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Keywords:

  • WB-F344 cells;
  • cell-cycle checkpoints;
  • gamma radiation;
  • absorbed dose;
  • cell growth;
  • G2/M-delay;
  • flow cytometry

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

Background

Apparently normal rat liver epithelial cells (WB-F344) have been widely used in studies pertaining to carcinogenesis. Ionizing radiation, a well known carcinogen, is known to perturb cell-cycle progression in a dose-dependent manner, thereby causing delay in cell proliferation. However, for WB-F344 cells, there is a paucity of such data, which are of substantial importance in understanding their radiation response. Here, the distribution of phases in the cell-cycle and the proliferation ability of WB-F344 cells are characterized at various time points after the cells have been irradiated with different doses of γ-rays.

Methods

After WB-F344 cells reached 100% confluence, they were trypsinized and suspended at 3.5 × 105 cells/ml in culture medium. Cells were irradiated in suspension with 137Cs γ-rays at doses from 1–10 Gy. After irradiation, 1 × 105 cells were plated into 60 × 15-mm culture dishes and incubated at 37°C, with 2% CO2 and 98% air. At 12, 24, 36, 48, and 60 h postirradiation, cells were harvested, counted, and subjected to flow cytometric cell-cycle analysis.

Results

Growth curves of WB-F344 cells irradiated with γ-rays started to separate at 36 h postirradiation. By 60 h postirradiation, the growth curves for each of the 10 absorbed doses were distinctly separated. Drastic redistributions of control and irradiated cells within G0/G1-, S-, and G2/M-phases of the cell cycle were observed during the first 36 h of cell growth. At each time point postirradiation, cell-cycle phase profiles of irradiated cells were altered in a dose-dependent manner. In general, there was a strong correlation between the percentage of G2/M-phase cells and absorbed dose, with the exception of 24 h postirradiation. The percentage of G2/M-phase cells increased as a function of time postirradiation, suggestive of delays in the passage of cells through the G2 cell-cycle checkpoint.

Conclusions

This work provides a general description of cell cycle redistribution and repopulation kinetics of WB-F344 cells at various times postirradiation of quiescent cells that were subsequently allowed to proliferate. In general, growth inhibition and delays in progression through G2/M-phase correlated well with radiation dose. These data should be of considerable significance in the design of experiments that examine the radiation response of these cells. © 2004 Wiley-Liss, Inc.

The normal rat liver epithelial cell line, WB-F344, is used widely in the study of carcinogenesis. Several reports from the laboratory of Dr. J.E. Trosko (Michigan State University, East Lansing, MI), including Oh et al. (1), Jou et al. (2), Esinduy et al. (3), Rae et al. (4), and Trosko and Ruch (5), have reported on extensive studies of the capacity of these cells to communicate with one another via gap junctional intercellular communication (GJIC) and the impact of GJIC on carcinogenesis. Kaufmann et al. (6) have examined the influence of the number of in vitro passages (generations) of WB-F344 cells on their G1 and G2 checkpoint function following exposure to ionizing radiation (IR). This cell line has also been used to examine effects imparted by irradiated cells on their unirradiated neighbors, also known as bystander cells. Specifically, Azzam et al. (7) used these cells to provide direct evidence for the participation of GJIC in the transmission of damage signals from α-particles irradiated to bystander cells. Using WB-F344 cells, radiation-induced bystander effects have also been observed by Gerashchenko and Howell (8, 9). It was found that WB-F344 cells that were irradiated with γ-rays stimulated proliferation in unirradiated bystander cells (8). Close proximity between the irradiated and bystander cells was a prerequisite for the proliferative response of the bystanders (9).

While WB-F344 cells have been used in a variety of studies related to radiation carcinogenesis, there is a paucity of data that characterizes the impact of ionizing radiation on their proliferation and cell-cycle progression. Irradiated cells are commonly characterized by G1/S- and G2/M-delays in the cell cycle, among which G2/M-delay is most frequently observed. These delays are thought to provide cells with additional time to repair damaged DNA before further cell-cycle progression. It has been well documented that G2/M-delay and its duration depend on the type of cells, type of IR, absorbed dose, dose rate, and cell-cycle stage during which cells were irradiated (10–22). Cell-cycle progression is controlled by a family of checkpoint genes modulated in response to IR and other DNA damaging agents (6, 23, 24). In this work, we used flow cytometry (FCM) and cell counting to monitor WB-F344 cell-cycle progression and growth after they were irradiated at plateau phase with various doses of γ-rays (1–10 Gy), and were subsequently subcultured at low density in fresh growth medium. Cells to be subjected to FCM cell-cycle analysis and cell counting were collected over a period of 60 h postirradiation at 12-h intervals. A total of 11 dose points (including zero dose point) and six time points (including zero time point) were used in this study.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

