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Original Studies |
Departments of Surgery (M.B., S.R.N., A.R.M.), Medicine (Endocrinology) (D.W.B., L.J.D.), Pathology (C.B.), and Medicine (Gastroenterology) (J.M.C.), University of California, and San Diego Veterans Affairs Medical Center, La Jolla, California 92161
Address all correspondence and requests for reprints to: Dr. Michael Bouvet, Department of Surgery (112-E), University of California, Veterans Affairs Medical Center, 3350 La Jolla Village Drive, San Diego, California 92161. E-mail: mbouvet{at}ucsd.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results indicate that PTHrP is commonly expressed in human exocrine pancreatic adenocarcinoma even though these patients are usually eucalcemic. These findings may have important implications for the diagnosis, management, and treatment of pancreatic cancer.
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Materials and Methods |
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The following human pancreatic cancer cell lines were obtained from American Type Culture Collection (Manassas, VA): AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1. PANC-28, and PANC-48 cell lines were provided by Douglas B. Evans, M.D. Anderson Cancer Center (Houston, TX). The cells were maintained in DMEM supplemented with 10% FCS, 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin (Life Technologies, Inc., Grand Island, NY). All cell lines were incubated at 37 C in a 5% CO2 incubator.
Antibodies
Monoclonal and polyclonal antibodies to the amino-terminal (1–34), midregion (38–64), and carboxyl-terminal (109–141) peptides of PTHrP were respectively used to identify cellular PTHrP or secreted PTHrP and/or for Western blotting and immunocytochemical staining for PTHrP (16, 17).
Immunoassay of cell extracts and culture media
The cell lines were plated into 100-mm culture dishes and incubated at 37 C for 24 h. The conditioned medium was then collected and frozen until further processing. The cell extracts were prepared as follows. The cells were trypsinized, pelleted, and sonicated in lysis buffer [0.25 mol/L Tris (pH 7.4), 0.25% Nonidet P-40, 2 mmol/L ethylenediamine tetraacetate (EDTA), 0.2 mmol/L phenylmethylsulfonylfluoride, 1 µmol/L leupeptin, and 1 µmol/L pepstatin). The cell debris was pelleted by centrifugation at 14,000 x g for 15 min, and the supernatants were transferred to new tubes and frozen until further processing. PTHrP was measured by three immunoassays, using modifications of previously described methods (17). In brief, tyrosinated human PTHrP-(1–34), PTHrP-(38–64), and PTHrP-(109–141) were used to prepare tracer by chloramine-T radioiodination and as respective assay standards. Rabbit antisera to each of the respective peptides were used in a nonequilibrium immunoassay format. Lack of cross-reaction in the assay for at least a 100-fold excess of peptide was demonstrated for the noncorresponding PTHrP peptides, calcitonin, calcitonin gene-related peptide, and human and rat atrial natriuretic peptide and bone natriuretic peptide. All samples were assayed in multiple dilutions that paralleled the corresponding PTHrP standard. The intra- and interassay variations were between 7–12%, respectively (16). All synthetic peptides were purchased from Peninsula Laboratories, Inc. (Belmont, CA). Cell protein was measured using a modified Bradford protein assay with BSA as standard (Bio-Rad Laboratories, Inc. Hercules, CA).
Western blotting of cell extracts
Cells were lysed by sonication in a lysis buffer containing 10 mmol/L Tris-HCl (pH 7.5; Sigma, St. Louis, MO), and 1 mmol/L EDTA, 1 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 1 mmol/L dithioreitol, 1% Nonidet P-40, and protease inhibitor cocktail (Roche Molecular Biochemicals, Bavaria, Germany). Protein lysates were reduced by incubation for 10 min at 100 C in Laemmli’s sample buffer (Sigma) at a final concentration of 15% ß-mercaptoethanol. Polypeptides were resolved at 150 V on 10% gels and electrophoretically transferred to 0.45-µm nitrocellulose membranes (Millipore Corp., Bedford, MA) for 1 h at 100 V. Membranes were blocked for overnight in 20 mmol/L Tris (pH 7.5) and 250 mmol/L NaCl containing 3% (wt/vol) casein. Blots were then probed for 90 min with monoclonal antibody to PTHrP-(109–141) and ß-actin (control) and developed using species-specific secondary antibodies. Immunoreactive material was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturer’s instructions.
