Molecular Human Reproduction Vol.11, No.5 pp. 351–355, 2005
Advance Access publication April 29, 2005
doi:10.1093/molehr/gah172
Role of cathepsins and cystatins in patients
with recurrent miscarriage
Tamao Nakanishi1,4, Yasuhiko Ozaki1, Klas Blomgren2, Hisashi Tateyama3,
Mayumi Sugiura-Ogasawara1 and Kaoru Suzumori1
1
Departments of Obstetrics and Gynecology and 32nd Pathology, Nagoya City University Medical School, 1-Kawasumi, Mizuho-cho,
Mizuho, Nagoya, 467-8601, Japan and 2Perinatal Center, Institute of Physiology and Pharmacology, Göteborg University,
P.O.Box 432, SE-405 30, Göteborg, Sweden
4
To whom correspondence should be addressed: E-mail: [email protected]
In the implantation, trophoblasts penetrate maternal decidua by secreting proteases. It has been reported that cathepsins are
highly expressed in the mouse villi, and play an important role in normal embryonal growth and decidualization. In this study,
we evaluated cathepsins and their endogenous inhibitors, cystatins, in tissue and serum of patients with recurrent miscarriage.
Decidua and villi were surgically collected from 22 patients and 12 healthy women. Immunohistochemistry was performed
with antibodies against cathepsins, stefin A (cystatin A), stefin B (cystatin B) and cystatin C. The concentrations of cathepsins,
stefins and cystatin C were measured by Enzyme-linked immunosorbent assay. In addition, we measured the serum level of
cystatin C in 85 Japanese women with recurrent miscarriage. Staining of cathepsin B, D, H, L, stefin B and cystatin C was
observed in the cytoplasm of epithelial cells in decidua. Stefin A was expressed on the surface of the trophoblast. The concentration of cathepsin B and H in patients’ decidua was significantly higher than in control individuals. The serum level of
cystatin C was significantly lower in patients than in control individuals. Our findings suggest that the regulation of the
cathepsin –cystatin system may play an important role in patients with recurrent miscarriage.
Key words: cathepsin/cystatin/decidua/recurrent miscarriage/villi
Introduction
Implantation of the hatched blastocyst requires several steps including apposition, attachment, penetration and trophoblast invasion into
the endometrium during early pregnancy. Embryo development
follows those implantation steps, which involve various mechanisms
that have not been well clarified. It is known that failure of any of
these mechanisms may cause miscarriage. However, there are few
reports about the possible relation between proteases and
miscarriage.
Cathepsins belong to the family of lysosomal cysteine proteases
and are active in acidic environments. They have the ability to
degrade matrix molecules and they play an important role in intracellular proteolysis (Kirschke et al., 1998). Cathepsins have been
found to be involved in a variety of pathological conditions, such as
rheumatoid arthritis (Lenarcic et al., 1988), Alzheimer’s disease
(Henskens et al., 1996), asthma (Cimerman et al., 2001) and cancer
invasion and metastasis (Ebert et al., 1994; Kos et al., 2001). They
are regulated by their endogenous inhibitors, stefins, cystatins and
kininogens (Lah and Kos, 1998). We have previously reported that
cathepsin B and cystatin C may contribute to the mechanism of
invasion by ovarian cancer (Nishikawa et al., 2004).
The cystatins constitute a protein superfamily of enzyme inhibitors. Their main function is to ensure protection of cells and tissues
against the proteolytic activity of lysosomal peptidases that are
released during normal cell death, or intentionally by proliferating
cancer cells or by invading organisms, such as parasites. The type 1
cystatins, stefin A (cystatin A) and B (cystatin B), are mainly intracellular, the type 2 cystatins, such as cystatin C, are extracellular
and the type 3 cystatins are intravascular proteins (Abrahamson
et al., 2003). Cystatin C is the most widely studied cysteine protease
inhibitor of low molecular weight (13 kDa) and has been found in a
wide variety of human tissues and mainly in extracellular
body fluids, such as seminal plasma, urine and blood plasma
(Abrahamson et al., 1986, 1990; Jiborn et al., 2004).
