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Experimental Therapeutics |
Departments of Radiation Oncology [E. K., M. K. N., E. N., A. R., T. S. L.], Radiology and Biological Chemistry [L. D. S., B. D. R.], and Pharmacology [J. P.], University of Michigan, Ann Arbor, Michigan 48109-0010
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The two most widely studied enzymes in the enzyme/prodrug strategy are HSV-TK3 and bCD. HSV-TK converts GCV into a phosphorylated toxic metabolite (GCV-P) that inhibits DNA polymerase, resulting in DNA chain termination. CD deaminates the prodrug 5-FC into the cytotoxic and radiosensitizing agent 5-FU (1 , 2) . 5-FU is further metabolized to FdUMP, which inhibits thymidylate synthase resulting in depletion of TTP pools and eventually inhibition of DNA synthesis.
The CD/5-FC strategy has several potential advantages over the HSV-TK/GCV strategy when used for colon cancer treatment. First, the produced 5-FU is the chemotherapeutic agent of choice for treatment of colon cancer. Second, the CD/5-FC strategy appears to have a greater bystander effect. In contrast to GCV-P, 5-FU does not require gap junctional communication to kill nontransduced neighboring tumor cells (3) . This is a major advantage, as gap junctions are often lost in cancer cells (4 , 5) . Third, 5-FU is not only cytotoxic, but also has radiosensitizing properties (6) . Indeed, several in vitro and in vivo studies have now demonstrated that the CD/5-FC strategy is more effective than the HSV-TK/GCV system in lung and colon cancer cells (7 , 8) .
Although the CD/5-FC strategy holds promise, a severe limitation to its effectiveness has come from the fact that 5-FC is poorly deaminated by bCD (9) . An enhancement of the conversion efficiency for 5-FC would be expected to substantially improve the efficacy of the CD/5-FC strategy both as a cytotoxic and radiosensitizing treatment. To this end, we have purified and characterized yCD, which has a 22-fold lower Km for 5-FC compared with bCD. We have demonstrated that the use of yCD significantly improves the efficacy of the CD/5-FC treatment strategy in human colorectal cancer cells in vitro and in vivo (10) .
In the present study, we have extended our comparison of bCD and yCD to radiosensitization by 5-FC in CD-transduced human colon cancer HT29 cells in vitro and in vivo. In addition, we have analyzed the bystander effect of yCD- and bCD-transduced HT29 cells, as well as the radiosensitizing effect of 5-FC on bystander cells. Our results demonstrate that yCD produces greater radiosensitization and an increased bystander effect compared with bCD. yCD has, therefore, a high potential to improve the therapeutic outcome of the combined enzyme/prodrug strategy and radiotherapy in cancer patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human colon cancer cell line HT29 was grown as described previously (12) . Stable HT29 cell lines expressing either bCD or yCD were generated by viral infection (10) using the retroviral expression vector Lazarus (kindly provided by Gary Nolan, Stanford, CA).
Cell Survival Assay.
The radiosensitizing effect of 5-FC and 5-FU in HT29, HT29/bCD, and
HT29/yCD cells was determined using a standard clonogenic assay
(13)
. Cells were treated with 5-FC or 5-FU at various
concentrations for 24 h before irradiation at 37°C in media
containing 10% dialyzed serum. The radiation survival data were
corrected for plating efficiency using a nonirradiated plate treated
with 5-FC or 5-FU under the same conditions. The surviving fraction was
plotted against the radiation dose, and curves were fit using the
linear-quadratic equation. The radiation sensitivity was expressed as
the MID, which represents the area under the cell survival curve
(14)
. Radiosensitization was expressed as the ER, which
was defined as MIDcontrol/MIDtreated.
To determine the cytotoxic and radiosensitizing effect of 5-FC and 5-FU on bystander cells, cocultures of 90% bystander hygromycin-resistant HT29 cells and 10% puromycin-resistant CD-transduced HT29 cells were used. Cell survival of the hygromycin-resistant HT29 cells and puromycin-resistant CD-transduced HT29/cells was determined by plating the cells in selective media after treatment and assessed using a standard clonogenic assay as described above.
