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Int. J. Cancer: 67,492-497 (1996)
0 1996 Wiley-Liss, Inc.
I
Publicationof the InternationalUnion Against Cancer
Publicationde I Union InternationaleContre le Cancer
EXPRESSION OF CATHEPSIN E IN PANCREAS: A POSSIBLE TUMOR
MARKER FOR PANCREAS, A PRELIMINARY REPORT
Takeshi A z u M A ~Masamichi
.~,
HIRAI',Shigeji I T d , Kenji YAMAMOTO?,
R. Thomas TAGGART~,
Takao MATSUBA4,
Kiyoshi YASUKAWA4,Kouji U N d , Takanobu HAYAKUMO~
and Masatsugu NAKAJIMA5
lSecond Department of Internal Medicine, Fukui Medical School, Fukui 910-11, Japan; 2Department of Pharmacology, Kyushu
University,Faculty of Dentistry, Fukuoka 812, Japan; 3Divisionof Genetics, Children's Hospital of Buffalo, Buffalo, NY 14209, USA;
4 T ~ kResearch
y~
Center, Tosoh Corporation, Hayakawa, Kanagawa 252; sDepartment of Gastroenterology, Kyoto Second Red Cross
Hospital, Kyoto 602, Japan.
Ductal cancers of the pancreas frequently express markers of
gastrointestinal epithelial cells. Cathepsin E (CTSE) is a nonsecretory, intracellular, but non-lysosomal proteinase found in
the highest concentration in the superficial epithelial cells of the
stomach. The aims of our study were to examine the expression
of CTSE in the pancreas, t o establish an assay system of CTSE
and to evaluate the diagnostic usefulness of CTSE in the
pancreatic juice. Eleven patients with pancreatic ductal adenocarcinoma, 10 with mucin-producing adenoma, 3 with intraductal papillary hyperplasia and 43 with chronic pancreatitis
were examined. Surgically resected pancreatic tissues were
subjected t o irnmunohistochemistry for CTSE. Pancreatic juice
was collected from the patients and subjected t o sandwich
ELISA and Western analysis for detecting CTSE. Positive staining for CTSE was observed in pancreatic ductal adenocarcinoma by immunohistochemistry. CTSE was also expressed in
mucin-producing adenoma, intraductal papillary hyperplasia
and mucinous hyperplasia. CTSE in the pancreatic juice was
present in 8 of I I patients with pancreatic ductal adenocarcinoma. 5 of 10 patients with mucin-producing tumor, I of 3
patients with intraductal papillary hyperplasia and 4 of 43
patients with chronic pancreatitis. The detection frequency of
CTSE in the pancreatic juice was significantly higher in the
patients with pancreatic ductal adenocarcinoma than in the
patients with chronic pancreatitis. Our findings suggest that the
expression of CTSE is associated with the pathogenesis of
pancreatic ductal adenocarcinoma, that CTSE in the pancreatic
juice seems t o be a useful marker for a definitive diagnosis and
that CTSE may be expressed at a relatively early stage of
multistep carcinogenesis in pancreatic lesions.
o 1996 Wiley-Liss,Inc.
The incidence of pancreatic cancer continues to increase in
industrialized Western countries (Silverberg and Lubera, 1989).
Pancreatic carcinoma has the lowest 5-year survival rate. Late
diagnosis, high recurrence rates and a characteristic tumor
biology with regard to growth behavior are responsible for the
fact that most tumors of the pancreas, when diagnosed, have
spread beyond the reach of surgical cure. Only 10-20% of the
patients have a resectable tumor at the time of diagnosis and a
chance of being cured by surgery (Gudjonsson, 1987; Warshaw
and Castillo, 1992). Early diagnosis of pancreatic cancer is
presently the only means to increase the proportion of patients
detected at resectable and curable tumor stages (Warshaw and
Castillo; 1992). During the past few years, great efforts have
been made to render the pancreas visible with new o r improved imaging techniques. It can still be difficult to distinguish between pancreatic cancer and chronic pancreatitis,
since the two diseases may share many clinical and radiologic
characteristics (Kawai et al., 1987). A wide variety of tumor
markers have been proposed for use in the diagnosis of
pancreatic cancer. These include the serum levels of tumorassociated antigens, enzymes and hormones (Steinberg et al..
