|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G1 phase in CHO cells
Angélique van Opstal*, José Bijvelt*, Elly van Donselaar†, Bruno M. Humbel† and Johannes Boonstra*1
*Cellular Dynamics, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, and †Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.
Key words: apoptosis, cell cycle, Chinese-hamster ovary (CHO) cells, G1 phase, protein kinase B, restriction point
Abbreviations: CDK, cyclin-dependent kinase, CHO cells, Chinese-hamster ovary cells, DAPI, 4′,6-diamidino-2-phenylindole, DMEM, Dulbecco's modified Eagle's medium, FCS, fetal calf serum, MAPK, mitogen-activated protein kinase, PDK, phosphoinositide-dependent kinase, PH, pleckstrin homology, PI3K, phosphoinositide 3-kinase, PKB, protein kinase B
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Growth factors regulate cell cycle progression at least two major points in the cell cycle of CHO (Chinese-hamster ovary) cells. Here, we will be concerned with the transition from M to G1 phase and in late G1- to S-phases (Hulleman et al., 2004). Of special interest is the growth factor-dependent point at the M/G1 transition, when cells are programmed to progress through the cell cycle or undergo apoptosis (Hulleman et al., 2004). We have demonstrated that the PI3K (phosphoinositide 3-kinase) plays an important role as it prevents the induction of apoptosis at the M/G1 transition in cycling CHO cells (van Opstal et al., 2006). Inhibition of PI3K activity during and for 2 h after mitosis inhibited cell cycle progression, which eventually leads to apoptosis (van Opstal et al., 2006).
PKB (protein kinase B; also known as Akt) is one of the signalling molecules downstream of PI3K that promotes cell survival as well as blocking apoptosis (Franke et al., 1997; Coffer et al., 1998; Datta et al., 1999; Lawlor and Alessi, 2001; Scheid et al., 2002; Song et al., 2005; Duronio, 2008). Thus, it can be suggested that PKB plays an important role in the early G1 phase by inducing cycle progression and preventing apoptosis. PKB is activated by binding through its PH (pleckstrin homology) domain to 3′-phosphorylated PI (phosphatidylinositol) phospholipids (Alessi et al., 1996; Franke et al., 1997), which are formed by PI3K activity. In general, activation of PI3K results in the formation of PtdIns(3,4,5)P3 (phosphatidyl-inositol-3,4,5-triphosphate) and PtdIns(3,4)P2 (phosphatidyl-inositol-3,4,-biphosphate) by phosphorylating PI) in the 3′ position of the inositol ring. The PH domain-dependent membrane translocation step of PKB is followed by phosphorylation at Thr308 in the activation loop of the kinase domain, and at Ser473 in the C-terminal regulatory domain (Scheid et al., 2002). Phosphorylation of PKB at both amino acid residues is required for full activation because phosphorylation of PKB at one of the two sites only partially activates the serine/threonine kinase (Alessi et al., 1996). There is evidence that PDK1 (phosphoinositide-dependent kinase-1) phosphorylates PKB at Thr308, the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa (Alessi et al., 1996; Stephens et al., 1998). The kinase responsible for Ser473 phosphorylation remains unknown. As purified or recombinant PDK1 is only phosphorylated at Thr308 of PKB and not Ser473, it was assumed that the phosphorylation of Ser473 is catalysed by another protein kinase, tentatively termed PDK2 (Alessi et al., 1997). Other possibilities may be that the Ser473 residue of PKB is phosphorylated by PKB itself or that other kinases are involved, such as mTORC2 (Toker and Newton, 2000; Sarbassov et al., 2005). Recently, >90 kinases have the ability to phosphorylate PKB on Ser473 (Chua et al., 2009).
Once PKB has been activated, a significant fraction of endogenous PKB detaches from the plasma membrane and translocates to distinct subcellular compartments, including the nucleus (Borgatti et al., 2000; Kunkel et al., 2005; Zhu et al., 2007). The finding that activated PKB, which lacks a nuclear localization signal, accumulates in the nucleus suggests that translocation occurs by a protein providing the nuclear import signal (Meier et al., 1997). In the nucleus, active PKB phosphorylates numerous proteins involved in cell cycle progression and anti-apoptotic signalling, such as the forkhead family transcription factors, FKHRL1 (forkhead in rhabdosarcoma L1), FKHR and AFX (Neri et al., 2002; Baldin et al., 2003; Brunet et al., 1999) and structural proteins like lamin A (Cenni et al., 2008).
