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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3499-3508
Adenovirus-Mediated Cytotoxicity of Chronic Lymphocytic Leukemia Cells
By
Daniel J. Medina,
Wendy Sheay,
Lauri Goodell,
Pamela Kidd,
Eileen White,
Arnold B. Rabson, and
Roger K. Strair
From The Cancer Institute of New Jersey and the Department of
Pathology, Robert Wood Johnson Medical School, University of Medicine
and Dentistry of New Jersey, New Brunswick, NJ; the Howard Hughes
Medical Institute and the Department of Molecular Biology and
Biochemistry, Rutgers University, Piscataway, NJ; the Center for
Advanced Biotechnology and Medicine, University of Medicine and
Dentistry of New Jersey, Piscataway, NJ; and the Department of
Molecular Genetics and Microbiology, Robert Wood Johnson Medical
School, University of Medicine and Dentistry of New Jersey, Piscataway,
NJ.
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ABSTRACT |
We have studied adenovirus-mediated cytotoxicity after infection of
malignant cells obtained from patients with chronic lymphocytic leukemia (CLL). Our studies indicate that adenoviruses can infect primary CLL cells and that infection of CLL cells with a
replication-competent strain of human adenovirus 5 (Ad5dl309) results in cytotoxicity. Adenovirus-mediated
cytotoxicity was also seen after infection of CLL cells with a variety
of viruses attenuated by mutations in the adenovirus early region 1 (E1) or early region 2 (E2). Even viruses attenuated by deletion of the
entire E1 region resulted in cytotoxicity after infection of the CLL
cells obtained from some patients. Although there was variability in
the degree of cytotoxicity induced by different viruses in different
patients cells, a virus with a mutation in the E1B 19K gene resulted in the greatest degree of cytotoxicity in most of the CLL samples tested.
These studies demonstrate that infection of CLL cells by attenuated
adenoviruses with specific mutations in the E1 or E2 region results in
cell death. Attenuated adenoviruses should be developed further as
therapeutic agents for patients with CLL.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SEVERAL BIOLOGICAL processes may
contribute to cytotoxicity after infection by wild-type or mutant
adenoviruses. For example, productive infection, abortive infection,
abortive infection with the induction of apoptosis, or a combination
may contribute to the death of an infected cell.1-5 These
possible outcomes result from complex interactions between host and
virus that impact on virus replication, host cell cycle regulation,
virus and host gene expression, the induction of apoptosis, and host
cell survival. As the nature of some of these interactions have been
elucidated, mutant adenoviruses have been developed as cytotoxic agents
with specificity for malignant epithelial cells.6,7
The initial studies demonstrating specific cytotoxicity of mutant
adenoviruses in selected epithelial malignancies used an adenovirus
with a mutation in the E1B 55K gene product.6,7 The
rationale for the specificity of the cytotoxicity induced by infection
with that virus was based on the complementation of a genetic defect of
the virus (E1B 55K mutation) by a genetic feature of the malignant cell
(p53 mutation), which should allow the mutant virus to replicate and
induce cytotoxicity selectively in malignant, but not normal (p53
wild-type) cells. The selective cytotoxicity induced by this virus was
presumed to be a consequence of the requisite inactivation of p53 by
the E1B 55K polypeptide during a productive infection of normal cells.
In the absence of the E1B 55K polypeptide (and presence of wild-type
p53), productive infection may be reduced. However, the relationship
between virus replication, p53 expression, and cytotoxicity is complex,
and other studies have demonstrated enhanced cytotoxicity of this virus
in the presence of wild-type p53 under certain conditions.5
Although cells with p53 mutations (the targets of infection the E1B 55K
mutated adenovirus) are not as prevalent in hematologic malignancies as
they are in many other types of tumors, several of the lymphoid
malignancies are characterized by the altered expression of other
cellular proteins that might complement adenovirus mutants or interact
with adenovirus proteins to result in specifically enhanced
cytotoxicity. For example, malignant cells from patients with chronic
lymphocytic leukemia (CLL) have a spectrum of abnormalities, including
altered expression of Bcl-2, other Bcl-2 family members, cdk
inhibitors, cyclins, pRB, or p53.8-11 Each of these genetic alterations may result in a cellular milieu that could enhance susceptibility to adenovirus-mediated cytotoxicity after infection with
specific mutant adenoviruses. For example, the overexpression of
specific cyclins, the absence of specific cdk inhibitors, and mutations
in pRb might result in alterations of pRb function that would
complement mutations in E1A that alter pRb binding. These changes could
result in cytotoxicity after infection with viruses containing specific
mutations in E1A.
Because prior studies have indicated limited virus
production/cytotoxicity after adenovirus infection of normal lymphoid
cells,12,13 we initiated studies to assess the degree of
cytotoxicity induced by adenovirus infection of CLL cells. Despite the
fact that many malignancies may have alterations that make them
susceptible to adenovirus and adenovirus mutant-mediated cytotoxicity,
we have chosen to direct our study to CLL because (1) the disease is
incurable with standard dose chemotherapy; (2) there are documented
biological/genetic changes characteristic of the disease that might
facilitate selective cytotoxicity after infection with a mutant virus;
(3) previous studies by others have indicated that adenovirus infection
of normal lymphoid cells is generally nonproductive; and (4) there is
therapeutic rationale for the induction of in vivo or ex vivo cytotoxicity. In vivo studies may demonstrate a novel therapeutic with
selective cytotoxcity during systemic or local administration. Ex vivo
therapy could be undertaken in conjunction with high-dose chemotherapy/autologous hematopoietic stem cell rescue (HDC/HSCR) as a
means of purging autologous hematopoietic stem cell grafts of
contaminating malignant cells.
