Ionizing radiation induces genomic instability, which is transmitted through many generations after irradiation in the progeny of surviving cells. To detect delayed activation of p53, we constructed a reporter plasmid containing the p53-responsible promoter and the bacterial β-galactosidase (β-gal) gene and introduced it into human fibrosarcoma (HT1080) cells, which retain wild-type p53 function. The resultant clones induce β-gal protein after X-irradiation, and the induction kinetics were similar to those of p21WAF1/CIP1 protein. More than 90% of the cells were stained blue when the cells were incubated with X-gal 4 h after 6 Gy of X-rays, whereas very few control cells were β-gal positive. The primary colonies formed after 6 Gy of X-rays were collected, and they were subjected to secondary colony formation. We observed that a significant number of surviving colonies contained β-gal-positive cells, suggesting that delayed activation of p53 occurred in the progeny of irradiated cells. We also found higher frequency of phosphorylation of p53, NBS1, and CHK2/Cds1 in the progeny of surviving cells. Furthermore, foci formation of phosphorylated histone H2AX was detected in the progeny of surviving cells. These findings provide the possibility that the observed instability results from these DNA breaks, i.e., the breaks lead to delayed chromosome rearrangements, delayed cell death, and so forth, many generations after irradiation and that activation of p53 function may eliminate cells that have potentially accumulated genomic alterations.

Ionizing radiation induces genomic instability, which is transmitted through many generations after irradiation in the progeny of surviving cells (1, 2, 3, 4, 5). Induced genomic instability manifests as the induction of various delayed phenotypes such as delayed lethal mutation or delayed reproductive death (6, 7, 8), delayed chromosomal instability (9, 10, 11), and delayed mutation induction (12). Although genomic instability has been reported commonly in mammalian cells exposed to ionizing radiation, the mechanisms underlying the initiation and manifestation of radiation-induced genomic instability are not fully understood.

A previous study reported that X-rays, ethyl methane sulfonate, and restriction endonuclease HinfI, but not UV light, initiated delayed reproductive death (13). Another report showed that radiomimetic antibiotics, bleomycin, and neocarzinostatin were equally effective at inducing delayed chromosomal instability (14). These and other studies (15, 16) strongly suggest that DNA double strand breaks are the initiators. However, it was also noted that the restriction enzymes BsaHI, PvuII, EcoRV, and StuI do not induce delayed chromosomal instability, suggesting that DNA double strand breaks per se do not always lead to genomic instability (14, 17). Furthermore, other studies have suggested that an extranuclear target or a bystander effect may also be involved in the induction of instability (18, 19). Therefore, more than one mechanism may be involved in the initiation of radiation-induced genomic instability.

In contrast, very few studies have examined the mechanism of manifestation of radiation-induced genomic instability. Recent studies have proposed that persistent oxidative stress in the progeny of surviving cells contributes to the expression of instability (20, 21). In fact, our and other studies have shown that decreased oxygen concentration in culture reduces the delayed effects of radiation (22, 23). Previously, we hypothesized that the expression of delayed phenotypes resulted from unscheduled breakage of DNA (24). Ionizing radiation causes DNA double strand breaks, which are the initiators for reproductive death, chromosomal aberration, apoptosis, and mutation. Because all of these are manifestations of radiation-induced genomic instability, it is highly possible that delayed DNA damage is associated with delayed phenotypes. DNA double strand breaks are well known to accumulate and activate p53, a tumor suppressor protein (25, 26, 27). Recent studies have described that DNA double strand breaks are recognized by ATM, and ATM-mediated phosphorylation of p53 protein accumulates and activates p53 as a transcriptional factor (28, 29, 30, 31, 32, 33, 34, 35). It regulates transcription of the downstream genes such as p21Waf1/Cip1, gadd45, Reprimo, BAX, PIG-3, and p53AIP1 (36, 37, 38). As a result, activated p53 causes cell cycle arrest, apoptosis, and senescence-like growth arrest. It was also suggested that p53 is involved in base excision repair (39). It was estimated that one single DNA double strand break is enough to activate p53 (40), therefore, it is highly possible that DNA breaks arising in the progeny of surviving cells activate p53 protein. Moreover, radiation-induced genomic instability could be the driving force underlying the development of radiation-induced carcinogenesis by accumulating genetic alterations (41, 42, 43). If p53 functions is a guardian of the genome, there should be delayed p53 activation in the progeny of surviving cells.

