Combined radiation and p53 gene therapy of malignant
glioma cells
Behnam Badie,1 Chern Sing Goh,1 Jessica Klaver,1 Hans Herweijer,2 and David A. Boothman3
1
Neuro-oncology Laboratory, Department of Neurological Surgery, 2The Waisman Center, and 3Department
of Human Oncology, University of Wisconsin School of Medicine, Madison, Wisconsin 53792.
More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. Because p53 plays a key role
in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured
human glioma cells containing normal or mutated p53. Three established human glioma cell lines expressing the wild-type (U87
MG, p53wt) or mutant (A172 and U373 MG, p53mut) p53 gene were transduced by recombinant adenoviral vectors bearing human
p53 (Adp53) and Escherichia coli b-galactosidase genes (AdLacZ, control virus) before radiation (0 –20 Gy). Changes in p53, p21,
and Bax expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by
flow cytometry and nuclear staining. Survival was assessed by clonogenic assays. Within 48 hours of Adp53 exposure, all three cell
lines demonstrated p53 expression at a viral multiplicity of infection of 100. p21, which is a p53-inducible downstream effector
gene, was overexpressed, and cells were arrested in the G1 phase. Bax expression, which is thought to play a role in p53-induced
apoptosis, did not change with either radiation or Adp53. Apoptosis and survival after p53 gene therapy varied. U87 MG (p53wt)
cells showed minimal apoptosis after Adp53, irradiation, or combined treatments. U373 MG (p53mut) cells underwent massive
apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation. Surprisingly, A172 (p53mut) cells demonstrated
minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose. Survival of all
three cell lines was reduced dramatically after .10 Gy. Although Adp53 transduction significantly reduced the survival of U373
MG cells and inhibited A172 growth, it had no effect on the U87 MG cell line. Transduction with AdLacZ did not affect apoptosis
or cell cycle progression and only minimally affected survival in all cell lines. We conclude that responses to p53 gene therapy are
variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving
activation of cell cycle regulatory and apoptotic genes.
Key words: Adenoviral vectors; brain neoplasm; gene therapy; glioma; p53; radiation.
M
utations of the p53 tumor suppressor gene have
been reported in more than half of all human
cancers, including brain, lung, breast, prostate, thyroid,
bladder, colon, liver, leukemia, head and neck, and
ovarian cancers.1,2 p53 may play a central role in cell
cycle regulation, DNA repair, and programmed cell
death after DNA damage. Alteration of its function can
also lead to genomic instability, which is thought to be an
early event in glioma oncogenesis.3 Normal p53 function
is important in the cellular response to genotoxic agents
causing DNA damage. The p53 protein acts as a transcription factor, inducing growth arrest by modulating
specific downstream target genes, including p21, which
arrests cells in late G1 by inhibiting cyclin-dependent
kinase activity,4,5 or by up-regulating GADD45, which
Received November 5, 1997; accepted May 4, 1998.
Address correspondence and reprint requests to Dr. Behnam Badie,
H4/330 Clinical Science Center, Department of Neurological Surgery,
University of Wisconsin, 600 Highland Avenue, Madison, WI 53792-3232.
E-mail address: [email protected]
© 1999 Stockton Press 0929-1903/99/$12.00/10
Cancer Gene Therapy, Vol 6, No 2, 1999: pp 155–162
inhibits DNA synthesis and may activate nucleotide
excision repair.6 Alternatively, p53-mediated regulation
of apoptosis is not well understood, and some evidence
implies that transactivation of the bax gene (a member
of the Bcl-2 family that, in contrast with Bcl-2, has the
ability to induce apoptosis) plays a role.7 Loss of these
p53-dependent regulatory functions may, therefore, lead
to tumor radioresistance and account for the association
between p53 mutations and the poorer prognosis observed in some tumors.