The PKB family consists of three different isoforms, i.e. PKBα/Akt1, PKBβ/Akt2 and PKBγ/Akt3 respectively, encoded by three different genes (Datta et al., 1999). These isoforms share 85% amino acid sequence homology, but they have different functions. PKBα/Akt1 seems to play an essential role in cell proliferation and development (Cho et al., 2001a), whereas PKBβ/Akt2 has an important role in glucose homoeostasis (Bae et al., 2003; Cho et al., 2001b). PKBγ/Akt3 is required for normal development of brain size (Easton et al., 2005).
PKB controls cell cycle progression at distinct points in the cell cycle. Expression of a kinase-inactive PKB mutant (K179M) arrests the cells at the G2/M-phase (Lee et al., 2005). Conversely, the expression of a constitutively active form of PKB (myrPKB) overcomes cell-growth arrest at the G2/M-phase induced by DNA damage (Kandel et al., 2002). Other data show that constitutively active PKB stimulates progression from G0/G1-to-S phase, whereas inhibition of PKB activity by either inhibitors, using dominant-negative kinase or through dominant-negative phosphorylation mutants, results in inhibition of G0/G1 to S phase transition (Hu et al., 2004; Zhang et al., 2004). PKBα/Akt1 plays an essential role in G1/S phase transition in mouse embryo fibroblasts (Yun et al., 2009). We have investigated the role of PKB during the G1 phase of the ongoing cell cycle of CHO cells, using the highly selective PKB inhibitor, API-2 (Yang et al., 2004). PKB activity is required during and for the first 2 h after mitosis to allow cells to progress through the G1 phase into S-phase. In the presence of API-2, 3H-thymidine incorporation and the expression of the late G1/early S phase cell cycle regulator, cyclin A, was suppressed. Addition of the inhibitor during mitosis resulted in a rapid decrease in cyclin D1/D2 expression within 2 h after mitosis. Since phosphorylation of MAPK (mitogen-activated protein kinase), which is one of the earliest events after mitosis, occurs in the presence of the API-2, it seems likely that cells become arrested in early G1 phase and not in mitosis. Inhibition of PKB during mitosis leads to the expression of cleaved caspase-3, a central mediator of apoptosis. We also demonstrate that the nuclear translocation of PKB, phosphorylated at Ser473, occurs during early G1 phase in cycling CHO cells. The results show a role for PKB in the control of early G1 phase progression and inhibition of cell death.
2. Materials and methods
2.1. Cell culture, synchronization and treatment
CHO cells were grown in DMEM (Dulbecco's modified Eagle's medium; Gibco) containing 7.5% FCS (fetal calf serum; Gibco) and 5 mM l-glutamine at 37°C in a humidified 5% CO2 air atmosphere. Three days prior to synchronization, cells were transferred to Hepes (25 mM) buffered DMEM containing 7.5% FCS. One day before mitotic shake-off, cells were plated at 3×104 cells/cm2. Mitotic cells were obtained by shaking an asynchronously growing cell population firmly for 1 min and collecting the medium (Boonstra et al., 1981). The mitotic cells obtained after mitotic shake-off were plated at 15000 cells/cm2 on cell culture dishes, either in the absence or presence of the PKB inhibitor 10 μM API-2 (Biomol Research Laboratories Inc.).
2.2. Time-lapse cinematography
A 21 cm2 tissue culture dish containing exponentially growing cells was placed in an incubation chamber (Zeiss, CTI controller 3700) in a humidified 5% CO2 air atmosphere at 37°C under an Axiovert 35M inverted microscope attached with a Nikon DXM1200 time-lapse camera system using the ACT1 program, essentially as described (Zetterberg and Larsson, 1985; Larsson et al., 1985). The cells were incubated for at least 24 h and photographs were taken every 10 min, illumination being switched off during the intervals between the photographs. From these images, visible cells in the frame of the camera were assigned an actual cell cycle age, that is, the h after mitosis at the time of the addition of the PKB inhibitor, which was at 24 h after the start of the experiment. The cells continued to be incubated for at least for 48 h and again photographs were taken each 10 min. From each image, we established whether the cells could undergo mitosis in the presence of the inhibitor.
2.3. [3H]-thymidine incorporation assay
To measure DNA synthesis, cells obtained after mitotic shake-off were plated in the presence of [3H]-thymidine (3H-TdR at a specific activity 2 Ci/mmol; Amersham) at 1 μCi/ml. Cells were grown in 24-well plates either in the presence or absence of the PKB inhibitor API-2. After the completion of one cell cycle, cells were washed twice with PBS and disrupted in 0.1 M NaOH. Incorporated radioactivity of the samples was measured using a liquid scintillation counter (Beckman LS 6000SE).
