[Cancer Research 60, 86-91, January 1, 2000]
© 2000 American Association for Cancer Research
Experimental Therapeutics |
Re-Evaluating Gadolinium(III) Texaphyrin as a Radiosensitizing Agent1
Eric J. Bernhard2,
James B. Mitchell,
Dennis Deen,
Monika Cardell,
David I. Rosenthal and
J. Martin Brown
University of Pennsylvania, Department of Radiation Oncology, Philadelphia, Pennsylvania 19104 [E. J. B., D. I. R.]; Radiation Biology Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, Maryland 20892 [J. B. M.]; Brain Tumor Research Center, University of California San Francisco, San Francisco, California 94143 [D. D., M. C.]; and Stanford University School of Medicine, Department of Radiation Oncology, Stanford, California 94305 [J. M. B.]
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ABSTRACT
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Gadolinium(III) texaphyrin (Gd-tex) was recently proposed as a
radiosensitizing agent that combines preferential tumor uptake with
detection of drug localization by magnetic resonance imaging (S. W. Young et al., Proc. Natl. Acad. Sci. USA,
93: 6610–6615, 1996). In view of the initial report on
this compound, four radiobiology laboratories undertook independent
efforts to further study radiosensitization by Gd-tex. In addition to
repeating the previously reported studies on Gd-tex in HT-29 cells, we
tested five other human tumor cell lines (U-87 MG, U251-NCI,
SW480, A549, and MCF-7). These studies included a Gd-tex treatment
period of 24 h before irradiation (as in the original
publication), with concentrations of Gd-tex ranging from 20–500
µM. In neither the HT-29 cells nor any of the other five
human cell lines did we see radiation sensitization by Gd-tex. Two cell
lines (MCF-7 and U-87 MG) were further tested for radiosensitization by
Gd-tex under hypoxic conditions. No radiosensitization was observed in
either case. Finally, the radiation response of two tumor lines were
assessed in vivo. Neither HT-29 xenografts in severe
combined immunodeficient (SCID) mice nor RIF-1 tumors growing in C3H
mice demonstrated radiosensitization after Gd-tex treatment before
single or fractionated doses of radiation. Our results raise questions
about the efficacy of Gd-tex as a radiosensitizing agent.
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INTRODUCTION
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Developing a radiosensitizing agent specific for tumor cells has
long been a research goal of radiobiology.
Gd-tex3
was recently proposed as a radiosensitizing agent that combines
preferential tumor uptake with detection of drug localization by MRI
technique (1)
. The proposed mechanism of action of Gd-tex
as a radiosensitizer was that the molecule would capture hydrated
electrons formed by irradiation, thereby increasing the concentration
of hydroxyl radicals formed by radiation in areas where the drug was
present. This novel mechanism of sensitization, combined with selective
concentration in tumors and the ability to detect drug presence with
MRI, represented a potentially revolutionary approach to tumor cell
radiosensitization. In their report outlining the properties of Gd-tex,
Young et al. (1)
showed a significant
radiosensitization of the HT-29 colon carcinoma cell line in
vitro after incubation with Gd-tex and a prolonged growth delay of
EMT-6 tumors given fractionated irradiation combined with i.v.
administration of Gd-tex 2 h before irradiation. Improved survival
was also reported in DBA/2N mice bearing SMT-F tumors when mice were
treated with a single dose of 30 Gy after i.v. administration of 40
µg/kg Gd-tex. On the basis of these initial findings, a Phase I study
was conducted, demonstrating that the compound was well tolerated in
patients and that it led to selective enhancement of tumor imaging on
MRI (2)
.
These promising results prompted us to study the effects of Gd-tex on
radiosensitivity in the cell lines originally reported and to extend
the studies to other cell lines in vitro. In addition, the
radiosensitizing properties of Gd-tex were tested in vitro
under conditions of hypoxia such as occur in tumors. We also sought to
further define the activity of this compound as a radiosensitizer of
transplanted tumors in vivo. The studies reported here were
independently initiated in four separate laboratories. Communication
between the authors concerning the data presented here was initiated
only after the majority of the studies had been completed, at which
time it was decided that these results could best be presented in a
single report.
The results we obtained cast doubt on the potential of the Gd-tex as a
radiosensitizing agent. No radiosensitization was detected in the HT-29
cell line originally reported by Young et al.
(1)
or in an expanded panel of human and rodent tumor
lines in vitro or as tumors in vivo, irradiated
under a variety of conditions.
