U.S. patent application number 12/782641 was filed with the patent office on 2011-01-20 for method for diagnosing non-small cell lung cancers by trna-dihydrouridine synthase activity of urlc8.
This patent application is currently assigned to Oncotherapy Science, Inc.. Invention is credited to Yataro Daigo, Yusuke Nakamura, Shuichi Nakatsuru.
Application Number | 20110014615 12/782641 |
Document ID | / |
Family ID | 35559267 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110014615 |
Kind Code |
A1 |
Nakamura; Yusuke ; et
al. |
January 20, 2011 |
METHOD FOR DIAGNOSING NON-SMALL CELL LUNG CANCERS BY
tRNA-DIHYDROURIDINE SYNTHASE ACTIVITY OF URLC8
Abstract
The present invention features a method for determining t-RNA
dihydrouridine-synthase activity of a polypeptide and screening for
modulators of t-RNA dihydrouridine-synthase activity. The present
invention further provides methods or pharmaceutical compositions
for preventing and/or treating non-small cell lung cancer (NSCLC)
using such modulators. Furthermore, the present invention provides
methods for diagnosing non-small cell lung cancer (NSCLC) using the
t-RNA dihydrouridine-synthase activity of IMS-E21 (URLC8) protein
as an index. The present invention further provides methods for
predicting and prognosing lung squamous-cell carcinoma (SCC).
Inventors: |
Nakamura; Yusuke; (Tokyo,
JP) ; Daigo; Yataro; (Tokyo, JP) ; Nakatsuru;
Shuichi; (Kanagawa, JP) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Oncotherapy Science, Inc.
Kawasaki-shi
JP
|
Family ID: |
35559267 |
Appl. No.: |
12/782641 |
Filed: |
May 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11575812 |
Oct 8, 2007 |
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PCT/JP05/17915 |
Sep 21, 2005 |
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12782641 |
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60612937 |
Sep 24, 2004 |
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Current U.S.
Class: |
435/6.11 ;
435/6.13 |
Current CPC
Class: |
G01N 2333/99 20130101;
G01N 33/57423 20130101; C12Q 1/6886 20130101; G01N 2500/02
20130101; A61P 11/00 20180101; C12Q 2600/118 20130101; C12Q 1/533
20130101; C12Q 2600/136 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1.-9. (canceled)
10. A method of identifying an agent that modulates t-RNA
dihydrouridine-synthase activity, said method comprising the steps
of: a. incubating in the presence of a test compound under
conditions suitable for the synthesis of t-RNA dihydrouridine a
polypeptide selected from the group consisting of: i. a polypeptide
comprising the amino acid sequence of SEQ ID NO: 2 (URLC8); ii. a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2
wherein one or more amino acids are substituted, deleted, or
inserted, provided said polypeptide has a biological activity
equivalent to the polypeptide consisting of the amino acid sequence
of SEQ ID NO: 2; iii. a polypeptide encoded by a polynucleotide
that hybridizes under stringent conditions to a polynucleotide
consisting of the nucleotide sequence of SEQ ID NO: 1, provided the
polypeptide has a biological activity equivalent to a polypeptide
consisting of the amino acid sequence of SEQ ID NO: 2; b. detecting
a t-RNA dihydrouridine synthesis level; and c. comparing the t-RNA
dihydrouridine synthesis level to a control level, wherein an
increase or decrease in the t-RNA dihydrouridine synthesis level as
compared to said control level indicates that the test compound
modulates t-RNA dihydrouridine-synthase activity.
11. A method of screening for a compound for treating and/or
preventing non-small cell lung cancer (NSCLC), said method
comprising the steps of: a. identifying a test compound that
modulates t-RNA dihydrouridine-synthase activity by the method of
claim 10, and b. selecting a compound that decreases the t-RNA
dihydrouridine synthesis level as compared to a control level.
12.-20. (canceled)
21. The method of claim 10, wherein the polypeptide comprising the
amino acid sequence of SEQ ID NO:2 wherein 25 or less of amino
acids are substituted, deleted, or inserted, provided said
polypeptide has a t-RNA dihydrouridine-synthase activity.
22. The method of claim 21, wherein DSRM (double-strand RNA binding
motif) in the amino acid sequence of the polypeptide is
conserved.
23. The method of claim 10, wherein the polypeptide encoded by a
polynucleotide that hybridizes under stringent conditions to a
polynucleotide consisting of the nucleotide sequence of SEQ ID NO:
1, provided the polypeptide has a t-RNA dihydrouridine-synthase
activity, wherein the stringent condition comprises washing 3 times
in 2.times.SSC, 0.01% SDS at room temperature for 20 minutes, then
washing 3 times in 1.times.SSC, 0.1% SDS at 37.degree. C. for 20
minutes, and washing twice in 1.times.SSC, 0.1% SDS at 50.degree.
C. for 20 minutes.
24. The method of claim 23, wherein DSRM (double-strand RNA binding
motif) in the amino acid sequence of the polypeptide is conserved.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
11/575,812, filed Oct. 8, 2007, which is a U.S. National Stage of
PCT/JP2005/017915, filed Sep. 21, 2005, which claims the benefit of
U.S. Provisional Application Ser. No. 60/612,937, filed on Sep. 24,
2004, the contents of which are incorporated by reference herein in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to lung cancer, more
particularly non-small cell lung cancer, and the diagnosis and
treatment thereof.
BACKGROUND OF THE INVENTION
[0003] Lung cancer is one of the most common causes of cancer death
worldwide, and non-small cell lung cancer (NSCLC) accounts for
nearly 80% of those cases (Greenlee, R. T., et al., (2001) CA
Cancer J Clin, 51: 15-36.). Many genetic alterations associated
with the development and progression of lung cancer have been
reported, but the precise molecular mechanisms remain unclear
(Sozzi, G. Eur J Cancer, (2001) 37 Suppl 7: S63-73.). Over the last
decade, newly developed cytotoxic agents, including paclitaxel,
docetaxel, gemcitabine, and vinorelbine, have emerged to offer
multiple therapeutic choices for patients with advanced NSCLC;
however, each of the new regimens can provide only modest survival
benefits as compared to cisplatin-based therapies (Schiller, J. H.
et al. (2002) N Engl J Med, 346: 92-98.; Kelly, K., et al. (2001) J
Clin Oncol, 19: 3210-3218.). Hence, the development of new
therapeutic strategies, such as molecular-targeted agents and
antibodies, and cancer vaccines, are eagerly awaited.
[0004] Systematic analysis of expression levels of thousands of
genes on cDNA microarrays is an effective approach to identifying
unknown molecules involved in pathways of carcinogenesis, and can
reveal candidate targets for development of novel therapeutics and
diagnostics. The present inventors have been attempting to isolate
novel molecular targets for diagnosis, treatment and prevention of
NSCLC by analyzing genome-wide expression profiles of NSCLC cells
on a cDNA microarray containing 23,040 genes, using pure
populations of tumor cells prepared from 37 cancer tissues by
laser-capture microdissection (Kikuchi, T. et al., Oncogene, (2003)
22: 2192-2205.; Zembutsu, H. et al. (2003) Int J Oncol, 23: 29-39.;
Kakiuchi, S. et al. Mol Cancer Res, (2003) 1: 485-499.; Suzuki, C.
et al., (2003) Cancer Res, 63: 7038-7041.). Through this genome
wide cDNA microarray analysis, 642 up-regulated genes and 806
down-regulated genes have been identified as diagnostic markers and
therapeutic targets for NSCLC (WO 2004/31413).
BRIEF SUMMARY OF THE INVENTION
[0005] To verify the biological and clinicopathological
significance of the respective gene products, tumor-tissue
microarray analysis of clinical lung-cancer materials have been
performed. This systematic approach revealed that a novel gene,
tentatively named IMS-E21 (also known as URLC8: up-regulated in
lung cancer 8, Accession No. AB101210; formerly FLJ20399), was
frequently over-expressed in primary NSCLCs.
[0006] The URLC8 gene encodes a double-strand RNA binding motif
(DSRM) domain. The lowered expression of this gene in normal
tissues, elevated expression in NSCLCs, and reduced growth,
proliferation and/or survival of transfected cells by the
suppression of this gene together suggest that this gene might be
useful as a novel diagnostic marker and target for new drugs and
immunotherapy (WO2004/31413).
[0007] The URLC8 gene has been assigned to chromosome 16q22.2 and
encodes a protein of 493 amino acids with 30% homology to S.
cerevisiae Dus1 (dihydrouridine synthase 1), a member of UPF0034
(unclassified protein family 0034), which catalyses the reduction
of the 5,6-double bond of a uridine residue on D-loop in tRNA
(Xing, F. et al., RNA, (2002) 8: 370-381.), and contains conserved
DSRM (double-strand RNA binding motif).
[0008] 5,6-Dihydrouridine is a modified base found abundantly in
the D-loops of tRNA from Archaea, Bacteria, and Eukarya.
5,6-Dihydrouridine is thought to be formed post-transcriptionally
by the reduction of uridines in tRNA transcripts. The role of
dihydrouridine in tRNA was presumed to increase the conformational
flexibility of the tRNA. Interestingly, an increase in the level of
dihydrouridine was previously reported in tumor-specific
tRNA.sup.Phe purified from human malignant tissues (Kuchino, Y. and
Borek, E. (1978) Nature, 271: 126-129.), however, its precise
mechanism and biological contribution to tumorigenesis remain
unclear (Dalluge, J. J. et al., (1996) Nucleic Acids Res, 24:
1073-1079.).
[0009] Herein the present inventors report the identification and
functional characterization of a novel human tRNA-dihydrouridine
synthase (DUS) protein, URLC8, in human lung tumors. The results
herein suggest that overexpression of URLC8 plays a significant
role in development/progression of lung cancer and that this
molecule represents a potential target for development of novel
therapeutic drugs.
[0010] The present invention is based in part on the discovery of
the t-RNA dihydrouridine-synthase activity of URLC8, a polypeptide
which is involved in the proliferation of lung cancer cells.
[0011] Accordingly, the present invention provides a method of
diagnosing non-small cell lung cancer or a predisposition to
developing non-small cell lung cancer in a subject, comprising
determining a level of t-RNA dihydrouridine-synthase activity
and/or t-RNA dihydrouridine in a biological sample derived from the
subject, wherein an increase in said level as compared to a normal
control level indicates that said subject suffers from or is at
risk of developing non-small cell lung cancer.
[0012] The present invention also provides methods of predicting a
lung squamous-cell carcinoma (SCC) prognosis. In some embodiments,
the method comprises the steps of:
[0013] a. detecting a URLC8 expression level in a specimen
collected from a subject whose SCC prognosis is to be predicted,
and
[0014] b. indicating a poor prognosis when an elevated level of
URLC8 expression is detected.
[0015] In a further embodiment, the present invention features a
method of measuring t-RNA dihydrouridine-synthase activity by
incubating a polypeptide under conditions suitable for synthesis of
t-RNA dihydrouridine and detecting the t-RNA dihydrouridine
synthesis level. The polypeptide is a URLC8 polypeptide or
functional equivalent thereof. For example, the polypeptide may
comprise the amino acid sequence of SEQ ID NO: 2. Alternatively,
the polypeptide may comprise an amino acid sequence of SEQ ID NO: 2
where one or more amino acids are substituted, deleted, or
inserted, so long as the resulting polypeptide retains the
biological activity of the polypeptide of SEQ ID NO: 2. Biological
activities of the polypeptide of SEQ ID No: 2 include, for example,
the promotion of cell proliferation and the synthesis of t-RNA
dihydrouridine. Additionally, the polypeptide may comprise a
493-amino acid protein encoded by the open reading frame of SEQ.
ID. NO. 1, or a polynucleotide that hybridizes under stringent
conditions, e.g., low or high, to the nucleotide sequence of SEQ ID
NO: 1, so long as the resulting polynucleotide encodes a protein
that retains the biological activity of the polypeptide of SEQ ID
NO: 2. Examples of suitable low stringency conditions include, for
example, 42.degree. C., 2.times.SSC, 0.1% SDS, or preferably
50.degree. C., 2.times.SSC, 0.1% SDS. Preferably, a high stringency
condition is used. An example of a suitable high stringency
condition includes, for example, washing 3 times in 2.times.SSC,
0.01% SDS at room temperature for 20 min, then washing 3 times in
1.times.SSC, 0.1% SDS at 37.degree. C. for 20 min, and washing
twice in 1.times.SSC, 0.1% SDS at 50.degree. C. for 20 min.
However, several factors, such as temperature and salt
concentration, can influence the stringency of hybridization and
one skilled in the art can suitably select the factors to achieve
the requisite stringency.
[0016] In the context of the present invention, t-RNA
dihydrouridine-synthase activity is defined as the catalysis of the
reduction of the 5,6-double bond of a uridine residue on tRNA (FIG.
1c.). Synthesis of t-RNA dihydrouridine may be detected by
conventional methods, such as those using a radioactive tRNA
substrate or derivatives thereof. The substrate may be any compound
capable of reduction of the 5,6-double bond of a uridine residue.
An exemplary substrate is a tRNA having D-loop such as a
tRNA.sup.Phe or other tRNA. The co-factor, e.g., the hydrogen
donor, may be any compound capable of donating a hydrogen atom. For
example, the co-factor may be a reduced form of nicotinamide
adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide
phosphate (NADPH). Suitable conditions for synthesis include, for
example, basic buffer conditions know in the art such as
Tris-HCl.
[0017] The present invention further provides methods of
identifying an agent that modulates (e.g., increases or decreases)
t-RNA dihydrouridine-synthase activity by incubating a polypeptide
under conditions suitable for synthesis of t-RNA dihydrouridine in
the presence of a test agent and determining the t-RNA
dihydrouridine synthesis level. A decrease in the level of
synthesis as compared to a normal control synthesis level indicates
that the test agent is an inhibitor of t-RNA
dihydrouridine-synthase activity. Compounds that inhibit (e.g.,
decreases) t-RNA dihydrouridine-synthase activity are useful for
treating, preventing or alleviating a symptom of lung cancer. For
example, such compounds may inhibit the proliferation of lung
cancer cells. Alternatively, an increase in the level or activity
as compared to a normal control level indicates that the test agent
is an enhancer of t-RNA dihydrouridine-synthase activity. Herein,
the phrase normal control level refers to a level of t-RNA
dihydrouridine synthesis detected in the absence of the test
compound.
[0018] The present invention also encompasses compositions and
methods for treating or preventing of lung cancer by contacting a
lung cancer cell with a compound identified as described above. In
a further embodiment, the present invention provides for the use of
a compound identified as described above, for manufacturing a
pharmaceutical composition suitable for treating or preventing lung
cancer. For example, a method of treating lung cancer may involve
the step of administering to a mammal, e.g. a human patient having
been diagnosed with such a disease state, with a composition
containing a pharmaceutically effective amount of a compound
identified as described above and a pharmaceutical carrier.
[0019] The present invention also provides a kit for the detecting
the t-RNA dihydrouridine-synthase activity of a compound with a
t-RNA dihydrouridine-synthase polypeptide or living cell expressing
the polypeptide and reagent for detecting a t-RNA
dihydrouridine-synthase activity. The reagents are preferably
packaged together in the form of a kit. The reagents may be
packaged in separate containers and may include, for example, a
t-RNA dihydrouridine-synthase polypeptide, reagent for detecting a
t-RNA dihydrouridine-synthase activity, a control reagent (positive
and/or negative), and/or a detectable label. Instructions (e.g.,
written, tape, VCR, CD-ROM, etc.) for carrying out the assay are
preferably included in the kit. The assay format of the kit may
comprise any t-RNA dihydrouridine-synthase assay known in the
art.
[0020] In a further embodiment, the present method may include the
step of administering to a subject a small interfering RNA (siRNA)
composition. In the context of the present invention, the siRNA
composition reduces the expression of URLC8.
