Note: Descriptions are shown in the official language in which they were submitted.
CA 02356189 2001-06-18
Substance for Obtaining Highly Effective Tumor Medications
as well as a Process
Description
The invention relates to a substance as well as a process
for obtaining anti-tumor agents.
Gastric carcinoma is one of the most common types of cancer
worldwide. According to Lauren in "The Two Histological Main
Types of Gastric Carcinoma," Acta Path Microbiol Scand; 64: 331-
49, they are histologically divided into diffuse adenocarcinomas
and intestinal adenocarcinomas. Intestinal gastric carcinomas
are often accompanied by chronic gastritis B and especially by
intestinal metaplasias, which are considered to be precursors of
dysplastic alterations and of gastric carcinomas. Differences
between these two types are also indicated in that patients with
carcinomas of the diffuse type often belong to blood group A,
from which it can be deduced that genetic factors influence the
risk of cancer, while environmental factors, e.g., a Helicobacter
pylori infection, are possibly of importance for the development
of carcinomas of the intestinal type. It is noted that gastric-
adenocarcinomas are becoming less common in the West but are now
on the rise in the East.
Up until now, therapy has been limited to gastrectomy and
lymphadenectomy, but because of the still poor prognosis, there
is a need for a new accompanying therapy. Immunological studies
have shown that even in cases in which the immune system cannot
CA 02356189 2001-06-18
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effectively control malignant cells, a cellular and humoral
activity can be measured, which is not sufficient, however, to
destroy the tumor cells. A more effective effort is now to
isolate the antibodies that originate from the immune response of
the patient, to reproduce them in a suitable manner and to use
them therapeutically. Thus, for example, antibodies that
originate from patients with lung, esophageal and colon cancer
were isolated, and human monoclonal antibodies that influence,
e.g., direct differentiation and the growth of the tumor cells,
but which in most cases have the problem of interaction with
other tumors or healthy cells, were derived from them.
It is known that human monoclonal SC-1 antibodies can
trigger apoptosis in gastric carcinoma cells (Vollmers et al.,
Cancer 49 (1989), 2471-2476). The antibody reacts with almost
all adenocarcinomas of diffuse type and about 20% of the
adenocarcinomas of intestinal type (Vollmers et al., Cancer 76
(1995), 550-558; Vollmers et al., Cancer 79 (1997), 433-440). In
clinical studies, it was found that antibody SC-1 is able to
induce a tumor-specific regression and apoptosis in primary
stomach cancer without toxic cross-reactivity relative to normal
tissue (Vollmers et al., Oncol. Rep. 5 (1998), 549-552).
Apoptosis is the programmed cell death, suicide of cells, by
fragmentation of the DNA, plasmolysis and dilatation of the
endoplasmatic reticulum, followed by cell fragmentation and the
formation of membrane vesicles, the so-called apoptotic elements.
Apoptosis, the physiological form of cell death, ensures a quick
and smooth removal of unnecessary cells without triggering
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inflammatory processes or tissue damages as in the case of
necrosis. Under pathological conditions, it is also used to
remove malignant cells, such as, for example, cancer precursor
cells. It can be triggered by the most varied stimuli, such as,
for example, by cytotoxic T-lymphocytes or cytokines, such as
tumor necrosis factors, glucocorticoids and antibodies. It is
the most common cause of death of eucaryotic cells and occurs in
embryogeneses, metamorphoses and tissue atrophy. Apoptotic
receptors on the cell surface, such as that of the NGF/TNF
family, are predominantly expressed in lymphocytes, but are also
found in various other cell types, and thus they are not suitable
for cancer treatment. In in-vivo tests, ligands and antibodies
for these receptors have led in particular to liver damage.
Tumor-specific receptors with an apoptotic function are therefore
especially important.
The cellular receptor of monoclonal antibody SC-1 was
previously not known. Within the scope of the studies that
resulted in this invention, it was possible to identify this
cellular receptor. This identification turned out to be
difficult, however. On the one hand, monoclonal antibody SC-1
reacts with its receptor only under quite specific stringency
conditions in the Western-blot analysis. On the other hand,
unspecific reactions that are caused by denaturation artifacts
are found with a number of other proteins.
The cellular receptor of antibody SC-1 is an isoform of the
protein CD55/DAF that is specific for tumor cells, especially for
gastric carcinoma cells (Medof et al., J. Exp. Med. 160 (1984),
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1558-1578; Caras et al., Nature 325 (1987), 545-549; Bjorge et
al., Int. J. Cancer 70 (1997), 14-25), which does not occur in
normal tissue. The specific receptor properties of this isoform
are based on a special glycostructure that is connected with the
protein backbone via an N-linkage. The tumor-specific receptor
can be used in a screening process for identifying specific
binding partners. Specific partners for binding to the receptor
are those substances within the meaning of this invention that
bind selectively to a tumor-specific glycostructure but not
significantly to a glycostructure of CD55/DAF that occurs in
normal cells and preferably have the ability to induce apoptosis.
These specific binding partners can be used for the production of
therapeutic agents for inducing apoptosis and/or for combatting
tumors as well as for the production of diagnostic agents.
The binding of antibody SC-1 to the tumor-specific N-linked
glycostructure of the CD55/DAF protein induces a tyrosine
phosphorylation of three proteins and the activation of caspase-3
and caspase-8. In addition, it was found that the apoptosis
induced by antibody SC-1 leads to a transient increase of the
presentation of tumor-specific N-glycosylated CD55/DAF on the
surface of tumor cells. This increased presentation can be
caused by an increased expression and/or by an increased
glycosylation. The tumor-specific N-glycosylated CD55/DAF
protein then disappears from the cell membrane by endocytosis.
In addition, a cleavage of cytokeratin 18, an increased
expression of c-myc and a reduction of the expression of
topoisomerase IIa and thus an at least partial cell cycle arrest
CA 02356189 2001-06-18
are observed. The apoptotic processes that are induced by SC-1
do not result in an increased cleavage of poly (ADP-ribose)-
polymerase (PARP). In addition, an increase of the intracellular
CaZ+ concentration, which is released from an intracellular Caz+
pool, is found. An inhibition of the CaZ+ release inhibits the
apoptosis that is induced by SC-1.