Imbalance between cathepsins and cystatins has been associated
with various diseases and it has been suggested that their levels may
be useful for prognosis and diagnosis, especially of cancer (Ebert
et al., 1994; Henskens et al., 1996). Cathepsin and cystatin levels
were significantly different between asthma patients and normal
control individuals (Cimerman et al., 2000, 2001). This might reflect
airway inflammation and be related to abnormal cellular or extracellular protein metabolism, cells and tissues.
It has been reported that cathepsin B and L and cystatin C are
necessary for normal embryonic development and uterine decidualization in mice and that the expression of cystatin C was controlled
in coordination with implantation (Afonso et al., 1997). It has been
reported that cathepsin L has a significant function in the mouse
placenta (Hamilton et al., 1991). Using the cDNA microarray technique, cathepsin L was found to be expressed at a higher level in
human decidua than in villi (Chen et al., 2002).
Jokimaa et al. (2001) measured the mRNA of cathepsin B, H and
K in human endometrium. The mRNA levels of cathepsins H and K
were found to be significantly decreased in the mid-secretory phase
in comparison with the proliferative phase. Cysteine proteases are
expressed during the implantation window and may well play a role
in the differentiation of the endometrial stroma. The mRNA level of
Molecular Human Reproduction q The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and
Embryology. All rights reserved. For Permissions, please email: [email protected]
351
T.Nakanishi et al.
cathepsin H was significantly higher in the mid-secretory phase
endometrium of recurrent miscarriage than in normal endometrium
(Jokimaa et al., 2002).
It is necessary to investigate the practical function of those
proteinases and their inhibitors in early pregnancy. However, few
reports have evaluated the protein level in human early pregnancy
samples from patients with recurrent miscarriage. We evaluated the
protein level and assessed the role of cathepsins and cystatins in
patients with recurrent miscarriage.
Figure 1. Continued
Materials and methods
Study subjects
All patients were managed at Nagoya City University Hospital as patients
with recurrent miscarriage from April 2000 to October 2003. The patients
had a history of two or more spontaneous miscarriages. Hysterosalpingography, chromosome analysis for both partners, immunological tests for
parameters such as natural killer cell activity, b2 glycoprotein I-dependent
anticardiolipin antibodies and lupus anticoagulant, blood tests for hyperthyroidism, diabetes mellitus, hyperprolactinemia and infections such as
chlamydia, were performed on all patients before subsequent pregnancy. The
control group consisted of women without obstetrical complications or any
history of miscarriage. Informed consent, approved by the Institutional
Review Board, was obtained from all subjects before collection of any
materials.
Tissue samples
Figure 1. Immunohistochemistry for cathepsins and cystatins using a sample
from a patient. The binding of antibodies to cathepsins and cystatins was
detected as a brown precipitate using the streptavidin –biotin–immunoperoxidase method. Cathepsin B in decidua (A) and villi (B), cathepsin D in
decidua (C) and villi (D), cathepsin H in decidua (E) and villi (F) and cathepsin L in decidua (G) and villi (H), stefin A in decidua (I) and villi (J). Stefin B in decidua (K) and villi (L), cystatin C in decidua (M) and villi (N)
negative control in decidua (O) and villi (P), and haematoxylin–eosin staining in decidua (Q) and villi (R). Arrows show positive staining of each localization: a ¼ glandular cell of decidua; b ¼ decidual cell; c ¼ surface
epithelia of trophoblast of the villi. No remarkable difference was observed
in the staining pattern of any antibody between patients with recurrent miscarriage and controls. Scale bar in Figure 1R [for Figures from 1(A) to 1(R)]
shows 50 mm.
352
We collected the samples surgically from 22 patients diagnosed with missed
abortion and with recurrent miscarriage at Nagoya City University Hospital.
