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Experimental Therapeutics, Preclinical Pharmacology |
First Department of Surgery, Shiga University of Medical Science, Shiga 520-2192, Japan [T. I., M. Ko.]; Section of Molecular Oncology, First Department of Surgery [T. F., T. W., J. S., M. Ka.], and First Department of Surgery [N. T.], Okayama University Medical School, Okayama 700-8558, Japan; Division of Otolaryngology/Head and Neck Surgery, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7600 [W. G. Y.]; and Department of Neuro-Oncology [T-J. L.], and Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery [J. A. R.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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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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Overexpression of E2F-1 can activate CPP32, one of the most important apoptosis-inducing molecules, and can cause cleavage of the death substrate poly(ADP-ribose) polymerase, which suggests that activation of the caspase cascade may be a pivotal mechanism in E2F-1-mediated apoptosis (9) . Moreover, this pathway seems separate from the p53-mediated pathway, because E2F-1 dose not increase the expression level of bax protein (a putative p53-mediated apoptotic pathway; Ref. 9 ). However, in light of our previous finding that overexpression of the wt-p533 gene by recombinant, replication-deficient viral vectors induces apoptosis in a variety of human cancer cells of differing p53 status (10, 11, 12, 13, 14) , we have hypothesized that the combined transfer of wt-p53 and E2F-1 genes into human cancer cells could be therapeutically advantageous because two different but major apoptotic pathways would be activated.
Recent studies have identified physical interactions between E2F-1 and
ARF (human p14ARF and mouse
p19ARF; Refs. 15
, 16
), which shares
a portion of the p16INK4a coding region and has a
unique first exon (termed exon 1ß) originating approximately 20 kb
centromeric to p16INK4a exon 1 (termed exon 1
;
Ref. 17
). Potential E2F-1 binding sites have also been
detected in the sequence of the ARF promoter region (18)
.
A proto-oncogene MDM2, which can be induced by wt-p53
activity, binds to p53 and masks the p53 transcriptional activation
domain (19)
. MDM2 targets p53 protein for degradation in
the ubiquitin pathway, which results in abrogation of its
antiproliferative and apoptosis-promoting effects (20)
. In
addition, it has been shown that ARF binds to and induces the
degradation of the proto-oncogene MDM2, which results in
stabilization of p53 (21)
. Furthermore, cell-cycle arrest
mediated by ARF can be abolished in cells lacking functional p53
(22
, 23)
, which indicates that ARF may act upstream of
p53.
In the present study, we demonstrated that: (a) E2F-1 gene transfer can down-regulate MDM2 protein levels through activation of ARF and thereby enhance apoptosis induced by exogenously transfected wt-p53; and (b) sequential transduction of p53 and E2F-1 promote apoptosis more efficiently than does simultaneous introduction of these genes. Together, our data indicate that this combination strategy may be useful in treating human squamous cell carcinoma of the esophagus.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant Adenoviruses.
A recombinant, replication-deficient adenovirus vector that was capable
of expressing either human p53 (24)
or E2F-1
(25)
was previously constructed and characterized. The
resultant viruses were termed Ad5CMVp53 (Ad-p53; Refs.
10, 11, 12, 13, 14
) and Ad5CMVE2F-1 (Ad-E2F-1; Ref. 25
)
and have been described therein. An E1A-deleted adenovirus
vector lacking a cDNA insert (dl312) was used as a control vector.
RT-PCR.
