Cell Cycle Analysis and Synchronization of the Xenopus Cell Line XL2Rustem Uzbekov,*,† Isabelle Chartrain,* Michel Philippe,* and Yannick Arlot-Bonnemains*

*UPR 41 CNRS Universite de Rennes 1, Campus de Beaulieu, Avenue du General Leclerc, 35042 Rennes, France; and †Laboratory CellMotility, A. N. Belozersky Institute, Moscow State University, Vorobjevy gori, 119899 Moscow, Russia

We have determined the length of the cell cycle andits different phases in a permanent Xenopus tadpolecell line, XL2. Following BrdU labeling, the totallength of the cell cycle was estimated as 28 h. Thedifferent phases of the cell cycle, G1, S, G2, and M were,respectively, 14 h, 10 h 45 min, 2 h 30 min, and 54 min.Knowing these parameters, we were able to developmethods that selectively enrich cells in differentphases of the cycle. Treatment with aphidicolin re-sulted in a S phase block in which more than 85% ofthe cells showed S phase chromosomes. Almost 60% ofthe cells were arrested in mitosis after a double blockwith aphidicolin/nocodazole or aphidicolin/ALLN(acetyl-leucyl-leucyl-norleucinal) treatment. This syn-chronization protocol will greatly facilitate studies ofbiochemical events associated with specific gene reg-ulation through the cell cycle. Our synchronizationprotocol does not disturb cell metabolism as the ex-pression of cyclin B2 during the cell cycle is in agree-ment with the results obtained with mammalian cells.© 1998 Academic Press

INTRODUCTION

The amphibian Xenopus laevis embryo is a modelwidely used to study the molecules and the mecha-nisms which control early development. Fertilizationinduces a particular proliferative stage. The first cellcycle takes place in 90 min and is followed by subse-quent cell divisions every 30 min. These cleavages arerapid and synchronous, oscillating between DNA syn-thesis (S phase) and mitosis (M phase) until cycle 13[1]. G1 and G2 phases are absent or very short. Inaddition, these cycles occur in the absence of transcrip-tion. After the 12th cell cycle, zygotic transcription isinitiated, cleavages become asynchronous, and the cellcycle is extended and G1 and G2 become distinguish-able. These changes including cell motility have beentermed MBT (Mid-Blastula Transition). Thus, cell cy-cle regulation in early embryos before the MBT couldnot be the same as in the somatic cells where transcrip-tion is active.

A large number of key molecules regulating the cellcycle have been isolated in yeast [2], Drosophila [3],

Xenopus [4], and marine invertebrates [5]. Xenopus eggextracts present one of the most powerful in vitro sys-tems to study the regulation of the cell cycle at themolecular level in the absence of transcription. Manytools which have been characterized in Xenopus eggextracts are now available and can potentially be stud-ied in a Xenopus cell line. For example, in the earlyembryonic cell cycle, cdc2 kinase activity oscillates ineach cell cycle as do the levels of mitotic cyclins [6].Cdk2 activity also oscillates in cycling extracts in con-trast to cells in culture, the level of cyclin E does notchange [7]. Thus, it is of great interest to compare cellcycle control in early embryos or egg extracts with thatin the cell.

The XL2 cell line first described by Anizet, was ini-tiated from stage 35 tadpoles of Xenopus laevis andused for synchronization [8]. The length of differentphases of the cell cycle has not been studied in thesecell lines. We have now characterized the differentparameters of the cell cycle and secondly establishedreproducible methods for synchronizing these cells.This analysis now makes it possible to investigate bio-chemical or biological regulation in a permanent em-bryonic cell line.

Highly synchronized cell populations greatly facili-tate cell cycle analysis and many procedures now existto synchronize mammalian cells [9]. For example, se-rum starvation or contact inhibition was often used toarrest the growth of fibroblasts in Go or quiescence.Drugs that inhibit DNA replication, such as thymidine,hydroxyurea, mimosin, or aphidicolin, are often used tosynchronize mammalian cells in S phase [10–14].Among the drugs described, aphidicolin is the leastcytotoxic and produces the best synchrony for S phase[13]. To obtain mammalian cells in M phase, the cellsare first blocked with microtubule depolymerizingdrugs and then collected by ‘‘shake off ’’ [13, 15, 16].Colchicine, colcemid, or nocodazole depolymerize mi-crotubules and may be used to synchronize cells in Mphase. Other authors have reported that the neutralcystein protease inhibitor N-acetyl-leucyl-leucyl-nor-leucinal (ALLN) also arrests cells in mitosis. ALLNinhibits cyclin B degradation and therefore arrests thechromosomes in M phase without spindle damage.

