Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Biochimie, 69 (1987) 1217- 1225 © Soci~t~ de Chimie biologique/ElsevieL Paris

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Research article

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth Gilbert MORIS, Denise MEYER, Georges ORFANOUDAKIS, Nicole BEFORT, Jean-Pierre EBEL and Pierre REMY Institut de Biologie Moldculaire et Cellulaire du Centre National de la Recherche Scientifique, 15, rue Descartes, 67084 Strasbourg Cedex, France (Received 3-9-1987, accepted after revision 30-9-1987)

Summary -

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracc!lular pools ",f dinucleoside tetraphosphates (AP4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 AP4X and ATP levels was observed when the cells entered the G I phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the GI/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory [14] and the observed decrease in AP4X pool after release from mitotic-arrested HTC cells. These results suggest that the AP4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G 2 phase and after mitosis. u...u'L..~..vd| . . . . | ~ | d ~o . u~., tt ~~ ,g.l~f l-]-[hJAi l~UR~ hJ l . t i l a. l. L. .~

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R ~ s u m ~ - V a r i a t i o n s de b i s n u c l ~ o s i d e s t ~ t r a p h o s p h a t e s au cours du cycle cellulaire e~ d_e la c r o i s s a n c e de cellules t u m o r a l e s en culture, Des cultures de cellules A549 et H T C asynchrones et synchronisdes ont dtd utilis~es pour d6tecter des variations dventuelles de la teneur en bisnucl6osides tdtraphosphates (AP4X) spdcifiques du cycle cellulaire ou de la densitd cellulaire. Les mesures effectudes n'ont pas montrd de variation importante de ia teneur intracellulaire de ces composds pendant !a croissance celiulaire. Lors du ddblocage de cellules H T C arr~tdes en mitose, une diminution d'un facteur 3 environ du taux d ' A P 4 X et d ' A T P est observde ~ l'entrde en phase G 1. Cette diminution est essentiellement due au doublement du nombre de cellules. Lors du ddblocage de cellules A549 d'un arr~t ~ l'interphase G I/S, la teneur en A p 4 X augmente pendant la phase G 2, juste avant ia mitose. Cette observation est en accord avec des rdsultats antdrieurs de notre laboratoire ainsi qu'a~,ec la diminution observde aprbs le ddbiocage de cellules H T C arr~tdes en mitose. Ces rdsultats suggbrent que la concentration intracellulaire en A p 4 X comme celle de i'A TP est soumise ~ des variations faibles pendant le cycle ceilulaire, une en phase G 2, l'autre aprbs la mitose.

bisnucidosides t~traphosphates I cellules tumorales I croissance cellulaire I synchronisation I bioluminescence

Introduction

Diadenosine 5',5'"-tetraphosphate (AP4A), which is synthesized in vitro and presumably in vivo by

the reaction of an aminoacyladenylate with ATP has been reported to be implicated in the regulation of cell growth and DNA replication of eukaryotic cells [1,2]. The arguments in favor of

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G. Mo"!s e! al.

