U.S. patent application number 10/645094 was filed with the patent office on 2005-09-01 for carcinostatic agent.
Invention is credited to Iwamoto, Mitsunori, Jung, Sang-Kee.
Application Number | 20050191737 10/645094 |
Document ID | / |
Family ID | 15107420 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050191737 |
Kind Code |
A1 |
Iwamoto, Mitsunori ; et
al. |
September 1, 2005 |
Carcinostatic agent
Abstract
Novel proteins which have cell death inducing activity or
proliferation inhibitory activity on cancer cells; fragments of
such proteins; genes coding such proteins; monoclonal antibodies to
such proteins; reagents which induce apoptosis on cells including
cancer cells in vitro for investigation of the mechanism of cell
death induction, and which are obtained from the proteins including
the amino acid sequence of SEQ ID No. 1 of the sequence listing,
fragments thereof, genes coding these; and carcinostatic
agents.
Inventors: |
Iwamoto, Mitsunori;
(Karatsu-shi, JP) ; Jung, Sang-Kee; (Kyoto-shi,
KR) |
Correspondence
Address: |
DARBY & DARBY P.C.
P. O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Family ID: |
15107420 |
Appl. No.: |
10/645094 |
Filed: |
August 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10645094 |
Aug 21, 2003 |
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09912176 |
Jul 24, 2001 |
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6649739 |
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09912176 |
Jul 24, 2001 |
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09230388 |
Jan 22, 1999 |
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6291644 |
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Current U.S.
Class: |
435/184 ;
435/320.1; 435/325; 435/69.2; 536/23.2 |
Current CPC
Class: |
C07K 14/461 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/184 ;
435/069.2; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12N 009/99; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 1997 |
JP |
H. 9-133549 |
Claims
1-8. (canceled)
9. A carcinostatic agent comprising at least one protein selected
from the from the group consisting of: (a) a protein containing an
amino acid sequence of SEQ ID: 1; (b) a protein containing an amino
acid sequence homologous to the amino acid sequence of SEQ ID No. 1
and having proliferation inhibitory activity on cancer cells or
cell death inducing activity; and (c) a protein containing amino
acid residues 61-89 and 497-514 of SEQ ID No. 1 and having
proliferation inhibitory activity on cancer cells or cell death
inducing activity, as an active component.
10. (canceled)
11. A carcinostatic agent comprising at least one monoclonal
antibody selected from the group consisting of: (a) a monoclonal
antibody to a protein containing an amino acid sequence of SEQ ID
No. 1; (b) a monoclonal antibody to a protein containing an amino
acid sequence homologous to the amino acid sequence of SEQ ID No. 1
and having proliferation inhibitory activity on cancer cells or
cell death inducing activity; and (c) a monoclonal antibody to a
protein containing amino acid residues 61-89 and 497-514 of SEQ ID
No. 1 and having proliferation inhibitory activity on cancer cells
or cell death inducing activity, as an active component.
12. The carcinostatic agent of claim 11, wherein said monoclonal
antibody is selected from the group consisting of I38A (NATIONAL
INSTITUTE OF BIOSCIENCE AND HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL
SCIENCE AND TECHNOLOGY, International Deposit No. FERM BP-5872),
monoclonal antibody I32D (NATIONAL INSTITUTE OF BIOSCIENCE AND
HUMAN TECHNOLOGY, International Deposit No. FERM BP-5873), and
monoclonal antibody I310H (NATIONAL INSTITUTE OF BIOSCIENCE AND
HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY,
International Deposit No. FERM BP-5874).
Description
FIELD OF ART
[0001] The present invention relates to novel proteins which induce
death of cancer cells by inducing apoptosis of the cells, or which
exhibit cancer cell growth inhibitory activity, genes of such
proteins, monoclonal antibodies to such proteins, reagents for
qualifying cancer cell apoptosis, and carcinostatic agents.
PRIOR ART
[0002] Remarkable development in chemotherapy has been improving
survival rate and remedial rate of patients having neoplastic
diseases. On the other hand, however, strong side effects of
carcinostatic agents give serious damage on normal cells, which has
been recognized as a social problem. For prevention of side effects
of carcinostatic agents, agents are demanded which have excellent
selectivity for cancer cells or which are capable of controlling
proliferation of oncocytes.
[0003] In conventional chemotherapy for cancer, administration
dosage of agents is kept as low as possible since even the
treatment dosage may cause harmful effects. Thus, attempts have
been made to seek for potentiation by combination of a plurality of
carcinostatic agents with different mechanisms of actions, or to
improve carcinostatic effect by combination of a carcinostatic
agent with other substances. In the latter case, a carcinostatic
agent is usually combined with an immune activator to combine the
direct effect on oncocytes with antineoplastic effect obtained
through activation of immunocompetence of organism. Further, in
some cases, radiotherapy or surgical treatment is performed in
addition to these methods to improve the effect of the
treatment.
[0004] Recently, it has been revealed that there are two different
types of cell death, i.e., apoptosis (cell death governed by genes)
and necrosis (cell death not governed by genes). Apoptosis and
necrosis are usually distinguished by observing DNA fragmentation
through biochemical measurement. This measurement shows that
conventional carcinostatic agents exhibit carcinostatic effect by
inducing necrosis of oncocytes, so that they cannot genetically
control the death of oncocytes. On the contrary, active researches
have been made on substances which induce apoptosis of oncocytes
since such substances have possibility to control the death of
oncocytes. Combination of an apoptosis-inducing carcinostatic agent
with a conventional carcinostatic agent having different mechanism
of action is expected to improve antineoplastic effect.
DISCLOSURE OF THE INVENTION
[0005] It is an object of the present invention to provide novel
proteins which induce death of cells, in particular human cancer
cells, by inducing apoptosis of the cells, or which exhibits cancer
cell growth inhibitory activity; fragments of such proteins; genes
of such proteins; and monoclonal antibodies to such protein.
[0006] It is another object of the present invention to provide
reagents for apoptosis induction which induce apoptosis of cells,
in particular of cancer cells, in vitro for the study of mechanism
of cell death induction.
[0007] It is still another object of the present invention to
provide carcinostatic agents which have proliferation inhibitory
effect on human cancer cells or which have death inducing effect on
cancer cells.
[0008] The present inventors have conducted intensive researches on
peptides or proteins which induce apoptosis of cancer cells for
applying conventionally known apoptotic cell death to oncotherapy.
As a result, the inventors have found that some proteins purified
from mackerel's viscus have apoptosis-inducing activity not only on
blood cancer cells but also on a variety of tumor cancer cells,
then synthesized cDNA, using the mRNA isolated from mackerel's
viscus, determined the DNA sequence of the obtained cDNA, and
estimated the amino acid sequence thereof, thereby completing the
present invention.
[0009] In sum, according to the present invention, there are
provided proteins comprising the amino acid sequence of SEQ ID No.
1 of the attached sequence listing, or the amino acid sequence at
least partly homologous or analogous to the sequence of SEQ ID No.
1, and having proliferation inhibitory activity on cancer cells or
cell death inducing activity; and fragments of such proteins.
[0010] According to the present invention, there are also provided
proteins which have the amino acid sequence homologous to the amino
acid sequence of SEQ ID No. 1 of the attached sequence listing, and
which have proliferation inhibitory activity on cancer cells or
cell death inducing activity.
[0011] According to the present invention, there are also provided
proteins comprising amino acids 61-89 and 497-514 of SEQ ID No. 1
of the attached sequence listing, and having proliferation
inhibitory activity on cancer cells or cell death inducing
activity.
[0012] According to the present invention, there are further
provided genes which code the aforementioned proteins or their
fragments, and genes which code the aforementioned proteins or
their fragments and which comprise the DNA sequence of SEQ ID No. 2
of the attached sequence listing or the DNA sequence at least
partly homologous or analogous to that of SEQ ID No. 2.
[0013] According to the present invention, there are further
provided monoclonal antibodies to the aforementioned proteins or
their fragments.
[0014] According to the present invention, there are also provided
reagents for qualifying apoptosis comprising at least one member
selected from the group consisting of the aforementioned proteins,
their fragments, and the monoclonal antibodies thereto.
[0015] According to the present invention, there are also provided
carcinostatic agents comprising the aforementioned proteins and
their fragments as active components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a photograph showing the results of
silver-staining following SDS-PAGE of leukemia cell-killing active
fractions prepared in Example 1, wherein Lane 1 shows the result of
extract of mackerel's viscus, Lane 2 shows a fraction precipitated
with ammonium sulfate, Lane 3 shows an active fraction obtained by
gel filtration, Lane 4 shows a fraction adsorbed on Con A column,
Lane 5 shows a fraction adsorbed on Mono Q, and Lane 6 shows final
purified AIP (Apoptosis Inducing Protein).
