U.S. patent application number 11/702119 was filed with the patent office on 2007-06-28 for gankyrin.
Invention is credited to Jun Fujita.
Application Number | 20070148179 11/702119 |
Document ID | / |
Family ID | 17701459 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148179 |
Kind Code |
A1 |
Fujita; Jun |
June 28, 2007 |
Gankyrin
Abstract
Gankyrin having the amino acid sequence as set forth in SEQ ID
NO: 2, or modified gankyrin comprising an amino acid sequence
modified by the deletion and/or addition of one or a plurality of
amino acids and/or the substitution with other amino acids in the
amino acid sequence of SEQ ID NO: 2 and retaining the biological
activity of gankyrin, a gene encoding it, and a method of preparing
said protein and uses thereof.
Inventors: |
Fujita; Jun; (Kyoto-shi,
JP) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Family ID: |
17701459 |
Appl. No.: |
11/702119 |
Filed: |
February 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11083944 |
Mar 21, 2005 |
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11702119 |
Feb 5, 2007 |
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09509775 |
Mar 31, 2000 |
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PCT/JP98/04467 |
Oct 2, 1998 |
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11083944 |
Mar 21, 2005 |
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Current U.S.
Class: |
424/155.1 ;
514/44A; 530/388.8; 536/23.1 |
Current CPC
Class: |
C07K 16/303 20130101;
C07K 14/47 20130101; C07K 2319/00 20130101; C07K 2317/34 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
424/155.1 ;
514/044; 530/388.8; 536/023.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/02 20060101 C07H021/02; A61K 48/00 20060101
A61K048/00; C07K 16/30 20060101 C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 3, 1997 |
JP |
9-286214 |
Claims
1. An agent for treating cancer comprising an inhibitor to a
polypeptide having a biological activity of gankyrin (gankyrin
inhibitor).
2. An agent for treating hepatoma comprising an inhibitor to a
polypeptide having a biological activity of gankyrin.
3. An agent for treating cancer comprising a gankyrin inhibitor
which inhibits a function of a polypeptide having a biological
activity of gankyrin.
4. An agent for treating hepatoma comprising an gankyrin inhibitor
which inhibits a function of a polypeptide having a biological
activity of gankyrin.
5. An agent for treating cancer comprising a gankyrin inhibitor
which inhibits an expression of a polypeptide having a biological
activity of gankyrin.
6. An agent for treating hepatoma comprising an gankyrin inhibitor
which inhibits an expression of a polypeptide having a biological
activity of gankyrin.
7. An agent for treating cancer according to claim 3, wherein the
gankyrin inhibitor which inhibits a function of a polypeptide
having a biological activity of gankyrin is an antibody to a
polypeptide having a biological activity of gankyrin.
8. An agent for treating hepatoma according to claim 4, wherein the
gankyrin inhibitor which inhibits a function of a polypeptide
having a biological activity of gankyrin is an antibody to a
polypeptide having a biological activity of gankyrin.
9. An agent for treating cancer according to claim 5, wherein the
gankyrin inhibitor which inhibits an function of a polypeptide
having a biological activity of gankyrin is an antisense
oligonucleotide to a gene encoding a polypeptide having a
biological activity of gankyrin.
10. An agent for treating hepatoma according to claim 6, wherein
the gankyrin inhibitor which inhibits an function of a polypeptide
having a biological activity of gankyrin is an antisense
oligonucleotide to a gene encoding a polypeptide having a
biological activity of gankyrin.
11. An agent for treating cancer according to claim 7, wherein the
antibody is a monoclonal antibody.
12. An agent for treating hepatoma according to claim 8, wherein
the antibody is a monoclonal antibody.
13. An agent for treating cancer according to claim 7, wherein the
antibody is a humanized antibody.
14. An agent for treating hepatoma according to claim 8, wherein
the antibody is a humanized antibody.
15. An agent for treating cancer according to claim 9, wherein the
antisense oligonucleotide is an oligoncleotide hybridizing to a
part of the nucleotide sequence shown in SEQ ID NO: 1.
16. An agent for treating cancer according to claim 9, wherein the
antisense oligonucleotide is an oligoncleotide hybridizing to a
continuous at least 20 nucleotides in the nucleotide sequence shown
in SEQ ID NO: 1.
17. An agent for treating cancer according to claim 16, wherein the
continuous at least 20 nucleotides contain a translation start
codon.
18. An agent for treating cancer according to claim 1, wherein the
polypeptide having a biological activity of gankyrin is selected
from the group consisting of: (a) A polypeptide comprising an amino
acid sequence from Ala at position 14 to Gly at position 226 of SEQ
ID NO: 2 and having the biological activity of gankyrin; (b) A
polypeptide comprising an amino acid sequence modified by the
deletion and/or addition of one or a plurality of amino acids
and/or the substitution with other amino acids in the amino acid
sequence from Ala at position 14 to Gly at position 226 of SEQ ID
NO: 2 and retaining the biological activity of gankyrin; (c) A
polypeptide comprising an amino acid sequence from Met at position
1 to Gly at position 226 of SEQ ID NO: 2 and having the biological
activity of gankyrin; (d) A polypeptide comprising an amino acid
sequence modified by the deletion and/or addition of one or a
plurality of amino acids and/or the substitution with other amino
acids in the amino acid sequence from Met at position 1 to Gly at
position 226 of SEQ ID NO: 2 and retaining the biological activity
of gankyrin; (e) A polypeptide that is encoded by a DNA capable of
hybridizing under a stringent condition to a DNA having the
nucleotide sequence as set forth in SEQ ID NO: 1 and that has the
biological properties of gankyrin; (f) A polypeptide comprising an
amino acid sequence from Ala at position 14 to Met at position 231
of SEQ ID NO: 4 and having the biological activity of gankyrin; (g)
A polypeptide comprising an amino acid sequence modified by the
deletion and/or addition of one or a plurality of amino acids
and/or the substitution with other amino acids in the amino acid
sequence from Ala at position 14 to Met at position 231 of SEQ ID
NO: 4 and retaining the biological activity of gankyrin; (h) A
polypeptide comprising an amino acid sequence from Met at position
1 to Met at position 231 of SEQ ID NO: 4 and having the biological
activity of gankyrin; (i) A polypeptide comprising an amino acid
sequence modified by the deletion and/or addition of one or a
plurality of amino acids and/or the substitution with other amino
acids in the amino acid sequence from Met at position 1 to Met at
position 231 of SEQ ID NO: 4 and retaining the biological activity
of gankyrin; (j) A polypeptide that is encoded by a DNA capable of
hybridizing under a stringent condition to a DNA having the
nucleotide sequence as set forth in SEQ ID NO: 3 and that has the
biological properties of gankyrin; (k) A polypeptide comprising an
amino acid sequence from Ala at position 14 to Met at position 231
of SEQ ID NO: 6 and having the biological activity of gankyrin; (l)
A polypeptide comprising an amino acid sequence modified by the
deletion and/or addition of one or a plurality of amino acids
and/or the substitution with other amino acids in the amino acid
sequence from Ala at position 14 to Met at position 231 of SEQ ID
NO: 6 and retaining the biological activity of gankyrin; (m) A
polypeptide comprising an amino acid sequence from Met at position
1 to Met at position 231 of SEQ ID NO: 6 and having the biological
activity of gankyrin; (n) A polypeptide comprising an amino acid
sequence modified by the deletion and/or addition of one or a
plurality of amino acids and/or the substitution with other amino
acids in the amino acid sequence from Met at position 1 to Met at
position 231 of SEQ ID NO: 6 and retaining the biological activity
of gankyrin; and (o) A polypeptide that is encoded by a DNA capable
of hybridizing under a stringent condition to a DNA having the
nucleotide sequence as set forth in SEQ ID NO: 5 and that has the
biological properties of gankyrin.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel protein gankyrin,
and to a method of preparation and to uses thereof.
BACKGROUND ART
[0002] Hepatocellular carcinoma (HCC) is one of the most prevalent
cancers in the Orient and Southern Africa. In the past 10 years,
there have been significant advances in the diagnosis and treatment
of HCC patients, with a result that the cases of surgical treatment
are increasing (Arii, S. et al., Primary liver cancer in Japan,
Springer-Verlag (1992) 243-255; The Liver Cancer Study Group of
Japan, Primary liver cancer in Japan, Springer-Verlag (1992)
445-453). Despite the marked progress, however, the survival rate
remains low. One of the barriers to the lengthening of the survival
period appears to be the occurrence of intrahepatic regeneration of
the cancer after its complete removal on a macroscopic level (The
Liver Cancer Study Group of Japan, Ann. Surg. (1990) 211, 277-287;
Belghiti, J. et al., Ann. Surg. (1991) 214, 114-117).
[0003] In this connection, extensive efforts have been made to
determine a prognosis judging factor that affects the intrahepatic
regeneration and the lengthening of the survival period. Up to now,
the inventor of the present invention has analyzed the expression
of several genes of HCC (Mise, M. et al., Hepatology (1996) 23,
455-464; Furutani, M.et al., Hepatology (1996) 24, 1441-1445;
Furutani, M. et al., Cancer Lett. (1997) 111, 191-197). As a result
the present inventor has identified kan-1 (bile acid CoA: amino
acid N-acyl transferase) mRNA as a novel prognosis judging factor.
The expression of this factor is decreased in HCC with poor
prognosis (Furutani, M. et al., Hepatology (1996) 24,
1441-1445).
[0004] In addition to the above, novel molecular markers of HCC,
that add to the predicative value to conventional clinical
prognostic factors such as portal complications,
.alpha.-fetoprotein (AFP) levels, tumor size, the number of tumors,
and the like, are sought (The Liver Cancer Study Group of Japan,
Primary liver cancer in Japan, Springer-Verlag (1992) 445-453; The
Liver Cancer Study Group of Japan, Ann. Surg. (1990) 211, 277-287;
The Liver Cancer Study Group of Japan, Cancer (1994) 74, 2772-2780;
Franco, D. et al., Gastroenterology (1990) 98, 733-738; Calvet, X.
et al., Hepatology (1990) 12, 753-760).
DISCLOSURE OR THE INVENTION
[0005] In order to identify a molecular marker that is elevated in
HCC, the inventor of the present invention has subtracted cDNA
derived from the non-cancerous portion of the liver from cDNA made
from HCC of the same patient. As a result, the inventors have
isolated a novel gene, gankyrin, that consists of the ankyrin
repeat motif alone and that exhibits carcinogenicity in in vitro
and in in vivo assay systems.
[0006] Thus, the present invention provides a novel gankyrin
polypeptide, genes encoding it, a method of preparing said
polypeptide, an antibody against said polypeptide, and uses
thereof.
[0007] In order to solve the above problems, the present invention
provides a polypeptide comprising an amino acid sequence from Ala
at position 14 to Gly at position 226 of SEQ ID NO: 2 and having
the biological activity of gankyrin.
[0008] The present invention also provides a polypeptide comprising
an amino acid sequence modified by the deletion and/or addition of
one or a plurality of amino acids and/or the substitution with
other amino acids, in the amino acid sequence from Ala at position
14 to Gly at position 226 of SEQ ID NO: 2 and retaining the
biological activity of gankyrin.
[0009] The present invention also provides a polypeptide comprising
an amino acid sequence from Met at position 1 to Gly at position
226 of SEQ ID NO: 2 and having the biological activity of
gankyrin.
[0010] The present invention also provides a polypeptide comprising
an amino acid sequence modified by the deletion and/or addition of
one or a plurality of amino acids and/or the substitution with
other amino acids in the amino acid sequence from Met at position 1
to Gly at position 226 in SEQ ID NO: 2 and retaining the biological
activity of gankyrin.
[0011] The present invention also provides a polypeptide that is
encoded by a DNA capable of hybridizing under a stringent condition
to a DNA having the nucleotide sequence as set forth in SEQ ID NO:
1 and that has the biological properties of gankyrin. The stringent
condition as used herein means, for example, a condition provided
by 65.degree. C. in 0.1.times.SSC and 0.1% SDS.
[0012] The present invention also provides a signal-added
polypeptide, in which a signal sequence has been added to a
polypeptide encoded by a DNA that encodes a polypeptide comprising
an amino acid sequence from Ala at position 14 to Gly at position
226 of SEQ ID NO: 2 and having the biological activity of gankyrin,
a DNA that encodes a polypeptide comprising an amino acid sequence
modified by the deletion and/or addition of one or a plurality of
amino acids and/or the substitution with other amino acids in the
amino acid sequence from Ala at position 14 to Gly at position 226
of SEQ ID NO: 2 and retaining the biological activity of gankyrin,
or a DNA capable of hybridizing under a stringent condition to a
DNA that encodes a polypeptide having the nucleotide sequence as
set forth in SEQ ID NO: 1 and having the biological properties of
gankyrin. The stringent condition as used herein means, for
example, a condition provided by 65.degree. C. in 0.1.times.SSC and
0.1% SDS.
[0013] The present invention also provides a polypeptide comprising
an amino acid sequence from Ala at position 14 to Met at position
231 of SEQ ID NO: 4 and having the biological activity of
gankyrin.
[0014] The present invention also provides a polypeptide comprising
an amino acid sequence modified by the deletion and/or addition of
one or a plurality of amino acids and/or the substitution with
other amino acids in the amino acid sequence from Ala at position
14 to Met at position 231 of SEQ ID NO: 4 and retaining the
biological activity of gankyrin.
[0015] The present invention also provides a polypeptide comprising
an amino acid sequence from Met at position 1 to Met at position
231 of SEQ ID NO: 4 and having the biological activity of
gankyrin.
[0016] The present invention also provides a polypeptide comprising
an amino acid sequence modified by the deletion and/or addition of
one or a plurality of amino acids and/or the substitution with
other amino acids in the amino acid sequence from Met at position 1
to Met at position 231 of SEQ ID NO: 4 and retaining the biological
activity of gankyrin.
[0017] The present invention also provides a polypeptide that is
encoded by a DNA capable of hybridizing under a stringent condition
to a DNA having the nucleotide sequence as set forth in SEQ ID NO:
3 and that has the biological properties of gankyrin. The stringent
condition as used herein means, for example, a condition provided
by 65.degree. C. in 0.1.times.SSC and 0.1% SDS.
[0018] The present invention also provides a signal-added
polypeptide, in which a signal sequence has been added to a
polypeptide encoded by a DNA that encodes a polypeptide comprising
an amino acid sequence from Ala at position 14 to Met at position
231 of SEQ ID NO: 4 and having the biological activity of gankyrin,
a DNA that encodes a polypeptide comprising an amino acid sequence
modified by the deletion and/or addition of one or a plurality of
amino acids and/or tile substitution with other amino acids in the
amino acid sequence from Ala at position 14 to Met at position 231
of SEQ ID NO: 4 and retaining the biological activity of gankyrin,
or a DNA capable of hybridizing under a stringent condition to a
DNA that encodes a polypeptide having the base sequence as set
forth in SEQ ID NO: 3 and having the biological properties of
gankyrin. The stringent condition as used herein means, for
example, a condition provided by 65.degree. C. in 0.1.times.SSC and
0.1% SDS.
[0019] The present invention also provides a polypeptide comprising
an amino acid sequence from Ala at position 14 to Met at position
231 of SEQ ID NO: 6 and having the biological activity of
gankyrin.
[0020] The present invention also provides a polypeptide comprising
an amino acid sequence modified by the deletion and/or addition of
one or a plurality of amino acids and/or the substitution with
other amino acids in the amino acid sequence from Ala at position
14 to Met at position 231 of SEQ ID NO: 6 and retaining the
biological activity of gankyrin.
[0021] The present invention also provides a polypeptide comprising
an amino acid sequence from Met at position 1 to Met at position
231 of SEQ ID NO: 6 and having the biological activity of
gankyrin.
[0022] The present invention also provides a polypeptide comprising
an amino acid sequence modified by the deletion and/or addition of
one or a plurality of amino acids and/or the substitution with
other amino acids in the amino acid sequence from Met at position 1
to Met at position 231 of SEQ ID NO: 6 and retaining the biological
activity of gankyrin.
[0023] The present invention also provides a polypeptide that is
encoded by a DNA capable of hybridizing under a stringent condition
to a DNA having the nucleotide sequence as set forth in SEQ ID NO:
5 and that has the biological properties of gankyrin. The stringent
condition as used herein means, for example, a condition provided
by 65.degree. C. in 0.1.times.SSC and 0.1% SDS.
[0024] The present invention also provides a signal-added
polypeptide, in which a signal sequence has been added to a
polypeptide encoded by a DNA that encodes a polypeptide comprising
an amino acid sequence from Ala at position 14 to Met at position
231 of SEQ ID NO: 6 and having the biological activity of gankyrin,
a DNA that encodes a polypeptide comprising an amino acid sequence
modified by the deletion and/or addition of one or a plurality of
amino acids and/or the substitution with other amino acids in the
amino acid sequence from Ala at position 14 to Met at position 231
of SEQ ID NO: 6 and retaining the biological activity of gankyrin,
or a DNA capable of hybridizing under a stringent condition to a
DNA that encodes a polypeptide having the base sequence as set
forth in SEQ ID NO: 5 and having the biological properties of
gankyrin. The stringent condition as used herein means, for
example, a condition provided by 65.degree. C. in 0.1.times.SSC and
0.1% SDS.
[0025] The present invention also provides a fusion polypeptide
comprising the above polypeptide and another peptide or
polypeptide.
[0026] The present invention also provides a DNA encoding the above
polypeptide.
[0027] The present invention also provides a vector comprising the
above DNA.
[0028] The present invention also provides a host transformed with
the above vector.