Total RNA was isolated using RNAZOL (Cinna/Bio Tecx, Friendswood, TX)
in a single-step phenol-extraction method. One µg of total RNA was
subjected to RT using an oligodeoxythymidylate primer and avian
myeloblastosis virus polymerase (Promega) in a 40-µl reaction volume
for 10 min at 22°C and then for 20 min at 42°C. Then, by using a
PCR kit according to the manufacturer’s protocol (Perkin-Elmer Corp.,
Norwalk, CT), 2 µl of the RT reaction product was amplified by PCR in
total volumes of 50 µl using the forward and reverse primer for exon
1ß human p19ARF gene
(p14ARF) This yielded a 188-bp
fragment (26)
that was run on a 1% agarose gel and
visualized by ethidium bromide staining. The specific primer sequences
for the exon 1ß human ARF gene were as follows: forward primer
(sense), 5'-TACTGAGGAGCCAGCCGT-CTA-3'; reverse primer (antisense),
5'-AGCACCACCAGCGTGTC-3'. The amplification reaction involved 25 or 35
cycles of denaturation at 95°C for 1 min and annealing at 60°C for
1 min and at 72°C for 15 s in a Perkin-Elmer thermal cycler
(Perkin-Elmer Corp., Foster City, CA). As a control, human
ß-actin was amplified as described previously (13)
.
Western Blot Analysis.
For Western blot analysis, attached cells collected by trypsinization
and cells floating in the culture medium were combined in a 10-ml
conical tube and were washed twice in cold PBS. Cells were then lysed
in SDS solubilization buffer [0.5 M Tris (pH 6.8), 10%
SDS, and glycerol]. Equal amounts of proteins were boiled for 5 min
and electrophoresed under reducing conditions on a 12.5% (w/v)
polyacrylamide gel. Proteins were then electrophoretically transferred
to a Hybond-polyvinylidene difluoride (PVDF) transfer membrane
(Amersham, Arlington Heights, IL) and were incubated with primary
antibodies against E2F-1 (KH95; Santa Cruz Biotechnology, Santa Cruz,
CA), p53 (Ab-2; Oncogene Science, Manhasset, NY), MDM2 (SMP14; Santa
Cruz Biotechnology), and ARF (C-18; Santa Cruz Biotechnology) and with
mouse antihuman actin monoclonal antibody (Amersham). An Amersham
enhanced chemiluminescence chemiluminescent system was used to
detect secondary probes.
Flow Cytometric Analysis.
For flow cytometric analysis, cells were collected, washed twice with
cold PBS, and resuspended in PBS containing 0.1% Triton X-100 and 1
g/liter RNase for 5 min at room temperature. Samples were then stained
with propidium iodide at 50 mg/ml and analyzed for DNA content in a
cell sorter (FACScan; Becton Dickinson, Mountain View, CA).
Visualization of Apoptotic Nuclei.
To visualize apoptotic nuclei, T.Tn and TE8 cells grown on
coverslips were fixed with 1.0% glutaraldehyde and stained with DNA
intercalating dye Hoechst 33342 (1 mM in PBS). Fluorescence
was visualized using a fluorescence microscope (Zeiss, Oberkochen,
Germany).
Plasmid Transfection.
Cell transfections were carried out using the LIPOFECT ACE reagent
according to the manufacture’s instructions (Life Technologies, Inc.).
Cells were plated on 6-well plates at a density of approximately
106 cells/well and allowed to grow to 70%
confluence (24 h after seeding), at which time they were transfected
using LIPOFECT ACE in a total volume of 1 ml OPTI-MEN I (Life
Technologies, Inc.). The cells were transiently or stably transfected
with various amounts of ARF cDNA (pcDNA3-Myc-ARF; 21
) as
indicated in the figure legend for each experiment. After a 24-h
incubation with the DNA/lipid mixture, the cells were replenished with
growth media. Stably transfected T.Tn (T.Tn/ARF) and TE8
(TE8/ARF) cells were selected in medium supplemented with 500 µg/ml
of geneticin (Sigma Chemical Co., St. Louis, MO) for at least 30 days.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). HeLa-S3
cells, which constitutively express ARF (27)
as well as
stabilized p53 (28)
, were used as a positive control. As
revealed by Western blot analysis, infection of T.Tn cells with
Ad-E2F-1 led to substantial overexpression of E2F-1 protein and
subsequent increases in the levels of endogenous ARF protein in a
dose-dependent manner, whereas the levels of MDM2 protein clearly
decreased on induction of endogenous E2F-1 (Fig. 1B
).