A first aspect of the invention relates to a glycoprotein
that comprises at least one section of the amino acid primary
structure of CD55/DAF, especially the membrane-bonded isoform
DAF-B and a glycostructure that is specific for tumor cells,
especially such a glycostructure that reacts with monoclonal
antibody SC-1. In SDS-polyacrylamide-gel electrophoresis (under
reducing conditions), such a glycoprotein that can be obtained
from, for example, human adenocarcinoma cell line 23132 (DSM ACC
201) or from other human adenocarcinoma cell lines, such.as.3051
(DSM ACC 270) or 2957 (DSM ACC 240) or from primary tumor cells
of gastric adenocarcinoma patients has an apparent molecular
weight of about 82 kD. In addition to this 82 kD of protein, the
invention also relates to variants with deletions, insertions
and/or substitutions in the amino acid primary structure, which,
however, have a glycostructure that is analogous to the natural
protein, i.e. tumor-specific and preferably reactive with
antibody SC-1.
The glycoprotein according to the invention can be obtained
by membrane preparations being produced from cells that express a
protein with the desired glycostructure, e.g., from cells of
human adenocarcinoma cell line 23132 or from other human
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adenocarcinoma cell lines, and the glycoprotein is obtained from
this by chromatographic processes, e.g., by size-exclusion and/or
anion-exchange chromatography. The production of the membrane
preparations is carried out preferably by lysis of cells in
hypotonic buffer, ultrasound treatment and subsequent separation
of the nuclei. The membrane preparations can be isolated from
the remaining extract by centrifuging and further purified by
chromatographic methods.
The tumor-specific CD55/DAF-glycoprotein can be used in a
test process, in which the ability of a substance to bind to the
tumor-specific glycoprotein, especially to its glycostructure, is
determined. The test process can be automated as a high-
throughput-screening process. In this respect, the glycoprotein
can be used in isolated form, as a cell extract, in particular as
a membrane preparation or in the form of complete cells, in
particular of human adenocarcinoma cell line 23132 or another
human adenocarcinoma cell line, or a heterologous eucaryotic cell
that is transformed with the CD55 gene, e.g., a mammal cell,
which is able to produce a protein with the correct
glycostructure. As a control, the binding of the tested
substance to a non-tumor CD55/DAF-glycoprotein can be examined,
which can be obtained from normal human cells or cell lines.
Substances that bind selectively to the tumor-specific
glycoprotein are suitable for the production of therapeutic
and/or diagnostic agents.
In addition, the ability of the tested substance to induce
apoptosis, especially in tumor cells, and/or the ability to
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induce a phosphorylation cascade that is mediated by CD55/DAF is
preferably determined. The induction of the apoptosis can be
performed by morphological cell studies, by apoptosis test
processes, e.g., by an adhesion test (Vollmers et al., Cell 40
(1985), 547-557) determining the keratin-1 and DNA-fragmentation,
or by proliferation tests such as the MTT-proliferation test. As
an alternative, a determination of caspase activities, for
example activities of caspase-3 and/or caspase-8 or a
determination of the intracellular free calcium concentration can
also be carried out. Substances that selectively induce an
apoptosis of tumor cells can be used as anti-tumor-action
substances. The induction of the phosphorylation cascade can be
monitored by use of antibodies that are specific for phosphorus
groups, e.g., phosphotyrosine and/or phosphoserine groups.
Pharmacologically compatible substances are suitably tested.
These include low-molecular pharmacological active ingredients,
but especially peptides, peptide mimetic agents, antibodies,
e.g., polyclonal, monoclonal or recombinant antibodies, antibody
fragments or antibody derivatives. Other examples of ligands of
the CD55/DAF receptor are aptamers (NexStar Pharmaceuticals, 2860
Wilderness Place, Boulder, Colorado 80301, USA) and enantiomers
(Noxxon Pharma, Gustav-Meyer-Allee 25, 13355 Berlin). Especially
preferred, for example, are recombinant antibodies, such as, for
example, single-chain scFv-antibodies, as they can be produced
in, for example, bacteria cells such as, for example, E. coli
(Pliickthun, Bio/Technology 9 (1991), 545-551 and bibliographic
references that are cited therein) or else in eucaryotic host
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cells (Reff, Curr. Opinion Biotech. 4 (1993), 573-576 and Trill
et al., Curr. Opinion Biotech 6 (1995), 553-560 or bibliographic
references that are cited therein). In addition, human
antibodies, i.e., antibodies with human constant domains, are
preferred, as they are produced in the human body, e.g., of
carcinoma patients, or chimera and humanized antibodies, in which
originally present non-human constant domains and/or framework
regions were exchanged by corresponding human areas. Examples of
antibody fragments are Fab-, F(ab)2- or Fab'-fragments, as they
can be obtained by proteolytic cleavage of antibodies. The
antibody derivatives include, for example, conjugates of
antibodies with labeling groups and/or effector groups, for
example toxic substances such as, for example, choleratoxin or
pseudomonas Exotoxin A or radioactive substances.
Another aspect of the invention is the use of substances
that bind specifically to tumor glycoprotein CD55/DAF according
to the invention (with the exception of already known monoclonal
antibody SC-1) for the production of the apoptosis-inducing
agents and/or for the production of anti-tumor agents and/or for
the production of agents for tumor diagnosis. A tumor-specific
or tumor-selective binding within the context of this application
preferably means that in immunohistochemical detection, a
substance reacts with tumor cells but not significantly with
other cells. An induction of the apoptosis within the context of
this application means an increase of the apoptosis index, i.e.,
the proportion of apoptotic cells after treatment with the
substance compared to the proliferating cells is higher than
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without treatment, preferably higher than 50~. The spontaneous
apoptosis index without treatment is significantly below 10%,
whereby the detection of proliferating cells can be done with
antigen Ki67.
Still another aspect of the invention is a process for the
preparation of agents that induce apoptosis and/or anti-tumor
agents and/or for the production of agents for tumor diagnosis,
whereby a potentially active substance is tested on its ability
for specific binding to a glycoprotein according to the
invention, and in the case of a positive test result, the
substance is converted into a form for dispensing that is
suitable for pharmaceutical applications optionally together with
commonly used adjuvants, additives and vehicles.
