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Molecular and Cellular Biology, January 2000, p. 628-633, Vol. 20, No. 2
0270-7306/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
p53 Deficiency Increases Transformation by v-Abl
and Rescues the Ability of a C-Terminally Truncated v-Abl Mutant To
Induce Pre-B Lymphoma In Vivo
Xiaoming
Zou,1
Feng
Cong,1
Margaret
Coutts,2
Giorgio
Cattoretti,3
Stephen P.
Goff,1,2,4 and
Kathryn
Calame1,2,*
Departments of Biochemistry and Molecular
Biophysics,1
Microbiology,2 and
Pathology3 and Howard Hughes
Medical Institute,4 Columbia University College
of Physicians and Surgeons, New York, New York 10032
Received 6 April 1999/Returned for modification 28 April
1999/Accepted 15 October 1999
 |
ABSTRACT |
Abelson murine leukemia virus (A-MuLV) is an acute transforming
retrovirus that preferentially transforms early B-lineage cells both in
vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine
kinase related to v-Src but containing an extended C-terminal domain.
Many mutations affecting the C-terminal portion of the molecule block
the pre-B-transforming activity of v-Abl without affecting the
fibroblast-transforming ability. In this study we have determined the
abilities of both wild-type and C-terminally truncated (p90) forms of
v-Abl to transform cells from p53
/ mice. Lack of p53
increases the susceptibility of bone marrow cells to transformation by
v-Abl by a factor of more than 7 but does not alter v-Abl's preference
for B220+ IgM pre-B cells. p53-deficient mice
have earlier tumor onset, more rapid tumor progression, and decreased
survival time following A-MuLV infection, but all of the tumors are
pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl
transformation but play no role in conferring preferential
transformation of pre-B cells. Surprisingly, the C-terminally truncated
form of v-Abl (p90) transforms pre-B cells very efficiently in mice
lacking p53, thus demonstrating that the C terminus of v-Abl does not
determine preB tropism but is necessary to overcome p53-dependent
inhibition of transformation.
| |
INTRODUCTION |
Abelson murine leukemia virus
(A-MuLV) is an acutely transforming retrovirus which causes pro-B or
pre-B tumors in infected animals (9). In vitro, A-MuLV
transforms early B cells, cells from other hematopoietic lineages, and
a permissive subset of 3T3 fibroblasts (the P-3T3 subline)
(34). p160 v-Abl, the oncoprotein encoded by A-MuLV,
exhibits constitutive protein tyrosine kinase activity (35).
A C-terminally truncated mutant of p160, p90 v-Abl, is defective for
early B-cell transformation but retains the ability to transform P-3T3
cells (26, 35). The phenotype of this virus led to the
suggestion that the C terminus of v-Abl is important for the pre-B-cell
preference of A-MuLV.
Despite the presence of a strongly transforming oncogene,
A-MuLV-induced lymphomas are usually clonal or oligoclonal, suggesting that other events in addition to expression of v-Abl are required for
tumor formation (12). Furthermore, in some cells where v-Abl does not cause transformation, it has been shown to cause growth arrest
or apoptosis (31, 47). These facts suggest that tumor suppressors, such as p53, might play an important role in determining whether v-Abl expression leads to transformation or apoptosis and might
be important for the striking early B-cell preference shown by A-MuLV
in vivo.
p53 is a potent tumor suppressor which is mutated in more than half of
all human tumors (21). Mice with p53 gene deletions develop
normally but are highly prone to tumor development (8, 17).
Indeed, p53 is not required for normal cell growth but acts to prevent
proliferation under circumstances of cellular stress. Hence, the
normally low levels of p53 increase following DNA damage, certain
oncogenic insults, hypoxia, and a variety of other cellular stresses
(19). Activation of p53 prevents cell proliferation by
inducing either cell cycle arrest or apoptosis. p53 is a
sequence-specific DNA binding protein that activates transcription of
genes involved in cell cycle arrest and in apoptosis, like the
cyclin-dependent kinase inhibitor p21 (19).