2.4. Statistical analysis
Unpaired student's t- tests were performed using GraphPad Prism version 3.00 for Windows and GraphPad Software. Results are expressed as means±S.D.; n = 6 for each determination. Asterisks indicate values significantly different from the control cells (**P<0.05 and ***P<0.001).
2.5. Cell extraction and Western blotting
At the indicated times after replating, cells were washed in PBS and lysed in RIPA buffer [20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium desoxycholate, 0.1% SDS, 1 mM EDTA, 100 mM NaF, 1 mM benzamidine, 1 mM PMSF and 1 mM Na3VO4 (ortho-sodiumvanadate)]. Those that were not allowed to attach to the substratum (mitotic cell samples) were centrifuged at 1400 rev./min for 7 min prior to lysis. For Western blotting, cell lysates of 1.38×105 cells per slot were loaded on 10% polyacrylamide gels. Proteins were separated and electroblotted on to PVDF membranes (Roche Diagnostics). Membranes were blocked with PBS containing 4% (w/v) non-fat dried skimmed milk powder and 0.1% Tween 20 and incubated with one of the following antibodies: mouse anti-cyclin A (2.5 μg/ml; Oncogen Research products), rabbit anti-cyclin D1/D2 (1 μg/ml), mouse anti-MAPK-R2 [ERK2 (extracellular-signal-regulated kinase 2)] (0.5 μg/ml), mouse anti-PKBα/Akt (0.25 μg/ml) (from Transduction Laboratories), rabbit anti-phosphorylated-MAPK (1:1000), and cleaved caspase-3 (1:1000; New England Biolabs) and mouse anti-phosphorylated Akt/PKB Ser473 (1:500) (Cell Signaling Technology). Incubations were followed by three washes with blocking buffer and incubated either with 0.8 μg/ml diluted horseradish DAM-PO (peroxidase-conjugated donkey anti-mouse IgG) or DAR-PO (peroxidase-conjugated donkey anti-rabbit IgG) (Jackson ImmunoResearch Laboratories Inc.) diluted in blocking buffer. Membranes were washed three times in blocking buffer and twice in PBS, followed by the detection of bound antibody using Enhanced Chemiluminescence (Dupont).
2.6. DNA staining
Cells were fixed in 4% formaldehyde in PBS before being incubated with 2 μg/ml DAPI (4′,6-diamidino-2-phenylindole) in PBS for 5 min at 37°C. They were washed twice with PBS and fluorescent DNA–DAPI complexes were visualized with a CCD (charge-coupled-device) camera (Leica; model DC350F) using Leica Image Manager 50 software. Pictures were processed with Adobe Photoshop® 7.0.
At different time-points after mitosis, cells cultivated in the absence or presence of the PKB inhibitor API-2 were fixed by adding an equal volume of fixative (4% formaldehyde plus 0.4% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2) to the medium. After 10 min fixation at room temperature, the mixture of fixative and medium was replaced by fresh fixative (2% formaldehyde plus 0.2% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2) and left at room temperature for 30 min, followed by overnight incubation at 4°C. After washing three times with phosphate buffer (pH 7.2), 1.5 ml of 1% gelatin in phosphate buffer (pH 7.2) was added to the cells, which were collected by scraping with a rubber policeman. Following centrifugation for 1 min at 10000 rev./min, cells were suspended in 12% gelatin phosphate-buffered (pH 7.2) at 37°C. The gelatin was allowed to solidify on ice. Gelatin containing the cells was cut into blocks and prepared for sectioning according to Tokuyasu (1973). Briefly, the gelatin blocks were immersed in 2.3 M sucrose (Merck) in phosphate buffer (pH 7.2) overnight and mounted on aluminium pins to which they were fixed by freezing in liquid nitrogen. Sections were obtained on a Leica cryo-ultramicrotome (UC6/FC6).
