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Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine
Mutation Research, 207 (1988) 171-177
171
Elsevier
MTRL 093
Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine Shishir K. Das Department of Pathology SM-30, University of Washington, Seattle, WA 98195 (U.S.A.) (Accepted 14 December 1987)
Keyword~: AraC-treated V79 cells; V79 cells, araC-treated; Caffeine, araC-treated V79 cells; Cytosine arabinoside.
Summary 0.3-1.0 t~M araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1-2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 ~M araC-treated cells. The cell cycle activated by caffeine in 0.3/~M araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0/~M araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with < G1 DNA content.
In mammalian cells the growth cycle can be divided into well defined phases GI, S, G2 and M (Mitchison, 1971; Prescott, 1976; Pardee et al., 1978). Studies carried out in a wide variety of eukaryotes indicate that this division of the cell cycle is universal (Yanishevsky and Stein, 1981; Lloyd et al., 1982). Although mammalian cell mutants lacking a G1 and/or G2 period have been described in the literature and in early embryogenesis cells lack a GI period, the S phase and mitosis are necessary for the maintenance of DNA Correspondence: Dr. Shishir K. Das, Pulmonary Division, Box 257, New England Medical Center, 171 Harrison Avenue, Boston, MA 02111 (U.S.A.).
homeostasis (Prescott, 1976, 1978; Liskay, 1977; Rao and Sunkara, 1980). At the beginning of the S phase cells have a diploid DNA content per nucleus which becomes tetraploid at the end of the S phase (Yanishevsky and Stein, 1981; Lloyd et al., 1982). Following mitosis the DNA content per cell again becomes diploid and this cycle is repeated during cell multiplication. Interference with the process of DNA replication causes cells to experience a progression slow down in the S phase and a delay in the G2 phase (Young and Fischer, 1968; Tobey, 1972; Tobey and Crissman, 1975). araC has antiproliferative effects on mammalian cells and causes a cell cycle progression slow down by virtue of its ability to be
0165-7992/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
[72
incorporated into D N A (Cohen, 1966; RoyBurman, 1970). The S phase specificity of araC also indicates that it primarily influences the rate of D N A replication (Karon and Shirakawa, 1970). In the present study I show that upon araC treatment Chinese hamster V79 cells experienced cell cycle progression slow down in the S phase and were delayed in G2; upon treatment with 2 mM caffeine, the araC-induced cell cycle progression slow down was relieved and the cells rapidly traversed the cell cycle. Reacquisition of diploid D N A content following cell division indicated that the cell cycle progression induced by caffeine was well behaved and D N A homeostasis was maintained. Materials and methods
(a) Culturing o f V79 cells and drug treatment. Chinese hamster lung fibroblast line V79 cells were grown in minimal Eagle's medium supplemented with dialyzed/heat-inactivated fetal bovine serum (10%), 100 units/ml penicillin, and 100 ~g/ml streptomycin. Cultures were maintained at 37°C in humidified 5% CO2, 95% air mixture. Under these conditions V79 cells had a doubling time of approximately 12 h. Periodic checks for mycoplasma contamination were carried out and the cultures were found to be free of mycoplasma. 1 mM araC stock was prepared in sterile 0.9% sodium chloride solution and diluted immediately before use; 0.1 M caffeine (1,3,7-trimethylxanthine) solution was prepared in 0.9% sodium chloride and sterilized by filtration through a 2-~m filter (millipore) and was warmed to 37°C immediately before use.
ethanol. The filters were dried and counted in aquasol.
(c) Flow microfluorometric analysis. DNA content per nucleus was analyzed as described previously using standard techniques (Das et al., 1983). Cultures were prepared tbr analysis by staining with 4,6-diamino-2-phenylindole (DAPI) in presence of nonionic detergent essentially as described by Rabinovitch et al. (Rabinovitch et al., 1982). (d) Determination o f cell multiplication and cell size. For cell multiplication and cell-size determination 2 ml of trypsinized cell suspension was vortexed vigorously and diluted 10-fold with 18 ml of phosphate-buffered saline (PBS; 10 g/l NaC1, 0.25 g/1 KC1, 0.25 g/l KHzPO4, 3.6 g/1 NaHzPO4 7 H 2 0 adjusted to p H 7.5 with 6 N HC1) and used immediately. Size-distribution measurements were carried out with Coulter Counter Model ZM equipped with channelyzer and an X - Y recorder. The instrument was calibrated with latex microspheres of 5.0, 9.71 and 20.14 ~m diameters. Each sample was analyzed at least twice to account for instrument fluctuations which were found to be negligible as determined by near complete overlap of first and second determinations of size distributions. The average volume was calculated by the formula
~_, vidni/F, i
dni
i
where v~ and dni are the volume and number of cells in the i-th channel.