Suitable pharmaceutical forms for dispensing contain the
active ingredient in a therapeutically effective quantity,
especially in an anti-tumor-action quantity. The dose that is
administered to a patient and the treatment time depend on the
type and severity of the disease. Suitable dosages for the
administration of antibodies are described in, for example,
Ledermann et al. (Int. J. Cancer 47 (1991), 659-664) and Bagshawa
et al. (Antibody, Immunoconjugates and Radiopharmaceuticals 4
(1991), 915-922).
The active ingredient can be administered alone or in
combination with other active ingredients either simultaneously
or sequentially. In addition to the active ingredient, the
pharmaceutical composition can contain other pharmaceutically
common substances. The composition can be administered, for
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example, orally, nasally, via a pulmonary pathway or by
injection. Compositions that can be administered orally can be
present in the form of tablets, capsules, powders or liquids.
Compositions that can be administered by injection are usually in
the form of a parenterally compatible aqueous solution or
suspension.
In addition, the invention relates to a process for
combatting tumors, whereby an anti-tumor-action quantity of a
substance that can bind specifically to a glycoprotein according
to the invention with the exception of monoclonal antibody SC-1
is administered to a patient, especially a human patient.
Binding partners for tumor-specific CD55/DAF proteins can
also be used for diagnostic purposes, e.g., for tumor imaging.
Suitable methods for tumor imaging are described in, e.g.,
Steinstraesser et al. (Clinical Diagnosis and Laboratory Medicine
2 ((1989), 1-11). In this respect, the binding partners are
preferably used in the form of conjugates with labeling groups,
e.g., radioactive or fluorescent labeling groups. As an
alternative, the binding partners can also be incubated in
unconjugated form with the sample that is to be tested, and then
stained with a secondary binding reagent.
A subject of the invention is thus a process for the
diagnosis of tumors, whereby a sample that is to be tested, e.g.,
a bodily fluid or a tissue sample, or a patient can be brought
into contact with a substance that can be bonded to a tumor-
specific CD55/DAF glycoprotein, and the presence, the
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localization and/or the quantity of the glycoprotein in the
sample or in the patient can be detected.
The use of substances that specifically bind tumor
glycoprotein CD55/DAF to trigger a phosphorylation cascade is
also a subject of the invention. Still another subject of the
invention is the use of substances that bind specifically to
tumor glycoprotein CD55/DAF for transient increase of the
presentation of tumor glycoprotein CD55/DAF to the cell surface,
which can be caused by an increased glycosylation and/or
expression. The tumor-specific glycoprotein then disappears from
the cell surface. Still another subject of the invention is the
use of substances that bind selectively to tumor glycoprotein
CD55/DAF to increase the intracellular calcium level. Substances
that bind specifically to tumor glycoprotein CD55/DAF can also be
used as agents for cell cycle arrest. Finally, the invention
also relates to the use of substances that bind specifically to
tumor glycoprotein CD55/DAF to induce apoptotic processes that do
not include any cleavage of PARP. The substance can optionally
be used as conjugates with labeling groups and/or effector
.:;: ::
groups.
Still another subject of the invention is the use of
substances that bind specifically to tumor glycoprotein CD55/DAF,
especially antibody SC-1 for inducing apoptosis in dormant tumor
cells. As far as the inventor knows, this finding is not known
to date for any tumor-selective substance.
The substances that bind tumor-specific glycoprotein
CD55/DAF preferably contain multiple, i.e. at least two, binding
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sites for CD55/DAF. For example, the substances can contain
three, four, five, six, seven, eight, nine, ten or more binding
sites, so that a cross-linking is produced in binding to
intracellular tumor-specific CD55/DAF. To obtain substances with
multiple binding sites, binding molecules can optionally be
cross-linked. The cross-linking can be carried out by, for
example, chemical coupling with, e.g., bifunctional linker
molecules or with highly affine interactions, e.g.,
streptavidin/biotin. Even if the CD55/DAF binding molecules are,
for example, antibodies, e.g., IgG or IgM, that already contain
several binding sites, an improvement of the apoptosis induction
can be achieved by cross-linking with, e.g., anti-IgG or anti-IgM
antibodies. The use of cross-linked antibodies is therefore
preferred.
Cell line 23132 can be obtained from the Deutschen Sammlung
fur Mikroorganismen and Zellkulturen GmbH [German Collection of
Microorganisms and Cell Cultures Gmbh], Braunschweig [Brunswick],
under file number DSM ACC 201.
In addition, the invention is explained by the examples and
figures below. Here:
Figure 1 shows: the identification of antigens that are
reactive with antibody SC-1.
a, Purification of SC-1 antigens from
membrane extracts of gastric
carcinoma cell line 23132.
b. Sequencing of an 82 kD protein that
is identified as an SC-1 antigen by
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nanoelectrospray-tandem-mass
spectroscopy.
Figure 2 shows: the influence of a cleavage of GPI
anchors by phosphatidylinositol-specific
phospholipase C (PI-PLC) on a staining
with SC-1. Untreated gastric carcinoma
cells of cell line 23132 stained with
SC-1 (a) and anti-EMA (c); cells that
are treated with PI-PLC and stained with
SC-1 (b) and anti-EMA (d) (400 x
magnif ication) .
Figure 3 shows: the result of an MTT test with antibody
SC-1 in gastric carcinoma cells.
Control: untreated cells; SC-1: cells
treated with SC-1; SC-1, PIPLC: cells
treated with phospholipase and then with
SC-1.
Figure 4 shows: the result of an analysis of transient
transfixed cells with a CD55-antisense
w vector. Cells that were transfixed with
a control vector show a normal staining
pattern with SC-1 (a) and anti-CEA (c).
In cells that are transfixed with the
antisense vector, the staining with SC-1
is reduced (b), while no change in the
staining with anti-CEA (d) can be
detected.
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Figure 5 shows: the result of a Klenow fragmentation
test. Transfixed cells show no
apoptosis without induction with SC-1
(e) in comparison to a positive control
(f). After incubation with SC-1, the
cells that are transfixed with the
control vector indicate apoptosis (g),
while the majority of the cells that are
transfixed with the CD55 antisense
vector are resistant to apoptosis (h).
Figure 6 shows: a quantitative determination of the
apoptosis that is induced by SC-1.