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MATERIALS AND METHODS
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Radiosensitizers.
Gd-tex (Mr 1148.4) is a green-to-black
solid that is soluble in water to a concentration of 200 mg/ml (174
mM). The compound was supplied by Pharmacyclics
Inc. (Sunnyvale, CA) in powder form and dissolved in either sterile
distilled water at 100 mM or 1x Earle’s
balanced salt solution at 25 mM. The resulting
solution was filter-sterilized by passage through a 0.22
µM filter, stored at 4°C and protected from
light, and used within 15 days from the time the stock solution was
made. Working solutions of the compound were made from stock solutions
immediately before use by dilution into tissue culture medium. The
Gd-tex compound used in animal experiments at Stanford was supplied by
Pharmacyclics Inc. as a solution of 2 mg/ml and stored at 4°C until
needed. This compound corresponds to Gd-tex compound 2 reported by
Young et al. (1)
. The structure and properties
of SR2508 (tirapazamine) have been reported previously (3
, 4)
.
Cell Culture and Survival Determination.
HT-29, SW480, and A549 cells were maintained and assayed in DMEM with
high glucose supplemented with 10% fetal bovine serum and
penicillin-streptomycin. U251-NCI and U-87 MG were cultured in Eagle’s
minimal essential medium supplemented with nonessential amino acids,
glutamine, and 10% fetal bovine serum. MCF-7 cells and, in one
institution, HT-29 cells were cultured in RPMI 1640 with 10% fetal
bovine serum. Cultures were kept at 37°C in humidified incubators
with 5% CO2. Clonogenic survival was determined
by two methods: (a) clonogenic assay at varying radiation
doses from 1–10 Gy; and (b) limiting dilution cloning to
measure the surviving fraction at 2 Gy. For both assays, cells from
log-growth cultures were treated for 24 h before irradiation with
the indicated concentration of Gd-tex. For growth inhibition studies,
Gd-tex was added to cultures established 1 day earlier, and cell counts
were determined at the times indicated after drug addition. Gd-tex
toxicity was determined by exposing cells to Gd-tex for the time
indicated, followed by plating of treated cells for clonogenic survival
in the absence of drug.
Clonogenic assays were carried out on exponentially growing cultures.
To generate clonogenic survival curves, cells were treated with Gd-tex
alone or treated with the drug and irradiated immediately before
trypsinization and counting. Known numbers of cells were then seeded in
plastic Petri dishes and incubated in a 37°C humidified incubator
with 5% CO2 until colonies were ready for
scoring. In experiments on U251-NCI and U-87 MG, cells were plated onto
a feeder layer of lethally irradiated SF-126 cells. In experiments
measuring the surviving fraction at 2 Gy (Fig. 2, C and D)
and in clonogenic survival determinations for A549 and
SW480 (Fig. 3, C and D)
, cells that had been
pretreated were harvested, and a known number of cells were replated in
drug-free medium before irradiation. Cells were either irradiated on
ice with a Phillips orthovoltage therapeutic X-ray machine at a dose
rate of 1.3 Gy/min or irradiated at ambient temperature with a Mark I
cesium irradiator (J. L. Shepherd, San Fernando, CA) at a dose
rate of 1.6 Gy/min or with a cobalt-60 irradiator at a dose rate of 1.5
Gy/min. Colonies were stained and counted 14–21 days after
irradiation. The surviving fraction at a given dose is defined as the
ratio of the PE of irradiated cells to that of unirradiated cells
exposed to the same concentration of drug. Each point on the survival
curves represents the mean surviving fraction from at least three
dishes.
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Fig. 2. Effect of Gd-tex pretreatment on HT-29 radiation
sensitivity. A, HT-29 survival data obtained from three
centers in the present study ( , •, and  ) was compared to the
data for untreated HT-29 cells reported by Young et al.
( ; Ref. 1
). B, HT-29 cells were treated
with carrier ( and •) or 20 ( ), 25 (), 40 ( ), or
100 µM () drug before irradiation at the doses
indicated. Irradiation was carried out in the presence of Gd-tex before
trypsinization and replating in drug-free medium. Clonogenic survival
was scored 2 weeks after irradiation. C, clonogenic
survival was determined by limiting dilution analysis (see "Materials
and Methods"). Cells were treated with 20 µM drug (
and ) or an equal volume of carrier ( and •) for 24 h
before irradiation. Cells were trypsinized and plated for clonogenic
survival in drug-free medium before irradiation (filled
symbols). The SF2 in the presence of drug was 0.82.