(b) Measurement of f H ] T d R and [3H]uridine incorporation. After incubation with noted
(e) Uptake o f fH]araC by V79 cells in the presence and absence o f caffeine. 105 V79 cells
amounts of radioactive label for the indicated times, cell monolayers were trypsinized. These trypsinized cell suspensions were precipitated with 0.5 N HC104 containing 10 m M sodium pyrophosphate and chilled. The precipitate was collected on G F / C filters (Glass Fiber; Whatman) which were extensively washed with 0.01 N HC1 containing 10 m M sodium pyrophosphate followed by 95°70
were plated on 35-ram plates 24 h prior to the beginning of the experiment. At t = 0 h, caffeine and [3H]araC (spec. act. 2000 c p m / p m o l e ) were added and the cells were incubated for the indicated times at 37°C. Following this the radioactive medium was aspirated and the plates were washed 3 times with ice-cold PBS. The cell monolayers were suspended in 2 ml of ice-cold 0.1 N
173 TABLE
1
GROWTH
AND SIZE CHANGES
OF V79 CELLS
TREATED
WITH
araC AND CAFFEINE
Time
Treatment
t=
t = Oh
Control
(a)
(b)
(a)
4.72 k 0.06
1.1
9.4
t=24h
12h
* 0.71
(b)
(b)
(a)
1.1
15.3 t 0.13
1.1
+ caffeine
1 mM
8.00 k 0.6
1.1
12.8 10.11
1.1
+ caffeine
2 mM
7.5
f 0.91
0.9
11.o r 0.09
0.88
+ araC 0.3 CM
5.3
+ 0.46
1.66
5.7 2 0.07
1.9
+ araC 0.3 gM + 1 mM caffeine
7.3
+ 0.88
1.2
12.3 t_ 0.13
1.2
+ araC 0.3 rrM + 2 mM caffeine
6.9
+ 0.8
1.2
9.5 +- 0.8
1.2
+ araC
1.0 PM
4.9
+ 0.6
1.9
4.8 2 0.5
+ araC
1.0 pM + 1 mM caffeine
5.8
+ 0.6
1.45
6.2 k 0.7
1.6
+ araC
1.0 pM + 2 mM caffeine
6.7
f 0.7
1.2
8.8 * 0.9
1.2
(a) cell number
per 60-mm plate
x lo-‘;
(b) average
cell volume in picoliters
NaOH which was transferred to a tube. After vortexing the tube, 1 ml of this suspension was mixed with 10 ml Aquasol and radioactivity was determined by liquid-scintillation touting. Results
(a) Growth and size changes of V79 cells upon treatment with araC and caffeine. Treatment of V79 cells with 0.3 PM and 1.0 PM araC caused a near complete shutdown of cell multiplication over a 12-24 h period. While control cultures doubled and nearly quadrupled in 12 h and 24 h respectively very little increase in cell number was observed in araC-treated cultures (Table 1). This growth inhibition by araC was substantially nullified in presence of l-2 mM caffeine. In presence of 1 mM caffeine, 0.3 PM araC-treated cultures increased in cell number by a factor of 1.5 and 2.6 at t = 12 h and t = 24 h respectively. 2 mM caffeine, although slightly growth inhibitory itself, also caused a substantial stimulation of cell growth in araC-treated cultures (Table 1). In araC-inhibited cells, due to the inhibition of DNA replication (see below, [3H]TdR incorporation), there was little increase in cell number; however, cells continued to accumulate protein at a nearly normal rate and increased in cell volume (Table 1). In control cultures, 0.3 PM and
(lo-‘*
liters). araC and caffeine
were added
>2.0
at t = 0 h.
1.0 FM araC-treated the amount of protein/plate increased by a factor of two in 12 h (data not presented). This result is in agreement with the fact that low concentrations of araC selectively inhibit DNA replication without any substantial effects on RNA and protein synthesis (Roy-Burman, 1970; Karon and Shirakawa, 1970). The increase in cell volume was also largely nullified by caffeine indicating progression through the cell cycle followed by cell division. TABLE
2
EFFECT
OF araC AND CAFFEINE
TdR AND [‘HIURIDINE
ON THE RATE OF [3H]-
INCORPORATION
Cells were plated 24 h prior to the beginning at a density added uridine details
of 10’ cells/35
at t = 0 h. [3H]TdR were used
mm plate.
araC and caffeine
(+ 1.0 PM cold TdR)
at a concentration
are in Materials
IN V79 CELLS of the experiments were
and [‘HJ-
of 1.0 &i/ml.
Other
and methods.