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, ectopically expressed ARF
alone led to a dose-dependent reduction of the levels of MDM2 in T.Tn
cells. We next asked whether ARF-promoted MDM2 degradation results in
p53 stabilization and accumulation. In TE8 cells that were stably
transfected with a plasmid that expressed ARF, there was a
marked accumulation of endogenous wt-p53 (Fig. 2B
). In
contrast, the levels of p53 slightly increased in ARF-transfected T.Tn
cells presumably because the T.Tn cells express mut-p53. These results
suggest that overexpression of E2F-1 induced endogenous ARF expression,
which in turn promoted MDM2 degradation, thereby leading to the
stabilization of p53.
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. Control dl312 infection at 100 MOI
had no effect on the growth of T.Tn and TE8 cells (data not shown).
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Infection first with Ad-E2F-1 and then with Ad-p53 had no effect on the
growth of T.Tn and TE8 cells, whereas the reverse caused significant
loss of viability in both cell lines (Fig. 4A
, left panel, and
data not shown). In addition, because TE-8 cells are more sensitive
than T.Tn cells to this combination therapy, lower MOI (5 MOI) of
Ad-p53 plus Ad-E2F-1 showed a significant antitumor effect (Fig. 4A
, right panel). As revealed by phase-contrast
photomicrographs, T.Tn cells infected with Ad-p53/Ad-E2F-1 suffered a
rapid loss of viability attributable to massive cell death, as
evidenced by floating, highly light-refractile cells;
Ad-E2F-1/Ad-p53-infected cells suffered no such fate (Fig. 4B
). Simultaneous Ad-p53 and Ad-E2F-1 infection as
well as infection with either vector alone induced no growth
inhibition. The total MOI used for Ad-p53 or Ad-E2F-1 infection
alone was adjusted to 30 MOI with dl312 infection. To obtain more
details about the cell death observed in T.Tn cells that were infected
first with Ad-p53 and then with Ad-E2F-1, cells were stained with the
DNA-intercalating dye Hoechst 33342 and then were examined by
fluorescence microscopy. As this analysis showed,
Ad-p53/Ad-E2F-1-infected cells underwent apoptosis characterized by the
extreme condensation and partial fragmentation of nuclei (Fig. 4B
).
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).
Blockage of MDM2 Up-Regulation and Induction of p53 Accumulation by
Sequential Transfer of wt-p53 and E2F-1
Genes.
To further study the mechanism of apoptosis induced by sequential
infection with Ad-p53 and Ad-E2F-1, we determined the expression levels
of various regulatory proteins after transfer of p53 and/or
E2F-1 genes. We used higher MOI of Ad-p53 and Ad-E2F-1 for
these experiments, because detection of suboptimal p53 and E2F-1
proteins was difficult, presumably because of the low sensitivity of
the assay. When T.Tn and TE8 cells were infected with Ad-p53 at 50 MOI,
a substantial increase in the level of exogenous p53 was observed;
however, when the cells were coinfected instead with Ad-p53 and
Ad-E2F-1 or sequentially infected with Ad-E2F-1 first and Ad-p53
second, a decrease in the level of exogenous p53 gene
product was seen. In contrast, the level of p53 was clearly enhanced by
sequential infection with Ad-p53 first and Ad-E2F-1 second (Fig. 5A and B)
, a result
presumably attributable to a dramatic reduction in Ad-p53-induced MDM2
protein expression (Fig. 5A
).
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, ectopic
overexpression of E2F-1 activated endogenous ARF expression in a
dose-dependent manner, resulting in markedly decreased expression of
MDM2, which was up-regulated by the feedback loop of p53. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Normally, steady-state levels of p53 protein are regulated by
complexing the proteins with MDM2, which in turn promotes the rapid
degradation of p53. Thus, the cellular concentration of p53 protein is
kept low by MDM2, even in the absence of genotoxic stress.
