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Departments of Experimental Radiation Oncology [D. C., N. S., F. A., R. M., M. L. M.], Thoracic and Cardiovascular Surgery [J. A. R.], and Radiation Oncology [A. P.], University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
ABSTRACT
An adenovirus 5 vector containing wild-type p53 cDNA (Ad5-p53) and a cytomegalovirus promoter was used to generate p53 transgene expression. Control vector (Ad5-pA) contained the poly-adenosine sequence. PC3 cells (2 x 106) were injected s.c. into the legs of nude mice. Treatment with Ad5-p53 was initiated at a tumor volume of 200 mm3. Three intratumoral injections (days 1, 4, and 7) were given with 3 x 108 plaque-forming units, followed by 5 Gy pelvic irradiation (day 8) in one fraction using a cobalt-60 source. Tumor volume measurements were obtained every 2 days. LNCaP cells (2 x 106) were injected orthotopically into the prostates of nude mice, and tumor weight was approximated using serum prostate-specific antigen (PSA) obtained from weekly tail vein bleedings. The target PSA for the start of the studies was 5 ng/ml. The intraprostatic injections of Ad5-p53 were done twice (days 1 and 2) and followed by 5 Gy pelvic irradiation on day 3.
The PC3 tumor volume growth curves were log transformed and fitted using linear regression. The times (in days) for the tumors to reach 500 mm3 were calculated as 10.7 ± 0.7 (± SE) for the saline control (no virus), 9.8 ± 2.1 for Ad5-pA, 15.6 ± 1.6 for Ad5-p53, 14.6 ± 1.5 radiation therapy (RT; 5 Gy), 14.6 ± 1.5 for Ad5-pA plus RT, and 31.4 ± 5.3 for Ad5-p53 plus RT. The Ad5-p53 plus RT times were significantly different from the other groups. An enhancement factor of 3.4 was calculated, indicating supra-additivity.
LNCaP tumor growth was determined via weekly serum PSA measurements.
Treatment failure was determined using two PSA-based methods; a serum
PSA of >1.5 ng/ml or two rises in PSA during 6 weeks posttreatment.
The results were similar using either end point. Treatment with Ad5-p53
plus 5 Gy resulted in significantly fewer PSA failures (<30%), as
compared with Ad5-p53 alone (64–73%) and the other controls
(
80–100%) These results are also consistent with a supra-additive
inhibition of tumor growth. Tumor growth in vivo was
inhibited supra-additively when p53null and
p53wildtype prostate tumors were treated with Ad5-p53 and 5
Gy radiation.
INTRODUCTION
Patients at high risk of PSA4 relapse after external beam radiotherapy may be identified using the pretreatment clinical parameters of PSA, Gleason score, and stage (1 , 2) . The question then, is how best to treat this group. External beam radiotherapy to conventional doses is inadequate, and the main mechanism appears to be failure to completely eradicate the disease locally. Local persistence is evident in most patients that exhibit a rising PSA in this setting, because prostate biopsies are positive in the majority of those that are investigated. Although dose escalation results from a number of institutions indicate modest reductions in biochemical failure rates for high-risk patients (3, 4, 5) , dose-related improvements in outcome have been modest and are still wanting. One approach that holds promise is radiosensitization.
Recent clinical (6, 7, 8) and animal (9, 10, 11) studies have described improved results when androgen ablation is combined with radiation. The results suggest a supra-additive interaction between these treatments. The clinical gains from the combination have been encouraging to a limited degree but have been associated with significant long-term side effects. Clearly, a radiosensitization strategy that has fewer systemic side effects is desirable. The potential for radiosensitization using gene therapy is relatively untapped. Our approach has been to alter the intracellular molecular milieu such that cell death via apoptosis is favored over cell cycle delay and repair in response to radiation. This concept was manifest from in vitro experiments (12) involving two prostate cancer cell lines using a replication defective adenovirus 5 vector containing a p53wildtype cDNA construct (Ad5-p53). A key question was whether Ad5-p53 would sensitize prostate cancer cells that did not express p53 (PC3 line), as well as those that expressed p53wildtype (LNCaP line). The results showed that clonogenic survival was reduced and apoptosis enhanced supra-additively in both cell lines when Ad5-p53 was combined with radiation. Thus, p53 gene replacement was not the only mechanism responsible for the radiosensitization observed.