600014-4827/98 $25.00Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

EXPERIMENTAL CELL RESEARCH 242, 60–68 (1998)ARTICLE NO. EX984097

ALLN inhibits the proteasome, calpains, and cathepsinB. It is presumably through its action on the protea-some that ALLN blocks the exit from M phase [16, 17].Mitotic ‘‘shake off ’’ is generally a good technique forcollecting fibroblasts in M phase because cell contactwith the substrate is reduced during mitosis. As epi-thelial cells interact more strongly with the substrateanother technique is required to obtain XL2 cells in Mphase.

In eukaryotic cells, DNA replication and mitosis aretriggered by the activation and inactivation of cdk/cyclin complexes consisting of a cyclin and a cyclin-dependent kinase. Activation of a cdk is dependent onthe association with a cyclin. Cyclin destruction resultsin the inactivation of the kinase in human cell culture.Synthesis and degradation of the cyclins follow an or-dered sequence. Protein level of cyclin B2 is low in G1phase, increases in S and G2, and peaks at mitosis [18].Cyclin B2 associates with cdc2 in late S phase and isactive in M phase [19]. During G2 phase, cdc2 is main-tained inactive by tyrosine phosphorylation. It getsactivated by cdc25 at the onset of M phase. The level ofthis protein was analyzed in XL2-synchronized cells toconfirm the efficiency of our synchronization method-ology.

The XL2 line was chosen to study its cell cycle amongthe different existing Xenopus cell lines (XL 177, XTC,and KR) for the expression of different mRNAs (Eg1,Eg5, EF1a, ODC) identified in Xenopus embryos (un-published data). Using cell size as a marker of cyto-plasmic progression and BrdU labeling for identifica-tion of cells in S phase, we have determined cell cycleposition [20]. Coordination of cytoplasmic and nuclearevents during induction synchrony was also studied.To achieve high synchronization of XL2 cells, we testeddifferent chemical blockers like aphidicolin, nocoda-zole, or ALLN. Using cyclin B2 protein level as a controlfor cell cycle, our results demonstrate that XL2 cellsare an excellent experimental system for cell cycle syn-chronization and as such, will be useful for investigat-ing molecular mechanisms involved in somatic cycleprogression events.

MATERIALS AND METHODS

Chemicals and Drugs

Bromodeoxyuridine (BrdU), primary monoclonal anti-BrdU anti-body, anti-b tubulin, aphidicolin, nocodazole, trypsin-EDTA solu-tion were obtained from Sigma. ALLN (N-acetyl-leucyl-leucyl-norleucinal) was bought from Calbiochem. A Secondary Texas-red-conjugated antibody was obtained from Interchim. Leibovitz15 cell culture medium (L-15), antibiotic-antimycotic solution(penicillin-streptomycin-amphotericin) was supplied by GIBCOBRL laboratories. Fetal calf serum was obtained from Biotimes.Antibody to cyclin B2 was a gift from Tim Hunt’s laboratory(ICRF-Clare Hall, London).

Cell Culture

The XL2 cell line [8] was a gift from Professor J. Tata (MillHill-NIMR Laboratory, London). Cells were cultured in L-15 mediumsupplemented with 10% fetal calf serum (FCS), penicillin (100 Units/ml), streptomycin (100 mg/ml), and amphotericin (25 mg/ml) (fullmedium). Cells were incubated at 25°C and maintained in exponen-tial growth as described by Smith and Tata [21].

Immunofluorescence

For immunofluorescence experiments, cells were cultured on glasscoverslips in L-15 with 10% fetal calf serum and 40 mM BrdU(Sigma). We analyzed cells constantly incubated with BrdU and thecells incubated for 30 min before fixation with the same concentra-tion of BrdU. At the end of incubation, cells were briefly washed withPBS and fixed with 70% ethanol for 30 min. The coverslips were thenrinsed in PBS, immersed in 4 M HCl for 20 min, washed in PBS, andincubated for 60 min at room temperature with mouse anti-BrdU.This incubation was followed by addition of Texas-red-conjugatedimmunoglobulin antimouse for 60 min at room temperature. Thecoverslips were mounted in Mowiol (Calbiochem) and examined un-der a Zeiss Axiolab microscope equipped with phase-contrast andepifluorescence using 403/0.65 and 100/1.25 achroplan objectives.Photomicrographs were taken with an Olympus 0M-2 camera onIlford HP5 film (400 ASA).