Recent data from other laboratories provide evidence that p53 may play
a role in v-Abl transformation. More than 40% of early B-cell lines
resulting from A-MuLV transformation of bone marrow cells (BMCs) in
vitro develop p53 mutations (43). The susceptibility of the
permissive subline of 3T3 cells to transformation by A-MuLV correlates
with a failure to induce a p53 response to DNA damage in these cells
(16). These considerations led us to study the ability of
wild-type and mutant forms of v-Abl to transform bone marrow cells from
p53/ mice in vivo and in vitro. The results show that
BMCs lacking p53 are transformed more efficiently by v-Abl and that the
latency period for v-Abl-dependent tumor formation in vivo is reduced in mice lacking p53. Our data also show that neither the status of the
p53 gene nor the C-terminal portion of v-Abl is responsible for the
early B-cell tropism of A-MuLV.
| |
MATERIALS AND METHODS |
Cells and mice.
A-MuLV-transformed bone marrow cells and
tumor cells derived from A-MuLV-infected mice were maintained in RPMI
1640 medium supplemented to contain 10% heat-inactivated fetal calf
serum and 50 µM 2-mercaptoethanol. Tumorigenicity studies were
conducted with p53 mutant mice. The p53 mutant mice were obtained from
The Jackson Laboratory; they are derived from 129/SV ES cells and in a
129/SV × C57BL/6 mixed genetic background (17).
A-MuLV preparation and bone marrow transformation assay.
P160 and P90A A-MuLV virus stocks were prepared by rescuing virus from
transformed nonproducer NIH 3T3 cells with Moloney MuLV (M-MuLV) helper
virus (10, 35). The titers of the transforming virus in the
stocks were determined after infection of NIH 3T3 cells
(36). Primary BMCs were recovered from the femurs of 4- to
6-week-old p53 mice. The bone marrow transformation assay was performed
as described elsewhere (15, 33).
In vivo tumorigenicity study.
Offspring from heterozygote
crosses were obtained so that wild-type (p53+/+),
heterozygote (p53+/), and null (p53/)
littermates could be compared. The mice were genotyped by PCR 3 weeks
after birth. Neonatal mice (48 h or less postpartum) were injected
intraperitoneally with 5 × 104 focus-forming units
(FFU) of A-MuLV-P160 or A-MuLV-P90A. Infected mice were monitored
regularly for the development of symptoms of Abelson disease. Afflicted
mice were sacrificed by CO2 asphyxiation when the disease
became near terminal. Diagnosis of disease was based on gross
pathological examination, histochemical staining, and
fluorescence-activated cell sorting (FACS) analyses.
FACS analysis.
Preparation, staining, and analysis of cells
were performed as described previously (18). All antibodies
used were obtained from Pharmingen Inc. (San Diego, Calif.). The FACS
analysis was performed on cytometers from Becton Dickinson. The results
were analyzed by using CellQuest software (Becton Dickinson).
Western and Southern blotting.
Whole-cell extracts were
prepared from primary or cultured cells as described elsewhere
(46); equal amounts of cellular proteins were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
v-Abl proteins were detected by using rabbit anti-Abl antibody (C-19;
Santa Cruz Biotechnology) with the standard protocol.
Genomic DNA was extracted from primary tumor cells, digested to
completion with restriction enzymes (New England Biolab), and
electrophoresed in a 0.8% agarose gel. The DNA was denatured, transferred to nitrocellulose, and analyzed by the standard method of
Southern blotting (42). The probe used for detection of
A-MuLV proviral genomes was derived from the p160 v-Abl coding sequence and consisted of the 715-bp PvuII fragment.
| |
RESULTS |
p53-deficient mice have earlier onset, more rapid tumor
progression, and decreased survival time following infection with
A-MuLV-P160.
To evaluate whether the development of Abelson
disease was affected by the absence of p53, mating pairs of mice
heterozygous for the p53 null mutation were crossed to generate
p53+/+, p53+/, and p53/
littermates. Neonatal mice derived from these crosses were injected intraperitoneally with 5 × 104 FFU of A-MuLV within
48 h of birth. Infected mice were monitored regularly for the
appearance of disease symptoms. Animals were killed and autopsied when
signs of disease were evident. Mortality curves (Fig.