[0029] The present invention also provides a method of preparing
the above polypeptide, said method comprising culturing a host
transformed with an expression vector comprising a DNA encoding
said polypeptide and recovering the desired polypeptide from said
culture.
[0030] The present invention also provides an antibody that
specifically reacts to the above polypeptide. The antibody is
preferably a monoclonal antibody or a polyclonal antibody.
[0031] The present invention also provides a method of detecting or
determining a gankyrin polypeptide, said method comprising
contacting the above antibody to a sample expected to contain said
gankyrin polypeptide and detecting or determining the formation of
an immune complex between said antibody and said gankyrin
polypeptide.
[0032] The present invention also provides an antisense
oligonucleotide that hybridizes any of the sites of the nucleotide
sequence as set forth in SEQ ID NO: 1.
[0033] The present invention also provides an antisense
oligonucleotide to at least 20 contiguous nucleotides in the
nucleotide sequence as set forth in SEQ ID NO: 1. Said antisense
oligonucleotide to the at least 20 contiguous nucleotides
preferably have a translation initiation codon.
[0034] The present invention also provides an expression inhibitor
of a gankyrin polypeptide, said inhibitor comprising said antisense
oligonucleotide as an active ingredient.
[0035] The present invention also provides a method of screening an
agonist or an antagonist of the gankyrin polypeptide to the binding
of the gankyrin polypeptide and Rb, said method comprising
contacting a gankyrin polypeptide or a substance containing the
gankyrin polypeptide with a sample expected to contain the agonist
or the antagonist of the gankyrin polypeptide in the presence of
Rb, and detecting a free gankyrin polypeptide or Rb. The above
substance containing the gankyrin polypeptide is for example a cell
lysate that expresses gankyrin.
[0036] The present invention also provides a method of screening an
agonist or an antagonist of the gankyrin polypeptide to the binding
of the gankyrin polypeptide and NF.kappa.B, said method comprising
contacting a gankyrin polypeptide or a material containing the
gankyrin polypeptide with a sample expected to contain the agonist
or the antagonist of the gankyrin polypeptide in the presence of
NF.kappa.B, and detecting a free gankyrin polypeptide or
NF.kappa.B. The above substance containing the gankyrin polypeptide
is for example a cell lysate that expresses gankyrin.
BRIEF EXPLANATION OF DRAWINGS
[0037] FIG. 1 is a diagram showing the position of a gankyrin gene
on the human X chromosome.
[0038] FIG. 2 is a photograph showing that a gankyrin gene on the
chromosome in human lymphocytes was fluorescence-stained by the in
situ hybridization method and detected.
[0039] FIG. 3 is an electrophoregram showing the result that mRNAs
from the normal liver tissue (N) and the hepatic cancer tissue (T)
of 5 patients with hepatic cancer (1 to 5) were detected by the
Northern method using human gankyrin cDNA as a probe.
[0040] FIG. 4 is an electrophoregram showing the result that mRNAs
from various human cell lines were detected by the Northern method
using human gankyrin cDNA as a probe.
[0041] FIG. 5 is an electrophoregram showing the result that mRNAs
from various normal tissues were detected by the Northern method
using human gankyrin cDNA as a probe.
[0042] FIG. 6 is an electrophoregram showing the result that
gankyrin polypeptides in cell lysates from the normal tissue (N)
and the hepatic cancer tissue (T) of 3 patients with hepatic cancer
(1 to 3) were detected by the Western blot method using
anti-gankyrin polypeptide antibody.
[0043] FIG. 7 is an electrophoregram showing the result that
gankyrin polypeptides in cell lysates from various human cell lines
were detected by the Western blot method using anti-gankyrin
polypeptide antibody.
[0044] FIG. 8A is an electrophoregram showing the result that the
in vitro translated gankyrin gene products were detected in a
similar manner to FIG. 6, and FIG. 8B is an electrophoregram
showing the result that the in vitro translated gankyrin gene
products (non-labeled) were detected in a similar manner to FIG.
6.
[0045] FIG. 9 is an electrophoregram showing the result that fusion
polypeptides comprising gankyrin polypeptide and GST were expressed
in Escherichia coli (E. coli) and then were detected using various
antibodies.
[0046] FIG. 10 is an electrophoregram showing the result that
fusion polypeptides of gankyrin polypeptides and HA were expressed
in the 293 cells, immunoprecipitated and then were detected using
various antibodies.
[0047] FIG. 11 is an electrophoregram showing the result that
fusion polypeptides of gankyrin polypeptides and HA were expressed
in the 293 cells, immunoprecipitated and then were detected, using
various antibodies.
[0048] FIG. 12 is an electrophoregram showing the result that mRNAs
in various cell cycles of the NIH/3T3 cells were detected by the
Northern method using mouse gankyrin cDNA as a probe.
[0049] FIG. 13 is an electrophoregram showing the result that mRNAs
in the NIH/3T3 cells propagated at various concentrations were
detected in a similar manner to FIG. 12.
[0050] FIG. 14 is an electrophoregram showing the result that mRNAs
in the hepatic tissue during the process of liver regeneration
after partial hepatic resection in mice were detected in a similar
manner to FIG. 12.
[0051] FIG. 15 is a drawing that shows the position and the number
of repetition of ankyrin repeats in various proteins.
EMBODIMENT FOR CARRYING OUT THE INVENTION
[0052] In accordance with the present invention, a gankyrin
polypeptide means a polypeptide having the biological activity of
gankyrin. The biological activity of gankyrin is carcinogenicity,
of which specific effects include, as described in Example 4, the
elevation of colony-forming ability of cells in soft agar and the
induction of tumorigenicity and the suppression of apoptosis in
mice.
[0053] The gankyrin gene of the present invention and the cDNA
thereof can be obtained by screening hepatic cancer cells or
hepatic cancer tissue as its source to obtain the gene or the cDNA
thereof. As a method of screening or isolating the gene or cDNA
thereof, there can be used one that can selectively screen the gene
whose amount expressed varies, such as the subtraction method
(Nucleic Acids Research (1988) 16, 10937) and the differential
hybridization method (Cell (1979) 16, 443-452).
[0054] The gene encoding the gankyrin polypeptide of the present
invention can be obtained by the subtraction between a cDNA library
prepared from the normal liver tissue and a cDNA library prepared
from the hepatic cancer tissue to select cDNA derived from mRNA
that is expressed in the hepatic cancer tissue but not in the
normal liver tissue.
[0055] For example, in the subtraction method, cDNA obtained from
the hepatic cancer tissue or the normal liver tissue is amplified.
Primers to amplify the normal liver tissue are labeled with a
labeling compound, for example biotin. Then, an excessive amount of
double stranded cDNA derived from the normal liver tissue is mixed
with a small amount of double stranded cDNA derived from the
hepatic cancer tissue to form a mixture, which is then made
single-stranded by heat denaturation, and then returned to double
strands. Among the cDNA derived from hepatic cancer tissue, most of
those that were also present in the normal liver tissue come to
form double strands with the cDNA derived from the normal liver
tissue and to be labeled.
[0056] However, when cDNAs derived from the hepatic cancer tissue
form double strands with each other, they are not labeled.
Accordingly, by removing cDNA double stranded DNA having a label,
cDNA that is specific for the hepatic cancer tissue can be
obtained. By repeating this procedure, cDNAS specific for the
hepatic cancer tissue can be concentrated. The specific procedure
is shown in Example 1. By using cDNA fragments or full-length cDNAs
obtained as probes, it is also possible to conduct Northern
blotting on mRNA from cells or tissues that express gankyrin
polypeptides or from cells or tissues that do not express gankyrin
polypeptides and thereby to confirm that the selected gene
specifically expresses mRNA.
[0057] By screening cDNA libraries using cDNA or cDNA fragments
obtained as above, it is possible to obtain gankyrin genes from
different cells, tissues, organs or species. Furthermore, by
determining the nucleotide sequence of the cDNA obtained, it is
possible to determine the translation region that encodes a
gankyrin gene product, a polypeptide, and thereby to obtain the
amino acid sequence of this polypeptide. It is also possible to
isolate chromosomal DNA by screening genomic DNA libraries using
the obtained cDNA as a probe.
[0058] DNA libraries such as cDNA libraries or genomic DNA
libraries may be prepared by a method described, for example, in
Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor
Laboratory Press (1989), or commercially available DNA libraries
may be used.
[0059] The gene or DNA of the present invention can be obtained by
the PCR method using as a primer the nucleotide sequence or part
thereof, if it is known.
[0060] The gankyrin polypeptide of the present invention includes a
polypeptide that is encoded by a DNA that hybridizes to a nucleic
acid having the nucleotide sequence as set forth in SEQ ID NO: 1
under a stringent condition, and that has the biological activity
of gankyrin.
[0061] Such stringent conditions include, for example, a low
stringent condition. By way of example, a low stringent condition
is 50.degree. C. in 2.times.SSC and 0.1% SDS. More preferably,
there may be mentioned a high stringent condition. By way of
example, a high stringent condition is 65.degree. C. in
0.1.times.SSC and 0.1% SDS.
[0062] The above hybridizing DNA is preferably naturally occurring
DNA, and, for example, it may be cDNA or genomic DNA. Homology
search carried out on the amino acid sequence as set forth in SEQ
ID NO: 2 and the nucleotide sequence as set forth in SEQ ID NO: 1
using all the sequences contained in the known DNA databases
(GenBank, EMBL) and the protein database (SWISS-PLOT) did not give
any matches. From this result, it was revealed that the gene and
the gene product polypeptide of the present invention are novel
molecules.
[0063] As shown in Example 1, it was found that the gene that
hybridizes to the cDNA of the novel gankyrin polypeptide of the
present invention is widespread in non-human animals such as rats,
mice, and the like, and also in various tissues. Thus, the above
naturally occurring DNA may be cDNA or genomic DNA derived from the
tissues in which mRNA that hybridizes to cDNA of human gankyrin
polypeptide in, for example, Example 1 is detected.
[0064] The present invention also encompasses a DNA that hybridizes
to a nucleic acid having the nucleotide sequence as set forth in
SEQ ID NO: 2 and that encodes a polypeptide having the activity of
gankyrin. This DNA can also be expressed by the above-mentioned
method. In order to obtain such a gankyrin polypeptide, synthetic
oligonucleotide primers can be used to introduce the desired
mutation in the nucleotide sequence of a gankyrin gene (Mark, D. F.
et al., Proc. Natl. Acad. Sci. U.S.A. (1984) 81, 5662-5666; Zoller,
M. J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500;
Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et
al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79, 6409-6413).
[0065] In addition to being cDNA and genomic DNA that encodes the
gankyrin polypeptide, it may be a synthetic DNA. Specifically,
there may be mentioned a DNA that encodes gankyrin having the amino
acid sequence as set forth in SEQ ID NO: 2, and a DNA having the
nucleotide sequence as set forth in SEQ ID NO: 1 is used. These
DNAs may be produced using gene engineering technology that is
known per se.
[0066] An obtained transformant Escherichia coli containing the
plasmid pBS-t4-11 described hereinbelow in Example 1 was designated
as Escherichia coli DH5.alpha. (pBS-t4-11) and was internationally
deposited on Sep. 29, 1997, with the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology, MITI (Higashi 1-Chome 1-3, Tsukuba-shi, Ibaraki, Japan)
under the accession numbers FERM BP-6128.
[0067] As the gankyrin polypeptide of the present invention, there
can be mentioned a gankyrin polypeptide having an amino acid
sequence that is identical or substantially identical to the amino
acid sequence as set forth in SEQ ID NO: 2. Specifically, in
addition to gankyrin having the amino acid sequence as set forth in
SEQ ID NO: 2, there can be mentioned those in which one or more,
preferably 2 or more and 30 or less, more preferably 2 or more and
10 or less amino acids are deleted, one or more, preferably 2 or
more and 30 or less, more preferably 2 or more and 10 or less amino
acids are added to the amino acid sequence as set forth in SEQ ID
NO: 2; or one or more, preferably 2 or more and 30 or less, more
preferably 2 or more and 10 or less amino acids in the amino acid
sequence as set forth in SEQ ID NO: 2 are substituted with other
amino acids.
[0068] The present invention also includes a polypeptide that has
the biological activity of gankyrin and that is homologous to a
polypeptide having the amino acid sequence as set forth in SEQ ID
NO: 2. As used herein, "homologous polypeptides" refers to those
polypeptides that have an amino acid homology of at least 70%,
preferably at least 80%, more preferably at least 90%, and more
preferably at least 95% or more, generally, for at least 20,
preferably 30 contiguous amino acid residues to the amino acid
sequence as set forth in SEQ ID NO: 2.
[0069] The gankyrin polypeptides of the present invention differ in
amino acid sequence, molecular weight, isoelectric point, or
presence or form of sugar chains, depending on the cells or host
that produce the polypeptides, or methods of purification described
hereinbelow. However, the gankyrin polypeptides obtained are
included in the present invention as long as they have the activity
substantially equivalent to that of a naturally occurring gankyrin
polypeptide. As the activity that is substantially equivalent to
the gankyrin polypeptide as used herein, there can be mentioned
carcinogenicity as in Example 4 described below, such as the
elevation of colony-forming ability of cells in soft agar and the
suppression of tumorigenicity and of apoptosis induction in mice.
Substantially equivalent as used herein means that carcinogenicity
is equivalent in property.
[0070] As a partial peptide of the gankyrin polypeptide of the
present invention, there can be mentioned, for example, a partial
peptide comprising one or more than one region of the hydrophobic
region or the hydrophilic region estimated from the hydrophobic
plot analysis among the gankyrin molecules. These partial peptides
can include part or all of a hydrophobic region or part or all of a
hydrophilic region.
[0071] The partial peptide of the gankyrin polypeptide of the
present invention can be produced according to the peptide
synthesis method that is known per se or by cleaving the gankyrin
polypeptide of the present invention with a suitable peptidase. The
peptide synthesis method may be, for example, a solid phase
synthesis or a liquid phase synthesis.
[0072] After the reaction, the partial peptide of the present
invention can be isolated and purified by combining conventional
purification methods such as solvent extraction, distilation,
column chromatography, high performance liquid chromatography, and
recrystalization.
[0073] The DNA constructed as described above can be expressed by a
known method to obtain a gankyrin polypeptide. When mammalian cells
are used, expression may be accomplished using an expression vector
containing a commonly used useful promoter/enhancer, the gene to be
expressed, and DNA in which the poly A signal has been operably
linked at 3' downstream thereof or a vector containing said DNA.
Examples of the promoter/enhancer include human cytomegalovirus
immediate early promoter/enhancer.
[0074] Additionally, as the promoter/enhancer which can be used for
expression of gankyrin polypeptide, there can be used viral
promoters/enhancers such as retrovirus, polyoma virus, adenovirus,
and simian virus 40 (SV40), and promoters/enhancers derived from
mammalian cells such as human elongation factor 1.alpha.
(HEF1.alpha.).
[0075] For example, expression may be readily accomplished by the
method of Mulligan et al. (Nature (1979) 277, 108) when the SV40
promoter/enhancer is used, or by the method of Mizushima et al.
(Nucleic Acids Res. (1990) 18, 5322) when the HEF1.alpha.
promoter/enhancer is used.
[0076] In the case of Escherichia coli (E. coli), expression may be
effected by operably linking a conventionally used useful promoter,
a signal sequence for antibody secretion, and the antibody gene to
be expressed, followed by expression thereof. As the promoter, for
example, there can be mentioned the lacZ promoter and the araB
promoter. The method of Ward et al. (Nature (1098) 341, 544-546;
FASEB J. (1992) 6, 2422-2427) may be used when the lacZ promoter is
used, and the method of Better et al. (Science (1988) 240,
1041-1043) may be used when the araB promoter is used.
[0077] As the signal sequence for gankyrin polypeptide secretion,
when produced in the periplasm of E.coli, the pelB signal sequence
(Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379) can be
used.
[0078] As the origin of replication, there can be used those
derived from SV40, polyoma virus, adenovirus, bovine papilloma
virus (BPV) and the like. Furthermore, for the amplification of
gene copy number in the host cell system, expression vectors can
include as selectable markers the aminoglycoside transferase (APH)
gene, the thymidine kinase (TK) gene, E.coli xanthine
guaninephosphoribosyl transferase (Ecogpt) gene, the dihydrofolate
reductase (dhfr) gene and the like.
[0079] For the production of a gankyrin polypeptide, any production
system can be used. The production system of gankyrin polypeptide
preparation comprises the in vitro or the in vivo production
system. As the in vitro production system, there can be mentioned a
production system which employs eukaryotic cells and the production
system which employs prokaryotic cells.
[0080] When the eukaryotic cells are used, there are the production
systems which employ animal cells, plant cells, and fungal cells.
Known animal cells include (1) mammalian cells such as CHO cells
(J. Exp. Med. (1995) 108, 945), COS cells, myeloma cells, baby
hamster kidney (BHK) cells, HeLa cells, and Vero cells, (2)
amphibian cells such as Xenopus oosytes (Valle, et al., Nature
(1981) 291, 358-340), or (3) insect cells such as sf9, sf21, and
Tn5. As CHO cells, preferably dhfr-CHO (Proc. Natl. Acad. Sci.
U.S.A. (1980) 77, 4216-4220) that lacks the DHFR gene and CHO K-1
(Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275) may be used.
[0081] Known plant cells include, for example, those derived from
Nicotiana tabacum, which is subjected to callus culture. Known
fungal cells include yeasts such as the genus Saccharomyces, for
example Saccharomyces cereviceae, or filamentous fungi such as the
genus Aspergillus, for example Aspergillus niger.
