SEARCH

SEARCH BY CITATION

Keywords:

  • restriction point;
  • flow cytometry;
  • human peripheral blood lymphocyte;
  • cyclin E

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Although the restriction point (R-point) was proposed in animal cells several decades ago, its existence in normal cells is still controversial, because, in most studies, long-term cultured cell lines rather than primary normal cells were used. Furthermore, cell synchronization was generally applied, resulting in growth imbalance between DNA synthesis and protein expression in cells. Finally, R-point was originally proposed as a unique arrest point that may be in G0 phase; however, generally believed R-point locates within G1 phase. Thus, up to now, there is no solid experimental evidence that supports the existence of R-point in asynchronous primary normal cells. In this study, we used freshly purified peripheral human blood lymphocytes, as asynchronous primary normal cells, to confirm the existence of restriction point in G1 not G0 phase. Our findings may help uncover the mystery of the deregulation of cell cycle progression in malignant tumors. © 2013 International Society for Advancement of Cytometry

The restriction point in the G1 phase of the mammalian cell cycle is a well-known and widely accepted control element regulating cell growth and division cycle [1-3]. Pardee applied cell synchronization in long-term cultured cell lines and defined the restriction point (R-point) as a unique point in the cell cycle at which starved cells become arrested [1] and further made a speculation that R-point was lost in cancer cells, leading to their continued cycling under conditions insufficient for normal cell proliferation [2]. However, the evidence of R-point in normal unperturbed cells (i.e., asynchronous cells) is still lacking, such that some researchers still question its potential role in regulating cell growth [4-7]. In this study, we conducted the shifts from serum-containing to serum-deprived medium at different times in normal asynchronous human peripheral blood lymphocytes (PBLs) and determined the expression of cyclin E and detected DNA synthesis in the cells and found that freshly purified PBLs, which were cultured in serum-containing medium for 12 h and then shifted to the serum-free medium for a total of 48 h, although expressing cyclin E and Ki-67 failed to initiate DNA synthesis; however, the cells, which were cultured in complete medium for 18 h and shifted to serum-free medium for a total of 48 h, were able to enter into the S phase and complete whole cell cycle. Thus, we have provided the definitive evidence to support the existence of the R-point in G1 not G0 phase in asynchronous human PBLs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Cells and Materials

Human PBLs, obtained from healthy volunteers by venipuncture, were isolated by density gradient centrifugation as described [8, 9]. As human material was used for this study, we have got approval from the Ethical Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. In this study, ∼30 healthy donors provided PBLs. The cells were washed twice with buffered saline, resuspended at a density of 106 cells/mL in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics and cultured in the presence of phytohemagglutinin (PHA; Gibco, Grand Island, NY) as described [10]. The acute lymphocyte leukemia cell line MOLT-4 (ATCC, Manassas, VA) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin,100 μg/mL streptomycin, and 2 mM L-glutamine. All media, supplements, and sera were obtained from Gibco (Grand Island, NY). 5-bromo-2-deoxyuridine (BrdU) was purchased from Sigma (Cat# b5002; Sigma Chemical). The cultures were passaged by dilution to a cell concentration of 2−8 × 105/mL to maintain asynchronous and exponential growth, unless otherwise indicated. Antibodies used in this study included mouse mAb to Ki-67 (Cat#556003; clone B56;, BD Biosciences, San Jose, CA), mouse mAb to cyclin E (Cat#51-1459GR; clone HE12; BD Biosciences), mouse mAb to P21 (Cat#556430; clone SX118; BD Biosciences), IgG (purified mouse IgG1k isotype control; Cat#554121; BD Biosciences), mouse Ab to BrdU (Cat#317902; clone: MoBU-1; BioLegend, San Diego, CA), goat anti-mouse FITC (Cat# F0479; Dako, Denmark), FITC-Annexin V/PI kit (KeyGEN Biotech, Nanjing, China).

