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[Cancer Research 63, 7190-7196, November 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Functional p53 Increases Prostate Cancer Cell Survival After Exposure to Fractionated Doses of Ionizing Radiation1

Susan L. Scott, John D. Earle2 and Paul H. Gumerlock3

Division of Hematology/Oncology, Department of Internal Medicine [S. L. S., P. H. G.] and Department of Radiation Oncology [J. D. E.], University of California, Davis Cancer Center, Sacramento, California 95817


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
External beam radiation therapy is an effective therapy for localized prostate cancer, although failures occur at high rates. One variable that may affect the radiosensitivity of prostate tumor cells is their p53 status because this gene controls radiation-induced cell cycle arrest, apoptosis, and the repair of DNA damage. Using a system in which p53 function was conditionally restored to p53-null PC3 prostate cancer cells by stable transfection with a human temperature-sensitive p53 mutant allele, we tested the hypothesis that functional p53 increases cell cycle arrest and contributes to increased clonogenic survival after ionizing radiation (IR) of prostate carcinoma cells. Cell cycle arrest and clonogenic survival in response to single and multiple daily exposures to clinically relevant 2-Gy doses of IR were examined. Whereas the temperature-sensitive p53 protein was activated by phosphorylation after IR exposure at both the restrictive and permissive temperatures, Cdkn1/p21 was only induced by functional p53 (at the permissive temperature). In the presence of functional p53, the maintenance of G2 arrest was significantly longer (P < 0.01), and a small increase in cell survival measured by clonogenic assay was seen after exposure to a single 2-Gy dose of IR. However, functional p53 significantly increased clonogenic survival (P < 0.01) after exposure to daily doses of 2 Gy of IR and contributed to a more sustained G2 arrest and increased G1 arrest in response to the multifraction regimen. These studies implicate the presence of wild-type p53 with increased survival of prostate carcinoma cells after fractionated exposure to radiation. Additionally, the data provide evidence that wild-type p53 in prostate tumor cells may reduce the effectiveness of radiation therapy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CaP4 is the second leading cause of cancer death among American men, exceeded only by lung cancer (1 , 2) . Nearly 90% of patients will present with presumed localized disease, making them candidates for potential curative local therapy, which currently includes radiation therapy, radical prostatectomy, and, in some cases, active surveillance. As a treatment option, EBRT is effective for localized disease. However, using prostate-specific antigen levels as a marker for tumor control, patients who present with organ-confined disease have a 5-year failure rate of 10–40% after EBRT (3 , 4) .

Exposure to IR results in DNA double-strand breaks and other cellular injuries. Cellular responses to radiation-induced DNA damage include cell cycle arrest and DNA damage repair, responses needed for cell survival, and cell death, which occurs through both mitotic and apoptotic processes when damage is either unrepairable or misrepaired (5, 6, 7) . Thus, signaling pathways and control of cell cycle progression are closely linked to the intrinsic radiation sensitivity of cells (8 , 9) . These pathways may include targets that can be exploited to enhance the efficacy of radiation therapy, particularly for those tumor types such as CaP that are considered to be relatively radioresistant.

The p53 status of prostate tumor cells may affect their radiosensitivity. p53 is involved in cell cycle arrest, apoptosis, and the repair of DNA damage (10, 11, 12, 13, 14) . It has been well established that the p53 protein contributes to both G1 and G2 arrests through transcriptional regulation that includes the induction of CDKN1/p21 (G1 and G2 arrests), 14-3-3{sigma} (G2 arrest), and, most recently, REPRIMO (G2 arrest; Refs. 15, 16, 17, 18, 19 ). Some studies have demonstrated that cells lacking p53 or having a mutated p53 show more radioresistant phenotypes (20, 21, 22, 23) . Explanations for the increase in radioresistance in cells with mutant p53 include the lack of cell cycle checkpoint arrest and p53-dependent apoptosis, each of which can increase radioresistance (24 , 25) . Additionally, some mutations in p53 result in a gain of function, which may also result in an increase in radiation resistance (26) . However, other investigators have reported that cell lines that lack both wild-type p53 alleles or express mutant p53 either do not show significant changes in their radiosensitivity or are more sensitive to radiation (27, 28, 29, 30, 31) . Thus, relationships between the p53 status of cells and their radiosensitivity are less than clear and may vary as a function of cell type, the specific p53 mutation, or the profile of genomic alterations present in the different tumor cells.

