U.S. patent application number 15/997477 was filed with the patent office on 2018-12-06 for anti-tnf induced apoptosis (atia) diagnostic markers and therapies.
The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health & Human Servic, The United States of America, as represented by the Secretary, Department of Health & Human Servic. Invention is credited to Zhenggang Liu.
Application Number | 20180348219 15/997477 |
Document ID | / |
Family ID | 42830385 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180348219 |
Kind Code |
A1 |
Liu; Zhenggang |
December 6, 2018 |
ANTI-TNF INDUCED APOPTOSIS (ATIA) DIAGNOSTIC MARKERS AND
THERAPIES
Abstract
The invention features diagnostic and therapeutic methods and
compositions featuring Anti-TNF Induced Apoptosis (ATIA). AITA is
useful as a diagnostic marker for cancer, in particular for
glioblastoma. ATIA is also a therapeutic target in diseases such as
cancer. The invention encompasses combination therapies where
knockdown of ATIA is used in combination with other treatment. The
invention also features kits for use in the diagnostic and
therapeutic methods.
Inventors: |
Liu; Zhenggang; (Germantown,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health & Human Servic |
Rockville |
MD |
US |
|
|
Family ID: |
42830385 |
Appl. No.: |
15/997477 |
Filed: |
June 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13322863 |
Nov 28, 2011 |
9989533 |
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PCT/US2010/036394 |
May 27, 2010 |
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15997477 |
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61182072 |
May 28, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/56 20130101;
C07K 14/82 20130101; C07K 14/4747 20130101; G01N 2800/52 20130101;
G01N 33/57407 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 14/47 20060101 C07K014/47; C07K 14/82 20060101
C07K014/82 |
Claims
1. A method of diagnosing a subject as having, or having a
propensity to develop a neoplasia, the method comprising
determining the level of expression or biological activity of an
anti-TNF Induced Apoptosis (ATIA) polypeptide or nucleic acid in a
subject sample wherein an alteration in the level of expression or
biological activity relative to the expression or biological
activity in a reference indicates that the subject has or has a
propensity to develop a neoplasia.
2. The method of claim 1, wherein the neoplasia is selected from
the group consisting of: brain, pancreatic, and stomach
neoplasia.
3. A method of determining the progression of a brain neoplasia in
a subject, the method comprising determining the expression or
activity of an ATIA polypeptide or nucleic acid in a subject
sample, wherein an alteration in the level of expression or
activity relative to the level of expression in a reference
indicates the progression of the a brain neoplasia in the
subject.
4. The method of claim 1, wherein the method is used to determine
if a subject will be responsive to TRAIL therapy, chemotherapy, or
radiation treatment.
5. The method of claim 1, wherein the ATIA nucleic acid comprises a
sequence selected from the group consisting of: SEQ ID NO: 1, SEQ
ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID: NO: 8, or
fragments thereof.
6. The method of claim 1, wherein the ATIA polypeptide comprises a
sequence selected from the group consisting of: SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10, or
fragments thereof.
7. The method of claim 1, wherein the level of expression is
determined in an immunological assay, enzyme-linked immunosorbent
assay (ELISA), or immunohistochemical assay.
8. The method of claim 7, wherein the ELISA is used to detect the
extracellular portion of ATIA.
9. The method of claim 8, wherein the extracellular portion
comprises a sequence selected from the group consisting of: SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or fragments
thereof.
10. The method of claim 1, wherein the alteration is an
increase.
11. The method of claim 10, wherein the increase corresponds to an
increased sensitivity to TRAIL-induced cell death.
12. A polypeptide comprising an isolated ATIA protein, or fragment
thereof, wherein the protein is upregulated in a neoplasia.
13. The polypeptide of claim 12, wherein the ATIA protein comprises
the extracellular domain.
14. The polypeptide of claim 13, wherein the extracellular domain
comprises amino acid residues 600-673 of SEQ ID NO: 2.
15. The polypeptide of claim 12, wherein the ATIA polypeptide
comprises a sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10,
or fragments thereof.
16. An isolated ATIA nucleic acid molecule, wherein the nucleic
acid molecule encodes a polypeptide selected from claim 12; or an
isolated ATIA inhibitory nucleic acid molecule, wherein the
inhibitory nucleic acid molecule specifically binds at least a
fragment of a nucleic acid molecule encoding an ATIA protein; or An
inhibitory nucleic acid molecule corresponding to at least a
portion of an ATIA nucleic acid molecule that encodes an ATIA
protein, wherein the inhibitory nucleic acid molecule is capable of
altering the level of protein encoded by the ATIA nucleic acid
molecule.
17. A method of treating or preventing a neoplasia in a subject,
the method comprising administering to a subject in need thereof an
effective amount of a small molecule that alters expression of an
ATIA polypeptide.
18. A kit for the diagnosis of a neoplasia in a subject comprising
a primer set to detect an ATIA nucleic acid molecule, or fragment
thereof, and written instructions for use of the kit for diagnosis
of a neoplasia.
19. A method of altering the expression of an ATIA nucleic acid
molecule or ATIA polypeptide in a cell, the method comprising
contacting the cell with an effective amount of a compound capable
of altering the expression of the ATIA nucleic acid molecule or
ATIA polypeptide.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/322,863, filed 28 Nov. 2011, which is a 35
U.S.C. .sctn. 371 U.S. National Entry of International Application
PCT/US2010/036394 (International Application Publication No. WO
2010138709) having an International filing date of 27 May 2010,
which claims priority to U.S. Provisional Application No.
61/182,072, filed on 28 May 2009. The entire contents of the
aforementioned applications are incorporated herein by reference in
their entirety.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The term glioma refers to tumors that are derived from
normal glial cells (i.e., astrocytes, oligodendrocytes, and
ependymal cells). For each of these cell types, there is a
malignant counterpart (e.g., astrocytoma is derived from
astrocytes). Despite significant improvements in the early
detection of malignant gliomas, the median survival of patients
remains less than 12 months from the time of diagnosis. Malignant
gliomas rarely metastasize outside the central nervous system, but
they will diffusely invade the host brain. Astrocytic tumors
comprise over 80% of primary CNS tumors and are classified by the
type of cell they most closely resemble and according to their
clinical and biological behavior (i.e., tumor grade). The slower
growing lesions are commonly referred to as low-grade gliomas
(LGGs), while the more clinically aggressive tumors are classified
as high-grade gliomas (HGGs). HGGs are more common comprising
nearly 80% of all gliomas.
[0004] Astrocytic tumors, the most common type of neuroepithelial
tissue tumors (and are therefore sometimes loosely referred to by
the term "glioma"), can be further subdivided based on the severity
of the condition (i.e., WHO Grade 1 to 4, based on the severity of
the condition, with 4 being the most serious form of glioma). Grade
1 corresponds to pilocytic astrocytoma; Grade 2 corresponds to
diffuse astrocytoma; Grade 3 corresponds to anaplastic or malignant
astrocytoma; and Grade 4 corresponds to glioblastoma multiforme,
which is the most common glioma in adults and is considered the
most serious form of astrocytic tumor.
[0005] Treatment of CNS tumors depends on the multiplicity,
location, and grade of the tumor, and may include any of surgical
resection, stereotactic radiosurgery (SRS), whole brain
radiotherapy (WBRT) and chemotherapy or some combination thereof;
however the inability of many conventional chemotherapeutic agents
to cross the blood-brain barrier (BBB) has historically limited
their use in the treatment of CNS tumors. Glial tumors, the most
prevalent and morbid of which is astrcoytoma and its aggressive
derivative glioblastoma multiforme, are the most common cancers of
the adult central nervous system. They are also among the least
treatable cancers, with a 5 year survival after initial diagnosis
of <10% for tumors initially diagnosed at the grade 3
(anaplastic astrocytoma) or 4 (glioblastoma) stages. The currents
treatment of glioma and glioblastoma are lacking, and achieve only
palliation and short-term increments in survival. They include
surgical resection--following which ultimate recurrence rates are
over 90%--as well as radiation therapy, and chemotherapies that
include cisplatin, BCNU and other mitotic inhibitors. The benefits
of these current therapies are brief and temporary, and none are
curative.
[0006] Accordingly, a need remains to for more effective
compositions and methods for the detection and treatment of brain
tumors.
SUMMARY OF THE INVENTION
[0007] The present inventors have found that ATIA is a
hypoxia-inducible gene, and under hypoxia conditions ATIA protein
expression is considerably increased, and deletion of ATIA renders
cells sensitive to hypoxia induced apoptosis. The present inventors
have shown in vitro and that ATIA protein localizes in the cell
plasma membrane and the mitochondria and that ATIA mutant protein
targeted to mitochondria is capable of protecting cells against
TNF-induced apoptosis. In particular, the present inventors have
shown that in glioblastoma, knocking down ATIA expression
sensitizes the cells to hypoxia treatment, thus demonstrating that
ATIA is important for cancer cell survival by providing protection
from hypoxia-induced cell death.
[0008] Included in the present invention are diagnostic and
therapeutic methods and compositions featuring ATIA. AITA is useful
as a diagnostic marker for cancer, in particular for glioblastoma.
ATIA is also a therapeutic target in diseases such as cancer. The
invention encompasses combination therapies where knockdown of ATIA
is used in combination with other treatment. In one aspect, the
invention features a method of diagnosing a subject as having, or
having a propensity to develop a neoplasia, the method comprising
determining the level of expression or biological activity of an
anti-TNF Induced Apoptosis (ATIA) polypeptide in a subject sample
wherein an alteration in the level of expression or biological
activity relative to the expression or biological activity in a
reference indicates that the subject has or has a propensity to
develop a neoplasia.
[0009] In another aspect, the invention features a method of
diagnosing a subject as having, or having a propensity to develop a
neoplasia, the method comprising determining the level of
expression or biological activity of an ATIA nucleic acid in a
subject sample wherein an alteration in the level of expression
relative to the expression in a reference indicates that the
subject has or has a propensity to develop a neoplasia.
[0010] In one embodiment, the neoplasia is a solid tumor.
[0011] In one embodiment, the neoplasia is selected from the group
consisting of brain, pancreatic, and stomach.
[0012] In another aspect, the invention features a method of
diagnosing a subject as having, or having a propensity to develop a
brain neoplasia, the method comprising determining the level of
expression or biological activity of ATIA polypeptide in a subject
sample wherein an alteration in the level of expression or
biological activity relative to the expression or biological
activity in a reference indicates that the subject has or has a
propensity to develop a brain neoplasia.
[0013] In yet another aspect, the invention features a method of
diagnosing a subject as having, or having a propensity to develop a
brain neoplasia, the method comprising determining the level of
expression or biological activity of an ATIA nucleic acid in a
subject sample wherein an alteration in the level of expression
relative to the expression in a reference indicates that the
subject has or has a propensity to develop a brain neoplasia.
[0014] In a further aspect, the invention features a method of
monitoring a subject diagnosed as having a brain neoplasia, the
method comprising determining the level of expression or activity
of an ATIA polypeptide in a subject sample, wherein an alteration
in the level of expression or activity relative to the level of
activity in a reference indicates the severity of the brain
neoplasia in the subject.
[0015] In another aspect, the invention features a method of
monitoring a subject diagnosed as having a brain neoplasia, the
method comprising determining the expression of an ATIA nucleic
acid molecule in a subject sample, wherein an alteration in the
level of expression relative to the level of expression in a
reference indicates the severity of the brain neoplasia in the
subject.
[0016] In another aspect, the invention features a method of
determining the progression of a brain neoplasia in a subject, the
method comprising determining the expression or activity of an ATIA
polypeptide in a subject sample, wherein an alteration in the level
of expression or activity relative to the level of expression in a
reference indicates the progression of the a brain neoplasia in the
subject.
[0017] In a further aspect, the invention features a method of
determining the progression of a brain neoplasia in a subject, the
method comprising determining the expression of an ATIA nucleic
acid molecule in a subject sample, wherein an alteration in the
level of expression relative to the level of expression in a
reference indicates the progression of the a brain neoplasia in the
subject.
[0018] In one embodiment of the above aspects, the subject has, or
has a propensity to develop a brain neoplasia.
[0019] In another embodiment of the above aspects, the method can
be used to determine if a subject has a brain tumor. In a further
embodiment, the tumor is a glioblastoma. In another further
embodiment, the tumor is an astrocytoma.
[0020] In another aspect, the invention features a method of
diagnosing a subject as having, or having a propensity to develop a
glioblastoma, the method comprising determining the level of
expression or biological activity of an ATIA polypeptide in a
subject sample wherein an alteration in the level of expression or
biological activity relative to the expression or biological
activity in a reference indicates that the subject has or has a
propensity to develop a glioblastoma.
