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[Cancer Research 60, 2623-2631, May 15, 2000]
© 2000 American Association for Cancer Research

Biochemistry and Biophysics

High and Low Fluences of {alpha}-Particles Induce a G1 Checkpoint in Human Diploid Fibroblasts1

Edouard I. Azzam2, Sonia M. de Toledo, Anthony J. Waker and John B. Little

Department of Cancer Cell Biology, Laboratory of Radiobiology, Harvard School of Public Health, Boston, Massachusetts 02115 [E. I. A., S. M. d. T., J. B. L.], and Radiation Biology and Health Physics, Atomic Energy of Canada Ltd., Ontario K0J 1J0, Canada [A. J. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of exposure to high and very low fluence {alpha}-particles on the G1 checkpoint were investigated in human diploid fibroblasts irradiated and released from density-inhibited confluent cultures by the use of the cumulative labeling index method. Transient and permanent arrests in G1 occurred in fibroblast populations exposed to mean doses as low as 1 cGy, suggesting that nontraversed bystander cells may contribute to the low dose response. In cells exposed to high fluences, the G1 checkpoint is at least as extensive as in {gamma}-irradiated cells. In contrast to {gamma}-irradiated cells, neither repair of potentially lethal damage nor a reduction in the fraction of cells transiently or permanently arrested in G1 were observed in cells held in confluence for 6 h after {alpha}-particle irradiation. Studies with isogenic wild-type, p53–/–, and p21Waf1–/– mouse embryo fibroblasts exposed to either {gamma} or {alpha}-particle radiation revealed a total lack of G1 arrest in either p53–/– or p21waf1–/– cells, indicating that the G1 checkpoint in wild-type cells is p53-dependent and that p21Waf1 fully mediates the role of p53 in its induction. In contrast to human cells, mouse embryo fibroblasts do not undergo a permanent G1 arrest. Except under conditions favoring potentially lethal damage repair, a comparable expression pattern of p53, p21Waf1, and other cell cycle-regulated proteins (pRb, p34cdc2, and cyclin B1) was observed in {alpha}-particle or {gamma}-irradiated human fibroblasts.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian cellular responses to DNA damaging agents result in delays in progression through the cell cycle at several checkpoints (reviewed in Refs. 1 and 2 ). Such delays have been hypothesized to provide more time for repair of DNA damage. Delays in G1, S, and G2 have been described after exposure of rodent and human cells to low LET3 ionizing radiations such as {gamma}- or X-rays (reviewed in Refs. 3, 4, 5 ). Compared to low LET radiation, more extensive delays in the G2 and S phases have been reported to occur in mammalian cells exposed to high LET radiation such as {alpha}-particles (6, 7, 8) . However, the existence of a G1-phase delay in {alpha}-particle irradiated cells has not been as clearly established. In earlier cytofluorometric analyses of {alpha}-particle irradiated rodent and human fibroblasts, a G1 phase delay was not observed (7 , 9) . In a more recent study (10) , a G1 phase delay was observed in asynchronously growing human fibroblasts exposed to high fluences of {alpha}-particles; however, its magnitude and persistence were reduced when compared to the delay observed after exposure of the same cells to {gamma}-radiation.

Radionuclides emitting {alpha}-particles are currently being investigated in clinical cancer treatment because of their extreme toxicity (11 , 12) and because their effects have been shown to be independent of oxygen and dose-rate (13 , 14) . Their short range facilitates selective targeting of malignant cell populations (15, 16, 17) . As cell cycle checkpoints have been proposed to act as regulators of tumor sensitivity (18, 19) , the characterization of the existence and magnitude of the G1 delay in {alpha}-particle irradiated cells could have significant implications in the design of clinical radiotherapeutic protocols. Also, an understanding of how cell cycle transitions are altered in response to high LET radiation is important in the field of radiation protection, as these transitions can influence the overall cellular response to radiation-induced damage (1 , 14) . It is now known that a large component of the background exposure dose equivalent received by the general public results from {alpha}-particles emitted by radon and its progeny decay products (20) . It has been estimated that 10–14% of lung cancer deaths are linked to radon gas in the environment (21) .

The ATM, p53, and Waf1 genes have been implicated in the G1 arrest that occurs in {gamma}-irradiated diploid fibroblasts (22) . Part of the ability of the p53 protein to cause a G1-phase arrest in {gamma}-irradiated cells results from the activation of transcription of Waf1, the protein of which is a universal inhibitor of cyclin-dependent kinases that control entry into S phase (23 , 24) . Waf1 has also been shown to be induced by p53-independent mechanisms (25 , 26) . Other possible mediators of the G1 checkpoint have also been described; GADD45 and c-ABL caused a G1 arrest when overexpressed (27 , 28) . Recently various genotoxic agents have been shown to activate different signaling pathways to relieve stress (29) . Whereas p53 appears to be a universal sensor of genotoxic stress, c-abl was not activated by UV radiation, and c-Jun NH2-terminal kinase was strongly stimulated only by UV light and the alkylating agent methyl methanesulfonate. Similarly, p73 protein levels in a cell are differentially modulated by cisplatin and UV or X-irradiation (30) . However, the nature of the induced molecular pathways in {alpha}-particle irradiated cells is largely unknown.