1986; Friess et al., 1993; Warshaw et al., 1990; Robles-Diaz et
al., 1991). Interestingly, Sessa et al. (1990) have reported that
ductal cancers of the pancreas frequently express markers of
gastrointestinal epithelial cells. Cathepsin E (CTSE) is one of
four immunologically distinct groups of aspartic proteinases
found in the human gastric mucosa. In addition to CTSE, the
gastric aspartic proteinases include pepsinogen A, pepsinogen
C and cathepsin D. Unlike other gastric aspartic proteinases,
CTSE is a non-secretory, intracellular, but non-lysosomal
proteinase found in the highest concentration in the superficial
epithelial cells of the stomach (Samloff et al., 1987).
In the present study, we have described the expression of
CTSE in pancreatic ductal adenocarcinomas, established an
assay system of CTSE and evaluated the diagnostic usefulness
of the CTSE in the pancreatic juice.
MATERIAL AND METHODS
Subjects
Sixty-seven consecutive patients, in whom pancreatic disorders were diagnosed by ultrasound, computed tomography,
endoscopic retrograde cholangiopancreatography (ERCP) and
endoscopic ultrasound, were studied. Forty-three patients had
chronic pancreatitis (27 men and 16 women, age range 42-85
years, mean 61.8 years). All chronic pancreatitis patients were
followed up for 2 years after the testing, and the presumption
of the absence of malignant pancreatic disease was verified if
there was no new or further evidence suggesting carcinoma.
Eleven had pancreatic ductal adenocarcinoma (5 men and 6
women, age range 54-70 years, mean 61.3 years), 10 had
mucin-producing tumor (4 men and 6 women, age range 48-78
years, mean 66.0 years) and 3 had intraductal papillary
hyperplasia (2 men and 1woman, age range 50-69 years. mean
62.3 years). These patients underwent surgery, and the final
diagnosis was proved by histology. We categorized the mucinproducing tumor as belonging to the group of diseases that can
b e detected clinically by the pooling of mucus produced by
tumors inside the main or branch ducts of the pancreas, thus
causing dilatation of the main or branch ducts (Kozuka et a/.,
1979). In the present study all mucin-producing tumors were
adenomas.
Immunohistochemistry for CTSE
Surgically resected human pancreatic tumors were subjected
to immunohistochemistry to identify CTSE. All specimens
were fixed in formalin and embedded in paraffin. The antisera
against CTSE purified from the human erythrocyte membranes were raised in rabbits as described previously (Saku et
al., 1990). Immunohistochemical staining was performed by
the avidin-biotin-peroxidase complex technique (Vectastain
ABC kit, Vector, Burlingame, CA) described by Hsu et al.
(1981). All incubations were done at room temperature. Each
section (4 k m thick) was immersed in 0.3% H 2 0 2in methanol
for 30 min to destroy endogenous peroxidase activity, washed
for 10 min with 0.05 M Tris-buffered saline, pH 7.5, and then
hTo whom correspondence and reprint requests should be addressed, at the Second Department of Internal Medicine, Fukui
Medical School, Matsuoka-cho, Yoshida-gun, Fukui 910-11, Japan.
Fax: (81) 776-61-8110.
Received: Janualy 22, 1996 and in revised form April 2, 1996.
