromatid-type aberrations were scored in G 2 and S phase ceils. As expected, mitotic delay in the presence of caffeine, (measured by a shift in the portion of the fraction of differentially stained metaphases (FDM) curve and a reduced fall in the mitotic index) was largely cancelled, but there was a negligible increase in chromatid-aberration frequency (all categories), only achieving significance if the consistency of the whole 7-h sampling period was considered. Caffeine had no effect on the frequencies of light/dark chromatid involvement, nor in the completeness of chromatid interchanges. We conclude that the enhanced breakage frequency often observed with post-irradiation caffeine treatment is not necessarily causally related to the cancellation of delay.

There are a number of substances which, if given to cells after either an S-dependent or S-independent clastogen treatment, will increase the frequency of chromatid or chromosome-type aberrations, and so decrease cell survival. Popular amongst these are the methylated oxypurines, of which a potent example is caffeine, 1,3,7-tri-

*Corresponding author. SSDI 0027-5107(93)E0146-H

methylxanthine (Kihlman, 1977; Kihlman and Andersson, 1987). This compound has a number of other effects on cells. In particular, it is capable of mitigating to a large extent, radiation-induced mitotic delay (Darroudi and Natarajan, 1987; Grinfeld and Jaquet, 1988; Liicke-Huhle et al., 1983; Parshad et al., 1982). It is thought that DNA lesions are indirectly responsible for mitotic delay by preventing the synthesis of proteins responsible for the G2/mitotic transition e.g. those required for

204

chromosome condensation (Ai-Bader et al., 1978; Rao, 1980). The suggestion has also been made by a number of workers (Tanzarella et al., 1986; Kihlman et al., 1982; Liicke-Huhle et al., 1983) that the two effects, enhanced breakage and reduced delay, are linked. During the mitotic delay, some of the induced damage that could lead to chromosomal aberrations is repaired. Cancellation of delay reduces the time available for such damage "repair", so that cells come into division with a higher proportion of DNA damage (e.g. unrejoined "breaks") than would normally be the case. Occasional exceptions to this delay/reduced aberration link have, however been reported. For instance, in the human radiosensitive syndrome ataxia telangiectasia, fibroblasts irradiated in late G 2 and post-treated with caffeine show no enhanced frequency in spite of abolished delay (Hansson et al., 1984). A similar situation was found by Parshad et al. (1982) for malignant fibroblasts, but not for the normal parental cell line where enhanced aberration frequency was unaccompanied by delay cancellation. We report here an observation, made during some recent experiments in V79 Chinese hamster cells, which also clearly divorces these two effects, namely, a significant cancellation of delay without any accompanying increase in "breakage". Materials and methods

V79-379A cells (modal chromosome number = 21) were seeded for exponential growth into Falcon T25 flasks, using Eagle's MEM with 10% Foetal Calf Serum, L-glutamine and antibiotics. Bromodeoxyuridine (BrdU, final concentration 10 /zg/ml) was added to growing cultures for 17 h, at the end of which time, most of the cells would be in their second division with a TB/BB sisterchromatid differentiation. The flasks were divided into four groups for further treatment: (1) Control. (2) Caffeine, final concentration 400 /zg/ml (2.06 x10 -3 M). (3) 1.5 Gy X-rays (250 kVp, 14 mA, 0.7 Gy/min). (4) 1.5 Gy X-rays plus caffeine. Immediately after irradiation, the BrdU medium in all flasks was removed and replaced

with pre-warmed medium containing 10 /xg/ml thymidine (nonradioactive), and caffeine if applicable. Flasks from all four treatments were sampled every hour up to 7 h post-irradiation, each with 1 h colcemid (0.05/zg/ml). Air-dried metaphase spreads were carefully stained with a modified FPG method (Harvey and Savage, 1992) to achieve a three-way stain distinction for TT, TB and BB chromatin. Scoring was performed on coded, randomised slides. Each metaphase was first allocated to its cycle position, G 2 being recognised as "pure" harlequin and S-cells as harlequin marked with TT patches. All categories of chromatid-type aberrations (Savage, 1976) were then recorded and the presumptive "break-points" assigned to the light (BB) or dark (TB) chromatids. Scoring was continued until 200 cells had been scored at each point and if the break frequency was low then extra scoring was carried out until approximately 200 discontinuities ("breaks") had been obtained at each point. Results