[0017] FIG. 2 is a photograph showing the results of immunostaining
of leukemia cell-killing active fractions prepared in Example 1
with a monoclonal antibody to a leukemia cell-killing substance,
following transferring of the fractions to PVDF membranes, wherein
Lane 1 shows the result of extract of mackerel's viscus, Lane 2
shows a fraction precipitated with ammonium sulfate, Lane 3 shows
an active fraction obtained by gel filtration, Lane 4 shows a
fraction adsorbed on Con A column, Lane 5 shows a fraction adsorbed
on Mono Q, and Lane 6 shows final purified AIP (apoptosis inducing
protein).
[0018] FIG. 3 is a photograph showing the results of Western
blotting of extracts of mackerel's viscus prepared in Example 1
using each monoclonal antibody (I38A, I32D, and I310H) prepared in
Example 2, wherein Lane 1 shows immunostaining with monoclonal
antibody I38A, Lane 2 with monoclonal antibody I32D, and Lane 3
with monoclonal antibody I310H.
[0019] FIG. 4 is a graph showing the cell-killing activity of a
purified leukemia cell-killing substance on human leukemia cell
HL-60 confirmed in Example 3 by means of relationship between the
concentration measured by MTS assay and the percentage of viable
cells 16 hours after the treatment.
[0020] FIG. 5 is a graph showing the results of analysis in Example
3 in the percentage of the viable cells after treatment with a
leukemia cell-killing substance taken with the lapse of time.
[0021] FIG. 6 is a photograph showing the DNA fragmentation after
treatment with a leukemia cell-killing substance analyzed with the
lapse of time in Example 3, wherein the number on each lane
indicates the time lapsed in hours, and M indicates the 123 bp
molecular size marker.
[0022] FIG. 7 illustrates graphs showing DNA contents of cells
analyzed by flow cytometry 0 hour (FIG. 7(A)), 2 hours (FIG. 7(B)),
4 hours (FIG. 7(C)), and 12 hours (FIG. 7(D)) after the treatment
with a leukemia cell-killing substance in Example 3, wherein the
ordinate represents the cell count, and the abscissa represents the
florescence intensity.
[0023] FIG. 8 shows microscopy photographs, showing morphological
changes in cells treated with a leukemia cell-killing substance in
Example 3, wherein (A) indicates the result 0 hour after the
treatment, (B) indicates the result 2 hours after the treatment,
(C) indicates the result 4 hours after the treatment, and (D)
indicates the result 12 hours after the treatment.
[0024] FIG. 9 shows fluorescence microscopy photographs, showing
the results of fluorescent-staining with HOECHST 33258 of the
nuclei of the cells treated with a leukemia cell-killing substance
after the lapse of time as each shown in FIG. 8, wherein (A)
indicates the result 0 hour after the treatment, (B) indicates the
result 2 hours after the treatment, (C) indicates the result 4
hours after the treatment, and (D) indicates the result 12 hours
after the treatment.
[0025] FIG. 10 is a photograph showing the results of Western
blotting, using monoclonal antibodies prepared in Example 2, of
mutant AIPs obtained by transfection with AIP gene and its mutants
into African green monkey kidney cell line cos-7 in Example 5. Lane
1 represents complete AIP consisting of amino acids 1-524 of SEQ ID
No. 1, Lane 2 represents mutant AIP consisting of amino acids
1-496, and Lane 3 represents mutant AIP consisting of amino acids
1-514.
[0026] FIG. 11 is a photograph showing the results of Western
blotting of extract of parasite-infected mackerel's viscus (Lanes
1-5) and of non-infected mackerel's viscus (Lanes 6-10) performed
in Example 6, using the monoclonal antibodies prepared in Example
2.
DESCRIPTION OF THE INVENTION
[0027] The proteins and their fragments of the present invention
are: (1) those including the amino acid sequence of SEQ ID No. 1 of
the attached sequence listing; (2) those including an amino acid
sequence at least partly homologous or analogous to the sequence of
SEQ ID No. 1 and exhibiting proliferation inhibitory activity on
cancer cells or cell death inducing activity; (3) those having the
amino acid sequence homologous to the sequence of SEQ ID No. 1 of
the sequence listing, and exhibiting proliferation inhibitory
activity on cancer cells or cell death inducing activity; or (4)
those including amino acids 61-89 and 497-514 of SEQ ID No. 1 of
the attached sequence listing, and exhibiting proliferation
inhibitory activity on cancer cells or cell death inducing
activity.
[0028] In the present invention, "homologous to the sequence of SEQ
ID No. 1" in above (2) means having preferably not lower than 70%,
more preferably not lower than 90%, most preferably not lower than
95% homology to the sequence. A "sequence analogous to the amino
acid sequence" means that the sequence is substantially homologous
to the amino acid sequence of SEQ ID No. 1, and that the protein
having such amino acid sequence has proliferation inhibitory
activity on cancer cells or cell death inducing activity, similar
to that exhibited by the proteins having the sequence of SEQ ID No.
1. Such amino acid sequence may be the sequence of SEQ ID No. 1 in
which at least one amino acid is substituted; in which at least one
new amino acid is inserted; or in which at least one amino acid is
deleted.
[0029] In above (3), "having the amino acid sequence homologous to
the sequence of SEQ ID No. 1" means that the sequence is recognized
to be different from that of SEQ ID No. 1 only in a sequence
portion varying depending on the species of the animal from which
the protein derives, and that the protein has proliferation
inhibitory activity on cancer cells or cell death inducing
activity.
[0030] The proteins and their fragments of the present invention
are believed to catalyze aldehyde-generating reaction by oxidative
deamination shown below to cause generation of hydrogen peroxide,
using .epsilon.-amino groups of proteins or amino acids, or amino
groups of an amine compounds or amino acids as a substrate:
R--CH.sub.2NH.sub.2+H.sub.2O+O.sub.2.dbd.R--CHO+NH.sub.3+H.sub.2O.sub.2
[0031] Further, the proteins and their fragments of the present
invention are believed to require flavin as a coenzyme as one of
the factors for exhibiting the proliferation inhibitory activity on
cancer cells or the cell death inducing activity. Accordingly, it
is preferred that the proteins and their fragments have amino acids
61-89 of the amino acid sequence of SEQ ID No. 1, which show the
change similar to the typical change in absorption of the visible
spectrum observed in flavoprotein. It is also preferred that the
proteins and their fragments have amino acids 497-514 of the amino
acid sequence of SEQ ID No. 1, which are believed to be essential
for exhibiting cell death inducing activity.
[0032] In the proteins and their fragments of the present
invention, "exhibiting cell death inducing activity" means to
induce apoptosis, and this activity can be confirmed by such
methods as observing DNA fragmentation, i.e., breakage of chromatin
DNA into nucleosome units, by biochemical measurement; determining
DNA fragmentation caused by apoptosis using flow cytometry; or
directly detecting apoptosis by observation of morphological
features under a microscope. The cells of which death is to be
induced may include cancer cells, such as lung cancer, stomach
cancer, colon cancer, ovarian cancer, brain tumor, mammary cancer,
or renal cancer cells, adult T cell leukemia (ATL) cells, or cells
infected with human T cell leukemia virus (HTLV-1).
[0033] The proteins and their fragments having homologous or
analogous amino acid sequences to those of the present proteins and
their fragments may easily be produced by conventional methods-such
as ordinary recombination technique.
[0034] Other structural features of the proteins and their
fragments of the present invention are not particularly limited.
For example, they may be modified, for example, with sugar
chains.
[0035] The origin of the proteins and their fragments of the
present invention is not particularly limited. For example, the
protein having the sequence of SEQ ID No. 1 is isolated from viscus
extract of mackerels infected with parasites. Thus, proteins and
their fragments derived from mackerels (scientific name: Scomber
japonicus, generic name: Chub mackerel) may be used. However, as
will be discussed later in Examples, the proteins of the present
invention are not obtained from viscus extracts of mackerels not
infected with parasites. Therefore, the proteins are induced in
mackerels by stimulation accompanied by activation of helper
T.sub.2 cells (Th.sub.2 cells) such as parasite infection.
