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Unitat de Biologia Cel.lular i Molecular, Institut Municipal d’Investigació Mèdica, Universitat Pompeu Fabra, 08003 Barcelona, Spain [T. A., A. G-C., A. S., F. X. R.], and Department of Pathology, Dartmouth Medical School, Lebanon, New Hampshire 03756 [O. S. P., D. S. L.]
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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The target cell for carcinogenesis in the human pancreas, leading to the common ductal-type adenocarcinoma, is not known. Several candidates have been proposed (discussed in Refs. 12 and 13 ): (a) a multipotential stem cell presumably located in the pancreatic ducts; (b) acinar cells that lose their differentiated properties and acquire a ductal-type phenotype representing either dedifferentiation or transdifferentiation; (c) islet-derived cells that acquire a ductal phenotype; and (d) pancreatic ductal cells. The elucidation of this issue is hampered by the fact that it is not possible to follow histological changes in the human pancreas sequentially. Nevertheless, several observations suggest that pancreatic ductal cells are the target for carcinogens in humans: (a) severe dysplasia, a general hallmark of high risk for neoplasia, is rarely observed in acinar cells, even in tissue from patients with pancreas cancer (14) ; (b) ductal cell hyperplasia, flat or papillary, can often be identified in tissue from patients with pancreas cancer (15) . However, similar histological changes can also be identified in tissue from patients without cancer, in particular in old individuals and in patients with chronic pancreatitis (15 , 16) ; and (c) recent evidence shows that some of the genetic lesions that are associated with pancreas cancer can also be detected in putative preneoplastic lesions, such as flat and papillary ductal cell hyperplasia. For example, mutations in codon 12 of K-ras occur in a high proportion of pancreas cancers (17) and have also been reported in ductal cell hyperplasia associated with chronic pancreatitis and pancreas cancer (18 , 19) . Such evidence may not be considered strong because the mutation found in the tumor often, but not always, corresponds to the mutation found in putative preneoplastic lesions (18, 19, 20, 21) and because K-ras mutations are restricted to a few codons, thus increasing the likelihood that the presence of the same mutation in putative preneoplastic lesions and in tumor from a given patient may be attributable to chance. A stronger evidence comes from the study by Moskaluk et al. (21) , showing that in two cases with pancreas cancer, putative preneoplastic lesions (in one case a flat pancreatic intraductal lesion and in another case a papillary lesion with severe atypia) harbored the same mutation in p16INK4A as the tumor. Because mutations in p16 show a wider spectrum than mutations in K-ras, this finding is unlikely to result from chance.
Experimental models of pancreas cancer in rodents have shown consistent genetic and phenotypic characteristics and can be of help in the understanding of human pancreas cancer (12 , 13) . In the Syrian hamster, nitrosamines induce ductal-type tumors that morphologically resemble the common human pancreas cancer and harbor K-ras and p53 mutations as human tumors (22) . In the rat, azaserine induces acinar cell hyperplasia and acinar tumors lacking K-ras mutations (23) . From these tumors, several useful cell lines have been derived: AR42J cells, displaying mixed acinar and neuroendocrine features, have been extensively used in studies of the physiology and regulation of gene expression of acinar cells (24 , 25) ; ARIP cells were derived from the same tumor as AR42J but they lack these differentiation features (25) . Pettengill et al. (26) have derived two cell lines that were independently derived from a transplantable acinar cell carcinoma DSL6, established from a primary carcinoma of the pancreas induced by azaserine in Lewis rats. After 1–2 weeks in culture, exocrine enzyme production ceased and the long-term cell lines obtained no longer displayed acinar features. Upon s.c. injection in rats, DSL6A cells produced solid tumors containing duct-like structures and a dense stroma; DSL6B produced cells with mixed glandular, squamous, and mucinous areas. In both cell lines, ultrastructural analysis revealed the loss of zymogen granules (26) . In addition, transgenic mice in which c-myc is expressed in acinar cells under the control of the elastase promoter develop tumors that can progress from an acinar to a ductal phenotype (27) . These studies support the notion that ductal-like tumors with a phenotype very similar to that of the common pancreatic adenocarcinoma found in humans can evolve from acinar tumors.