In Vivo 19F-MRS.
In vivo conversion of 5-FC to 5-FU in tumors was monitored
by 19F-MRS. Mice bearing s.c. tumors in the rear
limb were injected i.p. with 1000 mg/kg 5-FC and restrained on a
specially constructed plastic jig (15)
to allow
positioning of the tumor under a 6.1-mm single-turn surface coil.
Spectra were performed at 282.3 MHz on a Varian Magnetic Resonance
System equipped with an 18.3-cm horizontal bore 7.0 Tesla magnet.
Spectra were acquired as the average of 277 transients (free induction
decays) collected in 8K data points using a 25-µs pulse width
(corresponding to a 90° flip angle at a depth of 2.7 mm), a 4.328-ms
repetition time, and and a 25-KHz spectral width; 20 min were required
for each spectrum. Metabolite concentrations were calculated by
normalizing their resonance-peak areas to that of a NaF chemical shift
and concentration standard in a microcell placed above the coil. A
scaling factor relating the NaF reference signal to
19F signals arising from the tumor volume was
obtained from MRS experiments using the NaF external standard
and a tumor phantom containing a known 5-FC concentration.
GC/MS.
For the determination of 5-FC and 5-FU levels in plasma, tumors, and
normal organs by GC/MS, tumor-bearing mice were injected i.p. with 1000
mg/kg 5-FC. After 90 min, mice were sacrificed, and plasma, tumors,
liver, colon and muscle were collected and snap-frozen. Frozen tissue
was pulverized and weighed, and homogenates were made in 1 ml of 1
M acetic acid using a polytron. Subsequently, samples were
prepared for GC/MS as described previously (16)
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Quantification of the derivatized products was performed using a Varian
Saturn 2000 GC/MS spectrograph in selected ion-monitoring mode. The
amounts of 5-FC and 5-FU were expressed as µmol/g tissue.
Mouse Model.
Nude female mice (Nu/Nu CD-1; Charles River Laboratories,
Wilmington, MA) 7–8 weeks of age were injected s.c. in the flank with
5 x 106 viable tumor cells.
Tumors were measured with calipers in 2 dimensions. Tumor volumes were
calculated using the formula: 
/6 (length x
width)2). When tumors measured an average
volume of 100–150 mm3
, treatment was started.
Mice were treated 5 days a week for 2 weeks with either 5-FC (500 mg/kg
i.p.), radiation (3 Gy), or a combination of both in which 5-FC was
injected 3 h before radiation. The results were plotted as the
average tumor volume (in which a cured tumor was included as a tumor of
zero size) relative to that at the start of treatment versus
time after initiation of the treatment. Differences in the efficacy
between treatments were expressed as the number of tumor cures, defined
as tumors that did not recur for 60 days, and as the average minimum
relative tumor volume for a given condition. Mice were handled
according to the established procedures of the University of Michigan
Laboratory Animal Maintenance Manual.
Irradiation Conditions.
Cells were irradiated at room temperature at 1–2 Gy/min using an AECL
Theratron 80 (60Co). Dosimetry was carried out
using a Baldwin ionization chamber connected to an electrometer system
that was directly traceable to a National Bureau of Standards
calibration (12)
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Tumor-bearing mice were restrained in a Lucite restrainer and placed beneath the primary collimator and a secondary heavy alloy collimating device on the (60Co) teletherapathy unit. Mice were positioned such that the apex of the tumor was at the center of a 2.4-cm aperture in the secondary collimator (17) .
Immunohistochemistry.