Cyclin E/DNA Multiparameter Flow Cytometry Assay

The cells harvested were fixed in ice cold 80% ethanol at −20°C for at least 24 h. Following overnight fixation, cells were washed in PBS and permeabilized with 0.5% Triton-X 100 in PBS for 5 min on ice. After centrifugation, the cells were incubated overnight in the presence of primary antibody to cyclin E. Cells were then rinsed and incubated with the secondary FITC-conjugated antibody for 30 min. Then, cells were resuspended in RNase and PI solution (50 μg/mL propidium iodide, 50 μg/mL RNase) and incubated at room temperature for 30 min, as described previously [11-14]. Cellular fluorescence was measured using the FACSVantage flow cytometer.

Ki-67 Staining Detection

The harvested cells were fixed in cold 70–80% ethanol at −20°C for at least 2 h. Then, the fixed cells were washed twice by PBS and permeabilized with 0.5% Triton-X 100 in PBS for 5 min on ice. After centrifugation, the cells were incubated overnight or at least 2 h at RT in the presence of primary antibody to Ki67. Cells were then rinsed and incubated with the secondary FITC-conjugated antibody for 30 min at RT in the dark. Then, cells were resuspended in RNase and PI solution (50 μg/mL propidium iodide, 50 μg/mL RNase) and incubated at RT for 30 min, as described previously [15]. Cellular fluorescence was measured using the FACSVantage flow cytometer.

P21/DNA Multiparameter Flow Cytometry Assay

The harvested cells were fixed in cold 70–80% ethanol at −20°C for at least 2 h. Then, the fixed cells were washed twice by PBS and permeabilized with 0.5% Triton-X100 in PBS for 5 min on ice. After centrifugation, the cells were incubated overnight or at least 2 h at RT in the presence of primary antibody to P21. Cells were then rinsed and incubated with the secondary FITC-conjugated antibody for 30 min at RT in the dark. Then, cells were resuspended in RNase and PI solution (50 μg/mL propidium iodide, 50 μg/mL RNase) and incubated at RT for 30 min. Cellular fluorescence was measured using the FACSVantage flow cytometer.

BrdU Assay

BrdU at a final concentration of 20 μM was introduced into medium 1 h before cell counting in cell proliferation assay. After counting, cells were harvested and fixed in ice cold 80% ethanol, kept in −20°C overnight. The fixed cells were washed in PBS, resuspended in 1 mL of 1.5M HC1 at 20°C for 40 min, washed twice with 5 mL of PBS, and resuspended for 1 h in PBS solution containing 0.5% Tween-20 (Sigma Chemical), 0.5% BSA, and a 1:200 dilution of monoclonal anti-BrdU antibody. Cells were then rinsed and incubated with the secondary FITC-conjugated antibody for 30 min. They were then resuspended in RNase and PI solution (50 μg/mL propidium iodide, 50 μg/mL RNase) and incubated at room temperature for 30 min, as described previously [16]. Cellular fluorescence was measured using the FACSVantage flow cytometer.

Annexin V-FITC/PI Detection

Aliquots of freshly collected cells suspended in PBS were centrifuged (200g, 5 min) and resuspended in binding buffer, pH 7.4, containing 10 mM Hepes (Sigma), 140 mM NaCl, and 2.5 mM CaCl2 to have approximately 106 cells/mL. Five microliters of FITC-Annexin V and 5 μL of PI at a concentration of 50 μg/mL in PBS were then added, and the cells were incubated for 20 min at room temperature in the dark, as described previously [17-19].

The Shifts From Serum-Containing to Serum-Deprived or Low-Serum Medium in PBLs

Freshly purified PBLs were cultured in complete medium (e.g., RPMI-10% FBS supplemented with 1% PHA). At different times such as 12, 18, and 24 h, the cells were collected respectively and shifted into serum-free RPMI medium or low-serum RPMI medium (e.g., RPMI-1% FBS), neither of them containing PHA, and were then cultured for a total of 48 h. At the endpoint, the cells were collected and analyzed for expression of cyclin E and/or P21 and/or Ki-67 staining and/or BrdU assay as described above.

