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Molecular Biology and Genetics |
Department of Pathology, University of Iowa College of Medicine, Iowa City, Iowa 52241 [C. M. K., G. M. J., Y. L.], and Department of Pathology and Medicine, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [S. J. K.]
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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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Studies of human malignancies have also implicated BAX as an important tumor suppressor. For example, in colorectal cancer (11) , gastric carcinoma (12) , and acute lymphoblastic leukemia, frameshift mutations in BAX are found frequently in tumors with the microsatellite mutator phenotype. Mutations in BAX have been described in a number of human hematopoietic tumor cell lines (13) as well as directly from gastrointestinal cancer (14) . Retrospective studies of human malignancies have examined the relationship between BAX expression by immunohistochemistry or immunoblotting and clinical outcome. Reduced expression of BAX is associated with poor clinical outcome in ovarian cancer (15) , metastatic breast adenocarcinoma (16) , and squamous cell carcinoma (17) . In contrast, increased Bax expression correlated with a high rate of relapse in childhood acute lymphoblastic leukemia (18) .
Thus, a number of lines of evidence in both mice and humans suggest BAX is a tumor suppressor because of its ability to promote apoptosis. However, these studies have limitations. To date, studies done in mice have used potent viral oncogenes to induce oncogenesis. The relevance of these models to spontaneously or naturally occurring tumors is unknown. Similarly, the studies in humans are correlative, retrospective studies that do not prospectively establish a causative role. In this study we asked whether the levels of Bax expression could directly influence tumor development in mice in the absence of viral oncogenes. We find that Bax-deficiency alone or in combination with p53-deficiency do not predispose to malignancy. In contrast, we also find that overexpression of BAX accelerates oncogenesis in a p53-deficient background. Before malignant transformation, BAX overexpression increases apoptosis while it results in an increased percentage of cycling cells. These findings extend previous observations that Bcl-2 family members regulate both apoptosis and cell division and indicate that the interplay of these activities affects malignant transformation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For developmental studies, Lckpr-Bax line 38 transgenic males were mated overnight to C57BL/6 females. The day of plug identification is defined as day 0.5. At the indicated time, the pregnant females were sacrificed and the embryos dissected. Each of the thymi were removed with the aid of a dissecting microscope and examined to ensure that both lobes could be identified. Thymi were placed in individual wells of a 24-well plate. Thymocytes were isolated by first shearing the gland with two needles and then passing the cells through a 22-gauge needle multiple times. The cells were centrifuged, and in some experiments, samples were treated with hypotonic lysis buffer [0.83% NH4Cl and 10 mM Tris (pH 7.2)] for 5 min. As the RBC lysis reduced the cell yield, all sample embryos from a given litter were treated identically and then normalized to the average for the control animals in the litter. The tails from the embryos were used for PCR genotyping.
For reasons which are not clear, the Lckpr-Bax line 1 demonstrated a phenotype that varied with the background strain (data not shown). The differences in thymus size and percentage of cycling cells was much more pronounced on the C3H/He background when compared with the C57BL/6 background. These differences were not observed for the other transgenic lines. The Lckpr-Bax mice were genotyped using PCR with a forward Bax cDNA primer (5'-GAGCTGATCAGAACCATCATG-3' and a reverse human growth hormone primer (5'-GTAGCCATTGCAGCTAGGTG-3'. The primers were used at 0.4 µM and produced a 500-bp product (Lines 1 and 8) or a 350-bp product (line 38) after 30 cycles (94°C x 1 min; 60°C x 1 min; and 72°C x 1 min). Bcl-2 exon III cDNA primers (0.2 µM) that amplify a 250-bp product were included in each tube to serve as a positive control for each reaction (5'-CTTTGTGGAACTGTACGGCCCCAGCATGCG ' and reverse 5'-ACAGCCTGCAGCTTTGTTTCATGGTACATC').
Aging Studies.
Mice of the appropriate genotypes were mated for two or more
generations to obtain animals for the aging studies. Upon entry into
the aging study, animals were examined weekly for signs of illness or
malignancy. Sick animals were monitored more frequently and euthanized
when necessary to prevent suffering. Bax-deficient mice were
monitored for a minimum of 1.5 years. Animals that survived the
duration of the study were killed, and a necropsy was performed.