In this study, we constructed a reporter plasmid by which the activation of p53 was examined in situ. Furthermore, we detected delayed phosphorylation of p53, NBS1, and CHK2/Cds1 protein, and histone H2AX, which is phosphorylated at the site of DNA breaks. The results suggest that the observed genomic instability result from these delayed DNA breakage, and activation of p53 function may eliminate cells that have potentially accumulated genomic alterations.

Cell Cultures and Irradiation.

Human fibrosarcoma (HT1080) cells were cultured in Eagle’s MEM supplemented with 10% fetal bovine serum (Trace Bioscience PTY Ltd., A.C.N., Australia). HT1080 cells were obtained from Japanese Cancer Research Resources Bank. Cells seeded in T25 flasks (25 cm2) were subcultured every 3–4 days to maintain exponential growth. For X-irradiation, cells were irradiated with X-rays from a X-ray generator at 150 kVp and 5 mA with a 0.1-mm copper filter. The dose rate for X-irradiation was 0.44 Gy/min.

Plasmid Construction.

PG13Pyβ-gal plasmid was kindly provided by Dr. Wafik S. El-Deiry of the Howard Hughes Medical Institute (44). It was digested with XhoI and XbaI, and the 3.5-kb XhoI-XbaI fragment was ligated with the pREP4 plasmid digested with XhoI and NheI. The resulting pREP-PG13-β-gal plasmid (Fig. 1) was transfected into HT1080 cells by electroporation, and HTHyR clones harboring pREP-PG13-β-gal plasmid were cloned in the presence of 800 μg/ml hygromycin B.

Experimental Protocol.

As shown in Fig. 2, exponentially growing HTHyR1 clone was divided into two culture dishes, and control and X-irradiated cells were plated onto six dishes to form ∼300 primary colonies. After 14 days of incubation, three dishes were fixed with methanol and stained with 3% Giemsa’s solution to determine plating efficiency. The primary colonies from the remaining three dishes were cloned, the number of cells were counted, and they were reseeded into six culture dishes for secondary colony formation. Alternatively, the primary colonies were collected and pooled, and they were replated into six culture dishes to perform secondary colony formation. Both unirradiated and irradiated populations were cultured for 14 days, and three dishes were used to determine plating efficiency. Colonies derived from the remaining three dishes were collected and used as cells 30–35 PDN3 after irradiation.

Western Blot Analysis.

Cells were lysed in radioimmunoprecipitation assay buffer [50 mm Tris-HCl (pH 7.2), 150 mm NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS] containing 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride as described previously (28). The cell lysate was cleared by centrifugation at 15,000 rpm for 10 min at 4°C, and the supernatant was used as total cellular protein. The protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL). Protein samples (16 μg) were electrophoresed on SDS-polyacrylamide gel. The proteins were electrophoretically transferred to a polyvinyl difluoride membrane in a transfer buffer (100 mm Tris, 192 mm glycine). After overnight incubation with blocking solution (10% skim milk), the membrane was incubated with anti-β-gal monoclonal antibody (Oncogene Research Products, Boston, MA). It was then incubated with a biotinylated secondary antibody and streptavidin-alkaline phosphatase. The bands were visualized after addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as substrate.

Assay for β-Galactosidase Activity in Situ.

In situ β-gal staining with X-gal was performed as previously described with slight modification (45). Briefly, cells were fixed in PBS containing 2% formaldehyde and 0.05% glutaraldehyde, washed twice with PBS, and then incubated with 1 mg/ml X-gal dissolved in PBS containing 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, and 2 mm MgCl2.

Immunocytochemical Staining.

