Gemcitabine (2′, 2′-difluoro-2′-deoxycytidine; dFdCyd) has been shown to be a potent radiosensitizer in tumor cells both in vitroand in vivo. We evaluated the ability of dFdCyd to enhance the radiosensitivity of two human glioblastoma cell lines. The results demonstrated that U251 cells were more sensitive to the cytotoxicity of dFdCyd, and that dFdCyd was able to radiosensitize these cells. In contrast, D54 cells were more resistant to the cytotoxic effect of dFdCyd, and no radiosensitization occurred at any concentration of dFdCyd tested. Because radiosensitization by dFdCyd has been correlated with its ability to deplete dATP pools through inhibition of ribonucleotide reductase by dFdCyd diphosphate,we evaluated the metabolism of dFdCyd in both cell lines. At equitoxic concentrations of dFdCyd, both cell lines accumulated similar levels of the cytotoxic metabolite, dFdCyd triphosphate, as well as similar levels of dFdCyd monophosphate in DNA. In U251 cells,radiosensitizing concentrations of dFdCyd (10 or 25 nm;IC10 or IC50) depleted dATP by ∼80% within 4 h. In contrast, 80 nm (IC50) was unable to deplete dATP by >30% within 4 h in D54 cells. Higher concentrations of dFdCyd or hydroxyurea, an inhibitor of ribonucleotide reductase that depleted dATP >90%, also did not produce radiosensitization in D54 cells. D54 cells were not resistant to radiosensitization because bromodeoxyuridine was able to induce radiosensitization. Because D54 cells express wild-type p53, whereas U251 cells express a mutant p53, the effect of dFdCyd and ionizing radiation on cell cycle progression was evaluated. Radiation alone produced a G1 block in D54 cells and a transient G2-M block in U251 cells. After a 24 h incubation with dFdCyd alone or in combination with ionizing radiation, U251 cells readily accumulated in S-phase, which remained elevated for at least 72 h, consistent with previous results in other mutant p53 cell lines. In addition, radiation enhanced the ability of dFdCyd to induce S-phase-specific cell death in U251 cells. In contrast, D54 cells showed a G1 block after dFdCyd and radiation exposure, with fewer cells in S-phase for at least 48 h after drug washout/irradiation. Furthermore, treatment with dFdCyd and/or radiation did not increase the amount of S-phase-specific cell death in D54 cells compared with control cells. These results suggest that the G1 block in D54 cells resulting from wild-type p53 induction prevented radiosensitization by dFdCyd.

dFdCyd3(gemcitabine) is a deoxycytidine analogue that has shown clinical activity in the treatment of solid tumors, including pancreatic and non-small cell lung cancer (1, 2, 3, 4, 5). As a nucleoside analogue, after transport into the cell (6), dFdCyd requires phosphorylation for its antitumor activity, with the initial phosphorylation by deoxycytidine kinase being the rate-limiting step in its activation (7, 8). Mechanistic studies have demonstrated that there are two major pathways through which dFdCyd is toxic to tumor cells: (a) formation of the triphosphate,dFdCTP, can inhibit DNA synthesis directly or interfere with replication through incorporation of dFdCMP into the elongating DNA strand; and (b) the diphosphate, dFdCDP, is a potent,mechanism-based inhibitor of ribonucleotide reductase that results in the depletion of the necessary deoxynucleoside triphosphates for DNA synthesis (9, 10, 11, 12, 13, 14). dFdCyd can potentiate its own cytotoxicity through several mechanisms. For example, the dFdCyd-mediated depletion of dCTP can enhance the incorporation of dFdCMP into DNA, and the ability of dFdCTP to inhibit dCMP deaminase can prevent its catabolism (13, 15, 16). The cytotoxicity of dFdCyd in different cell lines is dependent upon the extent to which each of these pathways is affected by dFdCyd nucleotides(17).

On the basis of the observation that other antimetabolites that inhibit ribonucleotide reductase can enhance radiation-induced cytotoxicity (18, 19, 20), we hypothesized that dFdCyd would function as a radiation sensitizer. Indeed, it has now been demonstrated that dFdCyd is among the most potent of radiosensitizers in vitro(21, 22, 23). Recent studies in vivo have confirmed these observations and have shown significant tumor growth delay with the combination of dFdCyd and ionizing radiation in animal models (24, 25, 26). These results have prompted a variety of clinical trials using dFdCyd as a radiosensitizer for tumors in which the lack of local control is the reason for clinical failure, such as head and neck cancer, pancreatic cancer, non-small cell lung cancer, and gastrointestinal malignancies(27, 28, 29).

Glioblastoma multiforme is an aggressive brain tumor with poor prognosis in patients because of its propensity to recur locally. Despite a multitude of efforts to impact the natural course of this disease with surgery, chemotherapy, and/or radiation therapy, the median survival for these patients is <1 year (30). One strategy to improve therapeutic outcome has been to use radiosensitizers (31, 32). Of these, the most promising agents were the halogenated thymidine analogues; however, this combination has proven toxic to patients (33). dFdCyd may be preferable to the halogenated thymidine analogues as a radiosensitizer based on its potent activity at low doses in vitro. In addition, dFdCyd can radiosensitize cells in vitro, even after a brief exposure (34), compared with the lengthy dosing required for the thymidine analogues(35), suggesting that the once-weekly dosing schedule of dFdCyd in patients will be sufficient for radiosensitization.

We have evaluated the ability of dFdCyd to enhance radiation-induced cytotoxicity in the U251 and D54 human glioblastoma cell lines. Although both cell lines were sensitive to the cytotoxic effects of dFdCyd, only the U251 cells were radiosensitized by the drug. We noted that the U251 cells expressed a mutant p53, as did the other cell types reported previously by us to be radiosensitized by dFdCyd (HT-29 human colon carcinoma, and the BxPC-3 and Panc-1 pancreatic cancer cell lines), whereas D54 cells express wild-type p53 (36, 37, 38). Mutations in p53 or allelic loss of chromosome 17p occur commonly in the development of human glioblastomas (39). Inactivating p53 mutations have been shown to occur in >40% of adult glioblastomas(40, 41). Because induction of wild-type p53 by DNA-damaging agents can lead to cell cycle arrest (42),and cell cycle position affects cytotoxicity induced by radiation, we considered the possibility that the p53 status of cells may affect their ability to be radiosensitized by dFdCyd. These two cell lines provided an opportunity to evaluate the metabolism of dFdCyd and its effects on cell cycle progression to gain a greater understanding of the factors necessary to produce radiosensitization with this nucleoside analogue. A preliminary description of these results was reported previously (43).