2.8. Immunofluorescence microscopy on cryosections
Cryosections of 500 nm were obtained at −100°C. Sections were picked up in a 1:1 mixture of 2% methylcellulose in distilled water and 2.3 M sucrose in phosphate buffer (pH 7.2) and dipped on glass slides, the latter being pretreated with 1% silan (3-aminopropyltrietoxysilan) prior to adhesion of the sections. Sections were mounted on glass slides and incubated with 0.02 M glycin in PBS to block free aldehyde groups. After 5 min incubation, sections were blocked twice for 5 min with PBS containing 1% BSA (Sigma) and subsequently incubated with mouse anti-phosphorylated Akt/PKB Ser473 (1:30) (from Cell Signaling Technology) or mouse anti-PKBα/Akt (25 μg/ml) antibodies (from Transduction Laboratories) in 1% BSA in PBS. The labelled sections were washed five times with 0.1% BSA in PBS, before the sections were incubated with the secondary antibody Alexa Fluor® 488 goat anti-mouse IgG or Alexa Fluor® 488 goat anti-rabbit IgG (0.01 mg/ml; Molecular Probes) in PBS containing 1% BSA. After 45 min secondary antibody incubation, sections were washed 5 times in PBS. DAPI was added to the first washing step in PBS for staining of nuclei. Labelled samples were mounted in Vectashield (Vector Laboratories), and photographed on a Zeiss Axioskop equipped with a UV or FITC filter set and recorded using a Nikon digital camera DXM 1200 with a Zeiss Plan neofluor ×40 objective and processed using Photoshop® CS Version 8.0.
3.1. PKB activity is required for progression through G1 phase
Inhibition of PI3K resulted in cell cycle arrest and induction of apoptosis at the M/G1 transition in cycling CHO cells (van Opstal et al., 2006). To investigate the potential role of PKB in regulation of the ongoing cell cycle, we analysed the effect of the highly selective PKB inhibitor triciribine (API-2, TCN, NSC 154020), which inhibits the phosphorylation and activation of PKBα, PKBβ and PKBγ (Yang et al., 2004), on PKB phosphorylation. CHO cells were synchronized by mitotic shake-off (Boonstra et al., 1981) and allowed to progress from M into G1 for the times indicated. Cells taken at these time-points were lysed using equal amounts of cells (1.36×105) for loading on to gels. By Western blotting examined on cell basis, PKBα was expressed at a relatively constant level for up to 8 h after mitosis (Figure 1A). During G1 phase, phosphorylated PKB was detected (Figure 1B). Addition of API-2 immediately after shake-off (i.e. in M phase), significantly decreased the expression of phosphorylated PKB 2 h after mitosis (lane 2+), while the levels were comparable to control values 4 and 6 h after mitosis (Figure 1B). These results demonstrate that API-2 prevents phosphorylation of PKB only during the early G1 phase.
In exponentially growing cultures photographed before and after addition of the inhibitor cells up to 4 h after mitosis were unable to undergo mitosis in the presence of the inhibitor, in contrast to cells in late G1 phase at 8 h or more after M (Figure 2).
API-2 was similarly added to synchronized CHO cells at different time-points after mitosis to determine 3H-thymidine-incorporation at 14 h to estimate DNA synthesis (Figure 3A) and establish the extent to which cells entered S phase. Addition of API-2 CHO cells 4, 6 or 8 h after mitosis, did not significantly reduce 3H-thymidine-incorporation compared with control cells. In contrast, cells incubated with the inhibitor during mitosis or 2 h after mitosis incorporated significantly less 3H-thymidine (Figure 3A). Increasing the concentration of API-2 10-fold made no difference (results not shown). Since 0 h and early G1 (2 h) cells spent longer in API-2 compared with the later G1 phase cells, mitotic cells were grown for 2 or 4 h in the presence of the PKB inhibitor and assessed for 3H-thymidine-incorporation at 14 h after mitosis. Cells incubated for only 2 or 4 h with the API-2 had reduced 3H-thymidine-incorporation (57 and 78% respectively) 14 h after mitosis compared with control cells (Figure 3B). This indicates that the low 3H-thymidine-incorporation measured in cells with API-2 during mitosis (0 h) or early G1 (2 h) phase is due to cell cycle arrest rather than toxicity of the longer exposure time. Thus, continuous cycling CHO cells require PKB activity during mitosis and early G1 phase for transition into S-phase.
3.2. Inhibition of PKB activity causes cell cycle arrest in early G1 phase
Progression through the cell cycle is largely dependent on cyclin/CDK (cyclin-dependent kinase) complexes. To investigate the cell cycle phase in which cells arrest in the presence of API-2, protein expression levels of G1 phase-specific cyclins were determined. Expression of cyclin A after mitotic selection in the absence or presence of API-2 added during mitosis is shown (Figure 4). In untreated cells, it began to increase 6 h after mitosis (van Rossum et al., 2001). At 8 h, this increase was significant (van Opstal et al., 2006). In contrast, cyclin A expression could not be detected in cells incubated in the presence of API-2, suggesting that inhibition of PKB arrests cycle progression before or at mid G1 phase.