Cells that were transfixed with the
control and the CD-55 antisense vector
were incubated with SC-1, and cytospins
of these cells were stained with the
Klenow DNA fragmentation kit. The
percentages of apoptotic cells were
determined by two different individuals
by counting apoptosis-positive and
negative cells in three different fields
with about 500 cells in each case.
Figure 7 shows: the action of a deglycosylation on the
binding of antibody SC-1.
a: Tumor cells incubated with buffer
and stained with SC-1;
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b: cells incubated with N-glycosidase
and with SC-1;
c: cells incubated with buffer and
anti-CD55 and
d: tumor cells incubated with N-
glycosidase and anti-CD55.
Figure 8 shows: the result of an MTT test with SC-1 in
gastric carcinoma cell line 23132.
a: Titration of SC-1;
b: Cross-linking of SC-1 with rabbit-
anti-human-IgM-antibodies;
Figure 9 shows: the change in intracellular calcium
concentration after induction of cell
line 23132 with SC-1. At point 1, the
addition of SC-1 or control antibodies
is carried out. At point 2, the cells
were washed with Ringer's solution.
Figure 10 shows: the expression and activity patterns of
caspase-3 and caspase-8 after induction
with SC-1.
a. Western-blot analysis of caspase-3
and caspase-8. The activation of
caspase-3 based on proteolytic
cleavage can be detected by the
production of the p20 cleavage
product.
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b. The result of an activity
determination of caspase-8. A
four-fold increase of caspase-8
activity was found 20 hours after
apoptosis was induced.
Figure 11 shows: the phosphorylation pattern of cell line
23132 after apoptosis is induced.
a: A quick phosphorylation of tyrosine
radicals in proteins with molecular
weights of about 110 kD and 60 kD
as well as the dephosphorylation of
a serine radical in a protein with
about 35 kD was found after
apoptosis was induced with SC-1.
b: An increase of phosphorylation of
a
tyrosine radical in a 75 kD protein
with a maximum after 10 minutes was
found after apoptosis was induced.
Figure 12 shows: an expression and mutation analysis of
p53.
a: 5 minutes after apoptosis was
induced by SC-1, a significant
increase of the mRNA concentration
was found, while the high p53
protein concentrations remain
unchanged.
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b: A sequential analysis of p53 showed
a mutation in codon 273, which
results in an amino acid exchange
from Arg to His.
Figure 13 shows: an expression analysis of p21.
After apoptosis is induced, an increase
of the p21 mRNA concentration was found.
Figure 14 shows: a Western-blot analysis of SC-1-induced
cells.
a: CD55/DAF expression (staining with
SC-1 )
b: cleavage of PARP (staining with
anti-PARP-antibodies)
c: staining with anti-topoisomerase
IIa-antibody as a marker for
cellular proliferation
d: c-myc expression (staining with
anti-c-myc-antibody)
Figure 15: the action of the caspase-3 inhibitor Ac-
:: DEVD-CHO on the SC-1-induced apoptosis.
Figure 16: the detection of a tumor cell-specific
apoptosis by in-situ nucleus staining
produced by administration of antibody SC-1
on a primary tumor.
Figure 17: the action of the administration of antibody
SC-1 on a primary tumor.
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a: Biopsy sample before administration of
SC-1 (in situ staining for apoptosis)
b: primary tumor after administration of
SC-1 (in situ staining for apoptosis)
c: biopsy before administration of SC-1
(histological regression analysis)
d: primary tumor after administration of
SC-1 (histological regression analysis).
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Examples
1. Material and Methods
1.1 Cell Culture
For all tests, the established gastric-adenocarcinoma cell
line 23132 was used (Vollmers et al., Virchows Arch. B. Zell.
Pathol. Incl. Mol. Pathol. 63 (1993), 335-343). The cells were
cultivated in RPMI-1640 with 10% fetal calf serum and
penicillin/streptomycin (both 1%) until a subconfluence occurred.
For the described test process, cells were dissolved with
trypsin/EDTA and washed twice with phosphate-buffered salt
solution (PBS) before use. Human hybridoma cell line SC-1 was
produced and cultivated as described in Vollmers et al. (Cancer
Res. 49 (1989), 2471-2476).
1 2 Purification of Antibody SC-1
Human monoclonal antibody SC-1 was purified from mass
cultures with use of cation-exchange chromatography followed by
gel filtration, as described in Vollmers et al. (Oncology Reports
(1998), 35-40).
1.3 Purification of the SC-1 Receptor
For preparation of membrane proteins, harvested cells in
hypotonic buffer (20 mM of HEPES, 3 mM of KC1, 3 mM of
MgClz) were resuspended, incubated for 15 minutes on ice and
ensonified for 5 minutes. The nuclei were pelletized by
centrifuging (10,000 g, 10 minutes). The membranes were
CA 02356189 2001-06-18
palletized by centrifuging (30 minutes, 100,000 g) and
resuspended in membrane lysis buffer (50 ~ of HEPES, pH 7.4,
0.1 mM of EDTA, 1 M of NaCl, 10% glycerol and 1% Triton X-100).
Complete~R~ protease inhibitor (Boehringer Mannheim, Germany) was
added to all solutions.
The purification of the antigens was carried out by column
chromatography with use of an FPLC unit (Pharmacia, Freiburg,
Germany). For size-exclusion chromatography, a Pharmacia
Superdex 200 column (XK 16/60) was loaded with 5 mg of membrane
protein preparation in buffer A (100 mM of Tris HC1, pH 7.5, 2
mM of EDTA, 40 mM of NaCl, 1% Triton X-100). The column
eluate was fractionated and studied in a Western-blot analysis in
a reaction with antibody SC-1. Positive fractions were loaded on
a monoQ-column with use of buffer A. The bonded proteins were
fractionated with a linear gradient with use of buffer B (100
mM of tris-HC1, pH 7.5, 1 M of NaCl, 2 mM of EDTA, 1% Triton
X100) and studied by SDS-polyacrylamide-gel electrophoresis and
staining with Coomassie or Western-blot analysis. Positive
strips were cut out from the gel and sequenced.
1.4 Preparation of Cell Lysates after Induction with SC-1
Cell line 23132 was cultivated in 100 mm cell culture dishes
until a subconfluence occurred. Antibody SC-1 was added in a
final concentration of 30 ~g/ml for the time period indicated in
each case. Then, the culture plates were washed once with PBS,
and the cells were lysed directly with SDS buffer (50 mM of
tris-HC1, pH 6.8, 10 mM of dithiothreitol, 2% (w/v) SDS, 10%
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(v/v) glycerol). The cell residues were collected with a rubber
scraper.
