Departments of Surgery and Cell Biology, The Vanderbilt-Ingram Cancer Center and Nashville Veterans Affairs Medical Center, Vanderbilt University, Nashville, Tennessee 37232-2736
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ABSTRACT |
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The mechanisms linking acinar
cell apoptosis and ductal epithelial proliferation remain unknown. To
determine the relationship between these events, pancreatic duct
ligation (PDL) was performed on p53(+/+) and p53(/
) mice. In mice
bearing a wild-type p53 allele, PDL resulted in upregulation of p53
protein in both acinar cells and proliferating duct-like epithelium. In
contrast, upregulation of Bcl-2 occurred only in duct-like epithelium.
Both p21WAF1/CIP1 and Bax were also upregulated in
duct-ligated lobes. After PDL in p53(+/+) mice, acinar cells
underwent widespread apoptosis, while duct-like epithelium underwent
proliferative expansion. In the absence of p53, upregulation of p53
target genes and acinar cell apoptosis did not occur. The absence of
acinar cell apoptosis in p53(
/
) mice also eliminated the
proliferative response to duct ligation. These data demonstrate that
PDL-induced acinar cell apoptosis is a p53-dependent event and suggest
a direct link between acinar cell apoptosis and proliferation of
duct-like epithelium in duct-ligated pancreas.
duct ligation; regeneration; Bcl-2; Bax; p21
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INTRODUCTION |
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THE MAMMALIAN PANCREAS IS characterized by low resting rates of cell division but a significant proliferative response to mitogenic signals or tissue injury (11). Pancreatic epithelial proliferation may be initiated by soluble growth factors (16, 31, 40), inflammatory cytokines (12), islet cell injury (24), partial pancreatectomy (30), or pancreatic duct obstruction (1, 6, 33). After either islet cell injury or partial pancreatectomy, pancreatic regeneration appears to be initiated by proliferation of duct-like epithelial cells, with subsequent differentiation of new islet and/or acinar tissue (24, 30). Although controversial, this regenerative capacity has led Bouwens (3) and Sharma et al. (30) to propose a stem cell capacity within the adult pancreas.
In the case of pancreatic duct obstruction, proliferation is one
component of a complex phenotype that involves a combination of
cellular inflammation, acinar cell loss, generation of
ductal-tubular complexes, and islet neogenesis (26,
34-36). Recent studies (36, 39) have
demonstrated that pancreatic duct ligation (PDL) in mice or rats
induces expression of multiple growth factors and cytokines, including
gastrin, transforming growth factor- (TGF-
), interleukin
(IL)-1
, IL-1
, IL-6, IL-10, tumor necrosis factor-
, and Fas
ligand. These events are associated with acinar cell apoptosis, which
appears to represent the predominant mechanism of acinar cell loss
after duct ligation (1, 6, 39). Coincident with the
initiation of acinar cell apoptosis, significant proliferation is
observed in duct-like epithelial cells, leading to the formation of
ductal-tubular complexes with focal areas of islet neogenesis (2,
3, 35).
These lineage-specific changes in cell survival, proliferation, and differentiation induced by PDL provide the opportunity to gain important insight into factors regulating pancreatic stem cell biology (2, 3). Because of the complexity of histological events induced by duct ligation, previous studies have not evaluated whether duct-like epithelial proliferation represents a primary response to duct ligation or a secondary regenerative response to acinar cell loss. In addition, the molecular mechanisms by which duct ligation selectively induces apoptosis in acinar cells and proliferation in duct-like cells remain unknown.
In many cellular systems, initiation of apoptosis involves
activation of the p53 tumor suppressor gene (5). p53 is a
nuclear transcription factor known to transactivate several
well-characterized target genes, including p21WAF/CIP, an
inhibitor of cyclin-dependent kinase activity, and Bax, a positive
regulator of apoptosis (8, 21). In the current study, we
sought to determine the role of p53 in acinar cell apoptosis after PDL
and also to evaluate the participation of candidate gene products known
to mediate p53-dependent events. To facilitate genetic analysis, we
refined a technique for PDL in the mouse and found that duct ligation
stabilized p53 protein in both acinar and ductal cell lineages. p53
activation after duct ligation was further documented by increases in
tissue expression of both p21WAF/CIP and Bax. These events
were associated with extensive acinar cell apoptosis, as evidenced by
changes in cellular morphology and positive terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling
(TUNEL) labeling. In contrast to the widespread activation of p53
observed in both acinar and ductal lineages, upregulation of the
antiapoptotic protein Bcl-2 was limited to highly proliferative duct-like epithelium. Using p53-null mice carrying homozygous deletions
in the p53 allele [p53(/
)], we found that the absence of p53
prevented not only the induction of apoptosis after PDL but also the
associated proliferative response. These findings confirm that acinar
cell apoptosis after duct ligation is a p53-dependent event and further
suggest a link between acinar cell apoptosis and proliferation of
duct-like epithelium.
