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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cytometry A. Author manuscript; available in PMC 2010 August 16.
Published in final edited form as:
PMCID: PMC2921902
NIHMSID: NIHMS68527

Proliferative Response of Bystander Cells Adjacent to Cells with Incorporated Radioactivity

Abstract

Background

In a recent study, we showed that cells irradiated with γ-rays stimulate cell growth of unirradiated (bystander) cells, when the two populations are co-cultured as a mixture (Cytometry 2003;54A:1–7). Direct cell-to-cell contact appears to be a prerequisite for the proliferative response of the bystander cells (Cytometry, 2003 56A:71–80). The aim of the current work is to investigate the possible proliferative bystander effects caused by intracellular irradiation with incorporated radionuclides, specifically the short-range β particle emitter, tritium (3H).

Methods

Subconfluent monolayers of rat liver epithelial cells (WB-F344) were incubated in the presence of (methyl-3H)thymidine (3HTdR) at concentrations ranging between 5.2 kBq/ml and 57.8 kBq/ml for 18 h. Radiolabeled cells, containing between 0.7 × 10−3 Bq/cell and 8.8 × 10−3 Bq/cell were mixed with unlabeled (i.e., bystander) cells in a ratio of 1:1 and cultured together for 24 h followed by an flow cytometry (FCM) study of their proliferation. In order to discriminate the two populations of co-cultured cells, one cell population (unlabeled bystander cells) was stained with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE), which metabolizes intracellularly. The absorbed doses received by the radiolabeled cells that contained 0.7 × 10−3, 2.5 × 10−3, and 8.8 × 10−3 Bq/cell were 0.14, 0.49, and 1.7 Gy, respectively.

Results

Cells that were not treated with tritiated thymidine (unlabeled cells), in the presence of radiolabeled cells that received absorbed doses from 0.14 – 1.7 Gy, showed enhanced cell growth by approximately 9 to 10%.

Conclusions

Cells labeled with 3HTdR can induce increased proliferation in neighboring unlabeled bystander cells. FCM provides an excellent basis for characterization of proliferative bystander effects in co-culture systems.

Keywords: bystander effects, tritium, tritiated thymidine, β particles, co-culture, proliferation, unlabeled cells, radiolabeled cells, flow cytometry

Since the late 1950s, when tritiated thymidine (3HTdR) became commercially available, it has been used extensively in biomedical research. This radiochemical is widely used for labeling newly synthesized DNA in proliferating cells, because its incorporation can be rapidly and reliably measured by liquid scintillation counting and autoradiography. Biological effects from incorporated 3HTdR are well documented (1,2). Suppression of cell proliferation (3), low colony counts (4), chromosome aberrations (57), DNA strand breaks (8), cell-cycle arrest (911), and cell death (1113) indicate that this radiochemical is deleterious. Induction of tumors with 3HTdR has been also reported (14). Tritium emits short-range β particles such that when it is localized in the cell nucleus, it generally does not significantly irradiate neighboring cells. This said, there is evidence of lethal damage to unlabeled bystander cells when tritium (3H) is localized in the DNA of Chinese hamster lung fibroblasts (V79) cells and nonuniformly distributed in a 3D tissue model (15,16). This finding is of substantial importance to risk estimation in diagnostic nuclear medicine and radiation protection, as well as clinical outcome in therapeutic nuclear medicine.

Radiation-induced bystander effects have been well documented, primarily with external beams of various types of ionizing radiation. Among the bystander responses observed are induction of sister chromatid exchanges, alterations in gene expression, mutations, neoplastic transformation, and even cell death (1722). An enhanced proliferation of bystander cells as a response to cells subjected to various types of ionizing radiation has been only recently reported (2326). Proliferative bystander responses have been observed with respect to cells externally irradiated with α particles (23), heavy ions (24), and γ-rays (25,26). However, there are no data to date on whether short-range ionizing radiations emitted by intracellularly incorporated radionuclides have an impact on proliferation of bystander cells.