Chemicals.

dFdCyd and dFdCTP were synthesized and generously provided by Eli Lilly and Co. (Indianapolis, IN). RNase A was purchased from Boehringer Mannheim (Indianapolis, IN). All other chemicals were of the highest purity available.

Cell Culture.

The human glioblastoma cell lines U251 and D54 were cultured in RPMI 1640 supplemented with 10% calf serum (Life Technologies, Inc., Grand Island, NY) and l-glutamine (Fisher Scientific, Pittsburgh,PA). Cells were maintained in exponential growth in a humidified atmosphere at 37°C and 5% CO2.

Cytotoxicity Assays.

Cytotoxicity was measured using a standard colony formation assay. Cell culture flasks (25 cm2) were plated with between 300,000 and 600,000 cells a minimum of 36 h prior to the addition of drug. Exponentially growing cells were incubated with drug for 4 or 24 h. At the conclusion of the drug incubation period, cells were washed with Dulbecco’s PBS, trypsinized, and counted using a Coulter(Hialeah, FL) electronic particle counter. Approximately 100 viable cells were plated into each 35-mm diameter well of a six-well culture dish and allowed to grow in the absence of drug for 10–14 days. At that time, the resulting colonies were fixed using a methanol:glacial acetic acid solution (3:1, v/v) and stained with 0.4% crystal violet. Colonies of >30 cells were counted, and survival was determined as a fraction of plating efficiency of untreated control cells. The control plating efficiency for both cell lines was ∼40%.

Radiosensitization Assays.

After drug and/or radiation treatment, cells were assessed for clonogenic survival as described above. Radiation survival data from drug-treated cells were corrected for plating efficiency by comparison to cells treated with drug alone. Cell survival curves were fit using the linear quadratic equation. Radiation sensitivity is expressed in terms of the mean inactivation dose, which represents the area under the cell survival curve (44). Radiosensitization is expressed as the RER, which is defined by the mean inactivation dose(radiation treatment)/mean inactivation dose (drug + radiation treatment).

Irradiation of Cells.

Monolayer cultures of either U251 or D54 cells were irradiated at 1–2 Gy/min using 60Co (AECL Theratron 80). Dosimetry was performed using an ionization chamber connected to an electrometer system that was directly traceable to a National Institute of Standards and Technology standard. All cells were irradiated at room temperature.

Analysis of dNTP Pools.

Cells were incubated with drug for 1–24 h, harvested by trypsinization, and counted. The nucleotides were extracted with ice-cold 0.4 n perchloric acid and neutralized with 10 n KOH. The majority of the ribonucleotides were removed from the deoxyribonucleotides by elution over a boronate affinity column as described previously (45). Deoxyribonucleotides were separated and quantitated by strong anion exchange HPLC using a Waters (Milford, MA) gradient system composed of two model 501 pumps, a U6K injector, and a model 996 photodiode array detector. This system was controlled by Millennium 2010 software. Before injection, each sample was centrifuged at 14,000 × g for 2 min and acidified to pH 2.8. Samples were then injected onto a 5-μm Partisphere 4.6 × 250-mm SAX column (Whatman Scientific, Hillsboro, OR) and eluted with a linear gradient of ammonium phosphate buffer ranging from 0.15 m (pH 2.8) to 0.6 m (pH 2.8–3.8) at a flow rate of 2 ml/min. Nucleotides were identified and quantitated by comparison to a known amount of authentic standards using their characteristic absorbance spectra over the range of 200–350 nm.

Western Blot Analysis.

Fifty μg (p53) or 200 μg (mdm-2) of protein were loaded per lane of a 7.5% acrylamide gel and separated using SDS-PAGE. Proteins were transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore) and blocked with 5% nonfat dry milk. Blots were probed with the respective primary antibodies: p53 (Oncogene Research Products; Ab-6, 1:500 dilution); mdm-2 (Oncogene Research Products;AB-1, 1:50 dilution). Primary antibodies were labeled with goat antimouse IgG, horseradish peroxidase-conjugated secondary antibody(Pierce; 31430, 1:20,000 dilution). The antibody-bound proteins were visualized using Pierce SuperSignal Chemiluminescent Substrate detection kit.

Cell Cycle Analysis.

Flow cytometric analysis was performed as described in Hoy et al.(46). Briefly, at the conclusion of the dFdCyd incubation, cells were pulse labeled with 30μ m BrdUrd for 15 min and then harvested by trypsinization, counted, and washed with PBS. Cells were then fixed in cold 70% ethanol at a concentration of 1,000,000 cells/ml, with samples not to exceed a total of 3,000,000 cells. Fixed cells were stored at 4°C for up to 10 days. Within 6 h prior to flow cytometric analysis, fixed cells were washed with PBS and resuspended in 1 ml of PBS containing 0.5 mg/ml RNase A and incubated for 30 min at 37°C. Cells were then washed with PBS, resuspended in 1 ml 0.1 n HCl containing 0.7% Triton X-100, and incubated for 10 min on ice. This was followed by another PBS wash,resuspension in 1 ml of sterile HPLC grade water, and incubation at 95°C for 15 min. The samples were immediately transferred to an ice-water bath for an additional 15 min. Cells were then washed with PBS containing 0.5% Tween 20. One hundred μl of PBS containing 0.5%Tween 20 and 5% calf serum (PBT) were added to each cell pellet,followed by the addition of 100 μl of anti-BrdUrd mouse IgG1 antibody (1:100 dilution; PharMingen, San Diego, CA)and incubation for 30 min at room temperature. After centrifugation,150 μl of FITC-conjugated, goat antimouse IgG antibody (1:20–35 dilution; Sigma Chemical Co, St. Louis, MO) were added to the pellet,mixed gently, and incubated for 30 min at room temperature. Samples were centrifuged and resuspended in 0.5 ml of 18 μg/ml PI containing 40 μg/ml RNase A. Trout erythrocyte nuclei (Biosure, Grass Valley,CA) were added as an internal standard. Treated cells were placed in the dark a minimum of 30 min prior to cell cycle analysis using a Coulter EPICS Elite ESP flow cytometer. Cell cycle data were further analyzed using WinMDI software (version 2.8.8) provided by Joseph Trotter of The Scripps Research Institute.