Despite recent advances in p53 biology, the relationship between p53 gene expression and cellular radiosensitivity remains unclear. Reports of increased, decreased, and no apparent differences in radiosensitivities
have all been associated with p53 function.8 –10 In a
normal cell, irradiation or other DNA-damaging agents
can induce a p53-dependent cell cycle arrest, which
presumably allows enough time to repair the damaged
cellular genome. Alternatively, if DNA damage is beyond repair, p53-dependent apoptosis may serve a protective role and prevent the transfer of new mutations to
daughter cells. In a neoplastic cell bearing the wild-type
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(wt) p53 gene, however, such a response could have both
beneficial and harmful therapeutic consequences. After
DNA damage (chemotherapy or irradiation), p53-dependent G1 arrest could confer therapeutic resistance by
providing tumor cells with enough time to undergo DNA
repair before cell division. Conversely, p53-dependent
apoptosis could confer therapeutic sensitivity to the
tumor cells. In a neoplastic cell bearing a nonfunctional
p53 gene, the opposite may be true: cells may become
sensitive by not arresting in G1 for repair or may become
resistant by averting the apoptosis induced by irradiation
or chemotherapy. An overexpression of p53 by gene
therapy techniques may, therefore, have both beneficial
and harmful therapeutic consequences and may depend
upon the cell type and endogenous nature of p53 and
other cell cycle and apoptotic regulatory genes.11
Although the antineoplastic role of p53 gene therapy
has been documented in various tumor models, its effect
on the radiosensitization of gliomas has not been studied
thoroughly. Adenoviral vectors encoding the human p53
gene (Adp53) have been used to significantly inhibit
p53mut gliomas cells in vitro and in vivo.12–15 When tested
on p53wt gliomas, however, Adp53 induced only a slight
growth inhibition.15,16 This lack of response in p53wt
tumors may prove to be a significant limitation in a
clinical application of p53 gene therapy. Because p53 is
thought to be a mediator of radiation-induced apoptosis,
we hypothesized that its overexpression may lead to the
sensitization of both p53mut- and also p53wt-bearing
gliomas.
In this study, Adp53 was used to transfer the wt
human p53 gene into human glioma cell lines with
differing p53 functions before radiation. Although p53
overexpression led to apoptosis and/or radiosensitization of p53mut cell lines (U373 MG and A172), it had
little effect in p53wt-expressing cells (U87 MG).
In vitro gene delivery
In vitro transduction was performed as described previously.17
Briefly, nonconfluent monolayers in six-well plates were incubated in 1.5 mL of sera-free culture medium with or without
the vectors (i.e., AdLacZ or Adp53) at a multiplicity of
infection of 100 for 60 minutes at 37°C. After this incubation,
1.5 mL of medium containing 10% fetal calf serum was added
to each plate. Some cells were then irradiated 4 hours later at
different intensities using a 137Cs irradiator (Sheppard, Glendale, Calif) at a dose rate of 5.94 Gy/min. Other mockirradiated cells were treated in a similar manner without
ionizing radiation. Cells were then collected by trypsinization
after 48 hours and stained for flow cytometry as described
below.
Flow cytometry
At 48 hours after irradiation, adherent cells were harvested by
trypsinization and resuspended with the floating cells in 10 mM
tris(hydroxymethyl)aminomethane (Tris) (pH 7.0) and 150
mM NaCl (Tris saline). After several washings, cells were
centrifuged and resuspended in 1 mL of fixative solution (100
mL of Tris saline and 900 mL of absolute ethanol) and stored
at 220°C. Before flow cytometry, cells were washed and
incubated for 15 minutes in phosphate citric acid buffer (0.192
M Na2HPO4 and 4 mM citric acid) and then resuspended in
propidium iodine (PI) solution (50 mg/mL PI, 1 mg/mL
ribonuclease A, 10 mM ethylenediaminetetraacetic acid, and
0.2% Nonidet P-40 in phosphate-buffered saline (PBS)). Cells
were incubated in the dark for 1 hour at 4°C and then analyzed
with a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, Calif) equipped with a 15-mW,
488-nm air-cooled argon ion laser. At least 10,000 events were
acquired using Lysis II software (Becton Dickinson). Orange
fluorescence (PI, FL2) was collected using a 585 6 42-nm BP
filter, and DNA content was measured using FL2 area versus
FL2 width for doublet discrimination. Data were analyzed
using Modfit software (Verity Software House, Topsham, Me).
Each experiment was performed in triplicate and repeated at
least twice.