Similarly, proteins similar to the proteins and their fragments of
the present invention, or proteins and their fragments having amino
acid sequences homologous to that of the present invention, may be
induced also from mammals through the same mechanism. Accordingly,
the proteins and their fragments of the present invention are not
limited to those derived from mackerels, but may be those derived
from mammals such as humans, mice, or rats. Such proteins derived
from mammals have amino acid sequences which have homology to the
sequence of SEQ ID No. 1 due to the difference in species.
[0036] The proteins having amino acid sequences homologous to that
of SEQ ID No. 1 may easily be prepared, for example, from the genes
cloned by hybridization using DNA containing the sequence of SEQ ID
No. 2 with genes selected from a gene library of mammals.
[0037] The genes (DNA) of the present invention are those which
code the proteins and their fragments mentioned above, and those
which code the proteins and their fragments mentioned above and
include the DNA sequence of SEQ ID No. 2 of the attached sequence
listing or the DNA sequence at least partly homologous or analogous
to that of SEQ ID No. 2. The "DNA sequence analogous to the
sequence of SEQ ID No. 2" means that the genes have the DNA
sequence of SEQ ID No. 2 in which at least one codon is
substituted; in which at least one new codon is inserted; or in
which at least one codon is deleted, and that the genes code the
amino acids of the proteins of the present invention.
[0038] Such genes may be obtained by conventional methods, such as
isolation from cDNA prepared by extracted mRNA, or isolation from
genome DNA, or chemical synthesis.
[0039] The monoclonal antibodies of the present invention may be
prepared by conventional methods, such as preparation of hybridoma
using the proteins or their fragments as antigens.
[0040] The monoclonal antibodies of the present invention may be
monoclonal antibody I38A (NATIONAL INSTITUTE OF BIOSCIENCE AND
HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY under
International Deposit No. FERM BP-5872, deposited Mar. 13, 1997),
monoclonal antibody I32D (NATIONAL INSTITUTE OF BIOSCIENCE AND
HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY,
under International Deposit No. FERM BP-5873, deposited Mar. 13,
1997), or monoclonal antibody I310H (NATIONAL INSTITUTE OF
BIOSCIENCE AND HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL SCIENCE AND
TECHNOLOGY, under International Deposit No. FERM BP-5874, deposited
Mar. 13, 1997).
[0041] The proteins and their fragments of the present invention
may be obtained by constructing the aforementioned genes into
appropriate expression vectors, delivering the vectors into host
cells for transformation or transduction thereof, followed by
proliferating the cells, and effecting intracellular or
extracellular secretion of the target protein. The expression
vectors to be used may be plasmids, viruses, and DNA fragments into
which the genes of the present invention can be incorporated and
which allow stable presence of the genes in the host cells to be
expressed. The expression vectors require promoters, enhancer
elements for controlling transcription, operator sequences,
appropriate sequences of ribosome binding sites, sequences for
controlling transcription and translation, and sequences for
replicating vector DNA. For effecting extracellular secretion,
secretory signal sequences are also required.
[0042] As the host cells, microorganisms, such as Escherichia coli
or yeast, are useful. Also, COS7 cells derived from fibroblast of
African green monkeys may be used. These may be commercially
obtainable.
[0043] It is sufficient for the reagents for apoptosis induction of
the present invention to contain at least one member selected from
the group consisting of the proteins of the present invention,
their fragments, and the monoclonal antibodies thereto of the
present invention. The reagents are capable of inducing apoptosis
of cells such as tumor cells, so that they may be used for kits to
investigate the cell morphology, gene expression, and intracellular
signal transduction mechanism of apoptotic cells.
[0044] The carcinostatic agents of the present invention contain
the aforementioned proteins and/or their fragments of the present
invention as active components, and optionally contain the
monoclonal antibodies of the present invention, if required. The
agents may be administered, for example, orally or by injection,
and may be formed into, for example, powders, granules, tablets,
capsules, liquid preparations, emulsions, or suspensions. The
carcinostatic agents of the present invention may optionally
contain pharmaceutically or galenical-pharmaceutically acceptable
additives. Appropriate dosage may vary depending on the kind of
cancers or other factors, but it is preferred to administer about
0.01-10 mg of active components per kilogram of patient's body
weight per day.
[0045] The proteins and their fragments of the present invention
are novel, have the activities to induce apoptosis of cells, in
particular human cancer cells, to induce death of cancer cells or
to inhibit proliferation of cancer cells, and are contained in
edible fish such as mackerels. Thus, they are useful for functional
foods for inhibiting cancer, carcinostatic agents, reagents for
apoptosis induction, and the like. Further, the monoclonal
antibodies of the present invention are useful for carcinostatic
agents, reagents for apoptosis induction, and the like.
[0046] The genes of the present invention are capable of coding the
proteins and their fragments of the present invention, so that they
are useful for mass production of these.
[0047] The reagents for apoptosis induction induce apoptosis of
cells, so that they are useful for investigating the cell
morphology, gene expression, and intracellular signal transduction
mechanism in apoptotic cells.
[0048] The carcinostatic agents of the present invention are
capable of inducing apoptosis of cancer cells, so that they can
control death of cancer cells to achieve carcinostatic effect,
while they keep the side effects on other cells at an extremely low
level.
EXAMPLES
[0049] The present invention will now be explained in detail with
reference to Examples, but the present invention is not limited to
these.
Example 1
[0050] Viscera were removed from mackerels, and homogenized in the
equivalent weight of cold water. The homogenized mixture was
centrifuged in a refrigerated centrifuge at 15000 G for 30 minutes
to remove lipid and precipitate. The clear extract thus obtained
was freezing-dried to obtain mackerel viscus dried powders. The
viscus powders thus prepared were dissolved in distilled water at
the concentration of 10 mg/ml, sufficiently cooled on ice, and
saturated with 55% by weight ammonium sulfate. Then the saturated
solution was centrifuged at 15000 rpm for 30 minutes to remove the
precipitate, and the obtained supernatant was saturated with 95% by
weight ammonium sulfate. The saturated solution was again
centrifuged under the same conditions, and the precipitate was
separated. To the precipitate thus obtained was added a small
amount of Tris buffer (20 mM Tri-HCl, pH 7.5, 0.3M NaCl) to
dissolve the precipitate, thereby obtaining a saturated
fraction.
[0051] 5 ml of the saturated fraction thus obtained as a sample was
applied to a column (trade name "HiLoad 16/60 Superdex 200",
manufactured by PHARMACIA FINE CHEMICALS CO.) which had been
equilibrated with Tris buffer previously (to perform the gel
filtration) at the flow rate of 60 ml/hour, and the active fraction
was collected in aliquots of 2.5 ml each. The active fraction thus
obtained was applied to a column (Con A-Sepharose column,
manufactured by PHARMACIA FINE CHEMICALS CO.) which had been
equilibrated with Tris buffer previously, and the column was washed
with Tris buffer until the absorption at 280 nm was no longer
observed.
[0052] The active fraction was eluted with Tris buffer containing
0.5M of methyl-.alpha.-D-mannopyranoside, dialyzed against bis-Tris
buffer (20 mM bis-Tri-HCl, pH 6.4, 100 mM NaCl), and concentrated
by Ultrafree-15 (trade name "Biomax-50", manufactured by MILLIPORE
Co.). The concentrate thus obtained was applied to a column (trade
name "Mono Q 5/5 column, manufactured by PHARMACIA FINE CHEMICALS
CO.) which had been equilibrated with bis-Tris buffer previously,
and the column was washed with bis-Tris buffer until the absorption
at 280 nm was no longer observed. Elution was performed with
increasing concentrations of NaCl under the conditions below. The
fraction containing the target peptides (proteins) was eluted at
around 300 mM.
[0053] Conditions:
[0054] Buffer A: 20 mM bis-Tris buffer, 100 mM NaCl, pH 6.4
[0055] Buffer B: 1M NaCl in Buffer A
[0056] Gradient:
[0057] 0% B for 5 minutes, 0-50% B for 20 minutes,
[0058] 50% B for 2 minutes, 50-100% for 5 minutes
[0059] Flow Rate: 1.0 ml/min.
[0060] Detection: 280 nm, 0.2 AUFS
[0061] The active fraction thus obtained was concentrated by
Ultrafree-15 (trade name "Biomax-50", manufactured by MILLIPORE
CO., LTD.), and 1 ml of the sample was applied to a column (trade
name "HiLoad 16/60 Superdex 200", manufactured by PHARMACIA FINE
CHEMICALS CO.) which had been equilibrated with a phosphate buffer
(trade name "Dulbecco PBS" manufactured by NISSUI PHARMACEUTICALS
CO.) previously (to perform gel filtration) at the flow rate of 60
ml/hour, and the active fraction was collected in aliquots of 1 ml
each, thereby purifying the target protein. Incidentally, the
samples were centrifuged at 15000 rpm for 10 minutes and passed
through a membrane filter with the pore size of 0.45 .mu.m before
each of the purification steps.