It is generally thought that the arrest maintenance role of p53 in response to radiation-induced DNA damage is, at least in part, to allow sufficient time for DNA damage repair before DNA replication (G1 arrest) or before entering mitosis (G2 arrest). If the cell cycle arrest functions of p53 are protective, they may be particularly significant for cell types in which apoptosis has a minor role in the overall response to IR. This may be the case in prostate cancer, as suggested by the low numbers of radiation-induced apoptotic cells detected in CaP cell lines (32, 33, 34) . Additionally, these survival mechanisms may be especially relevant to IR treatment of the ~65% of primary prostate tumors that retain wt p53 (35) . Thus, we hypothesized that functional p53 increases cell cycle arrest and contributes to increased clonogenic survival after IR of CaP cells. A model system was used in which p53 function was conditionally restored by stable transfection with a temperature-sensitive p53 mutant allele (P223L) isolated from a human prostate cancer cell line. This study demonstrates the restoration of p53 function in prostate cancer cells at the permissive temperature of 32°C and uses the PC3tsp53 prostate cancer model to examine cell cycle arrest and clonogenic survival in response to clinically relevant doses of IR.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Tissue Culture.
The prostate cancer cell line PC3 (p53 null) used in this study was obtained from the American Type Culture Collection. The PC3 cells are hemizygous for chromosome 17p, and their single copy of the p53 gene has a bp deletion at codon 138 that has caused a frameshift and a new in-frame stop codon at position 169 (36) . As a result, PC3 cells do not express p53 protein (37) . The PC3V2 vector control cell line (PC3-vector) containing an empty pCR3.1 vector (Invitrogen, Carlsbad, CA) was a generous gift from Dr. Xu-Bao Shi (University of California Davis Cancer Center). The PC3tsp53 stable transfectants were created as described below. Cells were routinely cultured in Ham’s F-12 media (BioWhittaker, Walkersville, MD) supplemented with 7% heat-inactivated fetal bovine serum (Omega Scientific, Tarzana, CA), L-glutamine, vitamins, fungizone, penicillin, and streptomycin. G418 antibiotic (500 µg/ml) was added to the medium to maintain selection for the transfected plasmids. Cells were maintained at 37°C in 5% CO2 in air for routine culture.

Stable Transfection of the P223L Allele into PC3 Cells.
PC3 cells were transfected with the pCR3.1 vector (Invitrogen) that contains the pCMV promoter and the P223L (proline -> leucine at codon 223) mutant p53 allele (a generous gift from Dr. Xu-Bao Shi) using a lipid-based DNA transfection reagent complexed with 6 µg of plasmid DNA in serum-free media (Lipofectin Reagent; Life Technologies, Inc., Gaithersburg, MD; Ref. 38 ). Cells were grown for 4 h at 37°C, 5% CO2 before replacing the media with standard media containing 7% fetal bovine serum. After 48 h, the transfected cells were grown under G418 (500 µg/ml) selection for approximately 1 month until individual colonies proliferated. Clones were picked and expanded in T-25 flasks before being transferred to T-75 flasks for routine culturing. A total of five clones were isolated, expanded, and screened for expression of p53. Two of the five G418-resistant clones were positive for expression of p53, with equivalent levels of p53 protein. Clone 5 was selected for use in the experiments reported here, and the subline was named PC3tsp53.