[0021] In another further aspect, the invention features a method
of diagnosing a subject as having, or having a propensity to
develop a glioblastoma, the method comprising determining the level
of expression or biological activity of an ATIA nucleic acid in a
subject sample wherein an alteration in the level of expression
relative to the expression in a reference indicates that the
subject has or has a propensity to develop a glioblastoma.
[0022] In one embodiment of any one of the above aspects, the
method is used to determine if a subject will be responsive to
TRAIL therapy.
[0023] In another embodiment of any one of the above aspects, the
method is used to determine if a subject will be responsive to
chemotherapy.
[0024] In still another embodiment of any one the above aspects,
the method is used to determine if a subject will be responsive to
radiation treatment.
[0025] In another embodiment of any one of the above aspects, the
ATIA nucleic acid comprises a sequence selected from the group
consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or
fragments thereof.
[0026] In a further embodiment of any one of the above aspects, the
ATIA polypeptide comprises a sequence selected from the group
consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or
fragments thereof.
[0027] In one embodiment of any one of the above aspects, the level
of expression is determined in an immunological assay. In a further
embodiment, the immunological assay is an enzyme-linked
immunosorbent assay (ELISA). In a related embodiment, the
immunological assay is an immunohistochemical assay. In still
another related embodiment, the ELISA is used to detect the
extracellular portion of ATIA.
[0028] In another preferred embodiment, the extracellular portion
comprises a sequence selected from the group consisting of SEQ ID
NO: 7, 8, 9 or 10, or fragments thereof.
[0029] In one embodiment of any one of the above aspects, the
expression of an ATIA nucleic acid molecule is detected using a
hybridization reaction comprising hybridizing the sample to one or
more primer sets. In one embodiment, the hybridization reaction is
a polymerase chain reaction.
[0030] In a related embodiment, the each one or more primer set
comprises a forward primer and a reverse primer, wherein the
forward primer is complementary to a nucleic acid sequence
corresponding to a nucleic acid sequence selected from SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8, or
fragments thereof, and the reverse primer is reverse complementary
to a nucleic acid sequence corresponding to a nucleic acid sequence
selected from SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, SEQ ID
NO: 7 and SEQ ID NO: 8 or fragments thereof.
[0031] In another embodiment of any one of the above aspects, the
subject is being treated for brain cancer.
[0032] In still another embodiment of any one of the above aspects,
the alteration is an increase. In a related embodiment, the
increase corresponds to an increased sensitivity to TRAIL-induced
cell death.
[0033] In another embodiment of any one of the above aspects, the
reference is a control subject sample. In another embodiment of any
one of the above aspects, the reference is a subject sample
obtained at an earlier time point. In another embodiment of any one
of the above aspects, the subject sample is a biological
sample.
[0034] In still another embodiment of any one of the above aspects,
the method is used to diagnose a subject as having a brain tumor.
In another embodiment of any one of the above aspects, the method
is used to determine the treatment regimen for a subject having a
brain tumor.
[0035] In yet another embodiment of any one of the above aspects,
the method is used to determine the prognosis of a subject. In a
related embodiment, a poor prognosis determines an aggressive
treatment regimen for the subject.
[0036] In another embodiment of any one of the above aspects, the
method further comprises obtaining a biological sample from the
subject. In a related embodiment, the sample is a blood sample.
[0037] In another aspect, the invention features an ATIA antibody
that specifically binds to an ATIA protein or fragment thereof.
[0038] In one embodiment, the ATIA protein or fragment thereof is
selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10, or fragments
thereof.
[0039] In another embodiment, the antibody binds to a an epitope
corresponding to amino acids 600-673 of epitope of an ATIA
polypeptide corresponding to SEQ ID NO: 2.
[0040] In another embodiment of any one of the above aspects, the
antibody is monoclonal.
[0041] In another aspect, the invention features a polypeptide
comprising an isolated ATIA protein, or fragment thereof, wherein
the protein is upregulated in brain neoplasia.
[0042] In one embodiment, the ATIA protein is at least 85%
identical to ATIA. In another embodiment, the ATIA protein
comprises the extracellular domain. In another related embodiment,
the extracellular domain comprises any one of SEQ ID NO: 7, 8, 9 or
10, a fragments thereof.
[0043] In another embodiment of any one of the above aspects, the
polypeptide is linked to a detectable amino acid sequence or an
affinity tag.
[0044] In another embodiment of any one of the above aspects, the
ATIA nucleic acid comprises a sequence selected from the group
consisting of: SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 and SEQ ID NO: 8 or fragments thereof.
[0045] In another embodiment of any one of the above aspects, the
ATIA polypeptide comprises a sequence selected from the group
consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
9 and SEQ ID NO: 10 or fragments thereof.
[0046] In another aspect, the invention features an isolated ATIA
nucleic acid molecule, wherein the nucleic acid molecule encodes a
polypeptide of any one of the aspects described herein.
[0047] In another aspect, the invention features an isolated ATIA
inhibitory nucleic acid molecule, wherein the inhibitory nucleic
acid molecule specifically binds at least a fragment of a nucleic
acid molecule encoding an ATIA protein.
[0048] In one embodiment, the invention features a vector
comprising a nucleic acid molecule encoding the nucleic acid
molecule of any one of the aspects as described herein.
[0049] In one embodiment of any one of the above aspects, the
vector is an expression vector. In another embodiment of any one of
the above aspects, the nucleic acid molecule is operably linked to
a promoter.
[0050] In another embodiment, the invention features a host cell
comprising a nucleic acid molecule of any one of the aspects as
described herein. In one embodiment, the cell expresses an ATIA
protein. In another embodiment, the cell is in vitro. In another
embodiment, the cell is in vivo. In a further embodiment, the cell
is a mammalian cell. In another further embodiment, the cell is a
human cell.
[0051] In another aspect, the invention features a double-stranded
RNA corresponding to at least a portion of an ATIA nucleic acid
molecule that encodes an ATIA protein, wherein the double-stranded
RNA is capable of altering the level of protein encoded by the ATIA
nucleic acid molecule.
[0052] In one embodiment, the RNA is an siRNA.
[0053] In another aspect, the invention features an antisense
nucleic acid molecule, wherein the antisense nucleic acid molecule
is complementary to an ATIA nucleic acid molecule that encodes an
ATIA protein, and wherein the antisense is capable of altering
expression from the nucleic acid molecule to which it is
complementary.
[0054] In still another aspect, the invention features an ATIA
biomarker purified on a biochip.
[0055] In another aspect, the invention features a microarray
comprising at least two nucleic acid molecules, or fragments
thereof, fixed to a solid support, wherein at least one of the
nucleic acid molecules is an ATIA nucleic acid molecule.
[0056] In still another aspect, the invention features a microarray
comprising at least two polypeptides, or fragments thereof, bound
to a solid support, wherein at least one of the polypeptides on the
support is an ATIA polypeptide.
[0057] In one embodiment of the above aspects, the ATIA nucleic
acid comprises a sequence selected from the group consisting of:
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID
NO: 8 or fragments thereof.
[0058] In one embodiment of the above aspects, the ATIA polypeptide
comprises a sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 4 SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10 or
fragments thereof.
[0059] In another aspect, the invention features a diagnostic kit
for the diagnosis of a brain neoplasia in a subject comprising a
primer set to detect an ATIA nucleic acid molecule, or fragment
thereof, and written instructions for use of the kit for detection
of a brain neoplasia.
[0060] In still another aspect, the invention features a diagnostic
kit for the diagnosis of cancer in a subject comprising a primer
set to detect an ATIA nucleic acid molecule, or fragment thereof,
and written instructions for use of the kit for detection of
cancer.
[0061] In still another aspect, the invention features a diagnostic
kit for the diagnosis of a brain neoplasia in a subject comprising
an antibody that specifically binds an ATIA polypeptide, or
fragment thereof, and written instructions for use of the kit for
detection of a brain neoplasia.
[0062] In one embodiment, the antibody binds to ATIA in a blood
sample.
[0063] In another aspect, the invention features a kit for
identifying a subject as having or having a propensity to develop a
brain neoplasia, comprising an adsorbent, wherein the adsorbent
retains an ATIA biomarker, and written instructions for use of the
kit for detection of a brain neoplasia.
[0064] In one embodiment of the above aspects, the ATIA nucleic
acid comprises a sequence selected from the group consisting of:
SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, SEQ ID NO: 7 and SEQ
ID NO: 8 or fragments thereof.
[0065] In one embodiment of the above aspects, the ATIA polypeptide
comprises a sequence selected from the group consisting of: SEQ ID
NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10
or fragments thereof.
[0066] In another aspect, the invention features a method of
altering the expression of an ATIA nucleic acid molecule in a cell,
the method comprising contacting the cell with an effective amount
of a compound capable of altering the expression of the ATIA
nucleic acid molecule.
[0067] In one embodiment, the compound is an antisense nucleic acid
molecule, a small interfering RNA (siRNA), or a double stranded RNA
(dsRNA) that inhibits the expression of an ATIA nucleic acid
molecule.
[0068] In another aspect, the invention features a method of
altering ATIA protein expression in a cell, the method comprising
contacting the cell with a compound capable of altering the
expression of an ATIA polypeptide.
[0069] In one embodiment, the cell is a human cell. In another
embodiment, the cell is a neoplastic cell.
[0070] In another aspect, the invention features a method of
treating or preventing cancer, the method comprising administering
to a subject in need thereof an effective amount of a small
molecule that alters expression of an ATIA polypeptide.
[0071] In one embodiment, the small molecule is an inhibitory
nucleic acid.
[0072] In another embodiment, the inhibitory nucleic acid is an
siRNA.
[0073] In another further embodiment, the small molecule is a
chemical inhibitor.
[0074] In a related embodiment, the chemical inhibitor is
cyclohexamide.
[0075] In another particular aspect, the invention features a
method of identifying a compound that inhibits cancer the method
comprising contacting a cell that expresses an ATIA nucleic acid
molecule with a candidate compound, and comparing the level of
expression of the nucleic acid molecule in the cell contacted by
the candidate compound with the level of expression in a control
cell not contacted by the candidate compound, wherein an alteration
in expression of the ATIA nucleic acid molecule identifies the
candidate compound as a compound that inhibits cancer.
[0076] In another aspect, the invention features a method of
treating or preventing a brain neoplasia, the method comprising
administering to a subject in need thereof an effective amount of a
pharmaceutical composition that alters expression of an ATIA
polypeptide.
[0077] In another aspect, the invention features a method of
identifying a compound that inhibits a brain neoplasia the method
comprising contacting a cell that expresses an ATIA nucleic acid
molecule with a candidate compound, and comparing the level of
expression of the nucleic acid molecule in the cell contacted by
the candidate compound with the level of expression in a control
cell not contacted by the candidate compound, wherein an alteration
in expression of the ATIA nucleic acid molecule identifies the
candidate compound as a compound that inhibits a brain
neoplasia.
[0078] In one embodiment of any one of the above aspects, the
alteration in expression is a decrease in transcription.
[0079] In one embodiment of any one of the above aspects, the
alteration in expression is a decrease in translation.
[0080] In one embodiment of any one of the above aspects, the
method further comprises comprising treating the subject with a
chemotherapeutic agent. In one embodiment of any one of the above
aspects, the method further comprises treating the subject with
radiation. In a related embodiment, the chemotherapeutic agent is
an agent that can cross the blood-brain barrier. In a further
related embodiment, the chemotherapeutic agent is selected from
etoposide or cisplatin.
[0081] In another aspect, the invention features a kit for of
treating or preventing a brain neoplasia, the kit comprising an
effective amount of a pharmaceutical composition that alters
expression of an ATIA polypeptide and instructions for use.
[0082] In another aspect, the invention features a kit for of
treating or preventing a brain neoplasia, the kit comprising an
effective amount of a pharmaceutical composition that alters
expression of an ATIA nucleic acid and instructions for use.
[0083] In one embodiment of any one of the above aspect, the kit
further comprises an additional chemotherapeutic agent.
[0084] In another aspect, the invention features a method of
identifying a compound that inhibits cancer, the method comprising
contacting a cell that expresses an ATIA polypeptide with a
candidate compound, and comparing the level of expression of the
polypeptide in the cell contacted by the candidate compound with
the level of polypeptide expression in a control cell not contacted
by the candidate compound, wherein an alteration in the expression
of the ATIA polypeptide identifies the candidate compound as a
compound that inhibits cancer.
[0085] In another aspect, the invention features a method of
identifying a compound that inhibits cancer, the method comprising
contacting a cell that expresses an ATIA polypeptide with a
candidate compound, and comparing the biological activity of the
polypeptide in the cell contacted by the candidate compound with
the level of biological activity in a control cell not contacted by
the candidate compound, wherein an alteration in the biological
activity of the ATIA polypeptide identifies the candidate compound
as a candidate compound that inhibits cancer.