[0082] When the prokaryotic cells are used, there are the
production systems which employ bacterial cells. Known bacterial
cells include Escherichia coli (E. coli), and Bacillus
subtilis.
[0083] By transforming these cells with the desired DNA and
culturing the transformed cells in vitro, the gankyrin polypeptide
can be obtained. Culturing is carried out in a known method. For
example, as the culture liquid, DMEM, HEM, RPMI1640, and IMDM can
be used, and serum supplements such as fetal calf serum (FCS) may
be used in combination, or serum-free culture medium may be used.
pH during the culture is preferably about 6 to 8. The culturing is
usually conducted at about 30 to 40.degree. C. for about 15 to 200
hours with optional medium exchange, aeration and agitation.
[0084] As in vivo production systems, there can be mentioned those
which employ animals and those which employ plants. The desired DNA
is introduced into an animal or a plant, and the gankyrin
polypeptide is produced in such an animal or a plant and then
collected. As used herein "host" encompasses these animals and
plants.
[0085] When animals are used, there are the production systems
which employ mammals and insects.
[0086] As mammals, goats, pigs, sheep, mice, and cattle can be used
(Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When
mammals are used, transgenic animals can also be used.
[0087] For example, an desired DNA is inserted into the middle of
the gene encoding protein which is inherently produced in the milk
such as goat .beta. casein to prepare fusion genes. DNA fragments
containing the fusion gene into which said DNA has been inserted
are injected into a goat embryo, and the embryo is introduced into
a female goat. The gankyrin polypeptide is obtained from the milk
produced by the transgenic goat born to the goat who received the
embryo or offsprings thereof. In order to increase the amount of
milk containing the gankyrin polypeptide produced by the transgenic
goat, hormones may be given to the transgenic goat as appropriate
(Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
[0088] When insects are used, silkworms, for example, can be used.
When silkworms are used, baculovirus into which the desired DNA has
been inserted is infected to the silkworm, and the desired gankyrin
polypeptide can be obtained from the body fluid of the silkworm
(Susumu, M. et al., Nature (1985) 315, 592-594).
[0089] When plants are used, tabacco for example can be used.
Moreover, when tabacco is used, the desired DNA is inserted into an
expression vector for plants, for example pMON 530, and then the
vector is introduced into a bacterium such as Agrobacterium
tumefaciens. The bacterium is then infected to tabacco such as
Nicotiana tabacum to obtain the desired polypeptide from the leaves
of the tabacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994)
24, 131-138). As methods of introducing an expression vector into a
host, there can be used a known method such as the calcium
phosphate method (Virolgoy (1973) 52, 456-467), the electroporation
method (EMBO J. (1982) 1, 841-845), and the like. Considering the
frequency of use of the host's codon for use in expression, a
sequence having a better efficiency of expression can be designed
(Grantham, R. et al., Nucleic Acids Research (1981) 9,
r43-r74).
[0090] That the gankyrin gene products thus obtained have the
biological activity of gankyrin can be confirmed, for example, in
the following manner. Using, for example, a method described in
Example 4 below, cells that produce a gankyrin polypeptide are
cultured in soft agar. Gankyrin polypeptide-expressing cells have
an elevated colony-forming ability in soft agar. Alternatively,
cells that express a gankyrin polypeptide are grafted to mice.
Cells that express a gankyrin polypeptide show an elevated
tumorigenicity. Alternatively, cells that express a gankyrin
polypeptide are placed under a apoptosis-inducing condition. Cells
that express a gankyrin polypeptide suppress the induction of
apoptosis.
[0091] Polypeptides obtained as described above can be isolated
from the inside or outside of the host as a substantially pure
homogeneous polypeptide. Separation and purification of the
gankyrin polypeptide may be accomplished by, but this is not
limited to, the separation and the purification methods
conventionally used for protein purification. For example, the
gankyrin polypeptide can be separated and purified by selecting and
combining, as appropriate, methods including, but not limited to,
chromatography columns, filtration, ultrafiltration, salting-out,
solvent precipitation, immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, isoelectric focusing, dialysis, and the like.
[0092] As chromatography, there may be mentioned, for example,
affinity chromatography, ion exchange chromatography, hydrophobic
chromatography, gel-filtration, reverse phase chromatography,
adsorption chromatography, and the like (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press,
1986). These chromatographies can be carried out using a liquid
chromatography such as HPLC and FPLC. Before or after purification,
gankyrin polypeptides may be modified by the action of a suitable
protein-modifying enzyme, or peptides can partially be removed. As
the protein-modifying enzymes, there may be used trypsin,
chymotrypsin, lysil endopeptidase, protein kinase, glucosidase, and
protein kinase, glucosidase.
[0093] The gankyrin polypeptide of the present invention is
important since it is used in a screening method. Thus, it is
important since it is used in a method of screening an agonist or
an antagonist of the gankyrin polypeptide, said method comprising
contacting a gankyrin polypeptide or a material containing the
gankyrin polypeptide with a sample expected to contain an agonist
or an antagonist of the gankyrin polypeptide in the presence of Rb,
and detecting or determining a free gankyrin polypeptide or Rb; or
a method of screening an agonist or an antagonist of the gankyrin
polypeptide, said method comprising contacting a gankyrin
polypeptide or a material containing the gankyrin polypeptide with
a sample expected to contain the agonist or the antagonist of the
gankyrin polypeptide in the presence of NF.kappa.B (Baeuerle, P. A.
et al., Cell (1988) 53, 211-217), and detecting or determining a
free gankyrin polypeptide or NF.kappa.B.
[0094] The gankyrin polypeptide for use in these screening methods
may be either a recombinant type or a natural type. It may also be
a partial peptide of the gankyrin polypeptide as long as it retains
the property of binding to Rb or NF.kappa.B. As the substance
containing the gankyrin polypeptide, there may be mentioned the
lysates of the cells that express gankyrin polypeptides.
[0095] Thus, the present invention relates to a method of screening
an agonist or an antagonist of the gankyrin polypeptide comprising
comparing a free gankyrin polypeptide or Rb detected or determined
when a gankyrin polypeptide or a material containing the gankyrin
polypeptide and a sample expected to contain the agonist or the
antagonist of the gankyrin polypeptide are contacted, and a free
gankyrin polypeptide or Rb detected or determined when a gankyrin
polypeptide or a material containing the gankyrin polypeptide and a
sample that does not contain the agonist or the antagonist of the
gankyrin polypeptide are contacted, both in the presence of Rb.
[0096] The present invention also relates to a method of screening
an agonist or an antagonist of the gankyrin polypeptide comprising
comparing a free gankyrin polypeptide or NF.kappa.B detected or
determined when a gankyrin polypeptide or a material containing the
gankyrin polypeptide and a sample expected to contain the agonist
or the antagonist of the gankyrin polypeptide are contacted, and a
free gankyrin polypeptide or NF.kappa.B deleted or determined when
a gankyrin polypeptide or a substance containing the gankyrin
polypeptide and a sample that does not contain the agonist or the
antagonist of the gankyrin polypeptide are contacted, both in the
presence of NF.kappa.B.
[0097] In order to detect or determine free gankyrin polypeptides,
Rb or NF.kappa.B in these screening methods, gankyrin polypeptides,
Rb or NF.kappa.B are labeled with, for example, biotin, avidin, a
radioisotope such as [.sup.125I], [.sup.35S], [.sup.3H],
[.sup.14C], a fluorescent substance, an enzyme such as horseradish
peroxidase and alkaline phosphatase, and then the label is detected
or determined. These labeling compounds are known and can be
labeled by conventional methods. Free gankyrin polypeptides, Rb, or
NF.kappa.B can also be detected or determined using antibodies to
gankyrin polypeptides, Rb, or NF.kappa.B.
[0098] Specifically, a gankyrin polypeptide is bound to a support
such as beads or a plate, to which a sample expected to contain an
agonist or an antagonist of the gankyrin polypeptide is added in
the presence of Rb or NF.kappa.B, and after incubation, Rb or
NF.kappa.B contained in the solution may be detected or determined
with an antibody. Alternatively, in order to detect or determine
free RB or NF.kappa.B, Rb or NF.kappa.B that is bound to the
gankyrin polypeptide immobilized on the plate may be detected or
determined.
[0099] At this time, a fusion polypeptide in which a gankyrin
polypeptide has been fused to another peptide or polypeptide
through gene engineering technology may be used. Such another
peptide or polypeptide that can be subjected to fusion include
hemaglutinin (HA), FLAG, and the like, and a free gankyrin
polypeptide can be detected or determined using antibodies to
another peptide or polypeptide that are subjected to fusion. Thus,
the fusion polypeptides in which a gankyrin polypeptide and another
peptide or polypeptide have been fused through gene engineering
technology are useful in the present invention.
[0100] Samples expected to contain an agonist or an antagonist for
use in the screening method of the present invention include, for
example, peptides, proteins, non-peptide compounds, synthetic
compounds, microbial fermentation products, marine organism
extracts, plant extracts, cell extracts, or animal cell extracts.
These samples may be novel or known substances.
[0101] The screening method of the present invention is useful for
detecting or determining an agonist or an antagonist having
carcinogenicity.
[0102] It was found in the present invention that gankyrin
polypeptides interact with Rb or NF.kappa.B. Since gankyrin
polypeptides have tumorigenicity, agonists or antagonists of
gankyrin polypeptides that modulate the binding of a gankyrin
polypeptide and Rb or NF.kappa.B are useful as pharmaceuticals.
[0103] Specifically, in order to conduct the above-mentioned
screening method, the gankyrin polypeptide of the present invention
or a sample expected to contain the gankyrin polypeptide is first
suspended in a buffer solution suitable for screening and then
immobilized on a plate thereby to prepare a gankyrin polypeptide
sample.
[0104] Any buffer solution may be used as long as it does not
inhibit the binding of a gankyrin polypeptide and, for example, a
phasphate buffer of pH 6 to 8, Tris-HCl buffer, PBS, and HBSS may
be used. In order to reduce non-specific binding, it is also
possible to add protein such as bovine serum albumin, a surfactant
such as CHAPS, Tween 80, digitonin, and the like. Furthermore, in
order to suppress the decomposition of the gankyrin polypeptide
with proteolyltic enzymes, inhibitors of proteolytic enzymes such
as PMSF, pepstatin, leupeptin, and the like can be added.
[0105] Then, to the gankyrin polypeptide sample are added Rb or
NF.kappa.B that has been labeled with a radioisotope and an
appropriate concentration of sample, which are reacted at about 0
to 50.degree. C. (preferably about 4 to 37.degree. C.) for about
0.5 to 24 hours (preferably about 0.5 to 3 hours). After the
reaction, it is washed with an appropriate amount of buffer and the
amount of radioactivity remaining in the gankyrin polypeptide
sample is counted by a gamma counter or liquid scintillation
counter. In order to determine non-specific binding at this time,
another polypeptide that does not interact with the gankyrin
polypeptide is similarly labeled and added to prepare a gankyrin
polypeptide sample. The gankyrin polypeptide sample to which a
buffer that does not contain the sample has been added is used as a
negative control.
[0106] The amount of non-specific binding subtracted from the
amount of remaining radioactivity give the amount of specific
binding. A sample that reduces the specific binding as compared to
the case when no sample was added to the reaction can be selected
as a candidate substance for an agonist or an antagonist of the
gankyrin polypeptide.
[0107] An agonist or an antagonist of the gankyrin polypeptide
obtained by the screening method of the present invention can be
applied to screening, for example, peptides, proteins, non-peptide
compounds, synthetic compounds, microbial fermentation products,
marine organism extracts, plant extracts, cell extracts, or animal
cell extracts using the screening method. These samples may be
novel or known substances.
[0108] An agonist or an antagonist of a gankyrin polypeptide is a
substance that inhibits the binding of the gankyrin polypeptide and
Rb or NF.kappa.B. Substances obtained by addition, deletion or
substitution of part of the structure of an agonists or an
antagonists of a gankyrin polypeptide obtained by the screening
method of the present invention is also included into agonists or
antagonists of gankyrin polypeptides obtained by the screening
method of the present invention.
[0109] When agonists or antagonists of gankyrin polypeptides
obtained by the screening method of the present invention are used
as medicaments for humans and mammals such as mice, rats, guinea
pigs, rabbits, chickens, cats, dogs, sheep, pigs, cattle, monkeys,
baboons, and chimpanzees, they may be used in a conventional
method.
[0110] For example, they may be used, as desired, orally as
sugar-coated tablets, capsules, elixirs, and microcapsules, or
parenterally in the form of sterile solutions with water or other
pharmaceutically acceptable liquids or in the form of injections as
suspensions. For example, agonists or antagonists of gankyrin
polypeptides are produced in unit dosage forms required for
generally accepted formulations by mixing with physiologically
acceptable carriers, flavoring agents, excipients, vehicles,
antiseptics, stabilizers, and binders. The amount of active
ingredients in these formulations is designed to provide an
indicated suitable range of doses.
[0111] Additives that can be blended into tablets and capsules
include, for example, binders such as gelatin, corn starch,
tragacanth, gum Arabic, excipients such as crystalline cellulose,
swelling agents such as alginate, lubricating agents such as
magnesium stearate, sweetening agents such as sucrose, lactose, and
saccharin, and flavoring agents such as peppermint, Gaultheria
adenothrix oil, or cherry. When the formulation unit form is a
capsule, liquid carriers such as lipids can be included to the
above materials. Sterile compositions for injection can be
formulated according to the conventional formulation method for
dissolving or suspending active substances in a vehicle such as
distilled water for injection, and natural plant oils such as
sesame oil and coconut oil.
[0112] As aqueous solutions for injection, there may be mentioned,
for example, isotonic liquids such as physiological saline, glucose
and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and
sodium chloride, and they may be used in combination with suitable
solubilizing agents such as alcohols, specifically ethanol,
polyalchohols including, for example, propylene glycol and
polyethylene glycol, nonionic surfactants such as polysorbate 80
(TM), and HCO-50.
[0113] An oily liquid includes, for example, sesame oil and soybean
oil, which may be used together with solubilizing agents such as
benzyl benzoate and benzyl alchohol. There may be also blended
buffers such as phosphate buffer and sodium acetate buffer,
analgesics such as benzalkonium chloride and procaine chloride,
stabilizing agents such as benzyl alchohol and phenol, and
antioxidants. Prepared injections are usually filled into suitable
ampoules.
[0114] The dosage of agonists or antagonists of gankyrin
polypeptides for a human adult (assuming the body weight of 60 kg)
is, when given orally, usually about 0.1 to 100 mg/day, preferably
about 1.0 to 50 mg/day, and more preferably about 1.0 to 20 mg/day,
though this may vary depending on the medical conditions.
[0115] When given parenterally, the dose per administration for a
human adult (assuming the body weight of 60 kg) of usually about
0.01 to 30 mg/day, preferably about 0.1 to 20 mg/day, and more
preferably about 0.1 to 10 mg/day in the case of injections is
conveniently administered via intravenous injection, though this
may vary depending on the subject, subject organ, medical
conditions, and the method of administration. For other animals
also, the amount converted in terms of the body weight of 60 kg may
be administered.
[0116] Anti-gankyrin polypeptide antibodies of the present
invention can be obtained as monoclonal or polyclonal antibodies
using known methods.
[0117] Monoclonal antibodies can be obtained by using a gankyrin
polypeptide as a sensitizing antigen, which is immunized in a
conventional method for immunization, by fusing the immune cells
thus obtained with known parent cells, and screening monoclonal
antibody-producing cells using a known screening method.
[0118] Specifically, monoclonal or polyclonal antibodies may be
generated as follows.
[0119] For example, though the gankyrin polypeptide to be used as a
sensitizing antigen for generation of antibodies is not limited by
the animal species from which the antibodies are obtained, it is
preferably derived from a mammal such as humans, mice, or rats.
These gankyrin polypeptides derived from humans, mice, or rats can
be obtained using the gene sequences disclosed in the present
invention.
[0120] According to the present invention, gankyrin polypeptides
that have the biological activity of all the gankyrin polypeptides
disclosed in the present invention can be used as the gankyrin
polypeptide for use as a sensitizing antigen. As fragments of
gankyrin polypeptides, there may be mentioned, for example,
C-terminal fragments of gankyrin polypeptides. As used herein
"anti-gankyrin polypeptide antibody" means an antibody that
specifically reacts to the full-length or fragments of a gankyrin
polypeptide.
[0121] Genes encoding a gankyrin polypeptide or fragments thereof
may be inserted to a known expression system to transform the host
cell described herein, and the desired gankyrin or the fragments
thereof are obtained by a known method from the inside or the
outside of the host cell and then the gankyrin polypeptide may be
used as a sensitizing antigen. Alternatively, cells. tbat express
gankyrin polypeptide or lysates thereof may be used as a
sensitizing antigen.
[0122] Mammals to be immunized with the sensitizing antigen are not
specifically limited, and they are preferably selected in
consideration of their compatibility with the parent cell for use
in cell fusion. They generally include rodents, lagomorphs, and
primates.
[0123] Rodents include, for example, mice, rats, hamsters, and the
like. Lagomorphs include, for example, rabbits. Primates include,
for example, monkeys. As monkeys, catarrhines (Old-World monkeys)
such as cynomolgi, rhesus monkeys, sacred baboons, chimpanzees etc.
are used.