D, HT-29 cells were treated as described in
B, but with 100 µM drug. The
SF2 in the presence of drug was 0.79. Control cells have a
SF2 of 0.78 (this control result is superimposed on the
curves in A () and the data shown in C
and D for comparison with drug-treated cultures).
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Fig. 3. Radiation survival of tumor cells after 24 h of
Gd-tex treatment. U251-NCI cells (A) and U-87 MG cells
(B) were pretreated with 0 (), 25 (), 50 (), or
100 µM () Gd-tex for 24 h. Cells were then
irradiated on ice before trypsinization and plating for clonogenic
survival in drug-free medium. In B, a replicate
experiment is shown for treatment with 0 () and 100 µM
(•) Gd-tex. PEs for U251-NCI cells were as follows: control, 0.47; 25
µM, 0.49; 50 µM, 0.56; and 100
µM, 0.36. The PEs for U-87 MG cells were as follows:
control, 0.38 and 0.3; 25 µM, 0.36; 50 µM,
0.48; and 100 µM, 0.34 and 0.12. SW480 cells
(C) were pretreated with 100 µM Gd-tex for
24 h before trypsinization and plating. Irradiation was carried
out after replating in drug-free medium. The PE was 0.51 for control
cells and 0.54 for Gd-tex-treated cells. A549 radiation survival
(D) was determined as described in C. The
PE was 1.2 for control cells and 0.96 for Gd-tex treated cells. Control
cells, ; 100 µM Gd-tex-treated cells, .
E, MCF-7 cells were pretreated with 0 (), 25
µM (), or 100 µM () Gd-tex for
24 h before irradiation and plating for clonogenic survival as
described in A. The PE was 0.72 for control MCF-7 cells
and 0.8 and 0.6 for Gd-tex-treated cells at 25 and 100
µM, respectively.
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The SF2 was also determined by limiting dilution
analysis for HT-29 cells. Cells were pretreated, trypsinized, counted,
and plated in 96-well dishes in 200 µl of medium. Dishes were
irradiated or sham-irradiated and cultured for 14–21 days, after which
individual wells were scored for the presence of colonies. Wells were
scored positive for colony formation if they contained one or more
colony of 20 or more cells. Linear regression analysis was performed on
the natural log of negative wells with the origin as the initial point
of the line. The slopes obtained from this regression analysis were
compared to determine the surviving fraction in the presence or absence
of inhibitor. SF2 was defined as the slope of the
linear regression obtained from irradiated cultures divided by the
slope of the linear regression obtained from unirradiated cultures
given the same treatment before irradiation.
Hypoxia was induced by flushing cultures in glass Petri dishes or
flasks with either 95% air:5% CO2 or 95%
nitrogen:5% CO2 four times, followed by the
addition of Gd-tex through gas-tight valves. After 1 h, cells were
irradiated in Gd-tex under hypoxia and subsequently replated for
clonogenic survival in drug-free medium.
Determination of Radiosensitivity in Vivo.
C3H or severe combined immunodeficient (SCID) mice were inoculated in
the flank by intradermal injection with RIF-1 or HT-29 tumor,
respectively, at approximately 15 weeks of age. The mice were bred and
housed under specific pathogen-free conditions in the American
Association for Laboratory Animal Science-approved facilities at
Stanford Medical Center. Tumors were segregated into experimental
groups when they attained a mean diameter of approximately 7 mm. Gd-tex
compound 2 was administered as an i.v. injection at a dose of 40
µmol/kg in a volume of 0.02 ml/g before irradiation. SR2508 was
dissolved at a concentration of 50 mg/ml and administered i.p. at a
dose of 1500 mg/kg. Mice were irradiated at the tumor site with a
Phillips 250 kVp orthovoltage therapeutic X-ray machine with a single
fraction of 20 Gy to determine the clonogenic survival of HT-29 or with
either a single 15-Gy fraction or three fractions of 6 Gy at 24-h
intervals to determine the regrowth delay in RIF-1 tumors. In
experiments with RIF-1 tumors, animals that were treated with both
irradiation and Gd-tex received injections of the drug 4 h before
each irradiation. Each treatment group consisted of five animals.
Regrowth of RIF-1 tumor was monitored in five mice/treatment group
until tumors attained three times their original volume.