[0086] In another aspect, the invention features a method of
identifying a compound that inhibits a brain neoplasia, the method
comprising contacting a cell that expresses an ATIA polypeptide
with a candidate compound, and comparing the level of expression of
the polypeptide in the cell contacted by the candidate compound
with the level of polypeptide expression in a control cell not
contacted by the candidate compound, wherein an alteration in the
expression of the ATIA polypeptide identifies the candidate
compound as a compound that inhibits a brain neoplasia.
[0087] In another aspect, the invention features a method of
identifying a compound that inhibits a brain neoplasia, the method
comprising contacting a cell that expresses an ATIA polypeptide
with a candidate compound, and comparing the biological activity of
the polypeptide in the cell contacted by the candidate compound
with the level of biological activity in a control cell not
contacted by the candidate compound, wherein an alteration in the
biological activity of the ATIA polypeptide identifies the
candidate compound as a candidate compound that inhibits a brain
neoplasia.
[0088] In one embodiment of any one of the above methods, the brain
neoplasia is a brain tumor. In a related embodiment, the tumor is a
glioblastoma. In another related embodiment, the tumor is an
astrocytoma.
[0089] In one embodiment of any one of the above methods, the cell
is in vitro. In one embodiment of any one of the above methods, the
cell is in vivo.
[0090] In one embodiment of any one of the above methods, the
alteration in expression is assayed using an immunological assay,
an enzymatic assay, or a radioimmunoassay.
[0091] In one embodiment of any one of the above methods, the ATIA
nucleic acid comprises a sequence selected from the group
consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7 and SEQ ID NO: 8, or fragments thereof.
[0092] In one embodiment of any one of the above methods, the ATIA
polypeptide comprises a sequence selected from the group consisting
of: SEQ ID NO: 2, SEQ ID NO: 4 SEQ ID NO: 6, SEQ ID NO: 9 and SEQ
ID NO: 10 or fragments thereof.
[0093] Other aspects of the invention are described in or are
obvious from the following disclosure, and are within the ambit of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
drawings, incorporated herein by reference. Various preferred
features and embodiments of the present invention will now be
described by way of non-limiting example and with reference to the
accompanying drawings in which:
[0095] FIG. 1 (A-C) shows cloning of the ATIA gene. (A) The
structural scheme of ATIA gene. (B) The expression levels of ATIA
in different tissues. (C) ATIA expression in TRAF2-/- mouse
embryonic fibroblasts (MEFs) examined by Northern blotting.
[0096] FIGS. 2 (A and B) are graphs that shows ATIA protects cells
against TNF-induced apoptosis. The graphs show ectopic expression
of ATIA protects TRAF-/- MEFS (A) and MCF7 cells (B).
[0097] FIG. 3 (A-C) shows the cellular localization of ATIA
protein. (A) Expression of ATIA full-length and ATIAc YF P fusion
proteins. (B) and (C) shows confocal microscopy of living wild-type
ME Fs transiently transfected with EY FP-tagged ATIA full-length
and ATIAc cons tructs. The bottom panel shows mitochondrial
staining that was performed by MitoTracker.
[0098] FIG. 4 shows the results of western blot experiments
examining ATIA localization by cell fractionation. Wild type MEF
cells were collected for fractionation before and after pronase
treatment. Western blotting was carried out with different
antibodies. * nonspecific band.
[0099] FIG. 5 (A-E) shows ATIA null mice are generated and more
sensitive to TNF toxicity. (A) is a schematic diagram of ATIA
knockout strategy. (B) and (C) show the generation of ATIA+/- ES
cells (B) and ATIA+/- and ATIA-/- mice (C) was confirmed by PCR.
(D) and (E) shows that ATIA KO mice are more sensitive to
TNF-induced apoptosis. Survival curves of wild-type (n=5) and ATIA
KO (n=8) after TNF treatment. (D) shows histological analysis of
liver tissue isolated 4 hours after administration of GaIN and
INFcc (E). Tissue sections were analyzed by H&E (left panels)
and TUNNEL (right panels) staining.
[0100] FIGS. 6 (A and B) are graphs that show ATIA-/- MEFs are more
sensitive to TNF-induced apoptosis. (A) ATIA-/- MEFs are more
sensitive to TNF-induced apoptosis. Wild-type and ATIA-/- MEFs were
treated with TNF.alpha. (30 ng/ml) and indicated concentrations of
cycloheximide for 16 hours. Cell survival was measured by MTT. (B)
shows that ATIA-/- MEFs are not more sensitive to UV (left) and
Staurosporine treatment (right).
[0101] FIGS. 7 (A and B) show that ATIA inhibits TGFB signaling.
(A) TNFb-induced Smad2 phosphorylation is increased in ATIA-/-
MEFs. (B) ATIA deletion has no effect on BMP4 signaling.
[0102] FIG. 8 shows that two forms of ATIA protein are glycosylated
differently.
[0103] FIG. 9 is a graph that shows reactive oxygen species (ROS)
generation is elevated in TIA-/- MEFs.
[0104] FIG. 10 shows the results of Western Blot showing that ATIA
protein is highly expressed in brain tumor.
[0105] FIG. 11 shows the results of Western Blot showing that ATIA
protein expression in brain tumor cell lines.
[0106] FIG. 12 shows a panel of human glioblastoma tissue stained
with anti-ATIA antibody.
[0107] FIG. 13 shows that the soluble form of ATIA is present in
the culture medium from A172 glioblastoma cells. The results shows
are from a Western blot assay.
[0108] FIG. 14 shows results from the tissue array.
[0109] FIG. 15 is a Table that shows the distribution of ATIA
positive cases in the glioblastoma array.
[0110] FIG. 16 shows representative panels of immunohistochemical
staining with anti vasorin (R and D as control) from the
glioblastoma tissue array. Glioblastoma stage IV samples are shown
in the panels on the right and adjacent normal brain tissues are
shown in the panels on the left.
[0111] FIG. 17 shows the distribution of ATIA positive cases in the
mixed tissue array.
[0112] FIG. 18 shows the western results of human tissue blots. The
top panel shows the results of vasorin protein detection in human
normal tissues. The bottom panel shows the results of human vasorin
protein detection in human tumor tissues.
[0113] FIG. 19 shows glioblastoma cells that express ATIA are more
sensitive to TRAIL-induced cell death. The left panel shows a
western blot showing ATIA expression in A172, U87MG and 251
glioblastoma cells. The graph shows cell viability after the cells
are treated with TRAIL for 24 hours.
[0114] FIG. 20 shows knocking down ATIA expression renders cells
sensitive to etoposide or cisplatinum treatment. The top panel
shows that siRNA knockdown reduces ATIA expression. The bottom
panel is a graph where ATIA expression was partially knocked down
and an MTT assay was used to determine cell proliferation.
[0115] FIG. 21 is a graph that shows knocking down ATIA expressing
renders cells sensitive to cisplatinum treatment. The graph shows
percent survival of cells.
[0116] FIGS. 22 (A and B) are graphs. (A) shows the 1-599 ATIA
mutant, which lost its mitochondrial localization, does not protect
cells against TNF-induced apoptosis. (B) shows the mitochondrial
localized 232-637 ATIA mutant protects cells against TNF-induced
apoptosis.
[0117] FIG. 23 shows that ATIA is induced following CoCl.sub.2
treatment. In FIG. 23, CoCl.sub.2 is a hypoxia mimic, which induced
hypoxia in treated cells. ATIA protein expression particularly in
the lower band (the mitochondrial ATIA), is considerably
increased). ATIA promoter has two HIF responsive sites.
[0118] FIG. 24 is a Western blot that shows ATIA is induced under
hypoxia conditions.
[0119] FIG. 25 is a graph that shows increased sensitivity to CoCl
induced Hypoxia in ATIA KO MEFs. Wt and ATIA-/- MEfs are treated
with CoCl2 and cell death is detected at different time points as
indicated in the figure. ATIA-/- MEFs are much more sensitive to
CoCl2-induced cell death.
[0120] FIG. 26 is a graph that shows increased sensitivity of A172
cells to CoCl.sub.2 treatment when ATIA is knocked down. Human
glioblastoma A172 cells are transfected with siRNAs of lamin or
ATIA and then treated with CoCl.sub.2. ATIA knock down renders
cells more sensitive to CoCl.sub.2 induced cell death.
[0121] FIG. 27 is a graph that shows increased sensitivity of A172
cells to hypoxia treatment when ATIA is knocked down. A172 cells
were transfected with siRNA targeting lamin or ATIA for o/n, cells
were then subjected to 24 h and 48 h of hypoxia (0% Oxygen). Cell
death was determined using Annexin V/PI staining method and
analyzed using flow cytometry.
[0122] FIG. 28 is two panels that show the effect of cyclohexamide
on ATIA stability and cell apoptosis. The top panel is a graph that
shows the number of annexin positive cells after cyclohexamide
treatment. The bottom panel is a blot that shows vasorin expression
after cyclohexamide treatment.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0123] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them unless specified otherwise.
[0124] By "antibody" is meant any immunoglobulin polypeptide, or
fragment thereof, having immunogen binding ability.
[0125] By "ANTI-TNF Induced Apoptosis (ATIA)" is meant to refer to
a type I membrane protein that binds TGF-beta. In preferred
embodiments, mouse ATIA corresponds to the nucleic acid sequence
set forth by NCBI reference No. NM_139307 (SEQ ID NO: 1) and the
corresponding amino acid sequence set forth by NCBI reference No.
NP_647468 (SEQ ID NO: 2). In other preferred embodiments, rat ATIA
corresponds to the nucleic acid sequence set forth by NCBI
reference No. NM_001109382 (SEQ ID NO: 3) and the corresponding
amino acid sequence set forth by NCBI reference No. NP_001102852
(SEQ ID NO: 4).
[0126] By "vasorin" is meant to refer to a type I membrane protein
that binds TGF-beta. In preferred embodiments, human vasorin
corresponds to the nucleic acid sequence set forth by NCBI
reference No. NM_138440 (SEQ ID NO: 5) and the corresponding amino
acid sequence set forth by NCBI reference No. AAO27704 (SEQ ID NO:
6).
[0127] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, for example, hydroxyproline,
gamma-carboxyglutamate, and O-phosphoserine, phosphothreonine.
[0128] By "biomarker" is meant any protein or polynucleotide having
an alteration in expression level or activity that is associated
with a disease or disorder, for example cancer, for example
glioblastoma.
[0129] By "detectable amino acid sequence" or "detectable moiety"
is meant a composition that when linked with the nucleic acid or
protein molecule of interest renders the latter detectable, via any
means, including spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include radioactive isotopes, magnetic beads, metallic beads,
colloidal particles, fluorescent dyes, electron-dense reagents,
enzymes (for example, as commonly used in an ELISA), biotin,
digoxigenin, or haptens.
[0130] A "labeled nucleic acid or oligonucleotide probe" is one
that is bound, either covalently, through a linker or a chemical
bond, or noncovalently, through ionic bonds, van der Waals forces,
electrostatic attractions, hydrophobic interactions, or hydrogen
bonds, to a label such that the presence of the nucleic acid or
probe may be detected by detecting the presence of the label bound
to the nucleic acid or probe.
[0131] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, bearing a series of
specified nucleic acid elements that enable transcription of a
particular gene in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, including
constitutive or inducible promoters, tissue-preferred regulatory
elements, and enhancers.
[0132] By "fragment" is meant a portion (e.g., at least 10, 25, 50,
100, 125, 150, 200, 250, 300, 350, 400, or 500 amino acids or
nucleic acids) of a protein or nucleic acid molecule that is
substantially identical to a reference protein or nucleic acid and
retains the biological activity of the reference. In some
embodiments the portion retains at least 50%, 75%, or 80%, or more
preferably 90%, 95%, or even 99% of the biological activity of the
reference protein or nucleic acid described herein.
[0133] A "host cell" is any prokaryotic or eukaryotic cell that
contains either a cloning vector or an expression vector. This term
also includes those prokaryotic or eukaryotic cells that have been
genetically engineered to contain the cloned gene(s) in the
chromosome or genome of the host cell.
[0134] By "inhibitory nucleic acid" is meant a double-stranded RNA,
siRNA (short interfering RNA), shRNA (short hairpin RNA), or
antisense RNA, or a portion thereof, or a mimetic thereof, that
when administered to a mammalian cell results in a decrease (e.g.,
by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a
target gene. Typically, a nucleic acid inhibitor comprises at least
a portion of a target nucleic acid molecule, or an ortholog
thereof, or comprises at least a portion of the complementary
strand of a target nucleic acid molecule.
[0135] The terms "isolated," "purified," or "biologically pure"
refer to material that is free to varying degrees from components
which normally accompany it as found in its native state. Various
levels of purity may be applied as needed according to this
invention in the different methodologies set forth herein; the
customary purity standards known in the art may be used if no
standard is otherwise specified.