In the present work, efforts were focused on the impact of tritium-labeled (radiolabeled) cells on proliferation of unlabeled bystander cells using a 2D cell culture model.1 Subconfluent monolayers of rat liver epithelial cells (WB-F344) were incubated in the presence of 3HTdR at concentrations ranging between 5.2 and 57.8 kBq/ml for 18 h. Radiolabeled cells containing between 0.7 × 10−3 and 8.8 × 10−3 Bq/cell were mixed with unlabeled cells of the same cell line (50% radiolabeled cells and 50% unlabeled cells) and cultured together for 24 h, and the proliferation of unlabeled cells was quantitatively determined by a two scheme FCM assay, as described in Gerashchenko and Howell (25). FCM can precisely discriminate the radiolabeled cell population from the unlabeled cell population by fluorescence staining one of the cell populations (e.g., unlabeled cells), and can rapidly quantify responses in a large cohort of cells in sufficient numbers for precise statistical certainty.

Materials and Methods

Radiochemical and Quantification of Radioactivity

Tritiated thymidine (methyl-3H-thymidine) was obtained from Perkin-Elmer Life Sciences (Billerica, MA) as a sterile aqueous solution at a concentration of 37 MBq/ml with a specific activity of 3,000 GBq/mmole. The activity of 3H was measured with a Beckman LS3800 automatic liquid scintillation counter (Fullerton, CA) by transferring aliquots of radioactive culture medium into 5 ml of Eco-Lume™ liquid scintillation cocktail (ICN Biomedical, Costa Mesa, CA). The detection efficiency for the β particles emitted by 3H was 0.50.

Cell Line

The rat liver epithelial cell line WB-F344 (27) was generously provided by Dr. J.E. Trosko (Michigan State University, East Lansing, MI). Cells were asynchronously grown in D-medium (Custom Formula No. 78-5470EF; Gibco-BRL, Grand Island, NY) in a 37°C humidified incubator containing 2% CO2 and 98% air. The medium was prepared by dissolving 8.97 g of D-medium powder in 400 ml of deionized H2O. This was supplemented with 0.835 g/l NaCl, 1.0 g/l glucose, 1 mM Na pyruvate, and 10 mM HEPES buffer (Gibco); 1 M NaOH was added drop-wise until pH 6.5. Finally, 25 μg/ml gentamicin and 5% FBS was added.

Radiolabeling

WB-F344 cells were seeded into 60 × 15 mm (P60) dishes (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) at an initial cell density of 9.6 × 105 cells per dish. After 30 h, when cell confluence was approximately 70 to 80% (20.6 × 105 cells per dish), cells were treated with 3HTdR at concentrations of 5.2, 19.6, and 57.8 kBq/ml (total volume of culture medium was 5.5 ml) for 18 h at 37°C in a CO2 incubator.

Experiments with Cocultured Cells

Scheme 1. Determination of percentages of unlabeled and radiolabeled cells in co-cultures

In order to discriminate unlabeled cells (cells that were not treated with tritiated thymidine) from radiolabeled cells, the unlabeled cells were stained with a membrane-permeant reactive tracer Vybrant™ 5- (and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFDA SE; Molecular Probes, Eugene, OR). CFDA SE passively diffuses into cells. This dye is colorless and nonfluorescent until its acetate groups are cleaved by intracellular esterases to yield highly fluorescent, aminoreactive carboxyfluorescein succinimidyl ester (28). Monolayers of unlabeled cells were stained with a 3 μM solution of CFDA SE (loading solution) prepared with prewarmed Dulbecco's PBS (DPBS) (37°C) for 15 min in a CO2 incubator. The loading solution was replaced with fresh, prewarmed medium, and cells were incubated for another 30 min at 37°C in a CO2 incubator. Radiolabeled cells were similarly handled except no dye was added. Monolayers of radiolabeled and unlabeled cells were washed twice with 5 ml of DPBS, trypsinized with Trypsin-EDTA (Gibco) containing 0.05% trypsin and 0.53 mM EDTA, and suspended in D-medium (total volume of cell suspension was 1.5 ml). According to hemocytometer counting, the concentration of unlabeled cells was 3 × 106 cells/ml. Equal aliquots (200 μl) of unstained radiolabeled and stained unlabeled cell suspensions were mixed (Fig. 1) and the percentage of cells in each population was precisely determined with a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Once the concentrations of radiolabeled cells were determined from this information, the stained unlabeled cells (3 × 105 cells) were then plated together with unstained radiolabeled cells (3 × 105 cells) into Falcon six-well 35-mm–diameter culture dishes (Becton Dickinson Labware, Lincoln Park, NJ) at a ratio 1:1 (Fig. 2). A control sample was prepared by mixing stained unlabeled cells and unstained unlabeled cells at a ratio 1:1. Cells were co-cultured in 4 ml of D-medium for 24 h at 37°C in a CO2 incubator,2 trypsinized, and washed with DPBS at 4°C, and fixed in 0.5 volume of ice-cold 3.7% formaldehyde (Sigma, St. Louis, MO) in DPBS (final concentration of formaldehyde was approximately 1.2%, and final volume of cell suspension was 1.5 ml) at room temperature for 5 min. After fixation, the cells were analyzed by FCM to determine the percentages of the co-cultured cells that arose from the unlabeled cells and radiolabeled cells, respectively, in accordance with the published procedure called Scheme 1 (25).