Cells plated onto 22 × 22-mm coverslips in 35-mm dishes, or colonies grown on the cover slips were fixed with 4% formaldehyde in PBS, treated with 0.5% Triton X-100 in PBS, washed extensively with a sufficient amount of PBS, and then incubated with rabbit polyclonal antibody against phosphorylated p53 at serine 15, phosphorylated NBS1 at serine 343, and phosphorylated CHK2/Cds1 at threonine 68 (Cell Signaling Technology, Beverly, MA) and phosphorylated histone H2AX (Upstate Biotechnology, Lake Placid, NY). The primary antibodies were visualized by incubating cells with FITC-conjugated antirabbit antibody (Amersham, Japan, Tokyo) for 2 h at 37°C. The data were taken from one experiment, and the statistical significance was determined using Student’s t test with P < 0.01 considered as statistically significant.

Establishment of the HT1080 Clones Containing the p53-inducible β-Gal Gene.

To examine p53 activation in situ, we constructed a reporter plasmid containing multiple p53-binding sites and the bacterial β-gal gene. Because the plasmid, pREP-PG-13-β-gal, contains a hygromycin B-resistant gene (Fig. 1), transfected clones were selected in the presence of 400 μg/ml hygromycin B. The resultant HTHyR clones were analyzed for their ability to induce β-gal protein in response to ionizing radiation. Among 31 hygromycin B-resistant colonies, 11 clones did not express β-gal protein, and 8 clones expressed basal levels of β-gal protein, but it could not be induced after X-irradiation. Among the 12 remaining clones, we selected a HTHyR1 clone for their low levels of basal expression and high inducibility.

As indicated in Fig. 3, p53 protein in HTHyR1 clone was accumulated after 6 Gy of X-rays. The increased level of p53 protein was detected 1 h after irradiation, and it lasted for >24 h after irradiation. Accumulation of p53 protein increased p21WAF1/CIP1 protein level, which was induced 1 h after irradiation. We detected very low levels of β-gal expression under nonirradiated conditions, but the level was significantly increased after irradiation. The induction kinetics were similar to those of p21WAF1/CIP1(Fig. 3,B). Four h after X-irradiation with 6 Gy, cells were stained with X-gal, and >90% of cells were stained blue, whereas only 0.2% were β-gal positive in the control culture (Fig. 3, C and D).

Radiation-induced Genomic Instability in HTHyR1 Clone.

Induction of genomic instability by X-irradiation was examined in HTHyR clones. As shown in Fig. 2, cell cultures were divided into two, and one culture was irradiated with X-rays. The surviving primary colonies were collected and subjected to secondary colony formation. The average number of cells per colony was calculated by dividing the number of total cells by the number of colonies collected, and the mean population doubling number was estimated as 15–20 for the primary colonies, and 30–35 for the secondary colonies. To check delayed reproductive death, HTHyR1 clone was irradiated with various doses of X-rays, and the plating efficiency of the secondary colonies was determined (Fig. 4). Although the plating efficiency obtained immediately after irradiation decreased with increasing X-ray dose, the plating efficiency of the secondary colonies was still lower than that of the unirradiated controls, indicating the induction of delayed reproductive death. A persistent reduction in the plating efficiency was also confirmed in the progeny of 6 Gy-surviving cells 30–35 PDN after irradiation in HTHyR1 clone (Table 1). The frequency of colonies containing more than five giant cells was higher in surviving colonies than in control colonies. Furthermore, the frequency of dicentric chromosomes was consistently higher in the progeny of surviving cells. These findings clearly indicate that genomic instability is induced in the progeny of HTHyR1 clone irradiated with X-irradiation.

Delayed Activation of p53 in the Progeny of Surviving Cells.

The secondary colonies were stained with X-gal. In the control colonies, very few cells were stained blue heavily, although there was some background staining. In contrast, cells in the colonies formed by the progeny of 6 Gy-surviving cells were frequently β-gal positive (Fig. 5). The staining intensity was comparable with that observed in cells 4 h after 6 Gy of X-rays (Fig. 3,D). The frequency of colonies containing more than six stained cells/colony was <5% in all control clones (Table 2), whereas it was significantly increased (P < 0.01) in 16 of 18 surviving clones.