Western blotting
MATERIALS AND METHODS
Cell culture and adenoviral vectors
The human glioma cell lines U87 MG, A172, and U373 MG
were obtained from the American Type Culture Collection
(Manassas, Va) and grown in Dulbecco’s modified Eagle
medium supplemented with 10% fetal calf serum (Life Technologies, Gaithersburg, Md), penicillin G (100 U/mL), and
streptomycin (100 mg/mL) at 37°C. These cell lines were
selected because of their known p53 gene status and efficient
transduction with adenoviruses. U87 MG expresses wt p53,
whereas A172 and U373 MG express mutant forms.15
Adenoviral vector propagation and isolation have been
described previously.12 The adenoviral vector AdHCMVsp1LacZ
(AdLacZ) contains an expression cassette encoding the Escherichia coli b-galactosidase gene, driven by the immediate early
cytomegalovirus promoter upstream from a polyadenylation
signal. This vector served as a control in our experiments and
was used to confirm the transduction efficiencies of the glioma
cell lines as reported by other investigators.15 Adp53 has a
similar construction, except that it contains a 1.4-kilobase pair
sequence encoding the human wt p53 protein instead of the
b-galactosidase gene.
Both adherent and floating cells were collected as described
above, washed in PBS, and lysed in RIPA buffer (37.5 mM
NaCl, 12.5 mM Tris base, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 mL/mL
of phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and
pepstatin) at 4°C for 30 minutes. Lysates were centrifuged at
10,000 3 g for 15 minutes, sample protein concentrations were
defined spectrophotometrically using a bovine serum albumin
protein assay (Pierce, Rockford, Ill), and a 50-mg aliquot of
total protein was separated in 10% (for p53 and p21) or 15%
(for Bax) SDS-polyacrylamide gel electrophoresis under reducing conditions after a 3-minute boil in SDS sample buffer.
Gels were then electroblotted onto Immobilon-P transfer
membranes (Millipore, Bedford, Mass). After blocking for
nonspecific binding with 5% milk and 0.05% Tween-20 in PBS,
membranes were incubated with either mouse monoclonal
anti-human p53 antibodies (Santa Cruz Biotechnology, Santa
Cruz, Calif), anti-human p21WAF1 antibodies (Oncogene Science, Uniondale, NY), or polyclonal anti-human Bax antibodies (Santa Cruz Biotechnology) as recommended by the supplier. Horseradish peroxidase-conjugated goat anti-mouse IgG
antibody was then used to visualize the protein signal using
SuperSignal chemiluminescent substrate (Pierce).
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BADIE, GOH, KLAVER, ET AL: RADIATION AND p53 THERAPY OF GLIOMA CELLS
157
Hoechst staining for apoptotic cells
Cells in monolayers were washed in PBS and fixed (150 mL
methanol and 50 mL glacial acetic acid) for $2 hours at 4°C
before incubating them with freshly made Hoechst 33258 stain
(10 mg/mL in PBS, Sigma, St. Louis, Mo). The stain was
removed after a 15-minute incubation, and cells were washed
with PBS and visualized with a fluorescent microscope under
N-propyl gallate (0.1 M solution in 80% glycerol).
Clonogenic survival assays
After treatments with various vectors and/or radiation (as
indicated), cells were harvested by trypsinization, replated at a
density of 3000 cells/100-mm Petri dish in triplicate, and grown
under the above culture conditions. After 2 weeks, plates were
washed with PBS, fixed, and stained with Coomassie blue. The
number of colonies containing .50 normal-appearing cells was
counted.
RESULTS
p53, p21, and Bax expression
Consistent with other reports,15 all three cell lines
demonstrated $80% transduction with AdLacZ at a
multiplicity of infection of 100 (data not shown). Effective viral transduction was also confirmed by the elevation of p53 in all cell lines within 48 hours of Adp53
exposure (Fig 1, lanes 1 and 6). Neither radiation nor
AdLacZ had any effect on endogenous or exogenous p53
expression (Fig 1, lanes 2– 4 and 7–9).
The biological activity of exogenous p53 was tested by
examining cellular p21 content, which is a known p53inducible cell cycle regulator.4 p53 overexpression led to
elevations in p21 protein in all three cell lines, suggesting
intact p53 transcriptional activity (Fig 1). Although
X-radiation had no effect on p21 levels in transduced
cells, it slightly increased endogenous p21 expression by
A172 and U87 MG cells, suggesting the presence of
p53-independent mechanisms in regulating this protein.18 Because Bax has been suggested to play a role in
p53-mediated apoptosis,7 we examined its expression in
all three cell lines and found little change in its steadystate protein levels after radiation or Adp53 treatments.