Fig. 1 shows the profile of the fraction of differentially-stained metaphase (FDM) ceils, with time after thymidine replacement, for the caffeine and non-caffeine control cultures. (In this case FDM represents S-cells with TT patches, which is equivalent to the fraction of labelled metaphases, FLM, obtained if tritiated thymidine

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is used.). Caffeine alone at 400 /~g/ml has had no significant influence on the progress of the cells towards division, except, perhaps, a slight speeding-up through early S. Fig. 2 shows the irradiated series. There is, as expected, a clear delay from the 1.5 Gy in the absence of caffeine (the FDM is shifted to the right), but this is largely, but not completely, cancelled when caffeine is present. Another indication of mitotic delay is the mitotic index (MI), the proportion of cells in mitosis (at a point in time) in a population of dividing cells. Fig. 3b shows the MI for the controls with and without caffeine. The considerable fluctuations indicate that the population is not in steady-state at this time and almost certainly reflects the perturbations introduced by the medium change and replacement of BrdU with thymidinc. Nevertheless, the mitotic depression following irradiation and its cancellation in the presence of caffeine are clearly seen in Fig. 3a. For simplicity, the aberration results are given per cell, disregarding G2//S distinctions at each sample time. Fig. 4 shows the yield-time profile for achromatic lesions ("gaps") following irradiation with and without the presence of caffeine. There is a marginal, but consistent increase in gaps when caffeine is present spanning all sample times. The overall means are 1.50 per cell without caffeine and 1.90 per cell with caffeine. Because the trend is present at all sample times the difference is significant (P = 0.002)

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Fig. 5 is the picture for discontinuities, i.e. clear breaks, most of which can be shown to be incomplete intra-arm intrachanges (Savage and 6.0 =

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Harvey, 1991). There has been a very slight enhancement by caffeine at some sample times. The overall means are 1.02 per cell without caffeine and 1.15 per cell with caffeine and the difference is significant ( P = 0.007). However, the caffeine enhancement for both gaps and breaks is clearly negligible compared with that normally obtained (e.g. Darroudi and Natarajan showed a 1.8 × increase in break frequency after 1.4 Gy incubated post-irradiation with 1 mM caffeine, Darroudi and Natarajan, 1987). Fig. 6 gives the profiles for chromatid interchanges. These show differences at the different sample times, but not in any consistent manner. Overall 0.19 per cell without caffeine and 0.21 with caffeine. These overall values are not signifi-

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cantly different ( P = 0.34). We also checked interchanges for differences in symmetry (A/S); 141/136 = 1.037 without caffeine; 155/168 = 0.923 with caffeine, ( P = 0.48) and for the level of incompleteness ( i n c o m p l e t e / t o t a l interchanges); 165/275 = 0.600 without caffeine; 174/319 = 0.545 with caffeine ( P = 0.52). Comparison of absolute frequencies is, however, very difficult, since at each sample time interval the sampling populations with and without caffeine do not contain the same cell mixtures because of differences in delay.