1A) illustrate that wild-type mice had
the longest survival time, with the median tumor latent period being 49 days; p53 heterozygotes had a shorter survival time, with the median tumor latent period being 40.5 days. In comparison, p53 null mice had
the shortest survival time, the median tumor latent period being only
31 days and the tumor incidence reaching 100% by 40 days. The disease
seemed to progress more rapidly in p53/ mice. The
infected p53/ mice, if not sacrificed, usually died 1 or 2 days after symptoms were observed, compared to several days and up
to a week for wild-type mice. These data show that lack of p53
increases susceptibility of mice to transformation by v-Abl.
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FIG. 1.
Influence of p53 phenotype on the survival of mice
infected with A-MuLV-P160 or A-MuLV-P90A. (A) Neonatal mice were
injected intraperitoneally with approximately 5 × 104
FFU of A-MuLV-P160. Infected mice were monitored daily for the
appearance of disease symptoms. When disease became terminal, mice were
sacrificed. The plot shows the probability of survival as a function of
time (days) after infection. A total of 27 wild-type, 47 heterozygous,
and 24 p53-deficient mice are represented. (B) Neonatal mice were
injected intraperitoneally with approximately 5 × 104
FFU of A-MuLV-P90A. The infected mice were analyzed as for panel A. The
plot shows the probability of survival as a function of time (days)
after infection. A total of 16 wild-type, 21 heterozygous, and 12 p53-deficient mice are represented. (C) Schematic diagram of wild-type
p160 v-Abl and C-terminally truncated mutant p90 v-Abl proteins. GAG,
retroviral Gag domain; SH2, Src homology domain 2; SH1, tyrosine kinase
domain; DB, DNA binding domain; AB, actin binding domain.
|
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The p53
+/ mice showed an intermediate phenotype with
respect to tumor formation. However, upon further analysis using a PCR
assay, we found that two of four tumors from p53
+/ mice
had lost the remaining wild-type allele. Using a functional
assay based
on induction of p21 in response to gamma irradiation
as described
previously (
43), we found that one of two tumors
which
retained a wild-type allele had lost p53 function as measured
by
induction of p21. Thus, three of four tumors from the
p53
+/ animals were functionally p53
/. A
similar observation was made recently by Unnikrishnan et al.
(
44).
The target cell of A-MuLV-P160 is the same in wild-type and
p53-deficient mice.
In most cases, the gross pathology of the
disease was typical of A-MuLV lymphoma, regardless of p53 genotype.
Histochemical analyses of infected mice showed high-grade lymphoblastic
lymphomas that were morphologically and histologically
undistinguishable in p53/ and p53+/+ mice
(data not shown).
Tumor cells from most of the p53-deficient mice and from representative
wild-type and heterozygous mice were analyzed by FACS
to determine
their lineages. Freshly isolated or briefly cultured
tumor cells were
stained with antibodies specific for a range
of surface markers.
Although A-MuLV-P160-infected p53
/ and
p53
+/ mice have earlier disease onset, more rapid disease
progression,
and shorter survival time, the surface markers on tumors
cells
derived from these mice were the same as those from infected
wild-type
mice. Surface marker analyses of tumor cells from
p53
/ mice showed an early B-cell phenotype:
B220
+ IgM
CD43
low (B-cell
markers), CD4
CD8
CD3
(T-cell
markers), and Mac-1
GR-1
(myeloid
lineage markers). Thus, while p53 deficiency increases
the
susceptibility to transformation by v-Abl in vivo, there is
no change
in the early B-cell tropism of A-MuLV. The results suggest
that while
p53-dependent mechanisms inhibit transformation by
A-MuLV, they are not
responsible for determining the pre-B cell
preference of the
virus.
Loss of p53 complements the A-MuLV-P90A mutant for transformation
in vivo.
A-MuLV-P90A lacks most of the C-terminal portion of v-Abl
(Fig. 1C). It transforms early B cells poorly both in vitro and in vivo
but retains the ability to transform P-3T3 cells efficiently. We wished
to test how p53 affected the transforming ability and lineage
preference of this mutant.
Neonatal mice derived from the crosses of p53 heterozygotes were
injected intraperitoneally with 5 × 10
4 FFU of the
A-MuLV-P90A within 48 h of birth. Infected mice were
analyzed as
described previously for A-MuLV-P160-infected mice.