To characterize cell cycle regulation after {alpha}-particle irradiation, we exposed human diploid fibroblasts synchronized in G0/G1 by confluent density inhibition of growth to isosurvival doses of either {alpha}-particle or {gamma} radiation and monitored their progression through the cell cycle after subculture to low density by autoradiographic techniques. We tested the effects of holding {alpha}-particle irradiated quiescent human cells at 37°C prior to subculture on the repair of potentially lethal damage and on progression into S-phase. To determine whether signaling pathways other than the p53 pathway can induce the G1 checkpoint in {alpha}-particle irradiated cells, we measured transient and permanent G1 arrests in irradiated wt, p53, or p21Waf1 null MEFs. We explored mechanisms that may underlie the biological effects of exposure to {alpha}-particles by investigating altered gene expression associated with the G1 checkpoint and compared it with that in {gamma}-irradiated human diploid fibroblasts. Finally, to assess the effects of exposure to very low fluences of {alpha}-particles, we measured the G1 checkpoint in cell populations exposed to a mean dose as low as 1 cGy, at which only a small fraction of the exposed population of cells would have their nuclei traversed by an {alpha}-particle.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Maintenance
Human Cells.
AG01521 and GM06419 human diploid skin fibroblasts were obtained from the Genetic Cell Repository at the Coriell Institute for Medical Research (Camden, NJ). Both cell strains were wt for p53 by PCR-SSCP analysis. Cells destined for {gamma}-irradiation were plated in 60-mm polystyrene dishes, and cells for {alpha}-particle irradiation were grown in 36-mm stainless steel dishes with 1.5-µm-thick replaceable mylar bottoms (31) at a seeding density of about 1.2 x 105 cells/dish. The cells were subsequently refed on days 5, 7 and 9 with Eagle’s MEM supplemented with 15% heat-inactivated FCS, 50 units/ml penicillin, and 50 µg/ml streptomycin. Experiments were started 48 h after the last feeding. At that time, 95–98% of the cells were in G0/G1 as determined by labeling with [3H]thymidine and/or flow cytometry. The cells were maintained in a 37°C humidified incubator in an atmosphere of 5% CO2 in air. Cells in passage 10 or 11 were used in the experiments. Except for the radiation exposure, all control cells were handled in parallel with the test cells.

MEFs.
Primary MEFs were kindly provided by Dr. Philip Leder (Harvard Medical School, Boston, MA). They were derived from wt, p53–/–, or p21–/– embryos. Details describing the generation of these cells have been described previously (23) . The cells were grown in MEM supplemented with 10% heat-inactivated FCS. For experiments, cells were seeded at a density of about 105 cells per 60-mm polystyrene dish or 36-mm stainless steel dish with 1.5-µm-thick replaceable mylar bottoms. Five days after seeding, the cells were refed with MEM supplemented with 0.1% serum, and experiments were started 48 h later.


   Irradiation
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
For {gamma}-irradiation, cells were exposed to 60Co-rays at 12 cGy/s in a model GR-12 irradiator (U. S. Nuclear). Cells for {alpha}-particle irradiation were exposed to a 238Pu collimated source inside a helium-filled Plexiglas box at a dose rate of 9.9 cGy/min as described previously (31) . Irradiation was carried out from below, through the mylar base, with {alpha}-particles with an average energy of 3.65 MeV. The source was fitted with a photographic shutter to allow accurate delivery of the specific radiation dose. Microscopic examination of pits etched in CR-39 plastic after a 1-min exposure showed no source hot spots or cold spots down to the 2500 µm2 level (31) .


   Cell Survival Analysis
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Human and mouse cell survival curves were generated in cells exposed to {gamma}- and {alpha}-rays by a standard colony formation assay. Confluent, density-inhibited cultures were immediately trypsinized after the exposure or after various holding periods at 37°C in 5% CO2 in air atmosphere (in PLDR experiments), and the cells were suspended in complete medium. The cells were counted, diluted, and seeded in 100-mm dishes at numbers estimated to give about 100 clonogenic cells per dish. Three or four replicates were done for each exposure point, and the experiments were repeated at least twice. After an incubation of 2–3 weeks, the plates were rinsed with PBS, fixed in ethanol, and stained with crystal violet. Survival curves from typical representative experiments are shown in "Results."


   Autoradiographic Measurement of Labeling Indices
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 
Irradiated and nonirradiated confluent, density-inhibited cells were trypsinized, seeded at low density in 30-mm dishes, and incubated continuously at 37°C in growth medium containing [3H]thymidine at a final concentration of 1 µCi/ml (specific activity 20 Ci/ml). At regular intervals thereafter, duplicate dishes were removed, and the cells were rinsed with PBS and fixed with ethanol. For autoradiographic examination, Kodak NTB2 nuclear emulsion was applied directly to the dishes. After a 2-week exposure at 4°C, the dishes were developed and stained. To determine the continuous labeling indices, 200-1000 cells were scored on each dish.