We have used a panel of adenoviruses to infect CLL cells obtained from
patients. These studies demonstrate adenovirus-mediated cytotoxicity
after infection of primary CLL cells with viruses attenuated by
mutations in E1A, E1A+E1B, E1B 19K, or E2A, raising the possibility
that mutated adenoviruses may have a therapeutic role in the treatment
of selected hematologic malignancies.
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MATERIALS AND METHODS |
Patients.
Eight B-cell CLL patients were included in this study. Diagnosis was
based on clinical examination, peripheral blood count, and the
morphology and immunophenotype of the leukemic cells (CD5+,
CD19+, and CD23+). Samples of blood were
obtained under a protocol approved by the Robert Wood Johnson Medical
School Institutional Review Board. Some of the patients were treated
and others were untreated. Some patient samples were available before
and after the initiation of treatment. Mononuclear cells were isolated
from 10 to 50 mL of fresh, heparinized peripheral blood by Histopaque
(Sigma, St Louis, MO) centrifugation. Isolated cells were
counted, resuspended in freezing medium (90% calf serum and 10%
dimethyl sulfoxide [DMSO]), and stored in liquid nitrogen until use.
Cells and viruses.
HeLa cells and 293 cells were grown in L modified Eagle's medium
supplemented with 2 mmol/L L-glutamine, 10% fetal bovine serum (FBS),
and antibiotics. CLL cells were maintained in RPMI-1640 medium
supplemented with 20% FBS, L-glutamine, antibiotics, and 50 U/mL of interleukin-4 (IL-4; Boehringer
Mannheim, Indianapolis, IN).
The viruses used in the experiments, Ad5dl309, E1A ,
Pac3, E1B, and Ad5dl337 were described
previously.14-16 The temperature sensitive (ts) virus
Ad5ts125 was generously provided by Daniel Klessig (Rutgers University,
Piscataway, NJ).17 All viruses, with the
exception of the Ad5ts125, were grown and titered on 293 cells at
37°C. The Ad5ts125 virus was grown at 33°C and was titered on
293 cells at 33°C and 37°C. For mock infections,
Ad5dl309 was heat-inactivated at 65°C for 30 minutes.
The recombinant adenovirus containing the green fluorescent protein
(GFP) gene was purchased from Quantum Biotechnologies (Montreal,
Quebec, Canada). That virus (AdGFP) contains the GFP gene, under the
control of a cytomegalovirus (CMV) promoter, in the E1A
region of an adenovirus 5 containing a deletion in the E3 region.
Infection.
CLL cells (2 × 106) were infected with the indicated
viruses at a multiplicities of infection (moi) of 100 (or as otherwise indicated) for 4 to 6 hours at 37°C. Infections with Ad5ts125 were
conducted at both the permissive temperature (33°C) and the nonpermissive temperature (37°C). After infection, the cells were washed 3 times with Dulbecco's phosphate-buffered saline to remove nonadsorbed virus. The cells were then pelleted by centrifugation at
500g for 10 minutes, resuspended in 2 mL of medium, and
cultured in 24-well plates. At the indicated times, samples were
removed for determination of cell viability by trypan blue staining,
virus yield, and apoptosis. Mock infections used heat-inactivated
Ad5dl309.
Cell viability.
At the indicated times, cells in the various replicate wells were
resuspended and 100 µL were removed and analyzed for cell viability
by trypan blue staining and cell count. Cytotoxicity was calculated by
a comparison of viable cell numbers in the infected samples as compared
with uninfected controls.
Virus yield assay.
To assess virus replication, cell-free supernatants were collected at
the indicated times. The virus titer was determined by plaque assay on
either 293 cells or Hela cells. Briefly, 1 mL of serial 10-fold
dilutions of the cell-free supernatant were added to 60-mm dishes
containing a confluent layer of either 293 cells or Hela cells followed
by incubation for 1 hour at 37°C. After incubation, the virus was
removed and the plates were overlaid with agar as
described.18 Viral plaques were counted on day 14 and the viral titer was determined.
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RESULTS |
Infection of primary CLL cells with adenovirus 5 (Ad5dl309).
The ability of adenoviruses to infect primary CLL cells was established
by infection of CLL cells with a recombinant adenovirus expressing the
GFP under the control of a CMV promoter (AdGFP; Quantum
Biotechnologies). Three days after infection, green fluorescence was
measured by flow cytometry. A marked shift in fluorescence was seen
after infection with AdGFP (Fig 1),
indicating that primary CLL cells could be infected by adenoviruses.
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| Fig 1.
CLL cells can be infected with an adenovirus encoding
GFP. CLL cells were infected with AdGFP, containing the GFP gene under
the control of a CMV promoter (Quantum Biotechnologies). Three days
later, cells were assayed for green fluorescence by flow cytometry
using a cytometer equipped with an argon laser at 488 nm. The heavy
line represents the fluorescence of infected cells. The light line
represents the fluorescence of uninfected cells.
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To determine if adenoviruses induce cytotoxicity in CLL cells, cells
were obtained from the blood of a series of patients. Each patient had
a lymphocytosis that was morphologically and immunophenotypically
characteristic of B-cell CLL. Populations of malignant cells at 90% to
99.9% purity were infected with Ad5dl309, a
replication-competent strain of human adenovirus 5 containing a
deletion in E3B,14 and cell viability was monitored over
the course of 20 days. The Ad5dl309 virus was chosen because it
has served as the parental strain for generation of well-characterized mutant adenoviruses.
In each CLL sample, cytotoxicity after infection was detected in
comparison to mock infected cells (Fig 2).