[0021] These and other objects and features of the invention will
become more fully apparent when the following detailed description
is read in conjunction with the accompanying figures and examples.
However, it is to be understood that both the foregoing summary of
the invention and the following detailed description are of a
preferred embodiment, and not restrictive of the invention or other
alternate embodiments of the invention.
[0022] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the present specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts the structure of IMS-E21 (URLC8) and
structural features of tRNA-dihydrouridine.
[0024] a, Schematic structure of IMS-E21 (URLC8) protein. A member
of UPF0034 (unclassified protein family 0034), Dus domain at
N-terminal end, and DSRM (double-strand RNA binding motif) at
C-terminal end were conserved in its protein structure, showing 30%
homology to S. cerevisiae Dihydrouridine synthase 1 (DUS1), which
catalyses the reduction of the 5,6-double bond of a uridine residue
on tRNA. b, Alignment of the predicted amino acid sequences of the
IMS-E21 (URLC8). Shading indicates homologues residues. c, The
structure of dihydrouridine in tRNA. d, Two-dimensional
representation of a generic tRNA with the D-loop nucleotides.
[0025] FIG. 2 depicts the expression of IMS-E21 (URLC8) in lung
tumors and normal tissues.
[0026] a, Expression of IMS-E21 (URLC8) in clinical samples of
NSCLC and corresponding normal lung tissues, examined by
semi-quantitative RT-PCR. b, Expression of IMS-E21 (URLC8) in
lung-cancer cell lines by semi-quantitative RT-PCR. c, Expression
of IMS-E21 (URLC8) in human normal tissues, detected by
multiple-tissue Northern-blot analysis.
[0027] FIG. 3 depicts the results of the immunohistochemical study
of IMS-E21 (URLC8) expression in NSCLC and inhibition of growth of
NSCLC cells by siRNA against IMS-E21 (URLC8).
[0028] a, Immunohistochemical evaluation of representative samples
from surgically-resected NSCLC tissues using anti-IMS-E21 (URLC8)
polyclonal antibody on tissue microarrays. b, Kaplan-Meier analysis
of tumor specific survival in lung SCC patients according to
IMS-E21 (URLC8) expression. c, mRNA knock down effect in response
to si-IMS-E21 (URLC8) or control siRNAs in LC319 cells, (left
panels) analyzed by semi-quantitative RT-PCR (upper panels) and
endogenous IMS-E21 (URLC8) protein knock down effect by western
blotting (middle panels), and (lower panels) colony-formation
assays. (right panels) MTT assays of LC319 cells transfected with
specific siRNAs or control plasmids (EGFP, Scramble, or
Luciferase).
[0029] FIG. 4 depicts the characterization of IMS-E21 (URLC8)
function in NSCLC cells.
[0030] a, Subcellular localization of endogenous IMS-E21 (URLC8) in
LC319 cells; staining is visible mainly in cytoplasm (Rhodamine).
Co-localization of myc-tagged-IMS-E21 (URLC8) (Rhodamine) and
endogenous PDI (Protein Disulfide Isomerase), which was an abundant
protein of endoplasmic reticulum (ER) (FITC) was observed. b,
Identification of EPRS as an IMS-E21 (URLC8) interacting protein.
Reciprocal co-immunoprecipitation of IMS-E21 (URLC8) and EPRS, both
of which had been transfected into LC319 cell extracts.
Western-blot analysis of the cell extracts immunoprecipitated with
anti-FLAG M2 monoclonal antibodies, detected myc-tagged-IMS-E21
(URLC8) protein in the immunoprecipitate. Western-blot of extracts
immunoprecipitated with anti-myc antibodies, detected FLAG-tagged
EPRS protein in the immunoprecipitate. c, Co-localization of
myc-tagged-IMS-E21 (URLC8) and FLAG-tagged EPRS in LC319 cells. d,
Reduction of tRNA dihydrouridine content in A549 and LC319 cells
treated with siRNAs against IMS-E21 (URLC8).
DETAILED DESCRIPTION OF THE INVENTION
Overview:
[0031] Although advances have been made in development of
molecular-targeting drugs for cancer therapy, the ranges of tumor
types that respond as well as the effectiveness of the treatments
are still very limited (Ranson, M., et al. (2002) J Clin Oncol, 20:
2240-2250.; Blackledge, G. and Averbuch, S. (2004) Br J Cancer, 90:
566-572.). Hence, it is urgent to develop new anti-cancer agents
highly specific to malignant cells with minimal or no adverse
reactions. A powerful strategy toward these ends would combine
screening of up-regulated genes in cancer cells on the basis of
genetic information obtained on cDNA microarrays with
high-throughput screening of their effect on cell growth, by
inducing loss-of-function phenotypes with RNAi systems, and with
validation of the potential drug targets by analyzing hundreds of
clinical samples on tissue microarray (Sauter, G., et al. (2003)
Nat Rev Drug Discov, 2: 962-972.; Kononen, J., et al. (1998) Nat
Med, 4: 844-847.). By pursuing such a strategy, the present
inventors have demonstrated herein that IMS-E21 (URLC8) is not only
frequently co-over-expressed in clinical NSCLC samples and cell
lines, but also that the high levels of expression of the gene
products are indispensable for the disease progression as well as
growth of NSCLC cells.
[0032] IMS-E21 (URLC8) is assigned to chromosome 16q22.2 and
encodes a protein of 493 amino acids with 30% homology to S.
cerevisiae Dihydrouridine synthase 1 (DUS1), a member of UPF0034
(unclassified protein family 0034), which catalyses the reduction
of the 5,6-double bond of a uridine residue on tRNA (Xing, F., et
al. (2002) RNA, 8: 370-381.), and with conserved double-strand RNA
binding motif, DSRM. Several studies have revealed that tRNA
modification enhances structural stabilization (Bjork, G. R., et
al., (1987) Annu Rev Biochem, 56: 263-287.). In contrast, much less
attention has been paid to modifications that potentially decrease
regional stability and promote conformational flexibility of
individual nucleotide residues. Dihydrouridine is the single most
common form of post-translational modification in tRNA from
bacteria and eukaryotes (Sprinzl, M., et al., (1998) Nucleic Acids
Res, 26: 148-153.). The widespread presence of 5,6-dihydrouridine
in the D-loop of tRNA has been known for decades, and some DUS
enzymes have been identified in S. cerevisiae and E. coli, but the
DUS enzyme with, double-strand RNA binding motif in mammalian cells
has not been previously identified (Xing, F., et al., (2002) RNA,
8: 370-381.; Kuchino, Y. and Borek, E. (1978) Nature, 271:
126-129.).
[0033] With regards to dihydrouridine in tRNA in tumor cells,
interestingly, an increase in the level of dihydrouridine was
previously reported in tumor-specific tRNA.sup.Phe purified from
human malignant tissues. The role of dihydrouridine in tRNA may
increase conformational flexibility of the tRNA, but its precise
function remains unclear. To elucidate the biological function of
IMS-E21 (URLC8) and its contribution to lung carcinogenesis, the
sub-cellular localization of IMS-E21 (URLC8) protein in LC319 cells
was examined and found to be localized in ER. To clarify whether
native IMS-E21 (URLC8) protein is required for tRNA-DUS activity in
human NSCLC cells, IMS-E21 (URLC8)-siRNA vectors were transfected
to A549 and LC319 cell lines in which the IMS-E21 (URLC8) gene is
highly expressed. In those lung-cancer cells, endogenous IMS-E21
(URLC8) expression was suppressed significantly by siRNA, and
tRNA-DUS activity was reduced, resulted in suppression of growth of
the cancer cells. The results herein suggest that IMS-E21 (URLC8)
is likely to play an important role in synthesis of
tRNA-dihydrouridine of NSCLC cells, being possibly essential for
translation processes and, when over-expressed, for growth and
survival of NSCLC cells. The data herein strongly imply the
possibility of designing new anti-cancer drugs specifically inhibit
IMS-E21 (URLC8). Targeting the IMS-E21 (URLC8) enzyme activity
and/or the IMS-E21 (URLC8)-tRNA synthetase complex may be a
promising therapeutic and diagnostic strategy for treatment of
lung-cancer patients.
URLC8 and Its Functional Equivalents and Uses Thereof:
[0034] Herein, the words "a", "an", and "the" as used herein mean
"at least one" unless otherwise specifically indicated.
[0035] As noted above, the present invention is based in part on
the discovery of a novel t-RNA dihydrouridine-synthase, URLC8,
which is involved in the proliferation of lung cancer cells. The
present invention is also based on the finding that a high
expression level of URLC8 is associated with poor prognosis in lung
squamous-cell carcinoma (SCC) patients. In view of the evidence
provided herein, that URLC8 expression is associated with poor
prognosis of cancer patients, the present invention thus provides
methods for determining a prognosis for cancer patients. An example
of such a method comprises the steps of:
[0036] a. detecting a URLC8 expression level in a specimen
collected from a subject whose SCC prognosis is to be predicted,
and
[0037] b. indicating a poor prognosis when an elevated level of
URLC8 expression is detected.
[0038] In the context of the present invention, when the URLC8
expression level detected in a test specimen is higher than a
control level, then the test specimen is deemed to have an elevated
level of URLC8 expression. An example of a useful control level in
the context of the present invention may comprise a standard value
of URLC8 expression level taken from a group associated with good
prognosis. The standard value may be obtained by any method known
in the art. For example, a range of mean.+-.2 S.D. or mean.+-.3
S.D. may be used as the standard value. Alternatively, poor
prognosis can be determined, when strong staining is observed by
immunohistochemical analysis of sample tissue.
[0039] In the context of the present invention, an expression level
of URLC8 may be detected by any one of the method selected from the
group consisting of:
[0040] (a) detecting an mRNA encoding the amino acid sequence of
SEQ ID NO: 2,
[0041] (b) detecting a protein comprising the amino acid sequence
of SEQ ID NO: 2, and
[0042] (c) detecting the biological activity of a protein
comprising the amino acid sequence of SEQ ID NO: 2.
[0043] In the context of the present invention, the mRNA, the
`protein, or biological activity of the protein may be detected by
any method. Methods for detecting a given protein, mRNA or
biological activity thereof are well known to those skilled in the
art. For example, mRNA may be detected using known PCR or
hybridization based technologies. Alternatively, any immunoassay
format may be applied for detection of a protein. Furthermore, the
biological activity of URLC8, e.g. t-RNA dihydrouridine-synthase,
or interactions between EPRS, may also detected using any suitable
assay method, such as those described herein.
[0044] In the context of the present invention, determination of a
poor prognosis may be used to determine further treatment, e.g., to
stop further treatments that reduce quality of life, to treat the
cancer in a different manner than previously used, or to treat the
cancer more aggressively. In other words, the prediction of
prognosis by URLC8 enables clinicians to choose in advance the most
appropriate treatment for an individual SCC patient without even
the information of conventional clinical staging of the disease,
using only routine procedures for tissue-sampling.
[0045] Further, the methods of the present invention may be used to
assess the efficacy of a course of treatment. For example, in a
mammal with cancer from which a biological sample is found to have
an elevated level of URLC8 expression, the efficacy of an
anti-cancer treatment can be assessed by monitoring the URLC8
expression level over time. For example, a decrease in URLC8
expression level in a biological sample taken from a mammal
following a course of treatment, as compared to a level observed in
a sample taken from the mammal before treatment onset, or earlier
in, the treatment, may be indicative of efficacious treatment.
[0046] As noted above, the present invention also provides kits for
predicting lung squamous-cell carcinoma (SCC) prognosis, comprising
any one component selected from the group consisting of:
[0047] (a) a reagent for detecting an mRNA encoding the amino acid
sequence of SEQ ID NO: 2,
[0048] (b) a reagent for detecting a protein comprising the amino
acid sequence of SEQ ID NO: 2, and
[0049] (c) a reagent for detecting a biological activity of the
protein comprising the amino acid sequence of SEQ ID NO: 2.
[0050] URLC8 has t-RNA dihydrouridine-synthase activity, and its
expression level is markedly elevated in lung cancer cells as
compared to non-lung cancer tissues. Thus, URLC8-mediated t-RNA
dihydrouridine-synthase activity is useful as a diagnostic
parameter of lung cancer, e.g. non-small cell lung cancer.
Accordingly, the present invention provides a method of diagnosing
non-small cell lung cancer or a predisposition to developing
non-small cell lung cancer in a subject, comprising the step of
determining an level of t-RNA dihydrouridine-synthase activity
and/or t-RNA dihydrouridine in a biological sample derived from the
subject, wherein an increase in said level, as compared to a normal
control level, indicates that said subject suffers from or is at
risk of developing non-small cell lung cancer. Accumulation of
t-RNA dihydrouridine in a cell reflects the presence of t-RNA
dihydrouridine-synthase activity. Thus, the cellular t-RNA
dihydrouridine-synthase activity can be evaluated through the
determination of t-RNA dihydrouridine in a cell. The t-RNA
dihydrouridine may be determined by any method known in the art.
For example, t-RNA dihydrouridine can be determined through
colorimetric assay using N-phenyl-p-phenylenediamine and
2,3-butadione monoxime (Jacobson, M. and Hedgcoth, C. (1970) Anal
Biochem, 34: 459-469.). In the present invention any sample derived
from a patient to be diagnosed may be used. An example of a
preferred sample for use in the context of the present invention is
a lung tissue obtained by biopsy or surgical-resection.
[0051] The present invention also provides a kit for detection of
t-RNA dihydrouridine-synthase of URLC8. Examples of components of
such kits include, t-RNA having D-loop as substrate, e.g.,
t-RNA.sup.Phe or other t-RNAs (excluding t-RNA.sup.Tyr and
t-RNA.sup.Glu), an antibody that binds to URLC8, and a detectable
label for detecting the antibody in a cell.
[0052] The URLC8 cDNA consists of 2,020 nucleotides that contain an
open reading frame of 1,479 nucleotides as set forth in SEQ. ID.
NO.: 1. The open reading frame encodes a 493-amino acid protein
having amino acid sequence as set forth in SEQ. ID. NO.: 2. The
amino acid sequence shows a 30% homology to S. cerevisiae Dus1
(dihydrouridine synthase 1), is member of UPF0034 (unclassified
protein family 0034), and contains a conserved DSRM (double-strand
RNA binding motif). S. cerevisiae Dus1 catalyses the reduction of
the 5,6-double bond of a uridine residue on D-loop in tRNA (Xing,
F., et al., (2002) RNA, 8: 370-381.). URLC8 localized mainly in
cytoplasm and co-localized with ER-abundant protein PDI.
[0053] The present invention is also based on the finding that
URLC8 has t-RNA dihydrouridine-synthase activity. To that end, one
aspect of the invention involves identifying test compounds that
regulate URLC8-mediated t-RNA dihydrouridine-synthase activity.
Accordingly, the present invention provides novel methods for
identifying compounds that slow or arrest the progression of, e.g.,
non-small cell lung cancer, by inhibiting URLC8-mediated t-RNA
dihydrouridine-synthase activity.
[0054] The invention thus provides a method of screening for a
compound that modulates URLC8 t-RNA dihydrouridine-synthase
activity. The method is practiced by contacting a URLC8, or a
functional equivalent thereof having t-RNA dihydrouridine-synthase
activity, with one or more candidate compounds, and assaying t-RNA
dihydrouridine-synthase activity of the contacted URLC8 or the
functional equivalent. A compound that modulates t-RNA
dihydrouridine-synthase activity of the URLC8 or functional
equivalent is thereby identified.
[0055] In the context of the present invention, the term
"functionally equivalent" means that the subject protein has t-RNA
dihydrouridine-synthase activity. Whether or not a subject protein
has the target activity can be determined in accordance with the
present invention. For example, t-RNA dihydrouridine-synthase
activity can be determined by incubating a polypeptide under
conditions suitable for synthesis of t-RNA dihydrouridine and
detecting the t-RNA dihydrouridine synthesis level.