this hypothesis are the following: AP4A levels are inversely related to doubling time [3]; AP4A stimulates the initiation of DNA synthesis when added to permeabilized G,-arrested, quiescent baby hamster kidney cells [4]; microinjection of AP4A into unfertilized eggs of Psammechinus miliaris and into X e a o p u s iaevis oocytes has been shown to induce an increase of DNA synthesis, which in the latter case is inhibited in the presence of aphidicolin, suggesting a role of D N A polymerase ~t in this process [5, 6]. The intracellular target of Ap4A has been reported to be the DNA polymerase a. Indeed, AP4A was reported to act as a primer in D N A synthesis by the DNA polymerase a of HeLa cells using a poly(dT) or a double-stranded synthetic octadecamer as template [7-9]. An Ap4A binding protein associated with a high molecular weight form of DNA polymerase a from HeLa cells or calf thymus has been purified [7-9]. HeLa cell DNA polymerase ~t has also been shown to be associated with tryptophanyl-tRNA synthetase, suggesting a link between amino acid activation and DNA replication in mammalian cells [10]. Weinmann-Dorsch et al. reported a 1000-fold increase of Ap4A pool in Gl-arrested mitogen-stimulated mouse 3T3 and baby hamster kidney fibroblasts [11]. Similarly, an 8-30-fold increase of the Ap4A level during the naturally synchronous cell cycle of the slime mold Physarum p o l y c e p h a l u m was described by the same authors [12]. However, these last results are highly controversial, since this AP4A accumulation preceding DNA synthesis could not be confirmed by other groups, either in P. polycephalum [13], or in cultured mammalian cells [14]. The absence of any variation in the AP4A content throughout the cell cycle was also reported for synchronized Escherichia coli cells [30]. Recently, Segal et al. [15] suggested that AP4A might be involved in cell contact inhibition rather than in cell growth stimulation, once more questioning the correlation between the cellular AP4A level and growth stimulation. Facing these contradictory observations concerning the possible role of AP4A in cell growth and DNA replication, we tried to contribute to the clarification of these problems by investigating the intracellular levels of AP4X and ATP during cell growth of two transformed mammalian cell lines. We also made use of different synchronization methods for arresting cellz at 3 different stages of the cell cycle in order to detect possible cell cyclespecific variations of the nucleotide pools, a study that had already been realized in our laboratory, using other cell lines and other synchronization methods [14]. Our present report indicates that the

AP4X as well as the ATP pools are subiected to a slight increase during the G 2 phase, followed by a decrease after mitosis. The described increase could correspond to the accumulation of nucleotides prior to cell division, while the drop after mitosis could mainly reflect cell doubling. These experiments once more rule out the existence of large variations in the AP4X pool throughout the cell cycle, further questioning the role of AP4A as a trigger for the proliferative state of eukaryotic cells.

Materials and methods Chemicals and enzymes 3-Aminophenylboronic acid hemisulfate and nocodazole were obtained from Aldrich (Steinheim, F.R.G.). BioRex 70 (100-200 mesh) was from BioRad Laboratories (Richmond, CA). The condensation of the resin with aminophenylboronic acid was performed as described in [16]. ATP, AP4A, thymidine, luciferase from Photinus pyralis (EC 1.13.12.7, 11 U/mg), bacterial alkaline phosphatase (EC 3.1.3.1, 11 U/rag, product number 4377) were purchased from Sigma (St. Louis, MO). Luciferin was purchased from Boehringer (Mannheim, F.R.G.) and snake venom phosphodiesterase (EC 3.1.15.1, 25 U/rag) from Cooper Biomedical (Marne-la-Vall6e, France). [Methyl-3H]-thymidine (42 Ci/mmol) was purchased from the Commissariat/l l'Energie Atomique (Saclay, France). All chemicals necessary for cell culture were purchased from GIBCO Laboratories (Paisley, Scotland). The tissue culture flasks were obtained from NUNC (Roskiide, Denmark). Cell lines and cultures HTC cells (hepatoma tissue culture cells) and A549 (human lung carcinoma) were grown as monolayers in complete medium i.e., Dulbecco's Modified Eagle's Medium (DMEM) containing 0.3070 glucose, 50 U of penicillin and 50 mg of streptomycin per ml and supF~emented with 10% newborn calf serum for HTC cells or !00/0 fetal calf serum for A549 cells [17, 18]. These epithelial cells were routinely replated on 80 cm2 tissue culture flasks before reaching confluence and grown at 37°C in a water saturated-5% CO2 atmosphere. For the cell growth study, 5 x 105 (HTC or A549 cells) were plated in 25 c m 2 culture flasks and grown under standard conditions until confluence. Cell synchronization HTC cells were blocked in mitosis by nocodazole treatment [19] under the following conditions: samples of 10 x 10 6 cells were plated in 180 c m 2 culture flasks with complete medium. After 24 h, fresh medium containing 3 mM thymidine was added in order to presynchronize the cells. After 20 h of thymidine incubation, cells were washed twice with phosphate buffered saline (PBS: 136mM NaCl; 2.6mM KCI; 6.5 mM Na2HPO4; 1.5 mM KH2PO4; pH 7.4) and refed with complete