Synchronization of Cells

G1 phase cells. Xenopus cells were cultured at a density of 105

cells in 75-cm2 flasks in L-15 medium with 10% FCS until cellattachment. The cells were then washed with PBS and cultured inserum-free medium for 24 h. Coverslips with XL2 cells were culti-vated in the same conditions and then incubated in medium contain-ing 10% FCS and 40 mM BrdU for 30 min.

S phase cells. The cells were cultured at a density of 105 cells perflask in medium containing 10% FCS until attachment and thenincubated for 24 h in the medium without serum. At the end of thisincubation, the cells were mostly in G1 phase. The cells were thenincubated in the presence of L-15 containing 10% FCS and 2 mg/mlaphidicolin for 30 h. The cells were washed with PBS after thistreatment and then cultivated for a period of 2 h in medium with 10%FCS.

G2 phase cells. After a preculture of 24 h without serum, cellswere incubated for 30 h in medium with 10% FCS and aphidicolin (2mg/ml). The cells were washed with PBS and cultured in mediumwith 10% FCS for 11 h. After a recovery time of 11 h in fresh fullmedium, cells on coverslips were incubated in medium with 10% FCSand 40 mM BrdU.

M phase cells. Cells in M phase were synchronized using a doubleblock, first by incubating the cells in the absence of serum and thenwith aphidicolin (2 mg/ml) for 30 h. The cells were washed threetimes with PBS and then incubated for 8 h in medium with 10% FCS.Finally, the cells were cultured in full medium in the presence of 0.5mg/ml nocodazole for 7 h, washed with PBS, and then incubated infresh full medium with 10% FCS for 1 h. M-phase cells were alsosynchronized using ALLN. Cells released from aphidicolin and incu-bated in medium with 10% FCS were then treated for 6 h in mediumwith 10% FCS and 40 mg/ml of ALLN. The cells were then washedwith PBS and cultured for 1 h in full medium before analysis.

Determination of the percentage of cells in each phase. Cells cul-tivated on coverslips were incubated in the same conditions and thentreated for 30 min in full medium in the presence of 40 mm of BrdU.The cells were then washed with PBS, fixed in 70% ethanol, andtreated with the anti-BrdU antibody as described above.

The percentage of cells that are in Go was calculated by firstincubating the cells for 30 h in medium with BrdU. All cells in thecell cycle after this procedure must be BrdU-positive when treated

61CELL CYCLE OF Xenopus CELL LINE XL2

with anti-BrdU antibodies. So the unstained cells were considered tobe in Go. The duration of S and M phase were calculated directly bymicroscope observation of samples which had been fixed every hourand stained with anti-BrdU antibodies. Mitotic cell numbers werecalculated from observations of cells culture under phase contrast.The percentage of G2 cells was calculated as %S0 (S max, 2 h afteraphidicolin removal) minus %Sn (time of calculation) when no Mcells were present in the preparation. When mitotic cells werepresent during the last hour (from 8 h after aphidicolin release), thepercentage of G2 cells was calculated as follows: %G2 5 %S0 2 %Sn 2%M. For cells in the cell cycle, calculations are: 100% 5 %G1 1 %S 1%G2 1 %M and so, %G1 1 %G2 5 100% 2 %S 2 %M.

FACS Analysis

Flow cytometry was used to determine the degree of cell synchro-nization. Cells were synchronized for each phase as described above.For each step of synchronization, the cells were incubated in 40 mMBrdU, trypsinized, and cell suspensions were centrifuged at 3000gfor 5 min, followed by fixation in ethanol at 220°C for 1 h. Fixed cellswere treated with antibody anti BrdU for 1 h (1/200) at room tem-perature and then with Texas-red-conjugated immunoglobulin anti-mouse (1/50) for 1 h. Samples were stored at 220°C until analysis.Cells were stained with 10 mg/ml propidium iodide (PI) and DNAcontent was analyzed on a FACS Becton–Dickinson with two fluo-rescence photomultipliers equipped with an argon laser operating at15 mW, generating a light of 488 nm wavelength. PI emission wasmeasured at wavelengths between 620 and 655 nm. Cell cycle anal-ysis was performed with multicycle program.