[0136] By "isolated nucleic acid molecule" is meant a nucleic acid
(e.g., a DNA, RNA, or analog thereof) that is free of the genes
which, in the naturally-occurring genome of the organism from which
the nucleic acid molecule of the invention is derived, flank the
gene. The term therefore includes, for example, a recombinant DNA
that is incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a
prokaryote or eukaryote; or that exists as a separate molecule (for
example, a cDNA or a genomic or cDNA fragment produced by PCR or
restriction endonuclease digestion) independent of other sequences.
In addition, the term includes an RNA molecule which is transcribed
from a DNA molecule, as well as a recombinant DNA which is part of
a hybrid gene encoding additional polypeptide sequence.
[0137] "Microarray" is meant to refer to a collection of nucleic
acid molecules or polypeptides from one or more organisms arranged
on a solid support (for example, a chip, plate, or bead).
[0138] By "neoplasia" as used herein is meant the abnormal
proliferation of cells. In preferred embodiments, when the growth
of a neoplasm exceeds, and is uncoordinated with, that of the
normal tissues around it may cause a lump or tumor. A neoplasia may
be benign, pre-malignant or malignant.
[0139] By "nucleic acid" is meant an oligomer or polymer of
ribonucleic acid or deoxyribonucleic acid, or analog thereof. This
term includes oligomers consisting of naturally occurring bases,
sugars, and intersugar (backbone) linkages as well as oligomers
having non-naturally occurring portions which function similarly.
Such modified or substituted oligonucleotides are often preferred
over native forms because of properties such as, for example,
enhanced stability in the presence of nucleases.
[0140] "Complimentary nucleic acid sequences" refer to contiguous
DNA or RNA sequences which have compatible nucleotides (e.g., A/T,
G/C) in corresponding positions, such that base pairing between the
sequences occurs. For example, the sense and anti-sense strands of
a double-stranded DNA helix are known in the art to be
complimentary.
[0141] By "protein" is meant any chain of amino acids, or analogs
thereof, regardless of length or post-translational
modification.
[0142] By "reference" is meant a standard or control condition.
[0143] By "siRNA" is meant a double stranded RNA. Optimally, an
siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has
a 2 base overhang at its 3' end. These dsRNAs can be introduced to
an individual cell or to a whole animal; for example, they may be
introduced systemically via the bloodstream. Such siRNAs are used
to downregulate mRNA levels or promoter activity. In certain
preferred embodiments, the siRNA downregulates ATIA levels. In
certain embodiments, ATIA siRNA are commercially prepared
siRNA.
[0144] By "specifically binds" is meant a molecule (e.g., peptide,
polynucleotide) that recognizes and binds a protein or nucleic acid
molecule of the invention, but which does not substantially
recognize and bind other molecules in a sample, for example, a
biological sample, which naturally includes a protein of the
invention.
[0145] By "substantially identical" is meant a protein or nucleic
acid molecule exhibiting at least 50% identity to a reference amino
acid sequence (for example, any one of the amino acid sequences
described herein) or nucleic acid sequence (for example, any one of
the nucleic acid sequences described herein). Preferably, such a
sequence is at least 60%, more preferably 80% or 85%, and most
preferably 90%, 95% or even 99% identical at the amino acid level
or nucleic acid to the sequence used for comparison.
[0146] Sequence identity is typically measured using sequence
analysis software (for example, Sequence Analysis Software Package
of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software
matches identical or similar sequences by assigning degrees of
homology to various substitutions, deletions, and/or other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. In an exemplary approach to determining
the degree of identity, a BLAST program may be used, with a
probability score between e-3 and e-100 indicating a closely
related sequence.
[0147] Other definitions appear in context throughout the
disclosure.
Methods of the Invention
[0148] The invention features diagnostics and therapeutic methods
based on the discovery of ATIA as a diagnostic marker and as a
potential therapeutic target for certain cancers, in particular
brain cancers, such as human glioblastoma or astrocytoma.
[0149] The present invention is based, in part, on the finding that
there is high expression of ATIA in certain types of cancers, in
particular in brain cancers, for example human glioblastoma and
astrocytoma. The present inventors have found that ATIA is a
hypoxia-inducible gene, and that under hypoxia conditions ATIA
protein expression is considerably increased, and deletion of ATIA
renders cells sensitive to hypoxia-induced apoptosis.
[0150] Reported herein is the novel finding of a soluble form of
ATIA that suggests that blood testing of ATIA level will provide an
easy, quick and early diagnosis of disease.
[0151] Tumors that begin in brain tissue are known as primary brain
tumors and are classified by the type of tissue in which they
originate. The most common brain tumors are gliomas, which begin in
the glial or supportive tissue. There are several types of gliomas.
Astrocytomas are brain tumors that arise from small, star-shaped
cells called astrocytes. They may grow anywhere in the brain or
spinal cord. In adults, astrocytomas most often arise in the
cerebrum. In children, they occur in the brain stem, the cerebrum
and the cerebellum. A grade III astrocytoma is sometimes called
anaplastic astrocytoma. A grade IV astrocytoma is usually called
glioblastoma multiforme. Brain stem gliomas are brain tumors that
occur in the lowest, stem-like part of the brain. The brain stem
controls many vital functions. Most brain stem gliomas are
high-grade astrocytomas. Ependymomas are brain tumors that usually
develop in the lining of the ventricles. They may also occur in the
spinal cord. Although these tumors can develop at any age, they are
most common in childhood and adolescence. Oligodendrogliomas are
tumors that occur in the cells that produce myelin, the fatty
covering that protects nerves. These tumors usually arise in the
cerebrum. They are rare, grow slowly and usually do not spread into
surrounding brain tissue. They occur most often in middle-aged
adults but have been found in people of all ages.
[0152] There are other types of brain tumors that do not begin in
glial tissue. For example, medulloblastomas were once thought to
develop from glial cells. However, recent research suggests that
these tumors develop from primitive or developing nerve cells that
normally do not remain in the body after birth. For this reason,
medulloblastomas are sometimes called primitive neuroectodermal
tumors (PNET). Most medulloblastomas arise in the cerebellum;
however, they may occur in other areas as well. These tumors occur
most often in children and are more common in boys than in girls.
Meningiomas are tumors that grow from the meninges, or membranes
that enclose the brain and spinal cord. They are usually benign.
Because these tumors grow very slowly, the brain may be able to
adjust to their presence. Meningiomas often grow quite large before
they cause symptoms. They occur most often in women between 30 and
50 years of age. Schwannomas are benign and begin in Schwann cells,
which produce the myelin that protects the acoustic nerve, or the
nerve of hearing. They occur mainly in adults. These tumors affect
women twice as often as men. Craniopharyngiomas are tumors that
develop in the region of the pituitary gland near the hypothalamus.
They are usually benign but are sometimes considered malignant
because they can press on or damage the hypothalamus, a region of
the brain, and affect vital functions. These tumors occur most
often in children and adolescents. Germ cell tumors are tumors that
arise from developing sex cells or germ cells. The most frequent
type of germ cell tumor in the brain is the germinoma. Pineal
region tumors are tumors that occur in or around the pineal gland,
a tiny organ near the center of the brain. The tumor can be slow
growing (pineocytoma), or fast growing (pineoblastoma). The pineal
region is very difficult to reach, and these tumors often cannot be
removed.
[0153] The symptoms of brain tumors depend on their size and
location in the brain. Symptoms often are caused by damage to vital
tissue and pressure on the brain as the tumor grows within the
limited space in the skull. They may be caused by swelling and a
buildup of fluid around the tumor, a condition called edema.
Symptoms also may be due to hydrocephalus, which occurs when the
tumor blocks the flow of cerebrospinal fluid and causes a build-up
in the ventricles. If a brain tumor grows very slowly, its symptoms
may not appear for some time.
[0154] The most frequent symptoms of brain tumors include headaches
that tend to be worse in the morning and ease during the day,
seizures or convulsions, nausea or vomiting, weakness or loss of
feeling in the arms or legs, stumbling or lack of coordination in
walking, abnormal eye movements or changes in vision, drowsiness,
changes in personality or memory, changes in speech.
Anti TNF Induced Apoptosis (ATIA)
[0155] The Anti-TNF Induced Apoptosis (ATIA) gene was cloned
through screening for proteins that protect cells against
TNF-induced apoptosis. Four years before the cloning of ATIA, the
human homolog of ATIA, vasorin, was reported as a TGF-beta binding
protein.
[0156] In certain embodiments, mouse ATIA corresponds to the
nucleotide sequence set forth by NCBI reference No. NM_139307,
shown below as SEQ ID NO: 1, and the corresponding amino acid
sequence set forth by NCBI reference No. NP_647468, shown below as
SEQ ID NO: 2.
TABLE-US-00001 SEQ ID NO: 1 1 agagaccagc ctcttacgag tcaacttcga
gtctggagcc ggagccagag accggggctg 61 ggaaacccca gcccgggacg
ggacgcagca gcctctggat cccgggaccc cggacctctc 121 aggaccggcc
agaggtgaag gactgaggcc ccactgaggc cttggaccgc accgcctggc 181
tccttcagcc gcagtcgtct cctgggacag aagatgcact ccaggagctg cctgccacct
241 ctcctgttgt tgcttctggt gctcctgggg tctggagtac agggttgccc
atcaggctgc 301 cagtgcaacc agccacagac agtcttctgc actgcccgtc
agggaaccac agtgccccga 361 gacgtgccac ctgacacagt gggcctgtac
atctttgaga acggcatcac gacacttgat 421 gtgggctgtt ttgctggcct
tccgggcctg cagcttctgg acttgtcaca gaaccagatc 481 actagcctgc
ccgggggcat ctttcagcca cttgttaacc tcagtaacct ggacctgact 541
gccaacaaac tgcacgagat ctccaacgag accttccgtg gcctgcggcg cctggagcgc
601 ctctacctgg gcaagaaccg aattcgccac atccaaccgg gtgccttcga
cgcgcttgat 661 cgcctcctgg agctcaagct gccagacaat gagcttcggg
tgttgccccc attgcacttg 721 ccccgcctgc tgctgcttga cctcagccac
aacagcatcc cagccctgga agccggaata 781 ctggataccg ccaatgtaga
ggcattgagg ttggctggcc tagggctgcg gcagctggat 841 gaggggcttt
ttggccgcct tctcaacctc catgacttgg atgtttctga caaccagttg 901
gagcatatgc catctgtgat tcaaggcctg cgtggcctga cacgcctgcg gctggctggc
961 aacacccgta ttgcccagat acggcccgag gacctcgctg gtctgactgc
cctacaggaa 1021 ttggatgtga gcaacctaag cctgcaggcc ctgcccagtg
acctctcgag tctctttccc 1081 cgcctgcgcc tcttagcagc tgccaggaac
cccttcaact gcttgtgccc cttgagctgg 1141 tttggtcctt gggtgcgtga
gaaccatgtt gtgttggcca gccctgagga gacgcgttgt 1201 cactttccac
ccaagaatgc tggccgactg ctcctggatc tggattatgc agattttggc 1261
tgcccagtca ccactaccac ggccacagta cctactataa ggtctactat cagggaaccc
1321 acactttcaa cttctagcca agctcccacc tggcccagcc tcacagagcc
aactacccag 1381 gcctccaccg tactatcgac tgccccacca accatgaggc
cagctcctca gccccaggac 1441 tgtccagcat ccatctgcct gaatggtggt
agctgccgtt tgggagcaag acaccactgg 1501 gagtgcctat gccctgaggg
cttcattggc ctgtactgtg agagtccagt ggagcaaggg 1561 atgaagccca
gctccatacc agacactcca aggccccctc cactgctgcc tctcagcatt 1621
gagccggtga gccccacctc cttgcgtgtg aagctgcagc gctacttgca gggtaacact
1681 gtgcagctac ggagcctccg gctcacctat cgcaacctgt ctggccctga
caaacgactg 1741 gtgacattac ggctgcctgc ttcacttgca gagtatacag
tcacccagct gcgacccaat 1801 gccacctatt ctatctgtgt cacacccttg
ggagctggac ggacacctga aggtgaggag 1861 gcctgtgggg aggccaacac
ttcccaggca gtccgctcta accatgcccc agttacccag 1921 gcccgtgagg
gcaacctgcc actcctcatt gcgcctgccc tggctgctgt acttctggct 1981
gtgttagccg ctgcaggggc agcctactgt gtgcggcggg cacgggcaac ttctacagct
2041 caggacaaag ggcaggtggg gccagggact ggacccctgg aactagaggg
ggtgaaagcc 2101 cctttggagc caggctccaa ggcaacagag ggaggtgggg
aggctttgtc aggtggtcct 2161 gaatgtgagg tgcctcttat gggctaccca
gggcccagcc ttcagggggt cctccctgct 2221 aagcactaca tttagactgg
tgagaaagag cagccagggg gtcaggcttt cagtcaccac 2281 cctcctgctg
ccacagaagg aagttctcag tatacaccac agtgcacgtg catgatggag 2341
ctgtgggacc ctctctgggc tgggtctcat ctgtaagctg ctacagccca gatgaactct
2401 gccagccgcc agtgcatcca gtacagcgcc tgccatcttg tgcaatgtgc
aaccctggga 2461 tgtgagccct gccatgtgct ggtaacatgg ctaggcatgt
tgggcttccc aaaccatgga 2521 gtctggtaac cagtgaagga agcccccaga
aataatgagt ggggaaggta ctagggcact 2581 ggccttggcc tcaaaagtgc
aggcacactt gaaactggaa aggaaggtgc tctgggcaca 2641 tgtggatttg
cttctattgt tttgttttgt tttttctaat gtatttataa aagatctttt 2701
cccatttatg ctgggaaagt gtttttcaaa ctcagtgaca aggactttgg tttttgtaag
2761 actgttgatg atatgaaggc cttttgtaag aaaataaaaa ataaagtaaa
ttgcctgtct 2821 ctctggttgg gcttgagatt taaggtctgt ggacatgcac
aggattggag ggctgctgcc 2881 ctgccattag aatgctctag ccatgggtcc
tgacccatgg taaggcttgc acttgggtgg 2941 ggccggaaaa tggacttgtt
aggtagctta ccctaggcta ggcctcctct tctgccagca 3001 ggaaccacag
tgcttaatgt ataaggcaga aaggggctca tagaaaacac agaacacaaa 3061
gggaggtcac atccctcctt gggtgttctg aaagtgcagt ccactatctt caactagaga
3121 agacagcctg gagcttcctc attctagagc ctaacagctg atcctgggac
caggtggctt 3181 ccagactgg SEQ ID NO: 2 1 mhsrsclppl lllllvllgs
gvqgcpsgcq cnqpqtvfct arqgttvprd vppdtvglyi 61 fengittldv
gcfaglpglq lldlsqnqit slpggifqpl vnlsnldlta nklheisnet 121
frglrrlerl ylgknrirhi qpgafdaldr llelklpdne lrvlpplhlp rlllldlshn
181 sipaleagil dtanvealrl aglglrqlde glfgrllnlh dldvsdnqle
hmpsviqglr 241 gltrlrlagn triaqirped lagltalqel dvsnlslqal
psdlsslfpr lrllaaarnp 301 fnclcplswf gpwvrenhvv laspeetrch
fppknagrll ldldyadfgc pvttttatvp 361 tirstirept lstssqaptw
psltepttqa stvlstappt mrpapqpqdc pasiclnggs 421 crlgarhhwe
clcpegfigl ycespveqgm kpssipdtpr pppllplsie pvsptslrvk 481
lqrylqgntv qlrslrltyr nlsgpdkrlv tlrlpaslae ytvtqlrpna tysicvtplg
541 agrtpegeea cgeantsqav rsnhapvtqa regnlpllia palaavllav
laaagaaycv 601 rraratstaq dkgqvgpgtg plelegvkap lepgskateg
ggealsggpe cevplmgypg 661 pslqgvlpak hyi
[0157] In other embodiments, rat ATIA corresponds to the nucleotide
sequence set forth by NCBI reference No. NM_001109382, shown below
as SEQ ID NO: 3, and the corresponding amino acid sequence set
forth by NCBI reference No. NP_001102852, shown below as SEQ ID NO:
4.