   Western Analysis
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
RESULTS
 DISCUSSION
 REFERENCES
 
After irradiation, confluent, density-inhibited cultures were either (a) held at 37°C in 5% CO2 atmosphere for various time intervals prior to harvesting for analysis; or (b) subcultured immediately at a 1:3 dilution in fresh growth medium, and cell samples were analyzed at various times thereafter. The cells were pelleted, rinsed in PBS, repelleted, and lysed in chilled RIPA buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] as described previously (32) . The RIPA buffer was supplemented with the following protease and phosphatase inhibitors: phenylmethylsulfonyl fluoride (1 mM), aprotinin (1 µg/ml), pepstatin (1 µg/ml), leupeptin (1 µg/ml), sodium fluoride (50 mM), and sodium orthovanadate (1 mM). The following antibodies against human proteins were used: anti-p21Waf1 (Ab-1), anti-p53 (Ab-6), and anti-pRb (Ab-6) were obtained from Oncogene Research Products; the anti-p34cdc2 antibody was from Santa Cruz Biotechnology; and anti-cyclin B (clone 18) was from Transduction Laboratories. Anti-{alpha}-tubulin (Ab-1) from Oncogene Research Products was used to verify whether the samples were equally loaded. An anti-mouse IgG second antibody conjugated with horseradish peroxidase and the enhanced chemiluminescence system from New England Nuclear were used to detect the various proteins.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
DISCUSSION
 REFERENCES
 
Survival of Confluent, Density-inhibited AG01521 and GM06419 Fibroblasts Exposed to {gamma}- or {alpha}-Particle Radiation.
To examine cell cycle progression of AG01521 and GM06419 cells exposed to doses of {alpha}-particles or {gamma}-rays yielding isosurvival levels, clonogenic survival curves were determined for the two cell strains and are shown in Fig. 1Citation . The doses required to reduce survival to the 10% level (D10) in G0/G1 phase AG01521 cells after {gamma}- or {alpha}-particle irradiation were 450 and 90 cGy, respectively (Fig. 1ACitation ), and in GM06419 cells, the doses were 400 and 80 cGy, respectively (Fig. 1BCitation ). The resulting relative biological effectiveness for {alpha}-rays was approximately 5 in either cell strain. Although a shoulder region is evident in the survival curves of {gamma}-irradiated cells, cell killing for {alpha}-particle irradiated cells appeared to be an exponential function of dose in a dose range below 1 Gy. Consistent with this finding, a lack of sublethal damage repair, as measured by split-dose experiments, was observed in {alpha}-particle irradiated human fibroblasts (Fig. 2Citation ). The data in Fig. 2Citation indicate a lack of increased clonogenic survival when total doses of either 85 or 20 cGy were delivered to AG01521 cells into two equal fractions separated by time intervals ranging from 0 to 200 min.



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  		Fig. 1. Clonogenic survival of confluent, density-inhibited AG01521 (A) or GM06419 (B) cells exposed to {gamma}-rays or {alpha}-particles.

 


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  		Fig. 2. Clonogenic survival of confluent, density-inhibited AG01521 cells exposed to {alpha}-particle doses of 85 or 20 cGy. The dose was delivered in two equal fractions by incubation (37°C) intervals ranging from 0 to 200 min.

 
Cell Cycle Progression of {alpha}-Particle Irradiated Human Diploid Fibroblasts after Subculture.
To examine the effect of {alpha}-particle irradiation on the progression of human diploid fibroblasts from G1 into S phase, cell populations synchronized in G0/G1 by confluent density inhibition of growth were irradiated and then immediately subcultured to low density in growth medium containing [3H]thymidine. The cells were incubated at 37°C to allow progression through the cell cycle. Movement into S phase was monitored autoradiographically by measuring the cumulative labeling indices at multiple time points up to 135 h after subculture. The effects of {alpha}-irradiation were compared with the kinetics of progression through the cell cycle in parallel cultures exposed to a dose of {gamma}-rays that yielded an isosurvival level (about 17%). The results in Fig. 3ACitation indicate a transient delay in the movement of {alpha}-irradiated (0.85 Gy) AG01521 cells into S phase. In addition, as compared to the nonirradiated control cells, a fraction of the {alpha}-irradiated cells never entered S phase as evidenced by the maximum CLI. These transient and apparently permanent arrests in G1 of {alpha}-irradiated cells are comparable to the arrests observed in {gamma}-irradiated (4 Gy) AG01521 cells (Fig. 3BCitation ). A delay of approximately 11 h in G1 was observed after either {alpha}-particle or {gamma}-irradiation, and about 11% of {alpha}-particle- or {gamma}-irradiated cells remained arrested in G1.