1 5 Gel Electrophoresis and Blots
The SDS-polyacrylamide-gel electrophoresis under reducing
conditions and the Western-blotting of proteins were performed
with use of standard protocols as described in Vollmers et al.
(Cancer 79 (1997), 433-440). Nitrocellulose membranes were
blocked with PBS with the addition of 0.1% Tween-20 and 2% skim
milk powder or 3% bovine serum albumin (for determination of
phosphorylation) and then incubated for one hour with the primary
antibody. The antibodies were used in the following dilutions:
SC-1 (human) l0 ~g/ml or 15 ~g/ml; anti-caspase-3 or -8 (goat)
(Santa Cruz, Heidelberg, Germany) 5 ~g/ml, streptavidin anti-
phosphotyrosine conjugate (clone PT-66) 1:20,000 and streptavidin
anti-phosphoserine conjugate (clone PSR-45) 1:30,000 (Sigma,
Munich, Germany), mouse-anti-topoisomerase IIa-antibody 1:1,000
(Neomarkers, Baesweiler, Germany), anti-c-myc-antibody 1:1,000
(Santa Cruz, Heidelberg, Germany) and anti-PARP-antibody 1:1,000
(Pharmingen, Heidelberg, Germany). The secondary antibody
peroxidase-rabbit-anti-human-IgM conjugate or rabbit-anti-goat-
antibody (Dianova, Hamburg, Germany) and peroxidase-conjugated
extravidin (Sigma) were detected with the SuperSignal
Chemiluminescence Kit of Pierce (KMF, St. Augustin, Germany).
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1 6 Protein Sequencing
A protein strip with an apparent molecular weight of 82 kD
was isolated by one-dimensional polyacrylamide gel
electrophoreses and made visible by staining with Coomassie. The
p82-strip was cleaved in the gel with trypsin (Boehringer
Mannheim, non-modified, sequencing quality) as described in
Shevchenko et al., (Anal. Chem. 68 (1996), 850-858). The non-
separated pool of tryptic peptides was sequenced by
nanoelectrospray-tandem-mass spectrometry as described by Wilm et
al. (Nature 379 (1996), 466-469). The sequencing was carried out
on an API III Triple Quadrupol Mass Spectrometer (PE Sciex,
Ontario, Canada). The sequences of the peptide fragments were
assembled with use of the tandem-mass spectrometric data and
categorized in the respective proteins by data bank research.
1.7 RT-PCR
The cDNA synthesis of the entire RNA of tumor cells 23132
was carried out with 5 ~g of total RNA with use of M-MLV reverse
transcriptase (Gibco BRL, Eggenstein, Germany) according to the
information of the manufacturer. The PCR reactions were
performed in a reaction volume of 25 ~1 with 1.75 ~ of MgClz,
0.4 pM of primer, 200 ~M of each dNTP and 1 U of Taq polymerase
(MBI Fermentas, St. Leon-Rot, Germany).
The following PCR products were produced:
CD55 (640 by fragment from the sequence range of by 382 to
1022), p53 fragment 1 (850 by fragment from the sequence range of
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91 to 940), p53-fragment 2 (800 by from the sequence range of 492
to 1294).
1 8 Cloning Procedures
The PCR products were purified from an agarose gel with use
of the Jetsorb gel-extraction kit (Genomed, Bad Oeynhausen,
Germany). The cloning of the PCR fragments was carried out with
the pCR script Amp SK (+) cloning kit (Stratagene, Heidelberg,
Germany).
The cloning of the antisense vector pHOOK2-CD55-anti was
carried out by smoothing of the CD55-PCR product with Pfu-
polymerase and cloning in the expression vector pHOOK2 that is
cut with Smal (Invitrogen, Leek, The Netherlands). A clone with
antisense direction of the insertion under control of the P~Mv
promoter was selected for the antisense experiment.
1.9 DNA 8equencinc
Eight positive clones were sequenced with use of the
DyeDeoxy Termination Cycle Sequencing Kit (Applied BioSystems,
Inc., Weiterstadt, Germany), and the automated DNA sequencer
ABIPrism 373 was analyzed. Both strands were sequenced with use
of T3 and T7 primers. The sequences were analyzed with use of
the computer program DNASIS and BLAST.
1.10 Transfection
For transfection experiments 2-5 x 107 dissolved cells in
tris-buffered salt solution (TBS) were washed and resuspended in
CA 02356189 2001-06-18
24
400 ~1 of TBS. After 10 ~g of plasmid DNA was added, the cells
were pulsed with 240 V, 960 nF with an electroporation device of
BioRad (Munich, Germany). 5 x 105 transfixed cells were
saturated on a 60 mm cell culture dish and incubated for 24 hours
as described above. The apoptosis was induced by adding 50 ~g/ml
of purified SC-1 antibody to the growth medium. After 24 hours,
the cells were treated with trypsin and used for the production
of cytospins.
1 11 Phospholipase Test
Dissolved and deleted cells were resuspended in RPMI-1640
with additives and incubated for 90 minutes at 37°C. After this
rest period, 20 mU/ml of PI-PLC (Boehringer Mannheim) was added,
and the cells were incubated for another 60 minutes. Finally,
the cells were washed and used for the production of cytospins.
1 12 Glycosidase Test
Dissolved and washed cells were resuspended in RPMI-1640
with 10% fetal calf serum, incubated for 1 hour in ice, then
counted, and cytospins were produced. After air drying, the
cytospin preparations were fixed with acetone (10 minutes),
washed and incubated with 20 ~U/ml of O-glycosidase or 5 mU/ml of
N-glycosidase (Boehringer Mannheim) for 4 hours at 37°C.
1 13 Immunohistochemical Staining
The following antibodies were used for the
immunohistochemical staining: purified antibody SC-1, anti-CEA-
CA 02356189 2001-06-18
antibody (DAKO, Hamburg, Germany), Anti-EMA-antibody (Loxo,
Dossenheim, Germany) and anti-CD55-antibody (Biozol, Eiching,
Germany). The acetone setting and staining of the cytospin
preparations were carried out as described by Vollmers et al.