Treatment
Relative
incorporation
(4-5 h pulse)
Control + araC + araC
[3H]TdR
[‘Hluridine
0.3 gM
1.0 0.2
k 0.13 kO.03
1.0 1.1
+ 0.08 kO.12
1.0 pM
0.06 + 0.01
0.9
k 0.07 kO.06
+ caffeine
2 mM
+ araC 0.3 gM + caffeine + araC 1.0 gM + caffeine
2 mM 2 mM
1.0
+ 0.16
1.0
1.1 0.8
+0.12 + 0.09
1.1 io.09 0.97 -c 0.11
174
a
b
¢
e
JL_. |i
Relative fluorescence Fig. 1. Induction of rapid ceil cycle progression in araC-treated V?9 cells by caffeine. (a) Control; (b) 0.3 #M araC for 0-10 h; (c) 0.3 #M araC for 0-18 h; (d) 0.3 #M araC for 0-14 h and 2 mM caffeine for 10-14 h; (e) 0.3 #M araC for 0-18 h and 2 mM caffeine for 10-18 h. The two peaks on the DNA-content profile in panel (a) represent diploid (GI) and tetraploid (G2/M) DNA contents respectively. Each panel represents 100% of the nuclei although the area under each curve has not been normalized. Peak locations showed an error of _+5% as was determined by repeated analysis. The relative proportion of cells in each phase is equal to the relative area of that particular phase in each curve individually.
ora C 1.0 xM
LA ara C 1.0p.M + caff 2 m M
Relative flu(Irescence Fig. 2. Formation of nuclei with < G I DNA content following exposure to 1.0 p.M araC and 2 mM caffeine. Top panel, araC 1.0 #M; bottom panel, araC 1.0 #M + 2 mM caffeine, araC was added at t = 0 h and caffeine was added at t = 18 h. a, 18 h; e, 20 h; b,f, 22 h; c,g, 26 h; d,h, 30 h. Other details are as in Fig. 1.
175 TABLE 3 UPTAKE OF [3H]araC BY V79 CELLS IN THE ABSENCEAND PRESENCE OF CAFFEINE Time
pmoles/105 cells araC 0.3 ~M
araC 1.0 #M
No caff.
+ 2 mM caff.
No caff.
+ 2 mM caff.
30min
0.26_+0.06
0.22_+0.05
0.81_+0.11
0.73_+0.10
1h
0.55 +_0.07
0.48_+0.06
1.71 _+0.12
1.62 _+0.17
2h
0.83 -+ 0.06
0.71 _+0.08
2.46 _+0.12
2.64_+0.17
4h
1.21_+0.11
1.05_+0.09
3.51_+0.2
3.87_+0.33
DEAE-Sephadex A-25 chromatographyshowed that >90% of the intracellular araC was in the form of araCTP (data not shown).
(b) Inhibition o f f H ] T d R incorporation in araC-treated cells and its reversal by caffeine. Table 2 shows the dose-response data of the effect o f araC on the rate of [3H]TdR incorporation. During a 4-5 h pulse 1.0 #M araC produced greater than 90% inhibition; this effect of araC was substantially reduced by 2 mM caffeine. The rate o f [3H]uridine incorporation was not significantly altered by these treatments (Table 2).
(c) Effect o f caffeine on araC uptake by V79 cells. In order to rule out the possibility that caffeine was producing its effect by directly inhibiting the uptake of araC, the kinetics of araC uptake was studied in presence and absence of caffeine. To be sure, at 0.3 t~M araC concentration there was a slight reduction in araC uptake in presence of 2 mM caffeine; however, this was not the case at 1.0 #M araC concentration. Furthermore, at 1 mM caffeine substantial reversal of the effect of araC (0.3 #M) was observed; 1 mM caffeine did not produce any effect on araC uptake at 0.3 ~M concentration (data not shown). We therefore believe that caffeine is producing its effect on araC-treated V79 cells mainly by indirect mechanism rather than by directly influencing the uptake (Table 3).
(d) Flow microfluorometric analysis o f the effect o f caffeine on cell cycle progression in araCtreated V79 cells. To investigate directly whether the caffeine effect in araC-treated V79 cells is due t
to the activation of cell cycle progression, cell cycle parameters were analyzed. Exponentially growing V79 cells had approximately 40%, 40% and 20°/o of the cells showing G I , S, and G 2 / M D N A content (Fig. 1). Multiple analysis showed that the error o f location of the G1 and G2 D N A peaks was + 5°7o. At t = 10 h with 0.3 ~M araC most of the cells accumulated in the early S phase and a good number had G 2 / M DNA content; at t = 18 h most cells were still in the S and G2 phases. When 2 mM caffeine was added to araC-treated cells at t = 10 h, rapid progression through the S phase was observed; a large number of cells had traversed the S phase and had either G1 or G 2 / M D N A content at t = 1 4 h . At t = 18 h m o s t cells had moved through the cycle and the cell number doubled. 2 mM caffeine by itself did not drastically alter the cell-cycle distribution at t = 8 h as has been shown by us previously (Das, 1985, 1987). At 1.0 #M araC concentration, massive micronucleation was observed after 2 mM caffeine treatment which gave raise to nuclei with < G1 D N A content (Fig. 2) (Das et al., 1984; Shiraishi et al., 1979).
