Mutagenesis, Vol. 17, No. 5, 419-424,
September 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Reversible G1 arrest by dimethyl sulfoxide as a new method to synchronize Chinese hamster cells
Centre for Evolutionary Genetics, CNR, c/o Department of Genetics and Molecular Biology, `La Sapienza' University, Via degli Apuli 4, 00185 Rome, Italy
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Abstract |
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Dimethyl sulfoxide (DMSO), a well-known differentiation inducer in several myeloid cells, also induces a reversible G1 arrest in many cell lines. We recently showed that DMSO induces a G1 phase arrest in Chinese hamster ovary (CHO) cells, by restoring contact inhibition and preventing high density-dependent apoptosis. CHO cells are frequently used in cell biology and mutagenesis studies due to their good growth capacity and ease of manipulation but are very difficult to synchronize by serum starvation since they detach from monolayers when they reach confluence. In this study we investigated the possibility of using DMSO to reversibly synchronize CHO cells in the G1 phase of the cell cycle and analysed whether toxic effects follow the arrest using growth curve, sister chromatid exchange and micronuclei assays. We carried out a kinetic analysis of the arrest by DMSO and re-entry into the cell cycle after drug release by cytofluorimetric analysis of DNA content and bromodeoxyuridine incorporation. We show that CHO cells are efficiently and reversibly arrested in G1 by DMSO in concentrations ranging between 1 and 2%. In our experiments, >90% of cells grown for 96 h in presence of the drug were arrested in G1 and synchronously re-entered S phase sim;8–12 h after release. Furthermore, expression levels of p27 were down-regulated during G1 progression and cyclin D3 and E expression patterns were similar to those observed after serum starvation. No detectable cytotoxicity or genetic damage were induced in G1 released cells as revealed by the tests employed. Our results show that DMSO is a very powerful inducer of G1 synchronization in CHO cells without detectable cytotoxic or genetic effects in cell populations released from G1 arrest. DMSO synchronization represents a model system in which to analyse protein activities regulating G1 progression and investigate the response of G1 cells to mutagen treatments.
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Introduction |
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The polar chemical dimethyl sulfoxide (DMSO) is a widely used reagent in cell biology. It is employed as an anti-inflammatory and bacteriostatic agent and as a cryoprotectant in preserving cells in freeze–thaw processes (Yu and Quinn, 1994
). It is also the most frequently used solvent in cell biology, due to its efficiency as a carrier for a wide range of chemicals, its low toxicity and good miscibility with the test medium (Maron et al., 1981). DMSO is known to elicit multiple biological effects (Jacob and Herschler, 1986). It is a powerful scavenger of oxygen radicals (Simic, 1988; Sapora et al., 1991) and an inducer of cell differentiation in several systems, including primary hepatocytes and mouse erythroleukemia (Friend) and promyelocytic leukemia (HL60) cells (Watson et al., 1997). In all these cell types, growth inhibition and exit from the cell cycle accompany cell differentiation. In human lymphoid cell lines (Sawai et al., 1990; Teraoka et al. 1991, 1996) and mouse fibroblasts (Srinivas et al., 1991) a reversible G1 arrest was obtained by exposure to DMSO. We recently showed that DMSO also induces a G1 phase arrest in cells growing as monolayers, such as Chinese hamster ovary (CHO) cells, and that the DMSO-induced growth arrest in these cells is associated with restored contact inhibition of growth and prevention of density-dependent apoptosis (Fiore and Degrassi, 1999).
