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Experimental Therapeutics |
Cell Transformation Group, Department of Pathology, Dunedin School of Medicine, Dunedin, New Zealand 9001
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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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In addition to these findings, our laboratory reported that cell death induced by wtAd5 was dependent on the presence of a wt p53 protein (4) . However, others reported that the induction of cell death by wtAd5 was independent of p53 status (5) , which is seemingly at odds with our findings. However, examination of these results indicates that the kinetics of cell death in the presence or absence of p53 are different. Cells expressing a wt p53 died more quickly than cells defective in p53 (5) . Such results suggest there may be multiple cell death processes: one requiring p53, which kills cells rapidly, which we have reported previously (4) , and at least one other, independent of p53, which kills more slowly. If true, this may explain the differences between our data and those of others and also explain how ONYX-015 can destroy tumor cells deficient in p53, although it does not explain its selectivity for tumor cells.
In this study, we explored this possibility by determining the kill rates of wtAd5 and ONYX-015 in a panel of human cell lines that differ in their p53 status. We show that cells expressing a wt p53 die more rapidly than either cells with a mutant p53 or cells that are null for p53, after infection with wtAd5. These data confirm the existence of kinetically distinct cell death processes. We also show that the rapid p53-dependent death is markedly delayed or absent after ONYX-015 infection, suggesting that E1b55k is also necessary for this cell death. Because E1b55k binds p53, we hypothesized that formation of this complex may be necessary for induction of this rapid death pathway. We confirmed this hypothesis using a panel of E1b55k mutant viruses, which contain mutations that disrupt binding to p53. Thus, the widely held view that binding of E1b55k to p53 abrogates p53 function is not correct in the context of viral infection.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenoviruses.
WtAd5 and mutants ONYX-015 (dl 1520), dl 338, F484, R443, H354, R309,
H180, A262, and H326 were used for the experiments described in this
paper. These viruses have been described previously, and the
appropriate references are shown below. Briefly, these viruses contain
the following mutations. ONYX-015 (dl 1520; Ref. 15
) and
dl 338 (16)
do not produce detectable E1b55k products.
F484, R443, H354, R309, H180, A262, and H326 viruses contain 12-bp
insertion mutations immediately following the codons indicated by the
mutant numbers, and all express stable E1b55k proteins
(17)
. All viruses were grown on 293 cells and titrated on
293 cells using a CPE assay. Virus titers were standardized in CPEUs,
the details of which have been described elsewhere (18)
.
CPEUs are approximately equivalent to plaque-forming units.
Cell Viability Assay.
Cells (3 x 104) were seeded into
each well of six-well tissue culture plates and incubated for 3–4 h in
DMEM + 10% FBS. After this, cells were mock infected or
infected with 10 CPEUs/cell of adenovirus and further incubated in
DMEM + 10% FBS. At indicated times postinfection, both
floating and trypsinized cells were harvested and suspended in their
existing medium. An equal volume of PBS containing 0.1% trypan
blue was added to the cell suspension. Viable and nonviable cells were
then determined by direct counting. All counts were performed on
triplicate samples, and means were plotted on all graphs, with SEs
shown when greater than 5% of the mean.
Cell Synchronization.
Cells (4 x 105) were seeded into
six-well tissue culture plates and incubated for 3–4 h in DMEM + 10% FBS. Medium was replaced with DMEM + 10% FBS containing
1.5 mM hydroxyurea and incubated for a further 16 h.
Medium was removed, cells were washed twice with PBS, and
replaced with DMEM + 10% FBS. Cells were incubated for a
further 5 h before being mock infected or infected with 10
CPEUs/cell of adenovirus. At indicated times postinfection, cells were
harvested, and viable and nonviable cells determined by trypan blue
staining and direct counting. All counts were performed in triplicate
and plotted as means, with SE shown if greater than 5% of the mean.
FACS Analysis.