Restoration of Functional p53 in the PC3tsp53.
p53 protein function was restored by shifting the culturing temperature from 37°C to 32°C, and thus, the PC3tsp53 can serve as its own control when examined at both temperatures. The PC3-vector cells were used as additional controls in some experiments and subjected to the same temperature and culture conditions as PC3tsp53. For each experiment, all cells were cultured at 37°C until irradiation, at which time the cells were either returned to 37°C or incubated at 32°C for the time period indicated for each experiment.

Western Blotting.
Protein extraction, gel electrophoresis, and Western blotting were performed as reported previously, with slight modifications of the methods of Laemmli (39, 40, 41) . Proteins for Cdkn1/p21 analysis were separated on 15% SDS-PAGE gels using a mini-gel system (Protean II; Bio-Rad Laboratories). ß-Actin levels were used as a standard to evaluate equivalent protein loading. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) at a constant 130 V for 80 min or at 40 V overnight. After blocking, blots were incubated either overnight at 4°C or at room temperature for 1 h with Cdkn1/p21 primary antibody (catalogue number 554228; PharMingen, San Diego, CA). After washing, blots were incubated in a 1:3,000 dilution of the horseradish peroxidase-conjugated secondary antibody [catalogue number W402B (antimouse); Promega, Madison, WI] for 1 h. Antibody detection was accomplished by chemiluminescent detection reagents (ECL; Amersham, Arlington Heights, IL). Finally, membranes were exposed to Kodak XAR film and developed, and results were evaluated.

Cell Irradiation.
Cells were irradiated in the University of California Davis Cancer Center Department of Radiation Oncology using a 6 MV linear accelerator (Varian, Palo Alto, CA) with a source to bolus distance of 100 cm and a 30 x 30-cm field. The culture dishes were placed on top of a 5-cm solid water (Gammes RI, Middleton, WI) for build up of the radiation dose.

Cell Cycle Analysis.
Cells were cultured in T-25 flasks to 50–70% confluence, irradiated, returned to the incubator (37°C or 32°C), and harvested for flow cytometry at the times indicated. Unirradiated controls were handled identically, except for the irradiation. For each fractionated dose experiment (3 x 2 Gy), the cells were irradiated with 1, 2, or 3 daily fractions of 2 Gy. The irradiated cells were harvested 24 h after their last dose of IR. The cells undergoing temperature down-shift were kept at 32°C until they were harvested. Temperature down-shift experiments were also performed on unirradiated cells.

Cell cycle analyses were performed using flow cytometric evaluation of DNA content. After harvesting by trypsinization, cells were collected in culture medium and pelleted by centrifugation. Cells were resuspended in 0.5 ml of 1x PBS (diluted from 25x PBS, catalogue number PBS010; ScyTeck, Logan UT), fixed in a final concentration of 70% ethanol, and stored at -20°C. Before cytometric analysis, cells were centrifuged, washed once in 1x PBS, resuspended in 900 µl of 1x PBS with 20 µl of DNase-free RNase (Roche Molecular Biochemicals, Indianapolis, IN), and incubated at 37°C for 30 min. After incubation, propidium iodide (Boehringer Mannheim Corp., Indianapolis, IN) was added to a final concentration of 0.05 mg/ml, and samples were allowed to stand at room temperature, protected from light, for a minimum of 15 min. Cell aggregates were removed by filtration into Falcon polystyrene tubes fitted with cell-strainer caps (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ). Propidium iodide fluorescence as an indicator of relative DNA content/cell was measured with the Coulter Epics XL flow cytometer (Beckman Coulter, Miami, FL). At least 10,000 cells were analyzed per sample with doublet discrimination. The percentages of cells in the G1, S, and G2-M phases of the cell cycle were estimated from the DNA content histogram obtained from each cell sample using the Phoenix Multicycle computer software (Phoenix Flow Systems, San Diego, CA). Unirradiated control cells were harvested at a cell culture time equivalent to the 24 h postirradiation time point or, in some experiments, at approximately the same time as the irradiation and thus represent logarithmically growing cells. Preliminary studies revealed that the cell cycle distribution of an untreated cell culture obtained at these times in any given experiment was essentially identical. All cell cycle experiments were performed at least twice.