The expression of acinar cell-specific genes, such as amylase and elastase, is under the control of a pancreas-specific transcriptional complex designated PTF13 , which binds to a bipartite site in the A element of the regulatory region of these genes (28) and whose activity closely parallels the expression of pancreas-specific gene products during development (29) . PTF1 is constituted by three different bHLH transcription factors: p64 and p75 (30) , which are ubiquitous, and p48 whose expression is restricted to the pancreas (31) . p64 is the product of REB, and p75 is the product of E2A (32) . Although p48 and p64 bind to DNA, p75 is involved in the transport of the p48/p64 heterodimer to the nucleus (33) . The bHLHregion of p48 shares sequence homology with myoD (31) , a transcription factor that specifies activation of myocyte differentiation in nonmuscle cells (34) . Such homology, together with the finding that p48 expression is restricted to the exocrine pancreas, suggests that p48 may be involved in the activation of an acinar differentiation program. However, acinar gene expression also requires the contribution of other tissue-restricted transcription factors such as HNF-3ß (35) and Pdx-1 (36) .
In this study, we have addressed two questions: (a) whether the phenotype of rat and human exocrine pancreatic tumors is related to the expression of p48; and (b) whether p48 is able to instruct an acinar differentiation program in pancreas cancer cells displaying a ductal phenotype. Our findings, together with previous work (31) , provide evidence that p48 is necessary, but not sufficient, for the activation of the acinar phenotype in ductal cells, even when they are derived from cells with acinar differentiation potential. Furthermore, we show that under certain circumstances, p48 is retained in the cytoplasm, thus losing its ability to form the PTF1 complex and contribute to the activation of acinar genes.
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Results |
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. Pettengill et al. (26)
have shown
that when these tumors are placed in culture, loss of acinar features
and acquisition of ductal characteristics take place. In this way, two
independent cell lines have been established, DSL6A and DSL6B from a
transplanted tumor. When these cells are injected s.c. or
orthotopically into male Lewis rats, ductal-like tumors develop
that are morphologically very similar to the common ductal
adenocarcinoma occurring in humans (Fig. 1, C and E)
. At the ultrastructural level, the serially transplanted
tumors have acinar features and contain cells with electron-dense
secretion granules (Fig. 1B)
, whereas the tumors induced by
DSL6A and DSL6B cell lines display epithelial features but lack
exocrine secretion granules (Fig. 1, D and F)
.
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Production of p48-specific, Affinity-purified Rabbit Antibodies.
Rabbit polyclonal serum against rat p48 produced in Escherichia
coli was first preabsorbed with nuclear extracts from a mixture of
rat tissues (see "Materials and Methods") and subsequently purified
by affinity chromatography with His-tagged rat p48. Fig. 2A
shows the reactivity of the affinity-purified
immunoglobulin fraction with nuclear extracts from AR42J cells and
nuclear extracts from a variety of normal rat tissues. A band of
Mr 
48,000 was specifically
identified in AR42J cells and in normal pancreas tissue. There was no
reactivity with nuclear or cytoplasmic extracts from nonpancreatic
tissues. To confirm the specificity of the antiserum,
immunohistochemical assays on frozen sections of normal rat and human
tissues were performed (Fig. 2B)
. A nuclear pattern of
staining was identified in the majority of acinar cells though the
intensity of staining showed some variation from cell to cell. Acinar
staining could be inhibited by preincubation of the antibody with
purified p48. All ductal cells were consistently unreactive with
anti-p48 antibodies; double labeling with antibodies detecting p48 and
antibodies detecting cytokeratins 7 and 19, which are restricted to
centroacinar and ductal cells in the pancreas (3, 4, 5)
,
revealed no coexpression (data not shown). p48 was not detected in
islet cells (Fig. 2B)
. Apart from the pancreas, there was no
reactivity with a large panel of normal tissues: esophagus, stomach,
small bowel, colon, gallbladder, liver, breast, ovary, cervix, larynx,
trachea, lung, kidney, and thyroid, indicating that p48 is selectively
expressed in normal acinar cells. Similar results were obtained using
normal rat and human tissues.