Serum Deprivation in Exponentially Growing Molt-4 Cells and Lymphocytes

Exponentially growing Molt-4 cells and lymphocytes were collected, shifted to serum-free medium, cultured, and harvested at different time points. At the endpoint, the cells were collected and analyzed for expression of cyclin E and/or Ki-67 staining and/or BrdU incorporation assay as described earlier.

Flow Cytometry Instrument and Data Analysis

Flow cytometry measurements were done by FACS Vantage (Becton Dickinson) equipped with 488 nm laser. We used the filters 530/30 for FITC and 585/42 for PI in our study. Cyclin E/DNA, Ki-67/DNA, P21/DNA, BrdU/DNA, and Annexin V/DNA multiparameter analysis by CELLQuest software.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Serum Deprivation of Exponentially Growing PBLs and MOLT-4 Cells Failed to Completely Synchronize the Cells Into the Quiescent State

In order to define the R-point in normal cells, not in long-term cultured cell lines, we cultured PBLs in RPMI-10% FBS medium supplemented with PHA for 48 h, and then, IL-2 was administered into the medium. After several subcultures, exponentially growing lymphocytes were obtained (Fig. 1A). Serum starvation is one of well-accepted methods in defining R-point to synchronize the exponentially growing cells into a quiescent state, often referred to as G0 phase. Thus, we first conducted serum deprivation in exponentially growing lymphocytes for 24 and 48 h, respectively; however, many PBLs were found to undergo apoptosis as measured by Annexin V/PI analysis (Fig. 1C and Supporting Information Fig. S1A). In order to confirm whether surviving PBLs were synchronized into a quiescent state, regardless of high apoptotic rate, we then used flow cytometry to analyze the expression of cyclin E and Ki-67 together with cell cycle distribution. Unexpectedly, the percentage of the cells in S and G2/M phase as determined by DNA content significantly increased after serum deprivation for 24 or 48 h (Figs. 1A and 1B, P < 0.001), although the expression peak of cyclin E (Fig. 1A) and percentage of G1 phase cells expressing cyclin E (i.e., G1-cyclin E+ cells) significantly decreased (Fig. 1B), and furthermore, many PBLs were positive for Ki-67 staining once deprived of serum for 24 or 48 h (Fig. 1D and Supporting Information Fig. S1B), which is generally used as an index for proliferation [20, 21]. These results suggest that serum deprivation, while promotes exponentially growing PBLs to undergo apoptosis, is unable to synchronize the cells into a quiescent state.

image

Figure 1. Proliferative and apoptotic status in exponentially growing human PBLs upon serum deprivation. A: Representative dot-plot distribution of cyclin E and DNA content in exponentially growing PBLs depleted of serum for 0, 24, and 48 h. The insets in each figure were their own IgG controls, and the broken line represents the level of the isotypic control (IgG). B: Summarization of the data originated from the above experiments into the bar graph (mean ± SD, 0 h vs. 24 h and 0 h vs. 48 h; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001); the experiments were repeated at least three times. G1-cyclin E, the PBLs with G0/G1 DNA content not expressing cyclin E, G1-cyclin E+, the PBLs with G0/G1 DNA content expressing cyclin E, S, the PBLs in S phase, G2/M, the PBLs in G2/M phase. C: Percentage of apoptosis measured by Annexin V/PI multiparameter flow cytometry in exponentially growing PBLs once deprived of serum (mean ± SD, ***P ≤ 0.001). Serum-containing, the PBLs cultured in complete media supplemented with PHA; serum-free, the PBLs cultured in serum-free medium in the absence of PHA. D: Percentage of surviving PBLs positive for Ki-67 staining measured by Ki-67/PI multiparameter flow cytometry in exponentially growing the cells once deprived of serum (mean ± SD, *P ≤ 0.05; ***P ≤ 0.001). Serum-containing, PBLs cultured in complete media; serum-free, PBLs cultured in serum-free media.