Sections (5 µm thick) of frozen tumor tissues were cut and mounted on
slides (Fisher Scientific, Pittsburgh, PA). Tissues were fixed in 4%
paraformaldehyde (Electron Microscopy Sciences, Washington, PA) for 10
min at room temperature. Endogenous peroxidase activity was blocked
with 0.6% hydrogen peroxide in 100% methanol for 15 min at room
temperature. After the slides were washed in water, nonspecific binding
sites were blocked with 10% normal goat serum in PBS for 30 min at
room temperature. Polyclonal anti-yCD and anti-bCD were generated in
rabbits by Berkeley Antibody Company (BabCO, Richmond, CA). Antibodies
were diluted 1:200 and 1:75, respectively, in PBS containing 1% BSA
and incubated with sections overnight at 4°C. After being washed in
PBS (twice for 10 min), sections were incubated with a 1:100 dilution
of goat antirabbit IgG (Southern Biotechnology Associates Inc.,
Birmingham, AL) for 30 min at room temperature. After 4 further PBS
washes, sections were incubated with AEC Histostatin (Zymed, South San
Francisco, CA) for 1–5 min at room temperature. The sections were
counterstained in Mayer’s hematoxylin for 3 min and mounted in aqueous
mounting solution. Randomly chosen regions from at least two tumors
were scored for CD-positive cells.
Statistics.
Averages are presented as the mean ± SE. Differences in
cytotoxicity and radiosensitivity between cell lines were statistically
analyzed with the Student’s t test for unpaired data as
were comparisons of minimum tumor size. Complete tumor regressions were
compared using the Fisher’s exact test. Statistical significance was
defined at the level of P < 0.05 (two-tailed).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At nontoxic concentrations, a 
3-fold lower concentration of 5-FC
was required to radiosensitize HT29/yCD cells to a similar extent as
HT29/bCD cells (P < 0.02).
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. At 10 µM, both cell lines were radiosensitized by 5-FU
to a similar extent. Additional control experiments showed that
HT29/bCD cells were more sensitive than HT29/yCD cells to radiation
alone (MID of 2.51 ± 0.19 Gy and 3.74 ± 0.35 Gy, respectively). These results show that the enhanced
radiosensitivity produced by 5-FC in HT29/yCD cells was not
attributable to a greater sensitivity to radiation or 5-FU.
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. For HT29/bCD cells, a small bystander effect was observed
when cocultures were treated with 300 µM 5-FC.
Increasing the concentration of 5-FC to 1000 µM
did not significantly decrease the surviving fraction of bystander
cells. In contrast, a concentration-dependent bystander effect was
observed for HT29/yCD cells when cells were exposed to 5-FC, which was
significantly greater than that observed for HT29/bCD cells
(P < 0.001).
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. Cocultures of 90% bystander and 10%
CD-transduced HT29 cells were treated with 300 or 1000
µM 5-FC for 24 h and subsequently
irradiated. No significant radiosensitization was produced in bystander
cells after exposure to 300 µM 5-FC when cells
were cocultured with HT29/bCD cells, whereas exposure to 1000
µM caused an ER of 1.6 ± 0.2 in
bystander cells. When bystander cells were exposed to 300
µM 5-FC in the presence of HT29/yCD cells,
significant radiosensitization was produced in bystander cells when
compared with HT29/bCD cells (P < 0.01).
Increasing the concentration of 5-FC to 1000 µM
further increased the radiosensitivity of bystander cells.
In Vivo Monitoring of the Conversion of 5-FC to
5-FU.
As the in vitro data provided strong evidence for the
superior radiosensitizing effect of 5-FC in HT29/yCD cells, we decided
to compare the effect of 5-FC on the radiation sensitivity of HT29/bCD
and HT29/yCD tumors grown in nude mice. To obtain a maximum
radiosensitizing effect by 5-FC, we first determined the in
vivo time course of the radiosensitizing metabolite FdUMP after
5-FC injection. Mice bearing HT29/yCD tumors were injected i.p. with
1000 mg/kg 5-FC and the levels of 5-FC, 5-FU, and metabolites were
monitored by 19F-MRS (Fig. 4A)
. Peak levels for 5-FC and 5-FU were observed 60 and 90 min
after injection, respectively, and rapidly declined thereafter with
half lives of 90 ± 9 (5-FC) and 75 ± 6
(5-FU) min. FNuc, of which FdUMP is the major component, were first
observed 60 min after injection, reached a steady state approximately
160 min after injection, and remained at that level for at least an
additional 4 h.