[0062] Purification of Human Leukemia Cell (HL-60) Killing
Substance
[0063] The leukemia cell-killing active fractions at each
purification step were subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), followed by silver-staining or
immunostaining with a monoclonal antibody to a leukemia
cell-killing substance following transcription to a PVDF membrane.
The results of the silver-staining were shown in FIG. 1, and the
results of the immunostaining were shown in FIG. 2. As shown in
Lane 6 in FIG. 1, the leukemia cell-killing substance purified by
the above method exhibited bands only at about 62 and 63 kDa on
SDS-PAGE. The proteins at 62 and 63 kDa were also detected by
immunostaining using three independent monoclonal antibodies which
are capable of completely absorbing the leukemia cell-killing
activity by immunoprecipitation (FIG. 3). Further, the bands at 62
and 63 kDa having the leukemia cell-killing activity were cut out
separately from PVDF membrane, and amino acid sequence of the
N-terminal of each protein was determined. As a result, the
sequence of the band at 62 kDa was found to be Glu His Leu Ala Asp
Xaa Leu Glu Asp Lys Asp Tyr Asp Thr Leu Leu Gln Thr Leu Asp Asn Gly
Leu Pro His Ile, and that of the band at 63 kDa was Glu His Leu Ala
Asp Xaa Leu Glu Asp Lys Asp Tyr Asp Thr Leu Leu Gln Thr Leu Asp.
Consequently, it was found that the amino acid sequences at
N-terminals of the two proteins at 62 and 63 kDa were identical.
This result gives possibility that the two proteins are the
products from the same gene. This leukemia cell-killing substance
was named "Apoptosis Inducing Protein (AIP)" for its apoptotic
activity to be described later.
Example 2
[0064] Preparation of Monoclonal Antibodies to AIP
[0065] 1) Immunization
[0066] 10 .mu.g of the purified AIP prepared in Example 1 was mixed
with the equal volume of complete Freund's adjuvant, and
subcutaneously injected to a five-week-old female BALB/c mouse.
After two weeks, the equivalent amount of the antigen was mixed
with the equal volume of incomplete Freund's adjuvant, and
intraperitoneally injected into the same mouse. After three weeks,
30 .mu.g of the antigen dissolved in a phosphate buffer solution
(PBS) was intravenously injected to the mouse.
[0067] 2) Fusion
[0068] Three days after boosting, splenic cells of the mouse and
myeloma FOX-NY were fused in the presence of polyethylene glycol
(PEG). The ratio of the splenic cells to the myeloma cells was 5:1.
As a selective medium, 5% FBS-RPMI1640 containing AAT
(7.5.times.10.sup.-5 M adenine, 8.times.10.sup.-7 M aminopterin,
1.6.times.10.sup.-5 M thymidine) insulin (10 mg/l), and transferrin
(10 mg/l) was used.
[0069] 3) Cloning of Hybridoma
[0070] Immunoprecipitation was used for selecting
antibody-producing hybridoma and for cloning. In the
immunoprecipitation, 1 .mu.l of AIP (40 .mu.g/ml) was mixed with 20
.mu.l of the supernatant of hybridoma culture, incubated at
4.degree. C. for 2.0 hours, mixed with 3 g of anti-mouse IgG+IgM
rabbit Igs, and further incubated for 1.0 hour. Subsequently, 50
.mu.l of 10% Staphylococcus aureus cell suspension (PANSORBIN
Cells) was added to the reaction liquid, and the resulting mixture
was incubated at 4.degree. C. for 1.0 hour, and centrifuged at
10000 rpm for 2 minutes. 5 .mu.l, 10 .mu.l, and 20 .mu.l each of
the supernatant were taken out, and their apoptotic activity to
human leukemia cells HL-60 were determined.
[0071] 4) Preparation of Monoclonal Antibodies to Leukemia
Cell-Killing Substance
[0072] At day 8 of the cell fusion, a total of 768 wells were
screened by enzyme-linked immunosorbent assay (ELISA) to select
wells containing anti-AIP antibody, and 56 ELISA positive wells
were obtained. At day 10 of the cell fusion, 56 ELISA positive
wells were subjected to immunoprecipitation and Western blotting to
select 9 wells which were positive in all of ELISA,
immunoprecipitation, and Western blotting. Three out of the 9 wells
which produced the largest number of antibodies were subjected to
cloning by three cycles of limiting dilution-cloning method to
obtain three independent clones. Each clone was named I38A
(NATIONAL INSTITUTE OF BIOSCIENCE AND HUMAN TECHNOLOGY, AGENCY OF
INDUSTRIAL SCIENCE AND TECHNOLOGY under International Deposit No.
FERM BP-5872), 132D (NATIONAL INSTITUTE OF BIOSCIENCE AND HUMAN
TECHNOLOGY, AGENCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY, under
International Deposit No. FERM BP-5873), and I310H (NATIONAL
INSTITUTE OF BIOSCIENCE AND HUMAN TECHNOLOGY, AGENCY OF INDUSTRIAL
SCIENCE AND TECHNOLOGY, under International Deposit No. FERM
BP-5874), respectively. The results of Western blotting of the
extract of mackerel's viscus using I38A, I32D, and I310H are shown
in FIG. 3. Isotypes of monoclonal antibodies I38A, I32D and I310H
were determined by immunostaining using isotype specific anti-mouse
antibodies. The results are shown in Table 1. Monoclonal antibodies
I38A, I32D, and I310H were useful in all of ELISA,
immunoprecipitation, and Western blotting.
1TABLE 1 Immuno- Immuno- Antibody Class ELISA precipitation
staining I38A IgG 1 (k) + + + I32D IgG 1 (k) + + + I310H IgG 1 (k)
+ + + +: usable
Example 3
[0073] Measurement of Apoptotic Activity of AIP
[0074] Myelomonocytic cell line HL-60 obtained from peripheral
blood of a patient with acute promyelocytic leukemia was used as
the cells. RPMI1640 medium (manufactured by MENEKI SEIBUTSU
KENKYUSHO, IBL) with 10% fetal bovine serum (FBS) was used as
medium. FBS has been heated at 56.degree. C. for 45 minutes to
inactivate the complement system before use.
[0075] First, the cell suspension was poured into a tube,
centrifuged at 1000 rpm for 5 minutes to remove the supernatant,
and then resuspended in an appropriate amount of 10% FBS RPMI1640.
The number of cells was adjusted to be 5.0.times.10.sup.5 cells/ml,
and the suspension was treated with the purified AIP prepared in
Example 1 which had been adjusted to various concentrations.
[0076] Cell death was measured by Cell Titer 96 (trade name
"Aqueous Non-Radioactive Cell Proliferation Assay Kit" manufactured
by PROMEGA CO.).
[0077] DNA fragmentation was determined by the following process.
250 .mu.l of the cell suspension (1.25.times.10.sup.5 cells) was
mixed with 62.5 .mu.l of a lysis buffer (2.0M NaCl, 10 mM EDTA, 50
mM Tris-HCl, pH 8.0, 1% SDS) and 4 .mu.l of protease K (20 mg/ml),
and dissolved at 56.degree. C. for 90 minutes. The resulting
solution was left still on ice for 5 minutes, mixed with 80 .mu.l
of 5M NaCl, left on ice for another 5 minutes, and centrifuged at
12000 rpm for 5 minutes. To 400 .mu.l of the supernatant thus
obtained, 4 .mu.l of RNaseA (20 mg/ml) was added, and the resulting
mixture was treated at 37.degree. C. for 60 minutes to digest RNA,
then mixed with 900 .mu.l of cold ethanol, and left till at
-20.degree. C. overnight. The resulting mixture was centrifuged at
15000 rpm for 20 minutes. The resulting precipitate was dissolved
in 10 .mu.l of TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), and
subjected to analysis by 2% agarose gel electrophoresis. As a
marker, 123 bp DNA ladder marker (LIFE TECHNOLOGIES) was used.