Fig. 1
Radiolabeling and determining the concentrations of radiolabeled cells. Tritiated thymidine was added to subconfluent monolayers of WB-F344 cells (20.6 × 105 cells per P60 dish). The controls were not treated with 3HTdR (0 kBq/ml). After 18 h ...
Fig. 2
Schematic representation of Scheme 1. Fluorescence-stained unlabeled cells (shown in red) and unstained tritiated cells containing various amounts of radioactivity per cell (Bq/cell) were plated together at a ratio of 1:1 and co-cultured. After a 24-h ...

Scheme 2. Comparison of cell populations in co-culture of unlabeled cells with co-culture of unlabeled and radiolabeled cells

This procedure was performed in accordance with the published procedure called Scheme 2 (25), with slight modifications. After monolayers of unlabeled and radiolabeled cells were washed twice with 5 ml of DPBS, trypsinized, suspended in D-medium (total volume of cell suspension was 1.5 ml), unlabeled cells (3 × 105 cells) and radiolabeled cells (3 × 105 cells) were plated together in the same manner described for Scheme 1; however, in Scheme 2, no cells were stained prior to incubation. After a 24-h incubation at 37°C, in 2% CO2 and 98% air, the co-culture of unlabeled cells was stained with CFDA SE (Fig. 3; (0 ↔ 0) Bq/cell; the double arrow (↔) indicates that the cells were co-cultured). The stained co-culture ((0 ↔ 0) Bq/cell) and the remaining co-cultures (Fig. 3; (0 ↔ X) Bq/cell) were washed twice with DPBS, harvested with trypsin, and suspended in ice-cold DPBS (total volume of cell suspension was 1.4 ml). Two hundred microliter aliquots of the stained control sample ((0 ↔ 0) Bq/cell) were mixed with equal aliquots containing unstained co-cultured cells ((0 ↔ X) Bq/cell) according to the protocol outlined in Figure 3. As shown in Figure 3, the resulting mixture is denoted by ([(0 ↔ 0) & (0 ↔ X)] Bq/cell; the ampersand (&) implies that the two cultures were mixed). The cell mixtures were washed with DPBS at 4°C and fixed in formaldehyde as described above. After fixation, the percentages of stained and unstained cells were analyzed by FCM.

Fig. 3
Schematic representation of Scheme 2. Tritiated cells containing various amounts of radioactivity per cell (Bq/cell) and unlabeled cells were plated together at a ratio of 1:1 and co-cultured. After a 24-h incubation, unlabeled cells in the control ((0 ...

Calculation of the Proliferation Ratio

The proliferation of unlabeled cells co-cultured with radiolabeled cells was quantified using the “proliferation ratio,” which was formulated in a previous communication (25). Briefly, the numbers of unlabeled cells in co-cultures of unlabeled and radiolabeled cells that have received a dose D compared to the number of unlabeled cells in a co-culture of unlabeled and radiolabeled cells that have received a zero dose, can be expressed as a proliferation ratio. FCM analysis of Scheme 1 was used to determine the percentages of unlabeled cells pU(D) and radiolabeled cells pI(D) in the co-culture (the prime denotes Scheme 1 and D denotes the absorbed dose delivered to the radiolabeled cells). It is obvious that pU(D)+pI(D)=100%. For Scheme 2, the percentages of stained cells ((0 ↔ 0) Bq/cell) and unstained cells ((0 ↔ X) Bq/cell) in the cell mixture, as analyzed by FCM, were designated pU(D) and pI(D), respectively. Note that the double prime denotes Scheme 2 and pU(D)+pI(D)=100%. The proliferation ratio for the unlabeled cells RU is defined in Gerashchenko and Howell (25) as:

RU(D)=number of unlabeled cells in a co-culture of unlabeled cellsand radiolabeled cells that have received a dose Dnumber of unlabeled cells in a co-culture of unlabeled cellsand radiolabeled cells that have received zero dose.
(1)

As derived in Gerashchenko and Howell (25), this proliferation ratio RU can be expressed in terms of the percentages measured by FCM for Schemes 1 and 2:

RU(D)=pU(D)pU(0)pI(D)pU(D).
(2)

The proliferation ratio RU was calculated and tabulated as a function of absorbed dose D for six replicate experiments. This ratio quantifies the enhancement in proliferation of unlabeled bystander cells.

FCM Analysis

FCM was performed on a FACScan flow cytometer (Becton Dickinson), equipped with a 15-mW argon-ion laser (488 nm). The FSC and SSC light scatter signals were collected in linear mode. The fluorescence from products of intracellularly metabolized CFDA SE was measured in the green fluorescence channel (FL1) through a 530/30-nm band pass filter with logarithmic amplification. At least 10,000 events were collected for each sample. Analysis of the data was performed with WinMDI software developed by Dr. J. Trotter (http://facs.scripps.edu/software.html). Cells were gated on FSC versus SSC dot plots to eliminate debris and aggregates from analysis as previously described (25).

Cell Sorting of Unlabeled Cells

To ensure that there was no significant uptake of radioactivity by the unlabeled cells in the co-culture with radiolabeled cells, the following procedure was performed. Briefly, after the 24 h co-culture, unlabeled (stained) and radiolabeled (unstained) cells were separated using a cell sorter (Becton-Dickinson FACS Vantage). The stained cells (100,000) were deposited into 12 × 75 mm polypropylene tubes. The sorted cells were then centrifuged, transferred to liquid scintillation vials containing 5 ml of EcoLume, and the 3H activity per cell was determined. The remaining unused cells were subjected to FCM analysis to check the purity of sorted cells. The sorting purity was greater than 99%.

Measurement of Cell Dimensions

It is well known that 3HTdR incorporates into the DNA in the cell nucleus. Therefore, to calculate the absorbed dose to the cell nucleus from decays within itself, knowledge of the dimensions of the cell nucleus is required. Because of the very short range of the 3H β-particles, when 3H is confined to the nucleus of the cell, the absorbed dose is not sensitive to the shape of the cell nucleus but rather to its mass (29). Accordingly, the dimensions were measured while in suspension, so that the cells were spherical. In order to avoid compressing the cells during measurements, a special chamber was created on a microscope slide consisting of the slide and three cover slips. Hoechst 33342 stained cells were injected into the chamber prior to measurements of cell dimensions. A Leitz (Wetzlar, Germany) (Model DIALUX 20) fluorescent microscope equipped with an eyepiece reticule was used. At 400× magnification, one small division of the reticule corresponded to 1 μm. The mean and SDs of the WB-F344 cell (n = 50) and nuclear (n = 53) diameters were 13.3 ± 1.2 μm and 9.5 ± 1.4 μm, respectively.

To assess the distances between the nuclei of neighboring cells, cell dimensions and internuclear distances were measured in monolayers of WB-F344 cells. Cells were grown on coverslips and a chamber for measuring cell dimensions was created in the same way as described in the protocol above. Culture medium containing Hoechst 33342 was injected into the chamber prior to measurements. Measurements of the mean internuclear distance (n = 50) and mean nuclear diameters (n = 94) yielded 24.6 ± 5.5 μm and 14.6 ± 2.6 μm, respectively. The increased diameter of the nucleus of WB-F344 cells grown in monolayer was due to stretching and flattening of the cells in the monolayer culture.