[0124] Immunization of animals with a sensitizing antigen is
carried out using a known method. A general method, for example,
involves the intraperitoneal or subcutaneous administration of a
sensitizing antigen to the mammal. Specifically, a sensitizing
antigen which has been diluted and suspended in an appropriate
amount of phosphate buffered saline (PBS) or physiological saline
etc. is mixed, as desired, with an appropriate amount of a common
adjuvant, for example Freund's complete adjuvant. After being
emulsified, it is preferably administered to the mammal for several
times every 4 to 21 days. Alternatively a suitable carrier may be
used at the time of immunization of the sensitizing antigen. After
such immunization, the increase in the desired antibody levels in
the serum is confirmed by a conventional method.
[0125] In order to obtain polyclonal antibodies to a gankyrin
polypeptide, the blood of the mammal that was sensitized with the
antigen is removed after the increase in the desired antibody
levels in the serum has been confirmed. Serum is separated from the
blood. As polyclonal antibodies, serum containing the polyclonal
antibodies may be used, or, as desired, the fraction containing the
polyclonal antibodies may be isolated from the serum.
[0126] In order to obtain monoclonal antibodies, immune cells of
the mammal that was sensitized with the antigen are removed and are
subjected to cell fusion after the increase in the desired antibody
levels in the serum has been confirmed. At this time preferred
immune cells that are subjected to cell fusion include, in
particular, the spleen cells.
[0127] The mammalian myeloma cells as the other parent cells which
are subjected to cell fusion with the above-mentioned immune cells
preferably include various known cell lines such as P3
(P3X63Ag8.653) (Kearney, J. F. et al., J. Immunol. (1979) 123:
1548-1550), P3X63Ag8.U1 (Yelton, D. E., et al., Current Topics in
Microbiology and Immunology (1978) 81: 1-7), NS-1 (Kohler, G. and
Milstein, C., Eur. J. Immunol. (1976) 6: 511-519), MPC-11
(Margulies, D. H. et al., Cell (1976) 8: 405-415), SP2/0 (Shulman,
M. et al., Nature (1978) 276: 269-270), FO (de St. Groth, S. F. and
Scheidegger, D., J. Immunol. Methods (1980) 35: 1-21), S194
(Trowbridge, I. S., J. Exp. Med. (1978) 148: 313-323), R210
(Galfre, G. et al., Nature (1979) 277: 131-133) and the like.
[0128] Cell fusion between the above immune cells and the myeloma
cells may be essentially conducted in accordance with a known
method such as is described in Milstein et al. (Kohler, G. and
Milstein, C., Methods Enzymol. (1981) 73: 3-46) and the like.
[0129] More specifically, the above cell fusion is carried out in
the conventional nutrient broth in the presence of, for example, a
cell fusion accelerator. As the cell fusion accelerator, for
example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like
may be used, and, in addition, an adjuvant such as dimethyl
sulfoxide etc. may be added, as desired, to enhance the efficiency
of the fusion.
[0130] The preferred ratio of the immune cells and the myeloma
cells to be used is, for example, 1 to 10 times more immune cells
than the myeloma cells. Examples of culture media to be used for
the above cell fusion include RPMI1640 medium and MEM culture
medium suitable for the growth of the above myeloma cell lines, and
the conventional culture medium used for this type of cell culture
&ad, besides, a serum supplement such as fetal calf serum (FCS)
may be added.
[0131] In cell fusion, predetermined amounts of the above immune
cells and the myeloma cells are mixed well in the above culture
liquid, to which a PEG solution previously heated to about
37.degree. C., for example a PEG solution with a mean molecular
weight of about 1000 to 6000, is added at a concentration of 30 to
60% (w/v), and mixed to obtain the desired fusion cells
(hybridomas). Then, by repeating the sequential addition of a
suitable culture liquid and centrifugation to remove the
supernatant, cell fusion agents etc. which are undesirable for the
growth of the hybridoma can be removed.
[0132] Said hybridoma is selected by culturing in a conventional
selection medium, for example, the HAT culture medium (a culture
liquid containing hypoxanthine, aminopterin, and thymidine).
Culturing in said HAT culture medium is continued generally for a
period of time sufficient to effect killing of the cells other than
the desired hybridoma (non-fusion cells), generally several days to
several weeks. Then, the conventional limiting dilution method is
conducted in which the hybridomas that produce the desired antibody
are screened and cloned.
[0133] In addition to obtaining the above hybridoma by immunizing
an animal other than the human with an antigen, it is also possible
to sensitize human lymphocytes infected with EB virus with a
gankyrin polypeptide, cells expressing a gankyrin polypeptide, or
their lysates in vitro, and to allow the resulting sensitized
lymphocytes to be fused with a human-derived myeloma cell having a
permanent division potential, for example U266, and thereby to
obtain a hybridoma producing the desired human antibody having the
activity of binding the gankyrin polypeptide (see Japanese
Unexamined Patent Publication (Kokai) No. 63(1988)-17688).
[0134] Furthermore, a transgenic animal having a repertoire of
human antibody genes is immunized with the gankyrin polypeptide,
cells expressing the gankyrin polypeptide or lysates thereof to
obtain the anti-gankyrin polypeptide antibody-producing cells,
which are used to obtain human antibody against the gankyrin
polypeptide using hybrodomas fused to myeloma cells (see
International Patent Publication WO 92-03918, WO 93-2227, WO
94-02602, WO 94-25585, WO 96-33735 and WO 96-34096).
[0135] The monoclonal antibody-producing hybridomas thus
constructed can be subcultured in the conventional culture liquid,
or can be stored for a prolonged period of time in liquid
nitrogen.
[0136] In order to obtain monoclonal antibodies from said
hybridoma, there may be employed a method in which said hybridoma
is cultured in the conventional method and the antibodies are
obtained as the culture supernatant, or a method in which the
hybridoma is administered to and grown in a mammal compatible with
said hybridoma and the antibodies are obtained as the ascites. The
former method is suitable for obtaining high-purity antibodies,
whereas the latter is suitable for a large scale production of
antibodies.
[0137] In addition to using a hybridoma to produce an antibody,
immune cells that produce the desired antibody, for example the
sensitized lymphocytes that have been immortalized with an
oncogene, may be used to obtain the antibody.
[0138] A monoclonal antibody thus produced can also be obtained as
a recombinant antibody by recombinant gene technology. For example,
an anti-gankyrin polypeptide antibody gene may be cloned from the
hybridoma or an immune cell such as a sensitized lymphocyte that
produces antibodies, and is integrated into a suitable vector which
is then introduced into a host to produce a recombinant antibody.
Recombinant antibodies may also be used in the present invention
(see, for example, Borrebaeck, C. A. K., and Larrick, J. W.,
THERAPEUTIC MONOCLONAL ANTIBODIES, published in the United Kingdom
by MACMILLAN PUBLISHERS LTD. 1990).
[0139] Specifically, mRNA encoding the variable region (V region)
of anti-gankyrin polypeptide antibody can be isolated from a
hybridoma that produces the anti-gankyrin polypeptide antibody. The
isolation of mRNA is conducted by preparing total RNA using a known
method such as the guanidine ultracentrifugation method (Chirgwin,
J. M. et al., Biochemistry (1979) 18, 5294-5299), the AGPC method
(Chomzynski, P. and Sacci, N., Anal. Biochem. (1987) 162, 156-159),
and then purifying mRNA from the total RNA using the mRNA
Purification Kit (Pharmacia) and the like. Alternatively, mRNA can
be prepared directly using the QuickPrep mRNA Purification kit
(Pharmacia).
[0140] The mRNA obtained is used to synthesize the cDNA of the gene
using a reverse transcriptase. The synthesis of cDNA can be
effected using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Kogyo), and the like. Alternatively, for
the synthesis and amplification of cDNA, the 5'-Ampli FINDER RACE
kit (CLONTECH) and the 5'-RACE method (Frohman, M. A. et al., Proc.
Natl. Acad. Sci. U.S.A. (1988) 85, 8998-9002; Belyavsky, A. et al.,
Nucleic Acids Res. (1989) 17, 2919-2932) that employs the
polymerase chain reaction (PCR) may be used.
[0141] A DNA fragment of interest may be prepared from the PCR
product thus obtained and ligated to a vector DNA. Furthermore, a
recombinant vector is constructed from this, which is then
introduced into E.coli for selection of colonies to prepare the
desired recombinant vector. The base sequence of the desired DNA
may be confirmed by a known method such as the dideoxy nucleotide
chain termination method. Once the desired DNA encoding the V
region of anti-gankyrin polypeptide antibody has been obtained, it
may be ligated to DNA encoding the constant region (C region) of
the desired antibody, which is then integrated into an expression
vector. Alternatively, the DNA encoding the V region of the
antibody may be integrated into an expression vector which already
contains DNA encoding the C region of the antibody. The C region of
antibody may be the one derived from the same animal species as the
V region or the one derived from the different animal species from
the V region.
[0142] In order to produce the anti-gankyrin polypeptide antibody
for use in the present invention, the antibody gene is integrated
as described below into an expression vector so as to be expressed
under the control of the expression regulatory region, for example
an enhancer and/or a promoter. Subsequently, the expression vector
may be transformed into a host cell and the antibody can then be
expressed therein.
[0143] For the expression of an antibody, DNA encoding the heavy
chain (H chain) or the light chain (L chain) of the antibody may be
separately integrated into an expression vector and the hosts are
transformed simultaneously, or DNA encoding the H chain and the L
chain may be integrated into a single expression vector and the
host is transformed therewith (see International Patent Application
WO 94-11523).
[0144] Antibodies for use in the present invention may be antibody
fragments or modified versions thereof as long as they bind to
gankyrin polypeptides. For example, as fragments of antibody, there
may be mentioned Fab, F(ab')2, Fv or single-chain Fv (scFv) in
which Fv's of the H chain and the L chain were ligated via a
suitable linker. Specifically antibodies are treated with an
enzyme, for example, papain or pepsin, to produce antibody
fragments, or genes encoding these antibody fragments are
constructed, and then introduced into an expression vector, which
is expressed in a suitable host cell (see, for example, Co, M. S.
et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz,
A. H., Methods in Enzymology (1989) 178, 476-496; Plucktrun, A. and
Skerra, A., Methods in Enzymology (1989) 178, 476-496; Lamoyi, E.,
Methods in Enzymology (1986) 121, 652-663; Rousseaux, J. et al.,
Methods in Enzymology (1986) 121, 663-669; Bird, R. E. et al.,
Trends Biotechnol. (1991) 9, 132-137).
[0145] scFv can be obtained by ligating the V region of the H chain
and the V region of the L chain of an antibody. In the scFv, the V
region of the H chain and the V region of the L chain are ligated
via a linker, preferably a peptide linker (Huston, J. S. et al.,
Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The V region
of the H chain and the V region of the L chain in the scFv may be
derived from any of the above-mentioned antibodies. As the peptide
linker for ligating the V regions, any single-chain peptide
comprising, for example, 12-19 amino acid residues may be used.
[0146] DNA encoding scFv can be obtained using DNA encoding the H
chain or the H chain V region of the above antibody and DNA
encoding the L chain or the L chain V region of the above antibody
as the template by amplifying the portion of the DNA encoding the
desired amino acid sequence among the above sequences by the PCR
technique together with the primer pair specifying the both ends
thereof, and by further amplifying the combination of DNA encoding
the peptide linker portion and the primer pair which defines that
both ends of said DNA are ligated to the H chain and the L chain,
respectively.
[0147] Once DNAs encoding scFv are constructed, an expression
vector containing them and a host transformed with said expression
vector can be obtained by the conventional methods, and scFv can be
obtained using the resultant host by the conventional methods.
[0148] These antibody fragments may be antibody fragments part of
which have undergone mutation, substitution, deletion, or
insertion. These antibody fragments can also be produced by
obtaining the gene thereof in a similar manner to that mentioned
above and by allowing it to be expressed in a host. "Antibody" as
used in the claim of the present application encompasses these
antibody fragments.
[0149] As modified antibodies, anti-gankyrin polypeptide antibodies
associated with various molecules such as polyethylene glycol (PEG)
can be used. "Antibody" as used in the claim of the present
application encompasses these modified antibodies. These modified
antibodies can be obtained by chemically modifying the antibodies
thus obtained. These methods have already been established in the
art.
[0150] The anti-gankyrin polypeptide antibody of the present
invention can be obtained as chimeric or humanized antibody using a
known method.
[0151] The antibody gene constructed as above can be expressed by a
known method to obtain the antibody. For example,
promoters/enhancers for production of the gankyrin polypeptide
described herein can be used.
[0152] For the production of anti-gankyrin polypeptide antibody for
use in the present invention, any production system can be used,
and the production system for the production of gankyrin
polypeptide described herein can be used. For example, the
production system for anti-gankyrin polypeptide antibody
preparation comprises the in vitro or the in vivo production
system. As the in vitro production system, there can be mentioned a
production system which employs eukaryotic cells and the production
system which employs prokaryotic cells. As the in vitro production
system, there can be mentioned methods that use animals or that use
plants. When animals are used, there are the production systems
which employ mammals and insects.
[0153] As mammals, goats, pigs, sheep, mice, and cattle and, for
example, transgenic animals thereof can be used (Glaser V.,
SPECTRUM Biotechnology Applications, 1993). Also as insects,
silkworms can be used. Furthermore, when plants are used, tabacco,
for example, such as Nicotiana tabacum can be used (Ma, J. K. et
al., Eur. J. Immunol. (1994) 24, 131-138).
[0154] When an antibody is produced in in vitro or in vivo
production systems, as described above, DNA encoding the H chain or
the L chain of the antibody may be separately integrated into an
expression vector and the hosts are transformed simultaneously, or
DNA encoding the H chain and the L chain may be integrated into a
single expression vector and the host is transformed therewith (see
International Patent Application WO 94-11523).
[0155] Antibodies produced and expressed as described above can be
separated from the inside or outside of the host cell and then may
be purified to homogeneity. Separation and purification of the
antibody for use in the present invention may be accomplished by,
but not limited to, the separation and the purification methods
conventionally used for protein purification.
[0156] For example, there can be mentioned chromatography columns
such as affinity chromatography, filtration, ultrafiltration,
salting-out, dialysis, SDS-polyacrylamide gel electrophoresis,
isoelectric foculsing and the like, from which methods can be
selected and combined as appropriate for separation and
purification of antibody (Antibodies: A Laboratory Manual, Ed
Harlow and David Lane, Cold Spring Harbor Laboratory, 1988).
[0157] As columns for use in affinity chromatography, there can be
mentioned Protein A column and Protein G column. Examples of the
carriers used in the Protein A column are Hyper D, POROS, Sepharose
F. F. (Pharmacia) and the like.
[0158] As chromatography other than the above-mentioned affinity
chromatography, there can be mentioned, for example, ion exchange
chromatography, hydrophobic chromatography, gel-filtration, reverse
phase chromatography, adsorption chromatography, and the like
(Strategies for Protein Purification and Characterization: A
Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring
Harbor Laboratory Press, 1986). These chromatographies can be
carried out using a liquid chromatography such as HPLC, FPLC.
[0159] The concentration of antibody obtained as above can be
determined by the measurement of absorbance or by the enzyme-linked
immunosorbent assay (ELISA) and the like. Thus, when absorbance
measurement is employed, the antibody obtained is appropriately
diluted with PBS and then the absorbance is measured at 280 nm. In
the case of human antibody, calculation is conducted using 1.40 OD
at 1 mg/ml, though the absorption coefficient varies depending on
the species and the subclass.
[0160] When the ELISA method is used, measurement is conducted as
follows. Thus, 100 .mu.l of goat anti-human IgG diluted to 1
.mu.g/ml in 0.1 M bicarbonate buffer, pH 9.6, is added to a 96-well
plate (manufactured by Nunc), and is incubated overnight at
4.degree. C. to immobilize the antibody. After blocking, 100 .mu.l
each of appropriately diluted antibody of the present invention or
a sample containing the antibody, or 100 .mu.l of human IgG as the
concentration standard is added, and incubated at room temperature
for 1 hour.
[0161] After washing, 100 .mu.l of 5000-fold diluted alkaline
phosphatase-labeled anti-human IgG is added, and incubated at room
temperature for 1 hour. After washing, the substrate solution is
added and incubated, followed by the measurement of absorbance at
405 nm using the MICROPLATE READER Model 3550 (Bio-Rad) to
calculate the concentration of the desired antibody.
[0162] Alternatively, BIAcore (Pharmacia) can be used for the
measurement of antibody concentration.
[0163] The activity of anti-gankyrin polypeptide antibody of the
present invention can be evaluated by a known method. For example,
the activity of anti-gankyrin polypeptide antibody of the present
invention can be evaluated by adding .sup.125I-labeled
anti-gankyrin polypeptide antibody to a plate on which gankyrin
polypeptide has been immobilized, washing the plate according to a
known method, and then measuring the radioactivity (Antibodies: A
Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor
Laboratory, 1988).
[0164] As methods for determining the antigen-binding activity of
anti-gankyrin polypeptide antibody for use in the present
invention, there can be used ELISA, EIA (enzymeimmunoassay), RIA
(radioimmunoassay), or the fluorescent antibody method.
[0165] When ELISA is employed, for example, a gankyrin polypeptide
is added to a plate onto which anti-gankyrin polypeptide antibody
has been immobilized, and then samples containing the desired
anti-gankyrin polypeptide antibody, for example a culture
supernatant of anti-gankyrin polypeptide antibody-producing cells
or purified antibody, are added thereto. The second antibody that
recognizes the anti-gankyrin polypeptide antibody labeled with an
enzyme such as alkaline phosphatase is added, and the plate is
incubated, washed. Then the enzyme substrate is added, and the
absorbance is measured to evaluate the antigen-binding activity. As
the gankyrin polypeptide, a fragment of the gankyrin polypeptide, a
fragment comprising the C-terminal thereof, or a fragment
comprising the N-terminal thereof may be used. For the evaluation
of the activity of the anti-gankyrin polypeptide antibody of the
present invention, BIAcore (Pharmacia) can be used.