Because these animals did not show overt signs of disease, tumors found
in these mice were censored from the analysis because it is not known
whether they would have died or become ill from the tumor. Despite
these efforts, the majority of Bax-deficient mice that died
while on study were not noted to be sick before their death. When
possible, necropsies were performed on dead animals to determine
whether the animals had gross evidence of tumors. Tumors were then
confirmed by fixation with formalin and histological examination after
H&E staining. A hematopathologist examined the sections and confirmed
the tumor type. All mice were maintained in the animal facility at
Washington University (St. Louis, MO) or at the University of Iowa
(Iowa City, IA). Statistical analysis was performed with the StatView
Program (SAS Institute Inc.) using Kaplan-Meier cumulative survival and
the log-rank (Mantel-Cox) test to determine whether differences in
survival were significant. Analysis was performed by comparing
Bax-deficient mice, Bax +/+, Bax +/-,
and both together. p53-deficient mice with and
without the Bax or Bcl-2 transgene were compared
with each other.
Cell Preparation and Analysis.
Single-cell suspensions were prepared from the thymi by dispersing the
organs between two glass slides in isotonic saline. When necessary,
RBCs were removed by a 5-min incubation in hypotonic lysis buffer
[0.83% NH4Cl and 10 mM Tris (pH
7.2)], and the viable cell counts were determined using a
hemocytometer and trypan blue exclusion (25)
. Cells were
cultured in RPMI 1640 supplemented with 10% FCS, Pen-Strep,
glutamine, and 2-mercaptoethanol (100 µM). Thymocyte
viability was determined using double staining with Annexin V-FITC
(Trevigen) and PI as instructed by the manufacturer. Viability
after irradiation was normalized to the untreated control samples by
the following equation: % viable with irradiation/% viable
without irradiation at the same time point. Cell cycle analysis was
performed by analyzing PI-stained nuclei on a flow cytometer equipped
for doublet discrimination (FACScan or FACSCalibur from Becton
Dickinson). Briefly, 
1 x 106 cells were pelleted and resuspended in
0.5 ml of Krishan reagent before analysis (26)
. The
percentage of cycling cells (%S/G2-M) was
determined by examining histograms after gating-out doublet events on
the basis of FL-2A versus FL-2W. Cellquest software
(Becton Dickinson) was used for both the acquisition and analysis.
BrdUrd (Sigma) uptake in thymocytes was determined after i.p. injection of BrdUrd (5 mg/mouse). Thymocytes were harvested 1 h after injection and a single-cell suspension was prepared as above. BrdUrd staining was performed using the PermaCyte-FP kit following the manufacture directions (BioErgonomics, St. Paul, MN). Briefly, thymocytes were washed in PBS, centrifuged (1000 x g), and resuspended in 70% ethanol for fixation (-20°C for 30 min). After centrifugation, the cells were resuspended in 4 N HCl and incubated at room temp for 30 min to denature the DNA and expose the BrdUrd. After treatment with permeabilization buffer, cells were stained with anti-BrdUrd FITC (Becton Dickinson) by incubation for 30 min at room temperature. After washing with permeabilization buffer, the cells were resuspended in PBS containing 20 µg/ml PI. Mice that were not injected with BrdUrd were used as negative controls to set the level of background staining. Acquisition and analysis was performed on the FACS Calibur (Becton Dickinson) using doublet discrimination (FL2 area versus width).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the decreased survival could not be attributed to
neoplasia because the tumor-free survival was no different between
groups (Fig. 1B)
. Necropsies performed on the mice, either
at the end of the study or at the time of their deaths, did not
identify an obvious cause of death in the majority of
Bax-deficient mice (Table 1)
.
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. One explanation for this result is that
p53-deficiency results in a substantial apoptosis defect
where Bax-deficiency provides no additional resistance to
death, consistent with Bax being downstream of
p53. As an additional test of this hypothesis, the ability
of Bcl-2 overexpression to promote tumor development in
p53-deficient mice was determined.