Apoptosis.

Apoptosis was determined by sub-G1 content, as indicated by flow cytometry. Briefly, adherent cells were harvested by trypsinization, counted, and washed with PBS. Cells were then fixed in cold 70% ethanol at a concentration of 1,000,000 cells/ml with samples not to exceed 3,000,000 cells. Cells were fixed a minimum of 1 h prior to the addition of 0.5 ml of 18 μg/ml PI containing 40 μg/ml RNase A. Trout erythrocyte nuclei were added as an internal standard.

Cytotoxicity of dFdCyd.

The sensitivity of U251 and D54 cells to dFdCyd alone was determined to select the appropriate concentration range in which to evaluate dFdCyd as a radiosensitizer in these two cell lines. Using a clonogenic survival assay, U251 cells (IC50, 21.4 ± 1.1 nm) were found to be at least 3-fold more sensitive than D54 cells (IC50, 78.3 ± 17 nm) after a 24 h incubation with dFdCyd. At higher concentrations, U251 cell survival was 1–2 logs less than that of D54 cells (Fig. 1).

Radiosensitization by dFdCyd.

Previous work from this lab has shown that dFdCyd is a potent radiosensitizer in solid tumor cells, such as human colon carcinoma and pancreatic cancer cells (22, 23). To determine the ability of dFdCyd to enhance the sensitivity of these cells to radiation,noncytotoxic (IC10) and cytotoxic(IC50) doses of dFdCyd for 24 h were evaluated. Survival at the IC10 (90.9 ± 9.3% survival) was not found to be significantly different from untreated cells (100.0 ± 3.2% survival), and it permitted standardization of the noncytotoxic dose. Consistent with previous studies in other solid tumor cell lines, U251 cells were radiosensitized after a 24 h exposure to either 10 nm(IC10) or 25 nm(IC50) dFdCyd. The RERs were 1.60 ± 0.03 and 1.77 ± 0.22, respectively (Fig. 2 and Table 1).

In contrast to the U251 cells, D54 cells were not radiosensitized using either noncytotoxic or cytotoxic doses of dFdCyd for 24 h (RERs<1.0; Fig. 2 and Table 1). Higher concentrations of dFdCyd incubated for only 4 h in D54 cells also failed to produce radiosensitization (Fig. 3). The lack of radiosensitization of D54 cells is not due to an apparent radioresistance. D54 cells are significantly more sensitive to the effects of ionizing radiation alone than U251 cells, as indicated by D bar values of 2.01 ± 0.06 and 2.61 ± 0.08 for D54 and U251 cells, respectively (P < 0.001; Ref. 44).

Metabolism of dFdCyd.

To determine whether an altered rate of metabolism of dFdCyd could account for the lack of radiosensitization in D54 cells, dFdCTP levels were measured in the two cell lines. Both cell lines were incubated with their respective 24 h IC50 of dFdCyd,and the dFdCTP pool was measured periodically. Under these conditions,dFdCTP accumulation was similar, with peak levels of 0.08 and 0.06 nmol/107 cells at 12 h for U251 and D54 cells, respectively (data not shown). In addition, dFdCMP incorporation into DNA was similar in both cell lines after a 24 h incubation with IC50 concentrations of dFdCyd(0.40 ± 0.04 versus 0.57 ± 0.07 pmol/107 cells in U251 and D54 cells,respectively).

Effect of dFdCyd on dATP.

Previous studies in human colon carcinoma and pancreatic cancer cell lines suggested that radiosensitization by dFdCyd was related to its ability to deplete the endogenous dATP in the cells by at least 90%because of inhibition of ribonucleotide reductase (22, 23). It was important to determine whether the lack of radiosensitization in D54 cells was attributable to an inability to deplete dATP. Both cell lines were treated for 24 h with the IC50 of dFdCyd, and the nucleotide pools were measured. Within 4 h, dATP was depleted to <0.04 nmol/107 cells, or to 20% of control levels in U251 cells. The amount of dATP continued to decrease, with 0.01 nmol/107 cells (4%) remaining at 12 h and 0.001 nmol/107 cells (0.5%) at 24 h. In D54 cells, dFdCyd depleted dATP by only 30% within 4 h and required 24 h to deplete dATP to a minimum level of 0.07 nmol/107 cells (12%; Fig. 4). In addition, dGTP was depleted to a greater extent in U251 cells than D54 cells under these conditions. However, by the end of the 24 h incubation, the dGTP level in U251 cells began to recover (data not shown). No significant differences in the other dNTP pools were observed after dFdCyd treatment in these two cell lines.

Because dFdCyd at the IC50 concentration was unable to deplete dATP to <10% of control levels in D54 cells, we examined the ability of hydroxyurea, another known radiosensitizer that also inhibits ribonucleotide reductase, to deplete dATP. Treatment of D54 cells with 0.6 mm hydroxyurea (24 h IC50) did not produce radiosensitization (RER,1.06 ± 0.04), although it did deplete dATP to <2%(<0.01 nmol/107 cells) of control levels within 2 h of drug addition (data not shown). In contrast, 3 mm hydroxyurea (24 h IC50) was able to radiosensitize U251 cells (RER, 1.39 ± 0.08). Hydroxyurea depleted dATP in U251 cells in a similar pattern and to a similar extent as dFdCyd (data not shown).