Dinucleoside tetraphosphates and the cell cycle medium without thymidine. 8 h later, nocodazole was added to a final concentration of 0.1/ag/ml (from a 10 mg/ml solution in dimethyl sulfoxide) and allowed to act for 6 h. Mitotic cells were recovered by gentle shaking, centrifuged, washed twice in fresh medium and replated at 2 x 10 6 cells in 25 cm 2 culture flasks. A549 cells were synchronized in the S phase by two consecutive thymidine treatments [20]. For the synchronization, 1.5× 10 6 cells were plated in 80 cm 2 culture flasks in complete medium and grown under standard conditions for 24 h. The medium was then replaced with fresh complete medium containing 3 mM thymidine. After 17 h, the cells were released from thymidine block and cultured without thymidine for 8 h. A second block of 3 mM thymidine led to the accumulation of all cells in the very early S 0hase. A549 cells were also synchronized in the Gt phase by serum deprivation. 2x 106 cells were plated in 80 cm 2 culture flasks in DMEM without serum for 48 h.

DNA labeling After synchronization, the progression of cells through the cell cycle was studied by [3H]thymidine incorporation. The rate of DNA synthesis was determined by labeling cells with 1-2 tzCi/ml of [3H]thymidine (42 Ci/mmol) for 30 min at different times after release from the respective synchronization blocks. After incubation, cells were washed, dissolved in 1 ml of 0.1 N NaOH and kept at 50°C for 30 min, in order to hydrolyze the RNA. DNA was precipitated in 5070trichloroacetic acid at 4°C, collected on glass fiber filters and washed 3 times with 507o trichloroacetic acid. The radioactivity was measured by scintillation counting in 3 ml of a 4070(w/v) solution of omnifluor in toluene.

Protein determination Protein contents were determined by Lowry's method [23] or Bradford's method developed for ELISA plates [24, 25]. Bovine serum a~bumin was used as the standard in each case.

Nucleotide extraction Cells were grown in 25 or 80 cm 2 culture flasks. After removal of the medium, cell layers were washed twice with PBS at 37°C before the addition of, respectively, 3 or 8 ml of 5°70 (w/v) trichloroacetic acid and further incubation for 15 min at 4°C. From the recovered supernatant, 200 tzl were kept at -20°C for ATP measurement, whereas the remaining solution was used to separate specifically the dinucleoside tetraphosphates from the remaining pool of nucleotides by affinity chromatography on dihydroxyboryl BioRex, according to the procedure published by Baker and Jacobson [16].

AP4X and A TP determinations AP4X and ATP levels were measured by a bioluminescence assay as described by Ogilvie et al. [21], Baltzinger et al. [22] or Orfanoudakis et al. [141.

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Results A P 4 X and A T P p o o l size variations in asynchronously growing cells H T C and A549 cells were allowed to grow until confluence, with or without a daily renewal of the culture medium. Fig. 1 shows that for HTC cells this renewal did not affect either Ap4X, or A T P intracellular conccntrations, since both pool sizes remained constant in each case during the whole experiment up to cell confluence ( l a - d ) . The average APaX intracellular concentration in exponentially growing H T C cells was found to be 0 . 5 + 0 . 0 7 5 pmol of AP4X/106 cells or 2_+ 0.25 p m o l / m g of protein (mean for 10 experiments). In A549 cells, the daily renewal of the culture medium led to a somewhat larger APaX pool size, but the general evolution remained the same in both cases i.e., the APaX pool compared to the cell number decreased between days 2 and 3, then remained constant for 2 days before another decrease between days 5 and 6, when cells reached confluence (Fig. l g). This variation in AP4X content is not significantly changed when compared to protein content, as shown in Fig. If. The average AP4X value (determined for 10 experiments) in exponentially growing cells was 0.4 + 0.08 pmol/106 cells or 1.5 + 0 . 2 p m o l / m g of protein (mean for 10 experiments). The: A T P pool size remained constant until day 3, then decreased slowly until confluence. There was no decrease in the A T P pool during the first 2 days of culture contrary to what was observed for the APaX pool (Fig. l h).

