|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Adenosine induces G2/M cell-cycle arrest by inhibiting cell mitosis progression
Kun‑Zhi Jia*, Bo Tang*, Lu Yu*, Wei Cheng*, Rong Zhang*, Jian‑Fa Zhang† and Zi‑Chun Hua*1
*Jiangsu Center of Hepatobiliary Diseases and the State Key Laboratory of Pharmaceutical Biotechnology, Affiliated Gulou Hospital, Nanjing University, Nanjing 210093, People's Republic of China, and †Center for Molecular Metabolism, Nanjing University of Science and Technology, Nanjing 210093, People's Republic of China
Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G2/M cell-cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis.
Key words: adenosine, cell cycle, cell division cycle 2 kinase (cdc2), cyclin B; mitosis
Abbreviations: cdc2, cell division cycle 2 kinase, HEK, human embryonic kidney, NBTI, S-(4-nitrobenzyl)-6-thioinosine, PI, propidium iodide
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Adenosine is a purine nucleoside produced and released into the extracellular medium by cells during normal intracellular ATP metabolization and degradation. Extracellular adenosine acts as a local modulator with a generally cytoprotective function in the body (Linden, 2005; Sitkovsky et al., 2008). Its effects on tissue protection and repair fall into four categories: (i) increasing the ratio of oxygen supply to demand; (ii) protecting against ischaemic damage by cell conditioning; (iii) triggering anti-inflammatory responses; and (iv) promoting angiogenesis (Kaiser and Quinn, 1996; Jacobson and Gao, 2006; Ashton et al., 2007).
Considering the fact that adenosine is accumulated and it can induce cell-cycle arrest in unfavourable environments (Vanbelle et al., 1987; Matherne et al., 1990; Brambilla et al., 2000; Fishman et al., 2000), we were interested in the relationship between adenosine and cell-cycle arrest, or how adenosine regulates cell-cycle arrest. Some researchers have reported previously that adenosine inhibited cell proliferation through the A3 receptor by inducing G1/S cell-cycle arrest (Brambilla et al., 2000; Fishman et al., 2000); others have reported that adenosine inhibited macrophages through induction of p27 expression (Xaus et al., 1999). Until the present study, few researchers have investigated the influence of adenosine on the cell cycle in detail. In the present study, adenosine was found to induce cell-cycle arrest, not only at the G1/S phase, but also at the G2/M phase by regulating the activity of cdc2 (cell division cycle 2) and inhibiting mitosis progression.
2. Materials and methods
Adenosine, NBTI [S-(4-nitrobenzyl)-6-thioinosine], RNase A, PI (propidium iodide) and DMSO were purchased from Sigma. Antibodies were purchased from Cell Signaling Technologies.
2.2. Cell culture
HEK (human embryonic kidney)-293T, MCF-7 (human breast cancer cell line) and A549 (human lung adenocarcinoma cell line) cells were from A.T.C.C., and were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) FBS (fetal bovine serum; Gibco). All cells were incubated under standard culture conditions (20% O2 and 5% CO2 at 37°C). Adenosine and NBTI were dissolved in DMSO. DMSO was used as a negative control. All cells, except in the time-course experiment, were harvested at 6 h after adenosine treatment.
2.3. Fluorescent microscopy analysis
HEK-293T cells were seeded on to glass coverslips which had been placed in six-well plates and incubated at 37°C in a 5% CO2-humidified chamber. Cells were fixed by incubating them with 4% paraformaldehyde at 4°C for 1 h. After two washes with PBS, cells were permeabilized using 0.5% Triton X-100 in PBS at room temperature (20°C) for 45 min. Cells were then washed twice with PBS on ice and blocked with 3% BSA in PBS for more than 2 h. Subsequently, cells were incubated with primary antibody in PBS containing 3% BSA at 4°C overnight. Cells were then washed with PBS three times and incubated with the secondary antibody in PBS containing 3% BSA at room temperature for 1 h. The secondary antibody used was FITC-conjugated anti-mouse IgG. Cells were imaged with a microscope (Carl Zeiss Axioplan 2).
2.4. Western blot analysis
Cells were washed with PBS and then lysed in extraction buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate and 1 mM PMSF] on ice for 60 min. Lysates were cleared by centrifugation (12000 g for 10 min). Equal amounts of cell extracts were then resolved by SDS/PAGE, transferred on to PVDF membrane, and probed with antibodies.
2.5. Flow cytometry analysis
Cells were collected by centrifugation (300 g for 5 min at 4°C), and the culture medium was removed. Cells were then fixed with ice-cold 70% ethanol overnight. The cells were suspended in PBS containing 25 μg/ml PI and 15 μg/ml RNase A. After 30 min incubation at 37°C, the cells were analysed with FACSCalibur (Bjorklund et al., 2006).