Lckpr-Bcl-2 transgenic mice were
chosen for this study because the overexpression of Bcl-2 in
developing and mature T cells results in dramatic resistance to a
number of apoptotic stimuli (24)
. Furthermore, these mice
progress to T-cell lymphomas, albeit with a long latency
(5)
. Because p53-deficient mice are also prone
to the development of T-cell lymphomas (22)
, the
Lckpr-Bcl-2/p53-deficient
mice were generated and monitored for tumor development. In contrast
with our findings with Bax-deficiency, Bcl-2 was
able to dramatically accelerate oncogenesis in p53-deficient
mice (Fig. 2B)
. Because Bcl-2 expression is
restricted to T cells in these transgenic mice, the incidence of
lymphoma-free survival is most pertinent. In this case, seven of seven
Lckpr-Bcl-2/p53-deficient mice
developed thymic lymphomas, whereas only four of seven of the
nontransgenic p53-deficient littermates developed thymic
lymphomas. When a Kaplan-Meier lymphoma-free survival curve was
analyzed using the log-rank test, the P was 
0.006 (data
not shown). These results clearly show that Bcl-2 can
promote tumor development in p53-deficient mice. This
finding is consistent with experimental evidence that Bcl-2
can block p53 independent as well as dependent deaths. This
also illustrates that overexpression of an anti-apoptotic member,
Bcl-2, would have a broader effect than loss of expression
of a single pro-apoptotic member, BAX.
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. Two of these lines (lines 1 and 38) were used for
additional studies. A number of experiments indicate the lymphopenia is
secondary to increased apoptosis. First, in vitro survival
of isolated thymocytes is decreased in cells overexpressing
Bax (Fig. 3B)
. In addition, freshly isolated
thymocytes showed increased evidence of apoptosis based on both
terminal deoxynucleotidyltransferase-labeling and quantitation
of hypodiploid DNA (data not shown). In vivo, the thymi
showed moderately increased terminal deoxynucleotidyl
transferase-mediated nick end labeling. Freshly isolated thymocytes
from transgenic mice have an increased percentage of cycling cells,
thereby making decreased generation unlikely (Fig. 6A)
.
Overall, these data demonstrate that overexpression of Bax
increases the susceptibility of thymocytes to cell death.
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. Assessment of lymphoma-free survival was also
significantly decreased in
Lckpr-Bax/p53-deficient
mice (Fig. 4B)
. Nearly identical results were found when
tumor-free survival was assessed in
Lckpr-Bax line
1/p53-deficient mice (Fig. 4C)
. Similar results
for two independent transgenic lines demonstrate the results are not
dependent on the integration site of the transgene.
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. In vivo, the lymphopenia of
Lckpr-Bax transgenic mice was also
independent of p53 (data not shown). Of note, overexpression
of Bax proved insufficient to confer sensitivity of
thymocytes to 
-irradiation in p53-deficient mice (Fig. 5B)
. These findings and previous results (20)
demonstrate that p53 and Bax are independent
regulators of cell death in thymocytes.
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. Because the animals are lymphopenic, one possibility is
that the increased proliferation serves as a compensatory adjustment to
altered homeostasis. However, previous studies of Bax
transgenic mice using the CD2 promoter noted increased proliferation in
the presence of normal numbers of thymocytes (33
, 34)
.
This suggests the proliferative defect is not secondary to lymphopenia.
If the increased percentage of cycling cells is secondary to
lymphopenia, we reasoned the lymphopenia would precede changes in
proliferation. The chronology of defects in proliferation and thymocyte
number was determined at various developmental ages. The thymocyte
number and proliferative index of
Lckpr-Bax transgenic embryos at E16.5
were not significantly different from nontransgenic littermates (Fig. 6B)
. Over the next two days of embryonic development, the
Lckpr-Bax transgenic mice
synchronously displayed alterations in cell cycle and cell number (Fig. 6, B and C)
. We conclude that Bax
overexpression alters both apoptosis and proliferation of developing T
cells.