Efficiency o f the synchronization methods The various synchronization methods used were chosen so as to allow the accumulation of cells at different stages of the cell cycle without affecting cell viability or generation time. The generation time and the respective le~lgths of the different phases of the cell cycle were determined, using the graphical analysis described by Okada [26] and are indicated in Table I. H T C cells, presynchronized by thymidine treatment, were arrested in mitosis by a nocodazole block and allowed to resume growth by replating them in normal medium without nocodazole. Nocodazole inhibits tubulin polymerization [19] required for mitotic spindle formation and therefore leads to the accumulation of cells in late prophase. DNA synthesis was studied at different times after release from mitotic arrest.

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Fig. 1. Ap4X and A T P cellular contents during growth of HTC and A549 cells, with ( I ) or without (D) a daily renewal of culture medium. Evolution of the HTC cell population (a); Ap4X contents of HTC cells compared to protein contents (b) and to cell number (c); A T P contents compared to cell number (d). Values represented in b, c and d are the averages of 2 independent bioluminescence measurements. Evolution of the A549 cell population (e); AP4X contents of A549 cells compared to protein contents (f) and to cell number (g); ATP contents compared tn cell number (h). Bars indicate the results of 2 independent bioluminescence measurements.

Table I. Lengths of the different phases of the cell cycle of HTC and A549 cells.

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Following the release from mitotic arrest, the percentage of dividing cells passed from 90°70 to 5 070 within 2 h, showing that cells were synchronized in a very small time lapse. [3H]Thymidine incorporation began 6 - 7 h later which is the time course observed in cells with a short G] phase (which is known to be the most variable phase of the cell cycle). The peak DNA synthesis was observed 12 h after mitosis. At this time, about 80070 of the cells were in S phase as shown by autoradiography [31]. 20 h after mitosis, [3H]thymidine incorporation had returned to its initial level in early G l phase.

Dinucleoside tetraphosphates and the cell cycle Cell growth curves indicated that the cell population doubled rapidly after release and then remained almost constant. The protein content of the cells increased slowly but regularly during the S phase (Fig. 2a-c). A549 cells were synchronized in the ~)ery early S phase by a double thymidine treatment. In~•ibition of DNA synthesis is due to the action of dTTP as an allosteric inhibitor of ribonucleotide reductase. The double treatment led to a high synchronization efficiency as shown in Fig. 3.3 h after the release of the thymidine block, DNA synthesis was at its maximum, with the incorporation of [3H]thymidine being about 2-fold higher than in exponentially growing cells. This reflects a good synchronization efficiency, since the maximum attainable stimulation factor is close to 2.5 (in asynchronous exponentially growing cells, about 40% of the cells are

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engaged in S phase). 10 h after release of the block, [SH]thymidine incorporation had returned to its initial level. A549 cells were also synchronized by a total serum deprivation for 48 h. After mitogenic stimulation upon serum readdition, [SH]thymidine incorporation increased regularly during the first 4 h of the study, decreasing later on, but without returning to its initial level during the whole experiment. The somewhat unusual look of the DNA synthesis curve was due to an accumulation of cells at different stages of the G] phase, leading to a continuotis entry of cells into the S phase (Fig. 4a). The early incorporation corresponded to the cells which had been arrested in the late G 1 phase, while the cells starting at an earlier stage of the G] phase were responsible for the continuous incorporation between 7 and 15 h.

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Fig. 2. AP4X and A T P contents of H T C cells after release from a nocodazole block. Evolution of protein contents (a), evolution of cell population (b). [3H]Thymidine incorporation into DNA compared to protein contents (¢). A T P contents compared to cell number (d), Ap4X contents compared to cell number (e) and to protein contents (f).