At 4 days postinfection (Fig 2A), a loss of cell viability in
comparison with uninfected and mock-infected was seen for samples from
patients no. 1 through 6. The extent of cell death ranged from 40% to
50% for samples no. 1, 2, 3, and 5 to approximately 80% for samples
from patients no. 4 and 6. Uninfected CLL samples from patients no. 1 to 6 exhibited a loss of viability by day 10, and the full extent of
adenovirus-induced cytotoxicity could not be assessed under conditions
allowing survival of uninfected or mock-infected cells. In
contrast, CLL cells from patient 7 persisted in culture for greater
than a 3-week period (>90% viability on day 20) and 100% of the
cells were killed by Ad5dl309 by day 20 (Fig 2B). One hundred
percent of the cells in the control and mock-infected cultures retained
the immunophenotype of CLL, indicating that Ad5dl309 infection
induced marked cytotoxicity for the CLL cells. The biological/genetic
correlates of the ability of CLL patient no. 7 cells to persist in
culture are not clear, because the patient from whom these cells were
obtained has a clinically, morphologically, and immunophenotypically
classic form of CLL with normal cytogenetics. The results from all 7 patients suggest that infection with adenoviruses leads to cytotoxicity of CLL cells. In some experiments, up to 10% of the cells did not have
the morphology or immunophenotype of CLL. Cytotoxicity of the CLL cells
in those samples was comparable to those of more homogenous patient
samples.
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| Fig 2.
Adenoviruses infection of CLL cells induces cytotoxicity.
Primary CLL cells from 7 patients were infected with Ad5dl309
or heat-inactivated Ad5dl309 (mock-infected). At the
indicated times, cell viability was determined by cell counting and
trypan blue staining and compared with the viability of uninfected and
mock-infected cells. (A) The degree of cytotoxicity seen 4 days after
infection of primary CLL with Ad5dl309. (B) The degree of
cytotoxicity seen 20 days after infection of primary CLL with
Ad5dl309. In both figures, control refers to uninfected cells
and mock refers to cells infected with heat-inactivated virus.
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Infection of CLL cells with Ad5dl309 mutants containing deletions of
E1A, E1A+E1B, or E1B 19K.
The cytotoxicity detected after infection of CLL cells with
Ad5dl309 raised the possibility that more attenuated viruses
might also induce cytotoxicity. We thus performed a series of
infections with a panel of mutated adenoviruses. In these studies, the
CLL cells from individual patients were infected with Ad5dl309
mutants containing deletions of either E1A (E1A), E1A+E1B
(Pac3), or E1B 19K (Ad5dl337)15,16,19 (see
Table 1). Four days after infection with the adenovirus containing a deletion of E1A
(E1A), CLL cells from patients no. 1, 3, and 5 exhibited 20% to
30% cytotoxicity, whereas CLL samples from patients no. 2, 4, and 6 demonstrated 50% to 75% cytotoxicity (Fig
3A). Infection with the adenovirus deleted in both E1A and E1B (Pac3)
also resulted in cytotoxicity with samples from patients no. 2, 4, 5, and 6, demonstrating a 40% to 60% loss in viability 4 days after
infection (Fig 3A). Unlike the CLL cells from patients no. 1 through 6, CLL cells from patient no. 7 persisted in culture more than 20 days,
and both the viruses with E1A deletion (E1A) and E1A+E1B
deletion (Pac3) resulted in complete eradication of the CLL cells under conditions that supported full viability of control (uninfected or
mock-infected) cells (Fig 3B). These infections demonstrate that even
highly attenuated viruses containing deletion of E1A and E1B are
capable of inducing cytotoxicty after infection of CLL cells.
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| Fig 3.
Attenuated adenovirus infection of CLL cells
induces cytotoxicity. Primary CLL cells from 7 patients were
infected with Ad5dl309, heat-inactivated Ad5dl309
(mock-infected), an E1A deleted adenovirus (E1A), or an adenovirus
with a deletion of E1A and E1B (Pac3). At the indicated times, cell
viability was determined by cell counting and trypan blue staining and
compared with the viability of uninfected and mock-infected cells. (A)
The degree of cytotoxicity seen 4 days after infection of primary CLL
with each of the adenoviruses. (B) The degree of cytotoxicity seen 20 days after infection of primary CLL cells with each of the
adenoviruses. In both figures, control refers to uninfected cells and
mock refers to cells infected with heat-inactivated virus.
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CLL cells from patient no. 7 persist in culture for more than 22 days,
allowing a time course of full cytotoxicity for this patient's cells
to be undertaken (Fig 4). In this
experiment, the parental virus (Ad5dl309), the highly
attenuated virus containing a deletion of E1A and E1B (Pac3), and the
virus containing an E1B 19K deletion (Ad5dl337) were used to
infect the CLL cells from patient no. 7. Ad5dl337 was studied
because it has been demonstrated to be highly cytotoxic to other cell
types, resulting in cell death by
apoptosis.2,3,16,20 Uninfected and
mock-infected cells were viable for more than 22 days and cytotoxicity
was seen after infection with each of the viruses (Fig 4). At each time point, Ad5dl337 induced the most pronounced cytotoxicity and by day 22, all of the viruses resulted in 100% cytotoxicity of patient no. 7 CLL cells.
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| Fig 4.
Time course of cytotoxicity induced by adenovirus
infection of CLL cells. CLL cells from patient no. 7 were infected with
Ad5dl309, Pac3, or Ad5dl337 and the degree of
cytotoxicity was monitored by cell counting and trypan blue staining
every 3 to 4 days. Control refers to uninfected cells and mock refers
to cells infected with heat-inactivated virus.