[0056] Methods for preparing proteins functionally equivalent to a
given protein are well known to those skilled in the art and
include conventional methods of introducing mutations into the
protein. For example, one skilled in the art can prepare proteins
functionally equivalent to the human URLC8 protein by introducing
an appropriate mutation in the amino acid sequence of the human
URLC8 protein via site-directed mutagenesis (Hashimoto-Gotoh, T. et
al. (1995), Gene 152, 271-275; Zoller, M J, and Smith, M. (1983),
Methods Enzymol. 100, 468-500; Kramer, W. et al. (1984), Nucleic
Acids Res. 12, 9441-9456; Kramer W, and Fritz H J. (1987) Methods.
Enzymol. 154, 350-367; Kunkel, T A (1985), Proc. Natl. Acad. Sci.
USA. 82, 488-492; Kunkel (1991), Methods Enzymol. 204, 125-139).
Amino acid mutations can occur in nature, too. The proteins
suitable for use in the context present invention include those
proteins having the amino acid sequences of the human URLC8 protein
in which one or more amino acids are mutated, provided the
resulting mutated proteins are functionally equivalent to the human
URLC8 protein. The number of amino acids to be mutated in such a
mutant is generally 25 amino acids or less, preferably 10 to 15
amino acids or less, more preferably 5 to 6 amino acids or less,
and even more preferably 2 to 3 amino acids or less. To maintain
t-RNA dihydrouridine-synthase activity, it is preferable to
conserve the DSRM (double-strand RNA binding motif) in the amino
acid sequence of the mutated protein.
[0057] Mutated or modified proteins, proteins having amino acid
sequences modified by deleting, adding and/or replacing one or more
amino acid residues of a certain amino acid sequence, are known to
retain the original biological activity (Mark, D. F. et al., Proc.
Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M. J. &
Smith, M., Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et
al., Science (1984) 224, 1431-1433, Dalbadie-McFarland, G. et al.,
Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
[0058] The amino acid residue to be mutated is preferably mutated
into a different amino acid in which the properties of the amino
acid side-chain are conserved (a process known in the art as
"conservative amino acid substitution"). Examples of properties of
amino acid side chains are hydrophobic amino acids (A, I, L, M, F,
P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S,
T), and side chains having the following functional groups or
characteristics in common: an aliphatic side-chain (G, A, V, L, I,
P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom
containing side-chain (C, M); a carboxylic acid and amide
containing side-chain (D, N, E, Q); a base containing side-chain
(R, K, H); and an aromatic containing side-chain (H, F, Y, W).
Note, the parenthetic letters indicate the one-letter codes of
amino acids.
[0059] An example of a protein to which one or more amino acids
residues are added to the amino acid sequence of human URLC8
protein (SEQ ID NO: 2) is a fusion protein containing the human
URLC8 protein. Fusion proteins suitable for use in the context of
the present invention include, for example, fusions of the human
URLC8 protein and other peptides or proteins. Fusion proteins can
be made using techniques well known to those skilled in the art,
for example by linking the DNA encoding the human URLC8 protein of
the invention with DNA encoding other peptides or proteins, so that
the frames match, inserting the fusion DNA into an expression
vector and expressing it in a host. There is no restriction as to
the peptides or proteins to be fused to the protein of the present
invention.
[0060] Known peptides that can be used as peptides that are fused
to the URLC8 protein include, for example, FLAG (Hopp, T. P. et
al., (1988) Biotechnology 6, 1204-1210), 6.times. His containing
six His (histidine) residues, 10.times. His, Influenza agglutinin
(HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment,
T7-tag, HSV-tag, E-tag, SV4OT antigen fragment, lck tag,
.alpha.-tubulin fragment, B-tag, Protein C fragment, and the like.
Examples of proteins that may be fused to a protein of the
invention include GST (glutathione-S-transferase), Influenza
agglutinin (HA), immunoglobulin constant region,
.beta.-galactosidase, MBP (maltose-binding protein), and such.
[0061] Fusion proteins can be prepared by fusing commercially
available DNA, encoding the fusion peptides or proteins discussed
above, with the DNA encoding a protein of the present invention and
expressing the fused DNA prepared.
[0062] An alternative method known in the art to isolate functional
equivalent proteins is, for example, the method using a
hybridization technique (Sambrook, J. et al., (1989) Molecular
Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press). One
skilled in the art can readily isolate a DNA having high homology
with a whole or part of the URLC8 DNA sequence (e.g., SEQ ID NO: 1)
encoding the human URLC8 protein, and isolate functional equivalent
proteins to the human URLC8 protein from the isolated DNA. The
proteins used for the present invention include those that are
encoded by DNA that hybridize with a whole or part of the DNA
sequence encoding the human URLC8 protein and are functional
equivalent to the human URLC8 protein. These proteins include
mammal homologues corresponding to the protein derived from human
or rat (for example, a protein encoded by a monkey, mouse, rabbit
and bovine gene). In isolating a cDNA highly homologous to the DNA
encoding the human URLC8 protein from animals, it is particularly
preferable to use tissues from testis or lung cancer.
[0063] The conditions of hybridization for isolating a DNA encoding
a protein functionally equivalent to the human URLC8 protein can be
routinely selected by a person skilled in the art. For example,
hybridization may be performed by conducting pre-hybridization at
68.degree. C. for 30 min or longer using "Rapid-hyb buffer"
(Amersham LIFE SCIENCE), adding a labeled probe, and warming at
68.degree. C. for 1 hour or longer. The following washing step can
be conducted, for example, for a low stringency condition.
Exemplary low stringency conditions include, for example,
42.degree. C., 2.times.SSC, 0.1% SDS, or preferably 50.degree. C.,
2.times.SSC, 0.1% SDS. More preferably, high stringency conditions
are selected. Exemplary high stringency conditions include, for
example, washing 3 times in 2.times.SSC, 0.01% SDS at room
temperature for 20 min, then washing 3 times in 1.times.SSC, 0.1%
SDS at 37.degree. C. for 20 min, and washing twice in 1.times.SSC,
0.1% SDS at 50.degree. C. for 20 min. However, several factors,
such as temperature and salt concentration, can influence the
stringency of hybridization. Selection of the factors necessary to
achieve a requisite level of stringency constitutes routine
optimization that is well within the purview of one skilled in the
art.
[0064] In place of hybridization, a gene amplification method, for
example, the polymerase chain reaction (PCR) method, can be
utilized to isolate a DNA encoding a protein functionally
equivalent to the human URLC8 protein, using a primer synthesized
based on the sequence information of the DNA (SEQ ID NO: 1)
encoding the human URLC8 protein (SEQ ID NO: 2).
[0065] Proteins that are functionally equivalent to the human URLC8
protein, encoded by the DNA isolated through the above
hybridization techniques or gene amplification techniques, normally
have a high homology to the amino acid sequence of the human URLC8
protein. In the context of the present invention, the term "high
homology" refers to a homology of 40% or higher, preferably 60% or
higher, more preferably 80% or higher, even more preferably 95% or
higher. The homology of a protein can be determined by following
the algorithm in "Wilbur, W. J. and Lipman, D. J. (1983) Proc.
Natl. Acad. Sci. USA 80, 726-730".
[0066] A protein useful in the context of the present invention may
have variations in amino acid sequence, molecular weight,
isoelectric point, the presence or absence of sugar chains, or
form, depending on the cell or host used to produce it or the
purification method utilized. Nevertheless, so long as it has a
function equivalent to that of a human URLC8 protein (SEQ ID NO:
2), it is useful in the context of the present invention.
[0067] The proteins useful in the context of the present invention
can be prepared as recombinant proteins or natural proteins, by
methods well known to those skilled in the art. A recombinant
protein can be prepared, for example, by inserting a DNA encoding a
protein of the present invention (for example, the DNA comprising
the nucleotide sequence of SEQ ID NO: 1) into an appropriate
expression vector, introducing the vector into an appropriate host
cell, obtaining the extract, and purifying the protein by
subjecting the extract to chromatography, for example, ion exchange
chromatography, reverse phase chromatography, gel filtration, or
affinity chromatography utilizing a column to which antibodies
against the protein of the present invention are fixed, or by
combining more than one of aforementioned columns.
[0068] In addition, when a protein useful in the context of the
present invention is expressed within host cells (for example,
animal cells and E. coli) as a fusion protein with
glutathione-S-transferase protein or as a recombinant protein
supplemented with multiple histidines, the expressed recombinant
protein can be purified using a glutathione column or nickel
column.
[0069] After purifying the fusion protein, it is also possible to
exclude regions, other than the objective protein, by cutting with
thrombin or factor-Xa as required.
[0070] A natural protein can be isolated by methods known to those
skilled in the art, for example, by contacting an affinity column,
in which antibodies binding to the URLC8 protein described below
are bound, with the extract of tissues or cells expressing a
protein of the present invention. The antibodies can be polyclonal
antibodies or monoclonal antibodies.
[0071] In the present invention, t-RNA dihydrouridine-synthase
activity of URLC8 or its functional equivalent can be determined by
methods known in the art. For example, URLC8 and a substrate, e.g.
tRNA having D-loop can be incubated with a hydrogen donor, under
suitable assay conditions for t-RNA dihydrouridine synthesis. In
the present invention, exemplary conditions for the synthesis of
t-RNA dihydrouridine include the steps of contacting the substrate,
hydrogen donor, with t-RNA dihydrouridine-synthase or sample, and
incubating them. A tRNA.sup.Phe and reduced form of nicotinamide
adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide
phosphate (NADPH) are examples of suitable substrates and hydrogen
donors, respectively. Preferably, the substrate or hydrogen atom of
the donor is labeled for tracing t-RNA dihydrouridine.
Specifically, in the context of the present invention, a radio
labeled tRNA is the preferred substrate. The reduced
radio-labeled-tRNA (t-RNA dihydrouridine) may be detected by any
suitable method. Increase of molecular mass of tRNA by addition of
hydrogen atoms can be detected, for example by suitable
chromatography, e.g.
[0072] thin layer chromatography. For example, suitable conditions
for t-RNA dihydrouridine synthesis are set forth below:
[0073] Reaction mixture:
[0074] 100 mM Tris-HCl (pH 8.0),
[0075] 100 mM ammonium acetate,
[0076] 5 mM MgCl.sub.2,
[0077] 2 mM DTT,
[0078] 0.1 mM EDTA,
[0079] 1 mM NADPH,
[0080] 1 mM NADH, and
[0081] 50,000 cpm of labeled transcript of tRNA (6 fmol)
[0082] The reaction mixture is mixed with a sample containing t-RNA
dihydrouridine-synthase to be determined, and incubated for 30 min.
at 30`0 C. In titration assays, extracts and purified proteins are
preferably diluted in buffer containing 50 mM Tris-HCl, pH 8.0, 250
.mu.g/mL bovine serum albumin, and 2 mM DTT. In some assays, 250
.mu.M of flavin adenine dinucleotide (FAD) may also be included.
Following the incubation, RNA is extracted from the mixture, and
treated with P1 nuclease. Then nucleotides are resolved by thin
layer chromatography using either cellulose plates developed in one
dimension with solvent containing ammonium sulfate (74 g/100 mL
H.sub.2O, pH 3.5):H.sub.2O:isopropanol (80:18:2, v/v/v), or using
PEI-cellulose plates developed in two dimensions with 1 M acetic
acid, pH 3.5, for the first dimension, and in buffer containing 74
g ammonium sulfate/100 mL H.sub.2O (adjusted to pH 3.5 with
H.sub.2SO.sub.4) for the second dimension (Bochner & Ames,
(1982) J Biol Chem 257:9759-9769). In titration assays, 1 U of
activity is defined as the amount of protein required to convert
half of the tRNA.
[0083] Alternatively, t-RNA dihydrouridine-synthase activity of
URLC8 can be estimated based on t-RNA dihydrouridine accumulation.
t-RNA dihydrouridine can be detected by a colorimetric method.
Furthermore, t-RNA dihydrouridine-synthase activity of URLC8 can be
determined by consumed NADH and/or NADPH during the reaction.
Suitable methods for determination of NADH and/or NADPH are well
known to a person skilled in the art. Alternatively, following the
reaction, either or both of tRNA and reduced tRNA can be detected
with mass spectrometry, e.g. MALDI-TOF-MS.
[0084] Various low-throughput and high-throughput enzyme assay
formats are known in the art and can be readily adapted for
detection or measuring of the t-RNA dihydrouridine-synthase
activity of URLC8. For high-throughput assays, the tRNA substrate
is preferably immobilized on a solid support, such as a multi-well
plate, slide or chip. Following the reaction, the reduced product
(t-RNA dihydrouridine) can be detected on the solid support.
Alternatively, the t-RNA dihydrouridine-synthase reaction can take
place in solution, after which the t-RNA dihydrouridine can be
immobilized on a solid support, and detected. In order to detect
the reduction of tRNA, for example, H.sup.3 labeled NADH and/or
NADPH may be used as a hydrogen donor. The reduced product (t-RNA
dihydrouridine) can be traced with radioactive H.sup.3. To
facilitate such assays, the solid support may be coated with
streptavidin and the t-RNA labeled with biotin. The skilled person
can determine suitable assay formats depending on the desired
throughput capacity of the screen.
[0085] Any test compound, including, but not limited to, cell
extracts, cell culture supernatant, products of fermenting
microorganisms, extracts from marine organisms, plant extracts,
purified or crude proteins, peptides, non-peptide compounds,
synthetic micromolecular compounds and natural compounds, can be
used in the screening methods of the present invention. The test
compound of the present invention can be also obtained using any of
the numerous approaches in combinatorial library methods known in
the art, including (1) biological libraries, (2) spatially
addressable parallel solid phase or solution phase libraries, (3)
synthetic library methods requiring deconvolution, (4) the
"one-bead, one-compound" library method and (5) synthetic library
methods using affinity chromatography selection. The biological
library methods using affinity chromatography selection are limited
to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145).
Examples of methods for the synthesis of molecular libraries can be
found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA
90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422;
Zuckermann et al. (1994) J. Med. Chem. 37: 2678; Cho et al. (1993)
Science 261: 1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.
33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:
2061; Gallop et al. (1994) J. Med. Chem. 37: 1233). Libraries of
compounds may be presented in solution (see Houghten (1992)
Bio/Techniques 13: 412) or on beads (Lam (1991) Nature 354: 82),
chips (Fodor (1993) Nature 364: 555), bacteria (US Pat. No.
5,223,409), spores (U.S. Pat. No. 5,571,698; 5,403,484, and
5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89: 1865) or phage (Scott and Smith (1990) Science 249: 386; Devlin
(1990) Science 249: 404; Cwirla et al. (1990) Proc. Natl. Acad.
Sci. USA 87: 6378; Felici (1991) J. Mol. Biol. 222: 301; US Pat.
Application 2002103360).
[0086] A compound isolated by the screening method of the present
invention is a candidate for drugs that inhibit the t-RNA
dihydrouridine-synthase activity of URLC8 and can be applied to the
treatment or prevention of NSCLC.
[0087] Moreover, a compound in which a part of the structure of the
compound inhibiting the t-RNA dihydrouridine-synthase activity of
URLC8 is converted by addition, deletion and/or replacement are
also included in the compounds obtainable by the screening method
of the present invention.
Treating and Preventing Lung Cancer:
[0088] The present invention provides compositions for treating or
preventing NSCLC comprising any of the compounds selected by the
screening methods of the present invention.