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Fig. 3. ApeX and ATP cellular contents in A549 cells after release from a thymidine block. [3H]Thymidine incorporation compared to cell number (a), evolution of cell number Co). ATP contents compared to cell number (e). Ap4X contents compared to cell number (d) and to the amount of cellular protein contents (e). Experimental data in c, d and e were obtained from 2 independent bioluminescence measurements indicated by the bars.

AP4X and A TP pool size variations after release o f mitosis-arrested H T C cells When HTC cells were released from mitotic arrest, the A P 4 X intracellular level underwent a net decrease within 2 h, by a factor of 3.4 when the A P 4 X c o n t e n t was compared to the cell number, and 1.8 when compared to the protein content. The reason for this decrease is partly due to cell division (since the AP4X content corresponds to twice as many cells). However, since the decrease is not completely erased when the Ap4X content is compared to protein, we conclude that there is a real decrease in the A P 4 X concentration. This level

then remains constant during the whole experiment (Fig. 2e and f). The A T P pool size was roughly subjected to a parallel evolution, i.e., it underwent a decrease just after mitosis and then remained unchanged for the duration of the exp,~riment (Fig. 2d).

Ap4 X and A TP pool size variations after release o f A549 cells arrested in very early S phase When A549 cells were released from a thymidine block, the A P 4 X level remained constant for 6 h and then underwent an increase (1.8-fold) corresponding to the entry of the cells into the G 2

Dinucleoside tetraphosphates and the cell cycle

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it did not exceed 25%, whereas experimental variation may be as high as 20°70. The AP4X concentration can therefore be regarded as constant. However, it must be stressed that during the first hours of complete serum deprivation, the AP4X content increases by a factor of 2.5, when compared to protein contents, with respect to control cells. The origin of this increase is unknown, but once more, this observation questions the relationship between high Ap4X contents and the proliferative state. This point will be further dealt with in the Discussion. The ATP pool, contrary to what was observed for the AP4X pool, drops by a factor of 2.5 in the first hours of serum starvation. When serum is added to the culture medium, the ATP concentration increases again up to the control level, which is reached after 2 - 3 h (Fig. 4d).

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Fig. 4. AP4X and ATP contents after mitogenic stimulation of A549 cells arrested in the Gt phase by total serum deprivation. DNA synthesis measured by [3H]thymidine incorporation (a). APnX contents compared to cell number (b) and to protein contents (c). ATP contents compared to protein contents (d).

This study was undertaken in order to detect possible variations of the AP4A pool size along a growth curve and throughout the cell cycle of mammalian cells. In fact, the coupled lucif e r a s e - p h o s p h o d i e s t e r a s e assay detects all dinucleoside tetraphosphates of general structure AP4X where X represents any nucleoside. However, as shown by high-performance liquid chromatography [13, 16, 27, 28], AP4A contributes f,'~ ~ - h ~ k;,-,,-~,...+

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phase, as shown by the decrease of labeled thymidine incorporation. After mitosis, the AP4X level compared to cell number drops by a factor of about 2, as observed in the case of mitosis-arrested HTC cells (see above) (Fig. 3d and e). The ATP pool increased slowly but regularly during the first 6 h before undergoing a stronger increase during the G 2 phase parallel to the one described for the Ap4X pool. After mitosis, the ATP intracellular concentration also returned to the value measured in the early G 2 phase (Fig. 3c).

AP4X and A TP pool size variations after mitogenic stimulation o f serum-deprived A549 cells Mitogenic stimulation of A549 cells which had been previously completely deprived of serum did not lead to any significant variation of the AP4X pool (Fig. 4b and c). There was an overall tendancy towards a decrease throughout the experiment, but

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so we can assume that our measured values reflect the AP4A level. Dinucleoside tetraphosphate contents were compared to either cell number or the total amount of proteins, since as the cell ~olume increases throughout the cell cycle, conversion to an intraceUular concentration would be difficult. Furthermore, cell counting could only be carried out on parallel culture flasks (since trichloroacetic acid extraction of nucleotides prevented subsequent trypsinization), whereas protein contents could be determined on the same cell population that was used for the extraction of nucleotides. In the study of nucleotide pool variations during cell growth, determinations were always carried out on two parallel flasks, one in which the medium had been changed every day and another in which the medium was not renewed during the whole experiment, so as to be able to detect a possible contribution of the modification of the culture medium to nucleotide pool sizes. Our study did not reveal any large variation of nucleotide pools during cell growth. In the case of HTC cells, the Ap4X level