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METHODS |
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Mice, PDL, and tissue harvest.
C57BL/6 × DBA male mice were utilized for both unoperated
controls and PDL in the setting of wild-type p53 [p53(+/+)]. All animals were fed standard murine chow and water ad libitum. Twelve mice
underwent PDL in the following manner. After intraperitoneal injection
with a mixture of ketamine (80 mg/kg) and xylazine (20 mg/kg), the
peritoneum was entered through a midline laparotomy and the stomach,
pancreas, and spleen were mobilized. The spleen was retracted
laterally, and the splenic vessels were separated from the pancreatic
tissue to prevent devascularization of pancreatic parenchyma. The
splenic lobe of the pancreas was ligated to the left of the portal vein
with a 5-0 monofilament polypropylene suture. The viscera were replaced
in anatomic position, and the incision was closed in two layers using
4-0 braided polyglycolic acid suture. A subcutaneous injection of 1 ml
sterile saline solution was given at the conclusion of the procedure.
To examine the effects of PDL in the absence of p53,
C57BL/6J-Trp53tm1Tyj [p53(/
)] male and
C57BL/6J-Trp53tm1Tyj [p53(+/
)] female mice were
obtained from Jackson Laboratories (Bar Harbor, ME). Offspring from the
p53(
/
) × p53(+/
) cross were genotyped by PCR as previously
described (18), and homozygous p53(
/
) mice
(n = 10) were subjected to PDL as described above.
Immunostaining. Immunohistochemical analysis was performed in the following manner. Sections (5 µm) were fixed to APS-coated glass slides, dewaxed, and rehydrated. After quenching of endogenous peroxidase activity using 3% H2O2 for 5 min, the slides were blocked in 5% normal serum for 60 min at room temperature. The slides were then treated with primary antibody overnight at 4°C. Primary antibodies and dilutions were as follows: rabbit anti-amylase, 1:1,000 (Sigma Chemical, St. Louis, MO); anti-BrdU, prediluted (Zymed Laboratory, San Francisco, CA); mouse anti-Bcl-2 clone 124, 1:100 (Dako); and rabbit anti-p53 CM5, 1:1,000 (Novo Castra, Burlingame, CA ). After washing in PBS, species-specific biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) was applied for 60 min at room temperature and followed by application of avidin-biotin complex-conjugated solution (Vector Laboratories) for 30 min at room temperature. 3,3'-Diaminobenzidine (DAB) chromogen was applied, and the slides were counterstained with hematoxylin.
Conjugated lectin staining was performed on paraffin-embedded sections in the following manner: after dewaxing and rehydrating, the tissues were permeabilized with 0.2% Triton X-100/PBS for 10 min, then blocked with 2% BSA solution for 1 h. As a marker for acinar cell apical membranes, rhodamine-conjugated peanut agglutinin (PNA) was applied for 1 h at room temperature (1:100, Vector Laboratories). After PBS washes, nuclei were counterstained with YO-PRO-1 for 5 min (1:50,000, Molecular Probes, Eugene, OR) and mounted with GVA mounting solution (Zymed Laboratory). As a structural marker of ductal epithelium, fluorescent staining was performed on frozen sections with rhodamine-conjugated dolichos biflorus agglutinin (DBA) (1:100, Vector Laboratories) as described above. Immunohistochemical analysis for apoptosis was performed with the Apoptag kit (Intergen, Purchase, NY). The slides were dewaxed and rehydrated, then treated with proteinase K (5 µl of 20 mg/ml stock solution in 5 ml PBS) for 15 min at room temperature. After quenching in 3% H2O2 for 5 min, the equilibration buffer was applied for 30 s, followed by treatment with the terminal deoxynucleotidyl transferase enzyme for 1 h at 37°C. Stop buffer was applied, and slides were washed. Anti-digoxigenin-peroxidase was applied for 30 min. The slides were then treated with DAB solution for 5 min and counterstained with methyl green. The slides were dehydrated, and coverslips were applied.Cell counts and statistical analysis.