Estimation of Cellular Absorbed Dose to Radiolabeled Cells

As indicated above, the WB-F344 cells were exposed to extracellular 3HTdR for 18 h, washed free of extracellular activity, mixed with unlabeled cells, plated into P60 dishes, and harvested after coculturing for 24 h. The 3HTdR localizes in the cell nucleus; therefore, according to the general formalism for cellular dosimetry given by equation 7 of Goddu et al. (29), the mean absorbed dose D to the cell nucleus is given by:

D=AS(NN)
(3)
A=AI+AP
(4)

where ÃI and ÃP are the cellular cumulated activities during the periods of incubation for cellular uptake and proliferation, respectively. The quantity S(N ← N) is the cellular S value (absorbed dose per unit cumulated activity) for the radionuclide when localized in the cell nucleus. As given above, the mean diameter of the cell nucleus of WB-F344 cells is 9.5 ± 1.4 μm. Using these dimensions, a log-log interpolation of the S value tables for 3H in Goddu et al. (29) gives S(N ← N) = 1.64 × 10−3 Gy Bq−1s−1. Because 3HTdR is taken up by the cells linearly in time (30), the cellular cumulated activity during the uptake period ÃI is given by:

AI=0.5tIAI
(5)

where AI is the average cellular activity (Bq/cell) at the end of the uptake period, and tI is the incubation time during which the radioactivity is taken up by the cells (tI = 18 h). The cumulated activity during the proliferation period (tP = 24 h) can be estimated as follows. It is evident that the proliferation of radiolabeled WB-F344 cells is at best minimal. Therefore, for the purposes of estimating the cumulated activity, it is assumed that the radiolabeled cells do not divide during the 24-h proliferation period. Accordingly, the cumulated activity during the proliferation period is given by:

AP=tPAI
(6)

The total cumulated activity is therefore:

A=(0.5tI+tp)AI
(7)

Finally, the absorbed dose to the cell nucleus is:

D=(0.5tI+tP)AIS(NN)=195AIGy,
(8)

where AI is in units of Bq.

Results

Effects of Radiolabeling and Uptake of 3HTdR

Table 1 gives the percentages of fluorescence-stained unlabeled cells and unstained radiolabeled cells after they were identically harvested from P60 dishes and equal aliquots of each were mixed and analyzed by FCM. The values for six independent replicate experiments were averaged and the mean values of the ratio of percentages of unlabeled and radiolabeled cells is shown in Figure 4A. These data indicate that incorporation of 3HTdR inhibited cell proliferation. In agreement with previous reports (3,9,11), the degree of inhibition depended on the concentration of radioactivity added to the culture medium. Note that for the highest concentration of 3HTdR (57.8 kBq/ml), the ratio of unlabeled to radiolabeled cells in the mixture was approximately two times less than in the control mixture (stained unlabeled cells and unstained unlabeled cells).

Fig. 4
A: FCM analysis of the percentages of unstained radiolabeled cells and stained unlabeled cells after they were harvested and mixed in equal aliquots as illustrated in Figure 1. Data shown are the mean ± SD of six separate replicate experiments ...
Table 1
Flow Cytometry Data

Quantification of intracellular radioactivity yielded mean values of 0.7 × 10−3, 2.5 × 10−3, and 8.8 × 10−3 Bq per cell in the cell populations treated with 5.2, 19.6, and 57.8 kBq/ml of 3HTdR, respectively.3 These data are plotted in Figure 5, where it is apparent that the cellular uptake of 3HTdR is linearly proportional to its concentration in the culture medium. A similar linear correlation has been observed for 3HTdR and its analog iododeoxyuridine in other cell lines (13,30). These cellular uptake data were used to calculate the mean absorbed dose D to the nucleus of the radiolabeled cells according to equation 8. The resulting mean absorbed doses are given in the third column of Table 2.

Fig. 5
Cellular uptake of 3HTdR. The plot shows the linear correlation between the concentration of 3HTdR initially added to culture medium (kBq/ml) and the amount of radioactivity per labeled cell (Bq/cell) after an 18-h treatment with radiochemical as described ...
Table 2
Flow Cytometry Data

Results of Scheme 1

The percentages of unlabeled cells pU(D) and radiolabeled cells pI(D) 24 h after initiating the co-culture are presented in Table 2, Scheme 1. The values of pU(D) and pI(D) are plotted as a function of absorbed dose D and 3H activity per radiolabeled cell in Figure 6. Absorbed doses to radiolabeled cells from 0.14–1.7 Gy resulted in drastic drops in the number of radiolabeled cells compared with unlabeled cells in the co-culture. This can be explained by the reduced plating efficiency and reduced proliferation of the radiolabeled cells.