To determine whether there was a difference in dATP levels in these two cell lines after the combined treatment of dFdCyd and ionizing radiation, both cell lines were treated for 4 h with higher concentrations of dFdCyd (4 h IC50) and then irradiated with 5 Gy. Immediately after irradiation, both cell lines were markedly depleted of dATP, but neither showed recovery of dATP or changes in the other dNTP pools with the next 4 h, the time period during which the majority of DNA double strand breaks induced by radiation are repaired (34).

Effects of BrdUrd.

Because D54 cells could not be radiosensitized by two agents that inhibit ribonucleotide reductase, it was important to determine whether D54 cells could be radiosensitized by agents that radiosensitize via alternate mechanisms. BrdUrd was chosen because it has been shown that incorporation of BrdUMP, 5′-monophosphate of BrdUrd, into DNA increases sensitivity to radiation damage (47). BrdUrd was evaluated as a radiosensitizer at both noncytotoxic and cytotoxic concentrations for 24 h. U251 cells were radiosensitized at a noncytotoxic dose(IC10; RER, 1.71 ± 0.19; Table 1). BrdUrd was also able to radiosensitize D54 cells at a noncytotoxic dose (IC10; RER, 1.19 ± 0.06),and a cytotoxic dose (IC50; RER, 1.84 ± 0.16). Thus, D54 cells were not resistant to all radiosensitizers.

Expression of p53 and mdm-2.

One notable difference between these cell lines is that U251 cells express a mutant p53, whereas D54 cells express wild-type p53(36, 37, 38). Because induction of wild-type p53 in response to DNA damage can alter the cell cycle distribution, we evaluated the effect of dFdCyd and radiation on p53 expression. U251 or D54 cells were treated with their IC50 concentrations of dFdCyd for 24 h, followed by 5 Gy of ionizing radiation, and Western blot analysis was performed using an antibody that can detect both mutant and wild-type p53. These studies verified that p53 was constitutively expressed at a relatively high level in the U251 cells,as expected for a mutant p53 cell line. In contrast, p53 was present at lower levels in untreated D54 cells as compared with untreated U251 cells (data not shown). Treatment of D54 cells with dFdCyd alone did not induce p53 expression during the 24 h exposure prior to irradiation (data not shown). However, after the subsequent irradiation, p53 was induced within 1 h (Fig. 5), indicative of functional p53. Irradiation without prior dFdCyd treatment also induced expression of p53.

mdm-2 is a negative regulator of p53 that blocks transcriptional activation and mediates degradation by binding to p53 and promoting ubiquitinization (48). mdm-2 is expressed at higher levels in human glioblastoma cell lines (49). The response of mdm-2 to the combined treatment of dFdCyd and radiation was measured by Western blot analysis. mdm-2 was not detected in U251 cells after drug and radiation treatment, as predicted for a mutant p53 cell line. In D54 cells, mdm-2 was expressed within 4 h of radiation treatment,after an increase in p53 protein (Fig. 5). At the time of mdm-2 induction, there also was a modest reduction in p53 protein levels,consistent with a functional feedback mechanism. mdm-2 levels remained elevated for 20 h after the initial induction after irradiation.

Cell Cycle Progression.

Because p53 is an important cell cycle checkpoint regulator, it was essential to determine whether this difference in p53 status altered the ability of cells to progress through the cell cycle after treatment with dFdCyd and radiation. U251 cells were treated with 25 nm dFdCyd for 24 h and/or 5 Gy of radiation. The medium was replaced immediately after radiation treatment, and the cell cycle was monitored by dual parameter flow cytometry, which measured both BrdUrd incorporation and DNA content, for the following 72 h. In response to 5 Gy (IC90), U251 cells show a typical G2-M block within 12 h, and G2-M remained elevated at 24 h (Fig. 6 and Table 2). This G2-M block was partially released by 48 h, as indicated by a decreased percentage of cells in G2-M, an increase in G1 and in S-phase, and an increased cell number. At 72 h,G2-M remained elevated above control levels, and the total cell number increased further. In contrast, after a 24 h treatment with dFdCyd alone, U251 cells accumulated in S-phase(>70%), with corresponding decreases in G1 and G2-M. After drug washout, U251 cells slowly began to progress through the cell cycle. The S-phase population decreased by 24 h after drug washout, and there was a large increase in G2-M after 48 h. The increase in SNI [non-BrdUrd incorporating cells with S-phase DNA content identified as dying/dead cells, similar to the findings of Pallavicini et al.(50)] to ∼12% at 24 h after drug washout indicated that S-phase-specific death was induced by dFdCyd in agreement with the observed loss in cell number(data not shown). U251 cells treated with 25 nmdFdCyd for 24 h, followed by 5 Gy, show almost the same cell cycle pattern as with dFdCyd alone; however, SNIincreased to ∼33% at 24 h after drug washout and was consistently higher at 48 and 72 h compared with cells treated with dFdCyd alone.

D54 cells treated with 5 Gy (IC95) displayed an increase in G2-M to ∼18% within 12 h after irradiation (Table 2). However, by 24 h, the majority of this population had progressed into G1. The increase in G1 and decrease in S-phase indicated that the cells were blocked in G1. D54 cells treated with 80 nm dFdCyd (IC50) for 24 h exhibited a cell cycle distribution similar to controls throughout the 72 h following drug washout. In response to the combination of dFdCyd and radiation, G1 increased to ∼74% within 24 h of irradiation/drug washout and remained elevated for the following 48 h, similar to treatment with radiation alone. As observed with radiation alone, the percentage of cells in S-phase decreased. The low S-phase percentage was associated with no change in cell number. Furthermore, the addition of radiation to dFdCyd treatment did not increase SNI. These results show that the majority of D54 cells were unable to enter into S-phase for at least 48 h after the combination treatment of dFdCyd and radiation.

Apoptosis.