Detection of Phosphorylation of p53, NBS1, CHK2/Cds1, and Histone H2AX in the Progeny of Surviving Cells.

Cells surviving 6 Gy of X-rays were immunostained by antibody recognizing phosphorylated p53, NBS1, CHK2/Cds1, and phosphorylated H2AX at 30–50 PDN level after irradiation. As shown in Table 3, frequency of phospho-p53, phospho-NBS1, and phospho-CHK2/Cds1-positive cells was significantly higher in the progeny of 6 Gy-surviving cells. In Fig. 6, we compared the frequency of cells whose nuclei were stained with antiphospho histone H2AX antibody. The cells containing more than five nuclear foci were counted. Although very few cells were phospho-H2AX positive in the control cells, a significant number of cells showed phospho-H2AX foci, and half of them contained >20 foci, which was not detected in the control cells (Table 4).

In this study, we constructed a reporter system by which p53 activation was determined in situ. The induction of β-gal protein after irradiation was comparable with the induction of p21WAF1/CIP1 protein, indicating that transcription of the β-gal gene was regulated similarly to that of the p53-downstream effectors. The transfected clones were irradiated and cultured for primary colonies. The surviving colonies were then collected and subjected to secondary colony formation. The cells were estimated to go through 30–35 PDN after irradiation in forming the secondary colonies. Induction of genomic instability was confirmed as cells showed various delayed phenotypes such as delayed reproductive death, delayed giant cell formation, and delayed chromosomal instability (Table 1). Delayed p53 activation was examined in the secondary colonies. As shown in Fig. 4 and Table 2, we observed frequent induction of the colonies containing β-gal-positive cells, which predicts activation of p53, in the colonies formed by the progeny of surviving cells but not in the control colonies. These findings provide the possibility that the induced genomic instability causes delayed p53 activation many generations after irradiation. Although p53 activation is induced by DNA strand breaks in addition to several other mechanisms (27), the present results suggest that delayed DNA breakage may be induced in the progeny of surviving cells. To test this possibility, we performed immunostaining of the cells using antibody against phosphorylated p53 protein, NBS1 protein, CHK2/Cds1 protein, and phosphorylated histone H2AX protein. It was reported that DNA double strand breaks induce phosphorylation of H2AX, one of three members of histone H2A in mammalian cells (46). The number of phosphorylated H2AX foci was well correlated with the number of DNA double strand breaks, indicating that histone H2AX is phosphorylated at sites of DNA damage (47, 48). More recently, it was reported that histone H2AX was phosphorylated in response to DNA double strand breaks through ATM (49). The frequency of cells with phospho-p53-, phospho-NBS1-, phospho-CHK2/Cds1-, and H2AX-positive nuclei increased significantly in the surviving cells and in the colonies formed by the progeny of surviving cells. In addition, some surviving cells contained H2AX foci, the numbers of which correspond to 1 Gy equivalent dose. These results suggest that DNA breakage may be induced frequently in the progeny of surviving cells, and this causes delayed activation of p53 through activation of ATM.

Persistent genomic instability, or a mutator phenotype, can explain the higher frequency of genetic alterations in cancer cells, which cannot be explained by random mutation (50). Therefore, it has been hypothesized that radiation-induced genomic instability could be the driving force underlying radiation carcinogenesis (41, 42, 43). However, no previous studies have shown that delayed DNA damage occurs in the progeny of surviving cells. We showed here that delayed DNA breakage was induced frequently up to 30–35 PDN after irradiation. These unscheduled DNA breaks may result in accumulation of genomic alterations. Because activated p53 protein in response to ionizing radiation has been thought to protect the integrity of the genome, delayed activation of p53 may also play a role as a guardian of the genome. Our results support that activated p53 causes delayed cell death, including delayed apoptosis as described previously (20, 21). We also found that some of the p53-positive cells were giant cells and that some of them induced senescence-associated β-gal, which has been identified as a marker for senescence-like growth arrest, one mode of reproductive death observed after ionizing radiation (51). Therefore, radiation-induced genomic instability causes accumulation of genetic alterations, but delayed activation of p53 plays an indispensable role in eliminating the damaged cells and maintaining genomic integrity.