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. In contrast to HT29/yCD tumors, in which all
compounds could be detected, only 5-FC was measurable in HT29/bCD and
HT29 control tumors.
To determine whether the absence of the 5-FU peak in HT29/bCD tumors
was attributable to a limitation of the detection sensitivity of
19F-MRS, we also measured 5-FC and 5-FU levels in
tumor tissue by GC/MS analysis (Fig. 4C)
. Tumor-bearing mice
were injected with 1000 mg/kg 5-FC, and 90 min after injection, mice
were sacrificed. Plasma, tumors, liver, colon, and muscle were prepared
for analysis by GC/MS. For both HT29/bCD and HT29/yCD tumors, 5-FU
could be detected, but the concentration of 5-FU in HT29/yCD tumors was
approximately 15-fold higher than that in HT29/bCD tumors. The measured
amount of 5-FU in HT29/yCD tumors was in correspondence with that
observed by 19F-MRS. In control HT29 tumors, only
5-FC was present.
Radiosensitizing Effect of 5-FC in HT29/bCD and HT29/yCD Tumors.
To study the radiosensitizing effect of 5-FC in vivo, mice
bearing HT29/bCD or HT29/yCD tumors were treated with either 5-FC,
irradiation, or a combination of both treatment modalities. On the
basis of the kinetic data obtained by 19F-MRS,
tumors were irradiated 3 h after 5-FC administration at the time
of maximum FdUMP levels in the tumors (see above).
In HT29/bCD tumors, 5-FC treatment caused a small growth delay, and no
regressions were observed (Fig. 5)
. Tumors treated with either radiation or a combination of 5-FC and
radiation showed regression, but most regrew after day 30. Tumor cures
were observed in 3 of 9 irradiated tumors and in 1 of 10 tumors treated
with a combination of 5-FC and radiation. The addition of 5-FC to
radiotherapy did not appear to result in a better therapeutic effect in
HT29/bCD tumors.
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. When HT29/yCD tumors were treated with a combination of 5-FC
and radiation, the therapeutic effect significantly improved compared
with radiation alone (P < 0.005), with seven
of nine tumors being cured. We also assessed the effect of 5-FC and
radiation on the relative tumor size. We found that the minimum
relative tumor size (compared with pretreatment values) of HT29/bCD
tumors treated with 5-FC and radiation was 0.55 ± 0.1,
compared with 0.01 ± 0.01 in HT29/yCD tumors
(P = 0.002). This improvement was not
attributable to a greater sensitivity of HT29/yCD tumors to radiation
alone, because their radiosensitivity was similar to that of HT29/bCD
tumors (minimum relative tumor volume 0.61 ± 0.21 and
0.65 ± 0.19). These findings confirmed our hypothesis
that yCD would produce a greater antitumor effect than bCD when
combined with radiation.
Radiosensitizing Effect of 5-FC on Bystander Cells in
Vivo.
As current gene-delivery systems do not transduce all tumor cells, the
therapeutic outcome of the CD/5-FC treatment strategy in combination
with radiotherapy is largely dependent on the radiosensitizing effect
of 5-FU on bystander cells. To study the effect of 5-FC on the
radiation response of bystander cells in vivo, we treated
mice with tumors consisting of a mixture of 90% bystander and 10%
CD-transduced HT29 cells with 5-FC, radiation, or a combination of both
modalities, as described above.
Mixed tumors containing HT29/bCD cells did not respond to 5-FC
treatment alone (Fig. 6)
. In addition, consistent with tumors derived entirely from HT29/bCD
cells, mixed tumors did not show an improved therapeutic outcome when
5-FC was combined with radiation. Tumor regression was observed with a
regrowth after day 35, but no tumor cures were observed. In tumors
containing HT29/yCD cells, treatment with 5-FC caused a tumor growth
delay, but no regressions were observed. However, the addition of 5-FC
to radiation resulted in significantly more tumor regressions when
compared with radiation alone, and 6 of 11 tumors were cured
(P < 0.025). Similarly, in mice bearing HT29
tumors containing 10% HT29/yCD cells, the combined treatment resulted
in a minimum relative tumor size of 0.20 ± 0.07
compared with 0.60 ± 0.1 in 10% HT29/bCD cells
(P < 0.001). These results show that 5-FC
had neither cytotoxic nor radiosensitizing effects on bystander tumor
cells in the presence of HT29/bCD cells, whereas a small cytotoxic
effect and a significant radiosensitizing effect of 5-FC were observed
in bystander cells in the presence of HT29/yCD cells.