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p48 mRNA was detected in AR42J cells but not in ARIP, DSL6A, or DSL6B
cells. p48 transcripts were detected in the tumors that were serially
transplanted and in those induced by DSL6B cells but not in tumors
induced by DSL6A cells (Fig. 3A)
. p48 protein was exclusively detected in serially
transplanted tumors (Fig. 3B)
. Amylase was detected only
whenever p48 protein was also detectable; in tumors from DSL6B cells,
low levels of p48 mRNA were present, but no p48 protein was detected by
western blotting (Fig. 3B)
. Using immunohistochemistry, p48
was detected in the nucleus of cancer cells in serially transplanted
tumors, and amylase was weakly detected, mainly in luminal areas (Fig. 4, A and B)
. By contrast, neither p48 nor amylase
were detected in tumors induced by cultured DSL6A or DSL6B cells (Fig. 4, C–F)
. Altogether, these results indicate that loss of
acinar features in cultured tumor cells and in tumor tissues is
associated with the loss of expression of p48.
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, indicating that lack of expression of these
ubiquitous bHLH transcription factors does not occur during the loss of
the acinar phenotype.
Expression of the PTF1 Components in Human Pancreas Tumors.
To examine p48 transcript expression in human pancreas tissue and
cultured cells, a partial cDNA encoding the bHLH domain was isolated by
RT-PCR using degenerate oligonucleotides. PCR products were cloned, and
both strands of DNA were sequenced. The comparison of the cDNA sequence
coding for the bHLH region of rat and human p48 is shown in Fig. 5
. The deduced amino acid sequence in this region is identical for rat
and human p48, and there is 79% nucleotide identity; all differences
between the two sequences correspond to third nucleotides of codons.
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. p48 transcripts were detected in normal pancreas tissue but were
undetectable, using a nested PCR, in all human pancreas cancer cell
lines displaying a ductal phenotype examined (n = 8;
Fig. 6A
). In agreement with these findings, p48 was not
detected in cell lysates of cultured pancreas cancer cells (Fig. 6B)
. Using immunohistochemistry on frozen tissue sections,
p48 was absent from all ductal pancreas adenocarcinomas examined
(n = 18; Fig. 7B
). By contrast, anti-p48 antibodies showed strong reactivity
with the three acinar pancreatic tumors examined. In two cases,
classified as "predominantly acinar-glandular" and "predominantly
solid," all cells showed nuclear staining (Fig. 7C)
; in
one additional tumor, classified as "mixed acinar-solid," p48 was
detected exclusively in the cytoplasm of 30% of tumor cells, and it
was detected in the nucleus of a small proportion of tumor cells
(<5%; Fig. 7D
). In these tumors, amylase expression was
analyzed using immunohistochemistry. In the acinar-glandular tumor,
strong expression of amylase was observed; by contrast, in the mixed
acinar-solid tumor, amylase was detected in a very low proportion of
cells (<5%; data not shown). Anti-p48 antibodies did not react with
any of the nonpancreatic tumors analyzed (Table 1)
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. Therefore,
the only component whose expression is absent in ductal pancreatic
tumors is p48.
Is Transfection of p48 cDNA into Ductal-like Cells Able to Induce
an Acinar Phenotype?
The DSL6 cells and tumors provide an excellent model to analyze whether
p48 can activate an acinar differentiation program because the
ductal-like DSL6A and DSL6B cells are derived from an acinar tumor. To
this end, DSL6A and DSL6B cells were transiently transfected with the
rat p48 cDNA, and the activity of the PTF1 complex was analyzed by
monitoring amylase expression in cell lysates, amylase activity
released to the culture medium, and the activity of a reporter plasmid
in which the hGH cDNA is under the control of a 6-mer of the A element
of the rat elastase gene (37)
. DSL6B cells could not be
transfected using Lipofectamine, but p48 was detected in transfected
DSL6A cells as well as in control AR42J and ARIP cells (Fig. 8A)
. Amylase was not detected in the supernatant or lysates of
DSL6A and ARIP cells transfected with p48 cDNA (Fig. 8A)
,
and no activity of the hGH reporter could be demonstrated (Fig. 8B)
. By contrast, transfection of AR42J cells led to an
increase in nuclear p48 levels and to a 2–4-fold increase in the
activity of the hGH reporter (Fig. 8B)
. In addition, a small
but reproducible increase in intracellular and secreted amylase was
observed. The increase in secreted amylase was not statistically
significant, likely because of the low efficiency of transient
transfections in AR42J cells. These results were confirmed in more than
three independent assays.