Although several studies have described the expression of delayed phenotypes, the molecular mechanism(s) involved in delayed induction of DNA damage have not been determined. We propose that potentially unstable chromosome regions resulting from DNA repair of double strand breaks are involved in delayed DNA breakage (24). A similar possibility was proposed previously, which implicated the interstitial telomere repeat-like sequences, or recombinational hotspots, in perpetuating chromosomal instability (1, 10). These regions may cause delayed breaks, although it is still possible that they are consequence of the instability phenotype. Another possibility is that persistent oxidative stress in the progeny of surviving cells causes delayed DNA damage (20, 21). In fact, our recent study showed that decreased oxygen concentration in culture reduces delayed effects (22). The third possibility is that DNA breakage is generated through a bridge-breakage-fusion cycle. Previous studies demonstrated that dicentrics were the hallmark of chromosomal instability, and they provided a chance to cause DNA breakage during anaphase (10). Although several mechanisms may be involved in the induction of delayed DNA breakage, it may be explained in part by the mechanism of manifestation of radiation-induced genomic instability. Additional studies are required to clarify the mechanism that causes delayed DNA breakage for prevention of accumulated genetic alterations in the progeny of surviving cells exposed to ionizing radiation.

Fig. 1.

Plasmid map of the reporter plasmid containing the p53-responsible promoter. A 3.5-kb XhoI-XbaI fragment containing a multiple p53 binding sites and the bacterial β-gal gene was ligated with the pREP4 plasmid. The selectable marker hygromycin is under control of the thymidine kinase gene promoter.

Fig. 1.

Plasmid map of the reporter plasmid containing the p53-responsible promoter. A 3.5-kb XhoI-XbaI fragment containing a multiple p53 binding sites and the bacterial β-gal gene was ligated with the pREP4 plasmid. The selectable marker hygromycin is under control of the thymidine kinase gene promoter.

Close modal
Fig. 2.

Schematic diagram of the experimental protocol used to examine the induction of genomic instability in HTHyR1 clone. Control and X-irradiated cells were plated onto culture dishes to form ∼300 primary colonies. After 14 days of incubation, the primary colonies were subjected to secondary colony formation as described in “Materials and Methods.”

Fig. 2.

Schematic diagram of the experimental protocol used to examine the induction of genomic instability in HTHyR1 clone. Control and X-irradiated cells were plated onto culture dishes to form ∼300 primary colonies. After 14 days of incubation, the primary colonies were subjected to secondary colony formation as described in “Materials and Methods.”

Close modal
Fig. 3.

Accumulation of p53 and induction of β-gal protein. Exponentially growing HTHyR1 cells were irradiated with 6 Gy of X-rays and incubated for the time indicated. A, levels of p53, p21WAF1CIP1, and β-gal proteins were determined by Western blot analysis. The amount of β-tubulin (β-tub) was determined as a loading control. B, the blot was scanned densitometrically, and relative amount of proteins was calculated. C, photograph of the control cells stained with X-gal in situ. D, cells irradiated with 6 Gy of X-rays, and incubated for 4 h before staining with X-gal. The cells densely stained blue were counted as β-gal-positive cells.

Fig. 3.

Accumulation of p53 and induction of β-gal protein. Exponentially growing HTHyR1 cells were irradiated with 6 Gy of X-rays and incubated for the time indicated. A, levels of p53, p21WAF1CIP1, and β-gal proteins were determined by Western blot analysis. The amount of β-tubulin (β-tub) was determined as a loading control. B, the blot was scanned densitometrically, and relative amount of proteins was calculated. C, photograph of the control cells stained with X-gal in situ. D, cells irradiated with 6 Gy of X-rays, and incubated for 4 h before staining with X-gal. The cells densely stained blue were counted as β-gal-positive cells.

Close modal
Fig. 4.