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. We found that the fraction of positively staining cells in the 10%
condition was equal for both the yCD and bCD mixed tumors (although the
actual fraction that stained positive was 14%, suggesting that both
HT29/yCD cells and HT29/bCD cells may grow faster than bystander cells;
Table 1
). Thus, the different responses between mixed tumors of
bystander cells with either HT29/yCD or HT29/bCD cells is not
attributable to a difference in the percentage of CD-expressing cells
at the time of 5-FC with or without radiation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a previous study we reported on the preferential killing of bCD-expressing tumor cells in response to 5-FC treatment compared with bystander cells, because of the high intracellular 5-FU concentration (3) . It was possible that the higher conversion efficiency of HT29/yCD cells for 5-FC, resulting in a higher intracellular concentration of 5-FU, could have killed the "factory" prematurely, thereby reducing the bystander effect of HT29/yCD cells. Our in vitro data showed, however, an increased bystander effect of HT29/yCD cells when compared with HT29/bCD cells, suggesting that the rapid and substantial 5-FU production was sufficient to kill both yCD-transduced producer cells and bystander cells. Nevertheless, temporarily sparing CD-transduced tumor cells from cytotoxicity by intracellular 5-FU might result in a further increase in bystander effect as more 5-FU can ultimately be produced. To increase temporarily the life span of transduced cells, previously we generated a secreted form of bCD.4 We found that tumor cells that expressed secreted bCD survived longer and produced a higher extracellular concentration of 5-FU in response to 5-FC treatment than cells that expressed intracellular bCD. Thus, we would anticipate that the use of a secreted form of yCD, which is currently being generated, might be even more effective than the intracellular enzyme.
Studies have reported on the radiosensitizing effect of 5-FC in tumor cells transduced with bacterial CD in vitro (18) as well as in vivo (19) . A maximal radiosensitizing effect of 5-FC in nude mice bearing WiDr human colon cancer xenografts transduced with bCD was observed after treatment with 5-FC for 6 consecutive days followed by a single radiation dose (19) . This is in contrast to our results with HT29/bCD tumors, as we did not observe a radiosensitizing effect of 5-FC. The improved efficacy of the combined treatment in WiDr/bCD tumors might be explained by a possible higher expression of bCD in WiDr cells resulting in a greater sensitivity to 5-FC. Indeed, treatment of WiDr/bCD tumors with 5-FC alone did result in a tumor growth delay, whereas we observed almost no growth delay in HT29/bCD tumors in response to 5-FC treatment. Gabel et al. (19) also showed that the combined treatment of 5-FC and radiation was more effective in tumor-growth inhibition than a similar treatment of the conventional combined treatment of 5-FU and radiation. By using yCD instead of bCD, this difference in efficacy is expected to be higher, which may validate the preferential use of the CD/5-FC strategy over 5-FU when combined with radiotherapy in cancer patients.
Another approach to increase the radiosensitivity of tumors is to combine radiotherapy with double suicide gene therapy, in which tumor cells are transduced with a fusion protein of CD and HSV-1 TK (20) . This combined treatment potentiated the antitumor effect when compared with that of the single treatment modalities alone. However, the efficacy of this approach in tumors containing only a small percentage of transduced cells (as will be the case in patients) remains to be determined, because the radiosensitizing and cytotoxic effect of the HSV-TK/GCV on bystander tumor cells is dependent on gap junctional communication, which is often eliminated in tumors.