Apoptosis
Nuclear fragmentation suggestive of apoptosis was most
evident in Adp53-exposed U373 MG cells (Fig 2A) and
in A172 cells receiving combined treatment (data not
shown). Flow cytometry was used to quantitate apoptosis by measuring the sub-G1 cell population. Regardless
of irradiation dose, p53 gene therapy induced profound
apoptosis in U373 MG cells (Fig 3), so that all cells died
within 48 hours of transduction. Alternatively, Adp53
induced only low levels of apoptosis in A172 cells when
administered alone; however, when given before radiation, Adp53 significantly increased cell death (Fig 3). In
contrast, U87 MG cells showed mild apoptosis with
Adp53, radiation, or combined treatment. In every cell
line, AdLacZ had mild cytopathic effects and nuclear
fragmentation.
Cancer Gene Therapy, Vol 6, No 2, 1999
Figure 1. Western immunoblots of U373 MG, A172, and U87 MG
cell lines demonstrating expression of p53, p21, and Bax. Mockinfected (lanes 1– 4), AdLacZ-infected (lane 5), and Adp53-infected
(lanes 6 –10) cells were irradiated at various intensities and were
checked for each protein after 48 hours.
Cell cycle
The effect of ionizing radiation and p53 gene therapy on
cell cycle distribution was examined by flow cytometry
(Fig 4). Within 48 hours of irradiation (20 Gy), a 3- to
6-fold increase in G2/M cells was noted in U87 MG and
U373 MG cells (Fig 5, A and B, respectively). This G2/M
checkpoint most likely represented a p53-independent
mechanism, because U373 MG cells express a mutant
p53 protein and U87 MG cells did not demonstrate
elevations in p53 protein after X-radiation. Alternatively, A172 cells demonstrated mild G1 arrest after
irradiation (Fig 5C). This G1 arrest may have been
mediated by elevations in p21 protein, which functions
as a G1 checkpoint regulator5 and was elevated in this
cell line after irradiation (Fig 1). Because A172 cells
have a mutation in the p53 gene, this observation
supports the presence of other p53-independent G1
arrest mechanisms, possibly working through p21.18
The effect of p53 gene therapy on cell cycle regulation
was evident in all three cell lines. A 30% increase in G1
arrest was noted in A172 cells after Adp53 treatment,
and this increase did not change with the addition of
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BADIE, GOH, KLAVER, ET AL: RADIATION AND p53 THERAPY OF GLIOMA CELLS
Figure 3. Flow cytometry was used to quantitate the apoptotic
response of each treatment by measuring the sub-G1 cell population. U373 MG cells underwent significant apoptosis after Adp53
infection. Although radiation had no effect on these cells, it potentiated the p53-induced apoptosis in A172 cells. U87 MG cells
showed no nuclear fragmentation even when both agents were
combined. Error bars represent SE.
mediated p21 expression, which plays a role in cell cycle
regulation after radiation.
Survival
Figure 2. Nuclear morphology of Adp53-treated U373 MG cells (A)
demonstrated significant fragmentation (arrows) that was not seen
in nonirradiated control cells (B) or irradiated (10 Gy) cells (C).
Hoechst staining was performed at 48 hours after Adp53 or X-radiation.
radiation (Figs 4 and 5C). In U87 MG and U373 MG
cells, Adp53 did not increase G1 arrest by itself. However, when administered before radiation, Adp53 did
increase the G1 cells by 2- to 4-fold (Fig 4 and Fig 5, A
and B). This observation is most likely due to Adp53-
Clonogenic assays were used to examine the long-term
significance of the observed apoptosis and cell cycle
changes before and after the treatments described
above. Adp53 transduction reduced the survival of U373
MG and A172 cells by 90% and 60%, respectively, but
did not effect the U87 MG cells (Fig 6). Moreover,
radiation did not significantly potentiate the growth
inhibition of Adp53, even in A172 cells, where it increased Adp53-mediated apoptosis (Fig 3).
DISCUSSION
Malignant brain tumors are ideal candidates for gene
therapy because they only recur locally and rarely me-
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BADIE, GOH, KLAVER, ET AL: RADIATION AND p53 THERAPY OF GLIOMA CELLS
159
Figure 4. Representative DNA histograms of glioma cells at 48 hours after treatments with irradiation (20 Gy) and/or Adp53. Numbers represent
the percentage of cells in the G0/G1 and G2/M phases.
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BADIE, GOH, KLAVER, ET AL: RADIATION AND p53 THERAPY OF GLIOMA CELLS
Figure 5. Effect of Adp53 and radiation on cell cycle distribution.
