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Experimental Therapeutics |
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0504
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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) .
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MATERIALS AND METHODS |
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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 x g for 2
min and acidified to pH 2.8. Samples were then injected onto a 5-µm
Partisphere 4.6 x 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 30
µM 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.
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RESULTS |
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and Table 1
).
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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
).
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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.
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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.
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. 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 nM
dFdCyd for 24 h, followed by 5 Gy, show almost the same cell cycle
pattern as with dFdCyd alone; however, SNI
increased to 33% at 24 h after drug washout and was
consistently higher at 48 and 72 h compared with cells treated
with dFdCyd alone.
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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.
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DISCUSSION |
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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 IC50
concentrations 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. 
2 To whom requests for reprints should be
addressed, at Department of Pharmacology, 4713 Upjohn Center,
University of Michigan Medical School, 1310 East Catherine, Ann Arbor,
MI 48109-0504. Phone: (734) 763-5810; Fax: (734) 763-3438; E-mail: dshewach{at}umich.edu
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.
Received 3/31/00. Accepted 8/28/00.
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  M. A. Morgan, L. A. Parsels, J. Maybaum, and T. S. Lawrence Improving Gemcitabine-Mediated Radiosensitization Using Molecularly Targeted Therapy: A Review Clin. Cancer Res., November 1, 2008; 14(21): 6744 - 6750. [Abstract] [Full Text] [PDF]
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 S. A. Flanagan, C. M. Krokosky, S. Mannava, M. A. Nikiforov, and D. S. Shewach MLH1 Deficiency Enhances Radiosensitization with 5-Fluorodeoxyuridine by Increasing DNA Mismatches Mol. Pharmacol., September 1, 2008; 74(3): 863 - 871. [Abstract] [Full Text] [PDF]
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T. Okazaki, L. Jiao, P. Chang, D. B. Evans, J. L. Abbruzzese, and D. Li Single-Nucleotide Polymorphisms of DNA Damage Response Genes Are Associated with Overall Survival in Patients with Pancreatic Cancer Clin. Cancer Res., April 1, 2008; 14(7): 2042 - 2048. [Abstract] [Full Text] [PDF]
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 S. A. Flanagan, B. W. Robinson, C. M. Krokosky, and D. S. Shewach Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers Mol. Cancer Ther., June 1, 2007; 6(6): 1858 - 1868. [Abstract] [Full Text] [PDF]
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D. E. Milenic, K. Garmestani, E. D. Brady, P. S. Albert, A. Abdulla, J. Flynn, and M. W. Brechbiel Potentiation of High-LET Radiation by Gemcitabine: Targeting HER2 with Trastuzumab to Treat Disseminated Peritoneal Disease Clin. Cancer Res., March 15, 2007; 13(6): 1926 - 1935. [Abstract] [Full Text] [PDF]
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C. Bianco, E. Giovannetti, F. Ciardiello, V. Mey, S. Nannizzi, G. Tortora, T. Troiani, F. Pasqualetti, G. Eckhardt, M. de Liguoro, et al. Synergistic Antitumor Activity of ZD6474, An Inhibitor of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor Signaling, with Gemcitabine and Ionizing Radiation against Pancreatic Cancer Clin. Cancer Res., December 1, 2006; 12(23): 7099 - 7107. [Abstract] [Full Text] [PDF]
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 M. A. Morgan, L. A. Parsels, J. D. Parsels, A. K. Mesiwala, J. Maybaum, and T. S. Lawrence Role of Checkpoint Kinase 1 in Preventing Premature Mitosis in Response to Gemcitabine Cancer Res., August 1, 2005; 65(15): 6835 - 6842. [Abstract] [Full Text] [PDF]
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B. Hofstetter, V. Vuong, A. Broggini-Tenzer, S. Bodis, I. F. Ciernik, D. Fabbro, M. Wartmann, G. Folkers, and M. Pruschy Patupilone Acts as Radiosensitizing Agent in Multidrug-Resistant Cancer Cells In vitro and In vivo Clin. Cancer Res., February 15, 2005; 11(4): 1588 - 1596. [Abstract] [Full Text] [PDF]
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 B. Pauwels, A. E.C. Korst, F. Lardon, and J. B. Vermorken Combined Modality Therapy of Gemcitabine and Radiation Oncologist, January 1, 2005; 10(1): 34 - 51. [Abstract] [Full Text] [PDF]
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 L. K. Kvols Radiation Sensitizers: A Selective Review of Molecules Targeting DNA and non-DNA Targets J. Nucl. Med., January 1, 2005; 46(1_suppl): 187S - 190S. [Abstract] [Full Text] [PDF]
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B. W. Robinson, M. M. Im, M. Ljungman, F. Praz, and D. S. Shewach Enhanced Radiosensitization with Gemcitabine in Mismatch Repair-Deficient HCT116 Cells Cancer Res., October 15, 2003; 63(20): 6935 - 6941. [Abstract] [Full Text] [PDF]
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D. Sun, R. Urrabaz, S. Kelly, M. Nguyen, and S. Weitman Enhancement of DNA Ligase I Level by Gemcitabine in Human Cancer Cells Clin. Cancer Res., April 1, 2002; 8(4): 1189 - 1195. [Abstract] [Full Text] [PDF]
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B. W. Robinson and D. S. Shewach Radiosensitization by Gemcitabine in p53 Wild-Type and Mutant MCF-7 Breast Carcinoma Cell Lines Clin. Cancer Res., August 1, 2001; 7(8): 2581 - 2589. [Abstract] [Full Text] [PDF]
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) 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.
) 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, 
) or D54 (
, not done.