[0078] Detection of DNA breakage by measuring DNA content was
carried out as follows. 1.times.10.sup.6 cells of HL-60 which had
been treated with 20 ng/ml of AIP for a predetermined period of
time to induce apoptosis were washed with PBS(-), mixed with 200
.mu.l of 70% cold ethanol, and fixed at 4.degree. C. overnight. The
mixture was centrifuged at 1500 rpm for 5 minutes, the supernatant
was removed, and the cells were washed with PBS(-) for removing
ethanol. The obtained cell pellet was suspended in 0.1 ml of
PBS(-), mixed with 2.5 .mu.l of RNaseA (20 mg/ml), and treated at
37.degree. C. for 20 minutes to digest RNA. After that, the cells
were collected by centrifugation, mixed with 0.5 ml of a 50
.mu.g/ml propidium iodide solution (0.1% sodium citrate, 0.1%
NP-40) to effect staining at 4.degree. C. for 10 minutes in dark,
then passed through 50 .mu.m nylon mesh, and subjected to a flow
cytometry for determination.
[0079] Observation of chromatin condensation by fluorescent
staining was carried out as follows. 1.times.10.sup.6 cells of
HL-60 which had been treated with 20 ng/ml of AIP for a
predetermined period of time to induce apoptosis were washed with
PBS(-), mixed with 100 .mu.l of a cell fixing solution (PBS(-)
containing 1% glutaraldehyde), and left still at room temperature
for 30 minutes for fixing. The resulting mixture was centrifuged at
1500 rpm for 5 minutes to remove the supernatant, and washed with
PBS(-). The resulting cell pellet was suspended in 20 .mu.l of PBS
(-), and mixed with 4 .mu.l of 1 mM HOECHST 33258 in PBS (-) A drop
of the cell suspension was put on a slide glass, covered with a
cover glass, and observed under a fluorescence microscope.
[0080] Carcinostatic effect was tested using cultured human cancer
cell panels in the following method. A total of 39 lines of
cultured cancer cells consisting of 38 lines of cultured human
cancer cells (7 lines of lung cancer, 6 lines of stomach cancer, 6
lines of colon cancer, 5 lines of ovarian cancer, 6 lines of brain
tumor, 5 lines of mammary cancer, 2 lines of renal cancer, and 1
line of melanoma) and mouse P388 leukemia cells were spread over
96-well plates. On the next day, AIP at various concentrations was
added to the wells, and after 48 hours, cell proliferation was
determined by colorimetric assay with sulforhodamine B.
[0081] Effect of AIP on proliferation of adult T cell leukemia
(ATL) cells and cells infected with human T cell leukemia virus-1
(HTLV-1) was examined in the following method.
[0082] 1.times.10.sup.5 cells each of three lines of ATL cells
(Maeda-V, Fukuda-V, Hara-V) and 2 lines of cells infected with
human T cell leukemia virus-1 (HTLV-1) (OYAJ-V, YAM-V) were mixed
with AIP so that the final concentration was 0, 2, and 22 ng/mg,
respectively; cultured at 37.degree. C. in 5% CO.sub.2 for 7 hours;
then mixed with 0.5 .mu.Ci 6-.sup.3H thymidine; and cultured for 5
hours. The resulting cultures were washed three times with PBS(-),
and the amount of 6-.sup.3H thymidine taken up by DNA was
determined by a liquid scintillation counter.
[0083] 1) Apoptotic Activity of Leukemia Cell-Killing Substance
[0084] Killing activity of the purified leukemia cell-killing
substance on human leukemia cell HL-60 was determined by MTS assay.
The relationship between the concentration and the percentage of
the viable cells 16 hours after the treatment is shown in FIG. 4.
The substance at the concentration of about 5 ng/ml killed about
50% of 5.times.10.sup.5 cells/ml, which indicated that the
substance had a very strong leukemia cell-killing activity. The
results of the analysis of the percentage of the viable cells with
the lapse of time after treatment with the leukemia cell-killing
substance at the concentration of 20 ng/ml are shown in FIG. 5. As
a result, it was found that death of about 50% of the cells was
induced in about 4 hours. The results of the analysis of DNA
fragmentation with the lapse of time after treatment with the
leukemia cell-killing substance at the concentration of 20 ng/ml
are shown in FIG. 6. Two hours after the treatment, DNA
fragmentation into nucleosome units was observed to start, which is
specific in apoptosis. It is generally known that, in cell death by
apoptosis, apoptosis-specific DNA fragmentation, i.e. breakage of
chromatin DNA into nucleosome units, is observed.
[0085] The DNA fragmentation in apoptosis may also be determined by
flow cytometry, wherein the apoptotic cells are detected as the
cells having lower DNA content than those in G1 phase. The results
of the analysis by flow cytometry of the DNA content of the cells
0, 2, 4, and 12 hours after the treatment with the leukemia
cell-killing substance at the concentration of 20 ng/ml,
respectively, are shown in FIGS. 7(A), 7(B), 7(C), and 7(D),
respectively. As a result, it was observed that the apoptotic cells
with lower DNA content increased with the lapse of time.
[0086] Apoptosis may also be detected directly under a microscope
by the morphological features. Thus, morphological changes of the
cells 0, 2, 4, and 12 hours after the treatment with the leukemia
cell-killing substance at the concentration of 20 ng/ml,
respectively, were observed under a microscope. The microscopy
photographies are shown in FIGS. 8(A), 8(B), 8(C), and 8(D),
respectively. In the nucleus of the apoptotic cells, chromatin
condensation and nucleus fragmentation were observed. Further, the
nucleus of the cells 0, 2, 4, and 12 hours after the treatment with
the leukemia cell-killing substance at the concentration of 20
ng/ml, respectively, as shown in FIG. 8 were subjected to
fluorescent staining with HOECHST 33258, and examined under a
fluorescence microscope. The results are shown in FIGS. 9(A), 9(B)
9(C), and 9(D), respectively. As a result, chromatin condensation
and/or fragmented nucleus image, which is specific for apoptosis,
was determined in the cells treated with the leukemia cell-killing
substance.
[0087] From the above results, it was decided that the cell death
caused by the treatment with the leukemia cell-killing substance
was apoptosis.
[0088] 2) Proliferation Inhibitory Activity of AIP on Human Cancer
Cells
[0089] Effect of AIP on proliferation of 43 kinds of human cancer
cells was determined by calorimetric assay with sulforhodamine B or
by uptake of 6-.sup.3H thymidine. The results are shown in Table 2.
It was confirmed that AIP inhibited proliferation of the cells
examined.
2TABLE 2 Inhibition Inhibition Inhibition of of of Cell
Proliferation Cell Proliferation Cell Proliferation HBC-4 + HCT-15
+ RXF-631L + BSY-1 + HCT116 + St-4 + MCF-7 + NCl-H23 + MKN1 +
MDA-MB-231 + NCl-H226 + MKN7 + U251 + NCl-H522 + MKN28 + SF-268 +
NCl-H460 + MKN45 + SF-295 + DMS273 + MKN74 + SF-539 + DMS114 +
HBC-5 + SNB-75 + LOX-IMVI + A549 + SNB-78 + OVCAR-3 + ACHN +
HCC2998 + OVCAR-4 + OYAJ-V + KM-12 + OVCAR-5 + YAM-V + HT-29 +
OVCAR-8 + Maeda-V + WiDr + SK-OV-3 + Fukuda-V + Hara-V +
Example 4
[0090] 1) Isolation of Total RNAs from Mackerel's Viscus
[0091] Total RNAs were isolated in accordance with AGPC (Acid
Guanidinium-Phenol-Chloroform) method (Anal. Biochem. 162,
p156-159).
[0092] Viscus of mackerels were dissolved and homogenized in 5 ml
of a solution consisting of phenol and guanidine thiocyanate,
ISOGEN (manufactured by Kabushiki Kaisha Nippon Gene), using
Polytoron homogenizer. To the homogenized solution thus obtained, 1
ml of chloroform was added, and thoroughly mixed with the contents.
The resulting mixture was left on ice for 15 minutes, centrifuged
at 4.degree. C. at 5000 G for 30 minutes to collect the upper phase
(aqueous phase). To this solution, equal volume of
phenol/chloroform solution was added to extract the aqueous phase,
to which isopropyl alcohol in the volume equal to the extracted
aqueous phase was added, and the resulting mixture was left at room
temperature for 10 minutes. The mixture was then centrifuged at
4.degree. C. at 5000 G for 30 minutes to precipitate RNA, which was
then washed with 70% ethanol. The RNA pellet was dissolved in 1 ml
of an elution buffer (10 mM Tri-HCl.sub.1, pH 7.4, 1 mM EDTA),
thereby obtaining 1 mg of all the RNAs.