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To further confirm the induction of cytotoxicity in CLL cells by
different mutated adenoviruses, cells from an eighth CLL patient were
infected with Ad5dl309, the E1A deleted adenovirus (E1A), Pac3, or Ad5dl337 at different moi. At an moi of
1 infectious unit per cell, the virus deleted in the E1B 19K gene
(Ad5dl337) was the only virus that exhibited cell killing at
day 8 (Fig 5). At higher mois,
Ad5dl309, the E1A-virus, and Pac3 all exhibited cytotoxicity;
however, at each moi, Ad5dl337 was the most efficacious in
inducing cell death (Fig 5).
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| Fig 5.
Ad5dl337 induces cytotoxicity after infection of
primary CLL cells at a low moi. CLL cells from patient no. 8 were
infected with Ad5dl309, Pac3, or Ad5dl337 at different
moi. Cell viability was determined by cell counting and trypan blue
staining 8 days after infection. Control refers to uninfected cells and
mock refers to cells infected with heat-inactivated virus.
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Infection of normal B cells and committed hematopoietic progenitors
with Ad5dl309, E1A-, Pac3, and Ad5dl337.
To determine if normal B cells demonstrated adenovirus-mediated
cytotoxicity after infection, preparations of mononuclear cells from a
human tonsil were infected with Ad5dl309, Ad5dl337, E1A, and Pac3. As demonstrated in
Fig 6, the total number of viable B cells
(as detected by CD19 expression) was unaffected by infection with
Ad5dl309, E1A, Pac3, or Ad5dl337 infection. In a
similar experiment, blood mononuclear cells obtained from patients
undergoing hematopoietic stem cell mobilization/harvesting were
infected with Ad5dl309, E1A, Ad5dl337, or Pac3
for 4 hours before plating in methylcellulose. Fourteen days later,
colony formation was assessed (Fig 7). No
reduction in colony formation was detected after infection with any of
the viruses. Therefore, the tested viruses are not directly cytotoxic
after infection of either normal B cells or committed hematopoietic
progenitors.
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| Fig 6.
Adenoviruses do not induce cytotoxicity after infection
of normal B cells. Cells obtained from a nonmalignant human tonsil were
infected with Ad5dl309, E1A, Pac3, and Ad5dl337. At
the indicated time after infection, the number of viable B cells was
determined by isolating viable cells and assaying for CD19 expression
by flow cytometry.
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| Fig 7.
Adenoviruses do not alter the ability of committed
hematopoietic progenitor cells to form colonies in methylcellulose.
Blood mononuclear cells obtained after treatment of patients undergoing
hematopoietic stem cell mobilization with granulocyte
colony-stimulating factor (G-CSF) were infected with Ad5dl309
at an moi of 100 for the indicated periods of time and plated in
methylcellulose (in the presence of IL-3, granulocyte-macrophage
colony-stimulating factor [GM-CSF], and erythropoietin
[Epo]) using commercially available media (Stem Cell
Technologies, Vancouver, British Columbia, Canada).
Colonies (colony-forming unit-erythroid [CFU-e], burst-forming
unit-erythroid [BFU-e], colony-forming unit-granulocyte-macrophage
[CFU-GM]) were counted at 14 days. () Uninfected; (+) infected
with Ad5dl309.
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To determine if adenoviruses were capable of infection of the B cells,
a preparation of human tonsil cells (containing more than 75% B cells)
was infected with AdGFP. No shift in fluorescence was seen after
infection with AdGFP, suggesting that primary B cells are not infected
(data not shown).
Infection of CLL cells with adenoviruses containing mutations in E2A.
These studies with primary malignant cells highlight
adenovirus-mediated cytotoxicity induced by highly attenuated viruses in a cell type (B cells) that we have demonstrated to be refractory to
adenovirus-mediated cytotoxicity. Others have also demonstrated that
lymphoid cells are generally a nonproductive host.12,13 As
a test of a further means of attenuation, a temperature-sensitive mutant containing a mutation in the E2A gene, Ad5ts12517
(see Table 1), was studied to determine if the virus would induce cytotoxicity at either the permissive or restrictive temperature. Such
a temperature-sensitive virus might ultimately have particular appeal
for ex vivo therapeutic use, because it would be attenuated at the in
vivo temperature of a patient.
The results of the infections with the E2A temperature-sensitive mutant
are demonstrated in Fig 8. As can be seen,
both Ad5dl309 and Ad5ts125 induced significant cytotoxicity.
Surprisingly, Ad5ts125 induced cytotoxicity at both 33°C and
37°C. Despite the cytotoxicity induced by Ad5ts125 at
the restrictive temperature (37°C), it is important
to note that there was a marked reduction in virus production after
infection with Ad5ts125 at this temperature (see below).
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| Fig 8.
Temperature-sensitive viruses induce cytotoxicity after
infection of CLL cells. CLL cells from patient no. 7 were infected with
a temperature-sensitive viruses containing mutations in the E2A gene
(Ad5ts125) at the restrictive (37°C) and permissive
(33°C) temperatures. Cytotoxicity was determined by
cell counting and trypan blue staining at various times after
infection. Control refers to uninfected cells and mock refers to cells
infected with heat-inactivated virus. ( ) Mock infection; ( )
infection with Ad5dl309; ( ) infection with Ad5ts125 at
33°C; ( ) infection with Ad5ts125 at
37°C.
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Virus production and apoptosis after infection of CLL cells.
As a preliminary analysis of the mechanisms responsible for
cytotoxcity, we have undertaken an analysis of the amount of virus present in the culture supernatant after infection of the CLL cells. In
these experiments, virus was readily detected after the infection. The
presence of significant amounts of virus with each of the samples
reflects the presence of low-level viral production throughout the
course of infection with each of the viruses.