Densitometry
The PTHrP and ß-actin Western bands were quantitatively analyzed with a digital imaging system (Alpha Innotech, San Leandro, CA). The densitometry results represent all a single band at approximately 17 kDa that was the least saturated, providing for more linear densitometry readings. The intensities of the bands were assigned integrated density values, which represent the sum of all pixel values in the box. All bands are quantitated in equal area boxes, and autobackground subtract was used to control for background signal. The band intensity was determined to be below saturation by a false color palette in the image analysis software.
Cell proliferation assay
AsPC-1 cells, chosen because of their relatively rapid growth, were plated at a density of 5000 cells/well in replicates of 6 wells/group into 96-well plates and allowed to attach in low serum conditions of 2% FBS. Four hours later, after cell attachment, the medium was changed to 0.1% BSA. The following day the cells were treated with varying concentrations of PTHrP-(1–34) in the standard growth medium. At specific time points in the experiment, the media were removed from the wells by inverting the plates, and the plates were frozen at 70 C until further processing. A fluorogenic double stranded DNA-binding dye, bisbenzimide H33258 (Calbiochem-Novabiochem Corp., La Jolla, CA), diluted in cell lysis buffer [10 mmol/L Tris (pH 7.4), 200 mmol/L NaCl, 1 mmol/L EDTA, and 0.01% Triton X-100] was added to the thawed plates to quantitate cell number in the wells. The plates were scanned in a fluorometric plate reader (Wallac, Inc., Gaithersburg, MD) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. A reference standard curve was generated to convert the sample fluorescence values into cell numbers. A t test was used to determine statistically significant (P > 0.05) differences in cell growth between controls and varying doses of PTHrP. The experiment was repeated.
Patients and specimens
Fourteen cases of well characterized ductal pancreatic
adenocarcinoma were selected from the archives at the University of
California-San Diego Medical Center. The median age of the patients was
64 yr (range, 29–75 yr). The diagnosis of pancreatic adenocarcinoma
was made by pathological evaluation of tissues obtained from surgical
resection. Staging of tumors was performed according to the AJCC
TNM staging system. All patients were eucalcemic at presentation
(Table 2
). Tissues obtained had been immediately formalin-fixed,
routinely processed, and embedded in paraffin.
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Immunohistochemical localization of PTHrP was performed by the streptavidin-horseradish peroxidase enzyme conjugate method using the PTHrP-(109–141) antibody as previously described (16). All procedures were performed at room temperature unless otherwise specified. Formalin-fixed, paraffin-embedded cell blocks or tissue sections (3–5 µm thickness) were deparaffinized and hydrated through a series of isopropyl alcohol solutions. The slides were then incubated with a 1% zinc sulfate solution and microwaved for two 5-min bursts to enhance antigenicity. After washing and blocking with a nonspecific protein solution (20% FBS, 0.25% gelatin, and 0.01% azide in phosphate-buffered saline), protein A-purified anti-PTHrP antibodies at 10 µg IgG/mL were added to the slides overnight in humidified chambers at 4 C. Biotinylated goat antimouse IgG antibody was then applied for 1 h, and the streptavidin-horseradish peroxidase enzyme complex was applied for 1 h. Positive staining was developed by incubating the slides with a developing solution [1.3 mmol/L 3, 3'-diaminobenzidine-4 HCl with 0.02% (vol/vol) H2O2 in 10 mmol/L Tris, pH 7.4] for 5–10 min. To determine the specificity of positive staining, the antibodies were preadsorbed with the corresponding antigen overnight. This antibody-antigen control solution was applied to serial tissue sections adjacent to the corresponding antibody-treated sections on the same slide.