[0166] By using such methods, a method of detecting or determining
a gankyrin polypeptide may be conducted, said method comprising
contacting said antibody to a sample expected to contain said
gankyrin polypeptide and detecting or determining an immune complex
between said antibody and said gankyrin polypeptide.
[0167] Specifically, when ELISA is employed, for example, a sample
containing a gankyrin polypeptide is added to a plate onto which
anti-gankyrin polypeptide antibody has been immobilized, and then
anti-gankyrin polypeptide antibody is added thereto.
[0168] The second antibody that recognizes the anti-gankyrin
polypeptide antibody labeled with an enzyme such as alkaline
phosphatase is added, and the plate is incubated and washed. Then,
after adding the enzyme substrate such as p-nitrophenyl phosphate
thereto and determining absorbance, the presence of the gankyrin
polypeptide in the sample can be evaluated. As the gankyrin
polypeptide, a fragment of the gankyrin polypeptide, a fragment
comprising the C-terminal thereof, or a fragment comprising the
N-terminal thereof may be used. For the evaluation of the activity
of the anti-gankyrin polypeptide antibody of the present invention,
BIAcore (Pharmacia) can be used.
[0169] The method of detecting or determining the gankyrin
polypeptide of the present invention is important in various
experiments that employ gankyrin polypeptides since it can
specifically detect or determine gankyrin polypeptides.
[0170] The present invention includes a nucleotide (DNA or RNA)
capable of selectively hybridizing the gene of the present
invention or a nucleotide derivative such as an antisense
oligonucleotide or ribozyme and the like. The present invention
also includes an antisense oligonucleotide that hybridizes any of
the sites in the nucleotide sequence as set forth in SEQ ID NO: 1.
The antisense oligonucleotide is preferably an antisense
oligonucleotide to at least 20 or more contiguous nucleotides in
the base sequence as set forth in SEQ ID NO: 1. More preferably, it
is an antisense oligonucleotide in which said at least 20 or more
contiguous nucleotides in the nucleotide sequence as set forth in
SEQ ID NO: 1 contain a translation initiation codon. For example,
the antisense oligonucleotide of the present invention contains SEQ
ID NO: 8. Furthermore, for example, the antisense oligonucleotide
of the present invention is one that contains SEQ ID NO: 9.
[0171] As used herein "antisense oligonucleotide" may contain one
or a plurality of nucleotide mismatches as long as nucleotides
corresponding to nucleotides constituting a given region of DNA or
mRNA are all compelementary and DNA or mRNA and the oligonucleotide
can selectively and stably hybridize to the base sequence as set
forth in SEQ ID NO: 1. "Selectively and stably hybridize" as used
herein means that they have a homology of at least 70%, preferably
80%, more preferably 90%, more preferably 95% or more of the base
sequence on at least 20, preferably 30 contiguous nucleotide
sequence regions.
[0172] According to one embodiment of the present invention, the
antisense oligonucleotide has the nucleotide sequence as set forth
in SEQ ID NO: 8. Furthermore, according to one embodiment of the
present invention, the antisense oligonucleotide has the nucleotide
sequence as set forth in SEQ ID NO: 9.
[0173] When the oligonucleotide derivative for use in the present
invention is a deoxyribonucleotide, each structure is as shown in
formula (I): ##STR1##
[0174] wherein X may be independently any of oxygen (O), sulfur
(S), a lower alkyl group, or a primary amine or a secondary amine;
Y may be independently any of oxygen (O) or sulfur (S); B is
selected from the group consisting of adenine, guanine, thymine,
and cytosine, and is mainly a complementary oligonucleotide to the
DNA or the mRNA of the human gankyrin gene; R is independently
hydrogen (H) or a dimethoxytrityl group or a lower alkyl group; and
n is 7 to 28.
[0175] Preferred oligonucleotide derivatives may be not only
unmodified oligonucleotides, but, as described hereinbelow,
modified oligonucleotides. Examples of such modifications include,
for example, lower alkyl phosphonate-modifications such as the
above-mentioned methylphosphonate type or the ethylphosphonate
type, and the phosphorothioate modifications or the
phosphoroamidate modifications.
[0176] Examples of ##STR2## are represented by the following
formula (II): ##STR3##
[0177] These antisense oligonucleotide derivatives can be obtained
by a conventional method as shown below. An oligonucleotide of
Formula (1) in which X and Y are O may be readily synthesized using
a commercially available DNA synthesizer (for example the one
manufactured by Applied Biosystems). Synthesis can be effected by
the solid phase synthesis using hydrogen phosphonate (T. Atkinson,
& M. Smith, in oligonucleotide Synthesis: A Practical Approach,
ed. M. J. Gait, IRL Press, 35-81 (1984); M. H. Caruthers, Science
230, 281 (1985); A. Kume, et al., J. Org. Chem., 49, 2139 (1984);
B. C. Froehler, et al., Tetrahedron Lett. 27, 469 (1986); P. J.
Garegg, et al., ibid, 27, 4051 (1986); B. S. Sproat, et al., in
oligonucleotide Synthesis: A Practical Approach, ed. M. J. Gait,
IRL Press, 83-115 (1984); S. L. Beaucage & M. H. Caruthers,
Tetrahedron Lett., 22, 1859-1862 (1981); M. D. Matteucci and M. H.
Caruthers, Tetrahedron Lett., 21, 719-722 (1980); M. D. Matteucci
& M. H. Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981)
[0178] A triester phosphate modification in which X is a lower
alkoxy group can be obtained by, for example, a conventional method
in which an oligonucleotide that has been obtained by chemical
synthesis is treated with a solution of tosyl chloride in
DMF/methanol/2,6-lutidiene (Moody H. M. et al., Nucleic Acids Res.,
17, 4769-4782 (1989)).
[0179] An alkyl phosphonate modification in which X is an alkyl
group can be obtained by, for example, using phosphoamidite (M. A.
Dorman, et al., Tetrahedron Lett. 40, 95-102 (1984); K. L. Agarwal
& F. Riftina, Nucleic Acids Res., 6, 3009-3024 (1979)).
[0180] A triester phosphorothioate modification in which X is S can
be obtained by a solid phase synthesis using sulfur (C. A. Stein,
et al., Nucleic Acids Res., 16, 3209-3221 (1988)), or a solid phase
synthesis using tetraethyltiraum disulfide (H. Vu and B. L.
Hirschbein, Tetrahedron Lett. 32, 3005-3008 (1991).
[0181] A phosphorodithioate modification in which both X and Y are
S can be obtained by, for example, a solid phase synthesis in which
a bisamidite is converted to a thioamidite, on which is acted
sulfur to yield said modification (W. K. -D. Brill, et al., J. Am.
Chem. Soc., 111, 2321-2322 (1989)).
[0182] A phosphoroamidate modification in which X is a primary
amine or a secondary amine can be obtained by, for example, a solid
phase synthesis in which hydrogen phosphonate is treated with a
primary or secondary amine (B. Froehler, et al., Nucleic Acids
Res., 16, 4831-4839 (1988)). Alternatively the amidite may be
oxidized with tert-butyl hydroperoxide to yield said modification
(H. Ozaki, et al., Tetrahedron Lett., 30, 5899-5902 (1989)).
[0183] Purification and the confirmation of purity can be carried
out by high performance liquid chromatography and polyacrylamide
gel electrophoresis. The confirmation of molecular weight can be
carried out by Electrospray Ionization Mass Spectrometry or Fast
Atom Bombardment-Mass Spectrometry. The antisense oligonucleotide
of the present invention may be obtained by any synthetic method or
from any source as long as it has a sequence that hybridizes to the
base sequence of DNA or mRNA encoding a human gankyrin
polypeptide.
[0184] The antisense oligonucleotide derivative of the present
invention acts on the human gankyrin polypeptide-producing cells,
as shown hereinbelow in Example 7, to bind to DNA or mRNA encoding
the human gankyrin polypeptide and thereby to inhibit its
transcription or translation and promote the decomposition of mRNA,
resulting in the suppression of human gankyrin polypeptide
expression. Eventually it exhibits an effect of suppressing the
actions of human gankyrin polypeptide. The actions of human
gankyrin polypeptide suppressed by the antisense oligonucleotide
derivative of the present invention includes, for example, the
suppression of the colony-forming ability in soft agar by the cells
described in Example 7.
[0185] The antisense oligonucleotide derivative of the present
invention can be mixed with an appropriate base to formulate an
external preparation such as a liniment, a cataplasm and the
like.
[0186] It can also be mixed, as desired, with an excipient, an
isotonic agent, a solubilizer, a stabilizer, an antiseptic, a
soothing agent or the like to formulate a tablet, powder, granules,
a capsule, a liposome capsule, an injection, a solution, a nasal
drop, and the like as well as a lyophilized preparation. They can
be prepared according to a conventional method.
[0187] The antisense oligonucleotide derivative of the present
invention may be applied to the patient by either directly
administering to the affected area of the patient or administering
into the blood vessel thereby allowing the substance to be
delivered to the affected area. Furthermore, an antisense
encapsulating material that enhances prolonged action and membrane
permeability may be used. There may be mentioned, for example,
liposome, poly-L-lysine, lipid, cholesterol, lipofectin or
derivatives thereof.
[0188] Preferably the dosage of the antisense oligonucleotide
derivative of the present invention can be adjusted as appropriate
depending on the condition of the patient to employ a preferred
amount. For example, preferred dosage is in the range of 0.1 to 100
mg/kg, preferably 0.1 to 50 mg/kg.
[0189] The antisense oligonucleotide of the present invention is
useful in inhibiting the expression of gankyrin polypeptide and
thereby in suppressing the biological activity of gankyrin
polypeptide. An inhibitor of the expression of gankyrin polypeptide
containing the antisense oligonucleotide of the present invention
can suppress the biological activity, i.e. carcinogenicity, of
gankyrin, and therefore, is useful as a therapeutic agent for
cancer or hypertrophic disorders.
EXAMPLES
[0190] The present invention is now explained in more detail with
reference to the following examples.
Example 1
cDNA Cloning by the Subtraction Method
[0191] Using the subtraction method (Nakayama, H. et al., Develop.
Growth Differ. (1996) 38, 141-151), cDNAs of the genes that are
specifically expressed in hepatic cancer were cloned.
[0192] From surgical specimens of a 55-year old male patient,
tissues of stage 3 hepatic cancer as defined in the code of
handling primary hepatic cancer (compiled by the Japan Hepatic
Cancer Study Group) and the normal liver tissue were removed. From
each tissue, total RNA was extracted using the TRIsol reagent
(manufactured by GIBCO BRL). From the total RNA, double stranded
cDNA was synthesized with oligo-dT primers using the cDNA synthesis
kit (manufactured by Pharmacia). After digesting the cDNA with a
restriction enzyme RsaI, a linker adapter (Nakayama, H. et al.,
Develop. Growth Differ. (1996) 38, 141-151) was added thereto, and
these cDNAs were amplified by the PCR method using pimers
(Nakayama, H. et al., Develop. Growth Differ. (1996) 38, 141-151).
When the cDNA from the normal liver tissue was amplified by the PCR
method, primers that were end-labeled with biotin were used.
[0193] An excessive amount of double stranded cDNA derived from the
normal liver tissue was mixed with a small amount of double
stranded cDNA derived from hepatic cancer, and the mixture was then
heat-denatured to make it single-stranded, followed by annealing to
double strands. Most of the cDNAs derived from hepatic cancer
tissue that were also expressed in the normal liver tissue
hybridize with cDNAs derived from the normal liver tissue and come
to have biotin labels. However, molecules that are specific for the
hepatic cancer tissue form double strands there between and thereby
do not come to have biotin labels. Accordingly, double stranded
cDNAs having biotin labels were eliminated and cDNA molecules
specific for the hepatic cancer tissue were concentrated.
[0194] cDNA molecules that are specific for the hepatic cancer
tissue were amplified by the PCR method and were concentrated by
repeating the same procedure by 5 times. This gave 250 bp of cDNA
fragments specific for the hepatic cancer tissue derived from the
human hepatic cancer tissue. In order to isolate the full-length
cDNA, a cDNA library was constructed from a human placenta, the
mouse NIH/3T3 cell line, and a rat placenta by a conventional
method and were ligated to the .lamda.ZAPII phage vector
(manufactured by Strategene). Using the above 250 bp human cDNA
fragments as a probe, the above placenta cDNA libraries were
screened under a highly stringent condition.
[0195] Thus, they were hybridized in a hybridization solution
(5.times.SSPE, 50% formamide, 5.times.Denhardt's solution, 0.5%
SDS, 100 .mu.g/ml denatured DNA, 10% dextran sulfate) at 42.degree.
C., followed by washing under a condition of 1.times.SSC, 1.0% SDS,
65.degree. C. (Sambrook, J. et al., Molecular Cloning, Cold Spring
Harbor Laboratory Press (1989)). As a result, 1542 bp of human cDNA
containing 678 bp Of ORF was obtained.
[0196] The 678 bp of ORF in this cDNA was PCR-amplified, which was
then used as a probe in a screening of cDNA libraries of the rat
placenta and mouse NIH/3T3 cell lines under a less stringent
condition. Thus, they were hybridized in a hybridization solution
(5.times.SSPE, 50% formamide, 5.times.Denhardt's solution, 0.5%
SDS, 100 .mu.g/ml denatured DNA, 10% dextran sulfate) at 37.degree.
C., followed by washing under a condition of 1.times.SSC, 1.0% SDS,
37.degree. C. (Sambrook, J. et al., Molecular Cloning, Cold Spring
Harbor Laboratory Press (1989)).
[0197] As a result, cDNAs derived from the rat and the mouse were
isolated. The nucleotide sequence of these cDNAs were determined
using a conventional method, and the nucleotide sequence in turn
was used to determine amino acid sequence. The estimated amino acid
sequences of human, rat, and mouse cDNAs are shown by a one-letter
code and are compared, as shown in the following Table 1. The
polypeptides having these amino acid sequences were designated as
gankyrin. The human gankyrin gene and the mouse gankyrin gene had a
90% homology on the base sequence level and a 93% homology on the
amino acid sequence level. On the other hand, human gankyrin gene
and the rat gankyrin gene had a 91% homology on the base sequence
level and a 94% homology on the amino acid sequence level.
TABLE-US-00001 TABLE 1 Human
MEGCVSNLMVCNLAYSGKLEELKESILADKSLATRTDQDSRTALHWACSAGHTEIVEFLL (SEQ
ID NO: 2) Mouse
MEGCVSNIMICNLAYSGKLDELKERILADKSLATRTDQDSRTALHWACSAGHTEIVEFLL (SEQ
ID NO: 3) Rat
MEGCVSNLMVCNLAYNGKLDELKESILADKSLATRTDQDSRTALHWACSAGHTEIVEFLL (SEQ
ID NO: 5) Human
QLGVPVNDKDDAGWSPLHIAASAGRDEIVKALLGKGAQVNAVNQNGCTPLHYAASKNRHE Mouse
QLGVPVNDKDDAGWSPLHIAASAGRDEIVKALLVKGAHVNSVNQNGCTPLHYAASKNRHE Rat
QLGVPVNEKDDAGWSPLHIAASAGRDEIVKALLIKGAQVNAVNQNGCTALHYAASKNPHE Human
IAVMLLEGGANPDAKDHYWATAMHRAAAKGNLKMIHILLYYKASTNIQDTEGNTPLHLAC Mouse
ISVMLLEGGANPDAKDHYDATAMHRAAAKGNLKMVHILLFYKASTNIQDTEGNTPLHLAC Rat
IAVMLLEGGANPDAKNHYDATAMHRAAAKHNLKMVHILLFYKASYNIQDTEGNTPLHLAC Human
DEERVEEAKLLVSQGASIYIENKEEKTPLQVAKGGLGLILKRMVEG Mouse
DEERVEEAKFLVTQGASIYIENKEEKTPLQVAKGGLGLILKRLAESEEASM Rat
SEERVEEAKLLVTQGASIYIENKEEKTPLQVAKGGLGLILKRIVESEEASM
[0198] The nucleotide sequence of human gankyrin is shown in SEQ ID
NO: 1 and the amino acid sequence thereof is shown in SEQ ID NO: 2.
The nucleotide sequence of mouse gankyrin is shown in SEQ ID NO: 3
and the amino acid sequence thereof is shown in SEQ ID NO: 4. The
nucleotide sequence of rat gankyrin is shown in SEQ ID NO: 5 and
the amino acid sequence thereof is shown in SEQ ID NO: 6. In
addition, it was estimated that in the amino acid sequences of
gankyrins, the region from amino acid Met at position 1 to amino
acid Leu at position 13 is a signal sequence.
[0199] The amino acid sequence of the human gankyrin polypeptide
thus obtained had 5.5 ankyrin repeats (Lambert, S. et al., Proc.