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Discussion

The mitigation of delay, (i.e. an overall reduction in G 2 transit time) which has been an obvious effect of the caffeine treatment in this experiment with V79 Chinese hamster cells, has not been accompanied by any really significant enhancement of any category of chromatid-type aberration during late Gz, early G 2 or S-phase. Whatever the mechanism of mitotic delay, the net result will be observed as an extended transit time through the cycle (or phases thereof), and this will show itself most obviously by a change in both the shape and displacement of the "fraction of labelled metaphases" curve (FLM) or the equivalent - - in our case the FDM. The amount of delay will vary widely from cell to cell, depending upon a variety of factors, but, in accordance with the usual application of F L M / F D M , a crude measure of average delay can be obtained by measurement at the 50% F D M level. Average control G 2 with or without caffeine is about 1.75 h. Following irradiation alone, this is increased to ~3.25 h (average delay 1 h / G y ) but in the presence of caffeine reduces to ~ 2.25 h. The mitotic index (MI) is a ratio of the time spent in mitosis to the time spent in interphase, and as a stand-alone parameter is not a valid measure of cell rate, certainly not in a perturbed cell population, If the MI falls to zero, (i.e. complete blockage of entry into mitosis) then the time to reappearance of divisions can be used as a crude measure of minimum delay. In our case following irradiation, the MI has fallen almost to zero within the first sample time. In contrast, no major fall is observed in the presence of caffeine,

207 the point being well within the observed fluctuation of the control MIs. Both these data sets confirm the mitigation of radiation mitotic delay by caffeine in this experiment. The dose of caffeine that we chose (400/zg/ ml) was well within the range usually used to produce an increase in aberration yield and abolish mitotic delay i.e. from 60 /xg/ml to 1000 /~g/ml (Kihlman and Andersson, 1987; Pincheira and Lbpez-S~ez, 1991). This concentration of caffeine, in the absence of radiation, does not appear to have any major perturbation consequences for the cell cycle, at least, the first posttreatment one, and the aberration frequency for the caffeine treatment alone was negligible. There is a slight indication of a narrowing of the FDM in the absence of radiation when caffeine was present, suggestive of a more rapid transit time. This might be consistent with the evidence that caffeine can increase the number of replication units by activating new origins (Lehmann, 1972; Tatsumi and Strauss, 1979; Painter, 1980) or by reactivating origins already used (Schn6s and Inman, 1982) thus shortening S-phase transit. Since the profile of the restored FDM (Fig. 2) does not differ markedly from the control we can infer that the bulk of the delay has occurred in G 2 cells. It has been suggested that caffeine causes the synthesis of a protein necessary for mitosis, the inactivation of which is the cause of delay. This protein can induce PCC (premature chromosome condensation), nuclear envelope breakdown, morphological "rounding up" and mitosis-specific phosphoprotein synthesis (Palitti et al., 1983; Schlegel and Pardee, 1986; Kihlman and Andersson, 1987). There is also evidence that indicates that caffeine can actually overcome the more permanent "interphase death", allowing ceils to enter mitosis which would normally be prevented because of an excessive aberration burden (Liicke-Huhle, 1982). In both cases a higher frequency of aberrations would be observed, the former because more unrepaired lesions would be fixed into aberrations, the latter because of an increased number of more heavily damaged cells. It is unlikely that the absorbed dose used here (1.5 Gy) produces much "interphase death" in CHO fibroblasts, so any obvious enhancement

would be expected from curtailed repair. This has not happened, the dramatic rise expected at the earliest sample times has not materialised. Instead all types of aberrations have been marginally affected, though the statistical significance of the effects arises solely from the fact that a slight increase is consistently present at almost all sample times. An effect of caffeine on all types of aberrations, not just breaks has been observed on several occasions (Kihlman and Andersson, 1987). One of our principle interests is to study the relative frequency of aberrations in light (BB) versus dark (TB) chromatids and to achieve this purpose, the chromatin was loaded with bromouracil, a condition not normally employed in caffeine experiments (thymidine, to induce T-F patches and allow recognition of S-cells was also used). Bromouracil is known to increase the radiosensitivity of cells, as measured by survival (Dewey and Humphrey, 1965; Ling and Ward, 1990), and its presence might have precluded the mechanism by which caffeine increases aberration frequency. This is amenable to test. We have shown before (Savage and Harvey, 1991) that sensitization at the chromatid level ( B B > T B ) is only observed for interchanges; "breaks", intrachanges and gaps are unaffected. This pattern was seen again in this experiment, and was not affected by caffeine (light/dark location ratio; "breaks", 0.98 without caffeine, 1.09 with caffeine: interchanges, 1.88 without caffeine, 1.55 with caffeine) showing that the aberration response at the chromosome level is unchanged. The purpose of this caffeine experiment was in fact to study light/dark ratios under conditions of enhanced breakage, and in this it failed. There are a number of reports in the literature of differences in response to caffeine between different cell strains. Musk and his colleagues (Musk and Steel, 1990; Musk, 1991) irradiated human tumour cells at various doses and survival was measured in the presence or absence of caffeine. In one line there was a significant reduction in survival when caffeine was present and mitotic delay (as measured by mitotic index recovery) was abolished. Musk also reports (Musk et al., 1990) in Muntjac cells, an increase in the lethal effects of caffeine with no apparent affect