TABLE-US-00002 SEQ ID NO: 3 1 ggagcccggg gttgggagac ccggacgcag
tagcctccgg atcccgggac cccggacctt 61 tcaggaccgg ccggaggcga
aggactgagg ccccattgag gccttgggcc gcaccgcccc 121 gctccctcag
ccacagtcgt ctcccgggac agaagatgca ctccaggagc tgcctgccac 181
ctcttctgtt gttgctcctg gtgctcctgg ggtctggagt acagagctgc ccatcaggct
241 gccagtgcaa ccaaccacag acagtcttct gcactgcccg tcagggaacc
acggtgcccc 301 gagacgtgcc gcctgacaca gtgggcctgt acatctttga
gaacggcatc actacacttg 361 atgtaggctg ttttgctggc ttcccaggcc
tgcagcttct ggacttgtca cagaaccaga 421 tcactagcct gcccggtggc
atctttcagc cacttgtgaa cctcagtaac ctggacctga 481 ctgctaacaa
actgcacgag atctccaacg agaccttccg tggcctgcgg cgcctcgaac 541
gcctctacct gggcaagaac cgcattcgcc acatccagcc tggtgccttc gatgcacttg
601 accacctcct ggagctcaag ctgccagaca atgagcttcg ggtgctgccc
ccactgcact 661 tgcctcgcct gctgctgctt gacctcagcc acaacagtat
cccagccctg gaagctggaa 721 tactggatac tgccaatgtg gaggcactgc
ggctggctgg cctcgggctg cggcagctgg 781 atgaggggct ttttggccgc
cttcgcaacc tccatgacct ggatgtttct gacaaccagt 841 tggggcacat
gccctccgtg attcaaggcc tgcgtggcct gacacgcctg cggctggctg 901
gcaacacccg gattgcccag atccggcccg aggacctcgc tggcctgact gccctacagg
961 aactggatgt gagcaacctg agcctgcagg ccctgcccag tgacctctcc
agtctctttc 1021 cccgcctgcg cctcctagca gctgcccgaa acccctttaa
ctgcttatgc cccttgagct 1081 ggtttggtcc ttgggttcgt gagagccatg
ttgtgctggc cagccctgag gagacacgtt 1141 gtcacttccc acccaagaac
gccggccgac tgctcctgga gctggattat gcagattttg 1201 gctgcccagt
caccactacc acagccacag ttcctactat aaggcctact gtcagggagc 1261
ccacaccttc aacttccagc caagctccca cctggcccag ccccacagag ccaactaccc
1321 aggcccccat cgtactgtcc actgccccac caaccatgag gccggctcct
cagccccagg 1381 actgtccagc atccatctgc ctgaatggtg gtagctgccg
tgtaggggca aaacaccacc 1441 tggagtgcct gtgccccgag ggcttcattg
gcctgtactg tgagagtccc gtggaacaaa 1501 ggacaaagcc cagctccata
ccggacaccc cacggccccc gcggctgctg cctctgcgca 1561 ttgagccggt
gagccccacc tccctgcgtg tggagctgca gcgctacctg cagggcaaca 1621
ccgtgcagct gcggagcctc cggctcacct accgcaacct gtctggccct gacaagcggc
1681 tggtgacgct gcggctgcct gcttcacttg cagagtacac agtcacccag
ctgcggccca 1741 atgccaccta ttctatctgt gtcacagccc tgggagctgg
gcggacacct gaaggtgagg 1801 aggcctgtgg ggaggccaac actccccagg
ccgtccgctc caaccatgcc ccagtcaccc 1861 aggcccggga gggcaacctg
ccactcctca ttgcacccgc cctggctgct gtgcttctgg 1921 ctgtgttggc
tgcctcgggg gcagtctact gtgtgcgacg ggcgcgggca agttccacag 1981
ctcaggacaa agggcaggtg ggaccaggga ccgggcccct ggaactagag ggggtgaaag
2041 tccccttgga gccaggctcc aaggcatcag agggaggcgg ggaggcccta
tcaggtggtc 2101 ctgaatgtga ggtgcccctc atgggctacc cagggcccag
tcttcagggg gtcctccctg 2161 ctcagcccta catttaagca cgtgagaagg
agcagccagg aggctgggct ttcagtctcc 2221 accctcctgc tgctacagaa
ggaagttctc aatgcgcacc acagtgcaca tgtgtgaccg 2281 gtgctgtggg
acagcagcca gtccccgacc ctctctgggc tgggtcatct gaaagctgct 2341
acagcccaaa tgaactccca gcaccagcat ccagtacaga gcctgctgcc ttgcgcagtc
2401 tgcagtcctg ggacgggaac cctgccatgt gctggtagca tggctaggat
gttgggcttc 2461 ccgggccctg gggtctggta accagtgaag gaagccccca
aaaatagtgg gtagggaagg 2521 cactagggcc gtggccgtgg ccccgaaagt
gcaggaacac ttgaaactgg aaaggaaggt 2581 gctctgggca cacgtggatt
tgcttctatt gttttgtttt tctcctaatg tatttataaa 2641 agatcttttc
ccgtttatgc tgggaaaaag tgtttttcaa actcagtgac aaggactttt 2701
ggtttttgta agactattga tgatatgaag gccttttgt SEQ ID NO: 4 1
mhsrsclppl lllllvllgs gvqscpsgcq cnqpqtvfct arqgttvprd vppdtvglyi
61 fengittldv gcfagfpglq lldlsqnqit slpggifqpl vnlsnldlta
nklheisnet 121 frglrrlerl ylgknrirhi qpgafdaldh llelklpdne
lrvlpplhlp rlllldlshn 181 sipaleagil dtanvealrl aglglrqlde
glfgrlrnlh dldvsdnqlg hmpsviqglr 241 gltrlrlagn triaqirped
lagltalqel dvsnlslqal psdlsslfpr lrllaaarnp 301 fncicplswf
gpwvreshvv laspeetrch fppknagrll leldyadfgc pvttttatvp 361
tirptvrept pstssqaptw psptepttqa pivlstappt mrpapqpqdc pasiclnggs
421 crvgakhhle cicpegfigl ycespveqrt kpssipdtpr pprllplrie
pvsptslrve 481 lqrylqgntv qlrslrltyr nlsgpdkrlv tlrlpaslae
ytvtqlrpna tysicvtalg 541 agrtpegeea cgeantpqav rsnhapvtqa
regnlpllia palaavllav laasgavycv 601 rrarasstaq dkgqvgpgtg
plelegvkvp lepgskaseg ggealsggpe cevplmgypg 661 pslqgvlpaq pyi
[0158] Vasorin is a typical type I membrane protein, containing
tandem arrays of a characteristic leucine-rich repeat motif, an
epidermal growth factor-like motif, and a fibronectin type III-like
motif at the extracellular domain. Expression analyses demonstrated
that vasorin is predominantly expressed in vascular smooth muscle
cells, and that its expression is developmentally regulated. The
vasorin gene is conserved in chimpanzee, cow, mouse, rat, and
zebrafish.
[0159] In certain embodiments, human vasorin corresponds to the
nucleotide sequence set forth by NCBI reference No. NM_138440,
shown below as SEQ ID NO: 5, and the corresponding amino acid
sequence set forth by NCBI reference No. AAO27704, shown below as
SEQ ID NO: 6.