Survival curve of the mixture of four HTHyR clones immediately after irradiation (•) and 30–35 PDN level after irradiation (○).

Fig. 4.

Survival curve of the mixture of four HTHyR clones immediately after irradiation (•) and 30–35 PDN level after irradiation (○).

Close modal
Fig. 5.

Photographs of the colonies stained with X-gal in situ. Control colony (A) and the colony formed by the progeny of 6 Gy-surviving cells (B) were stained with X-gal at 30–35 PDN level after irradiation (×100). A part of the 6 Gy-surviving colony was shown in C (×400).

Fig. 5.

Photographs of the colonies stained with X-gal in situ. Control colony (A) and the colony formed by the progeny of 6 Gy-surviving cells (B) were stained with X-gal at 30–35 PDN level after irradiation (×100). A part of the 6 Gy-surviving colony was shown in C (×400).

Close modal
Fig. 6.

Photographs of the cells immunostained with antiphospho histone H2AX antibody. Control cells (A) and 6 Gy-irradiated cells subcultured for 30–35 PDN after irradiation (B) were stained on the coverslips as described in “Materials and Methods.” Cells whose nuclei contained more than five foci were counted (indicated by arrowheads).

Fig. 6.

Photographs of the cells immunostained with antiphospho histone H2AX antibody. Control cells (A) and 6 Gy-irradiated cells subcultured for 30–35 PDN after irradiation (B) were stained on the coverslips as described in “Materials and Methods.” Cells whose nuclei contained more than five foci were counted (indicated by arrowheads).

Close modal

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1

This work was supported by a grant for scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.

3

The abbreviations used are: PDN, population doubling number; β-gal, β-galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ATU, ataxia telangiectasia mutated.

Table 1

Genomic instability induced by 6 Gy of X-rays at 30–35 PDN level after irradiation

CellsPlating efficiency (%)aNo. colonies with giant cells (%)bNo. of metaphases with dicentrics (%)
HT-HyR1    
0 Gy 53 ± 2 0/345 1/200 (0.5) 
6 Gy 31 ± 3 18/381 (4.7) 27/200 (13.5) 
CellsPlating efficiency (%)aNo. colonies with giant cells (%)bNo. of metaphases with dicentrics (%)
HT-HyR1    
0 Gy 53 ± 2 0/345 1/200 (0.5) 
6 Gy 31 ± 3 18/381 (4.7) 27/200 (13.5) 
a

Percentage of average plating efficiency ± SD.

b

Colonies containing more than five giant cells were counted.

Table 2

Delayed activation of p53 in HTHyR1 cells surviving 6 Gy of X-rays (30–35 PDN level)