CATHEPSIN E IN PANCREAS TUMORS
493
incubated for 30 min with normal goat serum to block Statistical analysis
non-specific Ig binding sites. The goat serum was removed, and
Comparisons of the detection frequencies of CTSE in the
the slides were incubated overnight with 1:1,000 dilution of the pancreatic juice among the patient groups were made by the x2
rabbit polyclonal antiserum specific for CTSE. This was test, with significance assigned to values below p < 0.05.
followed by a 30-min wash in Tris-buffered saline, a 30-min Comparisons of the ratio of CA19-9 to secretory protein
incubation with biotinylated goat anti-rabbit IgG, a repeat concentration in the pancreatic juice among the patient groups
wash in Tris-buffered saline, and a 30-min incubation with were made by Student’s t test for unpaired samples, with
avidin DH-biotinylated horseradish peroxidase. After a final significance assigned to values below p < 0.05.
wash in Tris-buffered saline, the slides were incubated for 2
min in a solution containing 0.05% diaminobenzidine and
0.015% H202.Nuclear staining was carried out with hematoxyRESULTS
lin. The specificity of the immunohistochemical reaction was
lrnrnunohistochernical
analysis
for CTSE
established first by showing that the non-immune rabbit serum
CTSE
was
expressed
in
pancreatic
ductal adenocarcinomas
did not reveal positive staining. In addition, the specificity of
the antibody was evaluated by absorption controls. The diluted examined in the present study. CTSE was stained diffusely in
antibody was incubated with the recombinant human gastric the cytoplasm of the carcinoma cells (Fig. la). Normal ductal
CTSE in concentrations of up to 10 nmol/ml for 24 hr at 4°C cells were not stained (Fig. lb). CTSE was also expressed in
mucin-producing adenomas, intraductal papillary hyperplasia
and then applied to the tissue section.
and mucinous hyperplasia (Fig. lc, d ) . In absorption tests, the
Enzyme-linked irnrnunosorbent assay for CTSE
staining of CTSE was inhibited by the recombinant human
Serum was obtained from patients after an overnight fast, CTSE.
and was stored at -80°C until analyzed. Pancreatic juice was
collected from patients during ERCP with an injection of CTSE in the serum and the pancreatic juice
secretin and was stored at -80°C until analyzed. CTSE in the
Figure 2 shows the standard curve of the ELISA. CTSE at a
pancreatic juice was assayed by sandwich ELISA. The capture
antibody was a mouse anti-human gastric CTSE monoclonal concentration as low as 1.75 ng/ml could be detected. We also
antibody (MAb) obtained from mouse ascites fluid (Samloff et examined the effects of exogenously added FCS or human
al., 1987), and the second antibody was the polyclonal rabbit pancreatic juice obtained from normal patients on the ELISA
anti-human CTSE antibody. Alkaline phosphatase-conjugated for CTSE. The presence of 10-100% (v/v) of FCS or pancreanti-rabbit IgG was used for detection in this assay, and the atic juice did not significantly alter the slope of the standard
recombinant human gastric CTSE was used to create the curve or the level of the background. The intraassay coefficient
standard curve (Yamada et al., 1994). Briefly, 96-well micro- of variation was 8.0% (n = 5 ) , and the interassay coefficient of
plates were coated with the capture antibody (100 p1 of variation was 11.0% (n = 5).
CTSE was not detected in the serum of the patients. In
500-fold dilution in PBS) at 4°C overnight. The plates were
then washed with PBS and blocked with 2% BSA at 4°C pancreatic juice, CTSE was present in 8 of 11 patients with
overnight. Serum or pancreatic juice samples (100 yl; if pancreatic ductal adenocarcinoma, 5 of 10 patients with
necessary samples were diluted in PBS) were allowed to mucin-producing adenoma, 1 of 3 patients with intraductal
remain at room temperature for 2 hr. The plates were washed papillary hyperplasia and 4 of 43 patients with chronic pancrewith PBS, and the second antibody (100 p1of 500-fold dilution atitis. The detection frequency of CTSE in the pancreatic juice
in PBS) was added and allowed to stand at room temperature was significantly higher in the patients with pancreatic ductal
for 2 hr. The bound antibodies were detected with sequential adenocarcinoma (72.7%) than in the patients with chronic
incubation with alkaline phosphatase-conjugated goat anti- pancreatitis (9.3%) (x2 test, x2 = 20.39, p < 0.005) (Fig. 3).