Cells were harvested, centrifuged at 400 x g
for 10 min, and resuspended in sample buffer (PBS containing 1 g/liter
glucose). Cells were washed twice more in sample buffer and fixed in
70% ethanol by incubation at 4°C for at least 12 h. Fixed cells
were resuspended in a solution of 50 µg/ml propidium iodide in sample
buffer containing 2 mg/ml RNase A and incubated at room temperature for
at least 30 min. Samples were analyzed within 2 h on a Becton
Dickinson FACScalibur analyzer. All samples from individual experiments
were analyzed at the same time, and 0-h, uninfected controls were used
to calibrate the FACS analyzer settings prior to reading the remaining
samples. Cells were gated as demonstrated in Fig. 3
using FL2 area
versus FL2 width to exclude doublets and cell aggregates.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A549, HepG2, and HT 1080 cells, which express a wt p53, underwent
rapid cell death, with less than 30% of cells remaining viable by day
4 (Fig. 1, a–c
). These results are similar to our previous
findings (4)
. However, T98G cells, which express a mutant
p53 protein, and IIICF/c and Saos 2 cells, which do not express p53,
retained cell viabilities of 70–90% by day 4 after infection with
wtAd5 (Fig. 1, d–f
). Death did eventually occur in T98G and
IIICF/c cells, but this was delayed considerably (Fig. 1, d and e
). In Saos 2 cells, death was not delayed as
extensively as in the other p53-deficient cell lines but still showed a
2–3-day lag time (Fig. 1f
). To address whether these
different death profiles might be cell type-specific events, we
performed an identical assay on RKO and RKO p53.13 cell lines. RKO
p53.13 cells arose by stable transfection of a dominant negative mouse
p53 into the RKO parental cell line thereby eliminating p53 function
(11)
. Whereas RKO cells demonstrated rapid death similar
to that induced in the other wt p53-containing cell lines, RKO p53.13
cells showed a delayed death profile similar in kinetics to the other
p53-deficient cell lines (Fig. 1, g and h
). These
data demonstrate the presence of two different death phenotypes based
upon the presence or absence of a wt p53 protein.
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). In all other cells examined,
ONYX-015 failed to induce cell death to a greater extent than
uninfected controls. Where there is overlap of cell lines (A549 and
Saos 2), our results are essentially identical for both wtAd5 and
ONYX-015 to those reported previously (5)
. These data show
that E1b55k promotes death, because wtAd5 kills more quickly than
ONYX-015 in either the presence or absence of p53. This suggests that
E1b55k may play an important role in enhancing the p53-dependent and
p53-independent cell death phenotypes.
To determine whether these data were truly reflective of infection of
normal human cells, and not an artifact of cell immortalization,
similar experiments were carried out in cultures of HFs. To achieve the
same extent of cell killing, a 30-fold greater titer of virus was
required (i.e., 300 CPEUs/cell), but again wtAd5 caused cell
death much faster than ONYX-015 (Fig. 1i
). Less efficient
infection of "normal" cells has been reported recently for a number
of early passage human cells (6)
.
These data lead us to conclude that adenovirus induces more than one cell death pathway that is dependent on the presence of E1b55k and p53.
E1b55k Relieves Growth Arrest.
In the course of carrying out the above experiments, we noticed that in
addition to remaining viable for much longer than their counterparts
infected with wtAd5 (see Fig. 1
), fewer cells remained after ONYX-015
infection. This suggested that ONYX-015 might be slowing or inhibiting
cell division. To test this directly, A549 cells were infected with
wtAd5 and with two E1b55k mutants (dl 338 and ONYX-015). At different
times after infection, total cell number was determined, and cell cycle
analyses were carried out by flow cytometry. Results demonstrate that
cells infected with wtAd5 (Fig. 2a
) died within 4 days, as demonstrated previously (Fig. 1a
; Ref. 4
). However, cells infected with the
two E1b55k-deficient viruses failed to proliferate (Fig. 2, b and c
), and there was no decline in viability
(as shown in Fig. 1
) over the time course of the experiment.