Mitotic Trapping.
Cells were cultured in T-25 flasks to 50–70% confluence and then irradiated with a single dose of 2 Gy. Nocodazole was added to the culture media 30 min after irradiation or temperature down-shift (unirradiated cells) at a final concentration of 0.1 µg/ml culture medium. The cells were then cultured in the presence of nocodazole for 24 h. Because mitotic cells can become easily detached from the culture flasks, the culture medium from each flask was removed and saved before the remaining cells were harvested by trypsinization to ensure that these cells were not lost from the analyses. Once the cells were detached from the culture flask, the saved medium was used to collect the trypsinized cells and transferred to tubes, and the cells were pelleted by centrifugation. The pelleted cells were washed once in 1x PBS and fixed and stained in a solution containing 3.7% formaldehyde, 0.5% Igepal, and 10 µg/ml Hoescht 33258 (Sigma Chemical Co., St. Louis, MO) in PBS. Fifteen µl of the stained cell suspension were placed on a microscope slide, and a coverslip was placed on top. Nuclei with condensed, evenly staining chromosomes were scored as mitotic. At least 300 cells were counted for each treatment from each experiment using fluorescent microscopy to visualize the nuclei.

Clonogenic Assays.
Cell survival after IR was measured by clonogenic assay. For all experiments, single cells were seeded into 60-mm culture dishes on day 0 and allowed to attach for 24 h at 37°C. The cells were then irradiated at the desired dose (day 1), immediately returned to the incubator, and cultured for approximately 6 doubling times. Controls were handled identically to the irradiated test cells, with the exception of the radiation exposure. Thus, in the experiments in which p53 function was restored by temperature down-shift, both the irradiated cells and unirradiated controls were incubated at 32°C for 24 h after the IR exposure and then incubated at 37°C for the remainder of the assay (starting at day 2). Colonies were fixed in 1.0% crystal violet and 0.5% glacial acetic acid in ethanol, and visible colonies containing approximately 50 or more cells were counted. The SF at 2 Gy (SF2) was calculated relative to the unirradiated control cells for each temperature to account for the plating efficiency of each cell line in each experiment (number of IR colonies/number of colonies in control).

Survival after Three Daily Doses of 2 Gy (3 x 2 Gy).
Cells were plated as described for the single-dose assays. The cells were then irradiated daily with 2 Gy of radiation (approximately 24 h between each dose) for 3 consecutive days. For cells undergoing temperature down-shift, both the irradiated and unirradiated controls were incubated at 32°C for 3 days beginning after the first 2-Gy dose. After the last dose of irradiation, the cells were incubated to the 50-cell colony stage. At this point, the cells were fixed, stained, and counted as above. The SF3 x 2 was calculated relative to the untreated controls maintained under the same temperature conditions to account for plating efficiencies (as described above for single-dose experiments).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To study the contribution of the p53 protein to the radiation response in prostate cancer cells, the p53-null PC3 cells were transfected with the P223L mutant p53 allele that encodes a temperature-sensitive protein. This approach was chosen because of the difficulties encountered in establishing a stable line that expresses wild-type p53 (38 , 42) . Those difficulties have been attributed to the ability of wild-type p53 to induce growth arrest or apoptosis in transformed cells. The P223L allele is one of two mutant p53 alleles found in the DU145 prostate cancer cell line (36 , 43) . The mutation, proline -> leucine at codon 223, lies in the core DNA binding domain and appears to be in a protein hinge region. Mutations in this region are known to result in temperature-sensitive proteins (44) . The heat sensitivity of the protein from this p53 allele was originally established by us in a yeast assay that was used to investigate the transactivational activities of different mutant p53 alleles found in prostate cancer (38) . Normal transactivational activity was seen in the yeast colonies grown at 25°C, although partial function was observed in the yeast grown at 35°C.