Control samples were obtained from 12 women with normal pregnancies,
undergoing elective surgical abortions. The mean age in the patient group
was 33.1 ^ 4.65 years (range 25–39). The average gestational week was
8.55 ^ 1.47 (range 6–12) in the patient group and 7.83 ^ 1.40 (range 6– 10)
in the control group.
Sera
Venous blood was collected from non-pregnant women: 85 patients and 32
controls. Serum was obtained by centrifugation and stored at 2408C until
use.
Cathepsins and cystatins in recurrent miscarriage
Immunohistochemistry
Samples were fixed in neutral buffered formaldehyde (‘Formalin neutral buffer water 10’, Ken-ei Seiyaku Ltd., Osaka, Japan) immediately after surgery.
The following primary antibodies were used for immunohistochemistry: anticystatin C (rabbit polyclonal immunoglobulin G, 10 mg/ml, Upstate Biotechnology, Lake Placid, NY, USA), anti-stefin A (C5/2, 5 mg/ml, KRKA, d.d.,
Novo mesto, Slovenia), anti-stefin B (A6/2, 20 mg/ml, KRKA, d.d.), anticathepsin B, D and H (goat, 2 mg/ml; Santa Cruz Biotechnology Inc., Santa
Cruz, CA, USA) and anti-cathepsin L (rabbit, 4 mg/ml; Santa Cruz Biotechnology, Inc.).
Formaldehyde-fixed and paraffin-embedded samples were cut into 4 mm
sections and collected on MAS coated glass slides (Matsunami Glass Ind.
Ltd., Osaka, Japan). Immunohistochemical staining was performed using the
streptavidin– biotin–immunoperoxidase method using Histofine immunostaining kits (Nichirei, Tokyo, Japan). To block endogenous peroxidase
activity, the slides were incubated with 3% H2O2 in phosphate-buffered
saline (PBS; WAKO Pure Chemical Industries Ltd., Osaka, Japan) for
10 min. Next, the sections were exposed to normal serum of another species
to block non-specific staining. After washing in PBS, the sections were incubated with primary antibodies diluted in PBS containing 1% bovine serum
albumin (WAKO Pure Chemical Industries Ltd.) at 48C overnight. Peroxidase activity was visualized with diaminobenzidine, followed by a light
counterstain with haematoxylin. For negative controls, the primary antibodies
were substituted with normal serum of appropriate concentrations.
Enzyme-linked immunosorbent assay
We separated decidua from villi macroscopically in buffer-I [20 mM Tris–
HCl, 5 mM EDTA-2Na, 1 mM EGTA, 10 mM 2-mercaptoethanol, 10 mM
p-APMSF, 150 mM NaCl, 0.25% protease inhibitor cocktail (Sigma, St
Louis, MO, USA) pH 7.5] and stored the tissues at 2808C until use.
The samples were sonicated in buffer-II [20 mM Tris– HCl, 5 mM EDTA,
1 mM EGTA, 10 mM 2-mercaptoethanol, 10 mM p-APMSF, 8 mM CHAPS,
0.25% protease inhibitor cocktail (Sigma) pH 7.5] using a Branson sonifier
(Branson Ultrasonic Corporation, Danbury, CT, USA) at a duty cycle of
50% and output control at 5 for 1 min at 48C. The samples were centrifuged
at 12 000 g for 30 min at 48C, the supernatant fraction was collected
and the protein concentration was measured by reading the absorbance at
235 and 280 nm using a plate reader (Whitaker and Granum, 1980).
Measurement of cathepsins and cystatins in tissue and cystatin C in serum
was performed using Enzyme-linked immunosorbent assay (ELISA) kits
(Schweiger et al., 1997; Cimerman et al., 2000; Kos et al., 2000) (cathepsin
B, H, L, stefin A, B and cystatin C; KRKA d.d., cathepsin D; Oncogene
Research Products, San Diego, CA, USA) as suggested by the manufacturer.
Each assay was performed in duplicate. A microplate reader (SPECTRAmax340, Molecular Devices, Sunnyvale, CA, USA) was used to
measure the absorbance.