Cyclin D1/D2 is expressed throughout the entire G1 phase in continuously cycling CHO cells (Hulleman and Boonstra, 2001). During G1 phase, the level of cyclin D1 fluctuates in untreated cells (van Opstal et al., 2006). Addition of API-2 during mitosis resulted in a rapid decrease of both cyclin D1 and D2 expression within 2 h after the addition of the inhibitor.
MAPK phosphorylation is one of the earliest events during the G1 phase of the ongoing cell cycle in continuous cycling CHO cells. Phosphorylation can be detected as early as 10 min after mitosis and is required throughout G1 phase for progression into S-phase (Hulleman et al., 1999a). In untreated cells, phosphorylated MAPK was not detected during mitosis, whereas 2 h after mitosis an increase in MAPK phosphorylation was measured (Figure 4). Interestingly, cells treated with API-2 also expressed phosphorylated MAPK. This observation suggests that inhibition of PKB activity arrests CHO cells at early G1 phase.
3.3. Inhibition of PKB activity induces apoptosis
Since cells in the presence of the PI3K inhibitor, LY294002, in the early G1 phase become apoptotic (van Opstal et al., 2006), inhibition of PKB activity results in early G1 phase arrest in CHO cells, and PKB activity blocks apoptosis by suppressing the activation of caspase-3 (Fujita et al., 1998), these findings suggest that PKB during early G1 phase protects cells from cell death. Therefore, we investigated the expression of activated caspase-3 in synchronized CHO cells after incubation with or without the PKB inhibitor for 24 and 48 h, and analysed by Western blotting using an antibody recognizing the 17/19 kDa fragment of activated caspase-3. Cultures without inhibitor did not express cleaved caspase-3, but it was expressed in cells treated with API-2 cells within 24 h (Figure 5). Thus, the Akt/PKB signal transduction pathway blocks apoptosis by inhibiting cleavage of caspase-3.
3.4. Phosphorylated PKB translocates to the nucleus during early G1 phase
Once PKB is phosphorylated at both Thr308 and Ser473, a fraction of active PKB detaches from the plasma membrane and translocates to distinct subcellular compartments, including the nucleus, resulting in different cellular responses. Since nuclear PKB phosphorylates substrates that are important for progression through the cell cycle and inhibition of apoptosis (Brunet et al., 1999; Neri et al., 2002; Baldin et al., 2003), the localization of PKB during the G1 phase of the ongoing cell cycle was determined by means of immunofluorescence microscopy in cryosections of cells from different phases of G1. Cryosections were used because they can be obtained from the same cell preparations as used for Western blotting and 3H-thymidine-incorporation, allowing the direct comparisons. Nuclear morphology was examined in DAPI stained cells. Immunostaining with anti-PKB antibody showed that PKB is evenly distributed throughout the mitotic cell (0 h) with the exception of the chromosomes (Figures 6A–6C). Some cells, however, had significantly higher levels of expression of PKB (Figure 6A), suggesting that it varies during the different stages of the M-phase. Cryosections of early (2 h; Figures 6D–6F) or mid-late (8 h; Figures 6J–6L) G1 phase cells possessed a punctate and diffuse distribution of PKB throughout the entire cell including the nucleus, with the exception of the nucleolus. Some cells had a greater amount of PKB in the nucleus than in the cytoplasm. Incubation of cells in the presence of the PKB inhibitor for 2 h (Figures 6G–6I) did not influence PKB localization, although nuclear localization seemed a little more pronounced.
Using antibodies that recognize Ser473 phosphorylated PKB demonstrated that phosphorylated PKB is homogeneously distributed throughout the cell during M-phase (0 h; Figure 7A). Similar to labelling using anti-PKB antibodies, some cells with condensed chromosomes had higher levels of Ser473 phosphorylated PKB. Labelling in cryosections of early (2 h; Figures 7D–7F) and mid-late G1 (8 h; Figures 7J–7L) phase showed the phosphorylated protein to be mainly localized in the nuclei, but not the nucleoli (Figures 7D and 7J). However, cells with decondensed chromatin (Figure 7A) had its PKB phosphorylated at Ser473 localized in the cytoplasm or the nucleus of cycling CHO cells. This indicates that the distribution of Ser473 phosphorylated PKB varies during G1 phase. Incubation of the cells in the presence of the PKB inhibitor for 2 h did not significantly affect the localization of Ser473 phosphorylated PKB, although clearly the fraction of cells with nuclear localization increased (Figures 7G–7I). The results suggest that API causes a cell cycle arrest in the early G1 phase.