TABLE-US-00003 SEQ ID NO: 5 1 gactccggag cccgagcccg gggcgggtgg
acgcggactc gaacgcagtt gcttcgggac 61 ccaggacccc ctcgggcccg
acccgccagg aaagactgag gccgcggcct gccccgcccg 121 gctccctgcg
ccgccgccgc ctcccgggac agaagatgtg ctccagggtc cctctgctgc 181
tgccgctgct cctgctactg gccctggggc ctggggtgca gggctgccca tccggctgcc
241 agtgcagcca gccacagaca gtcttctgca ctgcccgcca ggggaccacg
gtgccccgag 301 acgtgccacc cgacacggtg gggctgtacg tctttgagaa
cggcatcacc atgctcgacg 361 caggcagctt tgccggcctg ccgggcctgc
agctcctgga cctgtcacag aaccagatcg 421 ccagcctgcc cagcggggtc
ttccagccac tcgccaacct cagcaacctg gacctgacag 481 ccaacaggct
gcatgaaatc accaatgaga ccttccgtgg cctgcggcgc ctcgagcgcc 541
tctacctggg caagaaccgc atccgccaca tccagcctgg tgccttcgac acgctcgacc
601 gcctcctgga gctcaagctg caggacaacg agctgcgggc actgcccccg
ctgcgcctgc 661 cccgcctgct gctgctggac ctcagccaca acagcctcct
ggccctggag cccggcatcc 721 tggacactgc caacgtggag gcgctgcggc
tggctggtct ggggctgcag cagctggacg 781 aggggctctt cagccgcttg
cgcaacctcc acgacctgga tgtgtccgac aaccagctgg 841 agcgagtgcc
acctgtgatc cgaggcctcc ggggcctgac gcgcctgcgg ctggccggca 901
acacccgcat tgcccagctg cggcccgagg acctggccgg cctggctgcc ctgcaggagc
961 tggatgtgag caacctaagc ctgcaggccc tgcctggcga cctctcgggc
ctcttccccc 1021 gcctgcggct gctggcagct gcccgcaacc ccttcaactg
cgtgtgcccc ctgagctggt 1081 ttggcccctg ggtgcgcgag agccacgtca
cactggccag ccctgaggag acgcgctgcc 1141 acttcccgcc caagaacgct
ggccggctgc tcctggagct tgactacgcc gactttggct 1201 gcccagccac
caccaccaca gccacagtgc ccaccacgag gcccgtggtg cgggagccca 1261
cagccttgtc ttctagcttg gctcctacct ggcttagccc cacagagccg gccactgagg
1321 cccccagccc gccctccact gccccaccga ctgtagggcc tgtcccccag
ccccaggact 1381 gcccaccgtc cacctgcctc aatgggggca catgccacct
ggggacacgg caccacctgg 1441 cgtgcttgtg ccccgaaggc ttcacgggcc
tgtactgtga gagccagatg gggcagggga 1501 cacggcccag ccctacacca
gtcacgccga ggccaccacg gtccctgacc ctgggcatcg 1561 agccggtgag
ccccacctcc ctgcgcgtgg ggctgcagcg ctacctccag gggagctccg 1621
tgcagctcag gagcctccgt ctcacctatc gcaacctatc gggccctgat aagcggctgg
1681 tgacgctgcg actgcctgcc tcgctcgctg agtacacggt cacccagctg
cggcccaacg 1741 ccacttactc cgtctgtgtc atgcctttgg ggcccgggcg
ggtgccggag ggcgaggagg 1801 cctgcgggga ggcccataca cccccagccg
tccactccaa ccacgcccca gtcacccagg 1861 cccgcgaggg caacctgccg
ctcctcattg cgcccgccct ggccgcggtg ctcctggccg 1921 cgctggctgc
ggtgggggca gcctactgtg tgcggcgggg gcgggccatg gcagcagcgg 1981
ctcaggacaa agggcaggtg gggccagggg ctgggcccct ggaactggag ggagtgaagg
2041 tccccttgga gccaggcccg aaggcaacag agggcggtgg agaggccctg
cccagcgggt 2101 ctgagtgtga ggtgccactc atgggcttcc cagggcctgg
cctccagtca cccctccacg 2161 caaagcccta catctaagcc agagagagac
agggcagctg gggccgggct ctcagccagt 2221 gagatggcca gccccctcct
gctgccacac cacgtaagtt ctcagtccca acctcgggga 2281 tgtgtgcaga
cagggctgtg tgaccacagc tgggccctgt tccctctgga cctcggtctc 2341
ctcatctgtg agatgctgtg gcccagctga cgagccctaa cgtccccaga accgagtgcc
2401 tatgaggaca gtgtccgccc tgccctccgc aacgtgcagt ccctgggcac
ggcgggccct 2461 gccatgtgct ggtaacgcat gcctgggccc tgctgggctc
tcccactcca ggcggaccct 2521 gggggccagt gaaggaagct cccggaaaga
gcagagggag agcgggtagg cggctgtgtg 2581 actctagtct tggccccagg
aagcgaagga acaaaagaaa ctggaaagga agatgcttta 2641 ggaacatgtt
ttgctttttt aaaatatata tatatttata agagatcctt tcccatttat 2701
tctgggaaga tgtttttcaa actcagagac aaggactttg gtttttgtaa gacaaacgat
2761 gatatgaagg ccttttgtaa gaaaaaataa aagatgaagt gtgtttaaaa
aaaaaaaaaa 2821 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
SEQ ID NO: 6 1 mcsrvplllp lllllalgpg vqgcpsgcqc sqpqtvfcta
rqgttvprdv ppdtvglyvf 61 engitmldag sfaglpglql ldlsqnqias
lpsgvfqpla nlsnldltan rlheitnetf 121 rglrrlerly lgknrirhiq
pgafdtldrl lelklqdnel ralpplrlpr lllldlshns 181 llalepgild
tanvealrla glglqqldeg lfsrlrnlhd ldvsdnqler vppvirglrg 241
ltrlrlagnt riaqlrpedl aglaalqeld vsnlslqalp gdlsglfprl rllaaarnpf
301 ncvcplswfg pwvreshvtl aspeetrchf ppknagrlll eldyadfgcp
attttatvpt 361 trpvvrepta lssslaptwl sptepateap sppstapptv
gpvpqpqdcp pstclnggtc 421 hlgtrhhlac lcpegftgly cesqmgqgtr
psptpvtprp prsltlgiep vsptslrvgl 481 qrylqgssvq lrslrltyrn
lsgpdkrlvt lrlpaslaey tvtqlrpnat ysvcvmplgp 541 grvpegeeac
geahtppavh snhapvtqar egnlplliap alaavllaal aavgaaycvr 601
rgramaaaaq dkgqvgpgag plelegvkvp lepgpkateg ggealpsgse cevplmgfpg
661 pglqsplhak pyi
[0160] Various conserved regions have been identified in
ATIA/vasorin, including putative signal peptide, leucine rich
repeats (LRRs), EGF motif, fibronectin type III motif,
transmembrane sequence, and at least five putative N-glycosylation
sites.
[0161] The extracellular domain of ATIA corresponds to amino acids
600-673 of the full length sequence.
[0162] In certain embodiments, ATIA is in a soluble form.
[0163] The soluble form of mouse ATIA corresponds to residues 289
to 1792 of SEQ ID NO: 1, and is represented by SEQ ID NO: 7, or
fragments thereof, shown below:
TABLE-US-00004 SEQ ID NO: 7 tcaggctgcc agtgcaacc agccacagac
agtcttctgc actgcccgtc agggaaccac agtgccccga gacgtgccac ctgacacagt
gggcctgtac atctttgaga acggcatcac gacacttgat gtgggctgtt ttgctggcct
tccgggcctg cagcttctgg acttgtcaca gaaccagatc actagcctgc ccgggggcat
ctttcagcca cttgttaacc tcagtaacct ggacctgact gccaacaaac tgcacgagat
ctccaacgag accttccgtg gcctgcggcg cctggagcgc ctctacctgg gcaagaaccg
aattcgccac atccaaccgg gtgccttcga cgcgcttgat cgcctcctgg agctcaagct
gccagacaat gagcttcggg tgttgccccc attgcacttg ccccgcctgc tgctgcttga
cctcagccac aacagcatcc cagccctgga agccggaata ctggataccg ccaatgtaga
ggcattgagg ttggctggcc tagggctgcg gcagctggat gaggggcttt ttggccgcct
tctcaacctc catgacttgg atgtttctga caaccagttg gagcatatgc catctgtgat
tcaaggcctg cgtggcctga cacgcctgcg gctggctggc aacacccgta ttgcccagat
acggcccgag gacctcgctg gtctgactgc cctacaggaa ttggatgtga gcaacctaag
cctgcaggcc ctgcccagtg acctctcgag tctctttccc cgcctgcgcc tcttagcagc
tgccaggaac cccttcaact gcttgtgccc cttgagctgg tttggtcctt gggtgcgtga
gaaccatgtt gtgttggcca gccctgagga gacgcgttgt cactttccac ccaagaatgc
tggccgactg ctcctggatc tggattatgc agattttggc tgcccagtca ccactaccac
ggccacagta cctactataa ggtctactat cagggaaccc acactttcaa cttctagcca
agctcccacc tggcccagcc tcacagagcc aactacccag gcctccaccg tactatcgac
tgccccacca accatgaggc cagctcctca gccccaggac tgtccagcat ccatctgcct
gaatggtggt agctgccgtt tgggagcaag acaccactgg gagtgcctat gccctgaggg
cttcattggc ctgtactgtg agagtccagt ggagcaaggg atgaagccca gctccatacc
agacactcca aggccccctc cactgctgcc tctcagcatt gagccggtga gccccacctc
cttgcgtgtg aagctgcagc gctacttgca gggtaacact gtgcagctac ggagcctccg
gctcacctat cgcaacctgt ctggccctga caaacgactg gtgacattac ggctgcctgc
ttcacttgca gagtatacag tcacccagct gc
[0164] The soluble form of human vasorin corresponds to residues
231 to 1731 of SEQ ID NO: 5, and is represented by SEQ ID NO: 8, or
fragments thereof, shown below:
TABLE-US-00005 SEQ ID NO: 8 tccggctgcc agtgcagcca gccacagaca
gtcttctgca ctgcccgcca ggggaccacg gtgccccgag acgtgccacc cgacacggtg
gggctgtacg tctttgagaa cggcatcacc atgctcgacg caggcagctt tgccggcctg
ccgggcctgc agctcctgga cctgtcacag aaccagatcg ccagcctgcc cagcggggtc
ttccagccac tcgccaacct cagcaacctg gacctgacag ccaacaggct gcatgaaatc
accaatgaga ccttccgtgg cctgcggcgc ctcgagcgcc tctacctggg caagaaccgc
atccgccaca tccagcctgg tgccttcgac acgctcgacc gcctcctgga gctcaagctg
caggacaacg agctgcgggc actgcccccg ctgcgcctgc cccgcctgct gctgctggac
ctcagccaca acagcctcct ggccctggag cccggcatcc tggacactgc caacgtggag
gcgctgcggc tggctggtct ggggctgcag cagctggacg aggggctctt cagccgcttg
cgcaacctcc acgacctgga tgtgtccgac aaccagctgg agcgagtgcc acctgtgatc
cgaggcctcc ggggcctgac gcgcctgcgg ctggccggca acacccgcat tgcccagctg
cggcccgagg acctggccgg cctggctgcc ctgcaggagc tggatgtgag caacctaagc
ctgcaggccc tgcctggcga cctctcgggc ctcttccccc gcctgcggct gctggcagct
gcccgcaacc ccttcaactg cgtgtgcccc ctgagctggt ttggcccctg ggtgcgcgag
agccacgtca cactggccag ccctgaggag acgcgctgcc acttcccgcc caagaacgct
ggccggctgc tcctggagct tgactacgcc gactttggct gcccagccac caccaccaca
gccacagtgc ccaccacgag gcccgtggtg cgggagccca cagccttgtc ttctagcttg
gctcctacct ggcttagccc cacagagccg gccactgagg cccccagccc gccctccact
gccccaccga ctgtagggcc tgtcccccag ccccaggact gcccaccgtc cacctgcctc
aatgggggca catgccacct ggggacacgg caccacctgg cgtgcttgtg ccccgaaggc
ttcacgggcc tgtactgtga gagccagatg gggcagggga cacggcccag ccctacacca
gtcacgccga ggccaccacg gtccctgacc ctgggcatcg agccggtgag ccccacctcc
ctgcgcgtgg ggctgcagcg ctacctccag gggagctccg tgcagctcag gagcctccgt
ctcacctatc gcaacctatc gggccctgat aagcggctgg tgacgctgcg actgcctgcc
tcgctcgctg agtacacggt cacccagctg c
[0165] In other embodiments, the amino acid sequence of soluble
mouse ATIA corresponds to residues 26 to 526 of SEQ ID NO: 2, and
is represented by SEQ ID NO: 9, or fragments thereof, shown
below:
TABLE-US-00006 SEQ ID NO: 9
PSGCQCNQPQTVFCTARQGTTVPRDVPPDTVGLYIFENGITTLDVGC
FAGLPGLQLLDLSQNQITSLPGGIFQPLVNLSNLDLTANKLHEISNE
TFRGLRRLERLYLGKNRIRHIQPGAFDALDRLLELKLPDNELRVLPP
LHLPRLLLLDLSHNSIPALEAGILDTANVEALRLAGLGLRQLDEGLF
GRLLNLHDLDVSDNQLEHMPSVIQGLRGLTRLRLAGNTRIAQIRPED
LAGLTALQELDVSNLSLQALPSDLSSLFPRLRLLAAARNPFNCLCPL
SWFGPWVRENHVVLASPEETRCHFPPKNAGRLLLDLDYADFGCPVTT
TTATVPTIRSTIREPTLSTSSQAPTWPSLTEPTTQASTVLSTAPPTM
RPAPQPQDCPASICLNGGSCRLGARHHWECLCPEGFIGLYCESPVEQ
GMKPSSIPDTPRPPPLLPLSIEPVSPTSLRVKLQRYLQGNTVQLRSL
RLTYRNLSGPDKRLVTLRLPASLAEYTVTQL
[0166] In other embodiments, the amino acid sequence of human
soluble vasorin corresponds to residues 25 to 525 of SEQ ID NO: 6,
and is represented by SEQ ID NO: 10, or fragments thereof, shown
below:
TABLE-US-00007 SEQ ID NO: 10
PSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVFENGITMLDAGS
FAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNE
TFRGLRRLERLYLGKNRIRHIQPGAFDTLDRLLELKLQDNELRALPP
LRLPRLLLLDLSHNSLLALEPGILDTANVEALRLAGLGLQQLDEGLF
SRLRNLHDLDVSDNQLERVPPVIRGLRGLTRLRLAGNTRIAQLRPED
LAGLAALQELDVSNLSLQALPGDLSGLFPRLRLLAAARNPFNCVCPL
SWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYADFGCPATT
TTATVPTTRPVVREPTALSSSLAPTWLSPTEPATEAPSPPSTAPPTV
GPVPQPQDCPPSTCLNGGTCHLGTRHHLACLCPEGFTGLYCESQMGQ
GTRPSPTPVTPRPPRSLTLGIEPVSPTSLRVGLQRYLQGSSVQLRSL
RLTYRNLSGPDKRLVTLRLPASLAEYTVTQL
Methods of the Invention
[0167] The present inventors have found that ATIA is a hypoxia
inducible gene, and that under hypoxia conditions ATIA is
considerably increased. Human cancers are characterized by
intratumoral hypoxia, a significant reduction in oxygen
availability. Physiological responses triggered by hypoxia impact
many aspects of cancer progression, including immortalization,
transformation, differentiation, genetic instability, angiogenesis,
metabolic adaptation, autocrine growth factor signaling, invasion,
metastasis, and resistance to therapy.