Discussion araC produced cell-cycle progression slow down in V79 cells. This was indicated by the lack of increase in cell number and reduction in the rate of [3H]TdR incorporation i n araC-treated cultures. These effects of araC were substantially nullified
176
in presence of 1-2 mM caffeine. Lack of increase in cell volume due to araC treatment in presence of caffeine indicated that cells were cycling and going through mitosis. This result was confirmed by flow microfluorometric analysis; caffeine induced a wave of cell cycle progression in araC-treated cells. Following caffeine exposure, cells arrested in early S phase moved through the cell cycle and attained a G2 D N A content and divided. The evidence that after cell division in araC-treated cultures the D N A content per nucleus was equal to G1 D N A content indicated that the cell cycle induced in presence of caffeine was well behaved. For if (1) the cells had divided prior to attaining a G2 D N A content, the daughter cells (at least one of the pair) will have < G 1 D N A in their nuclei and if (2) the cells had continued to synthesized D N A after attaining G2 D N A content they will give rise to nuclei with > G2 DNA. The fact that practically the entire D N A distribution profile was contained within the G1 and G2 D N A content throughout the experiment indicated that majority of cells progressed through the cycle in a well behaved manner and D N A homeostasis was maintained. Lack of cell lethality after 0 . 3 / , M araC + 2 m M caffeine also substantiates this conclusion (data not shown). At 1.0/~M araC concentration, massive micronucleation was observed after 2 m M caffeine treatment which gave rise to micronuclei with < G 1 D N A content (Das et al., 1984; Shiraishi et al., 1979). However, at this araC concentration also there was a substantial G 2 / M D N A peak indicating that some of the cells attained G 2 / M D N A content before dividing. These results are to be contrasted with those of Pardee and colleagues who suggested an abberant activation of mitotic events by caffeine in B H K cells treated with high concentrations of aphidicolin and hydroxyurea (Schlegel and Pardee, 1986). However, these authors did not measure the a m o u n t of D N A in the nucleus and it is possible that the nuclei with condensed chromatin had G2 D N A content; indeed it has been shown that premature c h r o m o s o m e condensation which is Sphase like in appearance can be obtained with nuclei having G2 D N A content upon treatment of V79 cells with UV-light and caffeine (Cremer and
Gray, 1982), Alternatively, it is possible that activation of mitotic processes requires only initiation of late replicating DNA rather than completion of D N A replication. These effects can also be due to the fact that caffeine has multiple effects on m a m m a l i a n cells (Kiblmann, 1977). Previously 1 have shown that the cell cycle progression block produced by low concentrations of aphidicolin can be abrogated by caffeine (Das, 1985, 1987}. It was further demonstrated that caffeine produced this effect presumably by (1) increasing the rate of d C T P formation and (2) increasing the steady state level of d C T P (Das, 1985, 1987), However, the effect of caffeine on aphidicolin uptake was not rigorously ruled out. 111 this communication 1 have shown that the effect of caffeine is indirect in that araC uptake is not influenced by caffeine. The abrogation of cell cycle block due to araC by caffeine suggests that in these cells caffeine might be producing its effect in a manner analogous to its effect in aphidicolintreated V79 cells.
Acknowledgements This work was supported by grant 61-8894 from the U.S. Department of Energy. Thanks are due to Peter Rabinovitch for help with flow microfluorometric analysis and to Joan Hiltner for help with the preparation of the manuscript.
References Cohen, S. (1966) Introduction to the biochemistry of Darabinosyl nucleoside, Prog. Nucl. Acid Res. Mol. Biol., 5, 1-88.
Cremer, C., and J.W. Grey (1982) DNA content of cells with generalized chromosome shattering induced by ultraviolet light plus caffeine, Mutation Res., 94, 133-142. Das, S.K. (1985) Mechanism of caffeine action: Role of dCTP, Fed. Proc. Fed. Am. Soc. Exp. Biol., 44, 1141. Das, S.K. (1987) Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine, Biochim. Biophys. Acta, 928, 29-35. Das, S.K., E.P. Benditt and L.A. Loeb (1983) Rapid changes in deoxynucleoside triphosphate pools in mammalian cells treated with mutagens, Biochem. Biophys. Res. Commun., 114, 458-464.
177 Das, S.K., C.C. Lau and A.B. Pardee (1984) Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells, Mutation Res., 131, 71-79. Karon, M., and S. Shirakawa (1970) Effect of 1-B-D-arabinofuranosyl-cytosine on cell cycle passage time, J. Natl. Cancer Inst., 45, 861-867. Kihlmann, B.A. (1977) Caffeine and Chromosomes, Elsevier, Amsterdam. Liskay, R.M. (1977) Absence of a measurable G2 phase in two Chinese hamster cell lines, Proc. Natl. Acad. Sci. (U.S.A.), 74, 1622-1625. Lloyd, D., P.K. Poole and S.W. Edwards (1982) The Cell Division Cycle, Academic Press, New York. Mitchison, J.M. (1971) The Biology of Cell Cycle, Cambridge University Press, London. Pardee, A.B., R. Dubrow, J.L. Hamlin and R.F. Kletzien (1978) Animal Cell Cycle, Annu. Rev. Biochem., 47, 715-750. Prescott, D.M. (1978) Genetic analysis of the G1 period: Isolation of mutants (or variants) with a G1 period from a Chinese hamster cell line lacking G1, Proc. Natl. Acad. Sci. (U.S.A.), 75, 2873-2877. Rabinovitch, P.S., G.M. Martin and H. Hoehn (1982) Interphase flow-cytogenetics: Correlation of DNA fluorescence to aneuploidy in human fibroblast cultures, Human Genet., 61, 246-249.