Clonogenic survival of HT-29 tumors treated with irradiation and Gd-tex
was determined by excision and dissociation of tumors 24 h after
irradiation. The relative number of clonogenic cells/tumor was
calculated as the product of the clonogenic survival and the number of
cells extracted per treated tumor relative to the untreated tumors.
This value therefore takes into account any cells that die in the first
24 h after irradiation. HT-29 tumor-bearing animals received
Gd-tex at the times indicated before irradiation.
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RESULTS
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Assessing Gd-tex Cytotoxicity.
To determine whether Gd-tex had any toxicity to tumor cells when these
were exposed to the drug alone, clonogenicity and cell proliferation
were assessed in U251-NCI and U-87 MG cells after treatment with Gd-tex
at varying concentrations for up to 96 h. Gd-tex inhibited cell
proliferation in a dose-dependent manner over the course of 4 days, as
shown in Table 1
. The toxicity of Gd-tex was also measured by determining the clonogenic
survival of cells treated at 100–500 µM drug
concentrations for 24–96 h (Fig. 1)
. The clonogenic survival of treated cells was decreased in a manner
dependent on both the concentration of Gd-tex, and the duration of the
exposure. Because the drug itself exhibits some toxicity, the radiation
survival determinations reported below are corrected for any decrease
in PE observed in unirradiated controls exposed to Gd-tex alone.
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Table 1 Effect of 4-day continuous culture in Gd-tex on cell proliferation
Values shown represent the number of cells recovered after 4 days of
culture in the presence of the indicated concentration of Gd-tex
relative to the number of cells recovered from cultures that received
no drug.
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Radiation Survival after Gd-tex Treatment of Tumor Cells in
Vitro.
Because clonogenic survival in the presence of Gd-tex was initially
tested by Young et al. (1)
in the HT-29 cell
line, three of the laboratories contributing to this report attempted
to reproduce the previously reported results using this cell line (Fig. 2)
. The first notable finding was that although the three laboratories in
the current study reported very similar radiosensitivity for HT-29
cells as determined by standard clonogenic assay or by limiting
dilution analysis, the survival reported by Young et al.
(1)
was significantly lower at all doses (Fig. 2A)
. This finding was corrected by the authors while the
current manuscript was under review (5)
.
To test the radiosensitizing effect of Gd-tex on HT-29 cells, the
clonogenic survival of cultured cells exposed to this drug for 24 h before irradiation was determined. Log growth cell cultures were
treated with Gd-tex for 24 h, and then they were either irradiated
in drug-containing medium or harvested by trypsinization and washed in
medium without drug before irradiation as indicated. Cells pretreated
with Gd-tex and rinsed in drug-free medium before irradiation were
observed to retain a yellow color after removal of the drug, indicating
residual drug presence on the cells or uptake by the cells, although
the location of the residual drug (intra- versus
extracellular) was not determined. As seen in Fig. 2B
, the
radiation survival of HT-29 cells as determined by standard clonogenic
assay after a 24-h pretreatment with 20–100 µM
Gd-tex was not altered from that seen in control cultures. This finding
was reproduced independently in two laboratories. The
SF2 was also measured by limiting dilution
analysis in a third laboratory after a 24-h pretreatment with 20
µM (Fig. 2C)
or 100
µM (Fig. 2D)
Gd-tex. The
SF2 obtained by this method for control cells was
0.78, in accord with the values obtained by standard clonogenic assay
(Fig. 2A)
, and no reduction in this value was seen after
pretreatment with either 20 or 100 µM Gd-tex.
Thus, in three studies carried out independently in three established
radiobiology laboratories, no radiosensitization of HT-29 cells was
observed after treatment with Gd-tex.
To test whether Gd-tex could radiosensitize other tumor cell types,
clonogenic survival determinations were also carried out with an
extended panel of tumor cells of different origins. Two tumor lines
derived from central nervous system malignancies, U251-NCI (Fig. 3A)
and U-87 MG (Fig. 3B)
were examined for
radiation survival after pretreatment with 25–100
µM Gd-tex for 24 h. No radiosensitization
was observed in Gd-tex-treated U251-NCI cells. Slightly reduced
survival was seen in U-87 MG cells treated with 100
µM Gd-tex in one experiment, but this finding
was not reproduced in a replicate experiment, nor was it observed after
48 h pretreatment with Gd-tex (data not shown). Similar negative
results were obtained in these two cell lines after a 24-h pretreatment
with doses of 250 and 500 µM (data not shown).