[0168] In embodiments of the invention, a patient having cancer
will show an increase in the expression of ATIA nucleic acid
molecule. In particular embodiments, the patient may suffer from a
solid tumor that expresses elevated levels of ATIA. In further
particular embodiments, the patient may suffer from glioblastoma or
astrocytoma. Alterations in gene expression are detected using
methods known to the skilled artisan and described herein. Such
information can be used to diagnose cancer, and in particular
glioblastoma or astrocytoma. In another embodiment, an alteration
in the expression of ATIA nucleic acid molecule is detected using
polymerase chain reaction (PCR), for example, real time PCR or semi
quantitative real time PCR to detect changes in gene
expression.
[0169] Primers used for amplification of ATIA nucleic acid
molecule, including but not limited to those primer sequences
described herein, are useful in diagnostic methods of the
invention. The primers of the invention embrace oligonucleotides of
sufficient length and appropriate sequence so as to provide
specific initiation of polymerization on a significant number of
nucleic acids. Specifically, the term "primer" as used herein
refers to a sequence comprising two or more deoxyribonucleotides or
ribonucleotides, preferably more than three, and most preferably
more than 8, which sequence is capable of initiating synthesis of a
primer extension product, which is substantially complementary to a
locus strand. The primer must be sufficiently long to prime the
synthesis of extension products in the presence of the inducing
agent for polymerization. The exact length of primer will depend on
many factors, including temperature, buffer, and nucleotide
composition. Primers of the invention are designed to be
"substantially" complementary to each strand of the genomic locus
to be amplified and include the appropriate G or C nucleotides as
discussed above. This means that the primers must be sufficiently
complementary to hybridize with their respective strands under
conditions that allow the agent for polymerization to perform. In
other words, the primers should have sufficient complementarity
with the 5' and 3' flanking sequences to hybridize therewith and
permit amplification of the genomic locus. While exemplary primers
are provided herein, it is understood that any primer that
hybridizes with the target sequences of the invention are useful in
the method of the invention for detecting ATIA nucleic acid
molecules.
[0170] In one embodiment, ATIA-specific primers amplify a desired
genomic target using the polymerase chain reaction (PCR), for
example quantitative PCR or semi quantitative RT-PCR. The amplified
product is then detected using standard methods known in the art.
In one embodiment, a PCR product (i.e., amplicon) or real-time PCR
product is detected by probe binding. In one embodiment, probe
binding generates a fluorescent signal, for example, by coupling a
fluorogenic dye molecule and a quencher moiety to the same or
different oligonucleotide substrates (e.g., TaqMan.RTM. (Applied
Biosystems, Foster City, Calif., USA), Molecular Beacons (see, for
example, Tyagi et al., Nature Biotechnology 14(3):303-8, 1996),
Scorpions.RTM. (Molecular Probes Inc., Eugene, Oreg., USA)). In
another example, a PCR product is detected by the binding of a
fluorogenic dye that emits a fluorescent signal upon binding (e.g.,
SYBR Green (Molecular Probes)). Such detection methods are useful
for the detection of ATIA PCR product.
[0171] In another embodiment, hybridization with PCR probes that
are capable of detecting an ATIA nucleic acid molecule, including
genomic sequences, or closely related molecules, may be used to
hybridize to a nucleic acid sequence derived from a patient having
a cancer, and in particular glioblastoma or astrocytoma; however
any other cancer types that express elevated levels of ATIA are
envisioned as well. The specificity of the probe determines whether
the probe hybridizes to a naturally occurring sequence, allelic
variants, or other related sequences. Hybridization techniques may
be used to identify mutations indicative of a cancer, e.g.
glioblastoma, or may be used to monitor expression levels of these
genes (for example, by Northern analysis (Ausubel et al.,
supra).
Diagnostics and Prognostics
[0172] The present invention provides a number of diagnostic assays
that are useful for the identification, in particular the early
identification, or characterization of cancer, for example
glioblastoma or astrocytoma. In particular embodiments, the
invention provides methods for detecting ATIA levels in biological
fluids, for example using an ELISA-based assay. This ELISA based
blood test of ATIA level will provide an easy and early diagnosis
of the disease, which normally needs surgery to spot.
[0173] The invention also features methods of determining if a
subject will respond to tumour necrosis factor-related
apoptosis-inducing ligand (TRAIL) therapy. The method is based, in
part, on the finding that certain cells that express ATIA are more
sensitive to TRAIL-induced cell death than cells which do not
express ATIA, for example human glioblastoma are more sensitive to
TRAIL-induced cell death than cells which do not express ATIA.
Accordingly, ATIA expression can be used as a marker to determine
if a subject will respond to TRAIL therapy.
[0174] Potency and lack of toxicity to normal tissues make
activation TRAIL death receptor signaling an attractive target for
cancer therapy. For example, recombinant human (rh) TRAIL/Apo-2L, a
TRAIL-encoding adenovirus, and monoclonal antibodies directed
against TRAIL receptors R1 and R2 have been used to study
cytotoxicity of TRAIL therapy in NSCLC cells (108). Recombinant
TRAIL as well as agonistic anti-TRAIL-R1 and anti-TRAIL-R2
antibodies have entered clinical trials. Gene therapy approaches
using TRAIL-encoding adenovirus (Ad-TRAIL) are being developed. To
optimize gene therapy approaches, CD34+ cells transduced with
Ad-TRAIL (CD34-TRAIL+) have been investigated as cellular vehicles
for TRAIL delivery (109).
[0175] In certain embodiments, the method comprises determining the
level of expression or biological activity of ATIA in a subject
sample wherein an alteration in the level of expression or
biological activity relative to the expression or biological
activity in a reference indicates that the subject will respond to
TRAIL therapy.
[0176] The invention also features methods of determining if a
subject will respond to TRAIL therapy, the method comprising
determining the level of expression or biological activity of a
ATIA nucleic acid in a subject sample wherein an alteration in the
level of expression relative to the expression in a reference
indicates that the subject will respond to TRAIL therapy.
[0177] The invention also features methods of determining if a
subject will respond to TRAIL therapy, the method comprising
determining the amount of an ATIA protein in a subject sample
wherein an alteration in the amount of the protein relative to the
amount in a reference indicates that the subject will respond to
TRAIL therapy.
[0178] In preferred embodiments, the subject has cancer.
Preferably, the cancer is glioblastoma or astrocytoma; however any
cancer that has elevated levels of ATIA may be a candidate for
therapy.
[0179] In certain embodiments the invention features diagnostic
methods. For example a subject, for example a patient, may be
diagnosed for a propensity to develop cancer, e.g. brain cancer, by
direct analysis of the sequence of an ATIA nucleic acid molecule.
The sequence of an ATIA nucleic acid molecule derived from a
subject is compared to a reference sequence. An alteration in the
sequence of the ATIA nucleic acid molecule relative to the
reference indicates that the patient has or has a propensity to
develop cancer, e.g. brain cancer.
[0180] In another approach, diagnostic methods of the invention are
used to assay the expression of an ATIA variant polypeptide in a
biological sample relative to a reference (e.g., the level of ATIA
polypeptide present in a corresponding control sample, or in a
sample taken before a treatment, such as surgical treatment). In
one embodiment, the level of an ATIA polypeptide is detected using
an antibody that specifically binds an ATIA polypeptide.
[0181] Exemplary antibodies that specifically bind an ATIA
polypeptide are described herein. Such antibodies are useful for
the diagnosis of cancer. Methods for measuring an antibody-ATIA
complex include, for example, detection of fluorescence,
luminescence, chemiluminescence, absorbance, reflectance,
transmittance, birefringence or refractive index. Optical methods
include microscopy (both confocal and non-confocal), imaging
methods and non-imaging methods. Methods for performing these
assays are readily known in the art. Useful assays include, for
example, an enzyme immune assay (EIA) such as enzyme-linked
immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western
blot assay, or a slot blot assay. These methods are also described
in, e.g., Methods in Cell Biology: Antibodies in Cell Biology,
volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites
& Terr, eds., 7th ed. 1991); and Harlow & Lane, supra.
Immunoassays can be used to determine the quantity of ATIA in a
sample, where an increase in the level of ATIA polypeptide is
diagnostic of a patient having cancer, e.g. brain cancer.
[0182] In general, the measurement of an ATIA polypeptide or
nucleic acid molecule in a subject sample is compared with a
diagnostic amount present in a reference. A diagnostic amount
distinguishes between a diseased tissue or, for example a
neoplastic tissue, and a control tissue. The skilled artisan
appreciates that the particular diagnostic amount used can be
adjusted to increase sensitivity or specificity of the diagnostic
assay depending on the preference of the diagnostician. In general,
any significant increase (e.g., at least about 10%, 15%, 30%, 50%,
60%, 75%, 80%, or 90%) in the level of an ATIA polypeptide or
nucleic acid molecule in the subject sample relative to a reference
may be used to diagnose cancer, e.g. brain cancer. In one
embodiment, the reference is the level of ATIA polypeptide or
nucleic acid molecule present in a control sample obtained from a
patient that does not have cancer, e.g. brain cancer. In another
embodiment, the reference is the level of ATIA polypeptide or
nucleic acid molecule present in a control sample obtained from
subjects with a disease of less severity, e.g., early stage
non-aggressive brain cancer. In another embodiment, the reference
is a baseline level of ATIA present in a biologic sample derived
from a patient prior to, during, or after treatment for cancer,
e.g. brain cancer. In yet another embodiment, the reference is a
standardized curve.
Types of Biological Samples
[0183] The level of the soluble form of ATIA, the extracellular
portion of ATIA, can be measured in different types of biologic
samples. In one embodiment, the biologic sample is a biologic fluid
sample (e.g., blood, blood plasma, serum, urine, seminal fluids,
ascites, or cerebrospinal fluid). Preferably, the sample is a blood
sample that is obtained from the subject.
[0184] The level of ATIA nucleic acid molecule can be measured in
different types of biologic samples. In one embodiment, the
biologic sample is a tissue sample that includes cells of a tissue
or organ. Such tissue is obtained, for example, from a biopsy. In
another embodiment, the biologic sample is a biologic fluid sample
(e.g., blood, blood plasma, serum, urine, seminal fluids, ascites,
or cerebrospinal fluid).
[0185] In certain exemplary embodiments, the sample is from a brain
tumor. In preferred embodiments, the brain tumor is
glioblastoma.
[0186] In other certain exemplary embodiments, the sample is from a
subject undergoing treatment for brain cancer, e.g. glioblastoma or
astrocytoma.
Therapeutics
[0187] As described herein, the present invention is based, in
part, on the finding that there is high expression of ATIA in
certain types of cancers, in particular in human glioblastoma and
astrocytoma. Accordingly, the invention features, in preferred
embodiments, ATIA as a therapeutic target. In certain embodiments,
ATIA levels can be modulated, for example, by knocking down ATIA
expression by siRNA or blocking its function through use of a
neutralizing antibody or use of small molecules. The invention
features in other preferred embodiments combination therapies,
where eliminating ATIA function (for example, through siRNA knock
down, neutralizing antibody or use of small molecules) along with
ionizing radiation will improve existing treatment, and is based on
the finding that knocking down ATIA expression leads to increased
sensitivity of cells to apoptosis (cell death).
[0188] In certain embodiments, the present invention features a
method of treating a patient with small molecule therapeutics. In
particular, the patient will be determined to have elevated levels
of ATIA, for example elevated levels of soluble ATIA. In particular
preferred embodiments, the patient is treated with low levels of
cyclohexamide. Cyclohexamide is an antibiotic produced by
Streptomyces griseus that inhibits protein synthesis. Preferably,
cyclohexamide is used at low doses that do not show toxicity to the
patient, but are suitable to knockdown ATIA levels. Preferred doses
of cylohexamide are 0.1 ug-1.0 ug. A particularly preferred dose is
0.5 ug.