Statistical analysis
We compared the level of each cathepsin and cystatin in patients with controls, and the level of each cathepsin and cystatin in decidua with villi in
each cathepsins and cystatins. Also, we compared the serum cystatin C level
in patients with controls. Student’s unpaired t-test or Welch’s t-test was
applied for statistical comparison of differences using DA Stat, StatView-J
4.11 and an Apple Macintosh computer (OS 9.2). We chose the more appropriate analysis for each analysis. Statistical significance was assumed when
P , 0.05 was obtained.
Results
Immunohistochemistry
Immunostaining for cathepsins and cystatins in human decidua and
trophoblast sample revealed a widespread distribution of all these
proteins (Figure 1). No major difference was observed in the staining pattern of any antibody between recurrent miscarriage patients
and controls, although there were some differences in the staining
intensity of each antibody. Among these antibodies, cathepsin D
showed the strong immunostaining in the cytoplasm of the glandular
and surface epithelia and decidual cells of the endometrium and
trophoblasts of the villi in a granular pattern. The other cathepsins
(B, H and L) showed positive immunostaining in a similar pattern
as cystatin C: these were also detected in the cytoplasm of glandular
and surface epithelia and decidual cells of the endometrium and
trophoblasts of the villi. Stefin B expression was found in glandular
and stromal cells of decidua, but not found in the trophoblast. Stefin
A was found on the surface of the trophoblast, but not in decidua.
Enzyme-linked immunosorbent assay
Tissue samples
The level of each cathepsin, stefin and cystatin in human decidua
and villi are shown in Table I. The average cathepsin B and H level
in decidua was significantly higher in the patient group than in
controls. We also evaluated the difference in the level of each cathepsin, stefin and cystatin between villi and decidua. The concentration of cathepsin D and L were significantly higher in villi than
in decidua. On the other hand, the stefin B level was higher in
decidua than in villi. The level of cystatin C in patients was higher
in decidua than in villi. The stefin A level was out of range for the
ELISA analysis.
Sera
The average serum concentration of cystatin C was
50.63 ^ 14.70 nmol/l in the patient group and 60.84 ^ 18.59 nmol/l in
the control group. The serum level of cystatin C was significantly
lower in the patient group than in the control group (Table II).
Table I. The concentration of cathepsins, stefins and cystatin C in human decidua and trophoblast
Patients
Decidua
Villi
Controls
Decidua
Villi
Cathepsin B
(pmol/ml)
Cathepsin D
(ng/ml)
Cathepsin H
(pmol/ml)
Cathepsin L
(pmol/ml)
Stefin A
(pmol/ml)
Stefin B
(pmol/ml)
Cystatin C
(pmol/ml)
144.5 ^ 74.48a
(n ¼ 14)
93.28 ^ 43.69
(n ¼ 5)
6.99 ^ 4.19b
(n ¼ 15)
11.78 ^ 4.44b
(n ¼ 5)
19.73 ^ 6.04d
(n ¼ 15)
17.96 ^ 6.81
(n ¼ 5)
42.96 ^ 14.65e
(n ¼ 14)
67.62 ^ 20.09e
(n ¼ 5)
Out of range
(n ¼ 16)
Out of range
(n ¼ 5)
57.09 ^ 23.85g
(n ¼ 14)
20.08 ^ 10.41g
(n ¼ 5)
19.98 ^ 10.80i
(n ¼ 14)
9.12 ^ 3.17i
(n ¼ 5)
89.22 ^ 33.97a
(n ¼ 9)
117.62 ^ 59.8
(n ¼ 10)
4.98 ^ 2.16c
(n ¼ 10)
10.67 ^ 3.94c
(n ¼ 10)
13.11 ^ 1.77d
(n ¼ 9)
12.09 ^ 1.80
(n ¼ 9)
56.54 ^ 24.22f
(n ¼ 10)
95.21 ^ 37.82f
(n ¼ 10)
Out of range
(n ¼ 10)
Out of range
(n ¼ 10)
49.20 ^ 22.6h
(n ¼ 10)
22.73 ^ 10.30h
(n ¼ 10)
14.70 ^ 8.81
(n ¼ 8)
7.99 ^ 2.87
(n ¼ 9)
The concentration of cathepsins, stefins and cystatin C in human decidua and villi was measured using ELISA. Patients and controls are explained in Materials
and methods. The results are shown as mean ^ standard deviation (n ¼ number of samples).
a,b,c,d,e,f,g,h,i
P , 0.05.