In the present study we investigated the use of DMSO as a synchronizing agent in CHO cells, since few methods are available to arrest the cell cycle efficiently and reversibly in this cell line, due to the poor growth inhibition exerted on these cells by confluence. We also characterized the cell cycle re-entry of G1 arrested cells upon release from the contact inhibition-proficient state produced by DMSO using cytofluorimetric analysis of DNA content and bromodeoxyuridine (BrdUrd) incorporation and protein analysis. Determination of the intracellular levels of proteins involved in regulating quiescence and G1 progression demonstrated that the pattern of G1 cyclin expression was similar to that observed after release of adherent cells from serum starvation. A prerequisite for an efficient synchronization method is the absence of toxic effects. Few data exist on the toxicity of DMSO, although it has been found to be mutagenic in some strains used in the Ames test, but only at very high concentrations (Hakura et al., 1993). No detectable cell toxicity was induced in cells released from the DMSO-induced arrest in our experiments, as revealed by measurement of growth capacity by cell counting. Induction of subtle genetic damage was also excluded by showing that neither sister chromatid exchanges (SCE) nor micronuclei (MN), which are sensitive indicators of DNA damage at the chromosomal level (Albertini et al., 2000), were induced in the DMSO-exposed cell population.
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Materials and methods |
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Cell cultures and chemicals
CHO-K1 cells were cultured in Ham's F10 medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine at 37°C in a 5% CO2 humidified atmosphere. Under these conditions, the average cell cycle lasts 12 h. DMSO, BrdUrd, cytochalasin B, propidium iodide and colchicine were purchased from Sigma. DMSO was directly administrated in the medium, while for the other chemicals stock solutions were prepared and kept frozen. In each experiment 1x106 CHO-K1 cells were seeded in 25 cm2 flasks and exposed to 1, 1.5 and 2% DMSO or kept in complete medium for 96 h.
Flow cytometric analysis
Cells cultured in the presence of DMSO were washed and released in fresh medium at low density for 8, 12, 16, 20 and 24 h. Before harvest, samples were pulsed with 45 µM BrdUrd for the last 20 min, then collected by trypsinization, washed in phosphate-buffered saline without Ca and Mg (PBS) and fixed in a 1:1 PBS:absolute methanol mixture. Immunostaining with BrdUrd monoclonal antibody (Dako) was performed as previously described (Fiore and Degrassi, 1999). Flow cytometric analysis was carried out using a FACStar Plus flow cytometer (Becton Dickinson) equipped with a 5 W Innova 90 Coherent laser with a 488 nm wavelength excitation light. Red fluorescence (DNA content) was detected with a 600 nm wavelength long pass filter and green fluorescence (BrdUrd content) was measured at 510 nm bandpass filter. Ten thousand events were collected for each sample and biparametric analysis of total DNA content and BrdUrd incorporation was performed using WinMDI 2.7 software.
Western blotting analysis
For each experimental point 5x106 cells were washed twice with cold PBS plus 1 mM phenylmethylsulfonyl fluoride (PMSF) and lysed for 45 min in RIPA buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% NP40, 1 mM EGTA, 1 mM EDTA, 0.25% sodium deoxycolate, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mM sodium orthovanadate and 1 mM sodium fluoride) on ice. Equal amounts of proteins extracted from DMSO-exposed and untreated cells were separated by SDS–PAGE and then electrotransferred onto nitrocellulose membranes in a Trans-Blot apparatus. Membranes were then incubated overnight in Tris-buffered saline containing 0.1% Tween 20 (TBT) and 5% low fat dry milk and then incubated with antibodies against cyclin A (C-19; Santa Cruz), cyclin D3 (14781A; Pharmigen), cyclin E (HE-111; Santa Cruz), p27 (F-8; Santa Cruz) or p21 (C-19; Santa Cruz) for 90 min. After multiple washes in TBT and incubation for 45 min with the appropriate horseradish peroxidase-linked secondary antibodies, immunocomplexes were revealed with a chemiluminescence kit (Amersham).
Growth curve
After 96 h growth in DMSO-containing medium, samples were collected by trypsinization and 2.5x105 cells from each sample were plated in duplicate in 25 cm2 flasks with fresh medium. Samples of 2.5x105 cells were also plated from exponentially growing cultures as control samples. After 24, 48 and 72 h cells were harvested and the cell number was counted using a Coulter ZM cell counter. All experiments were repeated three times.