BrdUrd Uptake Is Increased in Lckpr-Bax
Thymocytes and Is Independent of p53 Status.
To further characterize the cell cycle abnormalities in
Lckpr-Bax transgenic mice, BrdUrd
uptake was used to determine the relative percentage of cells in S
phase. As expected from the findings from freshly isolated thymocytes
using PI (Fig. 6)
, we find that in vivo BrdUrd uptake is
significantly increased in
Lckpr-Bax transgenic mice (Fig. 7, A and B)
. Furthermore, both the small thymus and
the increased BrdUrd uptake occurred independently of p53,
inasmuch as they were both observed in p53-deficient mice
(Fig. 7, C and D)
. We conclude that
Bax overexpression results in increased DNA synthesis or
S-phase, and before malignancy, the thymic lymphopenia and
proliferative changes are p53 independent.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study Bax-deficiency alone (Fig. 2A)
or
in conjunction with p53-deficiency did not accelerate tumor
development. The Bax-deficient mice showed decreased overall
survival, but this could not be attributed to tumor development. Two of
forty-one Bax-deficient animals died at <6 months of age
and were found to have glomerulonephritis. However, given the low
prevalence of glomerulonephritis, it does not appear to account
entirely for the decreased survival of Bax-deficient mice.
In contrast with Bax deficiency, BCL-2 overexpression accelerates oncogenesis in p53-deficient mice. This observation extends previous work in mice that shows BCL-2 alone or in combination with MYC can promote tumor formation (5 , 35) . However, in a myc-induced model of hepatocellular carcinoma, BCL-2 inhibited tumor formation (36) . BCL-2 inhibition of tumor formation was correlated with decreased proliferation in this model. Because Lckpr-Bcl-2 also inhibits proliferation of T cells (25) , the ability of Bcl-2 to cooperate with p53-deficiency and accelerate lymphoma formation was not obvious. Our findings show that when combined with p53 deficiency, the anti-apoptotic effects of BCL-2 dominate the anti-proliferative activity in regulating lymphoma formation. These results are consistent with the observation that BCL-2, but not p53-deficiency, inhibits apoptosis of T cells in response to diverse apoptotic signals (24) .
Analysis of the Bax promoter suggests the p53 binding sites may not be functional in mice (29) . Consistent with this finding, and in contrast to a previous result (37) , we noted no differences in the levels of Bax in tissues derived from p53 +/+ and -/- mice.4 However, regulation of murine Bax by p53 has been demonstrated in cells or tissues that also express either the TAg or E1A.
To further study the role of Bax in p53 mediated
oncogenesis, mice overexpressing Bax in developing T cells
were generated. Similar to transgenic mice that overexpress
Bax using the CD2 promoter (33)
, our
Lckpr-Bax mice display T-cell
apoptosis (Fig. 3)
. In contrast with the CD2 promoter model, mice
overexpressing Bax driven by the
Lckpr-proximal promoter show marked thymic
hypoplasia. Contrary to transient transfection of Bax into
p53-deficient fibroblasts (10)
, overexpression
of Bax in thymocytes promotes death to the same extent
independent of the level of p53 (Fig. 5A)
.
Moreover, CD2-Bax transgenic mice (33)
and
Lckpr-Bax transgenic mice are unable
to confer sensitivity to -irradiation (Fig. 5B)
. Coupled
with the observation that Bax-deficient thymocytes are not
protected from -irradiation, these results demonstrate that
Bax is neither necessary nor sufficient for apoptosis of
thymocytes after -irradiation.
The most surprising finding was the accelerated tumor progression in
Lckpr-Bax/p53-deficient
mice. These differences were seen whether tumor-free or lymphoma-free
survival was examined. These findings are potentially even more
remarkable inasmuch as the "target" population of thymocytes is
substantially reduced from the overexpression of BAX (Fig. 3A)
. Our paradoxical findings are reminiscent of E2F-1, in
which the deficiency of this critical positive regulator of cell
division results in tumor induction (38)
.
A number of possibilities may explain our paradoxical findings
regarding Bax and tumor progression. We have no evidence
that BAX would have a paradoxical anti-apoptosis effect in thymocytes.