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  		Fig. 3. Cumulative labeling index as a function of time after release from confluent, density-inhibited growth of AG01521 cells that were either nonirradiated (——), irradiated (· · · · ·), or irradiated and held in confluence at 37°C for 6 h (- - - - -). A, {alpha}-particle irradiated; B, {gamma}-irradiated.

 
To examine whether the observed G1 delay and G1 arrest in {alpha}-irradiated AG01521 cells is a general phenomenon that occurs in other human diploid fibroblasts, similar experiments were carried out with GM06419 cells exposed to {alpha}-rays (0.75 Gy; Fig. 4ACitation ). This cell strain has been shown previously to exhibit substantial transient and permanent G1 arrests after {gamma}-irradiation (33) . The CLI data in Fig. 4Citation indicate that {alpha}-particle irradiation of these cells results in a more extensive transient delay and permanent arrest in G1 than occurs after exposure to a dose of {gamma}-ray (3.25 Gy) that yielded an isosurvival level (about 13%). Delays of 19 and 14.5 h in G1 were observed after {alpha}-particle and {gamma}-irradiation, respectively. Compared to nonirradiated control cells, the maximum CLI data indicate that 42 and 32% of the cells remained arrested in G1 for up to 135 h after {alpha}-particle and {gamma}-irradiation, respectively. Collectively, these results indicate that a G1 delay and a G1 permanent arrest occur in {alpha}-particle irradiated human diploid fibroblasts. At isosurvival levels, the magnitude of these arrests is at least as extensive as that observed in parallel cultures exposed to {gamma}-radiation.



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  		Fig. 4. Cumulative labeling index as a function of time after release from the confluent state of GM06419 cells that were either non-irradiated (——), irradiated (· · · · ·), or irradiated and held in confluence at 37°C for 6 h (- - - - -). A, {alpha}-particle irradiated; B, {gamma}-irradiated.

 
Lack of PLDR in {alpha}-Particle Irradiated Cells Correlates with a Lack of Reduction in the Fraction of Cells Permanently Arrested in G1.
A lack of PLDR after irradiation with {alpha}-particles has been reported previously in Chinese hamster ovary (34) and mouse 3T3 (35 , 36) cells. The results in Fig. 5Citation indicate that holding {alpha}-particle irradiated AG01521 fibroblasts at 37°C for 24 h also did not lead to increased survival (PLDR). In fact, a trend toward a decrease in survival was observed (significant at 5 and 7 h in Fig. 5Citation ) as a result of holding {alpha}-particle irradiated cells at 37°C. In contrast, significant PLDR was observed in parallel cultures exposed to {gamma}-irradiation (Fig. 5Citation ; similar results, not shown, were observed with GM06419 cells). Significantly, the lack of PLDR in {alpha}-particle irradiated human fibroblasts correlated with a lack of reduction in the transient G1 delay and the fraction of cells permanently arrested in G1 (Figs. 3Citation 4Citation ). Holding of {alpha}-particle irradiated AG01521 or GM06419 cells for 6 h after the exposure resulted in neither a reduction in the delay of entry into S phase nor an increase in the maximum CLI. In contrast, incubation of {gamma}-irradiated cells for the same period resulted in a reduction in the delay and in an increase in the maximum CLI (Figs. 3BCitation and 4BCitation ). At 50% of the maximum CLI, the G1 delay was reduced by 4.8 and 3 h in {gamma}-irradiated AG01521 and GM06419 cells, respectively; the maximum CLI increased from 83.5 ± 3.5 to 88.8 ± 3% in AG01521 cells and from 56 ± 1 to 65 ± 1.6% in GM06419 cells. These reductions in the fraction of {gamma}-irradiated cells permanently arrested in G1 correlate with the expression of PLDR in these cells after {gamma}-irradiation (Fig. 5Citation ) as described previously (37) .



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  		Fig. 5. Clonogenic survival of confluent AG01521 cells exposed to {gamma}-rays (4 Gy) or {alpha}-particles (0.85 Gy) and held in confluence at 37°C prior to subculture.

 
Cell Cycle Progression of {alpha}- or {gamma}-Irradiated wt, p53, or p21Waf1 null MEFs.
Next, we investigated whether the p53/p21Waf1signaling pathway is essential to induce the G1 checkpoint in {alpha}-particle irradiated cells or whether {alpha}-particle irradiation is able to induce a G1 checkpoint independently of p53. Quiescent wt, p53–/–, or p21Waf1–/– MEFs were exposed to isosurvival (about 10%) {alpha}-particle radiation doses of 1.75, 2.65, or 2.22 Gy, respectively. Immediately after irradiation, the cell populations were subcultured to low density as described for the human cells and CLI were measured at multiple time points up to 86 h after subculture. As for the human cells, the effects of {alpha}-particle irradiation were compared with the kinetics of progression through the cell cycle in parallel cultures exposed to {gamma}-ray doses (8, 10, and 12 Gy for wt, p53–/–, and p21Waf1–/– respectively) yielding an isosurvival level (about 10%). The data in Fig. 6, A and BCitation , indicate a transient G1 delay in the movement into S-phase of {alpha}-particle and {gamma}-irradiated wt cells. Transient delays of 8.4 and 10.4 h in G1 were observed in wt cells exposed to 8 or 1.75 Gy of {gamma}- or {alpha}-particle radiation, respectively. Interestingly, in contrast to human diploid fibroblasts, irradiated wt MEFs did not exhibit any first cycle permanent arrest in G1, indicating that the response of wt mouse cells differs from that of diploid human fibroblasts. After the transient delay in G1, {gamma}- or {alpha}-particle-irradiated wt MEFs reached maximal cumulative labeling indices similar to those of control nonirradiated wt cells.