Fig. 6
Percentage of cells in a co-culture that correspond to radiolabeled cells and unlabeled cells (data from Scheme 1). This plot shows that even low 3H activities per cell (0.7 × 10−3 Bq/cell) caused remarkable decreases in the percentages ...

Results of Scheme 2

For Scheme 2, the percentages of stained cells ((0 ↔ 0) Bq/cell) and unstained cells ((0 ↔ X) Bq/cell) in the cell mixture, as analyzed by FCM, are reported in Table 2, Scheme 2, columns pU(D) and pI(D), respectively. There is an increase in the percentage of stained (unlabeled + unlabeled) cells pU(D) relative to unstained (radiolabeled + unlabeled) cells pI(D).

Enhanced Proliferation of Unlabeled Cells in the Presence of Radiolabeled Cells

The proliferation ratios RU, calculated using equation 2 and the FCM data in Table 2, are tabulated in Table 3 as a function of 3H activity per radiolabeled cell and absorbed dose for six replicate experiments. Also presented in Table 3 are the average values of RU and their respective standard deviations. Finally, the P values for a two-tailed t-test assuming equal variances are presented in the last column of Table 3. The proliferation ratios, plotted in Figure 7, clearly show an enhanced growth of unlabeled cells when co-cultured with radiolabeled cells, containing activities ranging from 0.7–8.8 × 10−3 Bq/cell with corresponding absorbed doses of 0.14–1.7 Gy. Over this range of doses, unlabeled cells showed a statistically significant (P < 0.05, see Table 3) increase in the proliferation ratio that was approximately 9–10% greater than controls.

Fig. 7
Proliferation ratios for unlabeled cells in the co-culture with radiolabeled cells. There is enhanced proliferation of unlabeled cells in the presence of radiolabeled cells as evidenced by values >1. Cells containing activities ranging from 0.7–8.8 ...
Table 3
Proliferation Ratios for Unlabeled Bystander Cells RU

Cell Sorting of Unlabeled Cells

Following the 24-h co-culture of unlabeled and radiolabeled cells, a small amount of radioactivity was found in the sorted unlabeled cells. These activities were determined to be 0.008 × 10−3, 0.023 × 10−3, and 0.065 × 10−3 Bq/cell when the radiolabeled cells were treated with 5.2, 19.6, and 57.8 kBq/ml of 3HTdR in the culture medium, respectively. Figure 6 shows that these levels of cellular activity do not have a significant impact on proliferation. Therefore, the small amount of radioactivity found in the unlabeled cells will have no impact on the proliferation ratios in Figure 7.

Discussion

To ensure that the proliferative response observed in the unlabeled cells can indeed be attributed to bystander effects (induction of alterations in cells that did not receive hits from the emitted radiations), it is first necessary to show that bystander cells were not irradiated by radiolabeled cells. Tritium emits short-range β particles with a spectrum of energies ranging from 0–18.6 keV (31) with corresponding ranges in water from 0–7 μm. The mean energy of the β particles is only 5.7 keV, and it has a range of ≈1 μm in water. Accordingly, the probability that the β particles emitted from radiolabeled cells will hit the nucleus of adjacent unlabeled cells at the early stage of co-culture is very low, because the majority of unlabeled cells were far beyond the range of β particles emitted from radiolabeled cells (see micrograph of plating density in Fig. 4 of Gerashchenko and Howell [26]). Even upon expansion of cells in the co-culture, which leads to increased cell population density, the probability that the nuclei of unlabeled cells can be hit by β particles remains very low (Fig. 8). Measurements of the diameters of cell nuclei in subconfluent monolayers of WB-F344 cells is 14.6 ± 2.6 μm, and the distance between centers of nuclei of neighboring cells is 24.6 ± 5.5 μm. Therefore, the average distance between surface of the cell nucleus of a radiolabeled cells and the surface of an adjacent cell nucleus is about 10 ± 6 μm, which is greater than the range of the most energetic β particles emitted by 3H. Furthermore, the flattened configuration of the nuclei in adherent cells makes the solid angle subtended by the unlabeled cell nucleus very small (Fig. 8). Finally, the probability of significantly irradiating the cytoplasm of adjacent bystander cells is also low, considering that the shortest distance between the surface of the cell nucleus of the radiolabeled cell and the cell membrane of the unlabeled cell is f = (c − d)/2 = 5 ± 3 μm and that the relative yield of 3H β-particles with ranges of greater than 5 μm is very low. These arguments lend a high degree of confidence that the proliferative response of the unlabeled cells is indeed a bystander effect triggered by the self-irradiation of neighboring radiolabeled cells.