A-MuLV-P90A is
indeed defective for tumor induction in wild-type
mice. All of the
wild-type mice injected with A-MuLV-p160 developed
disease within 80 days (Fig.
1A), whereas only 38% (6 of 16) of
the p53
+/+
mice injected with A-MuLV-P90A developed disease during the 120-day
observation period (Fig.
1B). However, all p53
/ mice
injected with A-MuLV-P90A developed disease within 60 days.
The median
tumor latent period for p53
/ mice upon A-MuLV-P90A
infection was 37.5 days (Fig.
1B), close
to the mean time of 31 days
for the p160 strain in these mice
(Fig.
1A). Heterozygous mice also
developed disease with A-MuLV-P90A:
86% (18 of 21) of the infected
mice developed disease during the
120-day observation period, with a
mean latent period of 67 days
(Fig.
1B). These results suggest that the
C-terminal region is
required for efficient tumor induction in
wild-type mice but dispensable
in p53-deficient
mice.
We examined v-Abl expressed in tumors raised in p53-wild-type and
-deficient mice injected with A-MuLV-P90A to determine whether
animal
passage resulted in recombination to restore the C terminus
to p90
v-Abl. In previous studies, pre-B lymphomas formed from
A-MuLV-P90 were
shown to be revertants to larger forms of v-Abl
or to have undergone
further deletion (
26). Western blots showed
that the v-Abl
proteins in A-MuLV-P90A-induced tumors from p53
/ mice
were all 90 kDa (Fig.
2A). In contrast,
as previously observed
(
26), p90 tumors from
p53
+/+ mice contained either smaller or larger forms of
v-Abl (Fig.
2B). Two of them contained v-Abl protein of approximately
120
kDa; others contained v-Abl protein in the 70- to 80-kDa range.
Some tumors contained both 90-kDa and smaller or larger forms
of v-Abl;
we assume that this reflects tumor oligoclonality. Thus,
absence of p53
increases transformation efficiency, decreases
latency, and abrogates
recombination of A-MuLV-P90A.
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FIG. 2.
v-Abl expression in tumors raised in p53-wild-type and
-deficient mice injected with A-MuLV-P90A and clonality analyses of
v-Abl-induced tumors in p53/ mice. (A) Whole-cell
extracts were prepared from tumor cells and control cells. Ten
micrograms of each sample was resolved by SDS-PAGE on a 6% gel and
subjected to Western blot analysis using antibody against Abl protein.
Lanes: 1, A-MuLV-P160-induced tumor cells; 2, 1881 cells, which express
p120v-abl; 3, NIH 3T3 cells (negative control);
4 to 9, six different tumors induced by A-MuLV-P90A in p53-deficient
mice. (B) Samples were prepared and analyzed as for panel A. Lanes: 1, tumor induced by A-MuLV-P90A in p53/ mice; 2 to 6, five
different tumors induced by A-MuLV-P90A in p53+/+ mice. (C)
Genomic DNA was prepared from primary A-MuLV-induced tumor tissue and
subjected to Southern blot analysis. DNA (20 µg) was digested with
EcoRI, electrophoresed through 0.8% agarose gel, blotted to
nitrocellulose paper, and hybridized to the 32P-labeled
abl-specific probe. Lanes: 1, A-MuLV-P160-induced tumor in
p53/ mouse; 2 to 4, A-MuLV-P90A-induced
p53/ tumors from different individual mice. An arrow
indicates the 27-kb c-abl-specific band present in all
cells.
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|
Tumors induced by wild-type A-MuLV originate from one or a few
transformed cells and thus are monoclonal or oligoclonal (
12,
13). We examined the clonality of tumors induced by A-MuLV-P90A
in p53-deficient mice. Southern analysis of DNA from primary
A-MuLV-P90A-induced
tumor tissue probed with an
abl-specific
probe demonstrated that
these tumors are also monoclonal or oligoclonal
in origin (Fig.
2C). This finding suggests that genetic events in
addition to
expression of p90 v-Abl are necessary for tumor formation
in p53
/ mice.
A-MuLV-P90A targets the pre-B-cell in p53/
mice.