Radiation-induced G2/M arrest was seen in both U87 MG cells (A)
and U373 MG cells (B) but not in A172 cells (C). Error bars represent SE.
tastasize to other organs. At the present time, cancer
gene therapy can be accomplished through either: (a)
cytoreductive techniques, in which genes are delivered
to interrupt cancer cell growth either directly via suicide
genes or indirectly through immunomodulator genes, or
(b) corrective gene therapy, in which defective or abnormal genes in neoplastic cells are inactivated or supplemented with genes that can reverse the loss of growthcontrol mechanisms. Tumor suppressor genes and
antisense oncogenes are among the candidates for corrective gene therapy.
Inactivation of the p53 tumor suppressor gene may
play a role in the tumorigenicity of most cancers. The
p53 gene encodes a nuclear protein that binds to and
modulates the expression of other genes that are important for DNA repair,6 cell division,19 and cell death by
apoptosis.20 Reconstitution of the p53 gene into p53mut
tumor cells may, therefore, lead to a re-establishment of
these regulatory functions and may prove to be an
effective antineoplastic strategy.
Exogenous p53 gene expression has been found to
induce apoptosis or sensitize p53-negative cells to DNAdamaging agents in several tumor models, including
lung,21 cervical,22 prostate,23 and head and neck tumors.24,25 In gliomas expressing p53mut, Adp53 has been
shown to induce apoptosis in vitro and partially inhibit
tumor growth in vivo.12,13,15,16 When human glioma cells
were transduced with Adp53 in vitro, most p53mutexpressing cells showed significant apoptosis and growth
suppression. Similarly, Gjerset et al14 used Adp53 to
induce apoptosis and sensitize T98G glioma cells
(p53mut) to cisplatin in vitro. None of these studies,
however, showed any apoptosis in p53wt-expressing cells.
Moreover, some p53mut cells, such as A172, were more
resistant to such therapy.15 In this study, we tested the
possibility that p53 overexpression may increase the
sensitivity of these Adp53-resistant cells to X-radiation.
Three glioma cell lines with variable p53 activities and
responses to Adp53 were selected to investigate the role
of Adp53 radiosensitization. In U373 MG cells (p53mut),
Adp53 induced apoptosis and cell death within 48 hours
of viral exposure. These observations were consistent
with those reported previously.15 This p53-mediated
apoptosis was probably mediated through pathways independent of Bax, because no changes in the expression
of this protein were detected. Although radiation alone
did not induce apoptosis within this time frame, it did
result in G2/M arrest, confirming the lack of p53 function
as a G1/S checkpoint in this p53mut cell line. Moreover,
X-radiation significantly suppressed U373 MG growth in
vitro. Whether this growth arrest was due to delayed
radiation-mediated apoptosis that was not detected under these experimental conditions or due to the observed G2/M arrest cannot be concluded from our
studies. Because most radiation-sensitive cells such as
thymocytes and medulloblastomas undergo apoptosis
within a few hours of radiation exposure,20,26 it is more
likely that the observed G2/M arrest was responsible for
this growth inhibition.
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BADIE, GOH, KLAVER, ET AL: RADIATION AND p53 THERAPY OF GLIOMA CELLS
The administration of radiation after p53 treatment in
U373 MG cells did not potentiate apoptosis. This observation indicates either that p53 cannot act as a radiosensitizer in this cell line or perhaps that the apoptotic
pathways were already saturated due to p53 expression.
Adp53 treatment did, however, lead to G1 arrest in
nonapoptotic cells after X-radiation. This change in cell
cycle profile was most likely due to p53-mediated p21
expression, which is known to play a role in the G1/S
checkpoint. It is not clear why most transduced U373
MG cells underwent apoptosis while some were arrested
in G1. It is possible that these responses are dependent
upon the temporal relationship of X-radiation and p53
expression to the cell cycle.
In contrast to U373 MG cells, A172 cells, which also
express p53mut, demonstrated little apoptosis after
Adp53 treatment. This Adp53 resistance has been reported previously by Gomez-Manzano et al15, who noted
that A172 cells die 8 days after Adp53 exposure, compared with 3 days seen in the more sensitive U373 MG
cells. Because we did not detect any apoptosis within 48
hours of exposure to Adp53, we believe that this growth
suppression was, in part, due to p21-mediated G1 arrest.