Fig. 8
Geometry of adjacent cells in WB-F344 co-cultures containing unlabeled cells (U) and radiolabeled cells (R). Measurements of the cell nuclei in sub-confluent monolayers of WB-F344 cells yield a mean nuclear diameter d = 14.6 ± 2.6 μm. ...

In the present study with tritium labeled WB-F344 cells, we employed a two-scheme co-culture assay system that was almost identical to that described in our previous work, in which it was shown that γ-rays over a wide range of doses (0.5–20 Gy) enhance the proliferation of neighboring bystander cells by approximately 14–17% (25). Similar to the γ-ray studies, WB-F344 cells containing various amounts of 3HTdR per cell were co-cultured as a mixture with unlabeled cells of the same cell line. Cells containing activities ranging from 0.7–8.8 × 10−3 Bq/cell triggered a proliferative response of unlabeled bystander cells that was 9–10% higher than controls. These cells received absorbed doses of 0.14, 0.49, and 1.7 Gy, respectively. This shows that the proliferative response of bystander cells can be triggered by very low absorbed doses delivered by 3HTdR. This response occurs at much lower absorbed doses than when cells are irradiated with γ-rays (25). In fact, the maximum bystander response to γ-rays was not seen until 1 Gy (25), whereas in the case of 3HTdR, the maximum response was observed at only 0.14 Gy and it appears that the response might be seen at even lower absorbed doses.

It is possible that the differences in the bystander dose response between γ-rays and 3HTdR may be related to the higher relative biological effectiveness (RBE) that has been observed for 3HTdR compared to γ-rays (2,13,15,32). The RBE of 3HTdR compared to γ-rays ranges from about 2 to 9 for a variety of biological end-points; however, it is possible that the RBE for inducing the proliferative response of bystander cells may be considerably higher. This could explain the low-dose response to 3HTdR. With signaling mechanisms in mind, it is interesting to note that Marko et al. (33) have shown that the stress response to radiation insults results in different gene expression profiles in cells irradiated with γ-rays versus cells irradiated with low-energy β particles emitted by intracellularly localized 35S-methionine. The cellular response to β irradiation was greater not only with respect to the number of genes induced, but also the level of gene expression. Interestingly, β irradiation also led to a distinct gene induction profile that included a large number of cell adhesion proteins (33). Accordingly, a major challenge for future investigations is the study of signaling between the radiolabeled and unlabeled cell populations. Of importance to this effort is our finding that direct cell-to-cell contact is a prerequisite for the proliferative response of bystander cells to cells treated with γ-rays, and that neither gap junctional intercellular communication (GJIC) nor soluble extracellular factors released by irradiated cells into the culture medium appear to play significant roles in the proliferative response of bystanders (26).

While comparisons within the same experimental model are indeed important, it is also interesting to compare the 3HTdR-induced proliferative bystander response with other proliferative bystander responses that have been observed. Monolayers of cells exposed to a low-dose of α particles (1 cGy, in which only about 7% of the cells are hit) have also been shown to exhibit enhanced cell growth (23). A similar response was elicited when unirradiated cells were treated with supernatants from irradiated cells (23). Furthermore, Shao et al. (24) have reported that cells that were irradiated with heavy ions caused an increase in cell proliferation of bystander cells that were co-cultured at a distance from irradiated cells. Their results indicate that cells irradiated with 1–3 Gy of 13 keV/μm carbon ions release substances into the cell culture medium that cause an 8–10% increase in the proliferation of unirradiated bystanders. Somewhat higher and dose-dependent increases were observed for 100 keV/μm. Interestingly, the increased proliferation they observed is similar in magnitude to that observed in the present study with 3H β particles, which have a mean linear energy transfer (LET) of about 5.5 keV/μm (1). However, it should be noted that released factors do not play a role in the bystander effects observed in our model (26). Nevertheless, it is clear that a variety of types of ionizing radiation can induce proliferative responses in bystander cells through a variety of mechanisms. While it is unclear whether growth acceleration of bystander cells is indicative of undesirable changes within these cells, radiation-induced bystander effects have been manifested in the form of alterations in gene expression (3437), sister-chromatid exchanges (38), micronuclei (37), mutations (39), transformations (40,41), and cell killing (15,16,42). More specifically to the type of radiation used in this work, energetic β particles emitted by 90Y have been shown to induce transformations in bystanders (40), and 3HTdR has been shown to induce cell killing in bystanders (15,16). Whether the proliferative bystander response observed in this study will ultimately lead to detrimental effects in the bystanders remains to be seen and requires further investigation.