208

on the cell cycle, with a UV-sensitive Muntjac line showing a "typical" response to caffeine. Hansson et al. (1984) studied cells from patients with ataxia telangiectasia (AT) and showed, in the presence of caffeine, no potentiation of aberrations in AT cells but normal cells showed a 2-3-fold increase. Both showed a response to caffeine in the recovery of the mitotic index after irradiation. This is in contrast to Zampetti-Bosseler and Scott (1985) who showed recovery of the mitotic index and increased aberrations in both AT and normal cells. Malignant human skin fibroblasts were shown by Parshad et al. (1982) to show no increase in aberration frequency in contrast to the parent line which showed a four-fold increase in chromatid breaks. Mitotic delay was abolished by caffeine in the normal cells but not the malignant ceils which failed to exhibit mitotic delay. It may be that our cell line V79 379A is also unusual in its caffeine response. However, these reports, together with our results point to the fact that mitigated delay and enhanced aberration frequency are not necessarily causally related.

References Al-Bader, A.A., A. Orengo and P.N. Rao (1978) G 2 phase specific proteins of HeLa cells, Proc. Natl. Acad. Sci., (U.S.A.), 75, 6064-6068. Darroudi, F., and A.T. Natarajan (1987) Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells x r s 5 and x r s 6, I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine, Mutation Res., 177, 133-148. Dewey, W.C., and R.M. Humphrey (1965) Increase in radiosensitivity to ionizing radiation related to replacement of thymidine in mammalian cells with 5-bromodeoxyuridine, Radiat. Res., 26, 538-553. Evans, H.J., G.J. Neary and S.M. Tonkinson (1957) The use of colchicine as an indicator of mitotic rate in broad bean root meristems, J. Genet., 55, 487-502. Grinfeld, S., and P. Jaquet (1988) G 2 arrest in mouse zygotes after X-irradiation: reversion by caffeine and influence of chromosome abnormalities, Int. J. Radiat. Biol., 54, (2), 257-268. Hansson, K., A.T. Natarajan and B.A. Kihlman (1984) Effect of caffeine in G 2 on X-ray-induced chromosomal aberrations and mitotic inhibition in ataxia telangiectasia fibroblast and lymphoblastoid cells, Hum. Genet., 67, 329-335.