Western Blot Analysis

The synchronized cells were washed with PBS and lysed in lysisbuffer. Cell lysates were boiled for 10 min at 100°C and subjected to17% acrylamide bisacrylamide gel electrophoresis. Equal amounts ofprotein were electroblotted on nitrocellulose membranes (Amer-sham) by standard methods. Membranes were blocked overnight in5% dry milk in TBST (20 mM Tris, pH 7.5, 500 mM NaCl, 0.05%Tween 20, and 0.02% Sodium azide). Probing with the differentantibodies (Cyclin B2 1:1000; b-tubulin 1:15,000) was carried out asabove for 2 h at room temperature in the blocking buffer. The blotswere washed in 2.5% dry milk TBST, 1 h at room temperature, andthen incubated for 2 h with conjugate 1:30,000 in 2.5% dry milk inTBST. After extensive washing in TBST, the membranes were pro-cessed for enhanced chemoluminescence using ECL reagents accord-ing to the manufacturer’s instruction (NEN). ECL exposures werecarried out using Kodak XAR film.

RESULTS AND DISCUSSION

In this paper, we characterized the length and thephases of the XL2 cell cycle. Cells were constantlyincubated in L-15 containing 10% FCS, antibiotics, and40 mM of BrdU. Cells were fixed every hour for analysisafter labeling with anti BrdU antibody. DNA is syn-thesized during S phase so all cells in S phase willincorporate BrdU. After 33 h incubation with BrdUmore than 80% of nuclei from cells which were in thecell cycle were labeled (Fig. 1).

The percentage of marked cells increased from30.0% at the beginning of the experiment to 82.5%after 33 h of constant incubation with BrdU (Fig.2A). Crossing of the graphic with ordinate axisshowed a value of 30% labeled cells. This value cor-

responded to the percentage of cells which were in Sphase at the beginning of the experiment. At about17.5 h the linearity breaks down. This specific pointrepresents the percentage of cells which have incor-porated BrdU, the cells which were in S phase(78.5%). This value corresponds to the difference be-tween the total length of the cell cycle (T) minus theduration of S phase (T 2 S 5 17.5). Not all cellsincorporated BrdU because after 33 h of incubationin the presence of BrdU, only 82.5% of the cells werelabeled. The percentage of unmarked cells observedafter 33 h incubation in BrdU (17.5%) was probablydue to cells which were not cycling (Go).

Consequently, the length of the XL2 cell cycle takinginto account this value can be expressed as: -totallength of the cell cycle (T) minus S 5 17.5 and S/T 5percentage of cells in S phase (30%)/percentage of cellsin the cell cycle as indicated on the graph (78.5%) (Fig.2A). This gave T 2 S 5 17.5; S/T 5 0.30/0.785 5 0.382;and we calculated T 5 17.5/1 2 0.382 5 28.31 (28 h 15min) and S 5 0.382 3 28.31 5 10.80 (10 h 45 min).Duration of the cell cycle and the length of S phase

FIG. 1. XL2 cells were cultivated for 30 h in full medium sup-plemented with 40 mM BrdU. (A) Immunofluorescence staining ofthe cells with anti-BrdU antibody showing more than 80% of labelednuclei. (B) Same cells in phase contrast. Bar, 40 mm.

62 UZBEKOV ET AL.

were calculated by other methods which gave similarvalues [22].

In order to determine the length of the G2 phase, thecells were incubated in full medium with 40 mM BrdU

for 30 min and then subsequently fixed every half hour.For each time point, 200 mitoses were observed todetermine the percentage of cells labeled with theBrdU antibody (Fig. 2B). The first labeled cells were

FIG. 2. (A) Time course of labeling of the XL2 cells in the presence of BrdU. Cells were constantly incubated in full medium supplementedwith BrdU (40 mM). Cells were fixed every 3 h and treated with the anti BrdU antibody. After 33 h, 82.5% of cells were labeled and theunmarked cells (17.5%) were in Go phase. Each point is the mean of three determinations. (B) Percentage of marked mitoses as a functionof time. Fifty to sixty percent confluent cells were cultivated for 30 min in full medium supplemented with 40 mM BrdU. Cells were washedand further cultivated in full medium. Cells were fixed every 30 min and treated with anti-BrdU antibody. The percentage of marked mitoseswas determined under the microscope. (C) Density of the XL2 cells as a function of time obtained when cells were cultured in a mediumsupplemented with 10% fetal calf serum (1 FCS) or in a medium without serum (2 FCS). Cells were cultivated in both cases at an initialdensity of 540 cells/mm2. Cell density was then measured every 3 h.