Table 2 demonstrates virus titer at days 3 and 10 after infection with Ad5dl309, Pac3, E1A,
Ad5ts125, or heat-inactivated Ad5dl309. Viral titers for
Ad5dl309, the E1A virus, and Pac3 were more than
105 pfu/mL on days 3 and 10. Pac3 is listed twice in Table
2, because the supernatant from the infection on day 10 was titered on
both 293 cells and HeLa cells. The absence of detectable virus when the
supernatant was titered on HeLa cells reflects the inability of the
Pac3 virus, which contains a deletion in E1A and E1B, to generate
plaques on HeLa cells (Table 2). The Ad5ts125 is listed 4 times in
Table 2, because the supernatants from infections at 33°C and
37°C were each titered at both 33°C and
37°C. For example, infection of CLL cells with
Ad5ts125 at the restrictive temperature (37°C)
resulted in very small amounts of virus in the supernatant at day 3 or
10 (2.5 × 103 or 4.5 × 103 pfu/mL),
regardless of whether the supernatant was titered at 37°C or
33°C. In contrast, there was significantly more virus present in the supernatant after infection of CLL cells at the permissive temperature (33°C) when the supernatant
was titered at the permissive as opposed to the restrictive temperature
(1.5 × 105 pfu/mL and 2.5 × 103
pfu/mL, respectively, on day 3; Table 2). Therefore, Ad5ts125 infection
of CLL cells results in considerably more virus production at the
permissive as opposed to the restrictive temperature and the virus
produced at the permissive temperature retains the phenotype of
temperature sensitivity.
Table 3 demonstrates viral yields after a
series of infections with a different patient's CLL cells. In
agreement with the data presented in Table 2, the virus deleted of E1A
and E1B (Pac3) and Ad5dl337 result in comparatively high virus
yields in culture (more than 105 pfu/mL). In these
experiments, infection of HeLa cells and 293 cells with the viruses
produced during infection demonstrated the restricted host range and
temperature sensitivity anticipated for infection by the temperature
sensitive and Pac3 virus mutants.
Apoptosis, as measured by subdiploid DNA content, Hoechst 33258 staining, or Annexin V staining, was only detected in a small subset of
the cells during infection (data not shown). Even Ad5dl337, a
virus which readily induces apoptosis in other cell
types,2,3,15,16 only resulted in a small increment in the
number of cells with a subdiploid DNA content (as compared with
mock-infected cells) and an insignificant alteration of Hoechst 33258 staining or Annexin V staining. For example, at 6 days after infection,
12% of CLL sample no. 7 cells infected with Ad5dl337 had a
subdiploid DNA content compared with 7% of control or mock-infected
cells. These results imply that some of the cytotoxicity induced by
infection may be a consequence of apoptosis, but the degree of
apoptosis, as measured by the above-described studies, was far less
than anticipated if apoptosis was the sole or major mechanism of
cytotoxicity. Presumably, adenovirus gene expression and virus
production in CLL cells results in cytotoxicity that is only partially
a consequence of apoptosis.
| |
DISCUSSION |
A panel of mutated adenoviruses was used to infect B-cell CLL cells,
and even highly attenuated viruses were capable of inducing cytotoxicity. The degree of cytotoxicity induced by individual viruses
varied among patients with CLL, although the adenovirus with an E1B 19K
deletion, Ad5dl337, had the broadest range and extent of
cytotoxicity. Although, the cells from few CLL patients persisted in
culture long enough to establish the full extent of cytotoxicity under
conditions supporting the continued viability of uninfected or
mock-infected CLL cells, viral-induced cytotoxicty was clearly
demonstrated at early time points for all of the patient samples. These
studies demonstrate that adenoviruses are capable of infection of CLL
cells and that infection with specific attenuated viruses results in
marked cytotoxicity. These results indicate that attenuated
adenoviruses could ultimately have a role in the therapy of CLL.
The variability in cytotoxicity induced by different viruses in
different patient samples emphasizes the need to establish ex vivo
conditions that will allow the CLL cells to persist long enough to
allow a full assessment of cytotoxicity for all patients. The
limited viability of CLL cells ex vivo, with 6 of the 7 initial uninfected CLL samples dying rapidly after day 10, is characteristic of
CLL survival in culture. To further assess cytotoxicity under conditions reported to support primary cell viability in cell culture,
we have undertaken infections during coculture of the CLL cells with
irradiated CDw32 transfected L cells in the presence of IL-4 and
anti-CD40 antibody.21-23 Our results confirm that this coculture system prolongs the viability of primary CLL cells in culture, however, the irradiated transfected L cells were not viable
after infection with Ad5dl309, confounding an analysis of
adenovirus-mediated cytotoxicity in most of the CLL samples (data not
shown). Therefore, our assessment of cytotoxicity is based on early
time points for the majority of the CLL samples and longer time points
for those cells with greater viability in culture. The samples that
persist in culture for prolonged periods of time do demonstrate 100%
cytotoxicty after infection with Ad5dl309, Ad5dl337,
E1A, Pac3, and Ad5ts125 under conditions supporting the full
viability of uninfected or mock-infected CLL cells. Immunophenotypic
analysis has demonstrated that the viable cells in the uninfected
culture were all CLL cells.