To establish possible variations of Ser473 phosphorylated PKB in the early G1 phase, CLSM analysis was carried out for the shorter times after mitosis. Ser473 phosphorylated PKB was predominantly present in the membrane of mitotic as well as G1 phase cells (Figure 8A). Moreover, pro-metaphase cells had no more intense immunostaining than metaphase or G1 phase cells. At 30 min after mitosis, Ser473 phosphorylated PKB was evenly distributed throughout the cytoplasm of CHO cells (Figures 8D–8F). Cells at 2 h after mitosis had similar nuclear staining to those observed in cryosections (Figures 8G–8I). Thus, translocation of Ser473 phosphorylated PKB, to the nucleus occurs in a cycle-dependent way during early G1 phase of continuously cycling CHO cells.
API-2 suppresses DNA synthesis when added in M or early G1, a down-regulation that was not seen when added during mid or late G1 phase. Since the incubation with the inhibitor for 2 h, starting at mitosis, already caused a significant reduction in DNA synthesis, continuously cycling CHO cells require PKB activity during mitosis and early G1 phase. Mitotic cells treated with API-2 arrest cycle progression in early G1 phase, as shown by the results on phosphorylation of MAPK and the expression of cyclin D in the presence of the inhibitor. PKB is one of the signalling molecules downstream of PI3K that is capable of promoting cell survival as well blocking apoptosis (Coffer et al., 1998; Scheid et al., 2002; Lawlor and Alessi, 2001; Datta et al., 1999; Franke et al., 1997). The point in G1 phase at which CHO cells arrest in the presence of API-2 seems to correlate with the point at which cells (in the absence of growth factors as well as in the presence of the PI3K inhibitor, LY294002) become susceptible to apoptosis (Hulleman et al., 2004; van Opstal et al., 2006). It seems likely that PKB serves to protect cells from apoptosis during early G1 phase. As anticipated, the expression of cleaved caspase-3 was markedly increased in cycling CHO cells exposed to API-2 for 24 h starting at mitosis. Thus, the inhibition of apoptosis during early G1 phase is under control of PKB. Furthermore, we have provided evidence that the nuclear translocation of Ser473 phosphorylated PKB occurs during early G1 phase in cycling CHO cells. Unlike the strong nuclear localization that is observed for Ser473 phosphorylated PKB, the nuclear localization of total PKB during G1 phase seems less obvious: some cells have a higher amount of total PKB in the nucleus than cytoplasm.
It is clear that during early G1 phase cycling CHO cells are programmed to continue their progress through the cell cycle or to undergo apoptosis. Our findings confirm studies suggesting that PKB is required for progression through the G1 phase; our studies demonstrate that inhibition of PKB causes cell cycle arrest in early G1 phase and not in mid or late G1 phase, as was suggested in other reports that focused on cells entering G1 phase from the quiescent state, G0 (Hu et al., 2004; Kops et al., 2002). Progression through G1 and entry into S phase requires the expression of cyclin D. In G0 cells, cyclin D1 expression is very low, but strongly increases upon the addition of serum, thereby reaching a maximum level prior to DNA synthesis (Baldin et al., 1993). Induction of cyclin D expression by serum results from activation of a pathway that is dependent upon PKB phosphorylated on serine/threonine residues (Muise-Helmericks et al., 1998). In continuously cycling cells, depletion of growth factors during mitosis decreased the expression of cyclin D; however, depletion of growth factors at 2 h after mitosis did not influence cyclin D expression, indicating that continuously cycling cells do not require cyclin synthesis during mid-late G1 phase (Hulleman et al., 2004). Furthermore, the elimination of increased levels of CDK inhibitors is essential for progression from G0 through G1 and entry into S phase (Han et al., 1999). In many G0 cells, high levels of the p21 and p27 CDK inhibitors ensure that the cyclin D/CDK4,6 complexes that are present in the cell remain inactive (Sherr and Roberts, 1995). CDK inhibitors are substrates of PKB that, upon phosphorylation, become degraded and lose their inhibitory influence on cycle progression (Shin et al., 2005; Li et al., 2001). In continuous cycling cells, this regulation is not required, since no change in the expression of the p27 CDK inhibitor in the absence of growth factor occurs (Hulleman et al., 1999b). API-2 inhibits the mTORC2 pathway in T-cell acute lymphoblastic cells, resulting in PKB dephosphorylation and induction of caspase-dependent apoptosis (Evangelisti et al., 2011). In this respect, it is of interest to note that API-2 did not dephosphorylate PKB 4 and 6 h after mitosis. Since API-2 inhibits PKB phosphorylation through interaction with its PH domain, it is tempting to suggest that PKB phosphorylation in mid-G1 phase is due to kinases that do not require the PH domain for phosphorylating PKB. In most studies on the effect of API-2 on phosphorylation of PKB (Evangelisti et al., 2011), dephosphorylation was measured in the cells that had been incubated for prolonged times in the presence of the inhibitor. From our studies, it is clear that prolonged incubation of exponentially growing cells with API-2 will eventually result in cycle arrest in early G1 phase and a dephosphorylation of PKB.