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  		Fig. 6. Cumulative labeling indices as a function of time after release from quiescence of MEFs that were nonirradiated or exposed to {alpha}-particles or {gamma}-rays. A, wt, {alpha}-particles; B, wt, {gamma}-rays; C, p53–/–, {alpha}-particles; D, p53–/–, {gamma}-rays; E, p21Waf1–/–, {alpha}-particles; F, p21Waf1–/–, {gamma}-rays.

 
In contrast to wt MEFs, p53, and p21Waf1 null cells exposed to either {alpha}-particle or {gamma}-radiation progressed into S-phase without delay in G1 (Fig. 6, C–FCitation ). These results are different from those reported in previous studies (23 , 24) , which had indicated that exponentially growing p21Waf1 null MEFs exposed to {gamma}-rays exhibit a G1 delay that is intermediate in magnitude between wt and p53–/– cells. Our CLI data indicate that progression of control nonirradiated and {alpha}-particle and {gamma}-irradiated p21Waf1–/– cells into S phase was similar. Collectively, these data therefore indicate that similar to {gamma}-irradiation, p53 is essential in mediating the G1 checkpoint induced in {alpha}-particle irradiated cells and that its downstream effector p21Waf1 is the main mediator.

Induction of the G1 Checkpoint in Human Diploid Fibroblasts Exposed to Low {alpha}-Particle Fluences.
As GM06419 fibroblasts show a marked sensitivity to the ionizing radiation induced G1 checkpoint (Fig. 4Citation ), we investigated whether the effects of low-dose {alpha}-particle irradiation on cell cycle progression can be quantified in this cell strain. The data shown in Fig. 7Citation indicate that exposure to a mean dose as low as 1 cGy, at which about 9% of the nuclei are expected to be traversed by at least one {alpha}-particle, is able to transiently delay progression of the overall population into S-phase. Delays of 2 and 6 h in G1 were observed in two different experiments. Furthermore, a modest percentage of the cells (about 7%), comparable to the fraction of the cell population that is expected to be traversed by an {alpha}-particle track, appeared to be permanently arrested in G1. These data indicate that the end point of cell cycle progression is very sensitive and can be used to quantify the biological effects of exposure to low fluences of {alpha}-particles. Interestingly, compared to nonexposed cells, at 20–35 h after subculture, 15–20% of the cells in the exposed population were delayed in their progression into S, whereas only 9% are expected to have their nuclei traversed by an {alpha}-particle.



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  		Fig. 7. Cumulative labeling index as a function of time after release from the confluent state of nonexposed or {alpha}-particle-exposed (mean dose, 1 cGy) GM06419 cells.

 
To further investigate the observation that exposure to a mean dose of 1 cGy can induce a G1 checkpoint, we analyzed the expression levels of p53 and p21Waf1 proteins in confluent density inhibited GM06419 cell populations exposed to either 1 or 3 cGy and immediately subcultured to lower density (1:3). The data in Fig. 8Citation show that exposure to either dose results in induction of p21Waf1. Compared to control nonirradiated cells, a 1.5–2-fold increase was observed by densitometry 8 h after subculture in cells exposed to 1 or 3 cGy. These levels remained increased for about 16 h, a time that correlates with the onset of DNA synthesis in subcultured cells. The increased expression of p21Waf1 8–16 h after irradiation (Fig. 8Citation ) is consistent with the G1 delay observed in cell populations exposed to 1 cGy of {alpha}-particle irradiation (Fig. 7Citation ).



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  		Fig. 8. Western analyses of p21Waf1 expression in GM06419 cells exposed to {alpha}-particles. Confluent cells were exposed to mean doses of 0, 1, or 3 cGy and immediately subcultured (1:3). Samples harvested at 8–34 h were analyzed.

 
Expression Patterns of p53, p21Waf1, pRb, p34cdc2, and Cyclin B1 in {alpha}- or {gamma}-Irradiated Cells Released from Density-inhibited Growth and Correlation with the Onset of DNA Synthesis.
To characterize molecular events in the {alpha}-radiation induced G1 checkpoint, we examined and contrasted the expression patterns and kinetics of regulation of p53, p21Waf1, and other cell cycle-regulated genes in {alpha}-particle or {gamma}-irradiated G0/G1 cells that were subcultured and allowed to progress through the cell cycle. Confluent GM06419 or AG01521 cells were exposed to {alpha}-rays at 0.75 or 0.85 Gy, respectively, or {gamma}-rays at 3.25 or 4 Gy, respectively, and subcultured to low density immediately after the irradiation. Western analyses were performed with samples harvested 4–54 h later. (Fig. 9Citation 10Citation ). In both {alpha}- and {gamma}-irradiated GM06419 and AG01521 cells, up-regulation of p53 and p21Waf1 was detected within 4–6 h after subculture. The levels of these proteins remained elevated in the irradiated cells for the time course studied.