ClonesPlating efficiency (%)No. of β-gal-positive colonies/no. of colonies counted (%)a
Control clones   
 0-1 56.1 3/171 (1.8) 
 0-2 52.2 2/183 (1.1) 
 0-3 49.7 5/187 (2.7) 
 0-5 56.2 3/172 (1.7) 
 0-6 51.9 1/161 (0.6) 
 0-7 47.6 4/173 (2.3) 
 0-8 55.3 3/191 (1.6) 
 0-9 56.6 4/186 (2.2) 
 0-10 54.2 3/161 (1.9) 
 0-12 53.7 5/189 (2.6) 
 0-13 54.4 3/168 (1.8) 
 0-14 41.2 6/185 (3.2) 
 0-15 56.9 2/182 (1.1) 
6 Gy-surviving clones   
 6-1 36.2 33/161 (20.5)b 
 6-2 31.0 45/172 (26.2)b 
 6-3 46.2 13/187 (7.0)b 
 6-4 26.7 56/154 (36.4)b 
 6-5 30.4 41/169 (24.3)b 
 6-6 31.2 43/153 (28.1)b 
 6-7 30.5 43/171 (25.1)b 
 6-8 27.9 51/185 (27.6)b 
 6-10 34.1 47/176 (26.7)b 
 6-11 56.7 11/198 (5.6) 
 6-12 33.0 39/164 (23.8)b 
 6-13 30.5 47/176 (26.7)b 
 6-14 31.0 53/188 (28.2)b 
 6-15 36.9 39/173 (22.5)b 
 6-151 36.2 33/165 (20.0)b 
 6-16 36.9 25/174 (14.4)b 
 6-18 29.4 46/178 (25.8)b 
 6-20 26.1 59/156 (37.8)b 
ClonesPlating efficiency (%)No. of β-gal-positive colonies/no. of colonies counted (%)a
Control clones   
 0-1 56.1 3/171 (1.8) 
 0-2 52.2 2/183 (1.1) 
 0-3 49.7 5/187 (2.7) 
 0-5 56.2 3/172 (1.7) 
 0-6 51.9 1/161 (0.6) 
 0-7 47.6 4/173 (2.3) 
 0-8 55.3 3/191 (1.6) 
 0-9 56.6 4/186 (2.2) 
 0-10 54.2 3/161 (1.9) 
 0-12 53.7 5/189 (2.6) 
 0-13 54.4 3/168 (1.8) 
 0-14 41.2 6/185 (3.2) 
 0-15 56.9 2/182 (1.1) 
6 Gy-surviving clones   
 6-1 36.2 33/161 (20.5)b 
 6-2 31.0 45/172 (26.2)b 
 6-3 46.2 13/187 (7.0)b 
 6-4 26.7 56/154 (36.4)b 
 6-5 30.4 41/169 (24.3)b 
 6-6 31.2 43/153 (28.1)b 
 6-7 30.5 43/171 (25.1)b 
 6-8 27.9 51/185 (27.6)b 
 6-10 34.1 47/176 (26.7)b 
 6-11 56.7 11/198 (5.6) 
 6-12 33.0 39/164 (23.8)b 
 6-13 30.5 47/176 (26.7)b 
 6-14 31.0 53/188 (28.2)b 
 6-15 36.9 39/173 (22.5)b 
 6-151 36.2 33/165 (20.0)b 
 6-16 36.9 25/174 (14.4)b 
 6-18 29.4 46/178 (25.8)b 
 6-20 26.1 59/156 (37.8)b 
a

Colonies containing more than six stained cells were counted as β-gal positive.

b

Compared with control clones: P < 0.01.

Table 3

Frequency of cells positive for phospho-p53, phospho-NBS1, and phospho-CHK2/Cds1 30–35 PDN after irradiation

CellsNo. of phosphorylation-positive cells (%)No. of cells counted
Phospho-p53-positive cells   
 Control 12 (0.8) 1521 
 6 Gy 187 (12.4)a 1514 
Phospho-NBS1-positive cells   
 Control 14 (0.8) 1754 
 6 Gy 112 (6.7)a 1671 
Phospho-CHK2/Cds1-positive cells   
 Control 9 (0.6) 1557 
 6 Gy 142 (9.0)a 1578 
CellsNo. of phosphorylation-positive cells (%)No. of cells counted
Phospho-p53-positive cells   
 Control 12 (0.8) 1521 
 6 Gy 187 (12.4)a 1514 
Phospho-NBS1-positive cells   
 Control 14 (0.8) 1754 
 6 Gy 112 (6.7)a 1671 
Phospho-CHK2/Cds1-positive cells   
 Control 9 (0.6) 1557 
 6 Gy 142 (9.0)a 1578 
a

Compared with control clones: P < 0.01.

Table 4

Frequency of cells containing phospho-H2AX-positive cells 30–35 PDN after irradiation

CellsNo. of cells with phospho-H2AX-positive cellsNo. of cells counted
0–56–2021–4041–60a
Control 1035 
6 Gy 11 17b 14b 17b 1123 
CellsNo. of cells with phospho-H2AX-positive cellsNo. of cells counted
0–56–2021–4041–60a
Control 1035 
6 Gy 11 17b 14b 17b 1123 
a

Number of foci per nucleus.

b

Compared with control clones: P < 0.01.

We thank Dr. Wafik S. El-Deiry for providing the PG13Pyβ-gal plasmid. We also thank Dr. John B. Little and Dr. William F. Morgan for their critical reading of this manuscript.

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