Download figure to PowerPoint

Because R-point was speculated to be lost in malignant cells, we then carried out parallel experiments in exponentially growing MOLT-4 cells. In contrast to PBLs, the total percentage of the cells in G0/G1 phase (including G1-cyclin E and G1-cyclin E+ in Figs. 2A and 2B), which are supposed to contain quiescent cells, dramatically increased after serum deprivation for 24 and 48 h (Figs. 2A and 2B). However, many cells were still in S and G2/M phase (Figs. 2A and 2B), and furthermore, the percentage of G1 phase cells expressing cyclin E (i.e., G1-cyclin E+ cells), which are considered as proliferating cells, increased upon serum deprivation (Fig. 2B). We further extended the cultures to 72 and 96 h, high percentage of proliferating cells, such as G1-cyclin E expressing cells, S and G2/M phase cells were still detected (Fig. 2B and Supporting Information Fig. S2A). In addition, the surviving cells, not apoptotic cells upon serum deprivation, were further confirmed by Ki-67 staining, as shown in Figure 2C and Supporting Information Figure S2B, ∼50% of the cells were positive for Ki-67 staining once deprived of serum for 96 h, although the apoptotic rate gradually increased over time (Fig. 2D and Supporting Information Fig. S2C). These results suggest that serum deprivation is unable to completely synchronize the cells into a quiescent state in Molt-4, a malignant cell line.

image

Figure 2. Proliferative and apoptotic status in exponentially growing acute lymphocyte leukemia cell line Molt-4 upon serum deprivation. A: Representative dot-plot distribution of cyclin E and DNA content in exponentially growing Molt-4 cells depleted of serum for 0, 24, and 48 h. The insets in each figure were their own IgG controls, and the broken line represents the level of the isotypic control (IgG). B: Summarization of the data, which originated from the above experiments together with those from prolong cultures (i.e., 72 and 96 h) in serum-free media, into the bar graph (mean ± SD, 0 h vs. 24 h, 0 h vs. 48 h, 0 h vs. 72 h, and 0 h vs. 96 h; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001); the experiments were repeated at least three times. G1-cyclin E, Molt-4 cells with G0/G1 DNA content not expressing cyclin E, G1-cyclin E+, Molt-4 cells with G0/G1 DNA content expressing cyclin E, S, Molt-4 cells in S phase, G2/M, Molt-4 cells in G2/M phase. C: Percentage of apoptosis measured by Annexin V/PI multiparameter flow cytometry in exponentially growing Molt-4 cells once deprived of serum [mean ± SD, serum-containing, vs. serum-free at different time points (i.e., 24, 48, 72, and 96 h), *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.]. Serum-containing, Molt-4 cells cultured in complete media; serum-free, Molt-4 cells cultured in serum-free media in the absence of PHA. D: Percentage of surviving Molt-4 cells positive for Ki-67 staining measured by Ki-67/PI multiparameter flow cytometry in exponentially growing Molt-4 cells once deprived of serum [mean ± SD, serum-containing vs. serum-free at different time points (i.e., 24, 48, 72, and 96 h), ***P ≤ 0.001]. Serum-containing, Molt-4 cells cultured in complete media; serum-free, Molt-4 cells cultured in serum-free media.

Download figure to PowerPoint

Taken together, our findings indicate that serum deprivation is unable to fully synchronize PBLs and Molt-4 cells into a quiescent state, and moreover, almost similar patterns were observed after serum deprivation, although R-point is considered to be lost in malignant cells but exist in normal cells.

The PBLs Were Capable of Progressing to the Cycle in RPMI Medium Containing Low Serum