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  		Fig. 9. Western analyses of p53, p21Waf1, pRb, p34cdc2, and cyclin B1 in nonirradiated and irradiated GM0649 cells. Confluent cells were exposed to {alpha}-particles (0 or 0.75 Gy; A) or {gamma}-rays (0 or 3.25 Gy; B) and immediately subcultured after irradiation. Proteins were analyzed in both control (C) and irradiated ({alpha} or {gamma}) cells at 5–54 h after subculture.

 


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  		Fig. 10. Western analyses of p53, p21Waf1, pRb, p34cdc2, and cyclin B1 in nonirradiated and irradiated AG01521 cells. Confluent cells were exposed to {alpha}-particles (0 or 0.85 Gy; A) or {gamma}-rays (0 or 4 Gy; B) and immediately subcultured after irradiation. Proteins were analyzed in both control (C) and irradiated ({alpha} or {gamma}) cells at 4–32 h after subculture.

 
To investigate whether the induction of the p53/p21Waf1 signaling pathway results in similar downstream consequent changes in gene expression in cells exposed to either {alpha}-particle or {gamma}-radiation, we examined the patterns of expression of pRb, p34cdc2 and cyclin B1 in control and irradiated cells. The retinoblastoma protein is known to be permanently present in normal cells. In quiescent cells, it is hypophosphorylated and hinders the transcription of proliferation genes. In proliferating cells, its degree of phosphorylation rises in late G1, remains high in S and G2, and then falls back to a dephosphorylated state as the cell goes through mitosis (38) . The expression pattern of CDC2 and Cyclin B1 is regulated during the cell cycle (39 , 40) . Their levels start to rise in late G1, are maintained high in S/G2, and decrease in G2-M. The data in Figs. 9Citation 10Citation show that in subcultured nonirradiated GM06419 and AG01521 control cells, the levels of pRb, p34cdc2, and cyclin B1 proteins were observed to accumulate over a 32–54-h time course, and pRb and p34cdc2 became progressively phosphorylated. The increase in the levels of p53 and p21Waf1 proteins in subcultured irradiated cells was correlated with a lack of increase in the levels of phosphorylated and dephosphorylated pRb and down-regulation of p34cdc2 and cyclin B1 beginning at 11–16 h. Furthermore, the accumulation in the levels of these proteins was significantly delayed in both {alpha} and {gamma}-irradiated cells. The delay in the synthesis of these proteins in {gamma}- or {alpha}-particle irradiated cells is consistent with the G1 delay observed in cells exposed to either type of radiation. These results indicate that similar molecular events leading to the regulation of expression of these genes occur as a result of exposure to either {gamma}- or {alpha}-particle radiation and redistribution of the cells in the cell cycle.

Effect on Gene Expression of Holding {alpha}- or {gamma}-Irradiated Quiescent Cells at 37°C for a Period of Time Prior to Subculture.
Next, we investigated the effects on p53, p21Waf1, and p34cdc2 expression levels caused by the lack of reduction in the magnitude of the transient and permanent G1 arrests induced by {alpha}-radiation in cells held at 37°C for a period of time prior to subculture to lower density. Confluent, density-inhibited AG01521 cells were exposed to {alpha}- and {gamma}-ray doses resulting in about 10% clonogenic survival. Irradiated and control cells were then subcultured to lower density either immediately after the exposure or after a 6-h holding period, and the cells were harvested for Western blot analysis. Fig. 11Citation is a simplified illustration of the modulation of expression of p53, p21Waf1, and p34cdc2 at 10–40 h after subculture. As described in Fig. 10Citation , p53 and p21Waf1 levels were increased in irradiated cells harvested 10–19 h after subculture. These increased levels were attenuated in {gamma}-irradiated cells held at 37°C for 6 h prior to subculture. In contrast, in {alpha}-particle irradiated cells, no attenuation was observed. In nonirradiated control cells, p34cdc2 accumulated over the time course of analysis and became progressively phosphorylated. In both {gamma}- and {alpha}-particle-irradiated cells, p34cdc2 levels were significantly decreased at 33 h after subculture. Incubation for 6 h prior to subculture resulted in significant attenuation of p34cdc2 down-regulation in {gamma}-irradiated cells but not in {alpha}-particle irradiated cells. At 40 h postexposure, the decrease in p34cdc2 levels in {alpha}-particle irradiated cells was minimal. However, further increase in its levels because of the 6 h holding period was also minimal, whereas in {gamma}-irradiated cells, the increase remained significant. These results are therefore consistent with the lack of reduction in the G1 arrest (Figs. 3ACitation and 4ACitation ) and the absence of PLDR (Fig. 5Citation ) in {alpha}-particle irradiated cells held at 37°C for a period of time after the exposure.