From the transfected PC3 cells, five G418-resistant clones were isolated and screened for expression of p53 by Western blotting. As shown in Fig. 1ACitation , two of the five clones expressed the tsp53 protein. Clone 5 was used in these radiation studies and renamed PC3tsp53 (unfortunately, Clone 4 was lost in a liquid nitrogen freezer failure). Because the Cdk inhibitor Cdkn1/p21 (p21) is transcriptionally regulated by p53, p21 protein expression was assessed by Western blot analysis as a marker of restored function (Fig. 1BCitation ; Refs. 45 and 46 ). Culturing PC3tsp53 at 32°C resulted in an induction of p21 protein as early as 1 h after the temperature down-shift and increased in a time-dependent manner (1.1-fold at 1 h to 2.4-fold at 5 h compared with the 37°C control). This was in contrast to the PC3-parental and PC3-vector cells, each of which showed no induction of p21 during the same time period after temperature down-shift (data not shown). Thus, these data demonstrate that the transcriptional function of the tsp53 protein was rapidly restored when the cells were incubated at 32°C, and thus established it as the permissive temperature for the PC3tsp53 cell line. Additional Western blot analysis using phospho-specific antibodies showed that both serines 15 and 20 of the tsp53 protein were phosphorylated after exposure to IR, confirming that this protein is activated in response to radiation-induced damage (data not shown).



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  		Fig. 1. Expression and restored transactivational function of the tsp53 protein. A, Western blot analysis of p53 protein expression in the G418-resistant clones (Lanes 3–7). The p53-null PC3 parental cells were used as the negative control (Lane 1), and LNCaP cells (wt p53) were used as the positive control (Lane 2). Clone 5 was selected for additional studies and renamed PC3tsp53. B, Western blot analysis of Cdkn1/p21 induction in PC3tsp53. PC3tsp53 cells were incubated at 32°C for the times indicated to assess p53 transactivational function. ß-Actin was used as a loading control.

 
Temperature Effects on Cell Cycling.
Because the model chosen for these radiation studies required the PC3tsp53 cell line to be cultured at the nonphysiological temperature of 32°C (permissive temperature) to restore function to the tsp53 protein, it was important to establish what effects this lower culturing temperature would have on the distribution of cells in the G1, S, and G2-M phases of the cell cycle. PC3tsp53 cells were incubated at 32°C for up to 24 h, with cells harvested at 6-h intervals for flow cytometry analysis of cell cycle distribution. Representative histograms and mean percentages of cells in each cell cycle phase derived from multiple experiments are shown in Fig. 2ACitation . The histogram labeled Control-37°, against which the cells grown at 32°C were compared, represents logarithmically growing cells at 37°C. Incubation at 32°C with restoration of functional p53 resulted in little change in the percentage of cells in G1 over the time points examined. The predominant effects were seen in the percentage of cells in both S phase, which decreased to a nadir at 18 h followed by a recovery by 24 h, and G2-M, which increased and remained elevated from 18 to 24 h. Mitotic trapping using the microtubule inhibitor nocodazole revealed no statistical difference in the percentage of PC3tsp53 cells attempting cell division in a 24-h period for those cells cultured at 32°C compared with similarly treated cells cultured at 37°C (data not shown; Refs. 15 and 17 ). Whereas mitotic trapping showed that the effects of culturing the PC3tsp53 cells at 32°C were relatively transient over the 24-h period examined, there was some delay of cells moving through the G1-S transition, and an accumulation in the percentage of G2-M cells. Thus, these data establish that it is important for PC3tsp53 to be compared with itself at the two culturing temperatures for the purpose of the experiments presented here, which examine the role of functional p53 after exposure of the cells to IR.



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  		Fig. 2. Functional p53 increases the duration of the IR-induced G2 arrest. A, representative histograms of PC3tsp53 cells exposed to 2 Gy of IR and incubated at 32°C or 37°C for up to 24 h. Cell cycle analysis was performed on cells harvested every 6 h. The panel labeled Control-37° is representative of logarithmically growing cells at 37°C. Percentages of cells in each phase of the cell cycle are the means of at least two experiments for each time point. *, note reduced S phase and persistence of elevated G2-M at 24 h. B, duration of arrest was assessed by trapping the cells that entered mitosis during the 24 h post-IR period using the nocodazole (IR +32° versus IR + 37°). Columns represent the means of duplicate experiments; error bars = SD.