Sister chromatid exchange assay
Cells grown with DMSO for 96 h were detached from the flasks and plated at low density. After 9 h, 5 µM BrdUrd was added and cells were harvested 24 h after BrdUrd addition. Proliferating cells grown for 24 h in the presence of BrdUrd served as controls. Colchicine (5x10-7 M) was added 3 h before fixation. Slides with sister chromatid differentiated metaphase cells were obtained by the air drying method and the fluorescence plus Giemsa technique, as previously described (De Salvia et al., 1988). From each experimental point, 50 second mitoses (M2) for SCE analysis, 1000 cells for mitotic index (MI) measurements and 100 metaphases for first (M1) and second (M2) mitosis determinations were scored from coded slides. All experiments were repeated three times.
Micronucleus assay
At the end of the DMSO treatment, cells were detached from flasks and seeded on 20x20 mm coverslips at low density. Cells were allowed to attach for 9 h and then were grown in the presence of 3 µg/ml cytochalasin B for 18 h. Finally, cells were fixed in situ by the gradual addition of methanol:acetic acid (3:1) and slides were air dried and stained in 5% Giemsa for 10 min (Antoccia et al., 1991). For each experimental point 2000 binucleated cells were scored for MN determination and 200 cells were analysed to count the number of nuclei per cell. Statistical analyses were carried out for three independent experiments.
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Results |
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G1 arrest and cell cycle re-entry after DMSO exposure
CHO cells grown for 96 h in complete medium with DMSO were detached and seeded in fresh medium at low density. Samples were taken from 0 to 24 h to monitor cell cycle progression. Flow cytometric analysis of DNA content and BrdUrd incorporation in cells exposed to DMSO for 96 h showed that >90% of cells did not incorporate BrdUrd and displayed a G1 DNA content for all the tested concentrations, (Figure 1
, 0 h), i.e. cells were efficiently synchronized in G1. In contrast, cells grown for 96 h in complete medium without DMSO displayed a less efficient synchronization in the G1 phase of the cell cycle, accompanied by the presence of an apoptotic cell fraction showing a sub-G1 DNA content (96 h medium panel in Figure 1), as already reported (Fiore and Degrassi, 1999). Under our experimental conditions, logarithmically growing CHO cells displayed sim;40% of cells actively synthesizing DNA (proliferating cells panel in Figure 1).
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After re-seeding DMSO-arrested cells in complete medium, these progressed synchronously through the cell cycle. Cells were still found in early S phase after 8 h release for 1 or 1.5% DMSO-exposed cells and by 12 h release after 2% DMSO (Figure 1
, 8 and 12 h). At 12 and 16 h release time, cells exposed to 1 or 1.5% DMSO synchronously arrived in late S phase, while 2% DMSO exposed cultures were found in middle S phase (Figure 1, 12 and 16 h). After 20 h recovery in fresh medium >60% of the cells exposed to 1% DMSO entered the second S phase and by 24 h >70% of the cells were in the second S phase after release (Figure 1, 20 and 24 h).
Cell cycle protein expression during G1 progression
To investigate the effects of DMSO on cell cycle progression we studied changes in expression of cyclins and cyclin-dependent kinase (CDK) inhibitors by western blot analysis (Figure 2). After 96 h growth in the presence of 1.5% DMSO cells showed a significant increase in the CDK inhibitor p27 compared with proliferating cells or cells grown for 96 h in DMSO-free medium. p27 levels slowly decreased from 2 until 8 h release in free medium, reaching protein levels lower than proliferating cells at the last time. p21 levels were higher in cells grown for 96 h in the presence of DMSO than 96 h in normal medium. The maximum amount of this protein was observed at 4–6 h from release and started to decrease at 8 h.
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Cyclin D3 levels were higher in cells grown for 96 h in DMSO compared with cells grown without the drug. Following release from the DMSO-induced arrest, cyclin D3 levels were markedly increased even at 2 h and remained high up to 6 h. At 8 h cyclin D3 content dropped to a level comparable with that observed in the cultures grown for 96 h in DMSO. Variations in cyclin E level were less significant. However, a clear induction of this protein was observed during late G1 phase, i.e. from 4 until 8 h after DMSO removal. Significant amounts of cyclin A were observed at the time of S phase entry, i.e. 8 h after release, whereas no cyclin A was detected during G1 progression. Cyclin A was also absent in cells incubated for 96 h in the medium with or without DMSO. Under both conditions >90% of cells were found in the G1 phase of the cell cycle (Fiore and Degrassi, 1999
).