To obtain quantitative data regarding the rate of ductal-tubular
complex formation in ligated pancreatic lobes, duct-like structures
from control, p53(+/+) PDL, and p53(/
) PDL lobes were counted under
light microscopy. Means ± SE were obtained, and unpaired
t-tests were performed comparing the number of small ducts
per low-power field (×200) in both PDL and control tissues. To obtain
quantitative data for BrdU incorporation and TUNEL positivity, cells
were counted under ×200 light microscopic magnification. Means ± SE were obtained, and unpaired t-tests were performed. Statistical analysis was performed with Prism (GraphPad Software, San
Diego, CA).
Protein extraction and Western blotting. Proteins were extracted from minced pancreatic tissues by solubilizing in lysis buffer [50 mM Tris, pH 7.5, 100 mM NaCl, and 0.5% Nonidet P-40 supplemented with protease inhibitors (chymostatin, leupeptin, antipain, and pepstatin)] on a rotating platform at 4°C for 1 h. Insoluble debris was pelleted, and the protein concentration of the resulting supernatant was determined using the Bradford method. Fifty micrograms of total protein per lane were loaded and resolved on SDS-10% polyacrylamide gels, transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore), and probed with the following primary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA): mouse monoclonal anti-p53 antibody (DO-1, 1:500); rabbit polyclonal anti-Bax antibody (P-19, 1:1,000); and rabbit polyclonal anti-p21 antibody (1:1,000). The membranes were washed and incubated with horseradish peroxidase-conjugated species-appropriate secondary antibodies (Pierce, Rockford, IL), then developed with enhanced chemiluminescence reagents (Amersham Life Science, Little Chalfont, UK) and exposed to radiograph film.
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RESULTS |
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Acinar cell loss and expansion of duct-like epithelium after PDL.
Murine PDL resulted in phenotypic changes in a time-dependent
manner (Fig. 1). Three days after PDL,
the ligated lobe was edematous, and there was a marked inflammatory
cell infiltrate, consisting mostly of segmented neutrophils. The acinar
architecture was poorly organized, with loss of nuclear polarity and
irregular acinar borders. Apoptotic bodies were visible in some acinar
units. The total volume of acini appeared to be reduced, and there was expansion of a novel duct-like epithelium composed of low-cuboidal cells. The islets appeared to be unaffected by PDL. By 5 days after
PDL, there were few remaining acini, and these appeared to be poorly
organized. The gland was fibrotic and showed an extensive inflammatory
process composed predominantly of mononuclear cells. There were
numerous duct-like structures throughout the ligated lobe.
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Lineage-specific analysis of p53 and Bcl-2 expression after PDL. To determine the molecular mechanisms responsible for the divergent behavior of acinar and ductal lineages after PDL, we analyzed lineage-specific expression of Bcl-2 and p53 using immunohistochemistry on control and duct-ligated pancreas. Control pancreatic tissues did not stain positively for Bcl-2 (Fig. 1G). However, 3 days after PDL, the newly emerging duct-like epithelium showed positive supranuclear/cytoplasmic staining for Bcl-2. Some mononuclear cells were also Bcl-2 positive. In contrast, acinar cells showed no evidence of Bcl-2 expression after duct ligation (Fig. 1H). Five days after PDL, pancreatic tissue showed ongoing strong expression of Bcl-2 within the duct-like epithelium. The few acinar cells remaining at this point did not express Bcl-2, consistent with widespread induction of apoptosis in this cell type (Fig. 1I). Immunohistochemical analysis for p53 in control pancreas demonstrated little-to-no detectable p53 protein in acinar cell, ductal, or islet nuclei (Fig. 1J). Five days after PDL, however, there was strong nuclear positivity in nuclei of both acinar and duct-like cells, indicating stabilization of p53 protein (Fig. 1, K and L).
PDL induces p53-dependent activation of Bax and
p21WAF1/CIP1.