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  		Fig. 11. Western analyses of p53, p21Waf1, and p34cdc2 in cells that were nonirradiated, irradiated ({alpha}-particles or {gamma}-rays), or irradiated and held in confluence at 37°C for 6 h. Confluent AG01521 cells were immediately subcultured after the different treatments. Proteins were analyzed in cell samples harvested 10–40 h later. C, control nonirradiated and subcultured; 0, irradiated and immediately subcultured; 6, irradiated and held in confluence for 6 h prior to subculture.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
REFERENCES
 
The data described in this report provide clear evidence for the occurrence of p53-mediated G1 arrest in human diploid fibroblasts exposed to {alpha}-particles. At isosurvival levels, the magnitude of the observed transient arrest in G1 is as extensive as in {gamma}-irradiated cells. Also similar to {gamma}-irradiation, a permanent arrest in G1 occurred in {alpha}-particle irradiated human fibroblasts (Fig. 3ACitation ). The failure to observe a G1 arrest in earlier studies (7, 8, 9) may be attributable to the fact that cells with an abnormal p53 function were used and/or the fact that cell cycle analyses were done at limited sampling times after irradiation. Importantly, the G1 arrest is observed in cell populations exposed to mean doses as low as 1 cGy (Fig. 7Citation ).

The G1 checkpoint is presumed to ensure that DNA damage is repaired prior to DNA replication (1 , 2) . Hence, the previously described (36) inability of cells to repair chromosomal damage and damage leading to lethality after {alpha}-particle irradiation is not a consequence of an absent G1 checkpoint but is probably attributable to the complex nature of the damage (41, 42, 43, 44) and the inability of the cell to process it with accuracy and fidelity. Whereas energy deposits along ß particle tracks resulting from {gamma} and X radiation are separated by at least several tenths of a micrometer, experiments with {alpha}-particles indicate interaction distances of the order of nanometers, resulting in mainly DNA dsbs that are somewhat different in quality (e.g., type of endgroups and multiply damaged sites) or distribution (e.g., clusters of dsbs; Refs. 41, 42, 43, 44 ). Recent studies by Newman et al. (45) indicate that for every {alpha}-particle track that induces a dsb, there is a 44% probability of inducing a second break within 300 kbp, whereas for electron tracks, the probability is 10%. Furthermore, dsbs induced by {alpha}-particles have been reported to be repaired more slowly than those induced by X-rays, and a higher fraction remain unrepaired even after a long incubation (46) .

Our results indicate that holding nonproliferating human diploid fibroblasts at 37°C after {alpha}-particle irradiation led to neither a reduction in the G1 delay or in the fraction of cells permanently arrested in G1 nor to increased cellular survival upon release from the confluent state (Figs. 3Citation 4Citation 5Citation ). Consistent with previous results showing no reduction of chromosomal aberrations in rodent cells (36) , we also found no reduction in micronucleus formation in {alpha}-particle irradiated human fibroblasts held in confluence after the exposure (data not shown). These effects might have resulted from exposing the cells to a dose that causes a level of damage that is beyond the repair capacity of the cell. To test this possibility, we exposed human fibroblasts to doses of {alpha}-particles resulting in moderate cell killing (survival levels greater than 10%). Results similar to those obtained at high {alpha}-particle fluences were observed (data not shown). Furthermore, our protein expression data show that compared to irradiated cells that were immediately subcultured, the holding of {alpha}-particle irradiated cells under nonproliferation conditions for 6 h did not result in a decrease in the level of p53 or p21Waf1 proteins upon subculture (Fig. 11Citation ), as it did in {gamma}-irradiated cells. Similarly, as described here and in other reports (37) , PLDR expression, reductions in the fraction of cells permanently arrested in G1 and in chromosomal aberrations occurred in {gamma}-irradiated human diploid fibroblasts upon postexposure holding at 37°C. Collectively, our results therefore suggest that in normal human fibroblasts, only damage processing leading to increased survival allows cells to exit from permanent arrest in G1 and reenter the cell cycle. Our protein expression data emphasize the role of p53/p21Waf1 in ensuring that cells harboring severe DNA damage are prevented from proliferating.