353
T.Nakanishi et al.
Table II. The concentration of cystatin C in serum (nmol/l)
Patients (n ¼ 85)
Controls (n ¼ 32)
50.63 ^ 14.70
60.84 ^ 18.59
P , 0.05
The concentration of serum cystatin C was measured using ELISA. Patients
and controls are explained in Materials and methods, samples collected
from non-pregnant patients and controls. The results are shown as
mean ^ standard deviation (n ¼ number of samples).
Discussion
There have been few papers published studying the correlation
between proteases and miscarriage. Previous studies have revealed
that tumour and trophoblast invasiveness are mediated by shared
factors, like metalloproteases and laminin (Divya et al., 2002).
Many investigators have revealed that cathepsin B and L in tissue
were related to various aspects of cancer: especially metastasis
(Sloane et al., 1981; Lah et al., 1995). Based on our hypothesis that
some proteases relate to miscarriage and that an imbalanced cathepsin – cystatin system plays an important role in miscarriage, we evaluated for the first time the protein expression of cathepsins and
cystatins in human decidua and villi from patients with recurrent
miscarriage.
First of all, we were able to confirm the presence and determine
the localization of cathepsins and cystatins in human decidua and
trophoblast using immunohistochemistry. Our work revealed that
cathepsin B, D, H, L and cystatin C are expressed in human decidua
and villi, stefin A is expressed on the surface of the trophoblast and
stefin B is expressed in decidua. These findings are in accordance
with the fact that cathepsin B and L and cystatin C were demonstrated in the mouse trophoblast as shown by Northern blot analysis,
Western blotting, immunohistochemistry and in-situ hybridization
(Afonso et al., 1997, 2002). The localization of cathepsin B and L
and cystatin C in human decidua and villi were the same as in mice,
namely in epithelial gland cells, decidualizing stroma and on the
surface of trophoblast (Afonso et al., 1997).
There were some reports about the expression of various cathepsins and cystatins in mouse decidua and human endometrium
(Hamilton et al., 1991; Afonso et al., 1997). We could observe
these cathepsins and cystatins in human decidua and trophoblast,
suggesting that they have some function such as in differentiation
or proliferation in those tissues. However, we could not find any
remarkable difference in the staining pattern of any antibody
between recurrent miscarriage patients and controls. Both cathepsin
B, D, H and L and cystatin C were highly expressed on the surface
of cells of decidua and trophoblast. This indicates that those cathepsins and cystatin C interact between maternal decidua and fetal trophoblast. We speculate that cystatins of decidua control and regulate
the hyper invasiveness of trophoblast mediated by cathepsins to
maintain normal early pregnancy course.
Furthermore, we used the ELISA method to evaluate the
expression of cathepsins and cystatins in these tissues. The ELISA
analysis revealed that the concentrations of cathepsin B and H were
significantly higher in decidua of patients than in that of controls,
and that villi expressed cathepsin D and L more highly than
decidua. There was no significant difference in the cathepsin L level
of villi between control and patients group. This result speaks
against the previous study, which found, using the cDNA microarray
technique, that cathepsin L is high in human decidua (Chen et al.,
2002). However, we think that in the field of proteases, evaluation
of the protein level using the ELISA method is more relevant than
measuring the mRNA level.
354
It is widely known that cathepsin B plays an important role in
cancer metastasis. In this study, the cathepsin B level of decidua
was significantly higher in patients than in controls. We speculate
that a high level of cathepsin B in decidua might cause a disordered
decidualiziation and dysfunctioning decidual cells. A damaged
decidua would impair the invasion by trophoblast.