 
Functional p53 Increases the Duration of the IR-Induced G2 Arrest.
To examine the effects of functional p53 on the response to IR exposure, the PC3tsp53 cells were exposed to a single 2-Gy dose of IR and cultured at either the permissive (Fig. 2ACitation , IR +32°) or restrictive (Fig. 2ACitation , IR +37°) temperature for up to 24 h post-IR. Cell cycle responses were assessed every 6 h as described above, and the results are shown in Fig. 2ACitation . The predominant effect of exposure to IR in the presence of functional p53 (IR +32°) was a sustained G2-M accumulation (+ ~23% versus control) from 12 to 24 h compared with the cells incubated at the restrictive temperature post-IR (IR +37°). At the restrictive temperature, the percentage of cells in G2-M was also elevated by 12 h; however, this accumulation quickly declined so that by 24 h, G2-M content approached that seen in the unirradiated control. At both the permissive and restrictive temperatures, exposure to 2 Gy of IR resulted in an initial decrease in G1 cells compared with the control. This decrease persisted in the cells cultured at 32°C up to 12 h, but by 24 h, the percentage of G1 cells was equivalent to control. In contrast, in the cells maintained at 37°C, the fraction of cells in G1 began to increase at 12 h, so that by 18 h and persisting to 24 h, the percentage of G1 cells exceeded that seen in the control (~60% versus ~46%, respectively). Reductions in S phase after 6 h were seen at both temperatures; however, the loss of S-phase cells was more substantial at 24 h when p53 was functional, demonstrating the contribution, albeit somewhat delayed, of p53 to G1 arrest.

The post-IR cell cycle changes seen after a single dose of IR suggest that the most potent influence of functional p53 is a more persistent G2 arrest. To further explore the contribution of functional p53 to the maintenance of IR-induced G2 arrest, mitotic trapping of the irradiated PC3tsp53 cells was performed as described above for the unirradiated cells. The PC3tsp53 cells were exposed to 2 Gy of IR followed by culturing at the permissive or restrictive temperature in the presence of nocodazole for 24 h. The percentage of cells that exited G2 and entered mitosis was significantly reduced (-23%; P < 0.01) in the presence of functional p53 compared with the cells incubated at 37°C (Fig. 2B)Citation . Thus, these data provide evidence that, when p53 was functional in PC3tsp53 cells, the IR-induced G2 arrest was more durable. These results likely explain the lower percentage of cells in G1 12–24 h post-IR at the permissive temperature because more cells are inhibited from completing the cell cycle and entering G1 than in the absence of functional p53. Additionally, these data are consistent with the reported role of p53 in the maintenance of G2 arrest after DNA damage (15 , 16 , 18 , 19 , 47, 48, 49) .

Functional p53 Increased Clonogenic Survival After Exposure to Fractionated IR.
The ability of prostate cancer cells not only to survive but also to retain reproductive potential after radiation underlies the problem of tumor recurrences seen in patients. To test the hypothesis that functional p53 contributes to increased clonogenic survival after IR of CaP cells, the PC3tsp53 and PC3-vector cells were exposed to a single dose of 2 Gy of IR and to a regimen of three consecutive daily doses of 2 Gy in separate experiments. Clonogenic survival was assessed using a standard colony formation assay that measures the loss of reproductive integrity after exposure to IR (50) . After IR, the cells were either cultured at 37°C for the entire assay or cultured at 32°C until 24 h after the last dose of 2 Gy (i.e., 24 h for single dose and 72 h for 3 x 2 Gy). The SFs were determined by comparing the irradiated cells with unirradiated controls cultured under the same temperature conditions. Thus, each cell line at each temperature served as its own control. As shown in Fig. 3Citation , there was little difference in SF at the permissive temperature compared with the restrictive temperature for both the PC3tsp53 and PC3-vector cells exposed to a single dose of 2 Gy of IR. However, when the cells were irradiated daily for 3 consecutive days, survival of the PC3tsp53 cells was increased compared with those cultured continuously at 37°C (Fig. 3ACitation ; SF = 0.28 versus 0.07, respectively; P < 0.01). No statistical difference was seen in the PC3-vector cells between those cultured at 32°C and those cultured at 37°C (Fig. 3BCitation ; P > 0.05).