At autopsy, the gross pathology of the diseased
p53/ animals infected by A-MuLV-P90A was the same as
that for mice infected with A-MuLV-P160 and typical for Abelson
disease. When analyzed by FACS, the tumors showed an early B-cell
phenotype: B220+ IgM CD43low
CD3 CD4 CD8
Mac-1 or Mac-1low GR-1.
Mac-1low expression was observed in four of nine cases of
A-MuLV-P90A-induced tumors in p53/ mice; its
significance is unclear at this time. A PCR-based assay for DNA
rearrangement showed that these tumor cells all completed DH to JH rearrangement, suggesting they are
B-lineage cells (data not shown). Histochemical staining also confirmed
that A-MuLV-P90A induced pre-B lymphomas in p53-deficient mice (data
not shown). The results demonstrate that the C-terminal region of v-Abl
is not responsible for the pre-B-cell tropism of A-MuLV, since the C-terminal deletion mutant virus, A-MuLV-P90A, still exclusively transforms pre-B cells in p53/ mice.
Loss of p53 increases the efficiency of A-MuLV-dependent
transformation in vitro.
To determine whether the absence of an
intact p53 gene affects v-Abl-induced lymphoid transformation in vitro,
BMCs from p53/ mice and p53+/+ littermate
controls were infected with A-MuLV-P160 or A-MuLV-P90A and then
cultured in soft agar. Transformed colonies were counted after 14 days.
A-MuLV-P160 induced colony formation in both p53+/+ and
p53/ BMCs, but it induced sevenfold more colonies in
p53/ BMCs (Table 1). In
comparison, A-MuLV-P90A did not induce colony formation in either
p53+/+ or p53/ BMCs (Table 1), even though
A-MuLV-P90A could induce pre-B lymphomas in p53/ mice
with good efficiency. A similar discrepancy between in vivo and in
vitro transformation was observed previously with some smaller
variants of A-MuLV-P90, which are highly oncogenic in vivo but do not
transform BMCs in vitro at high efficiency (26).
Representative colonies from A-MuLV-P160 infections were expanded in
liquid culture and analyzed by FACS to determine the
lineage and
differentiation stage of the transformed cells. The
results showed no
phenotypic differences between lines derived
from p53
+/+
and p53
/ BMCs. The four p53
+/+ and twelve
p53
/ lines were B220
+ IgM
CD43
low and negative for other lineage markers. Thus, lack
of p53 increases
the susceptibility of bone marrow cells to
transformation by wild-type
v-Abl, but it does not alter A-MuLV's
preference for B220
+ IgM
pre-B cells as
targets in
vitro.
| |
DISCUSSION |
In this study we have determined the ability of A-MuLV-P160 and
A-MuLV-P90A to transform BMCs from p53/ mice both in
vitro and in vivo. Our data lead to three important conclusions: (i)
p53-dependent mechanisms inhibit v-Abl transformation of early B cells;
(ii) the C-terminal region of v-Abl, which is missing from p90, is not
required for the early B-cell tropism of A-MuLV; and (iii) the
C-terminal region of v-Abl is required to counteract the inhibitory
effect of p53 for transformation in vivo.
v-Abl and p53-dependent inhibition of transformation.
A-MuLV
transformation of BMCs in vitro was more than sevenfold higher in
p53/ cells. In vivo onset of tumor formation and tumor
progression increased and survival time decreased following A-MuLV
infection in p53/ animals (Table 1 and Fig. 1). These
data show that a p53-dependent step(s) inhibits
v-abl-dependent transformation in normal cells. A similar
result has been reported for the related human oncogene bcr-abl (41). The data are also consistent with
previous reports that p53/ cells are more susceptible
to transformation by other oncogenes, including those activated by
M-MuLV insertion (2), Wnt-1 (7), E1A
(25), and oncogenic Ras (37). In addition, our
data are consistent with the finding that more than 40% of pre-B-cell
lines transformed in vitro with v-Abl develop mutations in p53
(43) and with the recent report that p53 is required for
apoptotic crisis during transformation of primary pre-B cells by A-MuLV (44).