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.
To examine whether lack of acinar gene activation and reporter activity
could be attributable to mislocalization of p48, as observed in one of
the acinar tumors, the subcellular distribution of p48 in transfectants
was examined by immunohistochemistry. p48 was found in the nucleus of
all transfected AR42J and ARIP cells (Fig. 8C, b and
c
) and in the cytoplasm of all transfected DSL6A and RWP-1
cells (Fig. 8C, a and d
).
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Until recently, p48 was thought to participate exclusively in exocrine pancreas differentiation. However, inactivation of the p48 gene in mice by homologous recombination leads to an apancreatic phenotype characterized by the lack of an exocrine pancreas and the presence of a reduced number of individual endocrine cells in the spleen (38) . These findings indicate that p48 plays a role both early and late in pancreas development and differentiation. However, little is known about the genetic mechanisms regulating the expression of p48. The p48 promoter has been cloned and sequenced, but its activity is not restricted to pancreatic cells, and putative tissue-specific control elements have not been identified in as much as 10 kb of 5'-flanking region. (39) . Several explanations can account for the finding that, upon in vitro culture, DSL6 cells displaying an acinar phenotype lose p48 expression: culture may select for populations lacking p48 expression and having some growth advantage; alternatively, the maintenance of p48 expression may require cellular interactions with the mesenchyme or with neural cells that cannot be maintained in vitro. In support of the latter hypothesis is the observation that cultures of normal human exocrine pancreas undergo an acinar-to-ductal phenotypic switch in vitro that is also accompanied by the loss of expression of p48 (40 , 41) . In these cells, a selective outgrowth of cells lacking p48 cannot be proposed because normal pancreatic cells undergo limited replication in vitro (40) . There is extensive evidence that mesenchymal-epithelial interactions play a major role in development (42) and differentiation (43) . For example, the LIM-homeodomain protein Isl-1 is expressed in all postmitotic islet cells as well as in mesenchymal cells surrounding the dorsal but not the ventral evagination of the gut endoderm during development. Inactivation of Isl-1 in the mouse results in the lack of Isl-1-expressing dorsal mesenchyme, lack of exocrine cell differentiation in the dorsal but not in the ventral pancreatic bud, and lack of endocrine cells in all of the pancreas. In vitro reconstitution experiments have shown that the wild-type pancreatic mesenchyme can support the normal development of the dorsal exocrine pancreas from Isl-1-/- mice (43) . A search for regulatory motifs in the 10 kb upstream of the p48 gene has failed to provide clues about tissue-specific transcription factors/complexes that may be involved in its regulation, although a putative Pdx-1 binding site has been identified (39) . The molecular mechanisms underlying the down-regulation of p48 gene expression in tumors remain unknown, although at least in the human tumors, the p48 gene is generally retained.4
Our findings support the contention that p48 is necessary, but not sufficient, for the activation of acinar genes in pancreatic cells. This conclusion, which is also supported by in vitro studies of promoter regulation (35 , 36) and by transfection of an antisense construct of p48 cDNA in AR42J cells (31) , is drawn from our transfection studies using both rat and human pancreas cancer cells. AR42J cells, which spontaneously express amylase and other acinar genes (25) , are equipped with the transcription factor machinery necessary for their expression, and transfection with p48 cDNA is accompanied by enhanced expression both in transient and stable assays (this work and data not shown). By contrast, transfection of p48 cDNA into other pancreatic cells did not result in the activation of the expression of amylase nor a reporter construct containing the A element of the elastase promoter, indicating lack of formation of an active PTF1 complex. Two types of mechanisms seem to contribute to the lack of effects of p48 overexpression. In DSL6A and RWP-1 cells, p48 was undetectable in the nucleus. p75, encoded by the ubiquitously expressed E2A gene, has been shown to