Rao, P.N., and P.S. Sunkara (1980) Correlation between the high rate of protein synthesis during mitosis and the absence of G1 period in V79-8 cells, Exp. Cell Res., 125, 507-511. Roy-Burman, P. (1970) Analogues of nucleic acid components, Rec. Res. Cancer Res., 25, 1-111. 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. Shiraishi, Y., K. Yamamoto and A. Sandberg (1979) Effects of caffeine on chromosome abberations and sister-chromatid exchanges, Mutation Res., 62, 139-149. Tobey, R.A. (1972) Arrest of Chinese hamster cells in G2 following treatment with the anti-tumor drug bleomycin, J. Cell Physiol., 79, 259-266. Tobey, R.A., and H.A. Crissman (1975) Comparative effects of three nitrosourea derivatives on mammalian cell cycle progression, Cancer Res., 35, 460-470. Yanishevsky, R.M., and G.H. Stein (1981) Regulation of the cell cycle in eukaryotic cells, Int. Rev. Cytol., 69, 223-259. Young, R.S.K., and G.A. Fischer (1968) The action of arabinosyl-cytosine on synchronously growing populations of mammalian cells, Biochem. Biophys. Res. Commun., 32, 23-29. Communicated by F.H. Sobels
171
Elsevier
MTRL 093
Activation of a well behaved cell cycle in araC-treated V79 cells by caffeine Shishir K. Das Department of Pathology SM-30, University of Washington, Seattle, WA 98195 (U.S.A.) (Accepted 14 December 1987)
Keyword~: AraC-treated V79 cells; V79 cells, araC-treated; Caffeine, araC-treated V79 cells; Cytosine arabinoside.
Summary 0.3-1.0 t~M araC (cytosine arabinoside) treatment of V79 cells produced inhibition of multiplication of cells which was accompanied by a large increase of cell size. In presence of 1-2 mM caffeine the inhibition of cell proliferation due to araC treatment was substantially reduced and cell-size increase was prevented; caffeine did not influence the uptake of araC by V79 cells. Flow microfluorometric analysis showed that caffeine induced a wave of cell cycle progression in 0.3 ~M araC-treated cells. The cell cycle activated by caffeine in 0.3/~M araC-treated cells was largely well behaved; this was indicated by the fact that (1) prior to cell division cells achieved a tetraploid DNA content and (2) following cell division they had diploid DNA content as a result of which DNA homeostasis was maintained. At 1.0/~M araC concentration, however, extreme micronucleation was observed which gave rise to a substantial fraction of micronuclei with < G1 DNA content.
In mammalian cells the growth cycle can be divided into well defined phases GI, S, G2 and M (Mitchison, 1971; Prescott, 1976; Pardee et al., 1978). Studies carried out in a wide variety of eukaryotes indicate that this division of the cell cycle is universal (Yanishevsky and Stein, 1981; Lloyd et al., 1982). Although mammalian cell mutants lacking a G1 and/or G2 period have been described in the literature and in early embryogenesis cells lack a GI period, the S phase and mitosis are necessary for the maintenance of DNA Correspondence: Dr. Shishir K. Das, Pulmonary Division, Box 257, New England Medical Center, 171 Harrison Avenue, Boston, MA 02111 (U.S.A.).
homeostasis (Prescott, 1976, 1978; Liskay, 1977; Rao and Sunkara, 1980). At the beginning of the S phase cells have a diploid DNA content per nucleus which becomes tetraploid at the end of the S phase (Yanishevsky and Stein, 1981; Lloyd et al., 1982). Following mitosis the DNA content per cell again becomes diploid and this cycle is repeated during cell multiplication. Interference with the process of DNA replication causes cells to experience a progression slow down in the S phase and a delay in the G2 phase (Young and Fischer, 1968; Tobey, 1972; Tobey and Crissman, 1975). araC has antiproliferative effects on mammalian cells and causes a cell cycle progression slow down by virtue of its ability to be
0165-7992/88/$ 03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
[72
incorporated into D N A (Cohen, 1966; RoyBurman, 1970). The S phase specificity of araC also indicates that it primarily influences the rate of D N A replication (Karon and Shirakawa, 1970). In the present study I show that upon araC treatment Chinese hamster V79 cells experienced cell cycle progression slow down in the S phase and were delayed in G2; upon treatment with 2 mM caffeine, the araC-induced cell cycle progression slow down was relieved and the cells rapidly traversed the cell cycle. Reacquisition of diploid D N A content following cell division indicated that the cell cycle progression induced by caffeine was well behaved and D N A homeostasis was maintained. Materials and methods
(a) Culturing o f V79 cells and drug treatment. Chinese hamster lung fibroblast line V79 cells were grown in minimal Eagle's medium supplemented with dialyzed/heat-inactivated fetal bovine serum (10%), 100 units/ml penicillin, and 100 ~g/ml streptomycin. Cultures were maintained at 37°C in humidified 5% CO2, 95% air mixture. Under these conditions V79 cells had a doubling time of approximately 12 h. Periodic checks for mycoplasma contamination were carried out and the cultures were found to be free of mycoplasma. 1 mM araC stock was prepared in sterile 0.9% sodium chloride solution and diluted immediately before use; 0.1 M caffeine (1,3,7-trimethylxanthine) solution was prepared in 0.9% sodium chloride and sterilized by filtration through a 2-~m filter (millipore) and was warmed to 37°C immediately before use.
ethanol. The filters were dried and counted in aquasol.