Although v-Abl sends multiple mitogenic signals (
49), it
also appears to activate p53-dependent paths which lead to senescence
or apoptosis. If this is so, it would provide an explanation for
the
p53-dependent inhibition that we have observed. Other activated
oncogenes, including
ras,
mek, and E1A, which
send strong mitogenic
signals, have recently been shown to activate p53
by a p19
ARF-dependent pathway (
6,
22,
27,
38). In these cases, whether
cells become transformed in response
to the oncogene depends in
part on how fully the
p19
ARF-p53 pathway is functioning (
6,
22,
24,
37). Previous
studies suggest that like Ras, Raf, and E1A,
v-Abl may activate
a p53-dependent growth arrest or apoptotic pathway
as well as
mitogenic pathways. For example, v-Abl was shown to have a
"lethal"
effect on BALB/c 3T3 cells (
47), and primary
mouse fibroblasts
are not transformed by the virus (
32).
Indeed, we have recently
found that v-Abl infection causes cell cycle
arrest in primary
embryonic fibroblasts (F. Cong, et al., submitted for
publication).
Furthermore, those variants of NIH 3T3 cells which are
susceptible
to v-Abl transformation have a deletion in the
INK4a locus and
a defect in their p53-dependent response to
DNA damage, suggesting
that defects in p19
ARF-
and/or p53-dependent pathways may make these cells susceptible
to v-Abl
transformation (
16,
23,
29). Also, a recent report
(
30) shows that absence of
INK4a locus products
(probably p19
ARF) increases v-Abl transformation
efficiency, providing a direct
link between
p19
ARF and p53 in A-MuLv transformation.
Finally, we have shown that
v-Abl causes induction of E2F-dependent
genes, including c-
myc,
and that this requires Ras and Raf
activation (
46,
50; M.
Coutts et al., submitted for
publication). Others have shown recently
that activated Ras
(
27) or overexpressed Myc (
48) can induce
p19
ARF and that p19
ARF
can be induced by E2F activators (
1). It seems likely,
therefore,
that when v-Abl activates E2F-dependent genes by a
Ras/Raf-dependent
pathway, conflicting signals may result: (i)
mitogenic signals
that induce c-
myc and S-phase genes and
(ii) growth-inhibitory
signals that induce
p19
ARF.
Other mechanisms could also be responsible for the increased
susceptibility of p53
/ cells to v-Abl-dependent
transformation. p53
/ mice have a higher percentage of
pre-B cells (B220
+ IgM
) in their bone marrow
compared to p53
+/+ mice, thus presenting more targets for
v-Abl (
40). However,
the percentage increase, which is less
than twofold (
40), cannot
fully explain the sevenfold
increase in transformation of p53
/ bone marrow cells in
vitro (Table
1). Another possibility is
that further mutational events
are required for v-Abl-dependent
transformation, and these mutations
may be more likely to occur
in p53
/ cells since they
are impaired for cell cycle regulation and apoptosis,
which normally
allow DNA repair or cell removal in response to
DNA damage
(
11). These mechanisms are not mutually exclusive
with each
other or with the mechanism discussed above of a v-Abl-dependent
activation of p53 via p19
ARF. We suspect that
multiple p53-dependent mechanisms may combine
to give the increased
susceptibility to v-Abl transformation which
we have
observed.
The ability of p90v-Abl to transform p53
/ early B cells
in vivo but not in vitro is intriguing because most previously studied
mutants of v-Abl have shown similar transformation efficiency
for pre-B
cells in vivo and in vitro. One explanation for the
difference between
in vitro and in vivo transformation is the
possibility that in vivo
transformation allows more time for additional
genetic events to occur
and that the particular functions missing
in the p90 mutant are
unusually dependent on these changes. We
also noted that Southern
analysis of one p53
/ pre-B cell tumor formed by p160
revealed that the tumor was clonal,
consistent with the idea that
genetic changes, in addition to
inactivation of p53-dependent
apoptosis, are required for v-Abl
transformation.
Early B-cell tropism of A-MuLV and role of the C-terminal region of
v-Abl.