In the present study, the effect of Ad5-p53 on the in vivo tumor growth response of PC3 and LNCaP cells to radiation was investigated. Whereas the in vitro data demonstrate radiosensitization by this vector under ideal conditions, these experiments are necessary to verify that p53 gene delivery plus radiation is effective in vivo.
MATERIALS AND METHODS
Cell Lines.
The PC3 and LNCaP cell lines were obtained from the American Tissue
Type Collection and were maintained in cell culture, using liquid
nitrogen for long-term storage. Cells were cultured for a period of
2 months, before taking a new aliquot from liquid nitrogen storage.
Both PC3 and LNCaP cells were cultured in a 5%
CO2 incubator at 37°C in DMEM/F12 supplemented
with 10% fetal bovine serum, 2 mM L-glutamine,
and 100 IU/ml Pen-Strep solution.
In Vivo Ad5-p53 Vector Treatment.
An adenovirus 5 vector containing wild-type p53 cDNA (Ad5-p53) and a
cytomegalovirus promoter was used to generate p53 transgene
expression (13)
. The main control vector used contained the
poly-adenosine sequence (Ad5-pA); however, an adenoviral-Luc vector
(Ad5-Luc) containing the cDNA for luciferase was also used as a control
in some studies. We have used these control vectors interchangeably and
have not seen a difference in clonogenicity or apoptosis
(12)
. PC3 cells (2 x 106) were
injected s.c. into the legs of nude mice. Treatment with Ad5-p53 was
initiated at a tumor volume of 200 mm3
. Three
intratumoral injections (days 1, 4, and 7) were given with 3 x
108 plaque-forming units, followed by 5 Gy
irradiation in one fraction using a cobalt-60 source. Tumor volume
measurements were obtained every 2 days.
LNCaP cells (2 x 106 in 24 µl) were injected orthotopically into the prostates of nude mice. Tumor weight was approximated using serum PSA obtained from weekly tail vein bleedings. There is a linear relationship between tumor (plus prostate) weight and serum PSA; linear regression results revealed that tumor weights of 0.15, 0.3, and 0.6 g correlated with PSAs of 1.1, 11.1, and 31.1 ng/ml. The target PSA for the studies was 5 ng/ml, which correlated with a tumor weight of 0.208 g, which was found at a median of 6 weeks after orthotopic injection. The animals were then anesthetized via s.c. injection of 100 µl of a 0.02 mg/µl solution of Ketamine in 0.9% saline, the prostate was surgically exposed, and 4.5 x 108 pfu injected in 24 µl. The intraprostatic injections were done twice (days 1 and 2), and 5 Gy pelvic irradiation using a cobalt-60 source was administered 24 h later (day 3).
Calculation of Enhancement Factor.
As a determination of supra-additivity in PC3 tumor volume growth delay
from the combination of Ad5-p53 + 5 Gy, an enhancement factor was
calculated (9)
. The tumor volume curves for each
tumor-bearing animal were first log-transformed, and the absolute delay
in tumor growth to 500 mm3
relative to the saline
control was calculated. These values were used to calculate the
enhancement factor [Abs delay (Ad5-p53 + RT - Ad5-p53)/Abs delay
(PBS + RT alone)], which measures the relative increase of the
combined treatment (taking into consideration the effects of the
Ad5-p53 vector) over radiation alone. The Ad5-pA controls were not
included because significant delays over the saline controls were not
observed. An enhancement factor of >1.0 is indicative of
supra-additivity between Ad5-p53 and radiation.
Measurement of Serum PSA.
Human PSA was measured in the serum obtained from tail vein bleedings.