1224

G. Moris et al.

only varies by roughly 30070 between day 1 when the cells are plated and day 5 when the cells reach confluence. Contrary to what could be expected if AP4X were a possible activator of the proliferative state [3], tt:~ lowest concentration of AP4X was observed during the exponential growth phase, the highest concentration being observed both at the beginning of growth curve (just after replating of the cells) and at the end of the growth period (when the cells reach confluence). For A549 carcinoma cells, similar results are observed, the highest concentration of AP4X being observed just after cell replating. During exponential grow[h, the AP4X concentration is roughly 40°70 lower, but this concentration continues to decrease up to day 5 of culture, contrary to what is observed for HTC cells. It has to be noted, however, that A549 cells still grow very efficiently after 120 h of culture, while HTC cell growth slows considerably after 80 h. This difference in the behavior of the two cell lines may reflect a higher sensitivity of HTC cells to the composition of the culture medium, as shown by the more rapid arrest of cell growth when the medium is not renewed (Fig. 1a and e). Clearly, the relationship between the AP4X concentration and cell growth is much more complex than suggested by the early studies (see also [15]) and needs to be more deeply investigated. This point will be further illustrated in the discussion of the effect of serum depletion (see below). To study the possible variations of AP4X and ATD

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pounds are in equilibrium inside the cell [29]. It should be emphasized that our experiments could not confirm either the initial observations reported in [11], showing a huge burst of AP4X at the GI/S boundary preceding DNA synthesis, or the cell cycle-dependent 8-30-fold variation of the Ap4X level in the autosynchronous mold Physarum polycephalum [12]. On the contrary, they confirm our earlier results [14] and are similar to those of Garrison et al. [13], who reinvestigated the variation of Ap4X in the P. polycephalum cell cycle, as well as those of Plateau et al. [30] who did not observe any variation of the AP4X level during the E. coli cell cycle. In the course of A549 cell synchronization experiments, it was observed that the complete deprivation of the growth medium in calf serum resulted in a substantial increase in the AP4X content (2.5-fold) as compared to control cells. This is once again in contrast to the idea that AP4X could be a positive regulator of cell growth, as were the observations that: 1) the AP4X level does not increase when phytohemagglutinin-stimulated human lymphocytes enter into cell division (results not shown); and 2) the Ap4X level in rapidly proliferating cells, such as Saccharomyces cerevisiae is not higher than those observed in naturally resting cells, such as Xenopus laevis oocytes or human lymphocytes [22, 29]o All these observations seem to question seriously the possibility that AP4A is a regulator of cell

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synchronization methods had to be used, in order to ensure that the effects observed would not result from the use of chemical synchronizing agents. Furthermore, the rapid desynchronization of the cells after release from the chemical block (especially during progression through the G 1 phase) required an arrest of the cells at different stages, so as to allow us to scan the whole cell cycle with rather synchronous populations. Here too, only moderate variations of the Ap4X level were observed, since the largest variation did not exceed a factor of 3.4 when compared to cell number or 1.8 when compared to total proteins. Our results indicate that the AP4X content slightly increases in the G 2 phase just before mitosis, and drops abruptly to its initial level after cell division. This observation is consistent with the idea that the cell is simply accumulating important compounds necessary for the development of the daughter cells, as suggested by our previous experiments [14]. It is not clear however if the AP4X variation has a significance per se, since the ATP pool varies roughly in the same way and since we could show that both com-

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Acknowledgments We wish to acknowledge the help of Dr. J. Bieth in providing us with the A549 carcinoma cell line.