rabbit IgG and the enzyme substrate solution. The plates were Although quantitative analysis was not proper for CTSE in the
washed with PBS, and the second antibody (100 pl of 500-fold pancreatic juice, in the CTSE-positive cases the ratio of CTSE
dilution in PBS) was added and allowed to stand at room to secretory protein concentration in the pancreatic juice was
temperature for 2 hr. The bound antibodies were detected higher in the patients with pancreatic ductal adenocarcinoma
with sequential incubation with alkaline phosphatase-conju- than in the patients with chronic pancreatitis, mucingated goat anti-rabbit IgG and the enzyme substrate solution. producing adenomas or intraductal papillary hyperplasia. Only
The plates were read on an ELISA reader at 405 nm.
one patient with chronic pancreatitis had a higher ratio than
The concentration of CA19-9 in the pancreatic juice was 30.0 pgl yg. In contrast, 5 of 11patients with pancreatic ductal
measured by a CA19-9 radioimmunoassay kit (Centocor, adenocarcinoma had values higher than 30.0 pg/ pg (Fig. 3).
Malvern, PA) according to the manufacturer’s instructions.
Protein in the pancreatic juice was measured by the Bradford CA19-9 in the pancreatic juice
method using a Bio-Rad protein assay (Richmond, CA). The
The ratio of CA19-9 to secretory protein concentration is
concentrations of CTSE and CA19-9 in the pancreatic juice
shown
in Figure 4. The mean of the ratio was higher in the
were expressed as pg/pg protein and U/yg protein, respecpatients with pancreatic ductal adenocarcinoma [41.45 2 76.88
tively.
U/pg (mean % SD)] than in the patients with chronic pancreatitis (4.15 2 8.66 Ulyg), in the patients with mucinWestern blot analysis
The pancreatic juice collected from the patients with pancre- producing adenomas (2.25 f 1.86 U/p,g) and in the patients
atic cancer was also subjected to SDS-PAGE, which was with papillary hyperplasia (2.29 r 2.46 U/kg). However, the
performed following the procedure of Laemmli (1970). For difference was not significant.
immunoblotting, proteins electrophoresed on SDS-PAGE gels
were transferred to nitrocellulose using the transblot appara- Western blot anaIysis of CTSE in pancreatic juice
tus (BioRad, Richmond, CA) according to the company’s
By Western blot analysis, CTSE was also detected in the
specifications. The transferred proteins were probed with the pancreatic juice from the patients who were positive in the
mouse anti-human CTSE MAbs. Goat anti-mouse IgG coupled ELISA. The molecular size of the CTSE in the pancreatic juice
to horseradish peroxidase was used as the second antibody of the patients was 84 kDa, which was larger than that of CTSE
followed by development using the ECL system ( h e r s h a m , purified from gastric mucosa and the recombinant CTSE (82
Aylesbury, UK) according to the company’s specifications.
kDa) (Fig. 5).
494
AZUMA ETAL.
FIGURE1 - Immunohistochemical staining for CTSE. CTSE was stained diffusely in the cytoplasm in the pancreatic ductal
adenocarcinoma (a). Normal ductal cells were not stained (b). The positive staining was also observed in the mucinous cell hyperplasia
(c) and intraductal papillary hyperplasia (d). Scale bar = 10 km.
OD
405nm
I
0.5t
FIGURE
2 - Standard curve of the ELISA for CTSE. Each point
was the mean of quintuple determinations.
DISCUSSION
Our immunohistochemical analysis showed that all human
pancreatic ductal adenocarcinomas expressed CTSE. Sessa et
al. (1990) also reported that most pancreatic ductal carcinomas express one or more antigens normally found in gastrointestinal epithelial cells, and that the antigens expressed most
commonly, M1 (an antigen associated with the peptide core of
much in gastric superficial-foveolar cells) and CTSE, were
each found in over 90% of the 88 differentiated ductal cell
carcinomas. Both antigens are constituents of normal gastric
superficial-foveolar cells. Therefore, we examined whether
CTSE could serve as a marker for pancreatic ductal carcinomas. The inherent problems for characterization of CTSE
have involved the difficulty in purification of the protein and
the inability to assay its activity distinct from other aspartic
proteinases. The lower concentration of this proteinase compared with that of the pepsinogens also contributed to the
relatively slow progress in the characterization of the enzyme.