Cell Line

The rat liver epithelial cell line WB-F344 (25) was generously provided by Dr. J.E. Trosko (Michigan State University, East Lansing, MI). Cells at passage 17, maintained in liquid nitrogen, were thawed and cultured in D-medium (formula No. 78-5470EF, Gibco-BRL, Grand Island, NY) in a 37°C humidified incubator containing 2% CO2, 98% air. The medium was prepared by dissolving 8.97 gg of D-medium powder in 400 ml of deionized H2O. This was supplemented with 0.835 gg/L of NaCl, 1.0 gg/L of glucose, 1 mmol of Na pyruvate, and 10 mmol of HEPES buffer (Gibco-BRL), and 1 mol NaOH was added drop-wise until the medium reached a pH of 6.5. In addition, 1 gg/L of NaHCO3 was added (final pH of 7.17) with deionized H2O to a total volume of 1 L, and the medium was filtered. Then 25 μg/ml of gentamicin and 5% fetal bovine serum (HyClone, Logan, UT) were added. Cells were subcultured two times per week. Experiments were carried out with cells at passage 20–25.

Irradiation

Confluent cell monolayers (density-inhibited culture) cultured in 75-cm2 tissue culture flasks (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) were washed twice with 10 ml of Dulbecco's phosphate buffered saline (DPBS; Gibco-BRL), trypsinized (0.05% trypsin and 0.53 mmol ethylene-diamine-tetraacetic acid; Gibco-BRL), and suspended at 3.5 × 105 cells/ml in growth medium. Aliquots of cell suspension (2 ml) were transferred to 11 14-ml polypropylene round-bottom tubes (Falcon; Becton Dickinson Labware) and irradiated at room temperature with 137Cs γ-rays at doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 Gy. The γ-rays were delivered by a J. L. Shepherd Mark I irradiator (San Fernando, CA) operating at a dose rate of 3.71 Gy/min. Irradiated cells (1 × 105 cells) were then plated into 60 × 15-mm (P60) dishes (Falcon; Becton Dickinson Labware) containing 4 ml of D-medium. As a control, 1 × 105 unirradiated cells (0 Gy dose point) were plated into P60 dishes (Fig. 1).

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Figure 1. Experimental protocol for characterization of cell-cycle progression and growth of irradiated WB-F344 cells. Suspensions of density-inhibited cells (7.0 × 105 cells in 2 ml of D-medium) were irradiated at room temperature with 137Cs γ-rays at doses ranging from 1–10 Gy. After irradiation, 1 × 105 cells were plated into P60 dishes containing 4 ml of D-medium. As a control, 1 × 105 unirradiated cells (0 Gy) were plated into P60 dishes, which are illustrated separately. At 12, 24, 36, 48, and 60 h after irradiation, cells were trypsinized, harvested, washed with ice-cold DPBS, and prepared for Coulter cell counting and FCM cell-cycle analysis, as described in Materials and Methods. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com].

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Cell Counting

At 12, 24, 36, 48, and 60 h after irradiation, cells were trypsinized, resuspended in growth medium, transferred to 5-ml polystyrene round-bottom tubes (Falcon; Becton Dickinson Labware), and centrifuged at 4°C for 5 min. After aspiration of the supernatant, the cell pellets were suspended in 2 ml of ice-cold DPBS and 150-μl aliquots of the cell suspensions were transferred to Dilu-Vials® (Fisher Scientific, Pittsburgh, PA) containing 20 ml of Coulter Balanced Electrolyte Solution - Isoton® II (Coulter, Miami, FL). Cell counting was performed on a Model ZM Coulter Counter (Coulter Electronics, Hialeah, FL).

Cellular Fixation and DNA Staining

The remaining cells were centrifuged and suspended in 100 μl of DPBS at 4°C. A total of 900 μl of absolute methanol (−70°C) was added drop-wise to the cell suspension while vortexing, followed by storage of the cells at −20°C. One day before FCM cell-cycle analysis, the stored methanol-fixed cell samples were centrifuged and washed twice with DPBS. A total of 300 μl of 50 μg/ml of propidium iodide (PI; Sigma, St. Louis, MO) containing 0.1% RNAse A (Sigma) in DPBS was then added to the cell pellets. Cells were stained overnight at 4°C.