The first in vivo studies of radiosensitization by 5-FC in adenovirally infected tumors have been published recently (21 , 22) . In mice bearing human squamous cell carcinoma xenografts (SQ-20B), adenoviral-directed bacterial CD/5-FC gene therapy did enhance the efficacy of fractionated radiotherapy in small and large tumors (21) . No tumor cures were observed in large tumors in response to the combined treatment, whereas three of seven small tumors were cured. Because this tumor type was resistant to 5-FU, the improved efficacy was considered to be attributable to an enhanced radiosensitization. In another study (22) , human cholangiocarcinoma xenografts (SK-ChA-1) infected with an adenovirus encoding for bCD also showed an improved response to the combined treatment of 5-FC and radiation. On the basis of the current study, the use of yCD would be expected to improve the therapeutic outcome of this treatment strategy in virally infected tumors.
One limitation of our study is that we used athymic nude mice bearing human tumor xenografts. Although this immunocompromised model is required to grow human tumors, it is possible that the immune system could ultimately play a role in tumor response, particularly because CD is not a mammalian enzyme. Additional experiments in an immunocompetent host will be required to address this issue in the future.
In conclusion, our results show an improved therapeutic effect of the combined treatment with 5-FC and radiation when tumor cells are transduced with yCD instead of bCD. This suggests that the combination of radiotherapy and the yCD/5-FC strategy has a high potential to increase the efficacy of radiation in cancer patients. Because the results in mixed tumors were promising, we are currently optimizing adenovirus-mediated delivery of yCD to tumors in a preclinical model of colorectal cancer.
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FOOTNOTES |
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1 Supported by a grant from the NIH (CA80145) and
a Munn Research Award from the University of Michigan Cancer Center. 
2 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, University of Michigan,
1500 East Medical Center Drive, UH-B2C490, Ann Arbor, MI 48109-0010.
Phone: (734) 647-9955; Fax: (734) 763-7371; E-mail: tsl{at}umich.edu
3 The abbreviations used are: HSV-TK, herpes
simplex virus-thymidine kinase; GCV, ganciclovir; CD, cytosine
deaminase; bCD, bacterial CD; yCD, yeast CD; 5-FC, 5-fluorocytosine;
5-FU, 5-fluorouracil; FdUMP, 5-fluoro-dUMP; MID, mean inactivation
dose; ER, enhancement ratio; MRS, magnetic resonance spectroscopy;
GC/MS, gas chromatography mass spectrometry; FNuc, fluorinated
nucleotide; ppm, parts per million.
4 A. Rehemtulla, E. Kievit, M. A. Davis, E. Ng,
and T. S. Lawrence. Extracellular expression of cytosine deaminase
results in increased 5-FU production from 5-FC treatment, submitted for
publication.
Received 3/24/99. Accepted 10/ 2/00.
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), 10 µM (
), or 100
µM (•) for 24 h before radiation. Cells were then
irradiated at various doses and assessed for clonogenicity. The average
values of surviving fractions of 3–4 experiments (± SE) are shown.
B, the radiosensitizing effect of 5-FC in HT29/bCD
(open bars) and HT29/yCD (closed bars)
cells. Cells were treated and assessed as described in Fig. 1
), and FNuc (
)
were monitored. Each point is the average of five animals. The
indicated times mark the start of the acquisition period ± 10 min. B, representative in vivo19F-MRS spectra of HT29, HT29/bCD, and HT29/yCD
tumors at 100 min after injection with 1000 mg/kg 5-FC.
1, FNuc (-45 ppm); 2, 5-FC (-48.5 ppm);
3, 5-FU (-49.6 ppm). C, determination of
5-FC (left) and 5-FU (right) levels in
plasma, tumors, liver, colon, and muscle in mice bearing HT29/bCD
(open bars), HT29/yCD (closed bars), or
HT29 (hatched bars) tumors by GC/MS. Tumor-bearing mice
were injected i.p. with 1000 mg/kg 5-FC. Ninety min after injection,
plasma, tumors, and organs were collected and processed for GC/MS. The
measured amount of 5-FC and 5-FU is expressed as µmol/g tissue. The
average values of 4–8 samples (± SE) are shown.
, n = 12–20) and
treated tumors (n = 9–16) are shown.