Nati. Acad. Sci. U.S.A. (1990) 87, 1730-1734). This is shown in the
following Table 2. TABLE-US-00002 TABLE 2 ANK consensus SEQ ID NO:
10 G TPLHLAAR GHVEVVKLLLD GADVNA TK A I SQ NNLDIAEV K NPD D V K T
MR Q SI N E 1st repeat SEQ ID NO: 11
DSRTALHWACSAGHTEIVEFLLQLGVPVNDKDD 2nd repeat SEQ ID NO: 12
AGWSPLHIAASAGRDEIVKALLGKGAQVNAVNQ 3rd repeat SEQ ID NO: 13
NGCTPLHYAASKNRHEIAVMLLEGGANPDAKDH 4th repeat SEQ ID NO: 14
YEATAMHRAAAKGNLKMIHILLYYKASTNIQDT 5th repeat SEQ ID NO: 15
EGNTPLHLACDEERVEEAKLLVSQGASIYIENK 6th repeat SEQ ID NO: 16
EEKTPLQVAKGGLGLILKRMVEG
[0200] In this table, the upper 3 lines represent ankyrin
sequences, and the bottom 6 lines represent ankyrin repeats in the
amino acid sequence of the gankyrin polypeptide of the present
invention.
[0201] Furthermore, FIG. 15 shows the site and the number of
ankyrin repeats in various proteins.
[0202] In order to determine the position of the gankyrin gene on
the chromosome, fluorescence in situ hybridization was conducted.
Thus, lymphocytes isolated from human blood were cultured in an
minimum essential medium (MEM) supplemented with 10% fetal bovine
serum and phytohemagglutinin (PHA) at 37.degree. C. for 68 to 72
hours. This lymphocyte culture was treated with 0.18 mg/ml BrdU
(manufactured by Sigma) to synchronize the cell cycle of the cell
population. The cells of which the cell cycle was synchronized were
washed three times with a serum-free medium. After the cell cycle
arrest was removed, they were cultured again in a MEM containing
2.5 .mu.g/ml thymidine (manufactured by Sigma) at 37.degree. C. for
6 hours. Cells were collected, subjected to a standard procedure
comprising a hypotonic treatment, fixation, and air drying to
prepare slides of chromosome specimens.
[0203] Phage DNA (Sambrook, J. et al. Molecular Cloning, supra)
having an about 8.0 kb gankyrin gene insert was biotin-labeled by
nick translation with DATP and biotin-16-dCTP at 15.degree. C. for
one hour using the BRL BioNick label kit according to the
instructions (Herg et al., Proc. Natl. Acad. Sci. U.S.A.
89:9509-9513 (1992)).
[0204] Using this as a probe, fluorescence in situ hybridization
(FISH) was carried out (Herg et al., Proc. Natl. Acad. Sci. U.S.A.
89:9509-9531 (1992); Herg et al., Chromosoma, 102; 325-332
(1993)).
[0205] Thus, a slide was first treated at 55.degree. C. for one
hour to attach the chromosome on the glass slides. After an RNase
treatment, the slide was denatured with 70% formamide in
2.times.SSC at 70.degree. C. for 2 minutes, and then dehydrated
with ethanol. The probe was denatured in a hybridization mixture
containing 50% formamide, 10% dextran sulfate and human cotI DNA at
75.degree. C. for 5 minutes. After incubation at 37.degree. C. for
15 minutes to suppress the repeat sequence, the probe was added to
the above denatured slide. After overnight hybridization, the slide
was washed with 50% formamide in 2.times.SSC at 37.degree. C., and
then in 1.times.SSC at 60.degree. C.
[0206] After biotin was detected with fluorescence-labeled
FITC-bound avidin (manufactured by Vector Laboratories), the slide
was stained with DAPI (manufactured by Sigma), a fluorescent
reagent for staining DNA, to generate a G/Q-band pattern on the
chromosome. Use of this method enables the generation of graded
band patterns peculiar to the chromosome with a fluorescent reagent
for staining DNA, and chromosome assigning and chromosome mapping
(location).
[0207] Using a cooled charge-coupled device (CCD) camera
(manufactured by Photometrics) that is a TV camera capable of
detecting a very weak light, 21 metaphase (mitotic period) images
were photographed. By over lapping the FISH signal and the DAPI
band-forming chromosome, the FISH map data was assigned to the
chromosome band (Hery et al., Methods in Molecular Biology: In situ
hybridization protocols (K. H. A Choo. ed), p. 35-49 (1994), Human
Press, Clifton, N.J.).
[0208] Under the condition used, hybridization efficiency for this
probe was about 81%. Thus, out of 100 mitotic figures tested, 80
figures have shown signals on a pair of chromosomes. Since DAPI
band formation was used in order to identify specific chromosomes,
signals from the probe were assigned to the long arm of the X
chromosome. Furthermore, detailed positions was determined by
putting together 10 photographs. The result is shown in FIG. 1.
Since no other loci were detected by the FISH under the condition
used, the probe T4-11 was assigned to the chromosome x region
q21.3-q22.2.
[0209] The result (fluorescence staining) of the in situ
hybridization obtained is shown in FIG. 2.
Example 2
A Study on the Expression Level of a Gankyrin Gene in the
Tissue
[0210] In order to study the expression of a gankyrin gene, various
tissues and cells were homogenized in the TRIzol reagent
(manufactured by GIBCO BRL). Total RNA (20 .mu.g) was denatured,
and was separated by electrophoresis in a 1.0% agarose gel
containing 2.2 M formaldehyde.
[0211] The gel was blotted to the Hybond N+ nylon membrane
(manufactured by Amersham), and was hybridized to
[.alpha.-.sup.32p]dCTP-labeled cDNA fragment (250 bp of human
gankyrin cDNA) in a rapid hybridization buffer (Rapi-hyb buffer,
manufactured by Amersham). After hybridization, the filter was
washed under a stringent condition comprising the wash buffer
containing 0.1.times.SSC and 0.1% SDS at 65.degree. C. for 30
minutes, and then was exposed to a film at -80.degree. C. The
filter was cut into strips, which were hybridized again to a probe
for 18S rRNA as an internal standard.
[0212] The expression level of RNA was evaluated by quantifying the
autoradiogram by a scanning densitometer (manufactured by Ato).
[0213] The result of samples from the hepatic cancer tissue (T) and
the hepatic non-cancer tissue (N) is shown in FIG. 3. The lower
part represents the result of the internal standard and the upper
part represents the result detected with the cDNA probe of human
gankyrin. It was shown that gankyrin mRNA is expressed in excessive
amounts in the hepatic cancer tissue alone.
[0214] The result (positive tests/total tests) for the human cancer
tissues other than the liver is shown below. TABLE-US-00003 RCC
(kidney cell carcinoma) 0/20 Testicular carcinoma 0/5 Ovary
carcinoma 0/5 Gastric cancer 4/4
[0215] As a result, gankyrin mRNA was expressed in excessive
amounts in the tissue of gastric cancer among the cancer tissues
tested.
[0216] The results for various cell lines, i.e. human cell line
HepG2 (lane 1), Hela (lane 2), K562 (lane 3), NC65 (lane 4), NEC8
(lane 5), T24 (lane 6), and IMR90 (lane 7) are shown in FIG. 4. The
expression of gankyrin mRNA was observed in some cell lines.
[0217] The result for various normal tissues, i.e. liver (lane 1),
spleen cells (lane 2), pancreas (lane 3), heart (lane 4), adrenal
(lane 5), thyroid (lane 6), placenta (lane 7), ovary (lane 8),
testis (lane 9), kidney (lane 10), and lung (lane 11) are shown in
FIG. 5. As shown in FIG. 5, there was little expression of gankyrin
mRNA in normal human tissues.
[0218] The above result confirmed the specific and high expression
of gankyrin mRNA in the cancer tissue.
Example 3
Preparation of Antibody to Gankyrin Polypeptide and
Immunohistochemical Analysis Thereof
[0219] For preparation of anti-gankyrin polypeptide antibody, a
peptide Met-Glu-Gly-Cys-Val-Ser-Asn-Leu-Met-Val-Cys-Asn-Leu-Ala-Tyr
(SEQ ID NO: 7) corresponding to the C-terminal region of gankyrin
was immunized. The peptide was linked to keyhole limpet hemocyanin,
and then was immunized to a rabbit. On day 14, 42, and 56 after the
immunization, the rabbit was immunized again to obtain
antiserum.
[0220] The antiserum obtained was affinity purified with the above
immobilized peptide to obtain polyclonal antibody (Fmoc Chemistry,
Research Gevetic Inc.). The reactivity and/or specificity to the
gankyrin polypeptide of the present invention were confirmed by the
Western blot analysis. Thus, an expression plasmid for a gankyrin
gene was added to a TNT expression system (manufactured by Promega)
to obtain a translation product (gankyrin polypeptide). At this
time, [.sup.35S]-methionine was added to the system to synthesize
[.sup.35S]-methionine-labeled human gankyrin polypeptide.
Non-labeled gankyrin polypeptide was also synthesized using the
same condition.
[0221] [.sup.35S]-methionine-labeled and non-labeled human gankyrin
polypeptides were subjected to polyacrylamide gel electrophoresis
(the condition is described below), and the migration pattern of
the non-labeled substance was analyzed by the Western blot method.
Thus, the migration gel of the non-labeled substance was
transferred to an Immobilon transfer membrane (manufactured by
Millipore), and was blocked in a Tris buffer containing 5% bovine
serum albumin (BSA). Then the above blotting membrane was incubated
with the polyclonal antibody diluted 1:2000 to 1:10000 in a Tris
buffer, 0.1% Tween 20, and BSA at 4.degree. C. for 16 hours.
[0222] The blotting membrane was repeatedly washed in the Tris
buffer and 0.1% Tween 20 and then was incubated with anti-rabbit
immunoglobulin antibody labeled with horseradish peroxidase as the
2nd antibody at room temperature for 1 hour. After washing, it was
allowed to develop color with an electrochemiluminescence reagent
(manufactured by Amersham).
[0223] On the other hand, for the migration gel for the labeled
substance, the migration pattern of gankyrin polypeptide was
confirmed by autoradiography. As a result, a Western blot band of
the non-labeled substance was observed corresponding to the
autoradiography band of the labeled substance, indicating that the
polyclonal antibody recognizes the gankyrin gene product.
[0224] In a Western blot analysis conducted simultaneously using a
TNT expression system containing no template DNA and a luciferase
cDNA expression product, the present polyclonal antibody, no such
specific bands were observed, confirming the specificity of the
polyclonal antibody to gankyrin polypeptide. FIG. 8A shows a result
on products of an in vitro-translated labeled gankyrin gene. The
template (-) (lane 1), positive control (luciferase cDNA
corresponding to about 60 kDa) (lane 2), and gankyrin (lane 3). The
result of Western blot analysis using anti-gankyrin polypeptide
antibody on the in vitro-translated gankyrin gene products
(non-labeled) is shown in FIG. 8B. Each lane represents the
template (-) (lane 1), positive control (luciferase cDNA
corresponding to about 60 kDa) (lane 2), and gankyrin (lane 3).
[0225] FIG. 6 shows a result of an experiment in which gankyrin
polypeptides in the lysates of the hepatic non-cancer tissue (N)
and the hepatic cancer tissue (T) of three patients with hepatic
cancer were detected by a Western blot method using anti-gankyrin
polypeptide antibody that was affinity-purified as described above.
Similarly the level of mRNA shown in Example 2, more gankyrin
polypeptides were detected in the hepatic cancer tissue than in the
hepatic non-cancer tissue.
[0226] FIG. 7 shows the result of an experiment in which gankyrin
polypeptides from the total cell lysates of human cell lines, i.e.
HepG2 (ATCC catalogue 1994 (American Type Culture Collection))
(lane 1), HeLa (ATCC catalogue 1994 (American Type Culture
Collection)) (lane 2), T24 (ATCC catalogue 1994 (American Type
Culture Collection)) (lane 3), NC65 (Hoehn, W. and Schroeder, F.
H., Invest. Urol. (1978) 16, 106) (lane 4), NEC8 (Human Science
Research Resource bank, Cell/gene catalogue, 2nd edition, 1995)
(lane 5), Jurkat (ATCC catalogue 1994 (American Type Culture
Collection)) (lane 6), 293 (ATCC catalogue 1994 (American Type
Culture Collection)) (lane 7) and COS-7 (ATCC catalogue 1994
(American Type Culture Collection)) (lane 8) were detected by a
Western blot method using the anti-gankyrin polypeptide antibody
that was affinity-purified as described above. As a result, the
expression of gankyrin polypeptide was confirmed in all cell
lines.
[0227] Total cell extracts and tissue extracts were prepared from
3.about.5.times.10.sup.6 cells lysated in a modified buffer for
radioisotope-labeled immunoprecipitation comprising 50 mM Tris-HCl,
pH 7.4, 400 mM NaCl, 1% SDS, 1% Triton X-100, 1% deoxycholic acid,
and 5 mM EDTA, and then subjected to ultrasonic treatment to shear
DNA. Immediately before use, a phosphatase inhibitor comprising 15
mM .beta.-glycerophosphate, 2 mM sodium pyrophosphate and 1 mM
Na.sub.3VO.sub.4, and a protease inhibitor comprising aprotinin,
leupeptin and phenylmethyl sulfonyl fluoride were added to all
extracts.
[0228] Samples were separated on a SDS-polyacrylamide gel
electrophoresis (PAGE), and the gel used in the SDS-PAGE was
transferred to an Immobilon transfer membrane (manufactured by
Millipore), followed by blocking in a Tris buffer containing 5%
bovine serum albumin (BSA). Then the above blotting membrane was
incubated together with the above primary antiserum or antibody
diluted to 1:2000 to 1:10000 in the Tris buffer, 0.1% Tween 20, and
BSA at 4.degree. C. for 16 hours. The blotting membrane was
repeatedly washed in a Tris buffer and 0.1% Tween 20. Then it was
incubated with anti-rabbit immunoglobulin antibody or anti-mouse
immunoglobulin antibody labeled with horseradish peroxidase as the
2nd antibody at room temperature for 1 hour. After washing, it was
allowed to develop color with an electrochemiluminescence reagent
(manufactured by Amersham).
Example 4
Characterization of Gankyrin Polypeptide
[0229] A 678 bp cDNA encoding human gankyrin polypeptide (Example
1) was ligated to pMKIT-NEO mammal expression vector (Shinsaibo
Kogaku Jikken Protocol (New Cell Engineering Experiment Protocol)
P. 259, The university of Tokyo, the Institute of Medical Science,
ed., Shujunsha) in a sense and antisense direction. The SRa
promoter in this vector can direct the constitutive synthesis of
RNA from the inserted DNA. The pMKIT-NEO vector has a neomycin
resistant gene suitable for selection of transformants.
[0230] Thirty micrograms of the plasmid construct was transfected
into NIH 3T3 cells (Jainchill, J. F. et al., J. Virol. (1969) 4,
549-553) by the calcium phosphate method. Forty eight hours after
the transfection, G418 was added to the culture medium to a
concentration of 1000 .mu.g/ml. Individual colonies were isolated
and were propagated for further analysis. Thus, 5 sense clones, 5
antisense lines, and 5 control clones were established, and these
clones were characterized by in vitro growth, morphology, cell
cycle, and tumorigenicity
[0231] The doubling time was determined from the growth curve. The
cells were cultured in a 2-layer soft agar comprising a bottom
layer (DMEM, 10% FCS, 0.6% agar) and an upper layer (DMEM, 10% FCS,
0.3% agar). A 35 mm soft agar plate was inoculated with
5.times.10.sup.3 cells, incubated at 37.degree. C. for 4 to 5
weeks, and then the cells were counted. The mean colony count of
the clonies comprising 15 or more cells was 25.+-.2 for the control
clone, and 123.+-.1 for the sense clone. Accordingly, the
enhancement in the ability of colony formation by the cells that
express gankyrin polypeptide in soft agar was demonstrated.
[0232] Tumorigenicity in the NIH 3T3 cell line was tested by
transferring subcutaneously 1.times.10.sup.6 cells to 4-week old
female nude mice (Flanagan, S. P. Genet. Res. (1966) 8, 295-309).
Each of 5 clonal cell lines comprising a mock construct and a
clonal cell line containing a sense human gankyrin construct was
subcutaneously transplanted to 3 mice (a total of 18 mice) at
1.times.10.sup.6 cells/mouse. Tumor formation was observed for 3
months after subcutaneous transplantation. Measurement of tumor was
conducted using a linear caliper in two right angles by the same
observer. For cell cycle analysis, flow cytometry was used.
[0233] As a result, tumor formation when the control
vector-containing cells were inoculated was 0 clone (no tumor
formation) out of 4 clones whereas tumor formation when the sense
vector-containing cell clones were inoculated was 3 clones out of 4
clones. It is evident, therefore, that the transplantation of
gankyrin polypeptide-expressing cells to mice shows tumorigenicity.
In one clone in which no tumor was formed, the expression level of
gankyrin mRNA was lower than other 3 clones.
[0234] In a cultured human kidney cell line 293, apoptosis is
induced by removing serum from the culture medium and dead cells
increase. Using this cell line, the effect of gankyrin gene on the
induction of apoptosis was investigated. Prior to the experiment,
each gene was transfected into the 293 cells by the calcium
phosphate coprecipitation method using 10 .mu.g of the above
pMKIT-NEO vector, and a plurality of clones in which each gene was
stably introduced were obtained, which were used for the subsequent
experiment.
[0235] It was shown, at this time, that the mean value of the
colony focus number obtained from the G418 selection medium in
three transfection experiments was 56.+-.4 for the control clone,
70.+-.4 for the sense clone, and 23.+-.3 for the antisense clone.
It was demonstrated, therefore, that gankyrin polypeptide is
involved in the promotion of cell growth and suppression of
apoptosis induction.
[0236] Using each 293 cell clone, 2.times.10.sup.5 cells were
plated to a 60 mm tissue culture plate (Nunc GmbH). After the
removal of serum, cells were trypsinized and the numbers of the
suspended cells and the attached cells, and the number of dead
cells were counted by trypan blue staining to determine the ratio
of the number of the dead cells to the number of total cells.