[0089] When administrating a compound isolated by a method of the
present invention as a pharmaceutical for humans and other mammals,
such as mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs,
cattle, monkeys, baboons, and chimpanzees, the isolated compound
can be directly administered or, alternatively, can be formulated
into a dosage form using conventional pharmaceutical preparation
methods. For example, according to the need, the drugs can be taken
orally, as sugar-coated tablets, capsules, elixirs and
microcapsules, or non-orally, in the form of injections of sterile
solutions or suspensions with water or any other pharmaceutically
acceptable liquid. For example, the compound can be mixed with
pharmaceutically acceptable carriers or media, specifically,
sterilized water, physiological saline, plant-oils, emulsifiers,
suspending agents, surfactants, stabilizers, flavoring agents,
excipients, vehicles, preservatives, binders, and such, in a unit
dose form required for generally accepted drug implementation. The
amount of active ingredients in these preparations makes a suitable
dosage within the indicated range acquirable.
[0090] Examples of additives that can be mixed to form tablets and
capsules include, for example, binders, such as gelatin, corn
starch, tragacanth gum and arabic gum; excipients, such as
crystalline cellulose; swelling agents, such as corn starch,
gelatin and alginic acid; lubricants, such as magnesium stearate;
sweeteners, such as sucrose, lactose or saccharin; and flavoring
agents, such as peppermint, Gaultheria adenothrix oil and cherry.
When the unit-dose form is a capsule, a liquid carrier, such as an
oil, can also be further included in the above ingredients. Sterile
composites for injections can be formulated following normal drug
implementations using vehicles such as distilled water used for
injections.
[0091] Physiological saline, glucose, and other isotonic liquids,
including adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and
sodium chloride, can be used as aqueous solutions for injections.
These can be used in conjunction with suitable solubilizers, such
as alcohol, specifically ethanol, polyalcohols such as propylene
glycol and polyethylene glycol, non-ionic surfactants, such as
Polysorbate 80.TM. and HCO-50.
[0092] Sesame oil and soy-bean oil are examples of suitable
oleaginous liquids and may be used in conjunction with benzyl
benzoate or benzyl alcohol as solubilizers. They may be further
formulated with a buffer, such as phosphate buffer or sodium
acetate buffer; a pain-killer, such as procaine hydrochloride; a
stabilizer, such as benzyl alcohol or phenol; and an anti-oxidant.
The prepared injection may be filled into a suitable ampule.
[0093] Methods well known to those skilled in the art may be used
to administer a pharmaceutical composition of the present invention
to patients, for example, as intra-arterial, intravenous, or
percutaneous injections and also as intranasal, transbronchial,
intramuscular or oral administrations. The dosage and method of
administration may vary according to the body-weight and age of the
patient and the selected administration method; however, one
skilled in the art can routinely select a suitable method of
administration and dosage. If said compound is encodable by a DNA,
the DNA can be inserted into a vector for gene therapy and the
vector can be administered to a patient to perform the therapy. The
dosage and method of administration may again vary according to the
body-weight, age, and symptoms of the patient; however, one skilled
in the art can suitably select them.
[0094] For example, although the dose of a compound that binds to
URLC8 and regulates its activity depends on the symptoms, a
suitable dose is generally about 0.1 mg to about 100 mg per day,
preferably about 1.0 mg to about 50 mg per day and more preferably
about 1.0 mg to about 20 mg per day, when administered orally to a
normal adult (weight 60 kg).
[0095] When administering parenterally, in the form of an injection
to a normal adult (weight 60 kg), although there are some
differences according to the patient, target organ, symptoms and
method of administration, it is convenient to intravenously inject
a dose of about 0.01 mg to about 30 mg per day, preferably about
0.1 to about 20 mg per day and more preferably about 0.1 to about
10 mg per day. Also, in the case of other animals too, it is
possible to administer an amount converted to 60 kg of
body-weight.
[0096] The present invention further provides a method for treating
a NSCLC in a subject. Administration can be prophylactic or
therapeutic to a subject at risk of (or susceptible to) a disorder
or having a disorder associated with aberrant the t-RNA
dihydrouridine-synthase activity of URLC8. The method includes
decreasing the function of URLC8 in a non-small cell lung cancer
(NSCLC) cell. Function can be inhibited through the administration
of a compound obtained by the screening method of the present
invention.
[0097] Also, an siRNA against a URLC8 gene can be used to reduce
the expression level. Herein, term "siRNA" refers to a double
stranded RNA molecule that prevents translation of a target mRNA.
Standard techniques for introducing siRNA into a cell are used,
including those in which DNA is a template from which siRNA is
transcribed. In the context of the present invention, the siRNA
comprises a sense nucleic acid sequence and an anti-sense nucleic
acid sequence against URLC8. The siRNA method of the present
invention can be used to alter the expression in a cell of an
up-regulated NSCLC gene, e.g., up-regulation resulting from the
malignant transformation of the cells. Binding of an siRNA to a
transcript corresponding to URLC8 in a target cell results in a
reduction in the protein production by the cell. The length of the
oligonucleotide is preferably at least 10 nucleotides and may be as
long as the naturally-occurring the transcript. Preferably, the
oligonucleotide is about 19-25 nucleotides in length. More
preferably, the oligonucleotide is less than 75, less than 50, or
less than 25 nucleotides in length. Examples of URLC8 siRNA
oligonucleotides which inhibited the expression in A549 and LC319
cells include the target sequence containing SEQ ID NO: 11.
[0098] An siRNA of the present invention can be constructed such
that a single transcript has both the sense and complementary
antisense sequences from the target gene, e.g., as a hairpin.
[0099] An siRNA of URLC8 hybridizes to a target mRNA and thereby
decreases or inhibits the production of URLC8 polypeptides by
associating with the normally single-stranded mRNA transcript,
thereby interfering with translation and thus, expression of the
protein. In order to enhance the inhibition activity of an siRNA,
nucleotide "u" can be added to 3'end of the antisense strand of the
target sequence. The number of "u"s to be added is at least 2,
generally 2 to 10, preferably 2 to 5. The added "u''s form single
strand at the 3'end of the antisense strand of the siRNA.
[0100] An siRNA of URLC8 can be directly introduced into the cells
in a form that is capable of binding to the mRNA transcripts.
Alternatively, a DNA encoding the siRNA may be carried in a
vector.
[0101] Vectors may be produced, for example, by cloning an URLC8
gene target sequence into an expression vector having
operatively-linked regulatory sequences flanking the sequence in a
manner that allows for expression (by transcription of the DNA
molecule) of both strands (Lee, N. S., et al., (2002) Nature
Biotechnology 20 : 500-505.). An RNA molecule that is antisense to
mRNA of URLC8 is transcribed by a first promoter (e.g., a promoter
sequence 3' of the cloned DNA) and an RNA molecule that is the
sense strand for the mRNA of URLC8 gene is transcribed by a second
promoter (e.g., a promoter sequence 5' of the cloned DNA). The
sense and antisense strands hybridize in vivo to generate siRNA
constructs for silencing of the URLC8 gene. Alternatively, the two
constructs can be utilized to create the sense and anti-sense
strands of an siRNA construct. Cloned URLC8 gene can encode a
construct having secondary structure, e.g., hairpins, wherein a
single transcript has both the sense and complementary antisense
sequences from the target gene.
[0102] A loop sequence consisting of an arbitrary nucleotide
sequence can be located between the sense and antisense sequence in
order to form the hairpin loop structure. Thus, the present
invention also provides siRNA having the general formula
5'-[A]-[B]-[A']-3', wherein [A] is a ribonucleotide sequence
corresponding to a sequence selected from the group consisting of
nucleotides of SEQ ID NO: 11.
[0103] [B] is a ribonucleotide sequence consisting of 3 to 23
nucleotides, and
[0104] [A'] is a ribonucleotide sequence consisting of the
complementary sequence of [A]. The region [A] hybridizes to [A'],
and then a loop consisting of region [B] is formed. The loop
sequence may be preferably 3 to 23 nucleotide in length. The loop
sequence, for example, can be selected from group consisting of
following sequences
(http://www.ambion.com/techlibitb/tb.sub.--506.html). Furthermore,
loop sequence consisting of 23 nucleotides also provides active
siRNA (Jacque, J. M., et al., (2002) Nature 418: 435-438.).
[0105] CCC, CCACC or CCACACC: Jacque, J. M, et al., (2002) Nature
418: 435-438.
[0106] UUCG: Lee, N. S., et al., (2002) Nature Biotechnology 20:
500-505. Fruscoloni, P., et al., (2003) Proc. Natl. Acad. Sci. USA
100: 1639-1644.
[0107] UUCAAGAGA: Dykxhoorn, D. M., (2003) Nature Reviews Molecular
Cell Biology 4: 457-467.
[0108] Examples of preferred siRNAs having hairpin loop structure
of the present invention are shown below. In the following
structure, the loop sequence can be selected from group consisting
of, CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. The preferred loop
sequence is UUCAAGAGA ("ttcaagaga" in DNA). Exemplary hairpin siRNA
suitable for use in the context of the present invention
include:
TABLE-US-00001 ugaggugcucagcacagug-[b]-cacugugcugagcaccuca (for
target sequence of SEQ ID NO: 10)
guuggcacagccuguguau-[b]-auacacaggcugugccaac (for target sequence of
SEQ ID NO: 11)
[0109] The regulatory sequences flanking the URLC8 genes can be
identical or different, such that their expression can be modulated
independently, or in a temporal or spatial manner. siRNAs are
transcribed intracellularly by cloning the URLC8 gene template into
a vector containing, e.g., a RNA polymerase III transcription unit
from the small nuclear RNA (snRNA) U6 or the human III RNA
promoter. For introducing the vector into the cell,
transfection-enhancing agent can be used. FuGENE6 (Roche
diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine
(Invitrogen), and Nucleofector (Wako pure Chemical) are useful as
the transfection-enhancing agent.
[0110] The siRNA of the present invention inhibits the expression
of a polypeptide of the invention and is thereby useful for
suppressing the biological activity of the polypeptide of the
invention. Also, expression-inhibitors, comprising siRNA of the
present invention, are useful in that they can inhibit the
biological activity of a polypeptide of the invention. Therefore, a
composition comprising an antisense oligonucleotide of the present
invention, such as an siRNA, is useful in treating NSCLC.
[0111] Thus, the present invention provides a composition for
treating or preventing non-small cell lung cancer (NSCLC),
comprising a pharmaceutically effective amount of small interfering
RNA (siRNA) against a URLC8 gene, wherein said small interfering
RNA comprises the nucleotide sequence 5'-TGAGGTGCTCAGCACAGTG-3'
(SEQ ID NO.10) and 5'-GTTGGCACAGCCTGTGTAT-3' (SEQ ID NO.11) as the
target sequence. Alternatively, the present invention further
provides a method for treating or preventing NSCLC, said method
comprising the step of administering a pharmaceutically effective
amount of the small interfering RNA.
[0112] Patients with tumors characterized as over-expressing URLC8
may be treated by administering URLC8-siRNA. siRNA therapy is used
to inhibit expression of URLC8 in patients suffering from or at
risk of developing non-small cell lung cancer (NSCLC). Such
patients are identified by standard methods of the particular tumor
type; for example, non-small cell lung cancer (NSCLC) is typically
diagnosed by tomography, ultrasound or biopsy.
[0113] Treatment is efficacious if the treatment leads to a
clinical benefit, such as a reduction in the expression of URLC8,
or a decrease in size, prevalence, or metastatic potential of the
tumor in the subject. When treatment is applied prophylactically,
"efficacious" means that the treatment retards or prevents tumors
from forming or prevents or alleviates a clinical symptom of the
tumor. Efficaciousness may be determined in association with any
known method for diagnosing or treating the particular tumor
type.
[0114] siRNA therapy is carried out by administering to a patient
an siRNA using standard vectors and/or gene delivery systems.
Suitable gene delivery systems may include liposomes,
receptor-mediated delivery systems, and viral vectors, such as
herpes viruses, retroviruses, adenoviruses and adeno-associated
viruses, among others. A reduction in URLC8 production results in a
decrease in URLC8 protein expression. A therapeutic nucleic acid
composition is preferably formulated with a pharmaceutically
acceptable carrier. The therapeutic composition may also include a
gene delivery system as described above. Pharmaceutically
acceptable carriers are biologically compatible vehicles which are
suitable for administration to an animal, e.g., physiological
saline. A therapeutically effective amount of a compound is an
amount which is capable of producing a medically desirable result,
such as reduced production of a URLC8 gene product, reduction of
cell growth, e.g., proliferation, or reduction in tumor growth in a
treated animal.
[0115] Parenteral administration, such as intravenous,
subcutaneous, intramuscular, and intraperitoneal delivery routes,
may be used to deliver URLC8-siRNA compositions.
[0116] Dosages for any one patient depends upon many factors,
including the patient's size, body surface area, age, the
particular nucleic acid to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. Suitable dosage for intravenous administration of
nucleic acids ranges from approximately 10.sup.6 to 10.sup.22
copies of the polynucleotide.
[0117] The polynucleotides of the present invention may be
administered by standard methods, such as by injection into the
interstitial space of tissues such as muscles or skin, introduction
into the circulation or into body cavities or by inhalation or
insufflation. Polynucleotides are preferably injected or otherwise
delivered to the animal in combination with a pharmaceutically
acceptable liquid carrier, e.g., a liquid carrier, which is aqueous
or partly aqueous. The polynucleotides of the present invention may
also beassociated with a liposome (e.g., a cationic or anionic
liposome). The polynucleotides of the present invention preferably
include the genetic information necessary for expression by a
target cell, such as promoters.
[0118] In another aspect, the present invention includes
pharmaceutical, or therapeutic, compositions containing one or more
therapeutic compounds described herein. Pharmaceutical formulations
may include those suitable for oral, rectal, nasal, topical
(including buccal and sub-lingual), vaginal or parenteral
(including intramuscular, subcutaneous and intravenous)
administration, or for administration by inhalation or
insufflation. The formulations may, where appropriate, be
conveniently presented in discrete dosage units and may be prepared
by any of the methods conventional in the art of pharmacy. All such
pharmacy methods include the steps of bringing into association the
active compound with liquid carriers or finely divided solid
carriers or both as needed and then, if necessary, shaping the
product into the desired formulation.
[0119] Pharmaceutical formulations suitable for oral administration
may conveniently be presented as discrete units, such as capsules,
cachets or tablets, each containing a predetermined amount of the
active ingredient; as a powder or granules; or as a solution, a
suspension or as an emulsion. The active ingredient may also be
presented as a bolus electuary or paste, and be in a pure form,
i.e., without a carrier. Tablets and capsules for oral
administration may contain conventional excipients, such as binding
agents, fillers, lubricants, disintegrant or wetting agents. A
tablet may be made by compression or molding, optionally with one
or more formulational ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active
ingredients in a free-flowing form, such as a powder or granules,
optionally mixed with a binder, lubricant, inert diluent,
lubricating, surface active or dispersing agent. Molded tablets may
be made by molding in a suitable machine a mixture of the powdered
compound moistened with an inert liquid diluent. The tablets may be
coated according to methods well known in the art. Oral fluid
preparations may be in the form of, for example, aqueous or oily
suspensions, solutions, emulsions, syrups or elixirs, or may be
presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may contain
conventional additives, .such as suspending agents, emulsifying
agents, non-aqueous vehicles (which may include edible oils), or
preservatives. Furthermore, the tablets may optionally be
formulated so as to provide slow or controlled release of the
active ingredient therein.
[0120] Formulations for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit dose or multi-dose containers, for example,
sealed ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline, water-for-injection,
immediately prior to use. Alternatively, the formulations may be
presented for continuous infusion. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0121] Formulations for rectal administration may be presented as a
suppository with the usual carriers, such as cocoa butter or
polyethylene glycol. Formulations for topical administration in the
mouth, for example, buccally or sublingually, include lozenges,
comprising the active ingredient in a flavored base, such as
sucrose and acacia or tragacanth, and pastilles comprising the
active ingredient in a base, such as gelatin and glycerin or
sucrose and acacia. For intra-nasal administration, the compounds
of the present invention may be used as a liquid spray or
dispersible powder or in the form of drops. Drops may be formulated
with an aqueous or non-aqueous base also comprising one or more
dispersing agents, solubilizing agents or suspending agents. Liquid
sprays are conveniently delivered from pressurized packs.