In patients with recurrent miscarriage, the proliferative phase
endometrium expresses higher levels of cathepsin H mRNA than in
normal control individuals and in cases of sterility (Jokimaa et al.,
2002). In this study, we reveal that cathepsin H was high in decidua
of patients with recurrent miscarriage. These results might show that
the level of cathepsin H in patients with recurrent miscarriage is
originally high from non-pregnant phase to early pregnancy.
We also tried to measure the serum concentration of cystatin C
because it is an extracellular protein. In this work, there was no significant difference in the cystatin C level in tissue, but the level in
serum was significantly lower in patients than in controls. This
might reflect the characteristic of cystatin C as an extracellular protein. Cystatin C is secreted by tissue into the periphery and does not
remain in the tissue. Our data suggest that an imbalance in the cathepsin– cystatin system, especially cathepsin B and H in tissue and
cystatin C in serum, plays an important role in miscarriage.
This study shows that the serum level of cystatin C was significantly lower in the patient group than in the control group. This
imbalance in turn causes uncontrolled proteolysis and can lead to
hyper invasion of trophoblast or regulatory failure of the normal
pregnancy course, and would cause a cytotoxic interaction in tissue
resulting in miscarriage. We suggest that an imbalance between
cathepsins and cystatins causes failure to maintain the pregnancy
and results in embryonal loss. However, it is difficult to judge
whether the result causes miscarriage or miscarriage causes it.
In conclusion, we have demonstrated for the first time the protein
expression of cathepsins and cystatins in human decidua and villi
from patients with recurrent miscarriage. These results suggest that
regulation of cathepsin – cystatin system plays an important role in
recurrent miscarriage.
Acknowledgements
We thank Kazufumi Aoyama, MD, PhD (Nagoya Municipal Midori Hospital), Kenji Asamoto, MD (Asamoto Clinic, Nagoya), Mr Seizo Nagaya (2nd
Pathology, Nagoya City University Medical School), Ulrika Hallin, PhD
(Perinatal Center, Institute of Physiology and Pharmacology, Göteborg University) and PHT Rm4428 for their technical advices and supports. This
work was supported by a Grant-in-Aid (12671622) for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology,
Japan.
References
Abrahamson M, Barrett AJ, Salvesen G and Grubb A (1986) Isolation of six
cysteine proteinase inhibitors from human urine. Their physicochemical
and enzyme kinetic properties and concentrations in biological fluids.
J Biol Chem 261,11282–11289.
Abrahamson M, Olafsson I, Palsdottir A, Ulvsback M, Lundwall A, Jensson
O and Grubb A (1990) Structure and expression of the human cystatin C
gene. Biochem J 268,287–294.
Abrahamson M, Alvarez-Fernandez M and Nathanson CM (2003) Cystatins.
Biochem Soc Symp 70,179–199.
Afonso S, Romagnano L and Babiarz B (1997) The expression and function
of cystatin C and cathepsin B and cathepsin L during mouse embryo
implantation and placentation. Development 124,3415–3425.
Afonso S, Tovar C, Romagnano L and Babiarz B (2002) Control and
expression of cystatin C by mouse decidual cultures. Mol Reprod Dev
61,155–163.
Chen HW, Chen JJ, Tzeng CR, Li HN, Chang SJ, Cheng YF, Chang CW,
Wang RS, Yang PC and Lee YT (2002) Global analysis of differentially
Cathepsins and cystatins in recurrent miscarriage
expressed genes in early gestational decidua and chorionic villi using a
9600 human cDNA microarray. Mol Hum Reprod 8,475–484.
Cimerman N, Brguljan PM, Krasovec M, Suskovic S and Kos J (2000)
Serum cystatin C, a potent inhibitor of cysteine proteinases, is elevated in
asthmatic patients. Clin Chim Acta 300,83–95.