Expression of wild-type p53 can lead to apoptotic cell death in response to dFdCyd, radiation, and other DNA-damaging agents (51, 52). Therefore, we evaluated the ability of dFdCyd and ionizing radiation to induce apoptosis in U251 and D54 cells at 0, 24, 48, and 72 h after drug/radiation treatment. In response to dFdCyd alone,U251 cells readily undergo apoptosis, as measured by sub-G1 DNA content determined with flow cytometry. Treatment with 10 nm (24 h IC10), 25 nm (24 h IC50), or 120 nm (24 h IC99) dFdCyd led to 16.9–21.1% of the cell population undergoing apoptosis 24 h after drug washout (data not shown). Seventy-two h after washout, the percentage of apoptotic cells was dependent on the severity of treatment (Fig. 7). U251 cells treated: with 10 nm dFdCyd showed a decline in apoptosis by 72 h; with 25 nm dFdCyd plateaued at 28%apoptotic cells; and with 120 nm dFdCyd displayed apoptosis in up to 46.5% of the population. U251 cells displayed lower levels of apoptosis with cytotoxic treatments of ionizing radiation. Neither 5 Gy(IC90) nor 10 Gy (IC99.5)produced >22.3% apoptosis during the 72 h after irradiation. Combinations of dFdCyd and radiation that produced radiosensitization in U251 cells produced apoptosis in up to 27.9% of the cell population; however, there was not even an additive increase compared with the individual treatments.

In contrast to the U251 cells, D54 cells do not readily undergo apoptosis in response to treatment with dFdCyd and/or radiation. Incubation with 80 nm dFdCyd produced similar levels of apoptosis, as seen in untreated cells, up to 72 h after drug removal. Increasing the dose of dFdCyd to 760 nm (24 h IC99) induced apoptosis to a greater extent(22.7%), but this required up to 72 h after the washout of dFdCyd. D54 cells showed an even less tendency to die through apoptosis after treatment with radiation than did U251 cells. Although these cells are more sensitive to radiation treatment, there was no appreciable increase (<5%) in apoptotic cell death in response to either 5 Gy (IC95) or 10 Gy(IC99.5) as compared with controls. Finally,combining dFdCyd with radiation in D54 cells still did not produce an increase in apoptotic cell death as compared with the individual treatments at any of the time points examined (Fig. 7).

dFdCyd is a promising radiosensitizing agent for the treatment of patients with solid tumors. In vitro studies have demonstrated that dFdCyd is a potent radiosensitizer, and in vivo studies in animals have shown that the combination of dFdCyd and ionizing radiation produces significant tumor growth delay(24, 25, 26). On the basis of these results, clinical trials have begun with the combination of dFdCyd and ionizing radiation(27, 28, 29). However, there has not been an extensive analysis of the mechanism by which dFdCyd is able to enhance radiation-induced cytotoxicity. Earlier work from this laboratory demonstrated a correlative relationship between the duration of dATP depletion in the cell and the extent of radiosensitization(22). Here we have extended these studies using two human glioblastoma cell lines, of which only one was radiosensitized by dFdCyd. The results suggest that, in addition to dATP depletion, the ability of cells to progress into S-phase after dFdCyd and radiation treatment may be key for radiosensitization to occur.

Both the U251 and D54 cell lines were sensitive to the cytotoxic effect of dFdCyd at low nanomolar concentrations. However, U251 cells were at least 3-fold more sensitive than the D54 cells. Analysis of the phosphorylation of dFdCyd indicated that similar levels of dFdCTP accumulated at equitoxic concentrations in the two cell lines,suggesting that the difference in cytotoxicity may be explained by altered rates of dFdCyd metabolism. Alternatively, noting the difference in p53 status and ability to undergo apoptosis between the two cell lines, it may be interesting to explore whether the lack of expression of wild-type p53 in the U251 cells sensitizes them to dFdCyd and facilitates p53-independent apoptosis. A recent report using glioblastoma cell lines indicated that sensitivity to dFdCyd cytotoxicity did not differ between cells expressing either mutant or wild-type p53 (53). However, this study used cell lines that did not originate from a single parental line or compared sensitivity after forced expression of wild-type p53 in a cell line with a mutant p53 background. It may be of interest to compare dFdCyd sensitivity in matched cell lines with wild-type or mutant p53 expression.

Evaluation of cytotoxicity from the combination of dFdCyd and ionizing radiation demonstrated that U251 cells were radiosensitized at both the IC10 and IC50concentrations of dFdCyd. However, attempts to radiosensitize D54 cells failed using a variety of dFdCyd doses and incubation periods. Initially, we hypothesized that this lack of radiosensitization was due to the inability of dFdCyd to deplete the dATP pool in D54 cells. Upon initial inspection, this appeared to be true, because there was a>80% reduction in dATP in U251 cells compared with <30% reduction in D54 cells within 4 h using equitoxic doses of dFdCyd (Fig. 4). This suggested that the remaining level of dATP in D54 cells after dFdCyd treatment was sufficient to prevent radiosensitization. However,further depletion of dATP to <10% of the control level using hydroxyurea failed to produce radiosensitization in D54 cells, although hydroxyurea was able to effect a similar decrease in dATP and radiosensitize U251 cells. Therefore, in D54 cells, dATP depletion alone was not sufficient to promote radiosensitization by dFdCyd or hydroxyurea. The D54 cells were not resistant to radiosensitization by all agents, because BrdUrd resulted in significant radiosensitization. Furthermore, incorporation of dFdCMP into DNA cannot explain the lack of radiosensitization because D54 cells were able to incorporate slightly more dFdCMP than U251 cells at equitoxic doses of dFdCyd.

Although a strong association has been made between dATP depletion and radiosensitization for dFdCyd in numerous cell lines (22, 23, 54), the mechanism by which low dATP levels may effect radiosensitization is not clear because radiosensitizing concentrations of dFdCyd in other cell lines did not produce DNA double strand breaks or inhibit their repair (34, 55). Low dATP levels may result in errors of replication, such as insertion of an incorrect nucleotide for the missing dATP, and the D54 cells may be able to prevent or correct this error. Thus, this cell line may be important in determining the molecular target for radiosensitization with dFdCyd.