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  		Fig. 3. Functional p53 increases clonogenic survival after fractionated IR exposure. (A) PC3tsp53 and (B) PC3-vector cells were exposed to either a single dose of 2 Gy or three consecutive daily doses of 2 Gy, incubated at 32°C for 24 h after the last dose of IR, and then switched to 37°C (dark gray bars) until colonies were counted or incubated at 37°C (light gray columns) for the entire post-IR growth period. Unirradiated controls were incubated after the same schedule as the IR-treated cells. The SFs of the irradiated cells were calculated by comparing them with their respective subline controls at each temperature. A statistically significant difference in survival was seen only in the presence of functional p53 after fractionated IR (P < 0.01). Columns represent the mean of at least two experiments. Error bars = SD. P shown only when significant.

 
Functional p53 Contributes to Increased G1 Arrest and Sustained G2 Arrest in Response to Fractionated IR.
To examine the relationship between cell cycle response and clonogenic survival after fractionated IR exposure, the PC3tsp53 cells were exposed to three consecutive daily doses of 2 Gy after a radiation schedule identical to the multifraction survival experiments. Cells were harvested for cell cycle analysis by flow cytometry 24 h after each 2-Gy fraction. Representative histograms are shown in Fig. 4Citation . Cell cycle analysis showed the percentage of G1 cells with functional p53 had increased only after the second and third IR fractions (60% and 55%, respectively, versus 45% in control). The persistent reduction in cells exiting G1 and entering S phase compared with the unirradiated control revealed the influence of functional p53 on G1 arrest. The sustained reductions of S phase (-30% after three fractions) were coincident with persistent elevations in the percentage of cells in G2-M (+21%). In contrast, without functional p53, S phase remained comparatively high (~30%) after each IR exposure, and the fraction of cells in G2-M did not approach the accumulations seen at the permissive temperature. Thus, the presence of functional p53 resulted in more durable IR-induced G1 and G2 arrests. Additionally, a substantially higher percentage of sub-G1 events were seen at the permissive temperature than at the restrictive temperature (24% versus 7%, respectively; control = 5%) 24 h after the third IR fraction, providing suggestive evidence that p53-dependent apoptosis was also restored.