We have therefore established that PKB is essential for progression through early G1 phase and can block apoptosis. Furthermore, PKB phosphorylated at Ser473 can be found in the nucleus during early G1 phase. However, how PKB exerts its anti-apoptotic effect within distinct subcellular compartments during early G1 phase of an ongoing cell cycle needs to be established.
Angelique van Opstal did most of the work and wrote the first draft of the paper as part of her Ph.D. thesis. Jose Bijvelt performed the time-lapse cinematography experiments presented in Figure 2. Elly van Donselaar and Bruno Humbel were responsible for the cryo-ultramicrotomy and subsequent labelling of the sections as shown in Figures 6 and 7. Cryo-ultramicrotomy is a very specialized technique and requires very experienced skills. Johannes Boonstra did provide the supervision of Angelique van Opstal and finalized the paper.
We thank Denise van Suylekom for her assistance in some experimental procedures and stimulating discussions and Frits Kindt and Ronald Leito for help with the time lapse cinematography. We also thank Elsa Regan-Klapisz for critical reading of the manuscript.
This work was supported by the University of Utrecht.
Alessi, DR, James, SR, Downes, CP, Holmes, AB, Gaffney, PR and Reese, CB (1997) Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα. Curr Biol 7, 261-9
Baldin, V, Theis-Febvre, N, Benne, C, Froment, C, Cazales, M and Burlet-Schiltz, O (2003) PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation. Biol Cell 95, 547-54
Boonstra, J, Mummery, CL, Tertoolen, LGJ, van der Saag, PT and de Laat, SW (1981) Cation transport and growth regulation in neuroblastoma cells. Modulations of K+ transport and electrical membrane properties during the cell cycle. J Cell Physiol 107, 75-83
Borgatti, P, Martelli, AM, Bellacosa, A, Casto, R, Massari, L and Capitani, S (2000) Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors. FEBS Lett 477, 27-32
Brunet, A, Bonni, A, Zigmond, MJ, Lin, MZ, Juo, P and Hu, LS (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857-68
Cenni, V, Bertacchini, J, Beretti, F, Lattanzi, G, Bavelloni, A and Riccio, M (2008) Lamin A Ser404 is a nuclear target of Akt phosphorylation in C2C12 cells. J Proteome Res 7, 4727-55
Cho, H, Thorvaldsen, JL, Chu, Q, Feng, F and Birnbaum, MJ (2001) Akt1/PKBα is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 276, 38349-52
Cho, H, Mu, J, Kim, JK, Thorvaldsen, JL, Chu, Q and Crenshaw, (2001) Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ). Science 292, 1728-31
Chua, BT, Galligo-Ortega, D, Ramirez de Molina, A, Ullrich, A, Lacal, JC and Downward, J (2009) Regulation of Akt(Ser473) phosphorylation by choline kinase in breast carcinoma cells. Mol Cancer 8, 131
Duronio, V (2008) The life of a cell: apoptosis regulation by the PI3K/PKB pathway. Biochem J 414, 333-44
Easton, RM, Cho, H, Roovers, K, Shineman, DW, Mizrahi, M and Forman, MS (2005) Role for Akt3/protein kinase Bγ in attainment of normal brain size. Mol Cell Biol 25, 1869-78
Evangelisti, C, Ricci, F, Tazzari, P, Chiarini, F, Battistelli, M and Falcieri, E (2011) Preclinical testing of the Akt inhibitor triciribine in T-cell acute lymphoblastic leukemia. J Cell Physiol 226, 822-31
Fujita, E, Kouroku, Y, Miho, Y, Tsukahara, T, Ishiura, S and Momoi, T (1998) Wortmannin enhances activation of CPP32 (Caspase-3) induced by TNF or anti-Fas. Cell Death Differ 5, 289-97
Hu, CL, Cowan, RG, Harman, RM and Quirk, SM (2004) Cell cycle progression and activation of Akt kinase are required for insulin-like growth factor I-mediated suppression of apoptosis in granulosa cells. Mol Endocrinol 18, 326-38