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) showed that by 48 h postinfection, cells infected with wtAd5
exhibited a decreased G1 peak, a subdiploid peak
indicative of cell death and a high percentage of cells in
S/G2. By 96 h postinfection, the
G1 population had declined substantially,
indicating that cells previously in S/G2 had not
cycled into G1. There also appeared to be a
general decline in the DNA profiles consistent with the decline in
viability (Fig. 1
). In addition, a general observation was that cells
appeared to traverse S phase and accumulate S/G2
DNA contents before cell death occurred. In contrast, ONYX-015-infected
cells did not show any evidence of a subdiploid peak at either 48 or
96 h postinfection. Instead, by 48 h postinfection, cells had
accumulated in late S/G2, and by 96 h
postinfection, the majority of cells had acquired a greater than 4N DNA
content. This increase in DNA content is not caused by doublet
formation or cell clumping as all doublets/clumps were "gated out"
of our DNA content analysis (see Fig. 3
). This shift is also not caused
by cell fusions because we would have observed integral shifts in DNA
ploidy (i.e., distinct peaks at 6N, 8N, and so forth). Thus,
the increased DNA content must be caused by a reinitiation of DNA
replication without an intervening cell division. Consistent with this
interpretation, addition of hydroxyurea (which inhibits initiation of
DNA replication) prevented ONYX-015-infected cells from acquiring
greater than 4N DNA contents (data not shown). This interpretation is
also consistent with several reports demonstrating that adenovirus
infection causes increases in cellular DNA content in rodent cells
(19, 20, 21)
. One of these reports specifically excluded
aggregates and cells with more than one nucleus using
microspectrophotometry (19)
. Similar cell cycle profiles
were observed in HepG2 cells after infection with ONYX-015 (data not
shown). These data provide further evidence for the existence of at least two distinct cell death phenotypes, one requiring E1b55k and one that does not. In addition, it appears that E1b55k functions in some way to relieve cell cycle abnormalities that lead to the accumulation of cells in late S/G2, and by so doing, it promotes cell death. However, the biochemical details of this cell death promotion are not yet clear and are the subject of ongoing investigation.
Formation of the E1b55k/p53 Complex Is Required for Cell Death.
E1b55k is known to form a stable protein complex with p53 in both
infected and adenovirus transformed cells (22, 23, 24)
.
Because death occurred most rapidly under conditions in which both p53
and E1b55k were present, we hypothesized that the E1b55k/p53 complex
may be important for enhancing the cell death pathway, despite reports
that E1b55k inhibits the action of p53 (25, 26, 27, 28)
. An
experiment was therefore carried out in which A549 cells were infected
with a number of adenovirus insertion mutants (17)
that
express full-length E1b55k proteins, some of which are defective in p53
binding. Mutants H354, R443, and F484 retain an ability to bind p53,
whereas mutants H180, A262, H326, and R309 are no longer able to bind
(17)
. Results (Fig. 4
) showed that mutants that retained the ability to complex with p53 were
able to induce cell death with kinetics similar to those of wtAd5. By
contrast, mutants defective for p53 binding induced cell death with
delayed kinetics, similar to those observed for ONYX-015 and for p53
mutant cells.
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) showed similar profiles to those of the E1b55k-deficient virus
ONYX-015 (Fig. 3
) with DNA contents mostly greater than 4N. These data
support the conclusion that in the absence of binding, cell death is
delayed and DNA replication continues without an intervening cell
division.
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indicate that wtAd5-infected cells
traverse S phase and acquire S/G2 DNA contents
prior to cell death. More detailed kinetic data using flow cytometry
confirm this interpretation (data not shown). In contrast,
ONYX-015-infected cells accumulate S/G2 DNA
contents but avoid death. These data suggest that adenovirus-induced
p53-dependent death might be cell cycle dependent. Thus, cell death as
a consequence of wtAd5 infection may be explained by cells traversing S
phase and becoming susceptible to death as they reach a critical point
in the cell cycle. If this is so, then synchronizing infected
cells such that a greater proportion traverse S phase as a cohort
should generate a synchronous wave of cell death.
To test this possibility, cells were synchronized by blocking at the
G1/S boundary with hydroxyurea and, upon release,
infecting them with wtAd5. Cell counts were performed over time periods
sufficient to encompass two cell doublings after release from the
block. These counts (Fig. 6
) indicate that the first cell doubling in both infected and uninfected
cells proceeds normally with no evidence of cell death, presumably
because they have insufficient time to express early viral proteins.