3.1. Adenosine induces G2/M cell-cycle arrest in HEK-293T cells
To investigate the influence of adenosine on the cell cycle, we added different concentrations of adenosine to HEK-293T cells. As shown in Figure 1(A), adenosine induces G2/M cell-cycle arrest in a dose-dependent manner, in addition to its effect on the G1/S-phase (data not shown). The higher the concentration of adenosine, the higher the percentage of cells arresting at G2/M-phase. We have also investigated the effects of adenosine on the cell cycle at different time points. The results show that adenosine can maintain cells in arrest phase for at least 24 h (Figure 1B), but the extent decreases with time, probably because of the existence of adenosine deaminase, which is responsible for the hydrolytic deamination of adenosine and 2′-deoxyadenosine into inosine and 2′-deoxyinosine (Blackburn et al., 2000).
3.2. Adenosine affects the cell cycle intracellularly
It is well documented that extracellular adenosine can be transported into cells across the cell membrane with the help of the NBTI-sensitive adenosine transporter (Thampy and Barnes, 1983; Wakade et al., 1995; Che et al., 1997). We investigated whether the effect of extracellular adenosine on the cell cycle is adenosine-transporter-dependent. The adenosine transporter inhibitor, NBTI (Huang et al., 1997), was added to HEK-293T cells together with adenosine to prevent extracellular adenosine from being transported into cells. The results showed that NBTI relieves the inhibitory effects of adenosine on the cell-cycle machinery (Figure 1C) and it implies that adenosine needs to be transported into cells to execute its inhibitory function on the cell cycle. In contrast, NBTI alone does not have a significant effect on the cell cycle of HEK-293T cells (Figure 1C).
3.3. Adenosine induces G2/M cell-cycle arrest by inhibiting the progression of cell mitosis
To explore how adenosine inhibits the cell-cycle machinery, we conducted double-staining experiments using the α-tubulin antibody for microtubule staining and Hoechst nuclear staining. The results show that more cells are arrested in the metaphase of mitosis after treatment with adenosine (Figure 2A). The activation of cdc2 is the pivotal indicator protein for transition of metaphase to anaphase. It is well known that the dephosphorylation of the Tyr15 residue of the cdc2 protein is essential for cdc2 activation (Borgne and Meijer, 1996). The phosphorylation level of the Tyr15 residue of cdc2 was examined using a phospho-specific antibody. The results show that phosphorylation of Tyr15 of cdc2 (Borgne and Meijer, 1996) was reduced upon adenosine treatment (Figure 2B), but the total cdc2 protein level remained the same (Figure 2B). These results indicate that adenosine may influence the transition of metaphase to anaphase of mitosis. To confirm this hypothesis, we further checked the protein level of cyclin B, which is the main regulator of cdc2 activity. The cellular protein level of cyclin B decreased after adenosine treatment (Figure 2B), which probably leads to impaired metaphase transition of HEK-293T cells to anaphase. Also the p21 protein level becomes higher, which is the negative regulator of cdc2 activity (Figure 2B). Thus our results suggest that adenosine induces G2/M cell-cycle arrest by down-regulating cdc2 activity through decreasing the expression of cyclin B and increasing the expression of p21.
3.4. Adenosine induces G2/M cell-cycle arrest in various cell lines
We have further investigated whether adenosine has a similar inhibitory effect on the cell cycle in other cells of different origin. Adenosine was added to MCF7 cells or A549 cells, and similar results were observed, which shows that adenosine can induce G2/M cell-cycle arrest (Figure 3), but the sensitivity differed depending on the cell line. This assay suggests a common mechanism that cell-cycle arrest is induced by adenosine under stress conditions.
Previous studies have shown that adenosine induces G1/S cell-cycle arrest (Brambilla et al., 2000; Fishman et al., 2000), consistent with our observations. In the present study, adenosine was also found to induce cell arrest at G2/M phase. These results probably reflect an effective self-control mechanism which cells use to protect themselves when they encounter an unfavourable environment.
Cdc2–cyclin B promotes the progression of mitosis. Adenosine was found to regulate cdc2 activity by modulating the protein level of some cell-cycle-related proteins, such as p21 and cyclin B, thus inhibiting the transition of metaphase to anaphase during mitosis (Figure 4). The inhibitory function of extracellular adenosine is cell-membrane-transportation dependent. How adenosine regulates the expression of the cell-cycle-related protein is also worthy of further study.
Taken together, the results of the present study suggests a novel mechanism through which adenosine imposes its inhibitory effect on mitosis in the cell cycle. By arresting the cell cycle at both G1/S- and G2/M-phases, adenosine effectively protects cells from the influence of environmental stress.
Kun-Zhi Jia performed most of the experiments in the manuscript. Bo Tang performed the immunofluorescent experiment with Kun-Zhi Jia, Lu Yu assisted with the FACS experiments, Wei Cheng helped to perform the cell-cycle experiments and Rong Zhang did some of Western blot analyses. Jian-Fa Zhang helped with the experimental design and provided some research tools. Zi-Chun Hua designed and directed the whole programme.
This work was supported by the
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Received 29 June 2009/13 August 2009; accepted 22 September 2009
Published as Cell Biology International Immediate Publication 22 September 2009, doi:10.1042/CBI20090136
© 2010 The Author(s) Journal compilation. © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)