FCM Cell-Cycle Analysis

PI-stained cells were analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA), equipped with a 15-mW argon-ion laser (488 nm). DNA-bound PI fluorescence was measured in the orange-red fluorescence channel (FL2) through a 585/42-nm bandpass filter with linear amplification. Analysis of cell-cycle distribution profiles was performed with ModFit LT software (Verity Software House, Topsham, ME). At least 15,000 events were collected for each sample. Cells were gated on FL2-Area versus FL2-Width plots to exclude aggregates and debris from analysis.

Colony Formation Assay

Density-inhibited cultures of WB-F344 cells were irradiated with 137Cs γ-rays at doses of 1.5, 3, 6, 9, and 12 Gy at the conditions described above. After irradiation, cells were serially diluted, and 1 ml of the appropriate dilutions (approximately 100 cells for control tubes) was seeded in triplicate into P60 dishes. The dishes were then placed in an incubator at 37°C with 98% air 2% CO2. After one week, the dishes were removed from the incubator, and the resulting colonies were washed with normal saline, fixed with absolute ethanol, stained with 0.05% crystal violet, and finally counted. The established criterion of counting colonies >50 cells was followed. The surviving fraction was determined compared to that for unirradiated control cells.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

Growth Curves

Growth curves of cells that received various doses of γ-rays are presented in Figure 2A. As expected, control cells (0 Gy) began to proliferate faster at an earlier time after plating than irradiated cells (i.e., on the 24-h time point of cell growth; Fig. 2A). It is apparent that the growth curves for cells irradiated with different doses begin to clearly separate in a dose-dependent manner by the 36-h time point, suggestive of sensitive checkpoint response (i.e., delays induced at low doses) (Fig. 2B). However, substantial differences in the growth curves are not seen until 48 h postirradiation. Maximum separation was observed at 60 h postirradiation, when significant differences were noted between each of the dose points. In this case, doses as low as 1 Gy had a significant impact on growth. These observations are more clearly seen in Figure 2B, in which the cell number is plotted as a function of absorbed dose for the 12-, 24-, 36-, 48-, and 60-h time points. A linear dose-response was observed, with the slope being indicative of the degree to which absorbed dose altered the growth measured at that particular time point relative to controls. These alterations in measured growth relative to unirradiated controls are a consequence of the combined impact of transient delays and cell death caused by IR (see below).

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Figure 2. A: Growth kinetics of WB-F344 cells irradiated with γ-rays over a wide range of doses (1–10 Gy). Sham-irradiated cells (0 Gy) represent control. Cells were collected over a period of 60 h of culture postirradiation and subjected to Coulter counting under the conditions described in Materials and Methods. Separation of growth curves began 36 h postirradiation. B: Regression lines show the dependence of cell number on absorbed dose for the 12-, 24-, 36-, 48-, and 60-h cultures. The arrows show that the slopes of the regression lines for the 36-, 48-, and 60-h cultures increased progressively. As expected, cell populations exposed to lower doses of γ-rays proliferated faster than cell populations exposed to greater doses.

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Cell Survival Curve

Figure 3 shows the survival curve for WB-F344 cells exposed to various doses of γ-rays (1.5–12 Gy). A least-squares fit of the data to the linear quadratic model (S = exp(–αD–βD2)), where D is the radiation absorbed dose, yields values for α and β of 0.196 ± 0.045 Gy−1 and 0.0114 × 0.0037 Gy−2, respectively. The character of this curve indicates that the WB-F344 cell line is relatively radioresistant. Furthermore, the growth curves in Figure 2A suggest that heavily irradiated WB-F344 cells did not undergo rapid cell death.

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Figure 3. Survival of WB-F344 cells after irradiation of density-inhibited cultures with γ-rays. The curve represents a least-squares fit of the data to the linear-quadratic model. Representative standard deviations are indicated by the error bars.

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Distribution of Cells in Phases of the Cell-Cycle

The delayed expansion of cultures of irradiated cells observed in this study was caused by perturbations in their cell-cycle progression. As shown in Figure 4A (at t = 0 h), cells were irradiated under conditions where the vast majority of them (≈90%) were quiescent (G0/G1). The percentage of cells in G0/G1, S, and G2/M are given as a function of time postirradiation in Figure 4A–C. Dramatic changes in the cell-cycle distribution of the harvested populations occurred during first 36 h of cell growth. Figure 4D shows the percentage of cells in G2/M as a function of dose.