Harvey, A.N., and J.R.K. Savage (1992) Simple cell-cycle analysis in second-division cells, Clin.Cytogenet. Bull., 2, 94-96. Kihlman, B.A. (1977) Caffeine and Chromosomes, Elsevier, Amsterdam. Kihlman, B.A., and H.C. Andersson (1987) Effects of caffeine on chromosomes in cells of higher eukaryotic organisms, Rev. Environ. Health, 7, (3 and 4), 279-381. Kihlman, B.A., K. Hansson, F. Palitti, H.C. Andersson and B. Hartley-Asp (1982) Potentiation of induced chromatid-type aberrations by hydroxyurea and caffeine in G2, in: A.T. Natarajan et al. (Eds.), Progress in Mutation Research, Vol. 4, Elsevier, Amsterdam. Lehmann, A.R. (1972) Effect of caffeine on DNA synthesis in mammalian cells, Biophys. J., 12, 1316-1325. Ling, L.L., and J.F. Ward (1990) Radiosensitization of Chinese hamster cells by bromodeoxyuridine substitution of thymidine: Enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution, Radiat. Res., 121, 76-83. Liicke-Huhle, C. (1982) Alpha-irradiation-induced G 2 delay: a period of cell recovery, Radiat. Res., 89, 298-308. Liicke-Huhle, C., L. Hieber and R.D. Wegner (1983) Caffeine-mediated release of alpha-irradiation-induced G 2 arrest increases the yield of chromosome aberrations, Int. J. Radiat. Biol., 43, 123-132. Musk, S.R.R. (1991) Reduction of radiation-induced cell cycle blocks by caffeine does not necessarily lead to cell killing, Radiat. Res., 125, 262-266. Musk, S.R.R., and G.G. Steel (1990) Override of the radiation-induced mitotic block in human tumour cells by methylxanthines and its relationship to the potentiation of cytotoxicity, Int. J. Radiat. Biol., 57, (6), 1105-1112. Musk, S.R.R., L. Pillidge, R.T. Johnson and C.S. Downes (1990) Action of caffeine on DNA replication after ultraviolet irradiation in Indian Muntjac cells: no connection with action on cell cycle delay, Biochim. Biophys. Acta, 1052, 53-62. Neary, G.J., H.J. Evans and S.M. Tonkinson (1959) A quantitative determination of the mitotic delay induced by gamma radiation in broad bean root meristems, J. Genet., 56, 363-394. Painter, R.B. (1980) Effect of caffeine on DNA synthesis in irradiated and unirradiated mammalian cells, J. Mol. Biol., 143, 289-301. Palitti, F., C. Tanzarella, F. Degrassi, R. De Salvia, M. Fiore and A.T. Natarajan (1983) Formation of chromatid-type aberrations in G 2 stage of the cell cycle, Mutation Res., 110, 343-350. Parshad, R., R. Gantt, Sanford K.K,. G.M. Jones and R.E. Tarone (1982) Repair of chromosome damage induced by X-irradiation during G z phase in a line of normal human fibroblasts and its malignant derivative, J. Natl. Cancer Inst., 69, 409-414. Pincheira, J., and J.F. L6pez-S~iez (1991) Effects of caffeine and cyciohexamide during G 2 prophase in control and X-ray-irradiated human lymphocytes, Mutation Res., 251, 71-77.

209 Rao, P.N. (1980) The molecular basis of G 2 arrest in mammalian cells, Mol. Cell Biochem., 29, 47-57. Savage, J.R.K. (1976) Annotation: Classification and relationships of induced chromosomal structural changes, J.Med.Genet., 13, 103-122. Savage, J.R.K., and A.N. Harvey (1991) Revell Revisited, Mutation Res., 250, 307-317. Schlegel, R., and A.B. Pardee (1986) Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells, Science, 232, 1264-1266. Schn6s, M., and R.B. Inman (1982) Caffeine induced reinitiation of phage DNA replication, J. Mol. Biol., 159, 457-465. Tanzarella, C., R. De Salvia, F. Degrassi, F. Palitti, H.C.

Andersson, H. Hansson and B.A. Kihlman (1986) Effect of post-treatments with caffeine during G2 on the frequencies of chromosome-type aberrations produced by X-rays in human lymphocytes during G o and G~, Mutagenesis, 1, 41-44. Tatsumi, K., and B.S. Strauss (1979) Accumulation of DNA growing points in caffeine-treated human lymphoblastoid cells J. Mol. Biol., 135, 435-449. Zambetti-Bosseler, F., and D. Scott (1985) The effect of caffeine on X-ray-induced mitotic delay in normal human and ataxia-telangiectasia fibroblasts, Mutation Res., 143, 251-256.