An alternative explanation for the difference in radiosensitization with dFdCyd is that the two cell lines respond to DNA damage in different ways. A variety of DNA-damaging agents can induce expression of wild-type p53, resulting in increased transcription of proteins such as p21, mdm-2, bax, and GADD45 which, in turn, can determine whether a cell will continue to progress through the cell cycle, arrest in G1 or G2, repair DNA damage, or die via apoptosis (42). As predicted for a wild-type p53 cell line, D54 cells exhibited a competent G1-S cell cycle checkpoint in response to dFdCyd and ionizing radiation. This may allow D54 cells time to repair DNA damage and/or prevent the replication of damaged cells after irradiation. In contrast, the mutant p53-expressing U251 cells continued to progress into S-phase and G2-M after dFdCyd treatment and irradiation. This cell cycle pattern is similar to that reported previously for HT-29 human colon carcinoma cells, a cell line that also expresses a mutant p53 protein, after a 2 h treatment with a noncytotoxic dose of dFdCyd (34). Reports in the literature suggest that the p53 status of a cell can affect its inherent radiosensitivity (56, 57, 58, 59, 60, 61). Here we have observed a difference in cell cycle progression between the radiosensitive U251 cells and the nonradiosensitized D54 cells, which may be attributable to their difference in p53. The ability of the U251 cells to continue to progress through the cell cycle after damage from dFdCyd and radiation may lead to the synergistic enhancement of cell death that presents as radiosensitization.

The observed difference in cell cycle progression resulted in a significant difference in cell cycle distribution of the two cell lines at the time of irradiation. Greater than 70% of the U251 cells were in S-phase, whereas <37% of the D54 cells were in S-phase. These results are similar to those reported previously by us using the HT-29 human colon carcinoma cell line (22, 34). In addition, a recent report using synchronized cell populations indicated that cells must be in S-phase to be radiosensitized by dFdCyd (62). Because cells in S-phase are generally more resistant to ionizing radiation than cells at the G1-S border or in G2-M, radiosensitization of U251 cells by dFdCyd is not attributable to the redistribution into a more radiosensitive phase (18). However, this redistribution may be important for radiation to enhance the S-phase-specific cell death induced by dFdCyd, as was observed prominently in the U251 cells but was noticeably absent in D54 cells. This enhancement of S-phase cell death may be responsible for radiosensitization by dFdCyd.

In the studies presented here, it is interesting to note that p53 was readily induced by ionizing radiation, yet a 24 h exposure to the IC50 of dFdCyd did not induce p53 in D54 cells. This is consistent with the lack of effect on the cell cycle distribution of D54 cells with drug treatment alone. A recent report indicated that dFdCyd alone induced wild-type p53 expression in H460 human lung cancer cell lines, although this was measured after a 72 h incubation with IC50 and IC80 concentrations of drug (63). It is possible that the ability to induce p53 by dFdCyd varies by cell line.

Several reports have demonstrated that dFdCyd can induce apoptosis in a variety of cell types (53, 63, 64). In addition, compared with lower grade malignant brain tumors, glioblastomas in patients are associated with higher levels of apoptotic cells (reviewed in Ref.65). Therefore, it was important to analyze the effect of dFdCyd and ionizing radiation on the ability of these cells to undergo apoptosis. Although a high percentage of U251 cells became apoptotic after treatment with dFdCyd alone, ionizing radiation was less able to induce apoptosis, and the combination of these agents did not increase apoptosis as compared with the single-agent treatments. These results are consistent with previous reports from other laboratories,indicating that radiation is not a strong inducer of apoptosis in nonlymphoid cell lines (reviewed in Ref. 66). D54 cells were less likely to undergo apoptosis after either dFdCyd or ionizing radiation than U251 cells, and the combination did not result in an increase in the amount of apoptotic cells in the D54 cell line. Thus,in both cell lines, the addition of radiation did not increase the ability of dFdCyd to produce apoptosis. Although these two cell lines differed in their ability to undergo apoptosis with dFdCyd and/or ionizing radiation, radiosensitization was associated with a decreased incidence of apoptosis in the U251 cells. Therefore, the lack of radiosensitization in D54 cells does not appear to be related to low induction of apoptosis.

The data presented here support the previous findings that, in addition to dATP depletion, progression of cells into S-phase after dFdCyd treatment is important for radiosensitization. When cells accumulated in S-phase, radiation enhanced the S-phase-specific cell death induced by dFdCyd. With the prominent G1 block observed after the combination of dFdCyd and ionizing radiation in the D54 cells and the difference in cell cycle distribution at the time of irradiation in these two cell lines, it is tempting to speculate that expression of wild-type p53 prevents radiosensitization through inhibition of progression into S-phase. However, in consideration of the myriad cellular effects triggered by p53, it is possible that the effects on cell cycle progression are secondary to a primary effect of p53 on another requisite but undefined pathway for dFdCyd-mediated radiosensitization. If expression of p53 is involved in the lack of radiosensitization in the D54 cells, then its inactivation should allow the cells to be radiosensitized. This could be accomplished by introducing the human papillomavirus E6 protein to promote degradation of p53 or by using a p53 antisense construct. This hypothesis is currently under investigation.

Fig. 1.

Cytotoxicity of dFdCyd in human glioblastoma cells. U251(▪) or D54 (•) cells were incubated with dFdCyd for 24 h and assayed for clonogenic survival. Values shown represent the mean of triplicate determinations calculated from a single experiment; bars, SE. Experiments were repeated at least three times.

Fig. 1.

Cytotoxicity of dFdCyd in human glioblastoma cells. U251(▪) or D54 (•) cells were incubated with dFdCyd for 24 h and assayed for clonogenic survival. Values shown represent the mean of triplicate determinations calculated from a single experiment; bars, SE. Experiments were repeated at least three times.

Close modal
Fig. 2.