The mechanisms contributing to cytotoxcity after infection by wild-type
and mutant adenoviruses are likely to be complex, involving direct
cytotoxicity induced by adenovirus gene products and apoptosis induced
by virus infection. Cytotoxicity after infection of malignant
epithelial cells (with mutations in p53) by viruses lacking E1B 55K may
be a consequence of virus replication, but the precise relationship
between p53 mutation, viral replication, and cytotoxicity is likely to
be complex and highly dependent on other host cell and virus
features.5-7,16 In contrast to the frequent mutation of p53
in epithelial malignancies, p53 mutation is not common in CLL, with
only 6% of standard CLL samples harboring mutations of
p53.8-11 However, a broad spectrum of genetic aberrations have been described and many of these mutations may impact on cell
cycle control or the regulation of apoptosis, and therefore, render the
malignant cell an excellent target for cytotoxicity after infection
with an attenuated adenovirus. For example, E1A function is essential
for productive infection, thus cellular mutations that activate
downstream targets of E1A (for example, mutations or deletions of pRb)
may be predicted to enhance replication of an E1A-adenovirus. E1A
itself has, under certain conditions, been demonstrated to mediate
apoptosis3,15,16,24-28 and infection with viruses
containing E1A in the absence of the antiapoptotoic functions of E1B
enhances apoptosis in many cells. Both the pRb and p300 binding domains
may be important for this induction of apoptosis in certain cells,
whereas pRb binding may be dispensable in other cell
types.16,24-27 Thus, specific E1A mutations may be
predicted to facilitate apoptosis and cytotoxicity in some cell types
and the proapoptotic functions of E1A may be offset by E1B. In
particular, E1B 19K is of critical importance in preventing E1A-mediated apoptosis, and viruses that do not encode E1B 19K may
facilitate cytotoxicity mediated by E1A and other viral genes, perhaps
in conjunction with cellular changes that result in alterations of
p300, pRb, Bcl-2 family members, or other cellular proteins, which
impact on the development of apoptosis.2,3,15,16,24-27 Therefore, viruses with mutations in E1A, E1B, or both may induce variable amounts of cytotoxicity, depending on the specific genotype of
the host.
While the potent cytotoxicity induced by viruses with deletions of E1A + E1B (Pac3) or mutation of E1B 19K (Ad5dl337) in CLL cells
from specific patients may at least partially be mediated by induction
of apoptosis, it has not been possible to measure high levels of
apoptosis during times of maximum cytotoxicity. Low-level virus
production was seen after most infections, even with viruses markedly
attenuated by mutations in E1A and E1B. Therefore, low-level productive
infection appears to be present during most of the infections and
cytotoxicity may be a consequence of a variety of processes, including
both productive infection and apoptosis. Ad5dl337 consistently
induced cytotoxicity in some samples of CLL to a greater extent than
the parental Ad5dl309 (containing wild-type E1A and E1B).
Presumably, the biological functions of E1B 19K inhibit adenovirus
cytotoxicity,2,3,15,16,24 and viruses with a mutation of
E1B 19K may be more cytotoxic than the parental virus in the context of
low-level virus production. The cytotoxicity of the Pac3 virus is also
interesting, considering the loss of both functional E1A and E1B,
making Pac3 as highly attenuated as many of the adenovirus vectors
commonly used for gene transfer. It is possible that other viral gene
products such as the E4 open reading frame 4 (E4orf4), which has been
demonstrated to be cytotoxic,29,30 may play a contributory
role in the induction of cell death in the absence of E1A. Therefore,
the specific cellular genotype and phenotype may complement
adenoviruses with mutations necessary for productive infection,
establish a milieu that fosters adenovirus-mediated apoptosis, or be
cytotoxic as a consequence of other mechanisms, such as toxicity
induced by adenovirus gene products. The cytotoxicity of the Pac3 virus
also demonstrates the capacity of a highly attenuated virus, similar to
those used as vectors for gene transfer in clinical trials of human
gene therapy, to replicate and induce cytotoxicity under certain conditions.
These studies, with CLL cells from multiple patients, emphasize an
advantage to screening for cytotoxicity induced by a spectrum of
adenoviruses. Despite identical morphologic and immunophenotypic features, there is genetic heterogeneity among CLL
samples.6-9 This heterogeneity presumably results in
different patterns of cytotoxicity after infection of cells from
different patients. This heterogeneity also emphasizes the importance
of studying primary malignant cells as opposed to cell lines.
Correlation of the cytotoxicity induced by each of the wild-type and
mutant adenoviruses with the individual clinical, genetic,
immunophenotypic, and cytogenetic features of the malignant cells will
be important, because it may provide important insights into
alterations of specific regulatory pathways in CLL cells. This may also
apply to other lymphoid malignancies . For example, malignant cells from a patient with mantle cell lymphoma demonstrated cytotoxicity after infection with Ad5dl309, but no cytotoxicity was seen
after infection with the Pac3 or E1A-viruses (unpublished
data). Understanding the nuances of the patterns of
cytotoxicity seen after infections with different mutated viruses may
lead to a better understanding of the functional state of the malignant
cell, particularly as it relates to the cell cycle regulatory control
and the proapoptotic/antiapoptotic functions of the adenovirus E1 region.
The potential ex vivo use of these viruses for hematopoietic stem cell
purging has been supported by preliminary studies from our laboratory
that have demonstrated that infection of hematopoietic stem cells with
Ad5dl309 and several of the adenovirus mutants used in this
study does not result in cytotoxicity to committed hematopoietic
progenitors, as measured by the capacity of the hematopoietic stem
cells to form colonies in methylcellulose. Recombinant adenovirus
vector infection of hematopoietic cells has also failed to induce
toxicity, as measured by colony formation and other
assays,31-37 including long-term culture
initiating cell assay.38 In fact, the E1A and Pac3
viruses used in our studies and demonstrated to be cytotoxic to CLL
cells contain the same degree of attenuation as many of the adenovirus
vectors used for gene transfer/therapy.31-38 Therefore,
selectively cytotoxic adenoviruses might ultimately be developed for
use ex vivo to purge hematopoietic stem cell grafts of CLL cells. The
concept of inducing cytotoxicity with Ad5ts125 may also be relevant to
this potential application of adenovirus-mediated cytotoxicity, as
temperature-sensitivity may ultimately be incorporated into the viruses
as an additional safety feature. Additional studies will be necessary
to demonstrate that the adenoviruses used in our studies are not
directly cytotoxic to primitive hematopoietic stem cells.