63CELL CYCLE OF Xenopus CELL LINE XL2

detected 90 min after addition of BrdU at the begin-ning of the experiment. The maximum BrdU labelingwas obtained 3 h after the appearance of the firstmarked mitosis. Consequently, the duration of the G2phase was about 2 h 30 min (Fig. 2B).

The length of the M phase was determined by ana-lyzing the unlabeled cells. Cells were cultivated in fullmedium and observed with an inverted light micro-scope. One cell in G2 phase was selected and observeduntil division was complete. This experiment was re-peated with 23 cells in the same preparation and theduration for each step of mitosis was determined. Thetime for the different steps was: 5.7 6 1.4 min forprophase, 6.7 6 1.6 min for prometaphase, 19.4 6 5.3min for metaphase, 5.7 6 1.2 min for anaphase, and16.6 6 3.2 min telophase and cytokinesis. The averagetime for mitosis was 54.1 6 6.8 min.

Thus, the parameters of the XL2 cell cycle can now besummarized. The total length of the cell cycle was about28 h 15 min with the S phase was 10 h 45 min, G2 phase2 h 30 min, M phase 54 min, and G1 phase 14 h.

The length of the XL2 cell cycle is quite long com-pared to the cell cycle of other cells. The total length ofthe Hela cell cycle is 19 h 30 min and G1, S, G2, and Mare, respectively, 8 h 15 min, 6 h 15 min, 4 h 30 min,and 30 min. Another example is shown by mesodermaland ectodermal cells of the rat embryo which have beendescribed as the most rapidly dividing vertebratescells, with a cell cycle time of 7 h to 7 h 30 min [23]. Thegreat difference observed in the length of the cell cyclebetween the above described cells and XL2 cells couldbe explained by the late embryonic origin of the XL2cells. Generally, during the embryonic cell cycle, totallength of the cycle is reduced by shortening S and G2phases as well as the G1 phase in contrast to the cellcycle of cells from later stages of development for whichduration is largely modulated by variations in thelength of G1.

G1 and S phases of the XL2 cell cycle were quite long,which makes it difficult to obtain a large quantity ofcells in a precise phase without synchronizing cells.Thus, it was highly desirable to find techniques tosynchronize cells for biochemical studies. A classic wayfor synchronizing cells is to deprive them of growthfactors, usually by exposing them to a low concentra-tion of serum. Other techniques involve treatment withcompounds which block cells in a specific phase of thecell cycle. For example, inhibitors of DNA synthesissuch as hydroxyurea, and methotrexate, do not directlyinhibit DNA polymerization but rather block the pre-cursor pathway. These compounds synchronize cells atthe G1–S boundary by inhibiting DNA synthesis,whereas drugs such as colcemid, nocodazole, taxol, orALLN block mitosis. Some of these drugs are suffi-ciently nontoxic to be of interest for cancer therapy aswell as useful for synchronizing cells in culture. If

inhibition is specific for one stage of the cell cycle, cellsin other phases move forward until they became ar-rested at the blocked point.

In order to develop methods for synchronizing XL2cells, we first tried to obtain cells in G1 phase. XL2 cellswere cultivated in two different flasks at an initialdensity of 535 cells/mm2 and the density of the cellswas observed every 24 h for 120 h. Figure 2C shows thenumber of XL2 cells as a function of time. The densityof the cells cultivated in the medium without serumvaried little from 535 to 740 cells/mm2 after 120 h. Incontrast, the quantity of cells in the serum-supple-mented medium increased from 535 to 7160 cells/mm2.The number of cells did not increase when they werecultured in serum-free medium during 120 h.

Cells cultivated in medium containing 40 mM BrdUwere treated with the antibody anti BrdU and ana-lyzed by microscopy in order to calculate the percent-age of S phase (Fig. 3). The percentage of cells in Sphase determined as a function of time decreases untilall cycling cells reach the G1 phase (data not shown).