Cell viability and cytogenetic damage after G1 arrest
To investigate possible harmful effects of growing cells in DMSO-containing medium we analysed the growth curves of DMSO-exposed cells. Cell number was counted at daily intervals after release from DMSO. The graph in Figure 3 shows the results obtained. At 24 h cell numbers in exposed cultures were between 60 and 40% of control cultures, depending on the DMSO concentration, while at 48 h cell numbers were sim;60% of the control value. At 72 h differences in cell numbers between cultures became negligible. This can be explained by the fact that DMSO-exposed cells were synchronized in early G1 phase, while control cultures were in exponential growth, with 40% of S phase cells at the seeding time. This might give a proliferation disadvantage to the DMSO-arrested cells, which lasted between 9 and 12 h, as judged by cytofluorimetric and protein analyses.
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Induction of MN or SCE was also investigated after release of G1 arrested cells in DMSO-free medium for sim;1 (MN) or 2 cell cycles (SCE). In our experiments, after cell growth in DMSO-containing medium at different concentrations, no statistically significant effects were found on MN or SCE frequencies in cells released from the G1 arrest, as compared with proliferating samples (Table I
). Interestingly, the frequencies of mitoses and the number of first and second mitoses in DMSO-exposed cells was not statistically different from the respective values in proliferating cells. We also investigated a possible DMSO effect on the frequency of polyploid and endo-reduplicated cells by chromosome counting. About 2% of metaphases were polyploid in control cultures and this frequency was unmodified in DMSO-exposed cells or in cells released from the DMSO arrest. No endo-reduplicated mitoses were observed in our scoring.
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Discussion |
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Several studies have demonstrated that DMSO, a well-known inducer of differentiation in myeloid cells (Watson et al., 1997
), also induces a reversible G1 arrest in cell lines of lymphoid origin with no signs of induction of differentiation (Sawai et al., 1990; Teraoka et al., 1991, 1996). We recently showed that incubation with DMSO induces a G1 arrest in adherent Chinese hamster CHO K-1 cells (Fiore and Degrassi, 1999). The G1 arrest was mediated by restoration of contact inhibition and prevention of high density-dependent apoptosis in DMSO-exposed cells. Moreover, a partial restoration of E-cadherin expression was also observed. These results indicate that DMSO not only arrests CHO cells in G1 but also stimulates cell differentiation, inducing CHO cells to acquire some features of differentiated epithelial cells (Fiore and Degrassi, 1999). In the present study we have demonstrated that DMSO-induced G1 arrest is efficiently reversed by incubation in fresh medium. Analysis of cell cycle progression by flow cytometry showed that cells are efficiently synchronized in early G1, since they progress synchronously through G1, enter S phase by 8 h and are found in middle S phase by 12 h after release from 1.5% DMSO. Interestingly, the synchronization is partially maintained through a second cell cycle, since a peak of early S phase was observed at 20 h release. Analysis of cell cycle-regulated proteins provided results consistent with the cytofluorimetric evaluation and corroborates the conclusion that cells are arrested in an early G1 stage. Indeed, DMSO growth-arrested cells are characterized by high levels of p27 protein, low levels of p21 and an intermediate cyclin D3 protein content, which corresponds to protein levels found in cells in early G1 stage (Sherr and Roberts, 1999). The time course of cell cycle-dependent protein fluctuation at early release times indicated that cells progress through G1 synchronously. This is confirmed by the concomitant increase in cyclin D3 content and decrease in p27 levels. Moreover, as usually found in response to growth factor, p21 levels increase during G1 progression in cells released from DMSO arrest (Sherr and Roberts, 1999). At the latest sampling time (8 h) cyclin E level reached its maximum, whereas cyclin D3 started to decrease. At this time point the p21 levels also drastically decreased as cells entered S phase. These results demonstrate that cells are accumulated at the G1–S transit at this recovery time, when cyclin D/CDK4 activity is overcome by cyclin E/CDK2 activity. At the same sampling time a sudden appearance of cyclin A was observed, concomitant with the first appearance of BrdUrd-positive cells by flow cytometry. These results suggest a tight regulation of DNA replication and cyclin A accumulation, in accordance with exhaustive immunofluorescent studies showing that cyclin A accumulation and the beginning of DNA replication occur simultaneously (Erlandsson et al., 2000).