Gd-tex was also tested as a radiosensitizer in SW480 colon carcinoma
cells (Fig. 3C)
, A549 lung cancer cells (Fig. 3D)
, and MCF-7 breast carcinoma cells (Fig. 3E)
after pretreatment with 20–100 µM Gd-tex for
24 h. None of these cell lines showed significantly reduced
clonogenicity as a result of Gd-tex treatment before irradiation at any
Gd-tex dose tested. A modest reduction in survival was noted for MCF-7
cells at 100 µM Gd-tex at lower radiation
doses.
Effect of Gd-tex on the Radiosensitivity of Hypoxic Cells.
Because the redox potential of Gd-tex was reported to be above the
threshold for electron affinic hypoxic cell senstizers, experiments
were carried out to test whether Gd-tex might act preferentially on
hypoxic cells. These experiments were carried out on both MCF-7 cells,
in which minor changes in survival had been noted at the highest Gd-tex
doses tested, and in U-87 MG cells. Cultures were pretreated with
Gd-tex for 24 h, subjected to hypoxia, and irradiated under
hypoxic conditions. As seen in Fig. 4
, hypoxia increased the radioresistance of these cells by a
dose-modifying factor of approximately 2.4. However, pretreatment for
24 h with 100 µM Gd-tex failed to alter the
radiosensitivity of either the hypoxic or aerobic cells. U-87 MG cell
radiosensitivity was also assessed by clonogenic survival after
pretreatment with 100–500 µM Gd-tex under oxic and
hypoxic conditions as detailed in "Materials and Methods." No
difference in radiosensitivity was detected in these cells under either
condition after pretreatment with up to 500 µM Gd-tex
(data not shown).
Radiation Survival after Gd-tex Treatment of Tumor Cells in
Vivo.
Because significantly increased survival of SMT-F tumor-bearing mice
and increased tumor growth delay of EMT-6 tumors were reported after
combined radiation plus Gd-tex treatment, we assessed the effect of
this drug on tumor cell radiosensitivity in vivo by
determining the clonogenic survival of tumor cells after irradiation
in vivo and the tumor regrowth delay. Clonogenicity of HT-29
tumor cells isolated from animals treated with a single dose of Gd-tex
administered at various times before irradiation with 20 Gy was
determined (Fig. 5)
. Irradiation alone reduced the survival of isolated tumor cells by 3
logs to 0.0018. Administering Gd-tex at 24, 8, 6, or 2 h before
irradiation showed no significant effect on the clonogenic survival of
the tumor cells compared to the effect of irradiation alone (0.0017-
0.0044). In contrast, survival was decreased by more than 50-fold (to
3 x 10-5) after treatment of
identical tumors in the same experiment with SR2508 1 h before
irradiation. Thus, Gd-tex failed to radiosensitize SMT-F tumors under
conditions where radiosensitization was observed using SR2508.
We then tested whether Gd-tex could contribute to radiation-induced
tumor growth delay in RIF-1 tumor-bearing animals (Fig. 6)
. Untreated tumors or those receiving either one or three injections of
Gd-tex reached three times the initial tumor volume by 5 to 6 days.
Irradiation with either a single fraction of 15 Gy or three fractions
of 6 Gy prolonged this interval to 23 and 22 days, respectively.
However, treatment with Gd-tex before irradiation had no significant
effect on the regrowth delay in either the 15-Gy single dose group or
in tumors treated with three 6-Gy fractions.
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Fig. 6. Regrowth delay after Gd-tex treatment and irradiation of
RIF-1 tumors. C3H mice bearing RIF-1 tumors were treated with Gd-tex
4 h before irradiation of the tumor with either 15 Gy or three
fractions of 6 Gy administered at 24-h intervals. Tumors were then
measured daily, and the volume was determined by the following
formula: ( /6) x a x b x c (where a, b, and
c are mutually perpendicular tumor diameters).
The growth of untreated tumors, tumors, and tumors exposed to either
drug or radiation alone are shown for comparison. The data represent
the geometric mean growth delay ± SE. Five
tumor-bearing animals were evaluated for each group.
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DISCUSSION
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This study presents a summary of the work of four different
laboratories that independently tested the potential of Gd-tex as a
radiosensitizer of tumor cells in vitro and in
vivo. The earlier publication of Young et al.