FIG. 3. Labeling of the XL2 cells incubated in serum-free me-dium for 24 h. (A) Cells were incubated in serum-free medium for24 h. Thirty minutes before the end of the incubation the cells werecultivated in serum-free medium supplemented with 40 mM BrdU.Cells were fixed and treated with the anti BrdU antibody and ana-lyzed by microscopy. (B) The same cells observed in phase contrast.Bar, 40 mm.

64 UZBEKOV ET AL.

The first lowest number of cells in S phase (4.3%) wasobtained 24 h after cultivating the cells without serum.The second lowest point (9.4%) was obtained 120 hlater. The mitotic index decreased from 4 to 0.8%, 24 hafter the beginning of the incubation and declined to apercentage of 0.35% in the next 24 h. This value did notchange during 120 h. In contrast, the mitotic index washigher (2.8%) for cells cultivated with serum. Thus,incubating cells in medium without serum allowed usto obtain cells which were mostly in G1 phase. Addingback serum to the cells released the cell cycle block.The resulting proliferating cell population lost syn-chrony while traversing G1 phase; thus, it was notpossible to collect cells in S phase. This led to difficul-ties in obtaining highly synchronized cells in S, G2, orM phase making it necessary to use drugs in order toobtain synchronized cells.

In order to synchronize the XL2 cells in S phase, wetested the effect of aphidicolin. Aphidicolin preventsDNA chain elongation by inhibiting DNA polymerase aand so cells which are incubated with aphidicolin re-main at the G1/S border. After a preculture for 24 hwithout serum, cells were incubated in full mediumwith different concentrations of aphidicolin. DNA rep-lication was analyzed by BrdU incorporation (Table 1).A minimal concentration of 2 mg/ml of aphidicolin for30 h was necessary to block the cells at the G1/Sboundary (Table 1; treatment A). After removal of thedrug and culturing the cells in fresh full medium for1 h (Table 1; treatment B), we observed an incorpora-tion of BrdU indicating the progression of the cells to Sphase (73.1% of BrdU-positive cells). Higher concentra-tions of aphidicolin (3 and 5 mg/ml) reduced this per-centage to 63.4 and 58.7%, respectively. The concen-tration of 2 mg/ml of aphidicolin appeared to be theoptimum for an efficient block of the cells and goodrecovery after removal of the drug. When cells werecultured 2 h in full medium after removal of aphidico-lin, 87.7% of the cells were in S phase (Fig. 4). Higher

concentrations of aphidicolin (3 and 5 mg/ml) stoppedthe incorporation of BrdU resulting in few S phasecells. This result may indicate a toxic action of aphidi-

FIG. 4. (A) Immunofluorescence staining of XL2 cells with anti-BrdU antibody after 30 h of incubation with aphidicolin (2 mg/ml)and 2 h in full medium without the drug. Thirty minutes beforefixation, cells were incubated in the presence of BrdU (40 mM). (B)The same cells observed in phase contrast. Bar, 40 mm.

TABLE 1

Effect of Different Concentration of Aphidicolin on DNA Replication

Aphidicolin concentration mg/ml 0.1 0.5 1 2 3 5

Treatment APercentage of BrdU-positive cellsafter 30 h of aphidicolin treatment 77.7 60.7 3.3 0 0 0

Treatment BPercentage of BrdU-positive cells1 h after washing aphidicolin 72.3 73.2 72.6 73.1 63.4 58.7

Note. Cells were cultured in a serum-free medium for 24 h and then in full medium complemented with different concentrations ofaphidicolin (0.1; 0.5; 1; 2; 3; 5 mg/ml). DNA replication was analyzed by adding BrdU to the medium 30 min before the end of incubation. Cellswere fixed and treated with the anti-BrdU antibody. Treatment A: The percentage of BrdU-positive cells was calculated after 30 h incubationwith aphidicolin. Treatment B: The percentage of BrdU-positive cells was calculated after 30 h incubation with aphidicolin followed by 1 hincubation in full medium without aphidicolin.

65CELL CYCLE OF Xenopus CELL LINE XL2

colin for the cells. In conclusion, a double treatmentincluding serum deprivation and aphidicolin preventscells from entering in S phase.