References 1 Zamecnik P.C., Stephensen M.L., Janeway C.M. & Randerath K. (1966) Biochem. Biophys. Res. Comrnun. 24, 91-97 2 Zamecnik P.C. (1983)Anal. Biochem. 134, 1-10 3 Rapaport E. & Zamecnik P.C. (1976) Proc. Natl. Acad. Sci. USA 73, 3984-3988 4 Grummt F. (1978) Proc. Natl. Acad. Sci. USA 75, 371-375 5 Weinmann-Dorsch C. & Grummt F. (1985) Exp. Cell Res. 160, 47-53 6 Zourgui L., Tharaud D., Solari A., Litvak S. & Tarrago-Litvak L. (1984) Dev. Biol. 1~3, 409-413 7 Grummt F., Waltl G., Jantzen H.M., I-lamprecht K., Huebscher U. & Kuenzle C.C. (1979) Proc. Natl. Acad. Sci. USA 76, 6081-6085

Dinucleoside tetraphosphates and the cell cycle

8 Rapaport E., Zamecnik P. & Baril E.F. (1981) J. Biol. Chem. 256, 12148-12151 9 Zamecnik P., Rapaport E. & Baril E.F. (1982) Proc. Natl. Acad. Sci. USA 79, 1791-1794 10 Rapaport E., Zamecnik P. & Baril E.F. (1981) Proc. Natl. Acad. Sci. USA 78, 838-842 11 Weinmann-Dorsch C., Hedl A., Grummt I., Albert W., Ferdinand J., Friis R., Pierron G., Moll W. & Grummt F. (1984) Eur. J. Biochem. 138, 179-185 12 Weinmann-Dorsch C., Pierron G., Wick R., Sauer H. & Grummt F. (1984)Exp. CellRes. 155, 171-177 13 Garrison P.N., Mathis S.A. & Barnes L.D. (1986) MoL Cell. BioL 6, 1179-1186 14 Orfanoudakis G., Baltzinger M., Meyer D., Befort N., Ebel J.-P., Befort J.-J. & Remy P. (1987) MoL Cell. BioL 7, 2444-2450 15 Segal E. & LePecq J.-B. (1986) Exp. CelIRes. 167, 119-126 16 Baker J.C. & Jacobson M.K. (1984) Anal Biochem. 141,451-460 17 Thompson E.B., Tomkins M. & Curran J.F. (1966) Biochemistry 56, 296-303 18 Giard D.S., Aaronson S.A., Todaro G.J., Arnstein P., Kersey J.H., Dosik H. & Parks W.P. (1973) J. NatL Cancer. Inst. 51, 1417-1423

1225

19 Brabander M.J., Van de Veire R.M.L., Aerts F.E.M., Borgers M. & Janssen P.A.J. (1976) Cancer Res. 36, 905-916 20 Firket H. & Mahieu P. (1966) Exp. Cell Res. 45, 11-21

21 Ogilvie A. (1981) Anal. Biochem. 115, 302-307 22 Baltzinger M., Ebel J.-P. & Remy P. (1986) Biochimie 68, 1231-1236 23 Lowry O.H., Rosebrough N.J., Farr A.L. & Randall R.J. (1951) J. BioL Chem. 193, 265-275 24 Bradford M.M. (1976) Anal. Biochem. 72, 248-254 25 Rylatt D.B. & Parish C.R. (1982) Anal. Biochem. 121,213-214 26 Okada S. (1967) J. Cell BioL 34, 915-919 27 Brevet A., Plateau P., Best-Belpomme M. & Blanquet S. (1985) J. BioL Chem. 260, 15566-15570 28 Baker J. & Jacobson M. (1986) Proc. NatL Acad. Sci. USA 83, 2350-2352 29 Gu6don G.F., Gilson G.J.-P., Ebel J.-P., Befort N. & Remy P.M. (1986) J. Biol. Chem. 261, 16459-16465 30 Plateau P., Fromant M., Kepes F. & Blanquet S. (1987) J. BacterioL 169, 419-422 31 Adams R.L.P. (1980) in: Cell Culturefor Biochemists Elsevier Biochemical Press, Amsterdam, pp. 136-161