To avoid using cell synchronization, we utilized freshly purified PBLs obtained from healthy volunteers. Freshly purified PBLs are considered to be in a quiescent state as no expression of cyclin D and cyclin E [22, 23]. The freshly purified PBLs were cultured in RPMI-10% FBS supplemented with 1% PHA, at different time points (e.g., 0, 12, 18, 24, and 48 h), the cells were harvested to determine the expression of cyclin E and cell cycle distribution. As shown in Figures 3A and 3B, consistent with previous studies [23, 24], the cells did not initiate DNA synthesis but expressed cyclin E upon stimulation by PHA for 24 h, suggesting that the cells remained in G1 phase, but some of the cells entered into S phase and G2/M phase upon stimulation by PHA for 48 h (Figs. 3A and 3B). The R-point was generally believed to be in late G1 phase of cell cycle [1, 2], suggesting that, in freshly purified PBLs, R-point might locate somewhere upon stimulation by PHA for more than 12 h. In addition, serum starvation (i.e., RPMI medium-containing 1% FBS) instead of serum deprivation (i.e., RPMI medium-containing no serum) is one of mostly applied blocking approaches. Thus, the freshly purified PBLs were cultured in RPMI-10% FBS supplemented with 1% PHA, at different time points (e.g., 12, 18, and 24 h), the cells were shifted into RPMI-1% FBS and cultured for a total of 48 h, and finally, expression of cyclin E and cell cycle distribution were analyzed by flow cytometry (Figs. 4A and 4B); ∼13–24% of S phase cells were found in all sets of shift experiment (Fig. 4B), suggesting that serum starvation (i.e., low serum) is able to drive the freshly purified PBLs into proliferating cycle. These results indicate that, in freshly purified PBLs, low-serum shift (i.e., serum starvation) is unable to define R-point.

image

Figure 3. Expression of cyclin E in freshly purified PBLs stimulated by PHA. A: Freshly purified PBLs were first cultured in RPMI-10% FBS supplemented with 1% PHA, at 0, 12, 18, 24, and 48 h, the cells were harvested, respectively, and then distributions of DNA content and expression of cyclin E in the cells were determined and analyzed by flow cytometry. The insets in each figure were their own IgG controls, and the broken line represents the level of the isotypic control (IgG). B: Summarization of the data originated from the above experiments into the bar graph (mean ± SD, 0 h vs. 12 h, 0 h vs. 18 h, 0 h vs. 24 h, and 0 h vs. 48 h, ***P ≤ 0.001); the experiments were repeated at least three times. G1-cyclin E, the PBLs with G0/G1 DNA content not expressing cyclin E, G1-cyclin E+, the PBLs with G0/G1 DNA content expressing cyclin E, S, the PBLs in S phase, G2/M, the PBLs in G2/M phase.

Download figure to PowerPoint

image

Figure 4. Expression of cyclin E and cell cycle distribution in freshly purified PBLs in the shifts from complete media to low serum (i.e., 1% serum) media. A: Freshly purified PBLs were cultured in complete media in the presence of 1% PHA for 12, 18, or 24 h, and then the cells were respectively shifted to 1% serum media for a total of 48 h; finally, cyclin E expression and DNA content in the cells were detected and analyzed by flow cytometry. The insets in each figure were their own IgG controls, and the broken line represents the level of the isotypic control (IgG). B: Summarization of the data originated from the above experiments into the bar graph (mean ± SD, 12 h vs. 18 h and 12 h vs. 24 h, *P ≤ 0.05; ***P ≤ 0.001); the experiments were repeated at least three times. G1-cyclin E, the PBLs with G0/G1 DNA content not expressing cyclin E, G1-cyclin E+, the PBLs with G0/G1 DNA content expressing cyclin E, S, the PBLs in S phase, G2/M, the PBLs in G2/M phase.

Download figure to PowerPoint

The Freshly Purified PBLs, Which Were Cultured in RPMI-10% FBS Supplemented With PHA for 18 and 24 H and Then Shifted to Serum-Free Medium, Were Able to Initiate DNA Synthesis and Complete the Cell Cycle