be responsible for the nuclear import of the p48/p64 complex (33) ; however, E2A is expressed in both cells. Similarly, the REB/HEB gene is also expressed in both lines. Thus, the molecular basis of the cytoplasmic accumulation of p48 remains to be elucidated. Id proteins, initially identified as inhibitors of differentiation of muscle and lymphoid cells (44 , 45) , are candidates to play a role in the subcellular distribution of p48. Id proteins, of which four members have been described thus far, contain an HLH domain that enables their dimerization with bHLH factors but lack the basic domain that allows DNA binding, therefore sequestering bHLH-type transcription factors and acting in a dominant-negative fashion (44, 45, 46) . Id1 has been directly implicated in the suppression of mammary cell differentiation (47) . There is currently no direct evidence that Id proteins interact with p48 nor that they can block its transcriptional activity. In addition to them, other yet unidentified proteins might interact with p48 and retain it in the cytoplasm.
The pathophysiological relevance of the subcellular distribution of p48 is underlined by our findings in human acinar tumors. Unlike normal acinar cells, one acinar tumor showed p48 expression exclusively in the cytoplasm. Furthermore, preliminary data indicate that p48 preferentially accumulates in the cytoplasm of acinar/ductular complexes in areas of chronic pancreatitis associated with pancreas cancer.4 The abnormal subcellular distribution of p48 might contribute to the disregulation of acinar gene expression and pancreatic insufficiency.
In ARIP cells, p48 did localize to the nucleus, but it did not activate acinar gene expression nor a reporter construct for the PTF1 complex. It has been shown that expression of acinar enzymes in AR42J cells requires the expression of the winged helix-loop-helix factor HNF-3ß (35) . Indeed, ARIP cells express low levels of HNF-3ß. Because transfection efficiency for these cells is very low, we have not been able to conclusively show the precise role of HNF-3ß by cotransfecting its cDNA with that of p48. Other mechanisms may account for the lack of activation of acinar genes. For example, the histone acetyltransferase PCAF and p300/CBP promote both MyoD-dependent transcription and myogenic differentiation in muscle cells (48) .
It is currently not known whether p48 may play a role in tumor progression. Acinar-to-ductal conversion has been demonstrated in other tumors of the exocrine pancreas in rodents, most notably the Ela-myc transgenic mouse. In these animals, acinar tumors progress to develop a ductal phenotype similar to that of the majority of human exocrine pancreas cancers (27) . It is conceivable that loss of acinar features and p48 expression might favor tumor progression, although this possibility has not been examined in detail. Studies on the role of the bHLH transcription factor MyoD in muscle differentiation support such contention. MyoD induces the expression of the cyclin-dependent kinase inhibitor p21 at the transcriptional level, leading to an arrest in G1 that allows the activation of a cell differentiation program in normal cells (49, 50, 51) . Such effects are abrogated in a high proportion of tumor cells (52) , despite that MyoD is expressed in the majority of rhabdomyosarcomas (53 , 54) . Furthermore, pRb favors both cell cycle exit and the expression of muscle-specific genes by cooperating with Myo-D, whereas in the absence of pRb, myogenic transcription factors are inactive (55) .
Finally, p48 is an excellent marker of the acinar cell differentiation in the pancreas. Although its selectivity is unlikely to be of clinical interest in the setting of human exocrine pancreatic cancers because acinar tumors are rare, the study of the expression, subcellular distribution, and transcriptional activity of p48 should shed light on the molecular mechanisms involved in the diseases of the exocrine pancreas.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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A serially transplanted tumor from the DSL6 transplantable tumor and DSL6A and DSL6B cell lines were obtained as described by Pettengill et al. (26) . DSL6A and DSL6B cells were cultured in Waymouth’s MB 752/1 medium supplemented with heat-inactivated fetal bovine serum (10%), L-glutamine, nonessential amino acids, penicillin, and streptomycin, and passed at weekly intervals.