However, at the second cell doubling, whereas uninfected cells doubled
in number as expected, approximately half the wtAd5-infected cell
population died. Although it is clear that these cells are no
longer in synchrony at the point at which they die, cell death begins
to occur in the wtAd5-infected population at the precise time that cell
division begins in the uninfected population. Importantly, these events
occur at similar rates.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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However, recent reports showing that ONYX-015 can replicate in cells expressing a wt p53 (5 , 6) and an apparent requirement for p53 in adenovirus-induced cell death (4) appear to be inconsistent with the model proposed for ONYX-015-induced death.
The data presented here demonstrate that at least two distinct cell death pathways exist after infection with adenoviruses. These are distinguished by the kinetics of cell death and by a requirement for both E1b55k and p53. One pathway results in rapid cell death and is dependent on the presence of a functional p53 protein. In the absence of p53, cell death is delayed considerably, with a minimum of a 2–3 day lag time compared with infection of wt p53 expressing cells. In the absence of E1b55k (e.g., infection with ONYX-015), cell death was delayed similar to that observed for p53-deficient cells infected with wtAd5. In the absence of p53, E1b55k-deficient adenoviruses were unable to induce cell death in the 10-day time period of the experiments we conducted. Such data do not support the selective targeting of p53-deficient cells by ONYX-015 but rather suggest that p53 and E1b55k are required for efficient induction of cell death by adenoviruses.
This is further supported by the finding that cells infected with
E1b55k-deficient viruses fail to proliferate after infection but rather
demonstrate an apparent block in cell division (Fig. 2, b and c
). This is not caused by an equilibrium being reached
between dying and living cells because viabilities remain high (Fig. 1
). FACS analysis of these populations demonstrated that
ONYX-015-infected cells accumulate with 4N and greater than 4N DNA
contents but fail to undergo cell death. The lack of a subdiploid peak,
present in the wtAd5-infected cells and characteristic of cell death,
suggests that ONYX-015 cannot induce rapid cell death. These data
support the conclusion that both p53 and E1b55k are necessary for the
induction of the rapid death pathway. In the absence of either of these
proteins, cell death is delayed considerably. These data also suggest
that both p53 and E1b55k play a positive role in promoting the cell
death process associated with adenoviral infection.
Because E1b55k has been shown previously to bind p53, we next
considered whether the formation of the complex was necessary for the
induction of cell death. To do this, we used a panel of insertion
mutants, some of which disrupt p53 binding and others of which still
bind p53 (17)
. Those mutants that retained an ability to
bind p53 demonstrated rapid cell death with kinetics similar to those
of wtAd5 (Fig. 4
). In contrast, those mutants in which p53 binding was
disrupted demonstrated a delayed cell death with kinetics that were
remarkably similar to those of ONYX-015 (Fig. 4
).
FACS analysis also demonstrated different death profiles dependent on
the ability of the E1b55k protein to bind p53. Viruses that expressed
E1b55k proteins that retained their ability to bind p53 accumulated in
late S/G2 prior to death. These profiles were
similar to those demonstrated for wtAd5 (Fig. 3
). Viruses that
expressed an E1b55k protein that was unable to bind p53 had DNA
contents of 4N or greater than 4N (Fig. 5
). These profiles are similar
to those shown for ONYX-015 (Fig. 3
) and suggest that cells are
continuing to replicate their DNA but are apparently unable to divide.
These data demonstrate that formation of the p53/E1b55k complex is
essential for the induction of rapid cell death by adenoviruses and are
the first evidence demonstrating a role for the E1b55k/p53 complex in
promoting cell death.
Previous reports have shown that the adenovirus E4ORF6 protein binds
E1b55k (32)
and that E4ORF6 and p53 binding domains
substantially overlap (24
, 33)
. Thus, disruption of the
p53 binding domain of E1b55k may equally affect the binding of both p53
and E4ORF6. However, the H180 virus, which disrupts p53 binding but
does not disrupt E4ORF6 binding (34)
, still demonstrates
an ability to abrogate the rapid cell death process (Fig. 4
). This
would argue against E4ORF6 being the protein responsible for the
induction of p53-dependent cell death.