The studies presented in this report extend prior studies of
adenovirus-mediated cytotoxicity by demonstrating that primary CLL
cells can be infected with adenoviruses and are subject to cytotoxicity
induced by even very highly attenuated mutant viruses. In addition, our
studies demonstrate that there is no cytotoxicity after infection of
normal B cells under identical conditions. These studies have
implications for the biology of CLL and for the use of adenoviruses as
vectors of gene-transfer and/or possible purging in lymphoid
malignancies. Furthermore, the phenotypic pattern of cytotoxicity
induced by different members of the panel of viruses will provide the
framework to correlate cytotoxicity with specific genetic features of
the malignant cells and provide additional information about the
viral-host cell relationships that mediate cell death and productive infection.
| |
FOOTNOTES |
Submitted February 26, 1999; accepted July 6, 1999.
Supported by the Elsa U. Pardee Foundation and National Institutes of
Health Grant No. R01 CA69281.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Roger K. Strair, MD, PhD, The Cancer
Institute of New Jersey, 195 Little Albany St, New Brunswick, NJ 08901;
e-mail: strairrk{at}umdnj.edu.
| |
REFERENCES |
1.
Horwitz MS:
Adenoviridae and their replication, in
Fields BN,
Knipe DM
(eds):
Virology. New York, NY, Raven, 1990, p 1679
2.
White E, Grodzicker T, Stillman B:
Mutations in the gene encoding the adenovirus early region 1B 19,000 molecular weight tumor antigen cause degradation of chromosomal DNA.
J Virol
52:410, 1984[Abstract/Free Full Text]
3.
White E:
Regulation of p53-dependent apoptosis by E1A and E1B.
Curr Top Microbiol Immunol
199:33, 1995
4.
Lucher LR:
Abortive adenovirus infection and host range determinants.
Curr Top Microbiol Immunol
199:119, 1995
5.
Hall AR, Dix BR, O'Carroll SJ, Braithwaite AW:
p53-Dependent cell death/apoptosis is required for a productive adenovirus infection.
Nat Med
4:1068, 1998[Medline]
[Order article via Infotrieve]
6.
Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Munna M, Ng L, Nye JA, Sampson-Johannes A, Fattaey A, McCormick F:
An adenovirus mutant that replicates selectively in P53-deficient human tumor cells.
Science
274:373, 1996[Abstract/Free Full Text]
7.
Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH:
ONYX-015, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and anti-tumor efficacy that can be augmented by standard chemotherapy agents.
Nat Med
3:639, 1997[Medline]
[Order article via Infotrieve]
8.
Hangaishi A, Ogawa S, Imamura N, Miyawaki S, Miura Y, Uike N, Shimazaki C, Emi N, Takeyama K, Hirosawa S, Kamada N, Kobayashi Y, Takemoto Y, Kitani T, Toyoma K, Ohtake S, Yazaki Y, Ueda R, Hirai H:
Inactivation of multiple tumor suppressor genes involved in negative regulation of the cell cycle, MTS1/p16INK4A/CDKN2, MTS2/p15INK4B, p53, and Rb genes in primary lymphoid malignancies.
Blood
87:4949, 1996[Abstract/Free Full Text]
9.
Gaidano G, Newcomb EW, Gong JZ, Tassi V, Neri A, Cortelzzi A, Calori R, Baldini L, Dalla-Favera R:
Analysis of alterations of oncogenes and tumor suppressor genes in chronic lymphocytic leukemia.
Am J Pathol
144:1334, 1994[Abstract]
10.
Catovsky D:
The search for genetic clues in chronic lymphocytic leukemia, in
Dighiero G,
Binet JL
(eds):
7th International Workshop on Chronic Lymphocytic Leukemia. Hematol Cell Ther 39:S5, 1997
11.
Kipps T:
Signal transduction pathways and mechanisms of apoptosis in CLL B-lymphocytes: Their role in CLL pathogenesis, in
Dighiero G,
Binet JL
(eds):
7th International Workshop on Chronic Lymphocytic Leukemia. Hematol Cell Ther 39:S17, 1997
12.
Silver L, Anderson CW:
Interaction of human adenovirus sertype 2 with human lymphoid cells.
Virology
165:377, 1988[Medline]
[Order article via Infotrieve]
13.
Horvath J, Weber JM:
Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection.
J Virol
62:341, 1988[Abstract/Free Full Text]
14.
Jones N, Shenk T:
An adenovirus type 5 early gene function regulates expression of other viral genes.
Proc Natl Acad Sci USA
76:3665, 1979[Abstract/Free Full Text]
15.
White E, Cipriani R, Sabbatini P, Denton A:
Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.
J Virol
65:2968, 1991[Abstract/Free Full Text]
16.
Chiou SK, White E:
p300 Binding by E1A cosegregates with p53 induction but is dispensable for apoptosis.
J Virol
71:3515, 1997[Abstract]
17.
Klessig DF, Brough DE, Cleghon V:
Introduction, stable integration, and controlled expression of a chimeric adenovirus gene whose product is toxic to the recipient human cell.