(Hum. Antibodies Hybridomas 7 (1996), 37-41).
For immunohistochemical staining of apoptotic cells, cells
that were cultivated until subconfluence occurred were incubated
with purified antibody SC-1 (diluted to 50 ~g/ml) in full growth
medium for up to 96 hours. Adherent and dissolved cells were
collected, centrifuged and resuspended in complete growth medium.
After cells were counted, cytospin preparations were produced and
dried overnight at room temperature. In studying the cleavage of
cytokeratin 18 in vivo, biopsies were taken from patients before
treatment with SC-1 and tissue sections after treatment and
gastrectomy as described in Vollmers et al., (Oncol. Rep. 5
(1998), 549-552).
The cytospins were blocked with bovine serum albumin (15
mg/ml) in phosphate-buffered salt solution (PBS) for 30 minutes.
Then, incubation was carried out for 1 hour with SC-1
.._ supernatant, M30 cyto death-antibody (Roche Biochemicals,
Mannheim, Germany) or mouse-anti-cytokeratin 18 antibody (DAKO,
Hamburg, Germany) diluted at 1:15. Then, it was washed for 30
minutes in PBS, followed by incubation with peroxidase-labeled
rabbit-anti-mouse or rabbit-anti-human conjugate (DAKO), diluted
at 1:25. After 30 minutes of washing with PBS, staining was
carried out with diaminobenzidine (0.05%) and hydrogen peroxide
(0.02%) for 3 minutes at room temperature. The reaction was
CA 02356189 2001-06-18
26
stopped with tap water, and the tissue sections were
counterstained with hematoxylin.
1 14 Apoptosis Tests
Cytospin preparations (5,000 cells/slides).were fixed in
acetone and then washed with TBS. Then, they were stained with
the FragEl-Klenow DNA-Fragmentation Kit (Calbiochem-Novabiochem,
Bad Soden, Germany) according to manufacturer information.
An ELISA for detection of apoptosis was performed with use
of the Cell Death Detection~R~ Kit (Roche Biochemicals) according
to the manufacturer's instructions.
1.15 MTT Test
The MTT proliferation test (Carmichael et al., Cancer Res.
47 (1987), 936-942) for determining the apoptosis activity of
antibody SC-1 on gastric carcinoma cells was performed as
described in Vollmers et al. (Cancer 76 (1995), 550-558). The
determination of cell growth was carried out by the mitochondrial
hydroxylase test (Mossmann, J. Immunol. Meth. 65 (1983), 55-63).
The percentage portion of apoptotic cells was determined from the
absorption of the cells that were induced with SC-1 and the
control that was not induced with SC-1 (Vercammen et al., J. Exp.
Med. 188 (1998), 919-930).
1 16 Caspase-3 and Caspase-8 Tests
The activation of caspase-8 and caspase-3 was determined
with the ApoAlertT" Caspase Fluorescence Test Kit (Clontech,
CA 02356189 2001-06-18
27
Heidelberg, Germany). In this connection, 1 x 106 cells with 40
~,g/ml of SC-1 were incubated for 7 or 20 hours. Then, the cells
were collected, resuspended in cell-lysis buffer, and the caspase
activity was determined according to manufacturer information.
1 17 Determination of Intracellular Free Calcium fCaZ+1
The determination of the intracellular free calcium
concentration was determined with use of the calcium-sensitive
dye Fura-2-AM as described by Grykiewicz et al. (J. Biol. Chem.
260 (1985), 3440-3450). In this connection, the cells were
incubated for 15 minutes with a Fura-2-AM in Ringer's solution
that contains a final concentration of 5 x 10-6 M (122.5 ~ of
NaCl, 5.4 mM of KC1, 1.2 mM of CaCl2, 0.8, mM of MgClZ, 1
of NaH2P04, 5.5 mM of glucose, 10 mM of HEPES, pH 7.4).
After flushing, the slides were examined with an Axiovert 100 TV
microscope (400-fold magnification). The fluorescence signal was
measured at 500 nm with excitation wavelengths that alternate
between 334 and 380 nm with use of a 100-W xenon lamp and an
automatic filter changing device (Zeiss, Germany). The
concentration of intracellular free calcium was calculated
according to the method of Grynkiewicz et al. (supra) with the
assumption of a dissociation constant of 225 nmol/1. The maximum
and minimum fluorescence ratios (R~X and Rfi~~) were measured after
calibrating solutions were added. R~X was determined after a
Ringer's solution with 3 mM Ca2+ and 10-6 M of ionomycin was
added. Rm~~ was determined in the presence of a CaZ+-free Ringer's
solution with 3 ~ of EGTA and 106 M of ionomycin.
CA 02356189 2001-06-18
28
1.18 Inhibition of Intracellular Calcium Release
Cells were washed once with phosphate-buffered salt solution
and washed for 24 hours in calcium-free DMEM medium without fetal
calf serum (FCS). Then, purified SC-1 antibody was added until a
final concentration of 40 ~g/ml was reached. As a control, the
same cells were used without SC-1. The cells were incubated in a
wet incubator for another 24 or 48 hours and then fixed with 3%
glutaric aldehyde. The cell culture plates were then examined
for morphological changes with the aid of a light microscope.
2. Results
2.1 Purification of the SC-1-Receptor CD55
In Western-blot analysis of extracts from total cell lysates
of gastric carcinoma cell line 23132, which had been produced
under low-salt conditions (100 mM of NaCl), antibody SC-1
reacted with a protein with a relative molecular mass of about 50
kD. By altering the stringency (1 M of NaCl) and with use of
membrane preparations, it was possible to detect other proteins
with approximately 70 kD and approximately 82 kD (Figure la,
trace 1). These proteins were isolated from the membrane
fractions and purified by sequential size-exclusion and anion-
exchange chromatography (Figure la, traces 2, 3). The molecules
were cut out from SDS-polyacrylamide gels and sequenced.
The 50 kD protein was identified as a dihydrolipoamide-
succinyltransferase (gene bank access no. L37418), and the 70 kD
protein was identified as the human Lupus p70 auto-antigen
protein (gene bank access no. J04611). These proteins are
CA 02356189 2001-06-18
29
cytoplasmatic or nuclear antigens. Since antibody SC-1 in
immunohistochemical studies binds only to cell surface antigens,
the reactivity can presumably be attributed to unspecific binding
based on the protein denaturation during the Western-blot
analysis.