To obtain grafted tumors, cryopreserved pieces of the DSL6 tumor or in vitro cultured DSL6A and DSL6B cells (2 x 106) were injected s.c. to male Lewis rats. Tumor development was monitored weekly, and tumors of 1–2 cm diameter were surgically excised for analysis.
Normal human tissues were obtained from surgical samples except for normal pancreas, which came from organ donors; tumor tissues were obtained from surgery performed at Hospital del Mar, Barcelona, Spain, except for the three acinar tumors which were kindly provided by Dr. A. Scarpa (Istituto di Anatomia e Istologia Patologia, Facolta di Medicina e Chirurgia, Verona, Italy).
Electron Microscopy.
Tissue fragments of approximately 1–2 mm3
were
fixed with 2% glutaraldehyde in PBS, postfixed with osmium tetroxide,
dehydrated in ethanol, and embedded in Epon 812 resin. Thin sections
were obtained using a ultramicrotome, stained with uranyl acetate and
lead citrate, and examined using a Philips 301 electron microscope.
Antibodies.
To generate p48-specific polyclonal antibodies, rat p48 cDNA was cloned
in pBUC and used to transform BL21 strain bacteria. Recombinant
colonies were individually analyzed. A positive colony was selected and
induced with isopropyl-1-thio-ß-D-galactopyranoside for
2.5 h at 37°C; cells were collected, snap frozen, thawed, and
sonicated. The fraction that was soluble in 8 M urea
contained recombinant p48. After centrifugation at 30,000 x
g for 20 min at 4°C, the supernatant was collected,
dialyzed against 1 M urea, and used to immunize
rabbits and to prepare an affinity chromatography matrix as indicated
below.
Rabbits were immunized with 50 µg of p48 [diluted in 20
mM HEPES (pH 7.9), 1 M urea, 0.1 M
KCl, 2 mM DTT, 10% glycerol, 0.1 mM EDTA, and
0.1% NP40 in the presence of protease inhibitors] mixed with complete
Freund’s adjuvant. Two, 4, and 6 weeks later, rabbits received boosts
with the same amount of protein mixed with incomplete Freund’s
adjuvant. Preimmune and postimmune blood samples were collected, and
their reactivity with recombinant p48 was analyzed by Western blotting.
Immune serum was preabsorbed three times with a mixture of nuclear
extracts from rat kidney, heart, skeletal muscle, and liver coupled to
Affigel 10 (Bio-Rad, Richmond, CA). Anti-p48 antibodies present in the
fraction that did not bind to this matrix were subsequently
affinity-purified on a Ni-NTA matrix to which His-tagged p48 had been
previously bound following the manufacturer’s recommendations (Qiagen,
Valencia, CA). Bound antibodies were eluted with 3.5 M
MgCl2; their reactivity with recombinant p48 and
specificity were analyzed by Western blotting as described below. This
antibody preparation recognizes exclusively a protein of
Mr 48,000 in nuclear extracts from
rat or human pancreas tissue. Rabbit polyclonal antibodies detecting
human and rodent amylase were purchased from Sigma Chemical Co. (St.
Louis, MO).
Western Blotting.
Normal rat tissues were immediately frozen at -80°C. To
prepare nuclear extracts, tissues were rapidly thawed and homogenized
with a Dounce homogenizer in 50 mM Tris (pH 7.5), 2
mM EDTA, 150 mM NaCl, 0.5 mM DTT,
and 0.3 M sucrose containing a cocktail of protease
inhibitors, filtered through a gauze, and separated through a 0.9
M sucrose cushion by centrifugation at 7000 rpm for 15 min
at 4°C. The supernatant was recentrifuged under the same conditions
to obtain cytoplasmic extracts. The pellet was resuspended in the
homogenization buffer, brought to 0.3 sucrose and 0.2% NP40,
rehomogenized, and recentrifuged through a sucrose cushion as described
above. The pellet, considered the nuclear extract, was used for Western
blotting after lysing and clearing by centrifugation. An aliquot of 40
µg was fractionated by 10% SDS-PAGE, transferred to nitrocellulose
filters, and incubated with Western blotting blocking buffer
(Tris-buffered saline containing 5% skim milk and 0.05% Tween 20).