Previously, we successfully isolated human CTSE cDNA clones
(Azuma et al., 1989) and established a CTSE production
system by the methylotrophic yeast Pichia pastoris (Yamada et
al., 1994). Using recombinant human CTSE, we established an
assay system for CTSE. CTSE was not detected in the serum of
the patients with pancreatic ductal adenocarcinoma. By contrast, CTSE in the pancreatic juice was present in 72.7% (8/11)
of the patients with pancreatic ductal adenocarcinoma. Although quantitative analysis of the CTSE in the pancreatic
juice was difficult in the present study, the increased detection
frequency of CTSE in the pancreatic juice was significantly
higher in the patients with pancreatic ductal adenocarcinoma
than in the patients with chronic pancreatitis.
Several reports have described the usefulness of CA19-9
detected in pancreatic juice. Chen et al. (1989) reported that
the CA19-9 content in the pancreatic juice was significantly
CATHEPSIN E IN PANCREAS TUMORS
495
CTSE
( P Q I 114
160
50
140
120
40
100
80
30
60
40
20
20
0
n-11
FIGURE
3 - Ratio of CTSE to secretory protein concentration in
the pancreatic juice. CTSE in the pancreatic juice was present in 8
of 11 (72.7%) patients with pancreatic ductal adenocarcinoma, 5
of 10 (50%) patients with mucin-producing adenoma, 1 of 3
(33.3%) patients with intraductal papillary hyperplasia and 4 of 43
(9.3%) patients with chronic pancreatitis.
increased in pancreatic cancer patients, whereas in chronic
pancreatitis patients it was normal, indicating that it is a
specific and valuable tumor marker in the differential diagnosis
of pancreatic cancer. In contrast, Schmiegel et al. (1985) have
shown that although the average juice CA19-9 concentration
was significantly higher in patients with pancreatic cancer than
in chronic pancreatitis patients, there was considerable overlap among the groups. Tatsuta et al. (1985) found that assay of
CA19-9 pancreatic juice concentration was not useful for the
diagnosis of pancreatic cancer, since no significant difference
was noted between the pancreatic cancer and chronic calcified
pancreatitis groups. However, Malesci et al. (1987) observed
that although the absolute CA19-9 juice concentration was not
likely to discriminate between patients with pancreatic cancer
and those with benign chronic pancreatic disorders, when the
CA19-9itotal protein concentration ratio was calculated, the
ratio in only 1 of 22 pancreatic cancer cases overlapped with
those found in non-neoplastic disorders. Additionally, they
have proposed that the measurement of the CA19-9itotal
protein concentration ratio in the pancreatic juice may be an
accurate diagnostic marker for pancreatic adenocarcinoma. In
the present study there was considerable overlap among the
groups of patients, although the ratio of CA19-9 to secretory
protein concentration was higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic
pancreatitis. The definitive diagnosis between pancreatic cancer and pancreatitis remains difficult. CTSE in the pancreatic
juice may be a useful marker for such distinction. However, our
work is a preliminary report in a small number of patients.
Further analysis with larger numbers remains necessary.
Multistep models of tumorigenesis, i e . , colon cancer, are
based on a defined progression from atypical hyperplasia and
dysplasia through benign tumors to malignant invasive tumors
(Vogelstein et al., 1988). Changes in the ductal epithelium,
ranging from papillary hyperplasia and atypia to carcinoma in
10
.
.
m
adenocarcin=1 1
n=10
n=43
FIGURE
4 - Ratio of CA19-9 to secretory protein concentration
in the pancreatic juice. There was considerable overlap among the
groups, although the ratio was higher in the patients with pancreatic ductal adenocarcinoma than in the patients with chronic
pancreatitis.