[0093] 2) Preparation of mRNA
[0094] Total RNAs thus obtained were treated at 65.degree. C. for 5
minutes, and rapidly quenched. The insolubles were removed by
centrifugation, and 0.2 ml of 3M NaCl solution was added to the
remaining to adjust the salt concentration to 0.5 M. The RNA
solution was applied to an oligo(dT) cellulose column (manufactured
by PHARMACIA FINE CHEMICALS CO.), and the fraction passed through
the column was again applied to the column. The column was then
washed with a solution of 10 mM Tri-HCl, pH 7.4, 1 mM EDTA, 0.5M
NaCl (in an amount for several columns) and with a solution of 10
mM Tri-HCl, pH 7.4, 1 mM EDTA, 0.1M NaCl (in an amount for several
columns), and the adsorbed RNA having poly(A) was eluted with an
elusion buffer. The RNA thus obtained was subjected to another
round of purification, mixed with {fraction (1/10)} volume of 3M
sodium acetate (pH 5.1) and 2 volumes of ethanol, and precipitated
at -20.degree. C., thereby obtaining about 35 .mu.g of mRNA having
poly(A)
[0095] 3) Synthesis of cDNA and Preparation of cDNA Library
[0096] 3 .mu.g of mRNA having poly (A) thus obtained was mixed with
distilled water to increase the volume to 7 .mu.l, mixed with 2
.mu.l of Not 1 primer-adapter (0.5 .mu.g/ml, LIFE TECHNOLOGIES),
treated at 70.degree. C. for 10 minutes, and then rapidly quenched.
To the resulting solution, 1 .mu.l of RNAse inhibitor (40
unit/.mu.l, manufactured by STRATAGENE CO.), 4 .mu.l of a buffer
consisting of 250 mM Tri-HCl, pH 8.3, 375 mM KCl, and 15 mM
MgCl.sub.2, 1 .mu.l of DTT, 1 .mu.l of 10 mM dNTP mixture, and a
reverse transcriptase (200 units/.mu.l, LIFE TECHNOLOGIES) were
added, thoroughly mixed, and reacted at 37.degree. C. for 1 hour.
After the reaction, 91 .mu.l of distilled water, 30 .mu.l of a
buffer consisting of 100 mM Tris-HCl, pH 6.9, 450 mM KCl, 23 mM
MgCl.sub.2, 0.75 mM .beta.-NAD+, and 50 mM
(NH.sub.4).sub.2SO.sub.4, 3 .mu.l of 10 mM dNTP mixture, 1 .mu.l of
E. coli DNA ligase (10 units/.mu.l), 4% 1 .mu.l of E. coli DNA
polymerase (10 units/.mu.l), and 1 .mu.l of E. coli RNaseH (2
units/.mu.l) were added, thoroughly mixed, and reacted at
16.degree. C. for 2 hours. After that, 2 .mu.l of T4DNA polymerase
(5 units/.mu.l) was added, and further reacted for 5 minutes. 10
.mu.l of 0.5M EDTA was added to the reaction mixture to terminate
the reaction, and subjected to extraction with phenol-chloroform
(1:1). The extracted upper aqueous phase was mixed with 0.5 volume
of 7.5M ammonium acetate and two volumes of ethanol. The resulting
mixture was centrifuged at 14000 G for 20 minutes, and the
separated pellet was carefully rinsed with 70% ethanol. After
drying, the pellet was dissolved in 25 .mu.l of distilled water, to
which 10 .mu.l of a buffer consisting of 250 mM Tri-HCl, pH 7.6, 50
mM MgCl.sub.2, 5 mM ATP, 5 mM DTT, and 25% (w/v) PEG8000, 10 .mu.l
of Sal 1 adapter (1 .mu.g/.mu.l), and 5 .mu.l of T4DNA ligase (1
unit/.mu.l) were added, and reacted at 16.degree. C. for 24 hours.
The resulting reaction product was subjected to extraction with
phenol-chloroform (1:1), followed by precipitation with ethanol.
The resulting precipitate was dissolved in 41 .mu.l of distilled
water, to which 5 .mu.l of a buffer consisting of 10 mM Tri-HCl, pH
7.5, 7 mM MgCl.sub.2, 150 mM NaCl, 7 mM 2-mercaptoethanol, and
0.01% TritonX-100, and 4 .mu.l of NotI (15 units/.mu.l) were added,
and reacted at 37.degree. C. for 2 hours. The resulting reaction
product was subjected to extraction with phenol-chloroform (1:1),
followed by precipitation with ethanol. The obtained DNA pellet was
dissolved in 100 .mu.l of a buffer consisting of 10 mM Tri-HCl, pH
7.5, 0.1 mM EDTA and 25 mM NaCl, and the solution thus obtained was
subjected to gel filtration through Sephacryl S-500 column (1 ml,
LIFE TECHNOLOGIES) which had been equilibrated with the same
buffer, thereby selecting the fraction containing cDNA of the
required size.
[0097] A mixture of 15 ng of the cDNA thus prepared and 50 ng of
pSPORT 1 plasmid vector (LIFE TECHNOLOGIES) which had been treated
with Not I-Sal I was mixed with distilled water to bring the volume
to 15 .mu.l, to which 4 .mu.l of a buffer consisting of 250 mM
Tri-HCl, pH 7.6, 50 mM MgCl.sub.2, 5 mM ATP, 5 mM DTT, and 25%
(w/v) PEG8000, and 1 .mu.l of T4DNA ligase (1 unit/.mu.l) were
added, and reacted at 22.degree. C. for 4 hours to ligate cDNA into
the vector. 5 .mu.l of yeast tRNA (1 .mu.g/.mu.l), 12.5 .mu.l of
7.5M NH.sub.4OAc, and 70 .mu.l of cold ethanol were added to effect
ethanol-precipitation, and the resulting precipitate was dissolved
in 5 .mu.l of distilled water. The vector cDNA thus obtained was
transformed into E. coli cells for electroporation (Electro MAX
DH10B Cell) using an electroporation system manufactured by BIORAT
CO. (1.8 kv, 25 mF, 200 .OMEGA.), thereby obtaining a cDNA library
of 2,300,000 transformants.
[0098] 4) Amplification of AIP Genes by RT-PCR (Reverse
Transcription Polymerase Chain Reaction)
[0099] Sense primer No. 1 corresponding to EDKDYDT of the amino
acid sequence in the N-terminal of AIP (EHLADXLEDKDYDTLLQTLDNGLPHI)
and antisense primer No. 2 corresponding to MIYDQAD in the internal
amino acid sequence (MIYDQADV) were chemically synthesized,
respectively, and used as amplification primers for AIP genes. To 5
.mu.g of all the RNAs obtained above, 1 .mu.l of
oligo(dT).sub.12-18 (0.5 ng/.mu.l) was added, and the resulting
mixture was incubated at 75.degree. C. for 10 minutes, and rapidly
quenched in ice. Then cDNA synthesis reaction was effected at
42.degree. C. for 1 hour in a solution containing 20 mM Tris-HCl
(pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 0.5 mM each of dNTP, 10 nM DTT,
20 units RNAse inhibitor, and 200 units of reverse transcriptase.
The reaction system was then heated at 70.degree. C. for 10 minutes
to terminate the reaction. 2 units of E. coli RNaseH was added and
treated at 37.degree. C. for 20 minutes, thereby digesting the
remaining RNA. In a solution containing {fraction (1/10)} amount of
the obtained cDNA, 100 pmole each of the two kinds of primers Nos.
1 and 2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl.sub.2, 0.2
mM each of dNTP, 10 mM DTT, and 2.5 units of Taq DNA polymerase,
PCR was performed by repeating 35 cycles of reactions at 94.degree.
C. for 1 minute, 56.degree. C. for 1 minute, and 72.degree. C. for
1 minute. After the completion of these cycles, another reaction
was effected at 72.degree. C. for 8 minutes, so that poly(A) was
incorporated at the 3' terminal of the PCR product.
[0100] 5) Cloning of PCR Product and Determination of DNA Sequence
Thereof
[0101] It was confirmed by 2% agarose gel electrophoresis that the
PCR product thus obtained was about 650 bp in size. The PCR product
was inserted into plasmid vector PCRII using T4DNA ligase and ATP,
and the DNA sequence was determined by a dye terminator method by
fluorescent-labeling of dideoxy nucleotide in accordance with the
instructions for a kit manufactured by PERKIN ELMER CO. (ABI PRISM
Dye Terminator Cycle Sequencing Ready Reaction Kit). As a result,
the amino acid sequence estimated from the DNA sequence was found
to include a sequence obtained by determination of a partial amino
acid sequence of AIP, and named pCRaip001.