Interestingly, the MDM2 gene, a proto-oncogene that is
amplified in 30–40% of human sarcomas, is known to be a
transcriptional target of p53, and the stress-induced up-regulation of
endogenous p53 protein is known to enhance MDM2 protein expression
through a feedback regulatory loop (32)
. Consistent with
these observations, we observed that adenovirus-mediated overexpression
of exogenous wt-p53 induced MDM2 protein accumulation (Fig. 5,
A and C
), which in turn may have inhibited the
ability of p53 to induce apoptosis. Indeed, it has been reported that
p53 degradation is accelerated when MDM2 cDNA is transfected into human
cells (33)
. Together, these results suggest that
disruption of the feedback regulatory loop between p53 and MDM2 may
enhance the cytocidal effect of wt-p53 gene transfer.
In seeking a strategy to specifically inhibit p53-mediated MDM2
up-regulation, we focused on ARF, a protein that can interact directly
with the MDM2 protein (21)
. ARF reportedly inhibits the
ubiquitin ligase activity of MDM2 (33)
and promotes MDM2
degradation (21)
. Although the biochemical mechanism
underlying ARF-mediated MDM2 degradation remains unclear, our present
data show that transfection of human esophageal cancer cells with a
plasmid designed to express an entire sequence of human ARF reduced the
levels of constitutive MDM2 expression (Fig. 2A
) and induced
p53 accumulation (Fig. 2B
). The mutant form of p53 in these
cells was also stabilized by ectopic ARF expression, consistent with a
previous report that a mutation of p53 did not affect the association
with MDM2 (34)
; mut-p53, however, could not activate the
transcription, which indicated that ARF function is dependent on the
status of the endogenous p53. Recently, it was shown that the ARF
promoter is highly responsive to E2F-1 overexpression and that E2F-1
directly activates expression of the human ARF in a p53-null human
osteosarcoma cell line (16)
, which is similar to our
finding in human esophageal cancer cells infected with Ad-E2F-1 of both
the mRNA and protein levels (Fig. 1, A and B
). In
addition, we showed the existence of an intact autoregulatory loop
between ARF, MDM2, and p53 in those same cells (Fig. 1B
).
Together, these results suggest that the possible interaction of p53
and E2F-1 may enhance growth inhibition and/or apoptosis induction,
although our finding that simultaneous infection with suboptimal
concentrations of Ad-p53 and Ad-E2F-1 could not lead to cell death
(Fig. 4B
), suggesting that this effect is not so
unambiguous. In fact, our finding is consistent with the finding by
Frank et al. that coexpression of wt-p53 and E2F-1 did not
enhance the growth-inhibitory effect in human SCCHN (35)
.
It is likely that the physical interaction of the p53 and E2F-1
proteins suppresses the antitumor effect of coinfection of Ad-p53 and
Ad-E2F-1 in SCCHN. In addition, as our data suggest, sequential
infection with Ad-E2F-1 followed by Ad-p53 infection apparently induces
no apoptotic cell death in human esophageal cancer cells (Fig. 4B
), whereas suboptimal Ad-E2F-1 infection 24 h after
suboptimal Ad-p53 infection induces an accumulation of p53 protein
(Fig. 5, A and B
), and the subsequent rapid
induction of apoptotic cell death (Fig. 4)
. Thus, our data clearly
suggest that the order of gene transfer may be important for mediating
p53 accumulation as well as for causing apoptosis in human esophageal
cancer cell lines.