However, it is important to acknowledge several reports in neuronal
systems, including transient Bax expression
(31)
, nigericin-induced death of neuronal cell lines
(39)
, and the infection of Bax-deficient mice
with a neurotropic virus (32)
suggest that Bax
may exert an anti-apoptotic effect. However, these examples of a
potential anti-apoptotic activity for BAX were all in neuronal cells,
and the applicability of these findings to lymphoid cells is not clear.
In fact, before malignant transformation, Bax promotes
apoptosis of thymocytes in the presence or absence of p53
(Fig. 3B)
.
A more likely explanation is that BAX-increased proliferation of thymocytes promotes transformation. In support, a number of studies have implicated BCL-2 in the regulation of cell cycle. Studies of T cells indicate that BCL-2 levels help dictate cell cycle entry from the quiescent state (25 , 40 , 41) . These findings have been extended to other cell types, including NIH 3T3 fibroblasts (40) and IL-3-dependent hematopoietic cell lines (42) . Other studies of BCL-2 suggest that inhibition of apoptosis is separable from cell cycle effects because two distinct mutations in BCL-2 result in normal anti-apoptotic function but the loss of anti-proliferation activity (42 , 43) . Downstream regulators of cellular proliferation appear to be altered in cells overexpressing BCL-2. BCL-2 decreased nuclear factor of activated T cells activation in mature T cells (25) . Other studies demonstrate that BCL-2 affects p27Kip1 and p130 levels and the cyclin-dependent kinase 2 activity (44 , 45) . However, the biochemical activity of BCL-2 that accounts for these downstream changes remains to be elucidated.
Unfortunately, less is known concerning a role for BAX in cell cycle
regulation. This reflects the difficulty of assessing cell cycle
regulations in cells committed to die. Cells from the
CD2-Bax transgenic mice do display accelerated entry into
the cell cycle after ConA stimulation (34)
. In addition,
thymocytes overexpressing Bax more rapidly degrade p27Kip1
and increase CDK2 kinase activity (44)
. These findings are
in agreement with our observation that BAX-induced changes in
proliferation and apoptosis occur simultaneously (Fig. 6B)
.
This interplay of BAX effects on cell cycle and apoptosis is likely
central to its unanticipated acceleration of tumorigenesis in the
absence of p53.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was initiated while C. M. K. was a
Pfizer Postdoctoral Fellow. This work was supported by Grant IN-122T
from the American Cancer Society (to C. M. K.) and CA49712 from the
NIH (to S. J. K.). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, University of Iowa College of
Medicine, Room 145 MRC, Iowa City, IA 52242. Phone: (319) 335-8147;
Fax. (319) 335-6555; E-mail: c-knudson{at}uiowa.edu
3 The abbreviations used are: TAg, large T
antigen; PI, propidium iodide; BrdUrd, bromodeoxyuridine.
4 C. M. Knudson and S. J. Korsmeyer, unpublished
observations.
Received 3/15/00. Accepted 11/10/00.
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transgenic mice show accelerated apoptosis in response to stimuli but do not show restored DNA damage-induced cell death in the absence of p53. EMBO J., 15: 1221-1230, 1996.[Medline]
perturbs T cell development and affects cell cycle entry of T cells. EMBO J., 15: 6991-7001, 1996.[Medline]
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),
heterozygous (+/-, 
), and wild-type (+/+, 
) mice is shown. All
animals were monitored weekly for signs of illness or tumor
development. Necropsies were performed on all mice (when possible) to
determine whether the animals showed evidence of tumor development.
Tumor-free survival of Bax-deficient mice was not
significantly different from that of wild-type or heterozygous mice. In
addition, heterozygous and wild-type mice were not significantly
different from each other. For disease-free survival,
Bax-deficient mice showed reduced survival relative
to heterozygous alone (P = .0263) and
heterozygous or wild type (P = 0.0353).
However disease-free survival compared with wild type alone did not
achieve significance in this cohort (P = 0.0644). Ps were derived using the log-rank (Mantel-Cox)
test and the StatView statistical package as described in "Materials
and Methods."
