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4050
DNA Double-Strand Breaks and H2AX-Phosphorylation
Contribute to FdUMP10-Induced Cell Death in PC3
Cells
William H. Gmeiner1, Robert E. Hamlin1, Mark
Willingham2, Yves Pommier3 1Department of Cancer
Biology and 2Department of Pathology, Wake Forest
University School of Medicine Winston-Salem, NC
27157 and 3Department of Molecular Pharmacology,
National Cancer Institute, Bethesda MD 20892
Materials and Methods Drugs FdUMP10 was
synthesized under an NCI contract to support the
RAID project (Gmeiner). Synthetic procedures
were similar to those described previously. Cell
Culture PC3 cells were obtained from ATCC and
grown in RPMI 1640 medium with 5 FBS, 2 mM
glutamine, penicillin (100 U/mL), and
streptomycin (100 U/mL) (37 C, 5 CO2). For
clonogenic assays 500 or 1,000 cells were plated
in 60 mm petri dishes and incubated with drug for
72 h followed by incubation in drug-free medium
for seven days.
Abstract We recently completed an in vivo study
evaluating the efficacy of FdUMP10 as a
single-agent and as a radiosensitizer for the
treatment of PC3 (human prostate cancer)
xenografts in nude mice. These studies go beyond
those described in the published abstract.
FdUMP10 is highly cytotoxic towards
hormone-refractory prostate cancer (HRPC) cells
(PC3 and DU145) and is an excellent
radiosensitizer of PC3 cells at sub-lethal drug
concentrations. FdUMP10 reduced the growth of
established tumors and significantly increased
survival. FdUMP10 was very-well tolerated in
vivo even with extensive dosing. The combination
of FdUMP10 and radiation shows exceptional
promise for the treatment of prostate cancer
(PC). Introduction Fluoropyrimidines (FPs) have
been evaluated for treatment of PC with limited
success. Data from the NCI 60 cell line screen
indicated that cellular models of HRPC were
highly responsive to FdUMP10. We performed an
in vivo study evaluating the safety and efficacy
FdUMP10 radiation for treatment of PC3
xenografts.
Results The results are summarized in Figures 1
6 and Tables 1 and 2. FdUMP10 is potent at
inhibiting the clonogenicity of PC3 at
concentrations greater than 1 nM and is a potent
sensitizer of PC3 cells to radiation at 1 nM and
0.1 nM. Cell death is preceded by massive DNA
damage consistent with a Top1-directed cytotoxic
mechanism. FdUMP10 inhibits growth of PC3
xenografts in vivo and significantly increases
survival. The combination of FdUMP10
radiation is highly potent at inhibiting tumor
growth. The results warrant evaluation of this
combination for the treatment of human prostate
cancer. Acknowledgements In addition to the
individuals listed as authors for this poster,
the following individuals contributed
significantly to the work presented Heather
Hatcher, Ph.D., Thomas Smith, M.D., Daniel
Bourland, Ph.D., Ralph DAgostino, Ph.D, Pradeep
Garg, Ph.D., Lance Warren, and Scott Cramer,
Ph.D. This work was supported by NCI CA 102532
and NCI RAID project and the CCCWFU.
Radiation Sensitization Radiation sensitization
of PC3 cells by FdUMP10 was evaluated using a
modified clonogenic assay. 5,000 PC3 cells were
plated in each well of a 24 well plate. Either
24 or 48 h into the 72 h drug-exposure period,
cells were irradiated (0 6 Gy) using a 137Cs
irradiator. Cells were incubated for 10 days
following drug/radiation exposure. Numbers of
viable cells were evaluated using an MTT assay.
The survival data were fit to a linear-quadratic
model and used to calculate radiobiological
parameters (Table 1). Synergy between FdUMP10
and radiation was determined using Calcusyn (v.
2.0 BioSoft). Jugular Vein Catheterization Male
NCr nude (nu/nu) mice were purchased from NCI.
Prior to tumor inncoculation, PE10 tubing was
used to cannulate the jugular vein. Catheters
were supplied with a heparin lock and were
heat-sealed following each procedure.
Establishment of Xenografts 3.5 x 106 PC3 cells
re-suspended in serum-free medium and mixed 11
with GFR Matrigel were injected s.c. into each
flank of male athymic (nu/nu) mice 8 weeks of
age. Palpable tumors were evident two weeks
post-inoculation and animals were randomized into
drug-treated and saline-treated groups.
Treatment Treatment was administered according
to the schedule shown in Figure 3. Mice treated
with FdUMP10 were administered 40 mg/kg/dose on
the indicated days via the catheter. Control
mice were injected with 100 mL of sterile saline
at the same time. The left flank of mice from
both groups was administered 3 Gy radiation on
the indicated days using an orthovoltage X-ray
irradiator (GE). Mice were anesthetized with
isoflurane during each injection and irradiation
procedure. Animal weights were measured each
week and tumors were measured twice per week.
Animals were sacrificed when tumor burden was
deemed excessive.
Statistical Analysis Survival data and tumor
growth curves were compared using parametric and
non-parametric methods using SAS. Mean tumor
volumes and SE values are shown for comparison
and to show trends. Comparisons were made
between the four groups using repeated measures
mixed model analysis. We also compared the mean
values at different time points to determine the
time when the four groups began to differ.
Median and ranges of tumor volume at a given date
were calculated to determine T/C ratios (Table 2)
and median volume scores were compared using
non-parametric two-sample median tests. Survival
curves were generated to compare groups. Mean
survival times were compared using two-sample
t-tests and median survival times were compared
via the log-rank test. Histological
Analysis Tumors and normal tissues were collected
following animal sacrifice. BrdU was injected
prior to sacrifice and tumors were analyzed for
BrdU and CD31.