[0237] To analyze the number of apoptosis cells, a histochemical
method was employed. Thus, cells of each clone were grown on a
cover slip for 48 hours until they reach a density of 60%
saturation. After the removal of serum, apoptosis cells were
stained by the ApopTa apoptosis detection kit (manufactured by
Oncor), and examined under a light microscope to count the ratio of
the number of the apoptosis cells to that of total cells.
[0238] In the above two experiments, the ratio (%) of the number of
total cells stained with trypan blue to the number of the total
cells and the ratio (%) of the apoptosis cells to the number of
total cells were 33.+-.5% and 45.+-.5% for the control clone, and
59.+-.6% and 30.+-.2% for the sense clone, respectively. It was
demonstrated, therefore, that apoptosis and cell death by gankyrin
gene products are suppressed.
[0239] For each clone, 2.times.10.sup.5 cells were plated to a 10
mm tissue culture plate. After incubation, serum was removed to
induce apoptosis. In order to analyze the fragmentation of gene DNA
between nucleosomes, which is characteristic to the apoptosis
cells, the above cells were scraped at predetermined times and the
supernatants were collected together with the attached cells. The
cells were resuspended in 0.25 ml of TBE (45 mM Tris-borate, 1 mM
EDTA, pH 8.0) containing 0.25% NP-40 and 0.1 mg/ml of RNase.
[0240] After incubating at 37.degree. C. for 30 minutes, the
extracts were further treated with 1 mg/ml of proteinase K at
37.degree. C. for 30 minutes. Then, 30 .mu.l of the extract was
subjected to a 1.7% agarose gel electrophoresis in the presence of
0.5 .mu.g/ml of ethidium bromide. As a result, the ladder-like
electrophoretic pattern due to DNA fragmentation between
nucleosomes was decreased in the sense gankyrin gene-introduced
cells as compared to the control cells. It was demonstrated from
these three experiments, therefore, that gankyrin gene products
acts on apoptosis induction in a suppressive manner. This is a
characteristics observed for many other tumorigenic genes, thereby
indicating that the gankyrin gene is a tumorigenic gene.
Example 5
Interaction of Gankyrin Polypeptides
[0241] The human gankyrin cDNA obtained in Example 1 was ligated to
a pCMV4-3HA' vector (Brockman, J. A. et al., Molecular and Cellular
Biology (1995) 15, 2809-2818) that has the cytomegalovirus
enhancer/promoter and having the nucleotide sequence of influenza
virus hemagglutinin (HA) epitope to construct a plasmid
pCMV4-3HA+gankyrin that expresses a fusion polypeptide comprising
gankyrin and influenza virus HA.
[0242] Furthermore, a human gankyrin coding sequence was inserted
to a vector GEX (manufactured by Pharmacia) to construct a plasmid
that expresses a fusion polypeptide comprising. glutathione
S-transferase (GST) and gankyrin. using 10 .mu.g of
pCMV4-3HA+gankyrin, the 293 cells were transiently transfected by
the calcium phosphate method. pGEX-gankyrin was introduced into
E.coli, and the production of GST-gankyrin fusion polypeptide was
induced with 1 mM IPTG. After collecting the cells by centrifuging
at 4.degree. C., the cells were dissolved by sonication in PBS
containing Triton X100. The cell lysate was mixed with the total
cell extract from the 293 cell transformant, which was incubated at
4.degree. C. for 16 hours.
[0243] The GST-fused polypeptide was collected on
glutathione-Sepharose 4B (manufactured by Pharmacia) and was
analyzed by the Western blot method using anti-Rb antibody,
anti-NF.kappa.B p50 antibody, and anti-NF.kappa.B p65 (all
manufactured by Santa Crutz).
[0244] The preparation of cell extracts and immunoprecipitates was
conducted as follows. To an IP buffer containing 50 mM HEPES (pH
7.5), 150 mM NaCl, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 10%
glycerol, a protease inhibitor, and a phosphatase inhibitor, the
cells were suspended, sonicated, and then centrifuged at
10000.times.g, 4.degree. C., for 10 minutes. Using Protein
A-Sepharose CL4b (manufactured by Pharmacia) precoated with anti-HA
antibody, anti-Rb antibody, anti-NF.kappa.B p50 antibody or
anti-NF.kappa.B p65 antibody, the supernatant was precipitated at
4.degree. C. for 16 hours.
[0245] Proteins that precipitated on the beads were washed ten
times in the IP buffer. The precipitate in the 2.times.SDS sample
buffer was separated by SDS-polyacrylamide gel electrophoresis, and
was analyzed by Western blot method with anti-HA antibody, anti-Rb
antibody, anti-NF.kappa.B p50 antibody or anti-NF.kappa.B p65
antibody.
[0246] The result is shown below.
[0247] FIG. 9 shows the result in which cell lysates were used in
vitro. GST alone or a fusion polypeptide of GST and gankyrin was
expressed in E. coli, which was then harvested. The GST alone or
the GST-gankyrin fusion polypeptide was mixed with human 293 cells,
and then precipitated with glutathione-bound Sepharose, which was
electrophoresed.
[0248] The result is shown that was obtained after the
electrophoresed gel was transferred to a nitrocellulose membrane
and was detected with (A) anti-Rb antibody, (B) anti-p50 antibody,
and (C) anti-p65 antibody. In (A), lanes indicate, from left to
right, the results of a human 293 cell lysate alone, a precipitate
obtained by mixing a human 293 cell lysate with the above E.
coli-expressed GST polypeptide and then by precipitating it with
glutathione-bound Sepharose, and a precipitate obtained by mixing a
human 293 cell lysate with an E.coli-expressed fusion polypeptide
and then by precipitating it with glutathione-bound Sepharose.
[0249] In (B), lanes indicate, from left to right, the results of a
human 293 cell lysate alone, a precipitate obtained by mixing a
human 293 cell lysate with an E. coli-expressed GST polypeptide and
then by precipitating it with glutathione-bound Sepharose, and a
precipitate obtained by mixing a human 293 cell lysate with an E.
coli-expressed fusion polypeptide and then by precipitating it with
glutathione-bound Sepharose. In (C), lanes indicate, from left to
right, the results of a human 293 cell lysate alone, a precipitate
obtained by mixing a human 293 cell lysate with an E.
coli-expressed GST polypeptide and then by precipitating it with
glutathione-bound Sepharose, and a precipitate obtained by mixing a
human 293 cell lysate with an E. coli-expressed fusion polypeptide
and then by precipitating it with glutathione-bound Sepharose.
[0250] The results of in vivo experiments on the cells are shown in
FIG. 10. By immunoprecipitating the lysate of human 293 cells that
express gankyrin fused to HA with (A) anti-Rb antibody, (B)
anti-p50 antibody, or (C) anti-p65 antibody, and then by detecting
with anti-HA antibody, an HA-fused gankyrin polypeptide was
detected. In (A), lanes indicate the results of a human 293 cell
lysate that was transformed with a vector containing no gankyrin
gene and precipitated with a non-specific immunoglobulin (lane 1)
or anti-Rb antibody (lane 2), which was then electrophoresed and
detected with anti-HA antibody, and a human 293 cell lysate that
was transformed with a vector containing a gankyrin gene and
precipitated with a non-specific immunoglobulin (lane 3) or anti-Rb
antibody (lane 4), which was then electrophoresed and detected with
anti-HA antibody.
[0251] In (B), lanes indicate the results of a human 293 cell
lysate that was transformed with a vector containing no gankyrin
gene and precipitated with a non-specific immunoglobulin (lane 1)
or anti-p50 antibody (lane 2), which was then electrophoresed and
detected with anti-HA antibody, a human 293 cell lysate that was
transformed with a vector containing a gankyrin gene and
precipitated with a non-specific immunoglobulin (lane 3) or
anti-p50 antibody (lane 4), which was then electrophoresed and
detected with anti-HA antibody, and a human 293 cell lysate that
was transformed with a vector containing a gankyrin gene and then
electrophoresed (lane 5) and detected with anti-HA antibody.
[0252] In (C), lanes indicate the results of a human 293 cell
lysate that was transformed with a vector containing no gankyrin
gene and precipitated with a non-specific immunoglobulin (lane 1)
or anti-p65 antibody (lane 2), which was then electrophoresed and
detected with anti-HA antibody, a human 293 cell lysate that was
transformed with a vector containing a gankyrin gene and
precipitated with a non-specific immunoglobulin (lane 3) or
anti-p65 antibody (lane 4), which was then electrophoresed and
detected with anti-HA antibody, and a human 293 cell lysate that
was transformed with a vector containing a gankyrin gene and then
electrophoresed (lane 5) and detected with anti-HA antibody.
[0253] The results of in vivo experiments on the cells are shown in
FIG. 11. By immunoprecipitating the lysate of human 293 cells that
express a fusion polypeptide comprising HA and gankyrin, and then
detecting with (A) anti-Rb antibody, or (B) anti-pSO antibody, Rb
and p65 were detected, respectively.
[0254] In (A), lanes indicate the results of a human 293 cell
lysate that was transformed with a vector containing no gankyrin
gene, electrophoresed, and detected with anti-Rb antibody (lane 1),
a human 293 cell lysate that was transformed with a vector
containing no gankyrin gene and precipitated with a non-specific
immunoglobulin (lane 2) or anti-HA antibody (lane 3), which was
then electrophoresed and detected with anti-Rb antibody, a human
293 cell lysate that was transformed with a vector containing a
gankyrin gene and precipitated with a non-specific immunoglobulin
(lane 3) or anti-HA antibody (lane 4), which was then
electrophoresed and detected with anti-Rb antibody.
[0255] In (B), lanes indicate the results of a human 293 cell
lysate that was transformed with a vector containing no gankyrin
gene and precipitated with a non-specific immunoglobulin (lane 1)
or anti-HA antibody (lane 2), which was then electrophoresed and
detected with anti-p65 antibody, a human 293 cell lysate that was
transformed with a vector containing a gankyrin gene and
precipitated with a non-specific immunoglobulin (lane 3) or anti-HA
antibody (lane 4), which was then electrophoresed and detected with
anti-p65 antibody, and a human 293 cell lysate that was transformed
with a vector containing a gankyrin gene and then electrophoresed
(lane 5) and detected with anti-p65 antibody.
[0256] These results indicated that the gankyrin polypeptide
interacts with Fb or NF.kappa.B in the cells (in vivo).
Example 6
Cell Cycle and Gankyrin Gene Expression
[0257] The cell cycle of NIH/3T3 cells was fixed at the early G1
period by serum starvation for 72 hours, and serum was added again
to synchronize cell cycle. Cells were lyzed, mRNA was extracted,
and detected using gankyrin cDNA as a probe. Thus, after mRNA was
amplified by the PCR method with cDNA of the coding region of mouse
gankyrin as a template, mRNA was detected by the Northern blot
method using .sup.32P-random primed labeled product as a probe.
[0258] Flow cytometry was used for the analysis of cell cycle by
various cell means. Thus, cells were washed in PBS containing no
Ca.sup.2+ or Mg.sup.2+, and were subjected to trypsin treatment.
Then after the cells were washed with DMEM containing 10% FCS and
collected, the cells were washed again in the sample buffer and
resuspended, and then were fixed in 70% ethanol. The cells were
stained with PI (Propiodium iodine), and determined by a flow
cytometer.
[0259] FIG. 12 shows the results. In FIG. 12, each lane represents
the result at 1 hour (lane 1), 3 hours (lane 2), 6 hours (lane 3),
9 hours (lane 4), 12 hours (lane 5), 15 hours (lane 6), 18 hours
(lane 7), 21 hours (lane 8), 24 hours (lane 9), 27 hours (lane 10),
30 hours (lane 11), and 33 hours (lane 12) after the re-addition of
serum. One to 9 hours (lane 1 to 4) corresponds to the G1 period,
12 to 18 hours (lane 5 to 7) corresponds to the S period, 21 to 24
hours (lane 8 to 9) corresponds to the G2+M period, and 27 hours or
after (lane 10) returns to the G1 period, again.
[0260] The result of the detection of mRNA expression in the cells
grown at various concentrations is shown in FIG. 13. In this
figure, the results of cell concentration 1.times.10.sup.6
cells/100 mm dish (lane 1), 2.times.10.sup.6 cells (lane 2),
3.times.10.sup.6 cells (lane 3), and 4.times.10.sup.6 cells (lane
4) are shown.
[0261] The result of detection of mRNA that is expressed during the
process of liver regeneration after partial hepatic resection in
mice is shown in FIG. 14. The results before partial hepatic
resection (lane 1), and 1 hour (lane 2), 6 hours (lane 3), 24 hours
(lane 4), 48 hours (lane 5), 72 hours (lane 6), and 168 hours (lane
7) after partial hepatic resection are shown.
[0262] When gankyrin mRNA was examined by the NIH3T3 cells for
which cell cycle was synchronized, gankyrin expression was found to
vary depending on the cell cycle. In the partially removed liver
also, expression was found to vary depending on the cell cycle.
These facts indicated that the expression of gankyrin, associated
with the progress of cell cycle, increases from the G1 phase to the
S phase, suggesting its association with cell cycle regulation.
Example 7
Inhibitory Effects of an Antisense Strand on Hepatic Cancer
Cells
[0263] Using a human hepatic cancer cell line HepG2, human gankyrin
antisense oligonucleotide derivative (sequence:
CCTGTCGCTTTACCTCCCCA) (SEQ ID NO: 8) and (TACCTCCCCACACACAGATT)
(SEQ ID NO: 9) were studied for their effect of suppressing the
growth of hepatic cells. As the culture medium, RPMI1640 (Nissui)
supplemented with 2% fetal calf serum (FCS) was used.
[0264] A 35 mm culture plate containing 1 ml culture liquid was
incubated overnight at 37.degree. C. in a CO.sub.2 incubator, to
which were added 1.times.10.sup.2 HepG2 cells that were made single
cells by a 0.25% trypsin treatment and pipetting. After 24 hours,
it was replaced with 1 ml of the culture liquied containing human
gankyrin antisense oligonucleotide (0, 2.5, and 10 .mu.g/ml) that
contains the initiation codon of a gankyrin gene, and was further
cultured for 4 days. A mass of 30 cells or more was defined as a
colony, and the number of colonies was counted under an inverted
microscope. Thus, the number of colonies when a sense
oligonucleotide was added was 95.+-.7%, whereas the number of
colonies when an antisense oligonucleotide was added decreased to
70.+-.5%, relative to 100% of the control to which distilled water
was added.
[0265] Human gankyrin antisense oligonucleotide had a suppressive
effect on the formation of colonies. As a result, it was revealed
that an antisense oligonucleotide containing the initiation codon
of gankyrin gene suppresses the growth of the hepatic cancer cell
line HepG2 cells.
INDUSTRIAL APPLICABILITY
[0266] Since the gankyrin polypeptide of the present invention
shows the elevation of colony-forming ability of cells, the
suppression of tumorigenicity, and apoptosis induction in mice, it
was shown to have carcinogenecity. The gankyrin polypeptide and DNA
encoding it are useful for elucidation of the mechanism of action
of oncogenesis. A screening method using a gankyrin polypeptide,
antibody to a gankyrin polypeptide, a method of detecting or
determininng a gankyrin polypeptide using it, and an antisense
oligonucleotide to DNA encoding a gankyrin polypeptide are also
useful for elucidation of the mechanism of action of
oncogenesis.