(c) Flow microfluorometric analysis. DNA content per nucleus was analyzed as described previously using standard techniques (Das et al., 1983). Cultures were prepared tbr analysis by staining with 4,6-diamino-2-phenylindole (DAPI) in presence of nonionic detergent essentially as described by Rabinovitch et al. (Rabinovitch et al., 1982). (d) Determination o f cell multiplication and cell size. For cell multiplication and cell-size determination 2 ml of trypsinized cell suspension was vortexed vigorously and diluted 10-fold with 18 ml of phosphate-buffered saline (PBS; 10 g/l NaC1, 0.25 g/1 KC1, 0.25 g/l KHzPO4, 3.6 g/1 NaHzPO4 7 H 2 0 adjusted to p H 7.5 with 6 N HC1) and used immediately. Size-distribution measurements were carried out with Coulter Counter Model ZM equipped with channelyzer and an X - Y recorder. The instrument was calibrated with latex microspheres of 5.0, 9.71 and 20.14 ~m diameters. Each sample was analyzed at least twice to account for instrument fluctuations which were found to be negligible as determined by near complete overlap of first and second determinations of size distributions. The average volume was calculated by the formula
~_, vidni/F, i
dni
i
where v~ and dni are the volume and number of cells in the i-th channel.
(b) Measurement of f H ] T d R and [3H]uridine incorporation. After incubation with noted
(e) Uptake o f fH]araC by V79 cells in the presence and absence o f caffeine. 105 V79 cells
amounts of radioactive label for the indicated times, cell monolayers were trypsinized. These trypsinized cell suspensions were precipitated with 0.5 N HC104 containing 10 m M sodium pyrophosphate and chilled. The precipitate was collected on G F / C filters (Glass Fiber; Whatman) which were extensively washed with 0.01 N HC1 containing 10 m M sodium pyrophosphate followed by 95°70
were plated on 35-ram plates 24 h prior to the beginning of the experiment. At t = 0 h, caffeine and [3H]araC (spec. act. 2000 c p m / p m o l e ) were added and the cells were incubated for the indicated times at 37°C. Following this the radioactive medium was aspirated and the plates were washed 3 times with ice-cold PBS. The cell monolayers were suspended in 2 ml of ice-cold 0.1 N
173 TABLE
1
GROWTH
AND SIZE CHANGES
OF V79 CELLS
TREATED
WITH
araC AND CAFFEINE
Time
Treatment
t=
t = Oh
Control
(a)
(b)
(a)
4.72 k 0.06
1.1
9.4
t=24h
12h
* 0.71
(b)
(b)
(a)
1.1
15.3 t 0.13
1.1
+ caffeine
1 mM
8.00 k 0.6
1.1
12.8 10.11
1.1
+ caffeine
2 mM
7.5
f 0.91
0.9
11.o r 0.09
0.88
+ araC 0.3 CM
5.3
+ 0.46
1.66
5.7 2 0.07
1.9
+ araC 0.3 gM + 1 mM caffeine
7.3
+ 0.88
1.2
12.3 t_ 0.13
1.2
+ araC 0.3 rrM + 2 mM caffeine
6.9
+ 0.8
1.2
9.5 +- 0.8
1.2
+ araC
1.0 PM
4.9
+ 0.6
1.9
4.8 2 0.5
+ araC
1.0 pM + 1 mM caffeine
5.8
+ 0.6
1.45
6.2 k 0.7
1.6
+ araC
1.0 pM + 2 mM caffeine
6.7
f 0.7
1.2
8.8 * 0.9
1.2
(a) cell number
per 60-mm plate
x lo-‘;
(b) average
cell volume in picoliters
NaOH which was transferred to a tube. After vortexing the tube, 1 ml of this suspension was mixed with 10 ml Aquasol and radioactivity was determined by liquid-scintillation touting. Results
(a) Growth and size changes of V79 cells upon treatment with araC and caffeine. Treatment of V79 cells with 0.3 PM and 1.0 PM araC caused a near complete shutdown of cell multiplication over a 12-24 h period. While control cultures doubled and nearly quadrupled in 12 h and 24 h respectively very little increase in cell number was observed in araC-treated cultures (Table 1). This growth inhibition by araC was substantially nullified in presence of l-2 mM caffeine. In presence of 1 mM caffeine, 0.3 PM araC-treated cultures increased in cell number by a factor of 1.5 and 2.6 at t = 12 h and t = 24 h respectively. 2 mM caffeine, although slightly growth inhibitory itself, also caused a substantial stimulation of cell growth in araC-treated cultures (Table 1). In araC-inhibited cells, due to the inhibition of DNA replication (see below, [3H]TdR incorporation), there was little increase in cell number; however, cells continued to accumulate protein at a nearly normal rate and increased in cell volume (Table 1). In control cultures, 0.3 PM and
(lo-‘*
liters). araC and caffeine
were added
>2.0
at t = 0 h.