[0102] 6) Labeling of Probe
[0103] pCRaip001 thus obtained was cleaved with restriction enzyme
Eco RI, separated by 1.5% agarose gel electrophoresis, and insert
DNA fragments of about 650 bp were eluted from the agarose gel. 60
ng of the purified DNA fragments were prepared into a template, and
labeled with [.alpha.-.sup.32P]dCTP by random primer method in
accordance with the instructions for a kit manufactured by
PHARMACIA FINE CHEMICALS CO. (Ready To Go DNA Labeling Kit).
[0104] 7) Screening of Complete AIP Genes from pSPORT 1 cDNA
Library
[0105] pSPORT 1 cDNA Library prepared above was subjected to
screening by colony hybridization method (Proc. Natl. Acad. Sci.
U.S.A. 72: 3961, 1975) using the probe obtained above. First, about
1,000,000 cells of the transformants obtained above were spread
over a 15 cm LB/ager plate containing ampicillin so that about
20000 colonies were formed, and cultured at 37.degree. C. for 12
hours. Over the plate on which the colonies had been formed, a
nylon filter (manufactured by AMERSHAM CO.) was placed so that no
air was captured therebetween. After 1 minute, the filter was
peeled off and dried. The filter with the side having the colonies
up was placed on a filter paper impregnated with 10% SDS, on a
filter paper impregnated with 0.5M NaOH and 1.5M NaCl, and on a
filter paper impregnated with a solution of 0.5M Tris-HCl, pH 7.5,
1.5M NaCl, respectively, for 3 minutes each in this order, washed
sufficiently with 2.times.SSC, and dried. DNA was fixed on the
filter using UV transilluminator. After the filter is immersed in
5.times.SSC, prehybridization was effected at 65.degree. C. for 2
hours in a mixture of 6.times.SSC, 5.times.Denhardt solution, and
0.5% SDS solution (hybridization solution). After the
prehybridization solution was removed, a mixture of a hybridization
solution which had been heated to 65.degree. C. in advance and the
probe DNA obtained above at 1000000 cpm/ml (after heated at
100.degree. C. for 5 minutes followed by rapid quenching) was
poured onto the filter, and shaken slowly at 65.degree. C. for 20
hours to effect hybridization. The membranes were washed three
times with 1.times.SSC and 0.1% SDS solution each for ten minutes,
and further washed three times at 65.degree. C. with 0.1.times.SSC
and 0.1% SDS solution each for 20 minutes. After the washed
membranes were allowed to dry, the membranes were subjected to
autoradiography at -80.degree. C. overnight, and 27 colonies which
had been strongly hybridized with the labeled probe were selected.
In the plasmid DNA of these, PCR product of about 650 bp was
confirmed by PCR mentioned above. These plasmids were named PSAIP
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, and 27. All of these contained DNA
sequences estimated from the amino acid sequence of AIP, and thus
are believed to contain AIP cDNA. Among these, PSAIP, which was
believed to have almost complete length, was subjected to dye
terminator method by fluorescent-labeling of dideoxynucleotide to
determine the entire DNA sequence of its open reading frame. The
DNA sequence is shown in SEQ ID No. 2 of the attached sequence
listing, and the amino acid sequence estimated from the DNA
sequence is shown in SEQ ID No. 1 of the sequence listing.
[0106] It was found that the cDNA sequence and the amino acid
sequence estimated therefrom include all of the sequences obtained
by determination of the partial amino acid sequence of AIP and
pCRaip001 inserts, and confirmed that the cDNA codes AIP. From the
comparison with the N-terminal amino acids of matured AIP, it is
believed that base 1-90 code the signal peptide, and base 91-1575
code the matured peptide of AIP. The estimated molecular weight of
the polypeptide which is coded by the cDNA is 58668 daltons (55243
daltons if the portion expected to be the signal peptide is
excluded), which is about 7500 daltons lower than the molecular
weight of AIP purified from viscus of mackerels. AIP is a
glycoprotein which is strongly adsorbed on Con A column, and its
sugar chain is believed to amount to about 7500 daltons.
Example 5
[0107] The purified AIP prepared in Example 1 was mixed and reacted
with L-lysine, poly-L-lysine, or poly-D-lysine, and examined with
coloring reagents (peroxidase and o-phenylene diamine) for
generation of hydrogen peroxide. As a result, the purified AIP was
found to oxidize L-lysine to generate about 2800 .mu.M of hydrogen
peroxide. On the other hand, when poly-L-lysine or poly-D-lysine
was mixed, only less than 20 .mu.M of hydrogen peroxide was
generated at most. Further, the purified AIP generates hydrogen
peroxide when not only lysine but also leucin, phenylalanine,
arginine, methionine, histidine, or the like is used as a
substrate. Accordingly, the purified AIP is believed to catalyze
aldehyde-generating reaction by oxidative deamination to generate
hydrogen peroxide.
[0108] Next, in order to determine the relation between the
hydrogen peroxide-generating reaction by the oxidative reaction and
the apoptotic activity of the purified AIP on cancer cells, the
apoptotic activity of the purified AIP on cancer cells was examined
with and without addition of catalase for catalyzing the
decomposition of hydrogen peroxide. The measurement was performed
in the same way as in the method for determining the apoptotic
activity of the purified leukemia cell (human leukemia cell HL-60)
killing substance in Example 3 except for addition of catalase, and
the percentage of the dead cells after 16 hours was measured. As a
result, the percentage of the cell death was 18% when catalase was
added in addition to the purified AIP, as the same time the
percentage of the dead cells was 100% when only the purified AIP
was added.
[0109] Therefore, it is believed that the apoptotic activity of the
purified AIP on cancer cells is at least partly attributed to the
reaction to catalyze the aldehyde-generating reaction by the
oxidative deamination to generate hydrogen peroxide.
[0110] In this regard, the purified AIP was subjected to motif
analysis to search for the domain exhibiting changes similar to the
typical change in absorption of the visible spectrum observed in
flavoprotein. As a result, it was found that amino acids 61-89 in
the amino acid sequence of SEQ ID No. 1 of the attached sequence
listing are the flavin-bonding domain. Consequently, it was
revealed that one of the amino acid sequences required for the
reaction to catalyze the aldehyde-generating reaction by oxidative
deamination to generate hydrogen peroxide was amino acids 61-89 of
the sequence.
[0111] Next, the AIP gene having the DNA sequence of SEQ ID No. 2
of the attached sequence listing was caused to vary by PCR method
to obtain mutant genes, and plasmid obtained by constructing the
mutant genes into an expression vector (pME18S) for mammal cells
was transfected into a African green monkey kidney cell line cos-7
to prepare mutant AIPs each with amino acids 1-514 or 1-496,
respectively, of SEQ ID No. 1 of the sequence listing. These mutant
AIPs and the purified AIP obtained in Example 1 were subjected to
Western blotting using the monoclonal antibodies prepared in
Example 2. The results are shown in FIG. 10. Further, the apoptotic
activity of the AIP and the mutant AIPs on human leukemia cells was
determined in the same way as in Example 3. It was found that the
complete AIP consisting of amino acids 1-524 and the mutant AIP
consisting of amino acids 1-514 exhibited apoptotic activity on
cancer cells, while the mutant AIP consisting of amino acids 1-496
has not apoptotic activity to be detected.
[0112] Consequently, it is understood that amino acids 497-514 of
the sequence are also essential to maintain the function of AIP of
the present invention.
Example 6
[0113] In order to detect the origin of the purified AIP prepared
in Example 1, the viscus of mackerels used were analyzed, which
were found to be infected with parasites Thus, extracts of viscera
were prepared from 5 mackerels infected with parasites and from 5
mackerels not infected with parasites, in the same way as in
Example 1. Each viscus extract thus obtained was subjected to
Western blotting using the monoclonal antibodies prepared in
Example 2. The results are shown in FIG. 11. In FIG. 11, Lanes 1-5
show the results of the viscus extracts from 5 mackerels infected
with parasites, while Lanes 6-10 show the results of the viscus
extracts from 5 mackerels not infected with parasites. Further,
apoptotic activity of these viscus extracts on human leukemia cells
was determined in the same way as in Example 3. The results are
shown in Table 3. In Table 3, sample Nos. 1-5 show the results of
the viscus extracts from 5 mackerels infected with parasites, while
sample Nos. 6-10 show the results of the viscus extracts from 5
mackerels not infected with parasites. Further, the specific
activity is indicated in relative values to the strength of the
apoptotic activity of the viscus extract sample No. 5 on human
leukemia cells, which is put as 1.