Another protein that is associated with and binds E1b55k is the
recently identified E1B-AP5 (34)
. This protein also
overlaps the p53 binding domain of E1b55k. Binding of E1B-AP5 is
disrupted in the F484 mutant (34)
; however, this mutant
retains its ability to bind p53, and our data demonstrate that it also
retains its ability to induce rapid cell death (Fig. 4
). These data
would argue against a role for E1B-AP5 in the induction of
p53-dependent cell death.
The data presented here and previously (4) indicate that rapid cell death requires the presence of both p53 and E1b55k and that these proteins must be able to complex. Thus, the original claim (2) that ONYX-015 was selective for tumor cells containing a mutant p53 is not supported by these data. Rather, they would suggest that wt p53-containing cells should be preferentially killed. However, ONYX-015 continues to show an ability to kill tumor cells in clinical trials (1 , 35) despite the absence of a functional E1b55k protein and, in many cases, the absence of wt p53.
The basis of this p53-independent cell death may be a reduced ability
of normal cells to either be infected or grow virus and
therefore to induce cell death. This interpretation is supported by
both our data (Fig. 1i
) and those of others (6)
showing that HFs require a much higher dosage of virus to induce
similar kill rates to cell lines derived from human tumors.
Alternatively, delayed death may be attributable to the apparent
quiescent state of normal cells compared with their tumorous
counterparts. It has been suggested previously (5)
that
cells are more readily infected when they are in S phase. Therefore, if
cells are cycling rapidly, a high percentage of cells will be infected
(because of their higher representation in S phase), whereas cells that
are quiescent will be infected rather poorly. A further possibility is
that death is simply a consequence of a delayed or impaired viral
replicative process. However, we would argue against this being a
simple relationship because replication of both wtAd5 and ONYX-015 has
been demonstrated in p53 defective cells and cells expressing a wt p53
protein (4
, 6)
. Also, similar replicative abilities have
been demonstrated for p53 binding mutants in A549 cells regardless of
whether or not they bind p53 (36)
.
Thus, the data presented here offer further evidence that ONYX-015 does not selectively kill tumor cells that are deficient for p53. In fact, the data suggest that the antitumor effects induced by ONYX-015 are attributable to the apparent selectivity of adenovirus infection for tumor cells. We would suggest that there is nothing unique about ONYX-015 in the context of tumor therapy. In fact, a more efficient destruction of tumor cells is likely to occur if wtAd5 were used as the therapeutic agent, because it should be able to induce cell death more efficiently in cells containing either a wt or mutant p53. The more efficient and faster death induced by wtAd5 should allow lower doses for equivalent results and therefore decrease the likelihood of possible side effects.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 B. R. D. was supported by The Cancer Society
of New Zealand; S. J. O. was supported by The University of Otago
Faculty Fund; C. J. M. was supported by The Health Research Council
of New Zealand; S. J. E. was supported by The Lottery Board of New
Zealand; and A. W. B. was supported by The University of Otago. 
2 To whom requests for reprints should be
addressed, at Cell Transformation Group, Department of Pathology,
Dunedin School of Medicine, P. O. Box 913, Dunedin, New Zealand 9001.
Phone: 64-3-479-7165; Fax: 64-3-479-7279; E-mail: antony.braithwaite{at}stonebow.otago.ac.nz
3 The abbreviations used are: wt, wild-type; Ad5,
adenovirus type 5; HF, human foreskin fibroblast; CPE, cytopathic
effect; CPEU, CPE unit(s); FBS, fetal bovine serum.
Received 8/13/99. Accepted 3/16/00.
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REFERENCES |
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)
or ONYX-015 (
) or mock infected (controls; 
). RKO
(g) cells contain a wtp53, and RKO p53.13 (h)
cells are RKO cells stably transfected with a dominant negative mouse
p53, which eliminates functional p53; these cells were infected
as outlined above. HF (i) cells were infected at 300
CPEUs/cell. Cell viability was assessed by trypan blue exclusion and
direct counting for 10 days postinfection. Data points,
means of triplicate counts; SE is shown if greater than 5% of the
mean.
) or mock
infected (