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  		Fig. 4. Functional p53 contributes to sustained G2-M arrest and increased G1 arrest in response to fractionated IR. PC3tsp53 cells were exposed to three consecutive daily fractions of 2 Gy of IR and incubated at 32°C or 37°C until harvest for flow cytometry. Cell cycle analysis was performed on cells harvested 24 h after their last dose of IR. This time point is equivalent to the time of the next dose of IR, so that the 1 x 2 Gy and 2 x 2 Gy histograms also represent the cell cycle distributions upon reexposure. Representative histograms are shown. Mean percentages of cells in each cell cycle phase and sub-G1 cells (indicated at the left of the G1 peak) determined from at least two experiments per time point are shown. *, note substantially reduced S phase and persistence of elevated G2-M.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the studies presented here, a mutant p53 allele (P223L) isolated from a human prostate cancer cell line that produces a temperature-sensitive protein was used to examine the contribution of p53 to cell cycle responses and clonogenic survival after exposure to clinically relevant doses of IR. Significantly increased clonogenic survival after exposure to multiple 2-Gy IR exposures was seen in PC3tsp53 cells when p53 function was restored. This increased survival was associated with a persistent elevation in the fraction of cells in G2-M, an increased G1 content, and markedly reduced S phase over the course of the three IR exposures, revealing a p53 component contributing to both G1 and G2 arrests. Additionally, the increased survival was not because of a loss of apoptosis as indicated by the higher percentage of sub-G1 events at the permissive temperature than at the restrictive temperature. Thus, in these cells, functional p53 contributed to both cell cycle arrest, a protective response necessary for repair of IR-induced DNA damage, and the elimination of irreparably damaged cells by apoptosis, with the overall outcome being one of higher clonogenic survival compared with these same cells without functional p53. What was not examined in these investigations was the third arm of the p53 response, DNA damage repair. An interesting finding of note was that exposure to IR resulted in activated p53, as defined by phosphorylation of serines 15 and 20, at both the restrictive (37°C) and the permissive (32°C) temperatures. However, transactivation function, as evidenced by the induction of CDKN1/p21, only occurred at the permissive temperature. This may be the first study to use the P223L mutant p53 allele and a restoration strategy to study the contributions of p53 to the radiation response; the most commonly used temperature-sensitive mutant alleles have been the murine p53, Val-135, or its human equivalent, Ala-143 (51, 52, 53, 54) .

The data presented here provide evidence that the presence of wt p53 in prostate tumor cells may reduce the effectiveness of radiation therapy and therefore constitute a therapeutic liability. Because in vivo animal model studies cannot be performed using a temperature-sensitive allele, these hypothesis-generating investigations set the stage for further investigations using strategies to down-regulate wt p53 or specific radiation response pathway downstream effectors. Specifically these might be ones that interfere with the protective functions of p53 while preserving p53 apoptotic activities. The downstream effectors of both p53-dependent G2 arrest, considered to be more vital to repair of radiation-induced DNA damage than G1 arrest, and DNA damage repair genes themselves may be effective targets to enhance the efficacy of radiation therapy, particularly in CaP with wt p53. Those preclinical studies, if supportive of these concepts, would then guide animal model studies necessary for clinical application.

EBRT plays an integral role in the management of primary prostate cancers. Loss of colony-forming ability is a critical event in IR-treated tumor cells. The clinical problem of tumor recurrence involves the few cells that not only survive the course of radiation treatments given but retain the ability to proliferate and repopulate the tumor. Because two-thirds of prostate cancer patients have wt p53 in their tumors, an important question to be addressed is whether the presence of wt p53 correlates with a higher rate of radiation therapy failure. This has been demonstrated in head and neck cancers using p53 sequence analysis (55) . In that study, the loss of p53 gene mutation in postirradiated tumors was associated with a significantly shorter disease-free interval and poorer prognosis. An appropriately powered examination of p53 status using sequence analysis of both pre- and post-IR prostate tissue samples to correlate with treatment outcome is needed.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Laurel Beckett (Professor and Head, Division of Biostatistics, Department of Epidemiology and Preventive Medicine, University of California Davis School of Medicine) for helpful discussions regarding statistical issues.


   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 Supported by a University of California Davis Health System Research Award (to P. H. G.) and University of California Davis Jastro-Shields Research and Floyd and Mary Schwall Dissertation Research Awards (to S. L. S.). Back

2 Present address: Department of Radiation Oncology, Mayo Clinic Jacksonville, Jacksonville, FL 32224. Back

3 To whom requests for reprints should be addressed, at University of California Davis Cancer Center, 4501 X Street, Sacramento, CA 95817. Phone: (916) 734-8614; Fax: (916) 734-2361; E-mail: paul.gumerlock{at}ucdmc.ucdavis.edu Back

4 The abbreviations used are: CaP, carcinoma of the prostate; IR, ionizing radiation; EBRT, external beam radiation therapy; wt, wild-type; tsp53, temperature-sensitive p53; SF, surviving fraction. Back

Received 12/31/02. Revised 8/ 4/03. Accepted 9/ 2/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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