Synchronization of XL2 cells in G2 phase was similarto the synchronization of cells in S phase except thatthe time for recovery was prolonged. To enrich cells inG2 phase, a combination of serum deprivation andaphidicolin block was followed by incubation in fullmedium for 11 h. Upon readdition of medium to thecells, growth was initiated in a semisynchronous fash-ion, reaching G2 phase in about 9 h. The percentage ofcells in S phase decreased 8 h after removing the drugwhile the percentage of cells in G2 phase increased.Taking sequential samples after release from theaphidicolin block showed that the highest percentageof G2 phase was obtained after 11 h. Although theenrichment of G2 was up to 49.8%, the final accumula-tion was not as high as for S phase. This result could beexplained by the prolonged recovery phase, allowingsome cells in G2 phase to transit from G2 to M, or thepresence of cells with a longer S phase and thus not yetin G2. Such results have already been described forsynchronized mammalian cells [24].

Synchronization of cells in M was achieved with adouble block with aphidicolin and nocodazole. Cells cul-tured in serum-free medium were treated with nocoda-zole and subsequently when released from this drug theywere cultivated for 8 h in serum-free medium in order toget the highest percentage of cells in G2. Sixty percent ofall cells were blocked in M phase by adding 0.5 mg/ml ofnocodazole to the complete medium.

Table 2 summarizes the percentages of the differentcell cycle phases obtained after synchronization. No-codazole was removed from the medium and the cellswere cultivated with complete medium for 6 h. DNAcontent in nocodazole-blocked cells was increased. Thehighest percentage of cell population in G1 (85%) wasobtained 6 h after release from nocodazole, but part of

the cell population started another cell cycle withoutundergoing cytokinesis. Polyploidization observed inXL2 cells has already been described for mammaliancells [24]. In order to prevent this phenomenon, wetried other drugs which do not effect microtubule po-lymerization.

Combining serum deprivation, aphidicolin treat-ment, and release for 8 h, we incubated the cells withALLN. Almost 57% of the cells were in M phase. Uponremoving the ALLN from the culture medium, cellswere cultivated in complete medium and polyploidywere not observed among G1 cells. These results wereconsistent with those obtained with CHO cells synchro-nized with ALLN [13]. The cells synchronized in Mwere able to undergo another cell cycle. The final en-richment of cells in M phase (57%) may be due to somecells in G2 phase having already passed the transitionpoint from G2 to M and G1 [13, 24]. We have success-fully synchronized XL2 cells in G1, S, G2, and Mphases. The variation of the percentages observed dur-ing the synchronization could be due to differentialsensitivity of the cells to the different drugs.

Flow cytometry DNA histograms of BrdU-stainednuclei of the different synchronized cells are shown inFig. 5. Analyzing the DNA distribution of the differentfractions shows that the fraction B (24 h serum depri-vation) is homogenous in G1. Two hours after releasefrom the aphidicolin block (C), an increasing propor-tion of cells in S phase is detectable. After aphidicolin/ALLN block (D), we observed that the proportion ofcells in G2/M phase increase. The percentage of cells ineach phase obtain by FACS analysis are 79% for G1,86% for S, and 59% for G2/M. The results obtained byFACS analysis showed values that confirmed our re-sults obtained by microscopic analysis. Even evalua-tion of the percentage of synchronized cells revealssome differences between flow cytometry and micro-scopic analysis, the two methods of analysis confirm

TABLE 2

Percentage of Cells Enriched in Specific Cell Cycle Phases in Response to the Specified Treatments

TreatmentSynchronization

for phase G1 S G2 M

No treatment Unsynchronized cells 50.8 37.3 8.8 3.1Medium without serum G1 90 7.7 1.7 0.6Aphidicolin treatment 30 h S 12.3 87.7 0 0Aphidicolin 30 h 111 h release G2 9.9 38.2 49.8 2.1Aphidicolin 30 h 18 h release

1Nocodazole 7 h M 27.8 9.7 2.2 60.3Aphidicolin 30 h 18 h release

1Nocodazole 16 h 15 h release G1 85.5 10.7 2.5 1.3Aphidicolin 30 h 18 h release

1ALLN 6 h M 36.6 4.9 1.2 57.3

Note. XL2 cells were treated with the indicated drugs under the conditions mentioned under Materials and Methods. Cells were allowedto recover in full medium.

66 UZBEKOV ET AL.

that our synchronization procedure is effective for ob-taining enriched cells in different phases.