[0122] For administration by inhalation, the compounds of the
present invention are conveniently delivered from an insufflator,
nebulizer, pressurized pack or other convenient aerosol spray
delivery means. Pressurized packs may comprise a suitable
propellant, such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount.
[0123] Alternatively, for administration by inhalation or
insufflation, the compounds of the present invention may take the
form of a dry powder composition, for example a powder mix of the
compound and a suitable powder base, such as lactose or starch. The
powder composition may be presented in a unit dosage form, in for
example, capsules, cartridges, gelatin or blister packs from which
the powder may be administered with the aid of an inhalator or
insufflators.
[0124] When desired, the above-described formulations, adapted to
give sustained release of the active ingredient, may be employed.
The pharmaceutical compositions of the present invention may also
contain other active ingredients, such as antimicrobial agents,
immunosuppressants or preservatives.
[0125] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of the present
invention may include other agents conventional in the art, having
regard to the type of formulation in question; for example, those
suitable for oral administration may include flavoring agents.
[0126] Preferred unit dosage formulations are those containing an
effective dose, as recited below, or an appropriate fraction
thereof, of the active ingredient.
[0127] For each of the aforementioned conditions, the compositions
may be administered orally or via injection at a dose ranging from
about 0.1 to about 250 mg/kg per day. The dose range for adult
humans is generally from about 5 mg to about 17.5 g/day, preferably
about 5 mg to about 10 g/day, and most preferably about 100 mg to
about 3 g/day. Tablets or other unit dosage forms of presentation
provided in discrete units may conveniently contain an amount which
is effective at such dosage or as a multiple of the same, for
instance, units containing about 5 mg to about 500 mg, usually from
about 100 mg to about 500 mg.
[0128] The pharmaceutical composition preferably is administered
orally or by injection (intravenous or subcutaneous), and the
precise amount administered to a subject will be the responsibility
of the attendant physician. However, the dose employed will depend
upon a number of factors, including the age and sex of the subject,
the precise disorder being treated, and its severity. In addition,
the route of administration may vary depending upon the condition
and its severity.
Examples
Example 1
Materials and Methods
(a) Lung Cancer Cell Lines and Tissue Samples.
[0129] The 19 human NSCLC and 4 SCLC cell lines used in this study
were as follows: lung ADC A427, A549, LC319, PC3, PC9, PC14, and
NCI-H1373; bronchioloalveolar cell carcinomas (BAC) NCI-H1666 and
NCI-H1781; lung adenosquamous carcinoma (ASC) NCI-H226 and
NCI-H647; lung SCC RERF-LC-AI, SK-MES-1, EBC-1, LU61, NCI-H520,
NCI-H1703, and NCI-H2170; a lung large-cell carcinoma (LCC) LX1;
and SCLC DMS114, DMS273, SBC-3, and SBC-5. All cells were grown in
monolayers in appropriate medium supplemented with 10% fetal calf
serum (FCS) and were maintained at 37.degree. C. in an atmosphere
of humidified air with 5% CO2. Human small airway epithelial cells,
SAEC were grown in optimized medium (SAGM) purchased from Cambrex
Bio Science Inc. (Walkersville, Md.).
[0130] Primary NSCLC samples, of which 22 were classified as ADCs,
14 as SCCs, and one as ASC, were originally obtained from 37
patients, with written informed consent, for a study described
elsewhere (Kikuchi, T. et al. (2003) Oncogene, 22: 2192-2205.). An
independent set of fourteen additional primary NSCLCs, including
seven ADCs and seven SCCs, were obtained along with adjacent normal
lung tissue samples from patients undergoing surgery.
[0131] A total of 292 NSCLC and adjacent normal lung tissue samples
used for immunostaining on tissue microarray and additional
statistical analysis were also obtained from patients who underwent
surgery.
(b) Semi-Quantitative RT-PCR Analysis.
[0132] Total RNA was extracted from cultured cells and clinical
tissues using the Trizol reagent (Life Technologies, Inc.)
according to the manufacturer's protocol. Extracted RNAs and normal
human tissue poly(A) RNAs were treated with DNase I (Nippon Gene)
and were reverse-transcribed using oligo (dT) primer and
SuperScript II reverse transcriptase (Invitrogen).
Semi-quantitative RT-PCR experiments were carried out with the
following synthesized gene-specific primers or with ACTB-specific
primers as an internal control:
(c) IMS-E21 (URLC8).
[0133] The following primers were used to isolated IMS-E21
(URLC8):
TABLE-US-00002 5'-GACCACATCCAACAGTATTCG-3' (SEQ ID NO. 3) and
5'-TGCCAGGACATCTAACTTCTG-3'; (SEQ ID NO. 4) ACTB,
5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO. 5) and
5'-CAAGTCAGTGTACAGGTAAGC-3'. (SEQ ID NO. 6)
[0134] PCR reactions were optimized for the number of cycles to
ensure product intensity within the logarithmic phase of
amplification.
(d) Northern-Blot Analysis.
[0135] Human multiple-tissue blots (BD Biosciences Clontech) were
hybridized with a .sup.32P-labeled PCR product of IMS-E21 (URLC8).
The cDNA probes of IMS-E21 (URLC8) were prepared by RT-PCR using
primers with the same above. Pre-hybridization, hybridization, and
washing were performed according to the supplier's recommendations.
The blots were autoradiographed with intensifying BAS screens
(BIO-RAD, Hercules, Calif.) at room temperature for 30 hours.
(e) Cloning of a Full-Length IMS-E21 (URLC8) cDNA and DNA
Sequencing.
[0136] Firstly, the IMS-E21 (URLC8) sequence was searched against
the EST database by BLAST program and found to be highly homologous
to an EST clone (FLJ20399, NM.sub.--017803). cDNA was generated
from total RNA derived from 12 normal organ mixed sample by random
priming using SuperScript II reverse transcriptase (Invitrogen)
according to the manufacturer's instructions. RT-PCR product was
cloned into the pcDNA3.1-myc-His vector (Invitrogen) and confirmed
by sequencing.
(f) Generation of Anti-IMS-E21 (URLC8) Antibodies.
[0137] Plasmids expressing IMS-E21 (URLC8) (full length) that
contain His-tagged epitope at the NH.sub.2-terminal of the
individual proteins were prepared using pET28 vector (Novagen,
Madison, Wis.). The recombinant protein was expressed in
Escherichia coli, BL21 codon-plus strain (Stratagene, LaJolla,
Calif.), and purified using TALON resin (BD Bioscience) according
to the supplier's protocol. The protein extracted in SDS-PAGE gel
was inoculated into rabbits, and the immune sera were purified on
affinity columns according to the standard methodology.
Affinity-purified anti-IMS-E21 (URLC8) antibodies were used for
western-blot analysis, immunoprecipitation, and immunostaining.
(g) Co-Immunoprecipitation and MALDI-TOF-MS Mapping of IMS-E21
(URLC8)-Associated Proteins.
[0138] Cell extracts from lung-cancer cell line LC319 transfected
with the plasmids expressing N-terminal FLAG-tagged and C-terminal
HA-tagged-pCAGGS-IMS-E21 (URLC8) vector or mock vector (control)
were pre-cleared by incubation at 4.degree. C. for 1 hour with 100
.mu.L of protein G-agarose beads in a final volume of 2 ml of
IP-buffer (0.5% NP-40, 50 mM Tris-HCl, 150 mM NaCl) in the presence
of proteinase inhibitor. After centrifugation at 1000 rpm for 5 min
at 4.degree. C., the supernatant was incubated at 4.degree. C. with
anti-Flag M2 agarose for 2 hours. The beads were then collected by
centrifugation at 5000 rpm for 2 min and washed six times with 1 ml
of each immunoprecipitation buffer. Then, the washed beads were
eluted by Flag-peptide (Sigma-Aldrich Co., St. Louis, Mo.). The
elution was incubated at 4.degree. C. with anti-HA agarose for 2
hours. The washed beads were resuspended in 50 .mu.L of Laemmli
sample buffer and boiled for 5 min, and the proteins were separated
by 5-10% SDS PAGE gels (BIO RAD). After electrophoresis, the gels
were stained with silver. Protein bands specifically found in
IMS-E21 (URLC8)-transfected extracts were excised and served for
matrix-assisted laser desorption/ionization-time of flight mass
spectrometry (MALDI-TOF-MS) analysis (AXIMA-CFR plus, SHIMADZU
BIOTECH, Kyoto, Japan).
(h) RNA Interference Assay.
[0139] A vector-based RNA interference (RNAi) system, psiH1BX3.0,
was previously established to direct the synthesis of siRNAs in
mammalian cells (Suzuki, C. et al. (2003) Cancer Res, 63:
7038-7041.). Herein, 10 .mu.g of siRNA-expression vector were
transfected, using 30 .mu.l of Lipofectamine 2000 (Invitrogen),
into NSCLC cell lines A549 and LC319, both of which over-expressed
IMS-E21 (URLC8) endogenously. More than 80% of the transfected
cells expressed the synthetic siRNA, and in those cells endogenous
expression of the IMS-E21 (URLC8) genes was effectively suppressed.
The transfected cells were cultured for seven days in the presence
of appropriate concentrations of geneticin (G418), after which cell
numbers and viability were measured by Giemsa staining and
triplicate MTT assays. The target sequences of the synthetic
oligonucleotides for RNAi were as follows:
TABLE-US-00003 control 1 (EGFP: enhanced green fluorescent protein
(GFP) gene, a mutant of Aequorea victoria GFP),
5'-GAAGCAGCACGACTTCTTC-3'; (SEQ ID NO. 7) control 2 (Luciferase:
Photinus pyralis luciferase gene), 5'-CGTACGCGGAATACTTCGA-3'; (SEQ
ID NO. 8) control 3 (Scramble: chloroplast Euglena gracilis gene
coding for 5S and 16S rRNAs), 5'-GCGCGCTTTGTAGGATTCG-3'; (SEQ ID
NO. 9) siRNA-IMS-E21-#2 (si-IMS-E21-#2), 5'-TGAGGTGCTCAGCACAGTG-3';
(SEQ ID NO. 10) siRNA-IMS-E21-#3 (si-IMS-E21-#3),
5'-GTTGGCACAGCCTGTGTAT-3'. (SEQ ID NO. 11)
[0140] The insert sequences of siRNA expression vectors were as
follows:
TABLE-US-00004 control 1 (EGFP); (SEQ ID NO. 12)
5'-TCCCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGC TGCTTC-3'; (SEQ ID
NO. 13) 5'-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGC TGCTTC-3';
control 2 (Luciferase); (SEQ ID NO. 15)
5'-TCCCCGTACGCGGAATACTTCGATTCAAGAGATCGAAGTATTCCG CGTACG-3'; (SEQ ID
NO. 16) 5'-AAAACGTACGCGGAATACTTCGATCTCTTGAAATCGAAGTATTCC
GCGTACG-3'; control 3 (Scramble); (SEQ ID NO. 18)
5'-TCCCGCGCGCTTTGTAGGATTCGTTCAAGAGACGAATCCTACAAA GCGCGC-3'; (SEQ ID
NO. 19) 5'-AAAAGCGCGCTTTGTAGGATTCGTCTCTTGAACGAATCCTACAAA GCGCGC-3';
siRNA-IMS-E21-#2 (si-IMS-E2-#2); (SEQ ID NO. 21)
5'-TCCCTGAGGTGCTCAGCACAGTGTTCAAGAGACACTGTGCTGAGC ACCTCA-3'; (SEQ ID
NO. 22) 5'-AAAATGAGGTGCTCAGCACAGTGTCTCTTGAACACTGTGCTGAGC ACCTCA-3';
siRNA-IMS-E21-#3 (si-IMS-E21-#3); (SEQ ID NO. 24)
5'-TCCCGTTGGCACAGCCTGTGTATTCAAGAGAATACACAGGCTGTG CCAAC-3'; (SEQ ID
NO. 25) 5'-AAAAGTTGGCACAGCCTGTGTATCTCTTGAAATACACAGGCTGTG
CCAAC-3';
(i) Immunohistochemistry and Tissue Microarray Analysis.
[0141] The tumor tissue microarrays using formalin-fixed NSCLCs
were constructed as published elsewhere. The tissue area for
sampling was selected based on a visual alignment with the
corresponding HE-stained section on a slide. Three, four, or five
tissue cores (diameter 0.6 mm; height 3-4 mm) taken from the donor
tumor blocks were placed into a recipient paraffin block using a
tissue microarrayer (Beecher Instruments, Sun Prairie, Wis.). A
core of normal tissue was punched from each case. 5-.mu.m sections
of the resulting microarray block were used for immunohistochemical
analysis. IMS-E21 (URLC8) positivity were assessed
semi-quantitatively, recording staining intensity as absent (scored
as 0), weak (scored as 1+) or strongly positive (scored as 2+), by
three independent investigators without prior knowledge of the
clinical follow-up data. Cases were accepted only as positive if
reviewers independently defined them thus.
[0142] To investigate the presence of IMS-E21 (URLC8) protein in
clinical tissue samples, the sections were stained using ENVISION+
Kit/HRP (DakoCytomation). Affinity-purified anti-IMS-E21 (URLC8)
antibody was added after blocking endogenous peroxidase and
proteins, and the sections were incubated with HRP-labeled
anti-rabbit IgG as the secondary antibody. Substrate-chromogen was
added and the specimens were counterstained with hematoxylin.
Example 2
Cloning of IMS-E21 (URLC8) Gene and Its Expression in Lung Tumors,
Cell Lines, and Normal Tissues
[0143] To obtain novel target molecules for development of
therapeutic agents and/or diagnostic markers for NSCLC, genes that
showed 5-fold higher expression in more than 50% of 37 NSCLCs
analyzed by cDNA microarray (Kikuchi, et.al. (2003) Oncogene, 22:
2192-2205.) were screened. Among 23,040 genes screened, one EST
transcript (FLJ20399, NM.sub.--017803) was identified as frequently
over-expressed in NSCLCs; its overexpression was confirmed--in
eight representative cases by semi-quantitative RT-PCR experiments.
To obtain a full-length clone of this transcript, the FASTA
database was screened using the cDNA fragment originally isolated
by cDNA microarray experiment and identified several ESTs.
Assembling DNA sequences of these clones, the entire coding
nucleotide sequence of this gene was determined. The cDNA consisted
of 2,020 nucleotides, including an open reading frame of 1,479
nucleotides that encoded a 493 amino acid peptide. A homology
search using the FASTA program revealed that this predicted protein
was homologous to proteins belonging to the family of
tRNA-dihydrouridine synthase (DUS), especially S. cerevisiae Dus1
(dihydrouridine synthase 1) which catalyses the reduction of the
5,6-double bond of a uridine residue on tRNA (30% identity in amino
acids, respectively; FIG. 1, a-c). Hence, this gene was tentatively
designated IMS-E21 (also referred to as URLC8: up-regulated in lung
cancer 8, Accession No. AB101210). Two motifs were identified using
SMART program (http://smart.embl-heidelberg.de/) (Letunic I, et
al., (2004) Nucleic Acids Res.;32(Database issue):D142-4; Schultz
J, et al., (1998) Proc Natl Acad Sci USA.;95:5857-64.) within the
sequence of IMS-E21 (URLC8); N-terminal Dus domain and C-terminal
DSRM, respectively (FIG. 1, a). The human IMS-E21 (URLC8) appeared
to conserve 90% identity in amino acids with the murine (M.
musculus and R. Norvegicus) products, 48% with the D. melanogaster
protein, and 40% with C. elegans protein (FIG. 1, b).