Following radiation (10 Gy), A172 cells demonstrated
a 30% increase in G1 cells and a 50% increase in G2/M
cells. These cell cycle changes may have been a result of
radiation-mediated p21 expression through a p53-independent mechanism that has been reported previously in
another cell line.27 Interestingly, p53 expression before
X-radiation significantly increased the apoptotic response to radiation in this cell line. This increase in
apoptosis, however, was not related to changes in Bax
expression, and other mechanisms such as activation of
CPP32b (caspase 3) may have played a role.28 Moreover, this radiosensitization did not translate into further
growth inhibition as detected by clonogenic assays, suggesting that activation of cell cycle checkpoints may be as
important as apoptosis in determining the radiation
sensitivity of gliomas.29
As reported by others,15,16 U87 MG cells showed the
least response to Adp53 treatment, probably due to lack
of apoptosis and weak G1 arrest. After X-radiation, U87
MG cells, like U373 MG cells, demonstrated a significant increase in G2/M arrest. This finding seemed to be
in contrast to the G1 arrest reported by Yount et al30 in
U87 MG cells expressing p53wt but not in those with an
inactivated p53 gene. The discrepancy in our findings
could be explained by the fact that we did not detect any
p53 up-regulation by these cells after radiation, confirming that expression of this gene is important for G1
arrest. In fact, when both p53 and p21 were up-regulated
by Adp53 transduction, this G1 checkpoint function was
re-established in these cells. Whether our U87 MG cells
have additional defects in radiation-mediated p53 expression (such as a mutation in the ATM gene) needs to
be investigated. Otherwise, our results are in agreement
with Yount et al, who also failed to observe any radiation-induced apoptosis or any radiosensitization of U87
MG cells when p53 was reactivated. This finding sug-
Cancer Gene Therapy, Vol 6, No 2, 1999
161
Figure 6. Clonogenic assay revealing growth inhibition by Adp53 in
U373 MG and A172 cells but not in U87 MG cells. The inability of
Adp53 to radiosensitize these cell lines under these conditions is
also demonstrated. Error bars represent SE.
gests that Adp53 therapy may not be appropriate for
p53wt tumors under these conditions.
We conclude that the response to combined Adp53
and radiation therapies may be variable among gliomas.
This therapy may not be an effective treatment for
p53wt-expressing tumors, and its efficacy on p53mut tumors may be limited not only by transduction efficiency
and levels of p53 expression in vivo but also by the
activation of other cell cycle regulatory and apoptotic
machinery.
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ACKNOWLEDGMENTS
We thank Pat Giuliani and Michael Liebman for editorial
support. This work was supported in part by National Cancer
Institute Grant P30 CA14520 and the University of Wisconsin
Comprehensive Cancer Center (to B.B.).
REFERENCES
1. Greenblatt M, Bennett W, Hollstein M, et al. Mutations in
the p53 tumor suppressor gene: clues to cancer etiology
and molecular pathogenesis. Cancer Res. 1994;54:4855–
4878.
2. Hollstein M, Sidransky D, Vogelstein B, et al. p53 mutations in human cancers. Science. 1991;253:49 –53.
3. Louis D, von Deimling A, Chung R, et al. Comparative
study of p53 gene and protein alterations in human
astrocytic tumors. J Neuropathol Exp Neurol. 1993;52:31–38.
4. El-Deiry WS, Tokino T, Velculescu VE, et al. WAF-1, a
potential mediator of p53 tumor suppression. Cell. 1993;
75:817– 825.
5. Xiong Y, Khang H, Beach D. p21 is a universal inhibitor of
cyclin kinases. Nature. 1993;366:701–704.
6. Smith ML, Chen IT, Zhan Q, et al. Interaction of the
p53-regulated protein GAAD45 with proliferating cell
nuclear antigen. Science. 1994;266:1376 –1380.
7. Miyashita T, Krajewski S, Krajewska M, et al. Tumor
suppressor p53 is a regulator of bcl-2 and bax gene
expression in vitro and in vivo. Oncogene. 1994;9:1799 –
1809.
8. MacIlwrath AJ, Vasey PA, Ross GM, et al. Cell cycle
arrests and radiosensitivity of human tumor cell lines:
dependence on wild-type p53 for radiosensitivity. Cancer
Res. 1994;54:3718 –3722.
9. Slichenmyer WJ, Nelson WG, Slebos RJ, et al. Loss of a
p53-associated G1 checkpoint does not decrease cell survival following DNA damage. Cancer Res. 1993;53:4164 –
4168.