Evaluation of immunohistology studies
Immunohistochemical staining of pancreatic tumors was evaluated by a surgical pathologist who was unaware of the clinical information or antibody treatment. Cells were considered positively stained if cytoplasm, cell membrane, or nucleus was distinctly colored compared with background and if staining was absent from adjacent control tissue sections. A negatively stained specimen was one in which the sections treated with antibody were not different from the corresponding control sections. For cell lines, the following scoring system was used: 1) the intensity of immunoreactive product (0 = no reaction, + = low intensity, 2+ = moderate intensity, 2++ = high intensity), b) the proportion of cells showing a positive immunoreactive product (1 = 0–0.9%, 2 = 1–24%, 3 = 25–49%, 4 = 50–74%, and 5 = 75–100%), and 3) index = (intensity score) x (% of positive cells). For patient tumors, a grading system of 1+ (least staining) to 3+ (most staining) was used to assess PTHrP staining intensity.
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Results |
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Cellular PTHrP was detected in all cell line extracts by RIA based
on PTHrP-(1–34) at concentrations ranging from 0.7–7.5 fmol/µg
protein (Fig. 1A). CFPAC-1, derived from
a pancreatic liver metastasis, had the highest concentration, and MIA
PaCa-2, derived from primary pancreatic adenocarcinoma, had the lowest.
Immunoassays based on PTHrP-(38–64) and -(109–141) also detected
PTHrP in each cell line extract at generally lower concentrations.
Secretion of PTHrP into cell medium was also measured by all three
assays for each cell line and, in general, paralleled the respective
cellular PTHrP levels.
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Evidence for differential processing of PTHrP was provided by
studies demonstrating the different patterns of PTHrP expression among
the cell lines when assessed by PTHrP immunoassays based on the three
distinct PTHrP peptides (Fig. 1A
). For example, although cellular PTHrP
levels were highest for CFPAC-1 using all three antibodies,
PTHrP-(38–64) secretion was highest for BxPC-3, whereas the highest
levels of secreted PTHrP-(109–141) occurred in both CFPAC-1 and
PANC-1.
Western blot analysis of pancreatic cancer cell line lysates
Western blotting of cell lysates confirmed the presence of PTHrP
in all eight cell lines (Fig. 1B). A distinct three-band pattern with
proteins at 17, 34, and 44 kDa was noted. Equal loading was controlled
with ß-actin. The most intense bands were seen in the CFPAC-1 cell
line, which was 6-fold higher on densitometry measurements compared
with PANC-48.
Dose-dependent PTHrP-(1–34) stimulation of cell growth
PTHrP-(1–34) was added to the medium of AsPC-1 cells in culture
at various concentrations. Growth of AsPC-1 cells was stimulated in a
dose-dependent manner by PTHrP-(1–34) (Fig. 2). By 48 h, statistically
significant differences in cell number were seen at all concentrations
of PTHrP compared with the control (by Student’s t
test).
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PTHrP was localized by immunocytochemical staining in the
cytoplasm in seven of eight cell lines (Table 1). Both nuclear and cytoplasmic
immunostaining were observed in the MIA PaCa-2 and PANC-1 cells.
Representative photographs are shown in Fig. 3, A–D.
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All 14 cases of pancreatic adenocarcinoma, including the 1
ampullary tumor, stained with antibody to the carboxyl-terminus of
PTHrP (Table 2). PTHrP staining of tumor
cells was noted in a background of stromal fibrosis typical of
pancreatic adenocarcinoma (Fig. 3, E–H). The staining of the cells was
predominantly cytoplasmic; however, in 2 patients, both cytoplasmic and
nuclear PTHrP immunoreactivities were noted. In general, staining
intensity was inversely related to differentiation of tumors. Both of
the well differentiated tumors had 1+ (least) staining for PTHrP, most,
but not all of the poorly differentiated tumors had 2+ or 3+ (most)
staining for PTHrP. In all cases of positive staining, incubation of
sections with the antibody-antigen control mixture described in
Materials and Methods showed no positive staining. As
previously reported (11), normal pancreatic elements
(pancreatic ducts and islets) stained positively to a variable
degree.