Effect of dFdCyd on the sensitivity of human glioblastoma cells to ionizing radiation. A, U251 cells were treated with either no drug (control, •) or 10 nm dFdCyd(IC10, □) for 24 h, followed by the indicated doses of radiation. B, D54 cells were treated with either no drug (control, •) or 80 nm dFdCyd (IC50, □)for 24 h, followed by radiation. Cell survival was determined by colony formation assay. The surviving fraction was corrected for cell death attributable to drug alone. Data shown are from a representative experiment. Each experiment was repeated at least three times.

Fig. 2.

Effect of dFdCyd on the sensitivity of human glioblastoma cells to ionizing radiation. A, U251 cells were treated with either no drug (control, •) or 10 nm dFdCyd(IC10, □) for 24 h, followed by the indicated doses of radiation. B, D54 cells were treated with either no drug (control, •) or 80 nm dFdCyd (IC50, □)for 24 h, followed by radiation. Cell survival was determined by colony formation assay. The surviving fraction was corrected for cell death attributable to drug alone. Data shown are from a representative experiment. Each experiment was repeated at least three times.

Close modal
Fig. 3.

Comparison of the radiation enhancement ratio in human glioblastoma cells. Either U251 (▪) or D54 (□) cells were treated with dFdCyd, as indicated, followed by radiation. Values are means of at least three determinations; bars, SE. ∗, P < 0.02 (value is significantly>1.0).

Fig. 3.

Comparison of the radiation enhancement ratio in human glioblastoma cells. Either U251 (▪) or D54 (□) cells were treated with dFdCyd, as indicated, followed by radiation. Values are means of at least three determinations; bars, SE. ∗, P < 0.02 (value is significantly>1.0).

Close modal
Fig. 4.

Effect of dFdCyd on the dATP pool in human glioblastoma cells. Exponentially growing U251 (▪) or D54 (•) cells were treated with 25 nm dFdCyd (IC50) or 80 nmdFdCyd (IC50), respectively, for 24 h. Cellular nucleotides were extracted with perchloric acid, eluted over a boronate affinity column to remove ribonucleotides, and analyzed by HPLC. Data are expressed as the means from at least two separate experiments; bars, SE.

Fig. 4.

Effect of dFdCyd on the dATP pool in human glioblastoma cells. Exponentially growing U251 (▪) or D54 (•) cells were treated with 25 nm dFdCyd (IC50) or 80 nmdFdCyd (IC50), respectively, for 24 h. Cellular nucleotides were extracted with perchloric acid, eluted over a boronate affinity column to remove ribonucleotides, and analyzed by HPLC. Data are expressed as the means from at least two separate experiments; bars, SE.

Close modal
Fig. 5.

Western blot analysis for p53 (top) and mdm-2 (bottom). Either U251 (left) or D54(right) cells were incubated with dFdCyd(IC50) for 24 h and then irradiated with 5 Gy of ionizing radiation. T = 0 represents the conclusion of the 24 h drug incubation when dFdCyd was washed out and the cells were irradiated.

Fig. 5.

Western blot analysis for p53 (top) and mdm-2 (bottom). Either U251 (left) or D54(right) cells were incubated with dFdCyd(IC50) for 24 h and then irradiated with 5 Gy of ionizing radiation. T = 0 represents the conclusion of the 24 h drug incubation when dFdCyd was washed out and the cells were irradiated.

Close modal
Fig. 6.

Effects of dFdCyd and ionizing radiation on cell cycle distribution of U251 (A) and D54 (B)cells. U251 cells were treated with 25 nm dFdCyd and/or 5 Gy of ionizing radiation. D54 cells were treated with 80 nmdFdCyd and/or 5 Gy of ionizing radiation. Control samples were obtained from untreated cells. Drug was added 24 h prior to irradiation. Cells were irradiated and/or drug was removed at T = 0 h. Thirty μmBrdUrd was added during the last 15 min prior to the indicated time points. Cells were prepared for dual parameter flow cytometry to determine DNA content and BrdUrd as described in “Materials and Methods.” Results of a single experiment are displayed. Experiments were repeated at least three times.

Fig. 6.

Effects of dFdCyd and ionizing radiation on cell cycle distribution of U251 (A) and D54 (B)cells. U251 cells were treated with 25 nm dFdCyd and/or 5 Gy of ionizing radiation. D54 cells were treated with 80 nmdFdCyd and/or 5 Gy of ionizing radiation. Control samples were obtained from untreated cells. Drug was added 24 h prior to irradiation. Cells were irradiated and/or drug was removed at T = 0 h. Thirty μmBrdUrd was added during the last 15 min prior to the indicated time points. Cells were prepared for dual parameter flow cytometry to determine DNA content and BrdUrd as described in “Materials and Methods.” Results of a single experiment are displayed. Experiments were repeated at least three times.

Close modal
Fig. 7.

Apoptosis in human glioblastoma cells in response to dFdCyd and ionizing radiation. Either U251 () or D54 (▪) were treated as indicated, and apoptosis was measured as sub-G1content by flow cytometry 72 h after irradiation and drug washout. Values are the means of at least three determinations; bars, SE. †, not done.

Fig. 7.

Apoptosis in human glioblastoma cells in response to dFdCyd and ionizing radiation. Either U251 () or D54 (▪) were treated as indicated, and apoptosis was measured as sub-G1content by flow cytometry 72 h after irradiation and drug washout. Values are the means of at least three determinations; bars, SE. †, not done.

Close modal

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 in part by Grant CA 83081 from the NIH, University of Michigan-Comprehensive Cancer Center NIH Grant CA46592, University of Michigan-Multipurpose Arthritic Center NIH Grant AR20557, and the University of Michigan Core Flow Cytometry facility.

3

The abbreviations used are: dFdCyd,2′,2′-difluoro-2′-deoxycytidine; BrdUrd, 5-bromo-2′-deoxyuridine;dFdCMP, 5′-monophosphate of dFdCyd; dFdCTP, 5′-triphosphate of dFdCyd;RER, radiation enhancement ratio; dNTP, deoxyribonucleoside triphosphate; HPLC, high-performance liquid chromatography; PI,propidium iodide.

Table 1

Radiosensitization of human glioblastoma cells

U251 or D54 cells were treated with the indicated doses of radiosensitizing agents for 24 h. Values represent a mean ± SE for at least three experiments.