After incubating with specific antibodies (anti-p48 or anti-amylase,
0.5 µg/ml in Western blotting blocking buffer), filters were washed
and incubated with peroxidase-labeled goat antirabbit immunoglobulin
(Dakopatts, Glostrup, Denmark), and reactions were developed with
enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL).
Cultured cells were lysed in 25 mM Tris (pH 7.5), 1 mM EGTA, 1 mM EDTA, and 1% SDS containing a protease inhibitor cocktail; lysates were boiled for 15 min and cleared, and protein concentration was determined. Proteins (50 µg) were fractionated by SDS-PAGE, and Western blotting was carried out as described above.
Immunohistochemistry and Immunocytochemistry.
Fresh tissues were immediately frozen in isopentane cooled at -80°C.
Five micron sections were fixed for 10 min with 4% paraformaldehyde,
incubated for 15 min with
H2O2 to block endogenous
peroxidase, and washed with PBS. After blocking with 1% BSA, 0.1%
saponin, and 0.1% Triton X-100 in PBS (blocking buffer) for 30 min,
sections were incubated with primary antibody diluted in the same
buffer. After washing with PBS, sections were incubated with
biotin-conjugated goat antirabbit immunoglobulin (Dakopatts), washed
with PBS, incubated with streptavidin-peroxidase (2 µg/ml; Pierce,
Rockford, IL), and washed with PBS. Reactions were developed using
diaminobenzidine as a chromogen. Affinity-purified rabbit anti-p48
antibodies were used at 1–5 µg/ml. Anti-amylase antibodies were used
at 10 µg/ml, and normal rabbit serum diluted to contain a comparable
concentration of IgG was used as control.
Immunocytochemical assays were performed on cells fixed with 4% paraformaldehyde, permeabilized with blocking buffer, and incubated with antibodies as described above. For these experiments, an irrelevant mouse monoclonal antibody (B12, to detect dextran) was used as control.
RNA Expression Analysis.
RNA from tissues and cultured cells was isolated using guanidinium
isothiocyanate as described by Chomczynski and Sacchi
(56)
. For RT-PCR, 4 µg of DNase-treated total RNA were
used to synthesize cDNA using a mixture of oligo-d(T) and random
hexamers. An aliquot of the cDNA products (1:16) was used for PCR using
amplification conditions optimized for each primer pair. The PCR
products were separated by agarose gel electrophoresis and visualized
by ethidium bromide staining. The primers and PCR conditions used are
shown in Table 2
. The identity of the amplified products was confirmed by digestion with
restriction enzymes or by direct sequencing.
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Transient Transfection and Reporter Gene Expression Assays.
Rat and human pancreatic cells (6 x
105) were seeded on six-well plastic plates
(Costar, Cambridge, MA) or on sterile coverslips. Twenty-four h later,
cells were transfected with 1 µg of linearized plasmid, either with
empty vector (pcDNA3) or with the same vector containing the
full-length rat p48 cDNA (pcDNA3.p48) using Lipofectamine
(Lipofectamine Plus Reagent; Life Technologies, Inc., Gaithersburg,
MD), following the manufacturer’s instructions. Cells were harvested
at different time points to determine the optimal time for analysis; in
general, assays were performed 48 h after transfection. For
Western blotting and immunocytochemical procedures, cell lysates were
prepared as described above. To determine the transcriptional activity
of the PTF1 complex, cells were cotransfected with one of the two
plasmids described above (0.2 µg) plus 0.2 µg of a plasmid, kindly
provided by G. Swift and R. MacDonald (University of Texas Southwestern
Medical Center, Dallas, TX), containing the hGH cDNA downstream from a
6-mer of the A element from the rat elastase promoter (6A26Elp.hGH;
Ref. 37
) and 0.1 µg of plasmid pGL2-control vector
(Promega Corp., Madison, WI), containing the luciferase cDNA, to
normalize for transfection efficiency. For these experiments,
105 cells were seeded in wells of 24-well plates
(Costar) in duplicates, and transfection was performed 24 h later
using the Lipofectamine Plus reagent. Forty-eight h later, hGH activity
was measured in the culture medium using a RIA (Nichols Institute
Diagnostics, San Juan Capistrano, CA), and amylase activity was
determined using the AMYL commercial kit (Boehringer Mannheim,
Mannheim, Germany). Cells were lysed and processed for luciferase
activity assays according to the manufacturer’s recommendations
(Promega).