situ, are frequent in cases of pancreatic ductal adenocarcinoma
(Chen et al., 1985; Cubilla and Fitzgerald, 1975; Morohoshi et
af., 1989). The multistep carcinogenesis theory in the case of
pancreas has been also proposed to occur via c-Ki-ra5 oncogene activation. The development of human pancreatic ductal
adenocarcinoma has been shown to be associated with the
c-Ki-rus oncogene activating mutation at high frequencies,
ranging from 75 to 100% (Almoguera et al., 1988; Smit et nl.,
1988; Tada et al., 1991). Mutation has been also shown to be a
frequent event in pancreatic ductal adenocarcinomas induced
by the carcinogen N-nitroso bis(2-oxopropy1)amine in Syrian
golden hamsters. The mutations were detected in 26% of
hyperplasia, 46% of papillary hyperplasia, 76% of carcinoma in
situ and 80% of adenocarcinomas (Cerny et al., 1992). These
findings suggest that mutation is an early event in pancreatic
multistep carcinogenesis. In the present study, CTSE in the
pancreatic juice was detected in the patients with mucinproducing adenomas (5 of 10). and in the patients with
intraductal papillary hyperplasia (1 of 3). By immunohistochemistry, CTSE was also expressed in mucin-producing adenomas,
intraductal papillary hyperplasia and mucinous cell hyperplasia of ductal epithelium. These findings suggest that expression
of CTSE is an important event in the pathogenesis of pancreatic ductal adenocarcinoma and that CTSE expression is an
event occurring at a relatively early stage of multistep carcinogenesis in the pancreas lesions in a similar way as with the
c-Ki-ras mutation.
CTSE is an intracellular aspartic proteinase consisting of 2
identical subunits with a molecular mass of about 42 kDa.
496
AZUMA E T A L
FIGURE
5 -Western blot analysis of CTSE in the pancreatic
juice. The molecular size of the CTSE in the pancreatic juice of the
patient with ductal adenocarcinoma was 84 kDa (lane 2), which
was larger than that of the recombinant CTSE (82 kDa) (lane I).
CTSE is known to be a glycoprotein and is considered to
contain oligosaccharide chains of both the high mannose and
the complex types (Yamamoto et al., 1978; Yonezawa et al.,
1990; Finley and Kornfeld, 1994; Fowler et al., 1995). There is
suggestive evidence that CTSE is synthesized as a high
molecular mass precursor and subsequently processed to the
mature form by the autocatalytic release of a NH2-terminal
prosegment. This suggests that CTSE is initially synthesized on
membrane-bound ribosomes and then transported to the
Golgi complex in a manner similar to lysosomal enzymes
(Athauda et al., 1991). Previously, we have reported the
expression of human CTSE cDNA in Chinese hamster ovary
(CHO) cells and the intracellular localization and processing
of the recombinant enzymes; we suggested that the recombinant CTSE was initially synthesized on membrane-bound
ribosomes as an N-glycosylated preproenzyme and that, after
cleavage of the signal segment, the 90 kDa proenzyme was
proteolytically processed to the intermediate (84 kDa) and
mature (82 kDa) forms by the transport system (Tsukubaet al.,
1993). Finley and Kornfeld (1994) also examined the subcellular localization and targeting of CTSE using murine L cells and
monkey Cos 1 cells that were transfected with CTSE cDNA,
and reported that the intracellular location of CTSE was the
endoplasmic reticulum. An interesting question is why CTSE is
present in the pancreatic juice of the patients with ductal
adenocarcinoma at a high frequency. CTSE is an intracellular
proteinase and is not secreted from cells. In this study, the
molecular weight of the CTSE in the pancreatic juice (84 kDa)
was larger than that of the mature form (82 kDa), and the
mature form was not detected in the pancreatic juice. These
findings suggest that the processing of the enzyme expressed in
pancreatic ductal tumors is different from that in normal
gastric superficial epithelial cells. The large molecular size
enzyme in the pancreatic juice may be tumor specific, and the
aberrant processing of CTSE might change the intracellular
localization of the enzyme, causing it to be released from
pancreatic tumor cells. Further analysis of the processing of
the CTSE in pancreatic ductal tumors and the intracellular
localization of the CTSE in the pancreatic ductal tumor cells is
necessary to answer these questions.
ACKNOWLEDGEMENTS
This work was supported by the grant-in-aid for General
Scientific Research no. 06670531.
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