From each blood draw, 30 µl of serum were diluted 1:5 in PSA diluent
(Abbott Labs, Abbott Park, IL) and analyzed for PSA concentration on an
IMX analyzer (Abbott Labs). The results are expressed in ng/ml.
Apoptosis and p53 Staining.
A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
assay was used to quantify apoptosis in tissue sections from PC3 and
LNCaP tumors injected in vivo with Ad5-p53 as described
above. The tumors were removed and fixed in 10% neutral formalin
overnight and embedded in paraffin. Sections were then mounted on
silane-coated slides as described previously (9
, 11)
. The
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
staining of apoptotic cells was accomplished using the ApopTag (Oncor,
Gaithersburg, MD) kit. The cells were counterstained with hematoxylin.
Positive controls were included with each group of samples stained.
The immunohistochemical staining of p53 was performed as outlined previously (14) . Briefly, paraffin-embedded tissue sections mounted on slides were deparaffinized, hydrated, and treated for 30 min with 0.3% H2O2. Antigen retrieval was accomplished with three high power microwave treatments of 5 min each. Nonspecific staining was blocked by incubating 15 min with 2% NHS in PBS (NHS-PBS). Primary Ab6 anti-p53 antibody (Calbiochem-Novabiochem Corp., San Diego, CA) was used at a 1:100 dilution in NHS-PBS, incubating on the slide overnight at room temperature. After rinsing the slide four times in PBS, biotinylated second antibody (1:200 in NHS-PBS) was added for 30 min. The biotinylated second antibody and other reagents for peroxidase staining were supplied in a kit from Vecta Laboratories (Vectastain ABC kit; Vecta Labs, Burlington, CA). After rinsing off the second antibody, the Vectastain Elite ABC reagent was added for 30 min, the slides were washed, peroxidase substrate solution was added for 20 min, and the cells were counterstained with Mayer’s hematoxylin.
RESULTS
The experiments with the PC3 line were designed to determine the
ability of intratumoral Ad5-p53 plus radiation to enhance tumor volume
growth delay over Ad5-p53 alone. The hypothesis was that the
administration of Ad5-p53 would replace p53 function in PC3 cells,
which are p53null. The replacement of p53
function would maximize the chance for apoptosis in response to
radiation. Injection of Ad5-p53 into PC3 tumors resulted in increased
p53 expression and apoptosis in portions of the tumor 24 h later
(Fig. 1)
, as compared with Ad5-Luc
control vector. The data indicate that Ad5-p53 treatment resulted in
functional p53 expression in vivo.
|
shows the tumor volume growth
delay response of PC3 cells grown in the legs of nude mice to three
Ad5-p53 intratumoral injections, with or without single-fraction 5 Gy
of 
-irradiation. There were a number of controls, including
injection of PBS alone, PBS + 5 Gy, Ad5-pA control vector alone, and
Ad5-pA + 5 Gy. The Ad5-p53 vector was administered alone and in
combination with 5 Gy. The results illustrate a substantial tumor
volume growth delay for Ad5-p53 plus radiation, as compared with the
other treatment groups, including Ad5-p53 alone. Table 1
summarizes the absolute time to reach
500 mm3
, which was calculated from the
log-transformed tumor volume growth curves from each animal. The
absolute delay was about three times that seen for the PBS alone and
Ad5-pA alone controls and was about two times that for the PBS + 5 Gy,
Ad5-pA + 5 Gy, and Ad5-p53 alone groups. One-way ANOVA (Scheffe test)
showed that absolute tumor growth delay from Ad5-p53 + 5 Gy was
significantly greater than from all of the other treatments. The
enhancement factor was calculated to be 3.4, indicating a
supra-additive affect on tumor growth.
|
|
, where a highly significant
relationship was found between serum PSA and tumor (plus prostate)
weight. Thus, serum PSA after treatment was used to determine the
failure rates for the various treatments tested.
|
. The average
pretreatment PSA was 4.86 ng/ml. There were no statistically
significant differences between the treatment groups in terms of
pretreatment PSA. Table 2
also displays the 6-week posttreatment PSAs.