Cell typeDrugDoseSurvival (%)RERMean inactivation dose (no drug)
U251 dFdCyd 10 nm 88.3 ± 11.4 1.60 ± 0.03a  
  25 nm 30.5 ± 15.5 1.77 ± 0.22a 2.61 ± 0.08 
 Hydroxyurea 3 mm 53.3 ± 1.1 1.39 ± 0.08a  
 BrdUrd 15 μm 78.8 ± 14.9 1.71 ± 0.19a  
D54 dFdCyd 10 nm 97.4 ± 9.3 1.26 ± 0.16b  
  80 nm 69.2 ± 12.0 0.94 ± 0.03b 2.01 ± 0.06 
 Hydroxyurea 0.6 mm 57.0 ± 7.3 1.06 ± 0.04b  
 BrdUrd 1.5 μm 81.7 ± 6.6 1.19 ± 0.06a  
  12 μm 58.3 ± 14.2 1.84 ± 0.16a  
Cell typeDrugDoseSurvival (%)RERMean inactivation dose (no drug)
U251 dFdCyd 10 nm 88.3 ± 11.4 1.60 ± 0.03a  
  25 nm 30.5 ± 15.5 1.77 ± 0.22a 2.61 ± 0.08 
 Hydroxyurea 3 mm 53.3 ± 1.1 1.39 ± 0.08a  
 BrdUrd 15 μm 78.8 ± 14.9 1.71 ± 0.19a  
D54 dFdCyd 10 nm 97.4 ± 9.3 1.26 ± 0.16b  
  80 nm 69.2 ± 12.0 0.94 ± 0.03b 2.01 ± 0.06 
 Hydroxyurea 0.6 mm 57.0 ± 7.3 1.06 ± 0.04b  
 BrdUrd 1.5 μm 81.7 ± 6.6 1.19 ± 0.06a  
  12 μm 58.3 ± 14.2 1.84 ± 0.16a  
a

P < 0.038 (significantly >1.0).

b

P > 0.083 (not significantly >1.0).

Table 2

Effect of dFdCyd and ionizing radiation on the cell cycle distribution of human glioblastoma cells

U251 and D54 cells were treated as indicated. Drug was added 24 h prior to T = 0 h (−24 h). T = 0 represents the time of irradiation and/or drug washout. DNA and BrdUrd content were measured using dual parameter flow cytometry. SNI represents cells that have S-phase DNA content by PI staining but do not incorporate BrdUrd(a measurement of S-phase-specific cell death). Data are from a representative experiment repeated at least three times.

TreatmentTime (h)G1 (%)S (%)SNI (%)G2 = M (%)
U251 cells      
Control 57.5 28.6 4.5 9.4 
5 Gy 61.2 27.2 2.6 9.0 
 12 11.1 68.3 1.7 18.9 
 24 48.9 16.3 2.2 32.6 
 48 57.6 20.6 4.1 17.7 
 72 52.3 26.1 4.8 16.8 
25 nm dFdCyd 16.8 70.2 10.5 2.6 
 24 48.0 35.7 11.9 4.3 
 48 24.6 44.4 13.1 18.0 
 72 33.9 45.0 5.0 16.2 
25 nm dFdCyd+ 5 Gy 13.8 72.0 10.9 3.3 
 24 33.0 28.3 33.0 5.8 
 48 24.9 32.7 20.4 22.1 
 72 28.1 39.0 11.5 21.4 
D54 cells      
Control 61.3 26.8 3.4 8.5 
5 Gy 64.8 24.9 1.7 8.7 
 12 74.8 5.3 1.0 18.9 
 24 77.0 11.6 2.0 9.4 
 48 71.4 15.9 2.3 10.5 
 72 65.1 20.9 2.3 11.7 
80 nm dFdCyd 54.3 35.3 1.4 8.9 
 24 56.9 31.8 2.2 9.0 
 48 66.5 24.3 2.4 6.8 
 72 64.4 25.8 2.0 7.9 
80 nm dFdCyd+ 5 Gy 54.1 36.1 1.7 8.1 
 24 74.3 10.7 2.8 12.3 
 48 71.9 14.8 2.3 11.1 
 72 61.9 22.3 4.1 11.7 
TreatmentTime (h)G1 (%)S (%)SNI (%)G2 = M (%)
U251 cells      
Control 57.5 28.6 4.5 9.4 
5 Gy 61.2 27.2 2.6 9.0 
 12 11.1 68.3 1.7 18.9 
 24 48.9 16.3 2.2 32.6 
 48 57.6 20.6 4.1 17.7 
 72 52.3 26.1 4.8 16.8 
25 nm dFdCyd 16.8 70.2 10.5 2.6 
 24 48.0 35.7 11.9 4.3 
 48 24.6 44.4 13.1 18.0 
 72 33.9 45.0 5.0 16.2 
25 nm dFdCyd+ 5 Gy 13.8 72.0 10.9 3.3 
 24 33.0 28.3 33.0 5.8 
 48 24.9 32.7 20.4 22.1 
 72 28.1 39.0 11.5 21.4 
D54 cells      
Control 61.3 26.8 3.4 8.5 
5 Gy 64.8 24.9 1.7 8.7 
 12 74.8 5.3 1.0 18.9 
 24 77.0 11.6 2.0 9.4 
 48 71.4 15.9 2.3 10.5 
 72 65.1 20.9 2.3 11.7 
80 nm dFdCyd 54.3 35.3 1.4 8.9 
 24 56.9 31.8 2.2 9.0 
 48 66.5 24.3 2.4 6.8 
 72 64.4 25.8 2.0 7.9 
80 nm dFdCyd+ 5 Gy 54.1 36.1 1.7 8.1 
 24 74.3 10.7 2.8 12.3 
 48 71.9 14.8 2.3 11.1 
 72 61.9 22.3 4.1 11.7 

We gratefully acknowledge Mark A. KuKuruga of the University of Michigan Flow Cytometry Core facility for excellent technical advice.

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