The 82 kD protein was identified as CD55 (DAF, gene bank
access no. M31516, Figure lb, sections 1 and 2). In humans, CD55
exists in two genetically specified isoforms (secreted DAF-A and
membrane-bonded DAF-B), which are produced by differential
splicing (Caras et al., Nature 325 (1987), 545-549). It was
found by RT-PCR analysis that cell line 23132 expresses only the
membrane-anchored DAF-B isoform.
2 2 Phos~holipase Treatment
The influence of a cleavage of the glycosidphosphatidyl-
inositol (GPI)-anchor on the bond of SC-1 was analyzed by
immunohistochemical studies and in the MTT-proliferation test.
In this connection, the GPI-anchor was cleaved by incubation with
phosphatidylinositol-specific phospholipase C (PI-PLC).
Cytospins of cells that were treated with PI-PLC and untreated
cells were stained immunohistochemically with SC-1, anti-CD55 and
anti-EMA (epithelial-membrane-antigen). A comparison with
untreated cells (Figure 2a) shows a loss in staining intensity in
cells that are treated with PI-PLC and stained with SC-1 (Figure
2b). In the case of staining with anti-EMA (Figure 2c, d), no
difference in staining was found, which indicates that the PI-PLC
treatment has no effect on non-GPI-anchored membrane proteins.
CA 02356189 2001-06-18
In the MTT test, a treatment of cells with phospholipase C
resulted in a significant reduction (p <_ 0.05) of the apoptotic
cells (Figure 3).
2.3 Transfection with Antisense-CD55 RNA
Cell line 23132 was transiently transfixed with the CD55
antisense-vector pHOOK2-CD55anti and the control vector pHOOK2 by
electroporation. First, cytospins of transfixed cells were
immunohistochemically stained with SC-1, anti-CD55 and anti-CEA
(carcino-embryonal antigen). The cells that were transfixed with
the control vector showed an intensive staining with SC-1 and CEA
(Figure 4a, c). In cells that were stained with the CD55
antisense vector, almost no staining with SC-1 was found (Figure
4b). The staining with anti-CEA-antibodies showed that the
expression pattern of the CEA (also GPI-anchored) is not affected
by the transfection with the antisense vector. Consequently, the
expression of CD55 was reduced specifically based on the
expression of the antisense RNA.
To analyze whether the expression of antisense-CD55 RNA also
inhibits the SC-1 induced apoptosis, the cells were incubated for
one day after the transfection with and without 30 ~g/ml of SC-1
for a period of 24 hours. Cytospins of cells that were
transfixed with the antisense vector and the control vector were
stained with the FragEl Klenow DNA Fragmentation Kit for the
detection of a DNA-fragmentation induced by apoptosis. While
untransfixed cells that are treated with two plasmids show almost
no spontaneous apoptosis (Figure 5e), a considerable reduction in
CA 02356189 2001-06-18
31
the apoptosis of cells that are transfixed with the CD55
antisense vector (Figure 5g) in comparison to cells that are
transfixed with the control vector (Figure 5h) is found after
incubation with SC-1.
A quantitative determination showed that spontaneous
apoptosis in transfixed 23132 cells occurred with a frequency of
6~, while 85% of the cells that were transfixed with the control
vector showed an apoptosis after incubation with SC-1. This
apoptotic reaction was reduced to 21% by transfection with the
CD55 antisense vector (Figure 6).
2 4 Glycosidase Treatment
The influence of a protein deglycosylation on the bond of
SC-1 to cell line 23132 was studied by incubation of cytospin
preparations with O- and N-glycosidases before the
immunohistochemical staining. A treatment of cells with N-
glycosidase resulted in a significant reduction of the SC-1
staining (Figure 7b), while a staining with anti-CD55, which
detects the proportion of protein in the SCR3 region, was not
influenced by protein deglycosylation (Figure 7d). Incubation
with phosphate buffer and a treatment with O-glycosidase had no
effect on the SC-1 bond. This shows that the specificity of SC-1
must be located in N-linked sugar radicals and not in the primary
protein sequence.
CA 02356189 2001-06-18
32
2.5 Cross-linkina of CD55/SC-1
The cells were incubated for 24 hours with increasing
quantities of SC-1 to determine the optimum apoptopic activity of
SC-1 (Figure 8a). Then, crosslinking was carried out at a
concentration of 40 ~g/ml of SC-1 with rabbit-anti-human IgM.
After incubation for 48 hours, a 47% higher portion of dead cells
than in the control cells that are incubated with SC-1 was found
(Figure 8b).
2.6 Calcium Level
To examine whether the apoptosis that is induced by SC-1 is
accompanied by changes of the calcium level, the intracellular
calcium concentration of cell line 23132 was determined after
induction with SC-1 and control antibodies (unspecific human
IgM). In this case, a significant increase of the intracellular
calcium concentration was found approximately 1 minute after SC-1
antibody was added, while the control antibody had no effect
(Figure 9).
2.7 Caspase Activity
It was found by Western-blot analysis that caspase-3 and
caspase-8 are regulated upward after induction of cell line 23132
with SC-1 (Figure l0a). A proteolytic cleavage that causes the
activation of caspases was detected for caspase-3 by identifying
cleavage product p20 (Figure l0a). In caspase-8, a four-fold
increase of the activity was found 20 hours after induction with
CA 02356189 2001-06-18
33
SC-1, which indicates a significant participation of this caspase
in the apoptosis process (Figure lOb).
The addition of the specific caspase-3 inhibitor AC-DEVD-CHO
(Alexis Biochemicals, Griinberg, Germany) showed, surprisingly
enough with increasing concentration, an increase of apoptosis in
the case of determination with the Cell Death Detection~R~ Kit
(Figure 15).
2.8 Protein Phosphorylation
After induction of cells with 40 ~,g/ml of SC-1 antibodies,
the phosphorylation pattern was examined by Western-blot analysis
of cytoplasmatic and membrane extracts. In this case, an early
tyrosine phosphorylation of 110 kD and 60 kD proteins was
found 30 to 60 seconds after apoptosis was induced (Figure 11).