Hulleman, E, Bijvelt, JJ, Verkleij, AJ, Verrips, CT and Boonstra, J (1999a) Nuclear translocation of mitogen-activated protein kinase p42MAPK during the ongoing cell cycle. J Cell Physiol 180, 325-33
Hulleman, E, Bijvelt, JJ, Verkleij, AJ, Verrips, CT and Boonstra, J (1999b) Integrin signaling at the M/G1 transition induces expression of cyclin E. Exp Cell Res 253, 422-31
Hulleman, E, Bijvelt, JJ, Verkleij, AJ, Verrips, CT and Boonstra, J (2004) Identification of a restriction point at the M/G1 transition in CHO cells. Cell Mol Life Sci 61, 600-9
Kandel, ES, Skeen, J, Majewski, N, Di Cristofano, A, Pandolfi, PP and Feliciano, CS (2002) Activation of Akt/protein kinase B overcomes a G(2)/M cell cycle checkpoint induced by DNA damage. Mol Cell Biol 22, 7831-41
Kops, GJ, Medema, RH, Glassford, J, Essers, MA, Dijkers, PF and Coffer, PJ (2002) Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors. Mol Cell Biol 22, 2025-36
Kunkel, MT, Tsien, RY, Zhang, J and Newton, AC (2005) Spatio-temporal dynamics of protein kinase B/Akt signaling revealed by a genetically encoded fluorescent reporter. J Biol Chem 280, 5581-7
Larsson, O, Zetterberg, A and Engström, W (1985) Cell-cycle-specific induction of quiescence achieved by limited inhibition of protein synthesis, counteractive effect of addition of purified growth factors. J Cell Sci 73, 375-87
Lee, SR, Park, JH, Park, EK, Chung, CH, Kang, SS and Bang, OS (2005) Akt-induced promotion of cell-cycle progression at G(2)/M phase involves upregulation of NF-Y binding activity in PC12 cells. J Cell Physiol 205, 270-7
Li, Y, Dowbenko, D and Lasky, LA (2001) AKT/PKB phosphorylation of p21Cip/WAF1 enhances protein stability of p21Cip/WAF1 and promotes cell survival. J Biol Chem 277, 11352-61
Meier, R, Alessi, DR, Cron, P, Andjelkovic, M and Hemmings, BA (1997) Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bβ. J Biol Chem 272, 30491-7
Muise-Helmericks, RC, Grimes, HL, Bellacosa, A, Malstrom, SE, Tsichlis, PN and Rosen, N (1998) Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J Biol Chem 273, 29864-72
Shin, I, Rotty, J, Wu, FY and Arteaga, CL (2005) Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry. J Biol Chem 280, 6055-63
Stephens, L, Anderson, K, Stokoe, D, Erdjument-Bromage, H, Painter, GF and Holmes, AB (1998) Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279, 710-4
van Opstal, AJM and Boonstra, J (2006) Inhibitors of phosphatidylinositol-3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells. Cell Mol Life Sci 63, 220-8
van Rossum, GSAT, Vlug, AS, van den Bosch, H, Verkleij, AJ and Boonstra, J (2001) Cytosolic phospholipase A(2) activity during the ongoing cell cycle. J Cell Physiol 188, 321-8
Yang, L, Dan, HC, Sun, M, Liu, Q, Sun, XM and Feldman, RI (2004) Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res 64, 4394-9
Yun, SJ, Tucker, DF, Kim, EK, Kim, MS, Do, KH and Ha, JM (2009) Differential regulation of Akt/protein kinase B isoforms during cell cycle progression. FEBS Lett 583, 685-90
Zetterberg, A and Larsson, O (1985) Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells. Proc Natl Acad Sci USA 82, 5365-9
Received 14 February 2011; accepted 18 January 2012
Published as Cell Biology International Immediate Publication 18 January 2012, doi:10.1042/CBI20110092
© The Author(s) Journal compilation © 2012 Portland Press Limited
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)
Figure 2 Effect of PKB inhibitor triciribine on cell cycle progression in exponentially growing CHO cells
Figure 3 Effect of PKB inhibitor triciribine on cell cycle progression in continuous cycling CHO cells
Figure 7 Localization of phosphorylated PKB at Ser473 and DNA in cryosections of synchronized CHO cells