[0144] Increased IMS-E21 (URLC8) expression was further confirmed
in 11 of 14 additional NSCLC cases (4 of 7 adenocarcinomas (ADCs);
7 of 7 squamous-cell carcinomas (SCCs) (FIG. 2, a), and documented
up-regulation of IMS-E21 (URLC8) in 21 of the 23 NSCLC and
small-cell lung cancer (SCLC) cell lines examined, but no
expression was found in SAEC cells derived from normal bronchial
epithelium (FIG. 2, b).
[0145] Northern blotting with IMS-E21 (URLC8) cDNA as a probe
identified a 2.4-kb transcript as a band, mainly seen in testis,
among the 23 normal human tissues examined (FIG. 2, c).
Example 3
Overexpression of IMS-E21 (URLC8) Protein is Associated with a
Worse Outcome in SCC
[0146] Immunohistochemical analysis was performed with
affinity-purified anti-IMS-E21 (URLC8) polyclonal antibodies in
tissue microarrays of available 292 NSCLCs. The study showed that
the number of cases that showed positive staining of IMS-E21
(URLC8) in the cytoplasm was 254 of 292 (87%) NSCLC total cases;
132 of 158 (84%) ADC cases, 89 of 99 (90%) SCC cases, 19 of 21
(90%) LCC cases, 10 of 10 (100%) BAC cases, and 4 of 4 (100%) ASC
cases, respectively. All of those tumors were surgically-resectable
NSCLCs, and no staining was observed in any of their adjacent
normal lung tissues (FIG. 3, a). A pattern of IMS-E21 (URLC8)
expression was classified on the tissue array, ranging from
absent/weak (scored as 0.about.1+) to strong (scored as 2+). Next,
the association of IMS-E21 (URLC8) expression with clinical outcome
was examined. Statistical analysis revealed no significant
correlation of any levels of IMS-E21 (URLC8) expression with pT- or
pN-factors among the lung-cancer patients examined. However, the
expression of IMS-E21 (URLC8) in SCC was found to be significantly
associated with tumor specific 5 year-survival using Kaplan-Meier
method (P=0.0091 by the Log-rank test) (FIG. 3, b). By univariate
analysis pT, pN, gender, and IMS-E21 (URLC8) expression (P=0.0111)
were each significantly related to a poor tumor-specific survival
among SCC patients. Furthermore, IMS-E21 (URLC8) staining was
determined to be an independent prognostic factors by multivariate
analysis using Cox proportional hazard model of SCC patients
(P=0.0234).
Example 4
Inhibition of Growth of NSCLC Cells by Specific siRNA Against
IMS-E21 (URLC8)
[0147] To assess whether IMS-E21 (URLC8) is essential for growth or
survival of lung-cancer cells, plasmids were designed and
constructed to express siRNA against IMS-E21 (URLC8) (si-IMS-E21)
and control plasmids (siRNAs for EGFP, Scramble, and Luciferase)
and transfected them into A549 and LC319 cells to suppress
expression of endogenous IMS-E21 (URLC8). The amount of IMS-E21
(URLC8) transcript and protein level in the cells transfected with
si-IMS-E21-#2, and -#3 were significantly decreased in comparison
with cells transfected with the control (FIG. 3, c, left panels);
transfection of si-IMS-E21-#2, and -#3 also resulted in significant
decreases of colony number in colony-formation assays (FIG. 3, c,
lower panels) and colony numbers measured by MTT cell viability
assay (FIG. 3, c, right panel).
Example 5
IMS-E21 (URLC8) Localized Mainly in Cytoplasm and Co-Localized with
ER-Abundant Protein PDI
[0148] To determine the subcellular localization of endogenous
IMS-E21 (URLC8) in lung cancer-cells, immunocytochemistry was
performed using affinity-purified anti-IMS-E21 (URLC8) polyclonal
antibodies. Confocal microscopy revealed the distribution of the
IMS-E21 (URLC8) protein in the cytoplasm of A549 cells (data not
shown). To further investigate the sub-cellular localization of
IMS-E21 (URLC8) proteins, we transfected LC319 cells with plasmids
that contained c-myc-His-epitope sequences (LDEESILKQE-HHHHHH) at
the C-terminal of the human IMS-E21 (URLC8) protein.
Co-immunostaining using anti-c-myc antibodies and antibodies
against endogenous Protein Disulfide Isomerase (PDI), which are
abundantly expressed in endoplasmic reticulum (ER) suggested that
IMS-E21 (URLC8) protein mainly distribute at ER (FIG. 4, a).
Example 6
Identification of EPRS as a Protein Interacting with IMS-E21
(URLC8)
[0149] To elucidate the function of IMS-E21 (URLC8) in lung-cancer
cells, protein(s) interacting with IMS-E21 (URLC8) were identified.
Lysate of LC319 cells transfected with N-terminal FLAG-tagged and
C-terminal HA-tagged-pCAGGS-IMS-E21 vector or mock vector (control)
were extracted and immunoprecipitated with anti-FLAG M2 monoclonal
antibody followed by immunoprecipitation with anti-HA monoclonal
antibody. The protein complex including IMS-E21 (URLC8) was stained
with SilverQuest (Invitrogen) on the SDS-PAGE gel. A 180-kDa band,
which was seen in immunoprecipitates of cell lysates transfected
with IMS-E21 (URLC8) expressing plasmids, but not seen in those
with mock plasmids was extracted and determined to be EPRS
(glutamyl-prolyl tRNA synthetase), by MALDI-TOF mass spectrometric
sequencing (data not shown). The interaction between endogenous
IMS-E21 (URLC8) and EPRS was confirmed by co-immunoprecipitation
and immunocytochemistry (FIG. 4, b and c), suggesting the
possibility of existence of IMS-E21 (URLC8)-aminoacyl tRNA
synthetase complex in an ER-dependent translation.
Example 7
tRNA-DUS Activity of IMS-E21 (URLC8) in Lung Cancer Cells
[0150] To test the hypothesis that IMS-E21 (URLC8) encodes a family
of dihydrouridine synthase enzyme, the effects of suppression of
IMS-E21 (URLC8) gene by siRNA on DUS activity in NSCLC cell line
were investigated. Total tRNA was purified from total RNA of A549
and LC319 cells transfected with siRNA constructs by HPLC technique
and analyzed the samples for dihydrouridine content of each cell
line (Jacobson, M. and Hedgcoth, C. (1970) Anal Biochem, 34:
459-469. Hunninghake, D. and Grisolia, S. (1966) Anal Biochem, 16:
200-205.). Most effective IMS-E21 (URLC8) siRNA constructs
(si-IMS-E21-#2), which could suppress transcript levels of IMS-E21
(URLC8) as stated above, also lead to reduce levels of
tRNA-dihydrouridine content when compared with their corresponding
control siRNA construct (si-EGFP) (FIG. 4, d). The deficiencies of
tRNA-dihydrouridine induced by si-IMS-E21-#2 in these tumor cells
were closely related to the tumor growth suppression (FIG. 3, c),
implicating that tRNA-DUS activity of IMS-E21 (URLC8) is
indispensable for tumor cell survival and/or progression.
INDUSTRIAL APPLICABILITY
[0151] The present inventors have shown that URLC8 has t-RNA
dihydrouridine-synthase activity, and the suppression of the
activity leads to the inhibition of cell proliferation of lung
cancer cells. Thus, agents that inhibit the t-RNA
dihydrouridine-synthase activity of URLC8 find therapeutic utility
as anti-cancer agents for the treatment of lung cancer, such as
NSCLC.
[0152] In addition, treatment of NSCLC cells with siRNA against
URLC8 suppressed its expression and also the activity of tRNA-DUS,
and suppressed growth of the cancer cells. Additional experiments
revealed that the URLC8 protein physically interacts with EPRS
(glutamyl-prolyl tRNA synthetase) in NSCLC cells. These data imply
that up-regulation of URLC8 function and increased tRNA-DUS
activity are common features of pulmonary carcinogenesis.
Accordingly, the selective suppression of URLC8 enzyme activity
and/or the formation of the URLC8-tRNA synthetase complex may be a
promising therapeutic strategy for treatment of lung-cancer
patients.
[0153] Alternatively, lung cancer can be detected using t-RNA
dihydrouridine-synthase activity of URLC8 as a diagnostic
index.
[0154] Furthermore, the present inventors revealed that a high
level of URLC8 (IMS-E21) expression was significantly associated
with poor prognosis for patients with lung squamous-cell carcinoma
(SCC). Accordingly, a prognosis of lung cancer can be predicted by
measurement of URLC8 (IMS-E21) expression level.
[0155] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety.
Furthermore, while the invention has been described in detail and
with reference to specific embodiments thereof, it will be apparent
to one skilled in the art that various changes and modifications
can be made therein without departing from the spirit and scope of
the invention.
Sequence CWU 1
1
3312020DNAHomo sapiensCDS(171)...(1652) 1gctcagtacg gtgtgtggag
ctggagcacc gtgaggaaga agcgaggttc tttttaagag 60ttcagctgcg agatatcaaa
caaagaatta ctctgtacaa agccagaaca catatatcaa 120agtaatcctg
aagtatcaga acaaaataat aggctgtaac agaggaggaa atg att 176 Met Ile
1ttg aat agc ctc tct ctg tgt tac cat aat aag cta atc ctg gcc cca
224Leu Asn Ser Leu Ser Leu Cys Tyr His Asn Lys Leu Ile Leu Ala Pro
5 10 15atg gtt cgg gta ggg act ctt cca atg agg ctg ctg gcc ctg gat
tat 272Met Val Arg Val Gly Thr Leu Pro Met Arg Leu Leu Ala Leu Asp
Tyr 20 25 30gga gcg gac att gtt tac tgt gag gag ctg atc gac ctc aag
atg att 320Gly Ala Asp Ile Val Tyr Cys Glu Glu Leu Ile Asp Leu Lys
Met Ile35 40 45 50cag tgc aag aga gtt gtt aat gag gtg ctc agc aca
gtg gac ttt gtc 368Gln Cys Lys Arg Val Val Asn Glu Val Leu Ser Thr
Val Asp Phe Val 55 60 65gcc cct gat gat cga gtt gtc ttc cgc acc tgt
gaa aga gag cag aac 416Ala Pro Asp Asp Arg Val Val Phe Arg Thr Cys
Glu Arg Glu Gln Asn 70 75 80agg gtg gtc ttc cag atg ggg act tca gac
gca gag cga gcc ctt gct 464Arg Val Val Phe Gln Met Gly Thr Ser Asp
Ala Glu Arg Ala Leu Ala 85 90 95gtg gcc agg ctt gta gaa aat gat gtg
gct ggt att gat gtc aac atg 512Val Ala Arg Leu Val Glu Asn Asp Val
Ala Gly Ile Asp Val Asn Met 100 105 110ggc tgt cca aaa caa tat tcc
acc aag gga gga atg gga gct gcc ctg 560Gly Cys Pro Lys Gln Tyr Ser
Thr Lys Gly Gly Met Gly Ala Ala Leu115 120 125 130ctg tca gac cct
gac aag att gag aag atc ctc agc act ctt gtt aaa 608Leu Ser Asp Pro
Asp Lys Ile Glu Lys Ile Leu Ser Thr Leu Val Lys 135 140 145ggg aca
cgc aga cct gtg acc tgc aag att cgc atc ctg cca tcg cta 656Gly Thr
Arg Arg Pro Val Thr Cys Lys Ile Arg Ile Leu Pro Ser Leu 150 155
160gaa gat acc ctg agc ctt gtg aag cgg ata gag agg act ggc att gct
704Glu Asp Thr Leu Ser Leu Val Lys Arg Ile Glu Arg Thr Gly Ile Ala
165 170 175gcc atc gca gtt cat ggg agg aag cgg gag gag cga cct cag
cat cct 752Ala Ile Ala Val His Gly Arg Lys Arg Glu Glu Arg Pro Gln
His Pro 180 185 190gtc agc tgt gaa gtc atc aaa gcc att gct gat acc
ctc tcc att cct 800Val Ser Cys Glu Val Ile Lys Ala Ile Ala Asp Thr
Leu Ser Ile Pro195 200 205 210gtc ata gcc aac gga gga tct cat gac
cac atc caa cag tat tcg gac 848Val Ile Ala Asn Gly Gly Ser His Asp
His Ile Gln Gln Tyr Ser Asp 215 220 225ata gag gac ttt cga caa gcc
acg gca gcc tct tcc gtg atg gtg gcc 896Ile Glu Asp Phe Arg Gln Ala
Thr Ala Ala Ser Ser Val Met Val Ala 230 235 240cga gca gcc atg tgg