The 60 kD protein was found only in the cytoplasma, while the 110
kD protein could be detected both in the plasma and in the
membrane extract. In addition, a slow tyrosine phosphorylation
of a cytoplasmatic 75 kD protein with a maximum was found after
minutes, and the complete disappearance of the serine
phosphorylation of a 35 kD protein was found 10 minutes after
induction.
2.9 Exuression andSectuencina of p53
To study the role of p53 in the case of SC-1-induced
apoptosis, the frequency of the mRNA by RT-PCR and the gene
product was determined by Western-blot analysis after induction.
In this case, a considerable increase of the mRNA concentration
CA 02356189 2001-06-18
34
was found. On the protein plane, however, a constant and not
significantly altered high concentration of the p53 gene product
was found (Figure 12a).
The DNA-sequence of p53 in cell line 23132 was determined by
amplification of two p53 fragments of cDNA with specific primers,
cloning of the PCR-fragments and sequencing of eight clones. All
clones with insertions spanning Exon 8 showed a mutation in codon
273, which resulted in an amino acid exchange of arginine to
histidine (Figure 12b). This is a dominant negative mutation,
which frequently occurs in gastric adenocarcinomas.
2 10 Expression of o21
Protein p21 is a molecule that is associated with the
expression of p53. A test of the expression of p21 in gastric
carcinoma cell line 23132 after treatment with SC-1 yielded an
increase after 5 minutes followed by a reduction after 60 minutes
(Figure 13).
2.11 Ext~ression of CD55/DAF after Apoptosis is Induced
The expression pattern of CD55/DAF was studied after
apoptosis was induced by 50 ~g/ml of SC-1 using
immunohistochemical staining of cytospin preparations with
antibody SC-1. While non-induced cells exhibit a slight membrane
staining with antibody SC-1, cells that were induced with SC-1
showed intensive membrane staining 12 hours after apoptosis was
induced. This indicates an increase of the CD55/DAF presentation
on the cell surface after the antibody is bonded to the cells.
CA 02356189 2001-06-18
This membrane staining disappears after 48 hours, and a diffuse
cytoplasmatic staining can be detected. This staining is also
found with reduced intensity after 96 hours. The increase in the
CD55/DAF expression was also found in a Western Blot analysis
with membrane extracts of apoptotic cells after SC-1 induction.
While CD55/DAF cannot be detected in non-induced cells, the
CD55/DAF expression increases 1 hour to 6 hours after induction.
After 24 hours, the expression of CD55/DAF decreases, but it is
always still higher than in non-induced cells (Figure 14a).
2 12 Cleavage of Cytokeratin 18
The degradation of apoptotic cells accompanies the
proteolytic cleavage of cytokeratin 18. The cleavage of
cytokeratin 18 in cell line 23132 after SC-1-induced apoptosis
and in primary tumors of patients who had been treated with 20 mg
of SC-1 for 48 hours before a tumor resection was studied. An
M30 cyto death staining showed a small quantity of apoptotic
cells without inducing apoptosis, while the number of apoptotic
cells increased up to 96 hours.
2.13 Molecular Analysis of SC-1-AyoDtosis
Consistent with the immunohistochemical staining, the
biochemical analysis showed an increase of the CD55/DAF molecule,
followed by a slight reduction after 24 hours of incubation with
SC-1 (Figure 14a). The cleavage of PARP was studied by Western
Blot analysis with use of total cell extracts from SC-1-induced
cells and murine anti-PARP antibodies. In five independent
CA 02356189 2001-06-18
36
experiments, no cleavage of PARP was found that would be detected
by the occurrence of an 85 kD cleavage product (Lazebnik et al.,
Nature 371 (1994), 346-347) (Figure 14b).
To study changes in the cell cycle after apoptosis is
induced, the expression of topoisomerase IIa by Western Blot
analysis was determined. Topoisomerase IIa is a key enzyme in
the cell cycle, which is involved in DNA replication (Watt and
Hickson, Biochem. J. 303 (1994), 681-695). The reduced
expression of topoisomerase IIa according to SC-1-induced
apoptosis therefore shows a cell cycle arrest for at least one
portion of cells (Figure 14c).
Transcription factor c-myc is involved in various apoptotic
processes and can induce an apoptosis by transfection in cells
(Evan et al., Cell 69 (1992), 119-128). A study of the
expression pattern of c-myc after SC-1-induced apoptosis showed
an increased expression 15 minutes after apoptosis was induced
followed by a reduction after 4 hours (Figure 14d).
2.14 Action of a Reduction of the Extracellular and
Intracellular Calcium Concentration in Apoptosis
It was examined whether Ca2+ ions from the cell culture
medium are taken up or are released from intracellular CaZ+
reservoirs. To determine whether CaZ+ is taken up from the
culture medium, the cells were incubated for 24 hours in serum-
free and Ca2+-free DMEM medium. Then, purified SC-1-antibody was
added at a final concentration of 40 ~g/ml and incubated for
another 24 hours. The cells were then fixed in 3% glutaric
CA 02356189 2001-06-18
37
aldehyde and studied in a rotated light microscope. Compared to
control cells (not induced with SC-1), SC-1-induced cells showed
morphological changes characteristic of an apoptosis and
comparable with cells that would have been incubated with SC-1 in
RPMI medium with the addition of 10~ FCS.
The effect of Ca2+ from intracellular Ca2+ reservoirs was
studied by incubation of cells (cultivated in serum-free DMEM
medium) for 5 hours with 50 ~M of the cell-permeable chelating
agent BAPTA (Alexis Biochemicals, Grunberg, Germany). The cells
were incubated for 24 hours with 40 ~g/ml of purified SC-1. No
detectable changes could be observed in the cell morphology,
which indicates that no apoptosis was induced. An inhibition of
the apoptosis produced by BAPTA could also be found by ELISA.
2 15 Detection of Apoptosis in Primary Tumors
The administration of antibody SC-1 to patients with stomach
cancer resulted in a clearly detectable tumor-cell-specific
apoptosis, as was detected by in-situ nucleus staining (Figure
16). While no apoptosis (Figure 17a) or the presence of an
intact tumor without regression (Figure 17c) was found in tumor
biopsies that were taken before SC-1 was administered, the
primary tumor showed strong apoptosis (Figure 17b) or a strong
regression (Figure 17d) after SC-1 was administered.