Although the mechanisms by which Ad-p53/Ad-E2F-1 infection selectively induces apoptosis are not completely understood, the possible existence of a regulatory feedback cycle controlling the level of each protein has important implications. Although Ad-E2F-1 infection promotes endogenous ARF up-regulation, the amount of constitutive MDM2 is very low in the absence of exogenous wt-p53; thus, the level of ARF may be rapidly suppressed through the feedback mechanism. In contrast, when Ad-p53 infection precedes Ad-E2F-1 infection, Ad-p53 activates the transcription of MDM2, which leads to the enhanced synthesis of MDM2 protein, which might in turn be effectively degraded by ARF protein induced by the later Ad-E2F-1 infection. Alternatively, the localization of each protein in the subnuclear compartments may be essential for the responses (36) . When Ad-E2F-1 infection precedes Ad-p53 infection, Ad-E2F-1 can induce expression of ARF, which may in turn sequester constitutive MDM2 in the nucleolus; however, the increased amounts of MDM2 resulting from the Ad-p53 infection may be sequestered in the nucleoplasm because of the localization of antagonistic ARF in the nucleolus, thereby preventing the transactivation in the nucleoplasm. But as our results show that this is not the case. When Ad-p53-infected cells were subsequently transduced with Ad-E2F-1, up-regulated nucleoplasmic MDM2 protein could be moved to the nucleolus by E2F-1-induced ARF, thus allowing the activation of p53 in the nucleoplasm. The precise mechanisms that mediate this cooperation, however, remain to be elucidated.
In light of our data, we conclude that wt-p53 and E2F-1 genes, sequentially transferred into human esophageal cancer cells, can cooperate to efficiently induce apoptosis by stabilizing p53 via ARF-mediated MDM2 degradation. We also conclude that, because combination treatment with suboptimal doses of adenovirus vectors is sufficient to achieve antitumor effects, this strategy may also minimize the risk of adenovirus toxicity. Thus, the sequential combination of Ad-p53 followed by Ad-E2F-1 as gene therapy may lead to the development of molecular therapies for human esophageal carcinoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by grants from
the Ministry of Education, Science, Sports and Culture, Japan; and by
grants from the Ministry of Health and Welfare, Japan [Health Sciences
Research grants (Research on Human Genome and Gene Therapy)]. 
2 To whom requests for reprints should be
addressed, at: First Department of Surgery, Okayama University Medical
School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Phone:
81-86-235-7257; Fax: 81-86-221-8775; E-mail: toshi_f{at}med.okayama-u.ac.jp
3 The abbreviations used are: wt-p53, wild-type
p53; mut-p53, mutant p53; CMV, cytomegalovirus; MOI, multiplicity of
infection; SCCHN, squamous cell carcinoma of the head and neck; RT,
reverse transcription.
Received 12/13/99; revised 3/16/00; accepted 4/ 6/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and p19ARF/p16ß is frequently altered in non-small cell lung cancer and correlates with p53 overexpression. Oncogene, 17: 2779-2785, 1998.[CrossRef][Medline]
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, mock-infected;
,
Ad-E2F-1/Ad-p53-infected; 
, Ad-p53/Ad-E2F-1-infected. Results were
expressed as the mean ± SD of triplicate experiments. Student’s
t test was used to compare viability between the
treatment groups. *, statistical significance (P < 0.05). B, phase-contrast photomicrographs of T.Tn
cells infected with indicated vectors (Ad-E2F-1, 20 MOI; Ad-p53, 10
MOI). Cell morphology was evaluated 48 h after the second
infection. Bottom row, right, T.Tn cells infected first
with Ad-p53 at 10 MOI and then with Ad-E2F-1 at 20 MOI were stained
with Hoechst 33342 and analyzed under a fluorescence microscope.
C, TE8 cells exposed first to Ad-p53 at 10 MOI and then
to Ad-E2F-1 at 20 MOI were harvested at 72 h posttreatment and
then were assayed for DNA content by propidium iodine staining and
subsequent flow cytometric analysis. Induction of apoptosis in these
cells and cells infected with the vectors in the reverse order was then
compared.