aac cca tct atc ttc ctc aag gag ggt ctg cgg 944Arg Ala Ala Met Trp
Asn Pro Ser Ile Phe Leu Lys Glu Gly Leu Arg 245 250 255ccc ctg gag
gag gtc atg cag aaa tac atc aga tac gcg gtg cag tat 992Pro Leu Glu
Glu Val Met Gln Lys Tyr Ile Arg Tyr Ala Val Gln Tyr 260 265 270gac
aac cac tac acc aac acc aag tac tgc ttg tgc cag atg cta cga 1040Asp
Asn His Tyr Thr Asn Thr Lys Tyr Cys Leu Cys Gln Met Leu Arg275 280
285 290gaa cag ctg gag tcg ccc cag gga agg ttg ctc cat gct gcc cag
tct 1088Glu Gln Leu Glu Ser Pro Gln Gly Arg Leu Leu His Ala Ala Gln
Ser 295 300 305tcc cgg gaa att tgt gag gcc ttt ggc ctt ggt gcc ttc
tat gag gag 1136Ser Arg Glu Ile Cys Glu Ala Phe Gly Leu Gly Ala Phe
Tyr Glu Glu 310 315 320acc aca cag gag ctg gat gcc cag cag gcc agg
ctc tca gcc aag act 1184Thr Thr Gln Glu Leu Asp Ala Gln Gln Ala Arg
Leu Ser Ala Lys Thr 325 330 335tca gag cag aca ggg gag cca gct gaa
gat acc tct ggt gtc att aag 1232Ser Glu Gln Thr Gly Glu Pro Ala Glu
Asp Thr Ser Gly Val Ile Lys 340 345 350atg gct gtc aag ttt gac cgg
aga gca tac cca gcc cag atc acc cct 1280Met Ala Val Lys Phe Asp Arg
Arg Ala Tyr Pro Ala Gln Ile Thr Pro355 360 365 370aag atg tgc cta
cta gag tgg tgc cgg agg gag aag ttg gca cag cct 1328Lys Met Cys Leu
Leu Glu Trp Cys Arg Arg Glu Lys Leu Ala Gln Pro 375 380 385gtg tat
gaa acg gtt caa cgc cct cta gat cgc ctg ttc tcc tct att 1376Val Tyr
Glu Thr Val Gln Arg Pro Leu Asp Arg Leu Phe Ser Ser Ile 390 395
400gtc acc gtt gct gaa caa aag tat cag tct acc ttg tgg gac aag tcc
1424Val Thr Val Ala Glu Gln Lys Tyr Gln Ser Thr Leu Trp Asp Lys Ser
405 410 415aag aaa ctg gcg gag cag gct gca gcc atc gtc tgt ctg cgg
agc cag 1472Lys Lys Leu Ala Glu Gln Ala Ala Ala Ile Val Cys Leu Arg
Ser Gln 420 425 430ggc ctc cct gag ggt cgg ctg ggt gag gag agc cct
tcc ttg cac aag 1520Gly Leu Pro Glu Gly Arg Leu Gly Glu Glu Ser Pro
Ser Leu His Lys435 440 445 450cga aag agg gag gct cct gac caa gac
cct ggg ggc ccc aga gct cag 1568Arg Lys Arg Glu Ala Pro Asp Gln Asp
Pro Gly Gly Pro Arg Ala Gln 455 460 465gag cta gca caa cct ggg gat
ctg tgc aag aag ccc ttt gtg gcc ttg 1616Glu Leu Ala Gln Pro Gly Asp
Leu Cys Lys Lys Pro Phe Val Ala Leu 470 475 480gga agt ggt gaa gaa
agc ccc ctg gaa ggc tgg tga ctactcttcc 1662Gly Ser Gly Glu Glu Ser
Pro Leu Glu Gly Trp 485 490tgccttagtc acccctccat gggcctggtg
ctaaggtggc tgtggatgcc acagcatgaa 1722ccagatgccg ttgaacagtt
tgctggtctt gcctggcaga agttagatgt cctggcaggg 1782gccatcagcc
tagagcatgg accaggggcc gcccaggggt ggatcctggc ccctttggtg
1842gatctgagtg acagggtcaa gttctctttg aaaacaggag cttttcaggt
ggtaactccc 1902caacctgaca ttggtactgt gcaataaaga caccccctac
cctcaaaaaa aaaaaaaaaa 1962aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 20202493PRTHomo sapiens 2Met Ile Leu
Asn Ser Leu Ser Leu Cys Tyr His Asn Lys Leu Ile Leu1 5 10 15Ala Pro
Met Val Arg Val Gly Thr Leu Pro Met Arg Leu Leu Ala Leu 20 25 30Asp
Tyr Gly Ala Asp Ile Val Tyr Cys Glu Glu Leu Ile Asp Leu Lys 35 40
45Met Ile Gln Cys Lys Arg Val Val Asn Glu Val Leu Ser Thr Val Asp
50 55 60Phe Val Ala Pro Asp Asp Arg Val Val Phe Arg Thr Cys Glu Arg
Glu65 70 75 80Gln Asn Arg Val Val Phe Gln Met Gly Thr Ser Asp Ala
Glu Arg Ala 85 90 95Leu Ala Val Ala Arg Leu Val Glu Asn Asp Val Ala
Gly Ile Asp Val 100 105 110Asn Met Gly Cys Pro Lys Gln Tyr Ser Thr
Lys Gly Gly Met Gly Ala 115 120 125Ala Leu Leu Ser Asp Pro Asp Lys
Ile Glu Lys Ile Leu Ser Thr Leu 130 135 140Val Lys Gly Thr Arg Arg
Pro Val Thr Cys Lys Ile Arg Ile Leu Pro145 150 155 160Ser Leu Glu
Asp Thr Leu Ser Leu Val Lys Arg Ile Glu Arg Thr Gly 165 170 175Ile
Ala Ala Ile Ala Val His Gly Arg Lys Arg Glu Glu Arg Pro Gln 180 185
190His Pro Val Ser Cys Glu Val Ile Lys Ala Ile Ala Asp Thr Leu Ser
195 200 205Ile Pro Val Ile Ala Asn Gly Gly Ser His Asp His Ile Gln
Gln Tyr 210 215 220Ser Asp Ile Glu Asp Phe Arg Gln Ala Thr Ala Ala
Ser Ser Val Met225 230 235 240Val Ala Arg Ala Ala Met Trp Asn Pro
Ser Ile Phe Leu Lys Glu Gly 245 250 255Leu Arg Pro Leu Glu Glu Val
Met Gln Lys Tyr Ile Arg Tyr Ala Val 260 265 270Gln Tyr Asp Asn His
Tyr Thr Asn Thr Lys Tyr Cys Leu Cys Gln Met 275 280 285Leu Arg Glu
Gln Leu Glu Ser Pro Gln Gly Arg Leu Leu His Ala Ala 290 295 300Gln
Ser Ser Arg Glu Ile Cys Glu Ala Phe Gly Leu Gly Ala Phe Tyr305 310
315 320Glu Glu Thr Thr Gln Glu Leu Asp Ala Gln Gln Ala Arg Leu Ser
Ala 325 330 335Lys Thr Ser Glu Gln Thr Gly Glu Pro Ala Glu Asp Thr
Ser Gly Val 340 345 350Ile Lys Met Ala Val Lys Phe Asp Arg Arg Ala
Tyr Pro Ala Gln Ile 355 360 365Thr Pro Lys Met Cys Leu Leu Glu Trp
Cys Arg Arg Glu Lys Leu Ala 370 375 380Gln Pro Val Tyr Glu Thr Val
Gln Arg Pro Leu Asp Arg Leu Phe Ser385 390 395 400Ser Ile Val Thr
Val Ala Glu Gln Lys Tyr Gln Ser Thr Leu Trp Asp 405 410 415Lys Ser
Lys Lys Leu Ala Glu Gln Ala Ala Ala Ile Val Cys Leu Arg 420 425
430Ser Gln Gly Leu Pro Glu Gly Arg Leu Gly Glu Glu Ser Pro Ser Leu
435 440 445His Lys Arg Lys Arg Glu Ala Pro Asp Gln Asp Pro Gly Gly
Pro Arg 450 455 460Ala Gln Glu Leu Ala Gln Pro Gly Asp Leu Cys Lys
Lys Pro Phe Val465 470 475 480Ala Leu Gly Ser Gly Glu Glu Ser Pro
Leu Glu Gly Trp 485 490321DNAArtificial SequenceAn artificially
synthesized primer sequence for RT-PCR 3gaccacatcc aacagtattc g
21421DNAArtificial SequenceAn artificially synthesized primer
sequence for RT-PCR 4tgccaggaca tctaacttct g 21521DNAArtificial
SequenceAn artificially synthesized primer sequence for RT-PCR
5gaggtgatag cattgctttc g 21621DNAArtificial SequenceAn artificially
synthesized primer sequence for RT-PCR 6caagtcagtg tacaggtaag c
21719DNAArtificial SequenceAn artificially synthesized target
sequence for siRNA 7gaagcagcac gacttcttc 19819DNAArtificial
SequenceAn artificially synthesized target sequence for siRNA
8cgtacgcgga atacttcga 19919DNAArtificial SequenceAn artificially
synthesized target sequence for siRNA 9gcgcgctttg taggattcg
191019DNAArtificial SequenceAn artificially synthesized target
sequence for siRNA 10tgaggtgctc agcacagtg 191119DNAArtificial
SequenceAn artificially synthesized target sequence for siRNA
11gttggcacag cctgtgtat 191251DNAArtificial SequenceAn artificially
synthesized oligonucleotide sequence for siRNA 12tcccgaagca
gcacgacttc ttcttcaaga gagaagaagt cgtgctgctt c 511351DNAArtificial
SequenceAn artificially synthesized oligonucleotide sequence for
siRNA 13aaaagaagca gcacgacttc ttctctcttg aagaagaagt cgtgctgctt c
511447DNAArtificial SequenceAn artificially synthesized hairpin
sequence for siRNA 14gaagcagcac gacttcttct tcaagagaga agaagtcgtg
ctgcttc 471551DNAArtificial SequenceAn artificially synthesized
oligonucleotide sequence for siRNA 15tccccgtacg cggaatactt
cgattcaaga gatcgaagta ttccgcgtac g 511652DNAArtificial SequenceAn
artificially synthesized oligonucleotide sequence for siRNA
16aaaacgtacg cggaatactt cgatctcttg aaatcgaagt attccgcgta cg
521747DNAArtificial SequenceAn artificially synthesized hairpin
sequence for siRNA 17cgtacgcgga atacttcgat tcaagagatc gaagtattcc
gcgtacg 471851DNAArtificial SequenceAn artificially synthesized
oligonucleotide sequence for siRNA 18tcccgcgcgc tttgtaggat
tcgttcaaga gacgaatcct acaaagcgcg c 511951DNAArtificial SequenceAn
artificially synthesized oligonucleotide sequence for siRNA
19aaaagcgcgc tttgtaggat tcgtctcttg aacgaatcct acaaagcgcg c
512047DNAArtificial SequenceAn artificially synthesized hairpin
sequence for siRNA 20gcgcgctttg taggattcgt tcaagagacg aatcctacaa
agcgcgc 472151DNAArtificial SequenceAn artificially synthesized
oligonucleotide sequence for siRNA 21tccctgaggt gctcagcaca
gtgttcaaga gacactgtgc tgagcacctc a 512251DNAArtificial SequenceAn
artificially synthesized oligonucleotide sequence for siRNA
22aaaatgaggt gctcagcaca gtgtctcttg aacactgtgc tgagcacctc a
512347DNAArtificial SequenceAn artificially synthesized hairpin
sequence for siRNA 23tgaggtgctc agcacagtgt tcaagagaca ctgtgctgag
cacctca 472450DNAArtificial SequenceAn artificially synthesized
oligonucleotide sequence for siRNA 24tcccgttggc acagcctgtg
tattcaagag aatacacagg ctgtgccaac 502550DNAArtificial SequenceAn
artificially synthesized oligonucleotide sequence for siRNA
25aaaagttggc acagcctgtg tatctcttga aatacacagg ctgtgccaac
502646DNAArtificial SequenceAn artificially synthesized hairpin
sequence for siRNA 26gttggcacag cctgtgtatt caagagaata cacaggctgt
gccaac 4627493PRTMus musculus 27Met Ile Val Asn Ser Leu Ser Leu Cys
Tyr His Asn Lys Leu Ile Leu1 5 10 15Ala Pro Met Val Arg Val Gly Thr
Leu Pro Met Arg Leu Leu Ala Leu 20 25 30Asp Tyr Gly Ala Asp Ile Val
Tyr Cys Glu Glu Leu Ile Asp Leu Lys 35 40 45Met Leu Gln Cys Lys Arg
Val Val Asn Glu Val Leu Ser Thr Val Asp 50 55 60Phe Val Ala Pro Asp
Asp Arg Val Val Phe Arg Thr Cys Glu Arg Glu65 70 75 80Gln Ser Arg
Val Val Phe Gln Met Gly Thr Ser Asp Ala Glu Arg Ala 85 90 95Leu Ala
Val Ala Arg Leu Val Glu Asn Asp Val Ala Gly Ile Asp Val 100 105
110Asn Met Gly Cys Pro Lys Glu Tyr Ser Thr Lys Gly Gly Met Gly Ala
115 120 125Ala Leu Leu Ser Asp Pro Asp Lys Ile Glu Lys Ile Leu Ser
Thr Leu 130 135 140Val Lys Gly Thr His Arg Pro Val Thr Cys Lys Ile
Arg Ile Leu Pro145 150 155 160Ser Leu Glu Asp Thr Leu Asn Leu Val
Lys Arg Ile Glu Arg Thr Gly 165 170 175Ile Ser Ala Ile Ala Val His
Gly Arg Asn Arg Asp Glu Arg Pro Gln 180 185 190His Pro Val Ser Cys
Glu Val Ile Arg Ala Ile Ala Glu Thr Leu Ser 195 200 205Ile Pro Val
Ile Ala Asn Gly Gly Ser His Asp His Ile Gln Gln His 210 215 220Val
Asp Ile Glu Asp Phe Arg Gln Ala Thr Ala Ala Ser Ser Val Met225 230
235 240Val Ala Arg Ala Ala Met Trp Asn Pro Ser Ile Phe Leu Lys Asp
Gly 245 250 255Leu Arg Pro Leu Glu Glu Val Met Gln Lys Tyr Ile Arg
Tyr Ala Val 260 265 270Gln Tyr Asp Asn His Tyr Thr Asn Thr Lys Tyr
Cys Leu Cys Gln Met 275 280 285Leu Arg Glu Gln Leu Glu Ser Pro Gln
Gly Arg Leu Leu His Ala Ala 290 295 300Gln Ser Ser Gln Glu Ile Cys
Glu Ala Phe Gly Leu Gly Ala Phe Tyr305 310 315 320Glu Glu Thr Ile
Arg Glu Leu Asp Ala Arg Arg Ala Asp Leu Leu Ala 325 330 335Lys Thr
Pro Glu Ala Val Glu Glu Pro Ala Glu Asp Thr Ser Gly Ile 340 345
350Ile Lys Met Ala Ile Arg Phe Asp Arg Arg Ala Tyr Pro Pro Gln Ile
355 360 365Thr Pro Lys Met Cys Leu Leu Glu Trp Cys Arg Arg Glu Lys
Leu Pro 370 375 380Gln Pro Val Tyr Glu Thr Val Gln Arg Thr Ile Asp
Arg Met Phe Cys385 390 395 400Ser Val Val Thr Val Ala Glu Gln Lys
Tyr Gln Ser Thr Leu Trp Asp 405 410 415Lys Ser Lys Lys Leu Ala Glu
Gln Thr Ala Ala Ile Val Cys Leu Arg 420 425 430Ser Gln Gly Leu Pro
Glu Gly Arg Leu Gly Glu Glu Ser Pro Ser Leu 435 440 445Asn Lys Arg
Lys Arg Glu Ala Pro Asp Gln Asp Pro Gly Gly Pro Arg 450 455 460Val
Gln Glu Pro Ala Leu Pro Gly Glu Ile Cys Lys Lys Pro Phe Val465 470
475 480Thr Leu Asp Ser Ser Glu Glu Asn Leu Leu Glu Gly Cys 485
49028531PRTRattus norvegicus 28Met Ile Val Asn Ser Leu Ser Leu Cys
Tyr His Asn Lys Leu Ile Leu1 5 10 15Ala Pro Met Val Arg Val Gly Thr
Leu Pro Met Arg Leu Leu Ala Leu
20 25 30Asp Tyr Gly Ala Asp Ile Val Tyr Cys Glu Glu Leu Ile Asp Leu