[0267] Reference to the microorganisms deposited under the Patent
Cooperation Treaty, Rule 13-2, and the name of the Depository
organ
Depository Organ
[0268] Name: the National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology [0269]
Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan Organism
(1) [0270] Name: Escherichia coli DH5.alpha. [pBS-t4-11] [0271]
Accession number: FERM BP-6128 [0272] Date deposited: Sep. 29, 1997
Sequence CWU 1
1
16 1 780 DNA Homo sapiens CDS (97)..(774) 1 tggtgaagct ctaacggctg
ttttgactgg cgtagccgga gccggcgacg tgaggcgggc 60 gttgctcgcg
cgacaagtag ttgctgggac agcgaa atg gag ggg tgt gtg tct 114 Met Glu
Gly Cys Val Ser 1 5 aac cta atg gtc tgc aac ctg gcc tac agc ggg aag
ctg gaa gag ttg 162 Asn Leu Met Val Cys Asn Leu Ala Tyr Ser Gly Lys
Leu Glu Glu Leu 10 15 20 aag gag agt att ctg gcc gat aaa tcc ctg
gct act aga act gac cag 210 Lys Glu Ser Ile Leu Ala Asp Lys Ser Leu
Ala Thr Arg Thr Asp Gln 25 30 35 gac agc aga act gca ttg cac tgg
gca tgc tca gct gga cat aca gaa 258 Asp Ser Arg Thr Ala Leu His Trp
Ala Cys Ser Ala Gly His Thr Glu 40 45 50 att gtt gaa ttt ttg ttg
caa ctt gga gtg cca gtg aat gat aaa gac 306 Ile Val Glu Phe Leu Leu
Gln Leu Gly Val Pro Val Asn Asp Lys Asp 55 60 65 70 gat gca ggt tgg
tct cct ctt cat att gcg gct tct gct ggc cgg gat 354 Asp Ala Gly Trp
Ser Pro Leu His Ile Ala Ala Ser Ala Gly Arg Asp 75 80 85 gag att
gta aaa gcc ctt ctg gga aaa ggt gct caa gtg aat gct gtc 402 Glu Ile
Val Lys Ala Leu Leu Gly Lys Gly Ala Gln Val Asn Ala Val 90 95 100
aat caa aat ggc tgt act ccc tta cat tat gca gct tcg aaa aac agg 450
Asn Gln Asn Gly Cys Thr Pro Leu His Tyr Ala Ala Ser Lys Asn Arg 105
110 115 cat gag atc gct gtc atg tta ctg gaa ggc ggg gct aat cca gat
gct 498 His Glu Ile Ala Val Met Leu Leu Glu Gly Gly Ala Asn Pro Asp
Ala 120 125 130 aag gac cat tat gag gct aca gca atg cac cgg gca gca
gcc aag ggt 546 Lys Asp His Tyr Glu Ala Thr Ala Met His Arg Ala Ala
Ala Lys Gly 135 140 145 150 aac ttg aag atg att cat atc ctt ctg tac
tac aaa gca tcc aca aac 594 Asn Leu Lys Met Ile His Ile Leu Leu Tyr
Tyr Lys Ala Ser Thr Asn 155 160 165 atc caa gac act gag ggt aac act
cct cta cac tta gcc tgt gat gag 642 Ile Gln Asp Thr Glu Gly Asn Thr
Pro Leu His Leu Ala Cys Asp Glu 170 175 180 gag aga gtg gaa gaa gca
aaa ctg ctg gtg tcc caa gga gca agt att 690 Glu Arg Val Glu Glu Ala
Lys Leu Leu Val Ser Gln Gly Ala Ser Ile 185 190 195 tac att gag aat
aaa gaa gaa aag aca ccc ctg caa gtg gcc aaa ggt 738 Tyr Ile Glu Asn
Lys Glu Glu Lys Thr Pro Leu Gln Val Ala Lys Gly 200 205 210 ggc ctg
ggt tta ata ctc aag aga atg gtg gaa ggt taaaca 780 Gly Leu Gly Leu
Ile Leu Lys Arg Met Val Glu Gly 215 220 225 2 226 PRT Homo sapiens
2 Met Glu Gly Cys Val Ser Asn Leu Met Val Cys Asn Leu Ala Tyr Ser 1
5 10 15 Gly Lys Leu Glu Glu Leu Lys Glu Ser Ile Leu Ala Asp Lys Ser
Leu 20 25 30 Ala Thr Arg Thr Asp Gln Asp Ser Arg Thr Ala Leu His
Trp Ala Cys 35 40 45 Ser Ala Gly His Thr Glu Ile Val Glu Phe Leu
Leu Gln Leu Gly Val 50 55 60 Pro Val Asn Asp Lys Asp Asp Ala Gly
Trp Ser Pro Leu His Ile Ala 65 70 75 80 Ala Ser Ala Gly Arg Asp Glu
Ile Val Lys Ala Leu Leu Gly Lys Gly 85 90 95 Ala Gln Val Asn Ala
Val Asn Gln Asn Gly Cys Thr Pro Leu His Tyr 100 105 110 Ala Ala Ser
Lys Asn Arg His Glu Ile Ala Val Met Leu Leu Glu Gly 115 120 125 Gly
Ala Asn Pro Asp Ala Lys Asp His Tyr Glu Ala Thr Ala Met His 130 135
140 Arg Ala Ala Ala Lys Gly Asn Leu Lys Met Ile His Ile Leu Leu Tyr
145 150 155 160 Tyr Lys Ala Ser Thr Asn Ile Gln Asp Thr Glu Gly Asn
Thr Pro Leu 165 170 175 His Leu Ala Cys Asp Glu Glu Arg Val Glu Glu
Ala Lys Leu Leu Val 180 185 190 Ser Gln Gly Ala Ser Ile Tyr Ile Glu
Asn Lys Glu Glu Lys Thr Pro 195 200 205 Leu Gln Val Ala Lys Gly Gly
Leu Gly Leu Ile Leu Lys Arg Met Val 210 215 220 Glu Gly 225 3 696
DNA Mus sp. CDS (1)..(693) modified_base (198) a, t, c or g 3 atg
gag ggg tgt gtg tct aac ata atg atc tgt aac ctg gcc tac agt 48 Met
Glu Gly Cys Val Ser Asn Ile Met Ile Cys Asn Leu Ala Tyr Ser 1 5 10
15 ggg aag ctg gat gag ttg aag gag cgc att ttg gct gat aaa tct ctg
96 Gly Lys Leu Asp Glu Leu Lys Glu Arg Ile Leu Ala Asp Lys Ser Leu
20 25 30 gct act aga act gat cag gac agc aga aca gct ttg cac tgg
gca tgc 144 Ala Thr Arg Thr Asp Gln Asp Ser Arg Thr Ala Leu His Trp
Ala Cys 35 40 45 tca gct ggc cat aca gaa att gtt gaa ttc ttg ctg
caa ctt gga gtg 192 Ser Ala Gly His Thr Glu Ile Val Glu Phe Leu Leu
Gln Leu Gly Val 50 55 60 cca gtn aat gat aaa gat gac gca ggt tgg
tct cct ctt cat att gct 240 Pro Val Asn Asp Lys Asp Asp Ala Gly Trp
Ser Pro Leu His Ile Ala 65 70 75 80 gcc tcc gct ggc cgg gat gag att
gta aaa gcc ctt ctg gtg aaa ggt 288 Ala Ser Ala Gly Arg Asp Glu Ile
Val Lys Ala Leu Leu Val Lys Gly 85 90 95 gca cat gtt aat tct gtc
aat caa aac ggc tgc act cca ctc cat tat 336 Ala His Val Asn Ser Val
Asn Gln Asn Gly Cys Thr Pro Leu His Tyr 100 105 110 gca gct tcg aag
aat agg cat gag att tct gtt atg tta cta gaa ggt 384 Ala Ala Ser Lys
Asn Arg His Glu Ile Ser Val Met Leu Leu Glu Gly 115 120 125 ggg gct
aac cca gat gcg aag gac cat tac gat gct aca gca atg cac 432 Gly Ala
Asn Pro Asp Ala Lys Asp His Tyr Asp Ala Thr Ala Met His 130 135 140
cgg gca gca gcc aag ggt aac ttg aag atg gtt cac atc ctt ctg ttc 480
Arg Ala Ala Ala Lys Gly Asn Leu Lys Met Val His Ile Leu Leu Phe 145
150 155 160 tac aaa gca tcc aca aac atc caa gac act gag ggt aac act
cct cta 528 Tyr Lys Ala Ser Thr Asn Ile Gln Asp Thr Glu Gly Asn Thr
Pro Leu 165 170 175 cac tta gcc tgt gat gaa gag aga gtg gaa gag gca
aaa ttt ctg gtg 576 His Leu Ala Cys Asp Glu Glu Arg Val Glu Glu Ala
Lys Phe Leu Val 180 185 190 act caa gga gca agt att tac att gag aat
aaa gaa gaa aag aca ccc 624 Thr Gln Gly Ala Ser Ile Tyr Ile Glu Asn
Lys Glu Glu Lys Thr Pro 195 200 205 ctg caa gtt gcc aaa ggg ggc ctg
ggt tta ata ctc aag aga cta gca 672 Leu Gln Val Ala Lys Gly Gly Leu
Gly Leu Ile Leu Lys Arg Leu Ala 210 215 220 gaa agt gaa gag gct tct
atg tag 696 Glu Ser Glu Glu Ala Ser Met 225 230 4 231 PRT Mus sp. 4
Met Glu Gly Cys Val Ser Asn Ile Met Ile Cys Asn Leu Ala Tyr Ser 1 5
10 15 Gly Lys Leu Asp Glu Leu Lys Glu Arg Ile Leu Ala Asp Lys Ser
Leu 20 25 30 Ala Thr Arg Thr Asp Gln Asp Ser Arg Thr Ala Leu His
Trp Ala Cys 35 40 45 Ser Ala Gly His Thr Glu Ile Val Glu Phe Leu
Leu Gln Leu Gly Val 50 55 60 Pro Val Asn Asp Lys Asp Asp Ala Gly
Trp Ser Pro Leu His Ile Ala 65 70 75 80 Ala Ser Ala Gly Arg Asp Glu
Ile Val Lys Ala Leu Leu Val Lys Gly 85 90 95 Ala His Val Asn Ser
Val Asn Gln Asn Gly Cys Thr Pro Leu His Tyr 100 105 110 Ala Ala Ser
Lys Asn Arg His Glu Ile Ser Val Met Leu Leu Glu Gly 115 120 125 Gly
Ala Asn Pro Asp Ala Lys Asp His Tyr Asp Ala Thr Ala Met His 130 135
140 Arg Ala Ala Ala Lys Gly Asn Leu Lys Met Val His Ile Leu Leu Phe
145 150 155 160 Tyr Lys Ala Ser Thr Asn Ile Gln Asp Thr Glu Gly Asn
Thr Pro Leu 165 170 175 His Leu Ala Cys Asp Glu Glu Arg Val Glu Glu
Ala Lys Phe Leu Val 180 185 190 Thr Gln Gly Ala Ser Ile Tyr Ile Glu
Asn Lys Glu Glu Lys Thr Pro 195 200 205 Leu Gln Val Ala Lys Gly Gly
Leu Gly Leu Ile Leu Lys Arg Leu Ala 210 215 220 Glu Ser Glu Glu Ala
Ser Met 225 230 5 696 DNA Rattus sp. CDS (1)..(693) 5 atg gag ggg
tgt gtg tct aac cta atg gtc tgt aac ctg gcc tac aac 48 Met Glu Gly
Cys Val Ser Asn Leu Met Val Cys Asn Leu Ala Tyr Asn 1 5 10 15 ggg
aag ctg gat gag ttg aag gaa agc att ttg gct gat aag tct ctg 96 Gly
Lys Leu Asp Glu Leu Lys Glu Ser Ile Leu Ala Asp Lys Ser Leu 20 25
30 gcc act aga act gat cag gac agc aga aca gca ttg cac tgg gca tgc
144 Ala Thr Arg Thr Asp Gln Asp Ser Arg Thr Ala Leu His Trp Ala Cys
35 40 45 tca gct ggt cat aca gaa att gtt gaa ttc ttg ctg caa ctt
gga gtg 192 Ser Ala Gly His Thr Glu Ile Val Glu Phe Leu Leu Gln Leu
Gly Val 50 55 60 cca gta aat gaa aaa gac gat gca ggt tgg tct cct
ctt cat att gct 240 Pro Val Asn Glu Lys Asp Asp Ala Gly Trp Ser Pro
Leu His Ile Ala 65 70 75 80 gct tcc gct ggc cgg gat gag att gta aaa
gcc ctt ctg ata aaa ggg 288 Ala Ser Ala Gly Arg Asp Glu Ile Val Lys
Ala Leu Leu Ile Lys Gly 85 90 95 gca caa gtg aat gcc gtc aat cag
aat ggc tgc acg gcc ctt cat tat 336 Ala Gln Val Asn Ala Val Asn Gln
Asn Gly Cys Thr Ala Leu His Tyr 100 105 110 gca gct tcc aag aat agg
cat gag att gct gtt atg tta cta gaa ggt 384 Ala Ala Ser Lys Asn Arg
His Glu Ile Ala Val Met Leu Leu Glu Gly 115 120 125 ggg gct aat cca
gat gct aag aac cat tat gat gct aca gca atg cac 432 Gly Ala Asn Pro
Asp Ala Lys Asn His Tyr Asp Ala Thr Ala Met His 130 135 140 cgg gca
gca gcc aag ggt aac ttg aag atg gtt cat atc ctt ctg ttc 480 Arg Ala
Ala Ala Lys Gly Asn Leu Lys Met Val His Ile Leu Leu Phe 145 150 155
160 tac aaa gca tcc aca aac atc caa gat act gag ggt aac act cct cta
528 Tyr Lys Ala Ser Thr Asn Ile Gln Asp Thr Glu Gly Asn Thr Pro Leu
165 170 175 cac tta gcc tgt gat gag gag aga gtg gaa gaa gca aaa ttg
ctg gtg 576 His Leu Ala Cys Asp Glu Glu Arg Val Glu Glu Ala Lys Leu
Leu Val 180 185 190 acc caa gga gca agt att tac att gaa aat aag gaa
gaa aag aca ccg 624 Thr Gln Gly Ala Ser Ile Tyr Ile Glu Asn Lys Glu
Glu Lys Thr Pro 195 200 205 ctg caa gtc gcc aaa ggg ggc ctg ggt tta
ata ctc aaa aga atc gca 672 Leu Gln Val Ala Lys Gly Gly Leu Gly Leu
Ile Leu Lys Arg Ile Ala 210 215 220 gaa agt gaa gag gct tct atg tag
696 Glu Ser Glu Glu Ala Ser Met 225 230 6 231 PRT Rattus sp. 6 Met
Glu Gly Cys Val Ser Asn Leu Met Val Cys Asn Leu Ala Tyr Asn 1 5 10
15 Gly Lys Leu Asp Glu Leu Lys Glu Ser Ile Leu Ala Asp Lys Ser Leu
20 25 30 Ala Thr Arg Thr Asp Gln Asp Ser Arg Thr Ala Leu His Trp
Ala Cys 35 40 45 Ser Ala Gly His Thr Glu Ile Val Glu Phe Leu Leu
Gln Leu Gly Val 50 55 60 Pro Val Asn Glu Lys Asp Asp Ala Gly Trp
Ser Pro Leu His Ile Ala 65 70 75 80 Ala Ser Ala Gly Arg Asp Glu Ile
Val Lys Ala Leu Leu Ile Lys Gly 85 90 95 Ala Gln Val Asn Ala Val
Asn Gln Asn Gly Cys Thr Ala Leu His Tyr 100 105 110 Ala Ala Ser Lys
Asn Arg His Glu Ile Ala Val Met Leu Leu Glu Gly 115 120 125 Gly Ala
Asn Pro Asp Ala Lys Asn His Tyr Asp Ala Thr Ala Met His 130 135 140
Arg Ala Ala Ala Lys Gly Asn Leu Lys Met Val His Ile Leu Leu Phe 145
150 155 160 Tyr Lys Ala Ser Thr Asn Ile Gln Asp Thr Glu Gly Asn Thr
Pro Leu 165 170 175 His Leu Ala Cys Asp Glu Glu Arg Val Glu Glu Ala
Lys Leu Leu Val 180 185 190 Thr Gln Gly Ala Ser Ile Tyr Ile Glu Asn
Lys Glu Glu Lys Thr Pro 195 200 205 Leu Gln Val Ala Lys Gly Gly Leu
Gly Leu Ile Leu Lys Arg Ile Ala 210 215 220 Glu Ser Glu Glu Ala Ser
Met 225 230 7 15 PRT Homo sapiens 7 Met Glu Gly Cys Val Ser Asn Leu
Met Val Cys Asn Leu Ala Tyr 1 5 10 15 8 20 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA 8 cctgtcgctt
tacctcccca 20 9 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic DNA 9 tacctcccca cacacagatt 20 10 28
PRT Artificial Sequence Description of Artificial Sequence ANK
consensus MOD_RES (3) P or A MOD_RES (6) L, I or V MOD_RES (8) A or
S MOD_RES (9) R, Q or K MOD_RES (10) G or N MOD_RES (11) H or N
MOD_RES (12) V, L or T MOD_RES (13) E or D MOD_RES (14) V, I or M
MOD_RES (15) V or A MOD_RES (16) K, E or R MOD_RES (17) L or V
MOD_RES (20) D, K, Q or E MOD_RES (23) D, N or S MOD_RES (24) V, P
or I MOD_RES (25) N or D MOD_RES (27) T, D or N 10 Gly Thr Xaa Leu
His Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Leu
Leu Xaa Gly Ala Xaa Xaa Xaa Ala Xaa Lys 20 25 11 33 PRT Homo
sapiens ankyrin repeat 11 Asp Ser Arg Thr Ala Leu His Trp Ala Cys
Ser Ala Gly His Thr Glu 1 5 10 15 Ile Val Glu Phe Leu Leu Gln Leu
Gly Val Pro Val Asn Asp Lys Asp 20 25 30 Asp 12 33 PRT Homo sapiens
ankyrin repeat 12 Ala Gly Trp Ser Pro Leu His Ile Ala Ala Ser Ala
Gly Arg Asp Glu 1 5 10 15 Ile Val Lys Ala Leu Leu Gly Lys Gly Ala
Gln Val Asn Ala Val Asn 20 25 30 Gln 13 33 PRT Homo sapiens ankyrin
repeat 13 Asn Gly Cys Thr Pro Leu His Tyr Ala Ala Ser Lys Asn Arg
His Glu 1 5 10 15 Ile Ala Val Met Leu Leu Glu Gly Gly Ala Asn Pro
Asp Ala Lys Asp 20 25 30 His 14 33 PRT Homo sapiens ankyrin repeat
14 Tyr Glu Ala Thr Ala Met His Arg Ala Ala Ala Lys Gly Asn Leu Lys
1 5 10 15 Met Ile His Ile Leu Leu Tyr Tyr Lys Ala Ser Thr Asn Ile
Gln Asp 20 25 30 Thr 15 33 PRT Homo sapiens ankyrin repeat 15 Glu
Gly Asn Thr Pro Leu His Leu Ala Cys Asp Glu Glu Arg Val Glu 1 5 10
15 Glu Ala Lys Leu Leu Val Ser Gln Gly Ala Ser Ile Tyr Ile Glu Asn
20 25 30 Lys 16 23 PRT Homo sapiens ankyrin repeat 16 Glu Glu Lys
Thr Pro Leu Gln Val Ala Lys Gly Gly Leu Gly Leu Ile 1 5 10 15 Leu
Lys Arg Met Val Glu Gly 20
* * * * *