Although Ad5-p53 + 5 Gy resulted in the lowest mean posttreatment PSA,
the only statistically significant difference between this group and
the others was with the PBS-only group. Mean posttreatment PSAs are not
an accurate reflection of response because once biochemical failure is
established, PSA rises quickly. Table 2
illustrates this variability in
posttreatment PSAs, showing that the Ad5-p53 + RT group had the lowest
median posttreatment PSA (0.5 ng/ml), and yet in one animal that
failed, the PSA rose to over 56 ng/ml. The more meaningful end points
of the threshold and rising PSA methods are reflective of clinical
biochemical end points that have been widely adopted clinically.
|
shows that both methods resulted
in similar estimates of treatment failure. Treatment with Ad5-p53 + 5
Gy resulted in significantly fewer PSA failures (<30%), as compared
with Ad5-p53 alone (64–73%) and the other controls (80–100%).
This trend was highly statistically significant. The determining
component was the distinctive reduction in biochemical failure from
Ad5-p53 plus radiation over Ad5-p53 alone and the other controls. In
univariate analysis (
2
), the Ad5-p53 plus
radiation failure rates by both the threshold and rising PSA methods
were significantly lower than all of the other groups, including the
Ad5-p53 alone group. There was a clear-cut advantage to the Ad5-p53
plus radiation combination. The reduction in biochemical failure from
this combination appeared to be greater than the additive effects of
the individual treatments.
|
The eradication of locally advanced or high-risk prostate cancer with radiation has proven more difficult than believed previously. The clinical application of PSA as an end point has been the principal factor leading to this realization. Although modern series document improved outcome with higher radiation doses, the gains have been modest and not without side effects (5 , 15 , 16) . The need for novel methods of radiosensitization is apparent. Androgen ablation has shown promise as a radiation sensitizer of androgen-sensitive cancer cells (9, 10, 11) ; however, the morbidity from prolonged androgen ablation in men with prostate cancer is significant. Novel approaches to radiosensitization with reduced systemic effects are more desirable, and gene therapy offers promise in this regard.
The p53 gene product has been shown to be a key factor in the radiation response pathways governing cell cycle arrest and repair and apoptosis (17, 18, 19) . A number of studies have indicated that p53 replacement in tumor cell lines with altered p53 expression reduces tumorigenicity and promotes apoptosis (20, 21, 22, 23, 24) and sensitizes tumor cells to radiation (25, 26, 27, 28) . These effects are less conclusive in cases of p53 transgene overexpression in p53wildtype tumors. For p53wildtype tumors treated with p53 gene therapy, the inhibition of tumorigenesis and promotion of apoptosis have ranged from significant (12) to nearly absent (29 , 30) . Likewise, the action of p53 gene transfer plus radiation on tumor cell lines with p53wildtype expression has been variable; some reports have described radiosensitization of p53wildtype tumors (29 , 31) , and others have not (30) . In our in vitro experience (12) , apoptosis was induced in the absence of radiation by p53 transgene expression in the p53wildtype LNCaP line to about the same degree as for the p53null PC3 line; adenoviral-mediated p53 radiosensitization using a clonogenic survival assay was also observed in these lines.