[0114] From the results in FIG. 11 and Table 3, the viscus extracts
of the mackerels infected with parasites were found to contain AIP
of the present invention, and to have apoptotic activity on cancer
cells. On the contrary, the viscus extracts of the mackerels not
infected with parasites were found to have no such activity.
Therefore, it was revealed that the expression of AIP was induced
in mackerels by infection with parasites.
[0115] From the above, it is decided that AIP is induced in
mackerels by stimulation accompanied by activation of helper T2
cells (Th2 cells) such as infection with parasites, and that
proteins having sequences homologous to AIP or having cell death
inducing activity may be induced in mammals in the similar
mechanism.
3 TABLE 3 Sample No. 1 2 3 4 5 6 7 8 9 10 Specific 38 38 4 38 1 0 0
0 0 0 Activity
[0116]
Sequence CWU 1
1
2 1 524 PRT Scomber japonicus 1 Met Asn Leu His Val Val Lys Trp Lys
Leu Ser Val Val Ser Val Leu 1 5 10 15 Ile Thr Leu Tyr Tyr Ser His
Thr Val Ala Leu Ser Leu Lys Glu His 20 25 30 Leu Ala Asp Cys Leu
Glu Asp Lys Asp Tyr Asp Thr Leu Leu Gln Thr 35 40 45 Leu Asp Asn
Gly Leu Pro His Ile Asn Thr Ser His His Val Val Ile 50 55 60 Val
Gly Ala Gly Met Ala Gly Leu Thr Ala Ala Lys Leu Leu Gln Asp 65 70
75 80 Ala Gly His Thr Val Thr Ile Leu Glu Ala Asn Asp Arg Val Gly
Gly 85 90 95 Arg Val Glu Thr Tyr Arg Asn Glu Lys Glu Gly Trp Tyr
Ala Glu Met 100 105 110 Gly Ala Met Arg Ile Pro Ser Ser His Arg Ile
Val Gln Trp Phe Val 115 120 125 Lys Lys Leu Gly Val Glu Met Asn Glu
Phe Val Met Thr Asp Asp Asn 130 135 140 Thr Phe Tyr Leu Val Asn Gly
Val Arg Glu Arg Thr Tyr Val Val Gln 145 150 155 160 Glu Asn Pro Asp
Val Leu Lys Tyr Asn Val Ser Glu Ser Glu Lys Gly 165 170 175 Ile Ser
Ala Asp Asp Leu Leu Asp Arg Ala Leu Gln Lys Val Lys Glu 180 185 190
Glu Val Glu Ala Asn Gly Cys Lys Ala Ala Leu Glu Lys Tyr Asp Arg 195
200 205 Tyr Ser Val Lys Glu Tyr Leu Lys Glu Glu Gly Gly Leu Ser Pro
Gly 210 215 220 Ala Val Arg Met Ile Gly Asp Leu Leu Asn Glu Gln Ser
Leu Met Tyr 225 230 235 240 Thr Ala Leu Ser Glu Met Ile Tyr Asp Gln
Ala Asp Val Asn Asp Ser 245 250 255 Val Thr Tyr His Glu Val Thr Gly
Gly Ser Asp Leu Leu Pro Glu Ala 260 265 270 Phe Leu Ser Val Leu Asp
Val Pro Ile Leu Leu Asn Ser Lys Val Lys 275 280 285 His Ile Arg Gln
Ser Asp Lys Gly Val Ile Val Ser Tyr Gln Thr Gly 290 295 300 Asn Glu
Ser Ser Leu Met Asp Leu Ser Ala Asp Ile Val Leu Val Thr 305 310 315
320 Thr Thr Ala Lys Ala Ala Leu Phe Ile Asp Phe Asp Pro Pro Leu Ser
325 330 335 Ile Ser Lys Met Glu Ala Leu Arg Ser Val His Tyr Asp Ser
Ser Thr 340 345 350 Lys Ile Leu Leu Thr Phe Arg Asp Lys Phe Trp Glu
Asp Asp Gly Ile 355 360 365 Arg Gly Gly Lys Ser Ile Thr Asp Gly Pro
Ser Arg Tyr Ile Tyr Tyr 370 375 380 Pro Ser His Ser Phe His Thr Asn
Glu Thr Ile Gly Val Leu Leu Ala 385 390 395 400 Ser Tyr Thr Trp Ser
Asp Glu Ser Leu Leu Phe Leu Gly Ala Ser Asp 405 410 415 Glu Glu Leu
Lys Glu Leu Ala Leu Arg Asp Leu Ala Lys Ile His Gly 420 425 430 Glu
Gln Val Trp Asp Lys Cys Thr Gly Val Ile Val Lys Lys Trp Ser 435 440
445 Ala Asp Pro Tyr Ser Leu Gly Ala Phe Ala Leu Phe Thr Pro Tyr Gln
450 455 460 His Leu Glu Tyr Ala Gln Glu Leu Phe Ser Ser Glu Gly Arg
Val His 465 470 475 480 Phe Ala Gly Glu His Thr Ala Phe Pro His Ala
Trp Ile Glu Thr Ser 485 490 495 Met Lys Ser Ala Ile Arg Ala Ala Thr
Asn Ile Asn Lys Val Ala Asn 500 505 510 Glu Glu Ser Thr Ile Glu His
Thr Lys Asp Glu Leu 515 520 2 1575 DNA Scomber japonicus 2
atgaatctgc atgtggtgaa atggaaatta tctgttgtca gtgtgctgat cacattgtac
60 tacagtcaca ctgttgctct cagcctgaag gaacatctgg ctgattgtct
tgaagacaaa 120 gactatgaca cgctgctgca gactctggat aacggtcttc
cacacattaa cacgtctcat 180 catgtggtta tagtcggagc tggcatggcc
ggactgacgg cggccaagtt actgcaagac 240 gcaggacaca cggtaaccat
attggaggct aatgatcgtg ttggaggacg tgtggagacc 300 tacaggaatg
aaaaagaagg ctggtatgct gaaatgggag ctatgaggat cccaagctct 360
caccgcatcg tccagtggtt tgtcaaaaag cttggggtcg agatgaatga gttcgtcatg
420 actgatgaca acacctttta cctggttaat ggggtgcggg agaggacata
tgttgttcaa 480 gaaaaccctg atgtcctgaa gtacaacgtg tcagaaagcg
agaagggaat ttcagccgat 540 gatctgctag atcgagcttt gcagaaggtg
aaagaggaag tggaagcaaa tggttgtaaa 600 gctgcactgg aaaaatacga
ccgctattct gtgaaggagt atctgaaaga agaaggtggt 660 ttgagtccag
gagcagtgag gatgattgga gacctgctga atgaacagag cctcatgtac 720
acagcgctga gtgagatgat ctacgaccag gctgacgtca atgacagtgt cacgtatcat
780 gaagtgacgg gtggatcaga tcttcttccc gaagcttttc tttctgtcct
ggatgtcccc 840 atcctcttaa actccaaagt caaacacatc aggcagtcag
ataaaggtgt aatcgtgtca 900 taccagacag gcaatgagtc ctctttgatg
gacctttctg ctgacattgt tctggtaaca 960 accacagcca aagcagccct
cttcatagac tttgatccac ctctctccat cagtaagatg 1020 gaggccctcc
ggtcagtcca ctatgacagc tccactaaaa tcctcctcac ctttcgcgat 1080
aagttctggg aggacgatgg catccgagga ggcaagagca ttaccgatgg accttctcgt
1140 tacatctact atcccagcca cagtttccat acaaatgaga ccattggagt
cctcctggca 1200 tcctacactt ggtctgacga gtccctcctc ttcctgggtg
caagcgatga agagctgaaa 1260 gagctggccc tgagagatct ggcaaaaatc
cacggtgagc aagtctggga taagtgcacg 1320 ggagtcatag tgaagaagtg
gagcgctgat ccttacagct tgggcgcctt cgctctcttc 1380 acaccctacc
aacacttgga gtacgctcag gagctcttca gcagcgaggg cagggtgcac 1440
tttgctggtg aacacacagc cttccctcat gcttggatcg aaacgtctat gaaatctgca
1500 atcagggctg ctacaaatat taataaagtg gcaaatgaag agtcaactat
agaacataca 1560 aaagatgagc tgtag 1575
* * * * *