Several reports provide firm evidence that bystander effects can be mediated via GJIC (15,34,35,43) and extracellular factors (23,4448). However, neither of these mechanisms appear to play a role in our experimental model (26). It is possible that extracellular signaling molecules are released by irradiated cells and directly delivered to adjacent bystander cells via the plasma membrane. Among the possible factors causing growth stimulation of bystander cells are elevated levels of reactive oxygen species (ROS) released by radiolabeled cells. It has been shown that metabolic radiolabeling with low-energy β-emitters, such as 35S-methionine, may be associated with p53-dependent ROS production (49). Very low levels of ROS are known to stimulate cell growth by mechanisms that are not well understood (5052). There is the mounting evidence that ROS play an important role in radiation-induced bystander responses (23,53). Although less information is available about possible role of reactive nitrogen species (RNS) in proliferative bystander responses, nitric oxide (NO) has been found as a potential mediator of these responses (24). Another potential mechanism of signal exchange via cell-to-cell contact between radiolabeled cells and bystander cells is transmembrane signaling mediated by membrane-bound proteins and receptors. The role of this mechanism in radiation-induced bystander effects remains to be explored. In addition, there is a room to speculate that alterations in the extracellular matrix of radiolabeled cells may initiate a proliferative response in adjacent bystander cells through membrane-associated signaling cascades with possible involvement of surface membrane NAD(P)H-oxidase, a producer of mitogenic superoxide radicals.

Finally, it is also important to not lose sight of the scientific tools that have been developed in this work. FCM not only can precisely discriminate cell populations of interest (e.g., fluorescence-stained unlabeled cells from unstained radiolabeled cells), but also can rapidly quantify responses in a large cohort of cells in sufficient numbers for precise statistical certainty. Therefore, use of FCM in the analysis of co-cultures of radiolabeled cells and unlabeled cells offers an excellent basis for the study of bystander effects induced by intracellularly incorporated radionuclides. Multicolor FCM and high-speed sorting technologies can be envisioned for the study of signaling between the radiolabeled and unlabeled cell populations.

Acknowledgments

We thank Drs. E.I. Azzam, S.M. de Toledo, P.V.S.V. Neti, and M. Pinto for their valuable commentaries and suggestions on the present work. We also thank Mr. D. Trivedi for his enthusiastic technical assistance and Ms. T. Mui for her excellent technical expertise in sorting the cells with the FACS Vantage. This work was supported in part by a UMDNJ Foundation 2002 Postdoctoral Fellowship and New Jersey Cancer Commission on Cancer Research Fellowship 03-2013-CCR-S2. Finally, the authors greatly appreciate the support provided by T. Denny and D. Stein at the core FCM facility, which is supported, in part, by USPHS shared instrumentation grant No. 1 S10 RR14753-01.

Footnotes

1Radiolabeled cells are those containing tritiated thymidine (3HTdR). Unlabeled cells are the bystander cells that do not contain 3HTdR. Stained cells are those that contain fluorescence dye. Unstained cells do not contain fluorescence dye.

2After 24 h of incubation, cells reached 80–90% confluence.

3The amount of radioactivity per cell was measured in triplicate by subtracting the amount of radioactivity in supernatant from the amount of radioactivity in cell suspension. Upon treatment of cells with 5.2, 19.6, and 57.8 kBq/ml of 3HTdR, the total uptake of 3HTdR by cells, compared to the total radioactivity added, was 9.9, 7.4, and 6.5%, respectively.

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