Lys 35 40 45Met Leu Gln Cys Arg Arg Val Val Asn Glu Val Leu Ser Thr
Val Asp 50 55 60Phe Val Ala Pro Asp Asp Arg Val Val Phe Arg Thr Cys
Glu Arg Glu65 70 75 80Gln Ser Arg Val Val Phe Gln Met Gly Thr Ser
Asp Ala Glu Arg Ala 85 90 95Leu Ala Val Ala Arg Leu Val Glu Asn Asp
Val Ala Gly Ile Asp Val 100 105 110Asn Met Gly Cys Pro Lys Glu Tyr
Ser Thr Lys Gly Gly Met Gly Ala 115 120 125Ala Leu Leu Ser Asp Pro
Asp Lys Ile Glu Lys Ile Leu Ser Thr Leu 130 135 140Val Lys Gly Thr
His Arg Pro Val Thr Cys Lys Ile Arg Ile Leu Pro145 150 155 160Ser
Leu Glu Asp Thr Leu Asn Leu Val Lys Arg Ile Glu Arg Thr Gly 165 170
175Ile Ser Ala Ile Ala Val His Gly Arg Asn Arg Asp Glu Arg Pro Gln
180 185 190His Pro Val Ser Cys Glu Val Ile Arg Ala Ile Ala Glu Thr
Leu Ser 195 200 205Ile Pro Val Ile Ala Lys Val Leu Ile Val Glu Gly
Leu Leu Lys Leu 210 215 220Thr Asp Asn Glu Arg Gln Arg Ser Ser Gly
Asn Thr Gly Arg Phe His225 230 235 240Tyr Gly Ile Leu Pro Asn Pro
Leu Leu Leu Phe Ser Gly Gly Ser His 245 250 255Asp His Ile Gln Gln
His Leu Asp Ile Glu Asp Phe Arg Gln Ala Thr 260 265 270Ala Ala Ser
Ser Val Met Val Ala Arg Ala Ala Met Trp Asn Pro Ser 275 280 285Ile
Phe Leu Lys Asp Gly Leu Arg Pro Leu Glu Glu Val Met Gln Lys 290 295
300Tyr Ile Arg Tyr Ala Val Gln Tyr Asp Asn His Tyr Thr Asn Thr
Lys305 310 315 320Tyr Cys Leu Cys Gln Met Leu Arg Glu Gln Leu Glu
Ser Pro Gln Gly 325 330 335Arg Met Leu His Ala Ala Gln Ser Ser Gln
Glu Ile Cys Glu Ala Phe 340 345 350Gly Leu Gly Thr Phe Tyr Glu Asp
Thr Ile Arg Glu Leu Asp Ala Arg 355 360 365Arg Ala Asp Leu Leu Ala
Lys Thr Pro Glu Ala Val Glu Glu Pro Ala 370 375 380Glu Asp Thr Ser
Gly Ile Ile Lys Met Ala Ile Arg Phe Asp Arg Arg385 390 395 400Ala
Tyr Pro Pro Gln Ile Thr Pro Lys Thr Cys Leu Leu Glu Trp Cys 405 410
415Arg Arg Glu Lys Leu Pro Gln Pro Val Tyr Glu Thr Val Gln Arg Pro
420 425 430Ile Asp Arg Met Phe Cys Ser Val Val Thr Val Ala Glu Gln
Lys Tyr 435 440 445Gln Ser Thr Leu Trp Asp Lys Ser Lys Lys Leu Ala
Glu Gln Thr Ala 450 455 460Ala Ile Val Cys Leu Arg Ser Gln Gly Leu
Pro Glu Gly Arg Leu Gly465 470 475 480Glu Glu Asn Pro Ser Leu Asn
Lys Arg Lys Arg Glu Ala Pro Asn Gln 485 490 495Asp Pro Gly Gly Pro
Arg Val Gln Glu Thr Ala Leu Pro Gly Glu Ile 500 505 510Cys Lys Lys
Pro Phe Val Thr Leu Glu Ser Ser Glu Glu Asn Leu Leu 515 520 525Glu
Gly Cys 53029473PRTDrosophila melanogaster 29Met Leu Arg Leu Pro
Thr Ile Leu Arg Lys Ser Phe Ser Met Lys Thr1 5 10 15Arg Gln Arg Leu
Asp Tyr Arg Asn Lys Leu Ile Leu Ala Pro Met Val 20 25 30Arg Val Gly
Thr Leu Pro Met Arg Leu Leu Ala Leu Glu Met Gly Ala 35 40 45Asp Ile
Val Tyr Thr Glu Glu Leu Val Asp Ile Lys Leu Ile Lys Ser 50 55 60Ile
Arg Arg Pro Asn Pro Ala Leu Gly Thr Val Asp Phe Val Asp Pro65 70 75
80Ser Asp Gly Thr Ile Val Phe Arg Thr Cys Ala Gln Glu Thr Ser Arg
85 90 95Leu Val Leu Gln Met Gly Thr Ser Asp Ala Gly Arg Ala Leu Ala
Val 100 105 110Gly Lys Leu Leu Gln Arg Asp Ile Ser Gly Leu Asp Ile
Asn Met Gly 115 120 125Cys Pro Lys Glu Phe Ser Thr Lys Gly Gly Met
Gly Ala Ala Leu Leu 130 135 140Ala Asp Pro Asp Lys Ala Ala His Ile
Leu Arg Thr Leu Cys Ser Gly145 150 155 160Leu Asp Ile Pro Val Thr
Cys Lys Ile Arg Ile Leu Pro Asp Val Glu 165 170 175Gly Thr Ile Asp
Leu Val Gln Lys Leu Ala Ala Thr Gly Ile Ala Ala 180 185 190Ile Gly
Val His Ala Arg Thr Arg Asp Glu Arg Pro Gln His Pro Ala 195 200
205His Pro Glu Val Leu Arg Ala Val Ala Gln Ala Val Asp Ile Pro Ile
210 215 220Ile Ala Asn Gly Gly Ser Lys Asn Met His Cys Tyr Asp Asp
Leu Arg225 230 235 240Lys Phe Gln Met Glu Cys Gly Ala Asp Ser Val
Met Val Ala Arg Ala 245 250 255Ala Gln Ile Asn Val Ser Ile Phe Arg
Pro Glu Gly Leu Leu Pro Met 260 265 270Asp Glu Leu Ile Glu Lys Tyr
Leu Arg Leu Cys Val Asp Tyr Asp Asn 275 280 285Ala Pro His Asn Ala
Lys Tyr Cys Val Gln Ser Ile Leu Arg Glu Leu 290 295 300Gln Glu Thr
Pro Arg Gly Lys Arg Phe Leu Gln Cys Gln Thr Leu Gln305 310 315
320Gln Ile Cys Glu Ile Trp Glu Leu Gly Asp Tyr Cys Arg Arg Lys Gln
325 330 335Arg Glu Leu Lys Thr Met Gly Asn Ser Gly Arg Ala Glu Val
Glu Pro 340 345 350Pro Glu Ala Leu Ala Lys Arg Gln Lys Leu Glu Asp
Ala Ala Ile Ala 355 360 365Ile Thr Asp Glu Tyr Asp Gly Ile Ile Cys
Arg His Met Pro Phe Leu 370 375 380Arg Ser Thr Tyr Pro Ser Asp Asn
His Leu Pro Lys Thr Gln Leu Tyr385 390 395 400Val His Ala Val Lys
Thr Gly Lys Ser Pro Pro Ala Tyr Glu Thr Gln 405 410 415Gln Cys Asp
Lys Leu Phe Arg Ser Ile Cys Thr Tyr Asp Gly Gln Arg 420 425 430Phe
Ser Ser Ser Phe Trp Glu Lys Asn Lys Lys Gln Ala Glu Gln Gly 435 440
445Ala Ala Leu Val Ala Leu Leu His Leu Gly Gln Leu Glu Ala Glu Val
450 455 460Leu Arg Asp Asn Gly Ser Leu Leu Asn465
47030436PRTCaenorhabditis elegans 30Met Ser Asp Leu Tyr Arg Asn Lys
Lys Ile Leu Ala Pro Met Val Arg1 5 10 15Ala Gly Arg Thr Pro Leu Arg
Leu Leu Cys Leu Lys Tyr Gly Ala Asp 20 25 30Leu Cys Tyr Thr Glu Glu
Ile Val Asp Lys Lys Leu Ile Glu Ala Thr 35 40 45Arg Val Val Asn Glu
Ala Leu Gly Thr Ile Asp Tyr Arg Asn Gly Asp 50 55 60Asp Ile Ile Leu
Arg Leu Ala Pro Glu Glu Lys Gly Arg Cys Ile Leu65 70 75 80Gln Ile
Gly Thr Asn Ser Gly Glu Lys Ala Ala Lys Ile Ala Gln Ile 85 90 95Val
Gly Asp Asp Val Ala Gly Ile Asp Val Asn Met Gly Cys Pro Lys 100 105
110Pro Phe Ser Ile His Cys Gly Met Gly Ala Ala Leu Leu Thr Gln Thr
115 120 125Glu Lys Ile Val Asp Ile Leu Thr Ser Leu Lys Ser Ala Ala
Lys Val 130 135 140Pro Val Thr Cys Lys Ile Arg Val Leu Asp Asp Pro
Glu Asp Thr Leu145 150 155 160Lys Leu Val Gln Glu Ile Glu Lys Cys
Arg Val Ser Ala Leu Gly Val 165 170 175His Gly Arg Arg Arg Asp Glu
Arg Gln Pro Asp Lys Cys Arg Ile Asp 180 185 190Glu Ile Arg Asp Val
Ala Gln Ala Val Gln Ser Ile Pro Val Ile Ser 195 200 205Asn Gly Leu
Ser Asp Glu Ile Glu Gln Tyr Ser Asp Phe Glu Lys Tyr 210 215 220Gln
Leu Leu Thr Glu Thr Ser Ser Thr Met Ile Ala Arg Lys Ala Leu225 230
235 240Ser Thr Pro Ser Ile Phe Arg Arg Glu Gly Cys Leu Asp Lys Tyr
Glu 245 250 255Asp Ile Arg Asn Phe Leu Glu Leu Ala Cys Gln Tyr Asp
Glu Ser Tyr 260 265 270Thr Met Thr Lys Tyr Val Val Gln Arg Ile Leu
Gly Ala Asp Gln Glu 275 280 285Tyr Asp Pro Arg Gly Lys Ala Thr Val
Ala Ala Gly Ser Val Leu Gln 290 295 300Ile Cys Lys Ala Phe Gly Met
Glu Asp Val Tyr Asp Lys Trp Arg Asp305 310 315 320Glu Arg Lys Lys
Lys Gln Ser Lys Lys Arg Ala Arg Val Asp Asp Asp 325 330 335Gly Val
Tyr Asn Ile Glu Val Ser Phe Pro Leu Lys Arg Leu Lys Asn 340 345
350Ser Val Gly Phe Ser Pro Thr Pro Lys Met Val Leu His Asp Tyr Cys
355 360 365Val Glu Thr Lys Ile Pro Lys Ala Thr Tyr Glu Val Val Lys
Arg Asp 370 375 380Asp Lys Arg Phe Val Ala Thr Ala Cys Ile Gly Asp
Lys Lys Tyr Arg385 390 395 400Ser Gly Ile Gly Gln Pro Asn Leu Arg
Met Ala Glu Gln Val Ala Ala 405 410 415Leu Ala Ala Leu His Gly Met
Asn Ile Arg Asn Leu Leu Val Gly Asn 420 425 430Trp Glu Glu Glu
43531384PRTSaccharomyces cerevisiae 31Met Val Thr Tyr Ala Gly Lys
Leu Val Leu Ala Pro Met Val Arg Ala1 5 10 15Gly Glu Leu Pro Thr Arg
Leu Met Ala Leu Ala His Gly Ala Asp Leu 20 25 30Val Trp Ser Pro Glu
Ile Ile Asp Lys Lys Leu Ile Gln Cys Val Arg 35 40 45Lys Glu Asn Thr
Ala Leu Gln Thr Val Asp Tyr Val Val Pro Ser Lys 50 55 60Val Gln Thr
Arg Pro Glu Thr Leu Val Phe Arg Thr Tyr Pro Lys Leu65 70 75 80Glu
Ser Ser Lys Leu Ile Phe Gln Ile Gly Ser Ala Ser Pro Ala Leu 85 90
95Ala Thr Gln Ala Ala Leu Lys Val Ile Asn Asp Val Ser Gly Ile Asp
100 105 110Val Asn Met Gly Cys Pro Lys His Phe Ser Ile His Ser Gly
Met Gly 115 120 125Ser Ala Leu Leu Arg Thr Pro Asp Thr Leu Cys Leu
Ile Leu Lys Glu 130 135 140Leu Val Lys Asn Val Gly Asn Pro His Ser
Lys Pro Ile Ser Val Lys145 150 155 160Ile Arg Leu Leu Asp Thr Lys
Gln Asp Thr Leu Gln Leu Val Lys Arg 165 170 175Leu Cys Ala Thr Gly
Ile Thr Asn Leu Thr Val His Cys Arg Lys Thr 180 185 190Glu Met Arg
Asn Arg Glu Gln Pro Ile Thr Asp Tyr Ile Ala Glu Ile 195 200 205Tyr
Glu Ile Cys Gln Ala Asn Asn Val Ser Leu Ile Val Asn Gly Ala 210 215
220Ile Arg Asp Arg Ser His Phe His Asp Leu Gln Ala Asn His Trp
Lys225 230 235 240Asn Thr Asn Ile Gly Gly Met Ile Ala Glu Cys Ala
Glu Arg Asp Pro 245 250 255Thr Val Phe Asp His Thr Ser Lys Pro Ser
Glu Asp Gly Pro Ser Trp 260 265 270Val Val Ala Cys Arg Glu Phe Ile
Gln Trp Ala Thr Lys Phe Asp Asn 275 280 285His Ile Gly Asn Thr Lys
Tyr Met Leu Ser Arg Ile Val Pro Gly Lys 290 295 300Ser Val Phe Phe
Gln Tyr Phe Ala Arg Cys Lys Ser Pro Glu Glu Val305 310 315 320Ser
Phe Val Leu Lys Gln Leu Asn Asp Asp Gly Ser Ala Gln Thr Asp 325 330
335Pro Ser Glu Tyr Leu Glu Asn Cys Arg Ala Gln Glu Lys Ala Leu Lys
340 345 350Asn Ala Asn Ala Ile Ala Lys Gln Lys Arg Lys Gln Thr Asp
His Ile 355 360 365Gly Ser Asp Thr Lys Lys Gln Lys Val Val Pro Leu
Pro Thr Asp Ile 370 375 38032423PRTSaccharomyces cerevisiae 32Met
Thr Glu Pro Ala Leu Ser Ser Ala Asn Asn Ala Leu Met Gln Lys1 5 10
15Leu Thr Gly Arg Gln Leu Phe Asp Lys Ile Gly Arg Pro Thr Arg Ile
20 25 30Val Ala Pro Met Val Asp Gln Ser Glu Leu Ala Trp Arg Ile Leu
Ser 35 40 45Arg Arg Tyr Gly Ala Thr Leu Ala Tyr Thr Pro Met Leu His
Ala Lys 50 55 60Leu Phe Ala Thr Ser Lys Lys Tyr Arg Glu Asp Asn Trp
Ser Ser Leu65 70 75 80Asp Gly Ser Ser Val Asp Arg Pro Leu Val Val
Gln Phe Cys Ala Asn 85 90 95Asp Pro Glu Tyr Leu Leu Ala Ala Ala Lys
Leu Val Glu Asp Lys Cys 100 105 110Asp Ala Val Asp Leu Asn Leu Gly
Cys Pro Gln Gly Ile Ala Lys Lys 115 120 125Gly His Tyr Gly Ser Phe
Leu Met Glu Glu Trp Asp Leu Ile His Asn 130 135 140Leu Ile Asn Thr
Leu His Lys Asn Leu Lys Val Pro Val Thr Ala Lys145 150 155 160Ile
Arg Ile Phe Asp Asp Cys Glu Lys Ser Leu Asn Tyr Ala Lys Met 165 170
175Val Leu Asp Ala Gly Ala Gln Phe Leu Thr Val His Gly Arg Val Arg
180 185 190Glu Gln Lys Gly Gln Lys Thr Gly Leu Ala Asn Trp Glu Thr
Ile Lys 195 200 205Tyr Leu Arg Asp Asn Leu Pro Lys Glu Thr Val Phe
Phe Ala Asn Gly 210 215 220Asn Ile Leu Tyr Pro Glu Asp Ile Ser Arg
Cys Met Glu His Ile Gly225 230 235 240Ala Asp Ala Val Met Ser Ala
Glu Gly Asn Leu Tyr Asn Pro Gly Val 245 250 255Phe Asn Val Gly Gln
Thr Lys Asn Lys Glu Lys Ile Phe Pro Arg Val 260 265 270Asp Lys Ile
Ile Arg Glu Tyr Phe Gln Ile Val Lys Glu Cys Gln Glu 275 280 285Ser
Lys Ala Ser Lys Thr Ala Met Lys Ser His Phe Phe Lys Ile Leu 290 295
300Arg Pro Phe Leu Pro His His Thr Asp Ile Arg Ser Thr Leu Ala
Thr305 310 315 320Met Asn Ala Lys Ala Thr Trp Glu Glu Trp Glu Glu
Gln Val Val Lys 325 330 335Pro Val Glu Lys Val Val Gln Glu Ile Phe
Glu Gln Pro Asp Ile Ala 340 345 350Ile Lys Asp Glu Ile Thr Ile Gly
Glu Lys Gln Ser Trp Gly Gly Ser 355 360 365Tyr Arg Thr Val Pro Tyr
Trp Arg Cys Gln Pro Tyr Phe Arg Pro Val 370 375 380Asn Gly Ile Thr
Gly Asp Lys Arg Val Met Gln Gly Leu Ile Asp Glu385 390 395 400Ser
Val Asn Lys Lys Arg Lys Ala Asp Val Pro Leu Glu Ser Ala Asp 405 410
415Lys Lys Lys Asp Val Lys Ala 4203310PRTArtificial
Sequencec-Myc-His epitope tag 33Leu Asp Glu Glu Ser Ile Leu Lys Gln
Glu1 5 10
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References