10. Smith ML, Chen IT, Zhan Q, et al. Involvement of the p53
tumor suppressor in repair of UV-type DNA damage.
Oncogene. 1995;10:1053–1059.
11. Gotz C, Montenarh M. p53: DNA damage, DNA repair,
and apoptosis. Rev Physiol Biochem Pharmacol. 1995;127:
65–95.
12. Badie B, Drazan KE, Kramar MH, et al. Adenovirusmediated p53 gene delivery inhibits glioma growth in rats.
Neurol Res. 1995;17:209 –216.
13. Badie B, Kramar MH, Lau R, et al. Adenovirus-mediated
p53 gene delivery potentiates the radiation-induced
growth inhibition of experimental brain tumors. J Neurooncol. 1998;37:217–222.
14. Gjerset RA, Turia ST, Sobol RE, et al. Use of wild-type
p53 to achieve complete treatment sensitization of tumor
cells expressing endogenous mutant p53. Mol Carcinog.
1995;14:275–285.
15. Gomez-Manzano C, Fueyo J, Kyritsis AP, et al. Adenovirus-mediated transfer of the p53 gene produces rapid and
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
generalized death of human glioma cells via apoptosis.
Cancer Res. 1996;56:694 – 699.
Köck H, Harris M, Anderson S, et al. Adenovirus-mediated p53 gene transfer suppresses growth of human glioblastoma cells in vitro and in vivo. Int J Cancer. 1996;67:
808 – 815.
Badie B, Hunt K, Economou JS, et al. Stereotactic delivery
of a recombinant adenovirus into a C6 glioma cell line in
a rat brain tumor model. Neurosurgery. 1994;35:910 –916.
Michieli P, Chedid M, Lin D, et al. Induction of WAF1/
CIP1 by a p53-independent pathway. Cancer Res. 1994;54:
3391–3395.
Kuerbitz SJ, Plunkett BS, Walsh WV, et al. Wild-type p53
is a cell cycle checkpoint determinant following irradiation.
Proc Natl Acad Sci USA. 1992;89:7491–7495.
Lowe SW, Schmitt EM, Smith SW, et al. p53 is required
for radiation-induced apoptosis in mouse thymocytes. Nature. 1993;362:847– 849.
Fujiwara T, Grimm EA, Mukhopadhyay T, et al. Induction
of chemosensitivity in human lung cancer cells in vivo by
adenovirus-mediated transfer of the wild-type p53 gene.
Cancer Res. 1994;54:2287–2291.
Hamada K, Alemany R, Zhang W-W, et al. Adenovirusmediated transfer of a wild-type p53 gene and induction of
apoptosis in cervical cancer. Cancer Res. 1996;56:3047–
3054.
Ko S-C, Gotoh A, Thalmann GN, et al. Molecular therapy
with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model. Hum Gene
Ther. 1996;7:1683–1691.
Clayman GL, El-Naggar AK, Roth JA, et al. In vivo
molecular therapy with p53 adenovirus for microscopic
residual head and neck squamous carcinoma. Cancer Res.
1995;55:1– 6.
Liu T-J, El-Naggar AK, McDonnell TJ, et al. Apoptosis
induction mediated by wild-type p53 adenoviral gene
transfer in squamous cell carcinoma of the head and neck.
Cancer Res. 1995;55:3117–3122.
Dee S, Haas-Kogan D, Israel M. Inactivation of p53 is
associated with decreased levels of radiation-induced apoptosis in medulloblastoma cell lines. Cell Death Different.
1995;2:267–275.
Akashi M, Hachiya M, Osawa Y, et al. Irradiation induces
WAF1 expression through a p53-independent pathway in
KG-1 cells. J Biol Chem. 1995;270:19181–19187.
Fuchs E, McKenna K, Bedi A. p53-dependent DNA
damage-induced apoptosis requires Fas/APO-1-independent activation of CPP32b. Cancer Res. 1997;57:2550 –
2554.
Haas-Kogan D, Yount G, Haas M, et al. p53-dependent
G1 arrest and p53-independent apoptosis influence the
radiobiologic response of glioblastoma. Int J Radiat Oncol
Biol Phys. 1996;36:95–103.
Yount GL, Haas-Kogan DA, Vidair CA, et al. Cell cycle
synchrony unmasks the influence of p53 function on
radiosensitivity of human glioblastoma cells. Cancer Res.
1996;56:500 –506.
Cancer Gene Therapy, Vol 6, No 2, 1999