1.0 FM araC-treated the amount of protein/plate increased by a factor of two in 12 h (data not presented). This result is in agreement with the fact that low concentrations of araC selectively inhibit DNA replication without any substantial effects on RNA and protein synthesis (Roy-Burman, 1970; Karon and Shirakawa, 1970). The increase in cell volume was also largely nullified by caffeine indicating progression through the cell cycle followed by cell division. TABLE
2
EFFECT
OF araC AND CAFFEINE
TdR AND [‘HIURIDINE
ON THE RATE OF [3H]-
INCORPORATION
Cells were plated 24 h prior to the beginning at a density added uridine details
of 10’ cells/35
at t = 0 h. [3H]TdR were used
mm plate.
araC and caffeine
(+ 1.0 PM cold TdR)
at a concentration
are in Materials
IN V79 CELLS of the experiments were
and [‘HJ-
of 1.0 &i/ml.
Other
and methods.
Treatment
Relative
incorporation
(4-5 h pulse)
Control + araC + araC
[3H]TdR
[‘Hluridine
0.3 gM
1.0 0.2
k 0.13 kO.03
1.0 1.1
+ 0.08 kO.12
1.0 pM
0.06 + 0.01
0.9
k 0.07 kO.06
+ caffeine
2 mM
+ araC 0.3 gM + caffeine + araC 1.0 gM + caffeine
2 mM 2 mM
1.0
+ 0.16
1.0
1.1 0.8
+0.12 + 0.09
1.1 io.09 0.97 -c 0.11
174
a
b
¢
e
JL_. |i
Relative fluorescence Fig. 1. Induction of rapid ceil cycle progression in araC-treated V?9 cells by caffeine. (a) Control; (b) 0.3 #M araC for 0-10 h; (c) 0.3 #M araC for 0-18 h; (d) 0.3 #M araC for 0-14 h and 2 mM caffeine for 10-14 h; (e) 0.3 #M araC for 0-18 h and 2 mM caffeine for 10-18 h. The two peaks on the DNA-content profile in panel (a) represent diploid (GI) and tetraploid (G2/M) DNA contents respectively. Each panel represents 100% of the nuclei although the area under each curve has not been normalized. Peak locations showed an error of _+5% as was determined by repeated analysis. The relative proportion of cells in each phase is equal to the relative area of that particular phase in each curve individually.
ora C 1.0 xM
LA ara C 1.0p.M + caff 2 m M
Relative flu(Irescence Fig. 2. Formation of nuclei with < G I DNA content following exposure to 1.0 p.M araC and 2 mM caffeine. Top panel, araC 1.0 #M; bottom panel, araC 1.0 #M + 2 mM caffeine, araC was added at t = 0 h and caffeine was added at t = 18 h. a, 18 h; e, 20 h; b,f, 22 h; c,g, 26 h; d,h, 30 h. Other details are as in Fig. 1.
175 TABLE 3 UPTAKE OF [3H]araC BY V79 CELLS IN THE ABSENCEAND PRESENCE OF CAFFEINE Time
pmoles/105 cells araC 0.3 ~M
araC 1.0 #M
No caff.
+ 2 mM caff.
No caff.
+ 2 mM caff.
30min
0.26_+0.06
0.22_+0.05
0.81_+0.11
0.73_+0.10
1h
0.55 +_0.07
0.48_+0.06
1.71 _+0.12
1.62 _+0.17
2h
0.83 -+ 0.06
0.71 _+0.08
2.46 _+0.12
2.64_+0.17
4h
1.21_+0.11
1.05_+0.09
3.51_+0.2
3.87_+0.33
DEAE-Sephadex A-25 chromatographyshowed that >90% of the intracellular araC was in the form of araCTP (data not shown).
(b) Inhibition o f f H ] T d R incorporation in araC-treated cells and its reversal by caffeine. Table 2 shows the dose-response data of the effect o f araC on the rate of [3H]TdR incorporation. During a 4-5 h pulse 1.0 #M araC produced greater than 90% inhibition; this effect of araC was substantially reduced by 2 mM caffeine. The rate o f [3H]uridine incorporation was not significantly altered by these treatments (Table 2).
(c) Effect o f caffeine on araC uptake by V79 cells. In order to rule out the possibility that caffeine was producing its effect by directly inhibiting the uptake of araC, the kinetics of araC uptake was studied in presence and absence of caffeine. To be sure, at 0.3 t~M araC concentration there was a slight reduction in araC uptake in presence of 2 mM caffeine; however, this was not the case at 1.0 #M araC concentration. Furthermore, at 1 mM caffeine substantial reversal of the effect of araC (0.3 #M) was observed; 1 mM caffeine did not produce any effect on araC uptake at 0.3 ~M concentration (data not shown). We therefore believe that caffeine is producing its effect on araC-treated V79 cells mainly by indirect mechanism rather than by directly influencing the uptake (Table 3).