Based on the uniform stabilization of p53 observed in both acinar and
ductal lineages after PDL, we next examined tissue expression of Bax
and p21WAF1/CIP1, two well-characterized p53 target genes
(8, 21). To confirm p53-dependent transactivation of
target genes, pancreatic tissue was harvested from duct-ligated
p53(+/+) and p53(/
) mice 5 days postligation, and protein lysates
were analyzed by Western blot. Pancreatic tissue from p53(+/+) control
mice showed low levels of detectable p53 protein. Five days after PDL,
pancreatic tissue from p53(+/+) mice demonstrated significant
upregulation of p53 protein, consistent with the immunohistochemical
staining pattern described above. As expected, no evidence of p53
protein expression was observed in tissue harvested from p53(
/
)
mice, even after duct ligation (Fig. 2).
Western blot analysis of Bax expression in p53(+/+) mice showed low
levels of Bax protein in control pancreas but significant upregulation
5 days after PDL. No evidence of Bax upregulation was observed after
PDL in p53(
/
) mice, confirming the p53-dependent nature of this
effect (Fig. 2B). Similarly, analysis of
p21WAF1/CIP1 protein in p53(+/+) mice demonstrated minimal
expression in control pancreas, but significant upregulation 5 days
after duct ligation. No upregulation of p21WAF1/CIP1
protein was observed after duct ligation in p53(
/
) mice (Fig. 2B). These results demonstrate induction of two known p53
target genes in pancreatic tissue after duct ligation, confirming
functional activation of p53 protein.
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PDL-induced acinar cell apoptosis is a p53-dependent event.
Based on the upregulation of p53 protein and transactivation of p53
target genes observed after PDL in p53(+/+) mice, we next sought to
determine whether PDL-induced acinar cell apoptosis represented a
p53-dependent event. The requirement for p53 in PDL-induced acinar cell
apoptosis was rigorously assessed by comparison of acinar cell markers
and TUNEL staining in control lobes, p53(+/+) ligated lobes, and
p53(/
) ligated lobes. Histological examination of p53(
/
)
pancreatic tissue 5 days after ductal ligation revealed fibrosis,
inflammatory cells, and interstitial edema throughout the ligated lobe,
but preservation of acinar cells. The acini were distorted, but the
nuclei retained their basal polarity compared with pancreatic tissue
from duct-ligated p53(+/+) mice (Fig. 3, C and B, respectively). Preservation of acinar
cell mass after duct ligation in p53(
/
) mice, but not p53(+/+)
mice, was confirmed using both immunostaining for amylase and
Cy3-conjugated PNA lectin staining to mark acinar cell membranes.
Control acini stained positive for amylase, whereas ducts and islets
did not (Fig. 3D). Five days after duct ligation, there were
only rare remaining amylase-positive cells in pancreatic tissue from
p53(+/+) mice (Fig. 3E). In contrast, the pancreatic tissue
from duct-ligated p53(
/
) mice retained amylase immunoreactivity
within preserved acini, confirming preservation of viable acinar cell
mass (Fig. 3F). PNA labeling of control pancreatic tissues
revealed specific staining of acinar cell apical membranes, with no
labeling of ductal, vascular, or islet tissues (Fig. 3G). In
contrast, pancreatic tissue from duct-ligated p53(+/+) failed to label
with PNA, confirming near total loss of acinar cells (Fig.
3H). In contrast, pancreatic tissue from duct-ligated
p53(
/
) mice retained PNA-positive acini, suggesting retention of
acinar apical membrane integrity in the absence of p53.
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PDL-induced epithelial proliferation does not occur in the absence
of acinar cell apoptosis.
The combination of acinar cell apoptosis and expansion of a novel
duct-like epithelium observed after PDL raises the question of whether
proliferation of duct-like epithelium represents a primary response to
duct ligation or, alternatively, a regenerative response to acinar cell
apoptosis. To address this question, we specifically analyzed
epithelial proliferation in duct-ligated p53(+/+) and p53(/
) mice.
Five days after PDL, p53([+/+) tissues demonstrated emergence of a
novel duct-like epithelium (Fig. 3B) while pancreatic tissue
from p53(
/
) mice displayed an attenuated number of duct-like
structures (Fig. 3C). Quantification of duct-like structures
per ×200 field revealed a mean of 9.8 ± 0.8 in pancreatic tissue
from control mice, 123.5 ± 3.9 in duct-ligated p53(+/+) pancreas
[P < 0.0001 vs. control and p53(
/
) PDL], and
20.8 ± 1.8 in duct-ligated p53(
/
) pancreatic tissue
(P < 0.0001 vs. control) (Table 1). These results
demonstrate an attenuated expansion of duct-like epithelium in the
absence of acinar cell apoptosis.