Our study extends previous studies on Raji lymphoma cells released from a DMSO-induced arrest in which similar kinetics of cell cycle re-entry and protein expression were observed (Takase et al., 1992, 1994; Ponzio et al., 1998), suggesting that the capacity of DMSO to induce a reversible cell cycle arrest is not confined to non-adherent cells of lymphoid origin but is also valid for adherent cells such as CHO cells, which are difficult to synchronize by serum starvation because of a weak inhibition of cell growth exerted by confluence on these cells. However, the control mechanisms that regulate the DMSO-induced growth arrest implicate different regulators of CDK activities in adherent versus suspension cells. In adherent CHO cells, DMSO exerts its synchronizing action primarily by stimulating contact inhibition-dependent growth arrest, which is known to be mediated by high levels of the CDK inhibitor p27 (Polyak et al., 1994; Kato et al., 1997), whereas in suspension cultures of Raji cells induction of p21 blocks cell cycle progression at an early time after DMSO addition (Ponzio et al., 1998).
Taken together, the expression patterns of growth-related molecules and the kinetics of cell cycle re-entry are similar to those observed in serum-starved adherent 3T3 fibroblasts stimulated to enter the cell cycle by serum addition (Matsushime et al., 1994; Botz et al., 1996). This indicates that the synchronization action of DMSO impinges on the same control mechanisms that regulate cell growth in response to the absence or presence of growth factors, as during serum starvation and cell cycle re-entry after serum restoration.
Our cytogenetic results on SCE, polyploid and endoreduplicated frequencies and MN induction show that cell synchronization by DMSO exposure does not produce any genotoxic effect in cell populations released from the G1 arrest. The growth capacity of recovering populations is also comparable with proliferating cells, as judged by growth curves and mitotic and proliferation indices. Interestingly, the growth curves of DMSO-exposed samples display similar kinetics to those of the control curve. However, the absolute cell numbers are lower due to the fact that cells grown in DMSO are efficiently synchronized in early G1 and spend the first 8 h after release passing through this stage. The elongation of the time cells spend in G1 after release from DMSO could be exploited to investigate the mechanism of action of potential mutagenic compounds by assessing the effects of treatments on homogeneous G1 cell populations and identifying a specific sensitivity of this cell cycle stage to different mutagens.
Our results show that DMSO exposure for 96 h could be a useful method to synchronize CHO cells in G1 as an alternative to mitotic shake off, which yields low numbers of cells (Zwanenberg, 1983), or treatments with other chemicals, such as L-mimosine or others, which usually produce toxic effects (Mikhailov et al., 2000). The system is characterized by a high degree of synchrony which is partially maintained at the second cell cycle without any growth inhibition or cytotoxic effects. The synchronous progression from early G1 to S phase of released cells renders the DMSO-induced arrest and re-entry a model system to synchronize cell lines showing poor contact inhibition-dependent growth arrest, to investigate protein activities regulating G1 progression in these cells and to perform mutagenicity testing on homogeneous G1 cell populations.
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Acknowledgments |
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The authors of this paper dedicate it to the memory of Franco Tatò.
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1 To whom correspondence should be addressed. Tel: +39 0644 57527; Fax: +39 0644 57529; Email address: f.degrassi{at}caspur.it
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References |
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Received on February 2, 2002; accepted on May 25, 2002.
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