Cimerman N, Mesko Brguljan P, Krasovec M, Suskovic S and Kos J (2001)
Serum concentration and circadian profiles of cathepsins B, H and L, and
their inhibitors, stefins A and B, in asthma. Clin Chim Acta 310,113–122.
Divya, Chhikara P, Mahajan VS, Datta Gupta S and Chauhan SS (2002)
Differential activity of cathepsin L in human placenta at two different
stages of gestation. Placenta 23,59–64.
Ebert W, Knoch H, Wetlw B, Trefz G, Muley T and Spiess E (1994) Prognostic value of increased lung tumor tissue cathepsin B. Anticancer Res
14,895–899.
Hamilton RT, Bruns KA, Delgado MA, Shim JK, Fang Y, Denhardt DT and
Nilsen-Hamilton M (1991) Developmental expression of cathepsin L and
c-rasHa in the mouse placenta. Mol Reprod Dev 30,285–292.
Henskens YM, Veerman EC and Nieuw Amerongen AV (1996) Cystatins in
health and disease. Biol Chem Hoppe Seyler 377,71–86.
Jiborn T, Abrahamson M, Wallin H, Malm J, Lundwall A, Gadaleanu V,
Abrahamsson PA and Bjartell A (2004) Cystatin C is highly expressed in
the human male reproductive system. J Androl 25,564–572.
Jokimaa V, Oksjoki S, Kujari H, Vuorio E and Anttila L (2001) Expression
patterns of cathepsins B, H, K, L and S in the human endometrium. Mol
Hum Reprod 7,73–78.
Jokimaa V, Oksjoki S, Kujari H, Vuorio E and Anttila L (2002) Altered
expression of genes involved in the production and degradation of endometrial extracellular matrix in patients with unexplained infertility and
recurrent miscarriages. Mol Hum Reprod 8,1111– 1116.
Kirschke H, Barrett AJ and Rawlings ND (eds) (1998) Lysosomal Cysteine
Proteases. Oxford University Press, Oxford, 131 pp.
Kos J, Krasovec M, Cimerman N, Nielsen HJ, Christensen IJ and Brunner N
(2000) Cysteine proteinase inhibitors stefin A, stefin B, and cystatin C in
sera from patients with colorectal cancer: relation to prognosis. Clin
Cancer Res. 6,505–511.
Kos J, Sekirnik A, Kopitar G, Cimerman N, Kayser K, Stremmer A, Fiehn
W and Werle B (2001) Cathepsin S in tumors, regional lymph nodes and
sera of patients with lung cancer: relation to prognosis. Br J Cancer 85,
1193–1200.
Lah TT and Kos J (1998) Cysteine proteinases in cancer progression and
their clinical relevance for prognosis. Biol Chem 379,125– 130.
Lah TT, Calaf G, Kalman E, Shinde BG, Russo J, Jarosz D et al. (1995)
Cathepsin D, B and L in breast carcinoma and in transformed human
breast epithelial cells (HBEC). Biol Chem Hoppe Seyler 376,21–24.
Lenarcic B, Gabrijelcic D, Rozman B, Drobnic-Kosorok M and Turk V
(1988) Human cathepsin B and cysteine proteinase inhibitors (CPIs) in
inflammatory and metabolic joint diseases. Biol Chem Hoppe Seyler
369,257–261.
Nishikawa H, Ozaki Y, Nakanishi T, Blomgren K, Tada T, Arakawa A and
Suzumori K (2004) The role of cathepsin B and cystatin C in the mechanisms of invasion by ovarian cancer. Gynecol Oncol 92,881– 886.
Schweiger A, Stabuc B, Popovic T, Turk V and Kos J (1997) Enzyme-linked
immunosorbent assay for the detection of total cathepsin H in human
tissue cytosols and sera. J Immunol Methods 201,165–172.
Sloane BF, Dunn JR and Honn KV (1981) Lysosomal cathepsin B: correlation with metastatic potential. Science 212,1151–1153.
Whitaker JR and Granum PE (1980) An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal
Biochem 109,156–159.
Submitted on January 13, 2005; accepted on March 30, 2005
355