In our experiments several concentrations of aphidico-lin have been tested. A concentration less than 2 mg/mlresulted in an incomplete suppression of S phase progres-sion, as assessed by BrdU incorporation. Incubation in 1mg/ml showed that only 3.3% of the cells were in S phase.A concentration of 0.1 mg/ml of aphidicolin lengthenedthe S phase. Incubation of cells in 2 mg/ml of aphidicolincompletely blocked DNA synthesis in XL2 cells. Our re-sults are consistent with those of Materly and coworkers,who described a S phase block with a concentration of 1.5mg/ml for mouse L1210 leukemia cells [14]. They ob-tained less than 20% inhibition of DNA synthesis in hu-man melanoma cells with 10 mg/ml of aphidicolin [25].Generally, for the majority of cell lines, 1–2 mg/ml ofaphidicolin is sufficient for full inhibition of nuclear DNAsynthesis [26–28, 14]. We incubated cells in aphidicolinfor 30 h, this is almost 1.1-fold the length of a mean cellcycle, in agreement with protocols described for mamma-lian cells [9, 14].

Biochemical cell analysis depends on obtaining syn-chronized cell populations and synchronization by se-

rum starvation and drug treatment could have toxiceffects or result in metabolic imbalance. In order tocontrol the viability of our cells in synchronizationexperiment, we determined the presence of cyclin B2 ateach step of the cell cycle. Western blot analysisshowed that the protein cyclin B2 was present at a verylow level in G1 phase and was maximum during the G2and M phases (Fig. 6). A control immunoblot with ananti-b tubulin antibody showed no distinct variation ofb tubulin during progression through the cell cycle.

The synchronization methods described in this re-port showed that drugs such as aphidicolin and nocoda-zole effectively block the progression of XL2 cells in thecycle. The cell cycle position is typically defined on thebasis of DNA content observed by BrdU incorporation.This technique is very useful for the determination ofG2 and M phases which is not really possible with fluxcytometry analysis. Our methods of synchronizationwere similar to those used for HeLa cells but the timeof recovery must be prolonged for Xenopus cells [24].Aphidicolin blocked more than 85% of the cells in Sphase and the combination of aphidicolin and nocoda-zole allowed a specific enrichment of around 60% of the

FIG. 5. Flow cytometry analysis of XL2 unsynchronized cells (A) and cells enriched in G1 (B), S (C), and G2/M (D) phase. (B) Cells serumdeprivated for 24 h; (C) cells serum deprivated (24 h), followed by aphidicolin treatment (2 mg/ml) for 30 h and by a phase recovery of 2 h;(D) cells serum deprivated, followed by aphidicolin treatment and 8 h recovery with a block of ALLN (6 h).

67CELL CYCLE OF Xenopus CELL LINE XL2

cells in M phase. ALLN is a potent inhibitor of XL2 cellcycle progression and the mitotic arrest correlated withan inhibition of cyclin B2 degradation.

We thank Louis Communier for photographic work and Dr. RebeccaHartley and Professor Dennis Webb for the critical reading of themanuscript. We are grateful to Professor Genetet’s Laboratory of im-munology, CHRU Pontchaillou Rennes, and her authorization to workon the flux cytometry analyzer and Mrs. Dejours for her precious tech-nical assistance for FACS analysis. This work was supported by theEuropean Economic Community (E. E. C. Grant CHRX-CT94-0568),the French government Ministere de la Recherche (ACC-SV4), theAssociation pour la Recherche contre le Cancer (ARC Grand-Ouest),and la Ligue Nationale contre le cancer (Comite d’Ille et Vilaine).

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Received September 5, 1997Revised version received March 6, 1998

FIG. 6. Western blot analysis of cyclin B2 and b-tubulin in thedifferent phases of the XL2 cell cycle. Cells were synchronized ineach phase (G1, serum deprivation; S, serum deprivation and aphidi-colin block; G2, serum deprivation/aphidicolin block and release fromthe block and M phase, serum deprivatio/aphidicolin block/releaseand block of ALLN) and proteins were extracted with 200 ml of lysisbuffer. Equivalent amounts of total protein were loaded on a 17%SDS–polyacrylamide gel. After transfer onto the nitrocellulose, themembrane was treated with the anti-cyclin B2 antibody (1/1000) andwith the anti-btubulin antibody (1/15,000). At the end of the incu-bation (2 h) the blots were incubated with a conjugate (1/30,000).Proteins were detected using an enhanced chemoluminescence sys-tem. Autoradiographies were performed using Kodak XAR film.

68 UZBEKOV ET AL.