Mol Cell Biol
4:1354, 1984[Abstract/Free Full Text]
18.
Hsiung GD:
Hsiung's Diagnostic Virology: As Illustrated by Light and Electron Microscopy. New Haven, CT, Yale, 1994
19.
Pilder S, Logan J, Shenk T:
Deletion of the gene encoding the adenovirus 5 early region 1B 21,000-molecular weight polypeptide leads to degradation of viral and host cell DNA.
J Virol
52:664, 1984[Abstract/Free Full Text]
20.
Debbas M, White E:
Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B.
Genes Dev
7:546, 1993[Abstract/Free Full Text]
21.
Banchereau J, De Paoli P, Valle A, Garcia E, Rousset F:
Long term human B-cell lines dependent upon interleukin-4 and anti-CD40.
Science
251:70, 1991[Abstract/Free Full Text]
22.
Johnson PWM, Watt SM, Betts DR, Davies D, Jordan S, Nortyon AJ, Lister TA:
Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40-stromal cell system.
Blood
82:1848, 1993[Abstract/Free Full Text]
23.
Buske C, Twiling A, Gogowski G, Schreiber K, Feuring-Buske M, Wulf GG, Hiddemann W, Wormann B:
In vitro activation of low grade non-Hodgkin's lymphoma by murine fibroblasts, IL-4, anti-CD40 antibodies and the soluble CD40 ligand.
Leukemia
11:1862, 1997[Medline]
[Order article via Infotrieve]
24.
Samuelson AV, Lowe SW:
Selective induction of p53 and chemosensitivity in RB-deficients cells by E1A mutants unable to bind the RB-related proteins.
Proc Natl Acad Sci USA
94:12094, 1997[Abstract/Free Full Text]
25.
de Stanchina E, McCurrach ME, Zindy F, Shieh SY, Ferbeyre G, Samuelson AV, Prives C, Roussel MF, Sherr CJ, Lowe SW:
E1A signalling to p53 involves the p19ARF tumor suppressor.
Genes Dev
12:2434, 1998[Abstract/Free Full Text]
26.
Querido E, Teodoro JG, Branton PE:
Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the induction of DNA synthesis.
J Virol
71:3526, 1997[Abstract]
27.
Thomas A, White E:
Suppression of the p300-dependent mdm2 negative-feedback loop induces the p53 apoptotic function.
Genes Dev
12:1975, 1998[Abstract/Free Full Text]
28.
Lowe S, Ruley HE:
Stabilization of the p53 tumor suppressor gene is induced by adenovirus e1a and accompanies apoptosis.
Genes Dev
7:535, 1993[Abstract/Free Full Text]
29.
Marcellus RC, Lavoie JN, Boivin D, Shore GC, Ketner G, Branton PE:
The early region 4 orf4 of human adenovirus type 5 induces p53-independent cell death by apoptosis.
J Virol
72:7144, 1998[Abstract/Free Full Text]
30.
Shtrichman R, Kleinberger T:
Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells.
J Virol
72:2975, 1998[Abstract/Free Full Text]
31.
Watanabe T, Kuszynski C, Ino K, Heimann DG, Shepard HM, Yasui Y, Maneval DC, Talmadge JE:
Gene transfer into human bone marrow hematopoietic cells mediatd by adenovirus vectors.
Blood
87:5032, 1996[Abstract/Free Full Text]
32.
Seth P, Katayose D, Li Z, Kim M, Wersto R, Craig C, Shanmugam N, Ohri E, Mudahar B, Rakkar AN, Kodali P, Cowan K:
A recombinant adenovirus expressing wild type p53 induces apoptosis in drug-resistant human breast cancer cells: A gene therapy approach for drug resistant cancers.
Cancer Gene Ther
4:383, 1997[Medline]
[Order article via Infotrieve]
33.
Chen L, Pulsipher M, Chen D, Sieff C, Elias A, Fine HA, Kufe DW:
Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources.
J Clin Invest
98:2539, 1996[Medline]
[Order article via Infotrieve]
34.
Neering SJ, Hardy SF, Minamoto D, Spratt SK, Jordan CT:
Transduction of primitive human hematopoietic cells with recombinant adenoviruses.
Blood
88:1147, 1996[Abstract/Free Full Text]
35.
Teoh G, Chen L, Urashima M, Tai Y-T, Celi LA, Chen D, Chauhan D, Ogata A, Finberg RW, Webb IJ, Kufe DW, Anderson KC:
Adenovirus-vector based purging of multiple myeloma cells.
Blood
92:4591, 1998[Abstract/Free Full Text]
36.
Garcia-Sanchez F, Pizzorno G, Fu SQ, Nanakorn T, Krause DS, Liang J, Adams E, Leffert JJ, Yin LH, Cooperberg MR, Hanania E, Wang WL, Won JH, Peng XY, Cote R, Brown R, Burtness B, Giles R, Crystal R, Deisseroth AB:
Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: A potential purging method for autologous transplantation.
Blood
92:672, 1998[Abstract/Free Full Text]
37.
Frey BM, Hackett NR, Bergelson JM, Finberg R, Crystal RG, Moore MAS, Rafii S:
High efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectors.
Blood
91:2781, 1998[Abstract/Free Full Text]
38.
Takahashi T, Yamada K, Tanaka T, Kumano K, Kurokawa M, Takahashi T, Hirano N, Honda H, Chiba S, Tsuji K, Yazaki Y, Nakahata T, Hirai H:
A potential molecular approach to ex vivo hematopoietic expansion with a recombinant epidermal growth factor receptor-expressing adenovirus vector.
Blood
91:4509, 1998[Abstract/Free Full Text]
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