(1)
and more recent unpublished observations from
Pharmacyclics Inc. (6)
report that this compound selectively
accumulates in transplanted mouse tumors, sensitizes tumor cells to
radiation cell kill in vitro, and potentiates the response
of transplanted tumors to both single and fractionated irradiation. The
fact that this compound is selectively localized in tumors and can be
imaged through MRI-contrasted enhancement because of the paramagnetic
nature of the agent makes this compound particularly appealing for use
in clinical trials (7)
. Indeed, the National Cancer
Institute bypass budget document (8)
features this
compound as a new agent to be used with radiotherapy. A multicenter
Phase III clinical trial of Gd-tex as a radiosensitizer combined with
radiotherapy for the treatment of patients with brain metastases is
being sponsored by Pharmacyclics Inc., the developer of this compound
(9)
. These characteristics and promising preclinical data
encouraged each of the four laboratories to undertake studies of the
compound as a radiation sensitizer. While the studies were conducted,
none of the laboratories was aware of activities in the other
laboratories, and only learned of the other data by chance after the
studies were largely completed. Because there was general agreement on
the results obtained with the cell lines tested in the four
laboratories, it was decided to combine the data into the present
publication. When the data were combined, the degree of similarity of
the results was remarkable. Three of the laboratories, for example, had
tested the HT-29 human colon carcinoma cells in vitro under
conditions similar to those described in the publication of Young
et al. (1)
. In the current study, cells were
irradiated before trypsinization in medium containing inhibitor as well
as after preincubation in inhibitor, followed by trypsinization and
replating as in the original report. The data for the control (and
Gd-tex-treated) HT-29 cells were essentially indistinguishable among
the three laboratories and are typical of those obtained for human
tumor cells, whereas the HT-29 cells of the earlier publication
(1)
were apparently hypersensitive to radiation, even more
so than cells with mutations in genes involved in DNA repair such as
ATM or DNA-PK (10
, 11)
. A
correction to the original report has since been published (Ref.
5
; discussed below).
In addition to attempting to repeat the earlier data showing
radiosensitization by Gd-tex in HT-29 cells, we tested five other human
tumor cell lines (U-87 MG, U251-NCI, SW480, A549, and MCF-7). These
cells were tested with a preirradiation Gd-tex treatment period of
24 h (as in the original publication) with concentrations of
Gd-tex from 20–500 µM (i.e., up to
concentrations more that 10 times higher than those used in
vivo). In neither the HT-29 cells nor any of the other five human
cell lines did we see any suggestion of radiation sensitization by
Gd-tex (Figs. 2
3
4)
.
Gd-tex was reported to have a redox potential above the upper threshold
proposed for electron affinic hypoxic cell radiosensitizers
(1)
, which led us to test whether the compound would be a
sensitizer of hypoxic cells. Although this was not tested specifically
in the earlier publication, the positive results obtained in
vivo could have been the consequence of sensitization of hypoxic
cells in the tumors. Also, if the compound were acting as an electron
affinic agent, as proposed (1)
, it would be expected to
sensitize hypoxic cells rather than aerobic cells (12)
.
However, the two cell lines tested under hypoxic conditions (U-87 MG
and MCF-7) showed no suggestion of any sensitization of hypoxic cells,
even at concentrations of up to 500 µM.
Finally, we tested Gd-tex for its ability to potentiate the antitumor
efficacy of radiation in the same way as that reported in the previous
study using two different tumor models assayed by two different
methods. Although it is unlikely that a compound would sensitize tumors
in vivo without radiosensitization in vitro, such
a phenomenon is not unprecedented. Nicotinamide, for example, does not
sensitize aerobic or hypoxic cells in vitro, but because it
improves tumor blood flow and oxygenation, it sensitizes tumors to
radiation in vivo (13)
. However, this was not
the case with the experiments we performed with HT-29 tumors growing in
SCID mice and irradiated with a large, single dose of 20 Gy. To assay
cell killing in this experiment, we measured the total number of
surviving cells in the tumor (by clonogenic assay) 24 h after
irradiation. As a positive control, we gave a large, single dose of the
2-nitromidazole hypoxic cell radiosensitizer SR2508, which sensitizes
tumors to irradiation by sensitizing hypoxic cells (3)
. As
shown in Fig. 5
, we saw no sensitization of the tumors to irradiation
with Gd-tex injected i.v. (at 40 µmol/kg) from 2–24 h before
irradiation. As a further test of the ability of the compound to
sensitize tumors in vivo, we performed a growth delay
experiment with the RIF-1 tumor-bearing mice injected 4 h before
either a single dose of 15 Gy or to three daily doses of 6 Gy to the
tumor. Under neither circumstance did we observe any change in the
growth delay of the tumors in mice injected with Gd-tex compared to
groups treated with irradiation only, despite the fact that larger
growth delays can be readily obtained at larger radiation doses
(14)
.