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Acknowledgments |
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Footnotes |
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1 Partially supported by Grant SAF94-0971 from the
Comisión Interministerial de Ciencia y Tecnología, Grant
PM97-0077 from the Dirección General de Enseñanza Superior
e Investigación Científica, Grant SGR-00433 Generalitat
de Catalunya, and Grant BMH4-CT98.3085 from the Biomed program. T. A.
and A. S. were supported by personal grants from Generalitat de
Catalunya. 
2 To whom requests for reprints should be
addressed, at Unitat de Biologia Cel.lular i Molecular, Institut
Municipal d’Investigació Mèdica, carrer del Dr. Aiguader,
80, 08003 Barcelona, Spain. Phone 34-93-2257586; Fax: 34-93-2213237;
E-mail preal@imim.es.
3 The abbreviations used are: PTF1, pancreas
transcription factor 1; bHLH, basic helix-loop-helix; HNF, hepatocyte
nuclear factor; hGH, human growth hormone; RT-PCR, reverse
transcription-PCR.
4 T. Adell, X. Molero, A. Skoudy, M. A. Padilla
and F. X. Real, unpublished observations.
Received for publication 9/14/99. Revision received 12/20/99. Accepted for publication 2/ 4/00.
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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and pancreas transcription factor 1 are required for efficient expression of the gene encoding pancreatic 
-amylase. Mol. Cell. Biol., 15: 1933-1941, 1995.Cbf. J. Biol. Chem., 271: 21993-22002, 1996.This article has been cited by other articles:
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  J. Pujal, M. Huch, A. Jose, I. Abasolo, A. Rodolosse, A. Duch, L. Sanchez-Palazon, F. J. D. Smith, W. H. I. McLean, C. Fillat, et al. Keratin 7 promoter selectively targets transgene expression to normal and neoplastic pancreatic ductal cells in vitro and in vivo FASEB J, May 1, 2009; 23(5): 1366 - 1375. [Abstract] [Full Text] [PDF]
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C. Bierkamp, S. Bonhoure, A. Mathieu, P. Clerc, D. Fourmy, L. Pradayrol, C. Seva, and M. Dufresne Expression of Cholecystokinin-2/Gastrin Receptor in the Murine Pancreas Modulates Cell Adhesion and Cell Differentiation in Vivo Am. J. Pathol., December 1, 2004; 165(6): 2135 - 2145. [Abstract] [Full Text] [PDF]
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S. Aguilar, J. M. Corominas, N. Malats, J. A. Pereira, M. Dufresne, F. X. Real, and P. Navarro Tissue Plasminogen Activator in Murine Exocrine Pancreas Cancer: Selective Expression in Ductal Tumors and Contribution to Cancer Progression Am. J. Pathol., October 1, 2004; 165(4): 1129 - 1139. [Abstract] [Full Text] [PDF]
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) or with p48 cDNA (
),
normalized for transfection efficiency. Panel C,
representative findings of the subcellular distribution of p48 in
transfected cells. In AR42J cells (b), two
subpopulations of cells expressing nuclear p48 are observed; endogenous
p48 is present in low levels, whereas transfected cells display high
levels of expression. In DSL6A (a) and RWP-1
(d) cells, cytoplasmic accumulation of p48 is observed.
In ARIP cells (c), p48 is exclusively localized in the
nucleus. x400.