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Figure 4. Dose- and time-dependent distribution of WB-F344 cells in the phases of the cell-cycle. Plateau-phase cells were irradiated with γ-rays over a wide range of doses (1–10 Gy), plated at low density (1 × 105 cells) into P60 dishes containing 4 ml of D-medium, and incubated under the conditions described in Materials and Methods. Sham-irradiated cells (0 Gy) represent control. Cells were collected up to 60 h postirradiation, fixed with methanol, stained with PI, and subjected to FCM cell-cycle analysis. A: Percentages of G0/G1-phase cells. The arrows denote dose-dependent decrease in the percentage of G0/G1 cells at 12 h postirradiation and dose-dependent increase in the percentage of G0/G1 cells at 24 h postirradiation. B: Percentages of S-phase cells. C: Percentages of G2/M-phase cells. D: Linear correlation between percentages of G2/M-phase cells and absorbed dose.

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12-h cultures.

A strongly dose-dependent change in the percentage of G2/M-phase cells was observed at 12 h postirradiation (Fig. 4C). A concordant change in the percentage of G0/G1-phase cells was observed at this time point (Fig. 4A). Interestingly, there was a linear relationship between the percentage of cells in G2/M-phase and the absorbed dose, with the greatest slope at 12 h postirradiation (Fig. 4D). There was some decrease in the percentage of G2/M-phase cells in the control cell population (0 Gy) at the 12-h time point (0.8%) compared to the 0-h time point (3.6%). This can be explained by rapid passage of G2/M-phase cells, and some fraction of S-phase cells (e.g., late S-phase cells), to the next stage of the cell-cycle, upon plating the slowly proliferating plateau-phase control cells at low density in fresh medium. At dose points of 3 Gy and higher, the accumulation of G2/M-phase cells at 12 h postirradiation suggests that passage of cells through the G2 cell-cycle checkpoint may have been delayed. This a well known phenomenon caused by ionizing radiation that is dependent on cellular radiosensitivity (26).

The flow cytometric cell-cycle phase distribution profile of the control cells at the 12-h time point revealed some growth in the fraction of cells at the G1-S boundary (Fig. 5). This indicates that some fraction of quiescent cells started to synthesize DNA within 12 h after plating. A noticeable depletion of S-phase cells in the irradiated cell populations (≤3.9%), compared to those in the control cell population (5.6%), implies that transition of G1-phase cells to S-phase is delayed to some extent at the G1 cell-cycle checkpoint (Fig. 4B). A similar emptying of the S-phase compartment following irradiation of WB-F344 cells with 8 Gy of γ-rays was observed by Kaufmann et al. (6).

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Figure 5. Cell-cycle phase distribution profile of sham-irradiated (0 Gy) WB-F344 cells 12 h after replating quiescent density-inhibited cells. The arrow indicates the growth of the fraction of cells at the G1-S boundary.

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24-h cultures.

According to Figure 2A, the number of control cells at the 24-h time point is double the initial number. In contrast, the number of irradiated cells is somewhat less, although the absorbed dose does not seem to have much of an impact on the number of cells (Fig. 2B). A redistribution of cells within the different phases of the cell cycle occurred between the 12- and 36-h time points. For doses less than 6 Gy, there was an increase in the percentage of G2/M-phase cells 24 h postirradiation (Fig. 4C). The sharp increase in the percentage of G2/M in the control cells was indicative of rapid proliferation. In contrast, there was a drop in the percentage of G2/M-phase cells in cultures that received doses of more than 8 Gy. Almost no changes in the percentage of G2/M-phase cells were observed for cells that received doses from 6–8 Gy. Surprisingly, at the 24-h time point of cell growth, the percentage of G2/M-phase cells in cell populations irradiated with wide range of doses (1–10 Gy) was about the same (≈10%; Fig. 4D). This phenomenon may be explained by differences in transition times to the next phase of the cell cycle for cells in S- and G2/M-phases of the cell-cycle that may take place prior to the first cell division. The percentage of S-phase cells at this stage of cell growth may also depend in part on the G1-checkpoint function that was affected by irradiation of quiescent cells. Figure 4B shows that the percentages of S-phase cells in all cell populations (0–10 Gy) rose dramatically by the 24-h time point of cell growth (25.9–37.2%), compared to those on the 12-h time point (1.5–3.9%). This is expected because the cells were released from their quiescent density inhibited state at the time of irradiation to more sparsely populated log-phase growth conditions. Accordingly, the percentages of G0/G1-cells within this time interval decreased (from 82.3–94.6% to 54.9–62.9%; Fig. 4A). This indicates that after 12-h time point of cell growth, considerably more cells were involved in proliferation. The switch from a dose-dependent decrease in G0/G1 at 12 h postirradiation to a dose-dependent increase in G0/G1 at 24 h postirradiation suggests that newly divided cells continued to move toward their second division, causing a drop in this fraction (Fig. 4A), with concomitant enrichment of the S-phase cells (Fig. 4B).