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Discussion |
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Although PTHrP expression has been reported in normal and malignant exocrine and endocrine cells of the pancreas, most studies have emphasized islet cells (7, 11, 12, 14, 15). Furthermore, pancreatic endocrine tumors, which normally are not associated with hypercalcemia, can express PTHrP (11, 15), and in case reports plasma PTHrP levels have been shown to fall after surgical resection of such tumors (18). Early studies evaluated PTH, but not PTHrP, as a tumor marker for exocrine pancreatic cancer (19), but it is now known that ectopic PTH production is rare (3, 14, 16). Such limited studies notwithstanding, relatively little is known about PTHrP expression by pancreatic exocrine cancers. In our study all 8 exocrine pancreatic cell lines produced and secreted PTHrP, and all 14 surgical specimens of pancreatic adenocarcinoma expressed PTHrP. The nuclear localization in some of the malignant cells is consistent with the recently appreciated nuclear mechanism of action for PTHrP (20).
Our studies also provided indirect evidence for PTHrP processing by pancreatic adenocarcinoma. Western blotting demonstrated a 17-kDa band as well as 34- and 44-kDa bands of immunoreactive PTHrP in the cell lines. It is possible, however, that these multiple bands may represent dimers or trimers of the native protein. We also observed different immunochemical patterns of PTHrP expression in different pancreatic exocrine cell lines that may be due to different processing of PTHrP by them. The complementary DNA-predicted amino acid sequence of PTHrP contains multiple basic amino acid motifs that would allow PTHrP to undergo extensive posttranslational processing before secretion (21, 22). One of the pancreatic cell lines, CFPAC-1, had high levels of all three PTHrP epitopes, suggesting that this cell line may process PTHrP in a different manner than the other cell lines. We recognize that there is a lack of correlation between the intensity of the bands and the reported immunoassayable material shown. This can be explained by the two different PTHrP-(109–141) antibodies used and the inherent differences between the two assay formats, i.e. denatured PTHrP (Western) vs. nondenatured PTHrP (RIA).
Although the function of PTHrP in pancreatic cancer is unknown, it appears to regulate growth in other tumor types (22). We previously demonstrated that the amino-terminal peptide, PTHrP-(1–34), stimulated thymidine uptake in prostate cancer cells more than 3-fold over control values under serum-free and steroid-free conditions; in addition, PTHrP-induced DNA synthesis was completely neutralized by our mouse monoclonal antibody against PTHrP-(1–34) (23). We also have demonstrated that PTHrP-(1–34) stimulates the growth of cultured type II epithelial cells (24). In this study we also demonstrated that PTHrP-(1–34) stimulates the growth of the AsPC-1 human pancreatic cancer cell line. Our findings of expression of PTHrP in exocrine pancreatic adenocarcinomas, much more common than its pancreatic endocrine tumor counterpart, warrants delineation of the functions of this important molecule in pancreatic exocrine cancer. The growth regulatory effects of PTHrP on AsPC-1 cells suggest that growth regulation may be one of these functions. However, more studies of the growth regulatory effects of PTHrP in pancreatic cancer are needed.
The absence of hypercalcemia in patients with PTHrP-expressing pancreatic adenocarcinomas is similar to that in prostate adenocarcinoma, but contrasts to breast adenocarcinoma where hypercalcemia is common (15, 23, 25, 26). There are many potential explanations for this phenomenon; for example, the PTHrP expressed by pancreatic adenocarcinomas may be degraded by this enzyme-rich tissue or processed into peptides that do not cause hypercalcemia (26). More extensive and systematic clinical studies are needed to document the relationship between hypercalcemia and PTHrP expression in pancreatic exocrine cancers (12, 14, 25).
In summary, we have demonstrated that PTHrP is commonly expressed and secreted in human exocrine pancreatic cell lines and in paraffin-embedded pancreatic adenocarcinoma tumor specimens. Further studies will be necessary to elucidate the role of PTHrP in the development of pancreatic cancer and to determine whether PTHrP could be useful in the early detection or clinical management of patients with this disease.
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Acknowledgments |
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Footnotes |
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Received March 24, 2000.
Revised July 31, 2000.
Revised September 5, 2000.
Accepted September 15, 2000.
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