(d) Flow microfluorometric analysis o f the effect o f caffeine on cell cycle progression in araCtreated V79 cells. To investigate directly whether the caffeine effect in araC-treated V79 cells is due t
to the activation of cell cycle progression, cell cycle parameters were analyzed. Exponentially growing V79 cells had approximately 40%, 40% and 20°/o of the cells showing G I , S, and G 2 / M D N A content (Fig. 1). Multiple analysis showed that the error o f location of the G1 and G2 D N A peaks was + 5°7o. At t = 10 h with 0.3 ~M araC most of the cells accumulated in the early S phase and a good number had G 2 / M DNA content; at t = 18 h most cells were still in the S and G2 phases. When 2 mM caffeine was added to araC-treated cells at t = 10 h, rapid progression through the S phase was observed; a large number of cells had traversed the S phase and had either G1 or G 2 / M D N A content at t = 1 4 h . At t = 18 h m o s t cells had moved through the cycle and the cell number doubled. 2 mM caffeine by itself did not drastically alter the cell-cycle distribution at t = 8 h as has been shown by us previously (Das, 1985, 1987). At 1.0 #M araC concentration, massive micronucleation was observed after 2 mM caffeine treatment which gave raise to nuclei with < G1 D N A content (Fig. 2) (Das et al., 1984; Shiraishi et al., 1979).
Discussion araC produced cell-cycle progression slow down in V79 cells. This was indicated by the lack of increase in cell number and reduction in the rate of [3H]TdR incorporation i n araC-treated cultures. These effects of araC were substantially nullified
176
in presence of 1-2 mM caffeine. Lack of increase in cell volume due to araC treatment in presence of caffeine indicated that cells were cycling and going through mitosis. This result was confirmed by flow microfluorometric analysis; caffeine induced a wave of cell cycle progression in araC-treated cells. Following caffeine exposure, cells arrested in early S phase moved through the cell cycle and attained a G2 D N A content and divided. The evidence that after cell division in araC-treated cultures the D N A content per nucleus was equal to G1 D N A content indicated that the cell cycle induced in presence of caffeine was well behaved. For if (1) the cells had divided prior to attaining a G2 D N A content, the daughter cells (at least one of the pair) will have < G 1 D N A in their nuclei and if (2) the cells had continued to synthesized D N A after attaining G2 D N A content they will give rise to nuclei with > G2 DNA. The fact that practically the entire D N A distribution profile was contained within the G1 and G2 D N A content throughout the experiment indicated that majority of cells progressed through the cycle in a well behaved manner and D N A homeostasis was maintained. Lack of cell lethality after 0 . 3 / , M araC + 2 m M caffeine also substantiates this conclusion (data not shown). At 1.0/~M araC concentration, massive micronucleation was observed after 2 m M caffeine treatment which gave rise to micronuclei with < G 1 D N A content (Das et al., 1984; Shiraishi et al., 1979). However, at this araC concentration also there was a substantial G 2 / M D N A peak indicating that some of the cells attained G 2 / M D N A content before dividing. These results are to be contrasted with those of Pardee and colleagues who suggested an abberant activation of mitotic events by caffeine in B H K cells treated with high concentrations of aphidicolin and hydroxyurea (Schlegel and Pardee, 1986). However, these authors did not measure the a m o u n t of D N A in the nucleus and it is possible that the nuclei with condensed chromatin had G2 D N A content; indeed it has been shown that premature c h r o m o s o m e condensation which is Sphase like in appearance can be obtained with nuclei having G2 D N A content upon treatment of V79 cells with UV-light and caffeine (Cremer and
Gray, 1982), Alternatively, it is possible that activation of mitotic processes requires only initiation of late replicating DNA rather than completion of D N A replication. These effects can also be due to the fact that caffeine has multiple effects on m a m m a l i a n cells (Kiblmann, 1977). Previously 1 have shown that the cell cycle progression block produced by low concentrations of aphidicolin can be abrogated by caffeine (Das, 1985, 1987}. It was further demonstrated that caffeine produced this effect presumably by (1) increasing the rate of d C T P formation and (2) increasing the steady state level of d C T P (Das, 1985, 1987), However, the effect of caffeine on aphidicolin uptake was not rigorously ruled out. 111 this communication 1 have shown that the effect of caffeine is indirect in that araC uptake is not influenced by caffeine. The abrogation of cell cycle block due to araC by caffeine suggests that in these cells caffeine might be producing its effect in a manner analogous to its effect in aphidicolintreated V79 cells.
Acknowledgements This work was supported by grant 61-8894 from the U.S. Department of Energy. Thanks are due to Peter Rabinovitch for help with flow microfluorometric analysis and to Joan Hiltner for help with the preparation of the manuscript.
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