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DISCUSSION |
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The distribution of mature cell types within epithelial tissues is
determined by relative levels of cell proliferation, differentiation, and programmed cell death. This balance may become altered during the
course of epithelial injury and repair, with certain cell types more
susceptible to programmed cell death and other cell types readily
contributing to tissue renewal (see Refs. 9 and 38 for review). The
molecular signals determining expansion or contraction of different
cell lineages during epithelial regeneration remain incompletely
understood. In the present study, we have utilized a murine model of
pancreatic duct obstruction to study lineage-specific changes in cell
survival and proliferation. After selective ligation of the splenic
lobe pancreatic duct, we observed widespread acinar cell apoptosis and
associated proliferation of small duct-like structures
(ductular-tubular complexes). PDL induced stabilization of the p53
tumor suppressor gene product in both acinar and duct-like epithelium,
as evidenced by immunohistochemical staining as well as transactivation
of known p53 target genes. Acinar cell apoptosis was found to be a
p53-dependent event, as neither transactivation of p53 target genes,
increased TUNEL labeling nor loss of acinar cell mass was observed in
C57BL/6J-Trp53tm1Tyj [p53(/
)] mice. Analysis of the
response to PDL in this p53-null genetic background further allowed us
the opportunity to examine PDL-induced proliferative events in the
absence of acinar cell apoptosis. Notably, proliferation of
Bcl-2-expressing duct-like epithelium was not observed after PDL in
p53(
/
) mice, confirming a direct link between acinar cell apoptosis
and the onset of epithelial proliferation in this model. These results
suggest that proliferation of duct-like epithelium represents a
regenerative event apparently initiated by loss of acinar cells, rather
than a direct response to duct ligation.
The current results confirm earlier reports indicating that acinar cell
apoptosis represents the dominant mechanism of acinar cell death after
PDL (1, 6, 13). Prior studies (13, 28) have
suggested species-to-species variability in the relative contributions
of apoptotic and necrotic acinar cell death after PDL, with apoptosis
predominating in the rat, while necrosis predominates in the opossum.
In the mouse, selective PDL is technically difficult, resulting in a
limited number of studies utilizing this species. Watanabe and
colleagues (37) initially reported the successful application of splenic lobe duct ligation in the mouse, resulting in
loss of acinar cell mass and ductal proliferation similar to that
observed in the rat. Two subsequent studies (1, 39) have
documented acinar cell apoptosis as the predominant mechanism of acinar
cell death after PDL in the mouse. In one of these studies (39), murine PDL was associated with increases in
expression of Fas ligand and IL-1-converting enzyme as assessed by
RT-PCR. However, the precise mechanism by which acinar cell
apoptosis is initiated after PDL has not previously been elucidated.
Our data suggest induction of a classical p53-dependent apoptotic pathway involving stabilization of wild-type p53 protein and
transactivation of p53 target genes, including p21WAF/CIP
and Bax. Both the p21WAF/CIP and Bax genes contain
consensus p53-binding sites in their regulatory elements (8,
21). p53 is also known to increase expression of Fas receptor
(4, 25), suggesting relevance between our findings and
those of Yasuda et al. (39). With respect to the ability
of Fas ligand and other signals to potentially induce apoptosis in a
p53-independent manner (7), the present results strictly
define PDL-induced acinar cell apoptosis as a p53-dependent event.
The present results also provide insight regarding the expansion of
duct-like epithelium observed after PDL. This epithelium has been
histologically well characterized in previous studies (34, 35,
37). In the rat, PDL induces expansion of ductular-tubular complexes characterized by high rates of cellular proliferation (33-35). Evidence of ductal differentiation has
previously been suggested based on positive immunohistochemical
staining for cytokeratin 20 (35). However, additional
studies (3, 35) have demonstrated similarities with
embryonic pancreatic epithelium, including expression of the glut-2
glucose transporter protein and initiation of islet neogenesis.
Bertelli and Bendayan (2) have reported the appearance of
intermediate cells displaying features of both endocrine and exocrine
differentiation after PDL in the rat. In this regard, the expanding
ductal epithelium induced by PDL shares features in common with several
other models of pancreatic epithelial proliferation, including islet
cell injury (24), transgenic overexpression of
interferon- (12), partial pancreatectomy
(30), and transgenic overexpression of TGF-
(31).