Therefore, the present data show no indication that Gd-tex is a
radiation sensitizer of aerobic or hypoxic cells in vitro or
of tumors in vivo. How can these data be reconciled with the
earlier study apparently showing highly significant radiosensitization
of tumor cells both in vitro and in vivo? In
terms of the in vitro data with HT-29 cells, Young et
al. (1)
reported a dose modification factor of 1.9
for HT-29 human colon cancer cells. We have commented above on the
unusually sensitive control survival curve published by these authors
(Fig. 1
of this report and Fig. 2
of Ref. 1
). During
review of our manuscript, a correction was published (5)
stating that the scale reported in Fig. 2
of the Young et
al. study (1)
was incorrect and that the experiments
had been repeated, giving "radiation enhancement comparable to our
initial findings at doses between 8 and 20 Gy." Because no other
details were given, it is impossible to assess this statement. However,
if the radiation sensitivity of the untreated HT29 cells were similar
to that found by us (Fig. 2A)
, the reported sensitization enhancement
ratio of 1.9 would have produced a surviving fraction at 10 Gy of less
than 10-10. This is well outside the range of
survival that can be measured using the clonogenic assay. However,
without additional details, it is not possible to reconcile the two
data sets.
The explanation that Gd-tex is an electron affinic sensitizer able to
"capture" the hydrated electron
(e-aq) could not account for
the sensitization because electron affinic radiosensitizers are
specific for hypoxic cells and show no radiosensitization of aerobic
cells (11)
. Because we found no radiosensitization of
either aerobic or hypoxic cells, this mechanism seems unlikely.
However, we feel that there is a likely explanation for the apparent
radiosensitization of the EMT-6 tumor transplanted into BALB/C mice and
given five consecutive doses of 1, 2, or 4 Gy/fraction with varying
doses of Gd-tex. This tumor is antigenic in this strain of mice, a
phenomenon that makes growth delay experiments hazardous
(15)
. In particular, any agent that changes the
cell-mediated immunity against this tumor could affect growth delay in
the absence of any change in the response of the cells to radiation.
Thus, a possible explanation for the positive results seen with Gd-tex
added to fractionated irradiation is that the concentration of the
compound in the tumor potentiated host cell immunity against the tumor,
thereby increasing growth delay. Consistent with this interpretation is
the large effect reported by Young et al. (1)
.
For example, the dose-response curve for 40 µmol/kg Gd-tex added to
5 x 1 Gy is similar to that of tumors given 5 x 4 Gy alone, implying a dose modification factor of 4 in these
tumors. This is much larger than that reported at even the highest
Gd-tex concentrations in vitro. Similar considerations may
have led to the positive results for mouse survival seen with SMT-F
tumors transplanted into DBA/2N mice and treated with 30 Gy.
Thus, our results differ substantially from those originally reported
by Young et al. (1)
, although conditions and
experimental systems did not exactly reproduce those of the original
report, and suggest that further independent study of Gd-tex as a
radiosensitizer is indicated.
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FOOTNOTES
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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 in part by National Cancer Institute
Grants CA73820 (to E. J. B.) and CA-15201 (to J. M. B.). Support
was also received from Pharmacyclics Inc., Sunnyvale, California
(D. D. and M. C.). 
2 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, 185 John Morgan
Building, University of Pennsylvania, Philadelphia, PA 19104-6072.
Phone: (215) 898-0078; Fax: (215) 898-0090; E-mail: bernhard{at}mail.med.upenn.edu
3 The abbreviations used are: Gd-tex,
gadolinium(III) texaphyrin; MRI, magnetic resonance imaging;
SF2, surviving fraction after 2 Gy irradiation; PE, plating
efficiency.
Received 3/ 5/99.
Accepted 10/22/99.
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Top
ABSTRACT
INTRODUCTION
). At the times indicated, cells were
rinsed, trypsinized, and replated in drug-free medium for clonogenic
survival determination. Values shown are the clonogenic survival
relative to untreated control cultures.