Next, the freshly purified PBLs were cultured in complete medium (i.e., RPMI-10% FBS supplemented with 1% PHA), at different time points (e.g., 12, 18, and 24 h), the cells were shifted to serum-free media instead of low-serum media and then cultured for a total of 48 h, and finally, expression of cyclin E and Ki-67 together with cell cycle distribution were analyzed by flow cytometry (Figs. 5A–5C and Supporting Information Fig. S3A). As shown in Figure 5B, ∼19% of PBLs initiated DNA synthesis and ∼2% of the cells were in G2/M phase when the cells were cultured in 10% serum in presence of PHA for 18 h before switching to serum free medium for a total of 48 h. Furthermore, more PBLs (∼23%) initiated DNA synthesis and ∼3.7% of the cells were in G2/M phase when the cells were shifted into serum-free medium at 24 h (Figs. 5A and 5B). By contrast, the PBLs grown in complete medium for 12 h before shifting to serum-free medium for a total of 48 h, although ∼75% of G1 phase cells expressing cyclin E, the cells failed to enter into S phase (Figs. 5A and 5B), and moreover, Ki-67 staining analysis (Fig. 5C and Supporting Information Fig. S3A) has also shown that ∼7.5% of cells with G0/G1 DNA content were positive for Ki-67 staining although the cells failed to proceed into S phase, suggesting that some, if not all, of cells progress into proliferating cycle. In order to further confirm DNA synthesis, we thus performed BrdU incorporation assay in the PBLs deprived of serum. As shown in Figures 5D and 5E, consistent with the results of cyclin E/DNA analysis, the PBLs shifted to serum-free medium at 18 h were detected with the synthesis of DNA at 48 h, while the PBLs shifted to serum-free medium at 12 h were not detected DNA synthesis. To explore the underlying mechanism(s) by which the G0/G1 phase PBLs, which were shifted to serum-free medium at 12 h, although expressing cyclin E at relative high levels, failed to enter into S phase, we then detected expression of P21 in the PBLs. P21, a CDK inhibitor, is capable of inhibiting the activity of CDK2 and thus prevents the cells from entering into S phase. As shown in Figure 5F and Supporting Information Figure S3B, indeed, P21 expressed more highly in the PBLs shifted to serum-free medium at 12 h when compared with 18 and 24 h. These results indicate that R-point exists in the PBLs and its location is within G1 phase, and P21 may be involved in maintenance of the R-point in the PBLs.

image

Figure 5. Proliferative status, DNA synthesis, and cell cycle distribution in freshly purified PBLs in the shifts from complete media to serum-free media. A: Freshly purified PBLs were cultured in complete media in the presence of 1% PHA for 12, 18, or 24 h, and then the cells were respectively shifted to serum-free media for a total of 48 h; finally, cyclin E expression and DNA content in the cells were determined by flow cytometry. The insets in each figure were their own IgG controls, and the broken line represents the level of the isotypic control (IgG). B: Summarization of the data originated from the above experiments into the bar graph (mean ± SD, 12 h vs. 18 h and 12 h vs. 24 h, *P ≤ 0.05; **P ≤ 0.01); the experiments were repeated at least three times. G1-cyclin E, the PBLs with G0/G1 DNA content not expressing cyclin E, G1-cyclin E+, the PBLs with G0/G1 DNA content expressing cyclin E, S, the PBLs in S phase, G2/M, the PBLs in G2/M phase. C: Percentage of PBLs positive for Ki-67 staining measured by Ki-67/PI multiparameter flow cytometry after shifts from complete media containing PHA to serum-free media (mean ± SD, 12 h vs. 18 h and 12 h vs. 24 h, **P ≤ 0.01; ***P ≤ 0.001). D: The DNA synthesis in PBLs was determined by BrdU incorporation assay after shifts from complete media containing PHA to serum-free media. Shown are representative dot-plot distributions of BrdU incorporation and DNA content by flow cytometer in PBLs after shifts from complete media containing PHA to serum-free media. E: Summarization of the data originated from the above experiments into the bar graph (mean ± SD, 12 h vs. 18 h and 12 h vs. 24 h, ***P ≤ 0.001); the experiments were repeated at least three times. F: The expression of P21 in PBLs was detected by P21/DNA multiparameter flow cytometry after shifts from complete media containing PHA to serum-free media, and the G0/G1 phase PBLs positive for P21 in whole G0/G1 phase cells are counted (mean ± SD, 12 h vs. 18 h and 12 h vs. 24 h, *P ≤ 0.05; **P ≤ 0.01); the experiments were repeated at least three times.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Although the R-point was proposed for several decades, there exist several shortcomings such that some researchers still question on it [4-7].