The prostate is amenable to direct intraprostatic injection of gene
therapy vectors (32)
. A foremost concern with such a
strategy is whether sufficient radiosensitization can be accomplished
with relatively few supplemental gene therapy treatments during
radiotherapy. The efficacy of intraprostatic gene therapy should be
established with two to three intraprostatic injections during a
radiation course because of cost, convenience, and potential morbidity
issues with more than three injections. The current investigation
establishes that two to three intratumoral injections results in
substantial sensitization in both p53null and
p53wildtype prostate cancer lines. The
enhancement in PC3 tumor growth inhibition by three daily intratumoral
injections of Ad5-p53, followed a day later by a single 5 Gy radiation,
was calculated to be >3-fold, relative to the controls. A similar
effect was observed for p53wildtype LNCaP cells
using serum PSA as a measure of failure to control tumor growth. The
rising PSA profile is the earliest and most sensitive end point in the
documentation of treatment failure in patients with prostate cancer and
is highly correlated with eventual clinical disease relapse. The
orthotopic LNCaP model used here is decidedly representative of human
prostate cancer, from the dependence on stromal growth factors for
tumorigenicity (33)
, to the secretion of PSA in proportion
to tumor weight (Fig. 3)
, as well as the sensitivity to radiation. With
two intratumoral injections of Ad5-p53 plus single-fraction radiation,
PSA response was sustained for >6 weeks in close to 80% by the rising
PSA method. Freedom from a rising PSA was seen in 36% of Ad5-p53 alone
control group and 20–30% of the control irradiated groups (Table 3)
.
Thus, the freedom from failure rate in the Ad5-p53 + 5 Gy group was
greater than the additive effect of the controls.
In conclusion, our results confirm the feasibility of sensitizing prostate cancer cells to radiation in vivo using adenoviral-mediated p53 gene therapy. By our estimation, based on prior in vitro (12) and in vivo data, the radiosensitization achieved in prostate cancer patients treated with Ad5-p53 and fractionated radiotherapy should be substantial. The data described here represent the minimum expected gain from combining Ad5-p53 and radiation, because all of the intratumoral injections were given before radiotherapy and only a single radiation fraction was used. The strategy currently being instituted in patients involves three injections of Ad5-p53 into the prostate at 2-week intervals during fractionated or low-dose-rate radiotherapy. Because transgene p53 expression lasts at least 5–7 days depending on cell type (34 , 35) , sensitization could occur for 35–45% of the daily radiation treatments, which typically ranges from 34 to 42 fractions over 6.8–8.5 weeks. Using intensity modulated radiotherapy and hypofractionation (36) , it may be possible to shorten overall treatment time without increasing side effects; this would facilitate sensitization by Ad5-p53 for >50% of the radiation fractions administered. Treatment of LNCaP cells in vitro (9) resulted in about a 2.5-fold reduction (0.187–0.072) in the surviving fraction at 2 Gy. If radiosensitization of this magnitude were sustained for even just 35–45% of the radiation fractions, tumor control probability would be expected to increase substantially (37) . Radiotherapy dose-escalation studies (3, 4, 5 , 38) have established that most radiation failures are attributable to local persistence of disease and that more aggressive local therapy is justified. Gene therapy is an ideal approach in this setting.
ACKNOWLEDGMENTS
We thank Mamta Sangha and Paul Hachem for technical assistance. We are grateful to Introgen Therapeutics, Inc. for supplying the adenovirus-p53 vector (RPR/INGN 201).
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported in part by Department
of Defense Grant DAMD 17-98-1-8483; NIH Grants CA 06294 and CA 16672
awarded by the National Cancer Institute, United States Department of
Health and Human Services; and the Prostate Cancer Research Program at
M. D. Anderson. D. C. and N. S. were supported in part by a Prostate
Cancer Research Program Fellowship. 
2 Present address: Institut Paoli Calmettes, 232
Boulevard Sainte Marguerite, Marseille 13009, France.
3 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, 1515 Holcombe Boulevard
(Box 97), Houston, TX 77030. Phone: (713) 792-0781; Fax:
(713) 794-5573; E-mail: Apollack{at}notes.mdacc.tmc.edu
4 The abbreviations used are: PSA,
prostate-specific antigen; Luc, luciferase; Ad5, adenovirus 5; pfu,
plaque-forming units; PBS, phosphate-buffered saline; NHS, normal horse
serum.
Received 5/ 1/00; revised 8/14/00; accepted 8/24/00.
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), Ad5-pA vector alone (
), Ad5-pA + 5 Gy RT (
), Ad5-p53
alone (
), Ad5-p53+5 Gy RT (
). The number of animals per group is
shown in Table 1