The role of the p53 tumor suppressor protein in regulating the G1 checkpoint is now firmly established (1 , 2 , 22) . p53 has been shown to be induced by a variety of DNA damaging agents and is part of a feedback control that arrests cells at the restriction point through its activation of p21Waf1 (23 , 24) . p21Waf1 has been shown to be induced also by p53-independent mechanisms (25 , 26 , 47) involving non-DNA damage signaling processes. Therefore, it was reasonable to verify whether {alpha}-particle irradiation can induce a G1 checkpoint by p53-independent mechanisms. However, the lack of G1 delay in {alpha}-particle irradiated p53 null MEFs indicate that p53 is essential for the induction of the G1 checkpoint by {alpha}-particle radiation. Interestingly, our data indicate that the p53-regulated G1 arrest caused by {alpha}-particle or {gamma}-radiation is entirely mediated by p21Waf1, as a total lack of G1 delay also occurred in irradiated p21Waf1 null MEFs. Previous reports (23 , 24) indicated that upon exposure to {gamma}-radiation p21–/– cells can induce a G1 checkpoint intermediate in magnitude between wt and p53–/– cells, implying that p21Waf1 partially mediates the role of p53 in response to {gamma}-irradiation. In those experiments, cells were exposed to considerably higher doses (20 Gy). In particular, cell cycle analysis was performed by flow cytometry 24 h after the exposure; therefore, the results could be biased by differences in generation time. Interestingly, measurement of cell cycle progression by the cumulative labeling index technique revealed basic differences between human and mouse fibroblasts, because wt mouse cells did not exhibit a permanent arrest in G1 as observed with human cells. Hence, caution should always be used when extrapolating mouse data to human.

We have shown recently that down-regulation of several cell cycle-regulated genes in {gamma} or X-irradiated quiescent or proliferating human diploid fibroblasts is p53-dependent and involves p21Waf1 as a negative factor in this regulation (32 , 48) . The similar pattern and kinetics of p53 and p21Waf1 induction, which was followed by down-regulation of p34cdc2 and cyclin B1 by {alpha}-radiation in proliferating (Figs. 9Citation 10Citation ) or quiescent cells (not shown), are similar to those that we have recently described in {gamma}-irradiated normal human fibroblasts. Therefore, these data suggest that {alpha}-radiation modulates the expression of cell growth genes by mechanisms similar to those of {gamma}-radiation. However, the attenuated response for p53, p21Waf1, and p34cdc2 expression observed when {gamma}-irradiated cells were held for 6 h prior to subculture did not occur in {alpha}-particle irradiated cells, consistent with the lack of repair after {alpha}- particle irradiation.

When populations of cells were exposed to mean doses as low as 1 cGy, a greater fraction (15–20%) of cells than those of which the nuclei would be traversed (9%) were arrested in G1 (Fig. 7Citation ). The determination of the fraction of cell nuclei traversed depends on the application of Poisson statistics and estimates concerning cell geometry and {alpha}-particle energy loss. Taking into account the observed variation in GM06419 cell nuclei cross-sectional area, the fraction of nuclei traversed at 1 cGy could be between 8 and 11%. The calculation of {alpha}-particle traversals also assumes that the energy lost by the {alpha}-particle is local and not transported beyond the cell being traversed. This assumption is valid, as {delta}-rays from the tracks of {alpha}-particles of the energy used in this study have an average energy range of 11 nm and a maximum range of 200 nm (49) . The extent of effects observed therefore suggest that a greater fraction of cells than those of which the nuclei were traversed contribute to the transient G1 delay observed. Gap-junction intercellular communication (50) , diffusible factors secreted by the {alpha}-particle traversed cells (51, 52, 53) , and/or cytoplasmic effects (54) could be contributing to these bystander effects. Recently, a p53-induced export of growth suppressive stimuli from damaged cells to neighboring cells has been described (55) . Our results support the hypothesis that the biological effects of {alpha}-particle irradiation are not restricted to the response of individual cells to the DNA damage that they directly receive from a particle traversal and may occur in neighboring bystander cells, as we have described by in situ techniques for gene expression (50) .

In conclusion, this study further demonstrates the complexity of {alpha}-particle induced damage. This is reflected in the exponential nature of the survival curves of exposed human fibroblasts, the absence of sublethal and damage repair and PLDR, the lack of reduction in the fraction of cells permanently arrested in G1, and different patterns of gene expression under conditions favoring cellular recovery. Overall, our data confirm that {alpha}-particles are highly toxic and support their use in cancer therapy (11 , 12) in which {alpha}-particle emitters are conjugated to antibodies to specific tumor cell antigens. Furthermore, our studies in cells exposed to low fluences indicate that the end points of cell cycle progression and gene expression are very sensitive and may be used to measure cellular effects of exposures relevant to radiation protection.


   ACKNOWLEDGMENTS
 
We thank Tamara B. Gooding for dedicated technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Center Grant ES-00002 and United States Department of Energy Research Grant DEFG02-89ER62685. Back

2 To whom requests for reprints should be addressed, at Department of Cancer Cell Biology, Laboratory of Radiobiology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. Back

3 The abbreviations used are: LET, linear energy transfer; CLI, cumulative labeling index; dsb, double strand break; MEF, mouse embryo fibroblast; PLDR, potentially lethal damage repair; wt, wild-type. Back

Received 10/26/99. Accepted 3/17/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Irradiation
 Cell Survival Analysis
 Autoradiographic Measurement of...
 Western Analysis
 RESULTS
 DISCUSSION
 REFERENCES
 

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