First, the long-term cultured cell lines rather than primary cells were used. The processes of culturing cell lines in vitro are considered as a sort of “clonal evolution” [25, 26]. Thus, the evidence generated from the long-term cultured cell lines may only represent the subgroups of cells that survived in the process of long term passaging in vitro. In our study, human PBLs were freshly obtained from healthy individuals and then used in experiments without the long-term culture.

Second, utilizing starvation/release synchronization experiments, R-point was originally proposed as a unique arrest point in the quiescent state. In fact, the unique arrest point is different from generally believed R-point, since the unique arrest point may be in G0 phase but generally believed R-point shall be within G1 phase, which is one of reasons why some researchers still question [4-7]. In our study, the freshly purified PBLs were first cultured in complete medium in presence of PHA for various time lengths and then shifted to suboptimal condition (i.e., serum-free medium). We found that the PBLs, which were cultured in complete medium supplemented with PHA for at least 18 h and then shifted into serum-free medium for a total of 48 h, were capable of initiating DNA synthesis and completing the cell cycle (Fig. 5); however, the PBLs, which were shifted to serum-free medium at 12 h, failed to initiate DNA synthesis and complete whole cell cycle although expressed cyclin E and were positive for Ki-67 staining, indicating that such cells, at least part of them have already entered into proliferating cell cycle [22, 23, 27]. Our results clearly demonstrate that R-point exists in PBLs; in addition, it locates within G1 phase not in G0 phase, and it locates in PBLs stimulated by PHA for between 12 and 18 h.

Third, in the experiments by Pardee, thymidine incorporation as DNA synthesis was applied, which can detect the DNA synthesis in subgroups of cells rather than single cells. In this study, flow cytometry was utilized to analyze the expression of cyclin E and DNA synthesis, which clearly demonstrates whether the individual cells progress into proliferating cycle and enter into S phase.

Fourth, cell synchronization in the cycle was applied, which introduces severe bias due to induction of growth imbalance between DNA synthesis and protein expression [28-30]. In the current study, we also tried to use the exponentially growing PBLs and synchronize them into quiescent state. Our results indicated that ∼78.1% and ∼71.2% of PBLs were in G0/G1 phase upon serum deprivation for 24 and 48 h, respectively. However, majority of PBLs in G0/G1 phase still expressed cyclin E (Figs. 1A and 1B) and were positive for Ki-67 staining (Fig. 1D and Supporting Information Fig. S1B), suggesting that the majority of cells with G0/G1 phase amount of DNA located de facto in G1 phase, and moreover, serum starvation failed to completely synchronize proliferating cells into a quiescent state. Freshly purified PBLs were shown to be in a quiescent state [31], in our study, we also found that no cyclin E expressed in freshly purified PBLs (Figs. 3A and 3B). So, we have chosen freshly purified PBLs as asynchronous normal cells in defining R-point.

The release of E2F initiation factors from phosphorylated Rb is the critical step to initiate the DNA synthesis, and Rb is phosphorylated sequentially by cyclin D/CDK4 (or CDK6) and cyclin E/CDK2. However, the activity of CDK is inhibited by CDK inhibitors [32]. Some studies have shown that serum depletion increases the expression of p21, which thus inhibits the activity of CDK2 [33]. In this study, we found that the freshly purified PBLs shifted to serum-free medium at 12 h, although expression of cyclin E increasing, were unable to enter into S phase (Figs. 5A and 5B). It may be interpreted that the G1 phase cells expressing elevated levels of cyclin E failed to enter into S phase due to increased expression of p21, an inhibitor of CDK2 (Fig. 5F and Supporting Information Fig. S3B).

Together, we have provided definitive evidence that the R-point exists in normal human unperturbed cells, and serum deprivation (i.e., the cells are cultured in serum-free medium) cannot synchronize the cells into quiescent state. However, whether the regulation of restriction point also exists in cycling cells (e.g., exponentially growing cells) needs to be addressed. Future work that permits sorted cells (i.e., G2/M phase cells) should allow us to provide direct evidence for the question.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
cytoa22341-sup-0001-suppinfo.docx1725KSupplementary Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.