36-h cultures.

By 36 h postirradiation, most of the irradiated cell populations had doubled (Fig 2A), although the number of cells present was only weakly dependent on the dose (Fig. 2B). The 36-h cultures were characterized by restoration of a dose-dependent distribution of the percentages of G2/M-phase cells (Fig. 4C). Moreover, as with all but the 24-h cultures, there was a linear correlation between absorbed dose and percentages of G2/M-phase cells (Fig. 4D). Some drop in the percentage of G2/M-cells in the control cell population at this time point (11.4%), compared to those of control cell population at the 24-h time point (13.7%), suggests that cells were passing through their second division, a fact showing that some degree of synchrony in the cell-cycle progression of control cells was still preserved (Fig. 4C). Unlike control cells, irradiated cells encountered delay in transition through the G2-checkpoint upon approaching their second division, causing a progressive increase in G2/M-phase cells (11.4–18.3%). Slow transition of irradiated cells through this cell-cycle checkpoint may have led to the deficiency in the supply of G0/G1-phase cells, and further deficiency in the supply of S-phase cells (dose-dependent decrease in the percentage of S-phase cells; Fig. 4B). G2/M-delay at this stage of cell growth may be responsible for initiating the separation of growth curves (Fig. 2A).

48- and 60-h cultures.

The irradiated cell populations showed continued slower growth rates at 48 and 60 h postirradiation as evidenced by the slopes of the curves in Fig. 2A. This is consistent with Figure 4C, which shows that G2/M-delay still persists in the 48- and 60-h cultures of irradiated cells. Similar to the 36-h cultures, these cultures have a linear correlation between absorbed dose and total number of cells (Fig. 2B) and percentage of G2/M-phase cells (Fig. 4D). Despite progressive increase in the total numbers of irradiated cell populations (Fig. 2A), the percentages of G2/M-phase cells in the 48- and 60-h cultures remained similar to those for the 36-h culture. This suggests that G2/M-delay may persist in the second division cycle, and even in the third cycle. This is supported by data on the frequency of radiation-induced chromosomal damage, which has been reported to decline during the first three postirradiation divisions (27, 28).

3-D Representation of G2/M-Delay

Figure 6A presents a 3-D representation of the radiation-induced G2/M-delay observed in this study. The percentages of G2/M-phase cells were plotted versus both absorbed dose and time postirradiation. The 3-D distribution of the percentages of G2/M-phase cells provides a facile means of visualizing major tendencies of the dose-dependence of the G2/M fraction in time. It also represents a unique topography that is characteristic for WB-F344 cells. It was clear that the increase in the percentage of G2/M-phase cells is dependent both on absorbed dose and time postirradiation. The general tendency of this increase is illustrated by the arrow in Figure 6B, which represents a projection of the 3-D plot in Figure 6A into the dose-time plane.

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Figure 6. A: Three-dimensional representation of G2/M-delay of WB-F344 cells that were subjected to irradiation with γ-rays over a wide range of doses (1–10 Gy). Sham-irradiated cells (0 Gy) represent control. The percentages of G2/M-phase cells were plotted versus absorbed dose and time elapsed after irradiation/plating of cells. B: Projection of the 3-D plot (A) into the dose–time plane. The arrow shows an increase in the percentage of G2/M-phase cells that correlates with absorbed dose and time elapsed after irradiation/plating of cells.

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In summary, the present data provide a general description of cell-cycle redistribution and repopulation kinetics of WB-F344 cells at various times postirradiation of quiescent density-inhibited cultures. These data should be of considerable use in the design of experiments that examine the radiation response of these cells, as well as in the design and interpretation of experiments that use WB-F344 cells to investigate radiation-induced bystander responses (7–9).

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED

We thank T. Denny and D. Stein for the support they provided at the core flow cytometry facility.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS AND DISCUSSION
  5. Acknowledgements
  6. LITERATURE CITED