In each of these models, an initiating stimulus results in loss of a
differentiated cell population, suggesting that associated epithelial
proliferation may represent a compensatory, regenerative response.
However, it remains unclear whether the observed proliferation is
directly triggered by differentiated cell loss as opposed to a direct
effect of the initiating stimulus. In the present study, the absence of
acinar cell apoptosis after PDL in p53(/
) mice represents a unique
opportunity to directly study the relationship between acinar cell
apoptosis and associated proliferation of duct-like epithelium.
Specifically, is proliferation in this model a direct effect of PDL or
a regenerative response initiated by loss of acinar cells? Our results
show that PDL-induced ductal proliferation does not occur in the
absence of acinar cell apoptosis, consistent with the view that ductal
proliferation represents a regenerative response to acinar cell loss.
This system also provides insight into factors determining lineage-specific influences on cell survival in pancreatic tissue. While our results do not directly identify the mechanism responsible for differential survival of acinar and ductal cells following PDL, selective upregulation of Bcl-2 in duct-like epithelium may act as an important survival signal. Bcl-2 acts as a regulator of mitochondrial membrane potential and is capable of preventing apoptosis induced by multiple stimuli, including direct activation of p53 (14, 19, 27). We have observed Bcl-2 upregulation in proliferating epithelium beginning on day 3 after PDL, with ongoing expression on day 5. During this time period, acinar cells show no detectable evidence of Bcl-2 expression and undergo widespread apoptosis. Similar results have been reported in the rat, in which increased Bcl-2 expression was noted as early as 6 h after PDL. Based on our additional observations suggesting activation of p53 in both acinar and ductal lineages, these findings suggest that Bcl-2 may exert an important survival influence for cells in the proliferating duct-like epithelium.
In several ways, the current results suggest analogy between the
epithelial response to PDL and the hepatic epithelial response to bile
duct ligation. Bile duct ligation in the mouse results in widespread
apoptosis among hepatocytes, accompanied by proliferation of duct-like
biliary epithelium (22-23). In this system,
hepatocyte apoptosis is apparently mediated by both Fas-dependent and
Fas-independent pathways (22). Changes in the expression
of both Bcl-2 and Bax have also been reported (17, 32),
although the requirement for p53 remains unknown. As in the case of
PDL, bile duct ligation in the rat results in increased expression of
specific growth factors, including TGF- (23). These and
other findings (17, 22, 23, 32) have led to the suggestion
that bile duct ligation induces expansion of a stem cell compartment.
In this regard, expansion of Bcl-2-expressing epithelium appears to represent a common component of epithelial regeneration and repair (10, 29). During embryonic development, Bcl-2 expression is frequently noted in cells undergoing morphological transition from undifferentiated stem cells to committed precursor cells (20). In the adult, Bcl-2 expression in many tissues is topographically restricted to progenitor cell zones, including the basal layer of skin, basal cells of prostate epithelium, and the lower half of crypts in small and large intestine (15, 20). It is, therefore, possible that the Bcl-2-expressing duct-like epithelium observed after PDL may represent a cell population with progenitor function. This interpretation is consistent with previous studies (3, 35) demonstrating similarities between these ductal complexes and embryonic epithelium.
In summary, we have demonstrated that murine PDL induces widespread activation of p53 and transactivation of p53 target genes in pancreatic tissue. This response is associated with extensive acinar cell apoptosis and proliferation of duct-like epithelial cells expressing high levels of Bcl-2. Acinar cell apoptosis in this system represents a p53-dependent event, and proliferation of duct-like epithelium is not observed in the absence of acinar cell apoptosis. These findings are consistent with induction of a regenerative response that may involve expansion of cells with progenitor function.
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ACKNOWLEDGEMENTS |
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We thank Dr. Daniel Beauchamp, Dr. Robert Coffey, and Dr. Howard Crawford for helpful discussions.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants DK-56211 (to S. D. Leach), F32 CA-79107 (to C. R. Scoggins), F32 CA-76698 (to I. M. Meszoely), DK-42502 (Pancreatic Morphology Core Services), and CA-68485 (Vanderbilt-Ingram Cancer Center).
Address for reprint requests and other correspondence: S. D. Leach, Dept. of Surgery, Johns Hopkins Hospital, 600 N. Wolfe St./Blalock 610, Baltimore, MD 21287.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 February 2000; accepted in final form 23 April 2000.
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