One of the fascinating paradoxes of v-Abl biology is that
although A-MuLV (pseudotyped by M-MuLV) can bind to and infect most cell types, tumors that develop from mice infected with A-MuLV are
almost exclusively pro- or pre-B-cell lymphomas (34). We originally suspected that since early B cells undergo a specific DNA
rearrangment of their immunoglobulin genes, p53 regulation might be
different in these cells and that differential regulation of p53 might
provide an explanation for the pre-B-cell tropism of A-MuLV. However,
our results clearly show that p53 is not required for the pre-B-cell
tropism of A-MuLV. Both in vitro and in vivo, A-MuLV retained its
pre-B-cell tropism in p53/ cells.
The C terminus of v-Abl is unique among nonreceptor tyrosine kinases;
it contains a nuclear localization signal, a proline-rich
region, a DNA
binding domain, and an actin binding domain (
20,
45). This
C-terminal region is also responsible for v-Abl's
association with
Abi-1/2 and Jak1/3 (
3,
4,
39). The observation
that
A-MuLV-P90 is severely defective for pre-B-cell transformation
both in
vivo and in vitro but retains the ability to transform
P-3T3 cells with
high efficiency (
26,
35) led to the widely
accepted view
that the C-terminal portion of v-Abl determined
pre-B-cell tropism.
However, there are data in the literature
that are not consistent with
this view. For example, both naturally
arising mutants and revertants
(
26,
28) and genetically engineered
mutants of v-Abl
(
14) which lack the C-terminal region but retain
B-cell
tropism have been described. In addition, studies using
viruses in
which chimeric oncogenes were engineered so that portions
of
v-
src were used to replace v-
abl showed that a
virus containing
the v-Src SH2 domain, and the v-Abl protein tyrosine
kinase domain
and lacking the v-Abl C-terminal regions retained B-cell
tropism,
suggesting that the protein tyrosine kinase domain, not the C
terminus, may confer B-cell tropism (
15). Our data clearly
show
that the C-terminal portion of v-Abl, which is missing in the
P90
virus, is not involved in B-cell tropism since tropism is
strictly
maintained upon infection of p53
/ mice with
A-MuLV-P90A. Thus, we conclude that the C terminus
does not confer
B-cell tropism although it appears to be important
for efficient
responses in pre-B cells. We suggest that future
studies aimed at
understanding B-cell tropism should focus on
the protein tyrosine
kinase
domain.
Although our data have ruled out a role for the C-terminal region of
v-Abl in B-cell tropism, they have identified an alternate
but
important role for the C terminus. A-MuLV-P90A is very defective
for
transformation in vivo in normal animals but regains the phenotype
of
an acutely transforming virus in p53
/ mice (Fig.
1B).
These data indicate that the C-terminal region
of v-Abl is important
for sending mitogenic signals which counteract
p53-dependent growth
arrest or apoptotic signals. This activity
of v-Abl may be required for
transformation of primary cells,
where p53-dependent apoptotic pathways
are intact, but not of
immortalized cells such as 3T3
cells.
It is clear that v-Abl activates multiple mitogenic signaling pathways.
Some of these, such as the E2F-Myc path, depend on
the SH2 and protein
tyrosine kinase portion of the protein (
46);
however, others
depend on the C-terminal portion and could include
activation of
Jak/STAT (
5) or other pathways. It seems likely
that
A-MuLV-P90A cannot send appropriate mitogenic signals to
overcome
p53-dependent growth arrest signals, and thus it is poorly
transforming. However, when the p53 pathway is defective, mitogenic
signals from the SH2 and tyrosine kinase domains are sufficient
to
cause transformation in vivo. It will be important to identify
the
C-terminal domains of v-Abl which are involved in sending
mitogenic
signals.
| |
ACKNOWLEDGMENTS |
We are grateful to members of the Calame and Goff laboratories
for helpful discussions and to Cristina Angelin-Duclos for critically
reading the manuscript. We thank Sharon Boast and Yuming Xu for expert
technical assistance.
This work was supported by grant PO1 CA75339 to both S.P.G. and
K.C. S.P.G. is an investigator of the Howard Hughes Medical Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Columbia University, 701 West 168th St., HHSC 1202, New York, NY 10032. Phone: (212) 305-3504. Fax: (212) 305-1468. E-mail: klc1{at}columbia.edu.
| |
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