Kits
[0189] The invention also provides kits for the diagnosis or
monitoring of cancer, e.g. brain cancer, in a biological sample
obtained from a subject. In one embodiment, the kit detects an
increase in the expression of an ATIA nucleic acid molecule or
polypeptide relative to a reference level of expression. In another
embodiment, the kit detects an alteration in the sequence of an
ATIA nucleic acid molecule derived from a subject relative to a
reference sequence.
[0190] In preferred embodiments, ATIA is the soluble portion, for
example as represented by SEQ ID NOs: 7-10.
[0191] In related embodiments, the kit includes reagents for
monitoring the expression of an ATIA nucleic acid molecule, such as
primers or probes that hybridize to an ATIA nucleic acid molecule.
In other embodiments, the kit includes an antibody that binds to an
ATIA polypeptide.
[0192] Optionally, the kit includes directions for monitoring an
ATIA nucleic acid molecule or polypeptide levels in a biological
sample derived from a subject. In preferred embodiments, ATIA is
the soluble portion, for example as represented by SEQ ID NOs:
7-10.
[0193] In other embodiments, the kit comprises a sterile container
which contains the primer, probe, antibody, or other detection
regents; such containers can be boxes, ampules, bottles, vials,
tubes, bags, pouches, blister-packs, or other suitable container
form known in the art. Such containers can be made of plastic,
glass, laminated paper, metal foil, or other materials suitable for
holding nucleic acids. The instructions will generally include
information about the use of the primers or probes described herein
and their use in diagnosing cancer, e.g. brain cancer. Preferably,
the kit further comprises any one or more of the reagents described
in the diagnostic assays described herein. In other embodiments,
the instructions include at least one of the following: description
of the primer or probe; methods for using the enclosed materials
for the diagnosis of cancer, e.g. brain cancer; precautions;
warnings; indications; clinical or research studies; and/or
references. The instructions may be printed directly on the
container (when present), or as a label applied to the container,
or as a separate sheet, pamphlet, card, or folder supplied in or
with the container.
Antibodies
[0194] Antibodies are well known to those of ordinary skill in the
science of immunology. As used herein, the term "antibody" means
not only intact antibody molecules, but also fragments of antibody
molecules that retain immunogen binding ability. Such fragments are
also well known in the art and are regularly employed both in vitro
and in vivo. Accordingly, as used herein, the term "antibody" means
not only intact immunoglobulin molecules but also the well-known
active fragments F(ab').sub.2, and Fab. F(ab').sub.2, and Fab
fragments which lack the Fc fragment of intact antibody, clear more
rapidly from the circulation, and may have less non-specific tissue
binding of an intact antibody (Wahl et al., J. Nucl. Med.
24:316-325 (1983). The antibodies of the invention comprise whole
native antibodies, bispecific antibodies; chimeric antibodies; Fab,
Fab', single chain V region fragments (scFv) and fusion
polypeptides.
[0195] The preparation and use of polyclonal antibodies are also
known the skilled artisan. The invention also encompasses hybrid
antibodies, in which one pair of heavy and light chains is obtained
from a first antibody, while the other pair of heavy and light
chains is obtained from a different second antibody. Such hybrids
may also be formed using humanized heavy and light chains. Such
antibodies are often referred to as "chimeric" antibodies.
[0196] In general, intact antibodies are said to contain "Fc" and
"Fab" regions. The Fc regions are involved in complement activation
and are not involved in antigen binding. An antibody from which the
Fc' region has been enzymatically cleaved, or which has been
produced without the Fc' region, designated an "F(ab').sub.2"
fragment, retains both of the antigen binding sites of the intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an "Fab'" fragment, retains one of the antigen
binding sites of the intact antibody. Fab' fragments consist of a
covalently bound antibody light chain and a portion of the antibody
heavy chain, denoted "Fd." The Fd fragments are the major
determinants of antibody specificity (a single Fd fragment may be
associated with up to ten different light chains without altering
antibody specificity). Isolated Fd fragments retain the ability to
specifically bind to immunogenic epitopes.
[0197] Antibodies can be made by any of the methods known in the
art utilizing ATIA polypeptides, or immunogenic fragments thereof,
as an immunogen. One method of obtaining antibodies is to immunize
suitable host animals with an immunogen and to follow standard
procedures for polyclonal or monoclonal antibody production. The
immunogen will facilitate presentation of the immunogen on the cell
surface. Immunization of a suitable host can be carried out in a
number of ways. Nucleic acid sequences encoding an ATIA
polypeptide, or immunogenic fragment thereof, can be provided to
the host in a delivery vehicle that is taken up by immune cells of
the host. The cells will in turn express the receptor on the cell
surface generating an immunogenic response in the host.
Alternatively, nucleic acid sequences encoding an ATIA polypeptide,
or immunogenic fragments thereof, can be expressed in cells in
vitro, followed by isolation of the receptor and administration of
the receptor to a suitable host in which antibodies are raised.
[0198] Using either approach, antibodies can then be purified from
the host. Antibody purification methods may include salt
precipitation (for example, with ammonium sulfate), ion exchange
chromatography (for example, on a cationic or anionic exchange
column preferably run at neutral pH and eluted with step gradients
of increasing ionic strength), gel filtration chromatography
(including gel filtration HPLC), and chromatography on affinity
resins such as protein A, protein G, hydroxyapatite, and
anti-immunoglobulin.
[0199] Antibodies can be conveniently produced from hybridoma cells
engineered to express the antibody. Methods of making hybridomas
are well known in the art. The hybridoma cells can be cultured in a
suitable medium, and spent medium can be used as an antibody
source.
[0200] Polynucleotides encoding the antibody of interest can in
turn be obtained from the hybridoma that produces the antibody, and
then the antibody may be produced synthetically or recombinantly
from these DNA sequences. For the production of large amounts of
antibody, it is generally more convenient to obtain an ascites
fluid. The method of raising ascites generally comprises injecting
hybridoma cells into an immunologically naive histocompatible or
immunotolerant mammal, especially a mouse. The mammal may be primed
for ascites production by prior administration of a suitable
composition; e.g., Pristane.
[0201] Monoclonal antibodies (Mabs) produced by methods of the
invention can be "humanized" by methods known in the art.
"Humanized" antibodies are antibodies in which at least part of the
sequence has been altered from its initial form to render it more
like human immunoglobulins. Techniques to humanize antibodies are
particularly useful when non-human animal (e.g., murine) antibodies
are generated. Examples of methods for humanizing a murine antibody
are provided in U.S. Pat. Nos. 4,816,567, 5,530,101, 5,225,539,
5,585,089, 5,693,762 and 5,859,205.
Polypeptides
[0202] The present invention encompasses ATIA polypeptides, for
example biomarker polypeptides (e.g., SEQ ID NO: 2, 4, 6, 9 and 10)
and fragments, variants, and derivatives thereof, that can be used
to diagnose or predict cancer, and in particular brain cancer, as
well as polynucleotides (e.g., SEQ ID NO: 1, 3, 5 7 and 8) encoding
these peptides or polypeptides. In accordance with the invention,
peptides can range in size from 5 amino acid residues to all but
one residue of the entire sequence. Accordingly, peptides include,
but are not limited to, fragments comprising at least 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 45, 55, 65, 75 or more
contiguous amino acids of any one of SEQ ID NO: 2, 4 or 6. In
certain embodiments, the peptides comprise antigenic fragments of
any one of SEQ ID NO: 2, 4 or 6.
[0203] The polypeptide, e.g. biomarker polypeptides, peptides, or
fragments or variants thereof, may be linked to short tags, e.g.,
epitope tags such as HA and the like, or to other proteins, such as
GST, GFP (e.g., GFP Y66F, GFP Y66H, GFP Y66W, wild type GFP, GFP
S65A, GFP S65L, GFP S65T, ECFP, EYFP, DsRed; BD Biosciences
CLONTECH, Palo Alto, Calif.), thioredoxin, maltose binding protein,
etc. Also provided by the invention are chemically modified
derivatives of the peptides and polypeptides of the invention that
may provide additional advantages such as increased solubility,
stability, and circulating time of the polypeptide. The chemical
moieties for derivitization may be selected from water soluble
polymers such as polyethylene glycol, ethylene glycol/propylene
glycol copolymers, carboxymethylcellulose, dextran, polyvinyl
alcohol and the like. The polypeptides may be modified at random
positions within the molecule, or at predetermined positions within
the molecule and may include one, two, three or more attached
chemical moieties.
[0204] In addition, amino acid sequence variants of the present
invention include, but are not limited to, variants that share at
least 40%, 50%, 60%, 61%, 67%, 70%, 74%, 76%, 80%, 81%, 84%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,
99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% nucleotide
sequence identity with any one of SEQ ID NO: 2, 4 or 6. For
variants that are functional equivalents, the percent amino acid
sequence identity is at least 61% or 67%. More preferably, the
percent amino acid sequence identity is at least 74% or 76%, still
more preferably, at least 81% or 84%, and even more preferably, at
least 90% to any one of SEQ ID NO: 2, 4 or 6. However, one of skill
in the art will appreciate that biological function need not be
maintained where a biomarker peptide comprises an antigenic
epitope.
[0205] Polypeptide and peptide variants include variants differing
by the addition, deletion, or substitution of one or more amino
acid residues. For example, to isolate biomarker polypeptides or
peptides, it may be useful to encode a tagged biomarker peptide or
polypeptide that can be recognized by a commercially available
antibody. In particular, a peptide or polypeptide can be fused or
linked to epitope tags (e.g., FLAG, HA, GST, thioredoxin, maltose
binding protein, etc.), or affinity tags such as biotin and/or
streptavidin. As one example, a system for the ready purification
of non-denatured fusion proteins expressed in human cell lines has
been described by Janknecht et al., (1991, Proc. Natl. Acad. Sci.
USA, 88:8972-8976). In this system, the gene of interest is
subcloned into a vaccinia recombination plasmid such that the open
reading frame of the gene is translationally fused to an
amino-terminal tag having six histidine residues. The tag serves as
a matrix-binding domain for the fusion protein. Extracts from cells
infected with the recombinant vaccinia virus are loaded onto an
Ni2+ nitriloacetic acid-agarose column and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0206] A peptide or polypeptide tagged with an epitope or protein
may also be engineered to contain a cleavage site located between
the binder coding sequence and the tag coding sequence. This can be
used to remove the tag, and isolate the biomarker peptide or
polypeptide. The biomarker peptides or polypeptides of the
invention can be covalently attached to chemical moieties via the
amino acid backbone. For these purposes, the peptides or
polypeptides may be modified by N- or C-terminal processing of the
sequences (e.g., proteolytic processing), deletion of the
N-terminal methionine residue, etc. The polypeptides may also be
modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein, as described in detail herein.
[0207] Also included are modified polypeptides and peptides in
which one or more residues are modified, and mutants comprising one
or more modified residues. Amino acid variants of the invention can
be generated by employing the techniques of gene-shuffling,
motif-shuffling, exon-shuffling, and/or codon-shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling can be
employed to generate peptides or polypeptides with altered
activity. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;
5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr.
Opinion Biotechnol., 8:724-33; Harayama, 1998, Trends Biotechnol.,
16(2):76-82; Hansson, et al., 1999, J. Mol. Biol., 287:265-76; and
Lorenzo and Blasco, 1998, Biotechniques, 24(2):308-313, the
contents of each of which are hereby incorporated by reference in
its entirety.
[0208] The peptides and polypeptides may be differentially modified
during or after translation, e.g., by derivatization with known
protecting/blocking groups, proteolytic cleavage, linkage to an
antibody molecule or other cellular ligand, etc. Useful
modifications may include glycosylation, amidation,
phosphorylation, sulfation, reduction/alkylation (Tarr, 1986,
Methods of Protein Microcharacterization, J. E. Silver, Ed., Humana
Press, Clifton, N.J., pp. 155-194); acylation (Tarr, supra);
chemical coupling (Mishell and Shiigi (Eds), 1980, Selected Methods
in Cellular Immunology, W H Freeman, San Francisco, Calif.; U.S.
Pat. No. 4,939,239); and mild formalin treatment (Marsh, 1971, Int.
Arch. of Allergy and Appl. Immunol. 41:199-215). Any of numerous
chemical modifications may be carried out by known techniques,
including but not limited, to specific chemical cleavage by
cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH4; acetylation, formylation, oxidation, reduction; metabolic
synthesis in the presence of tunicamycin; etc. Additional
post-translational modifications encompassed by the invention
include, for example, e.g., attachment of N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of prokaryotic host cell expression.
[0209] Additionally, D-amino acids, non-natural amino acids, or
non-amino acid analogs can be substituted or added to produce a
modified polypeptide. Furthermore, the polypeptides disclosed
herein can be modified using polyethylene glycol (PEG) according to
known methods (S. I. Wie et al., 1981, Int. Arch. Allergy Appl.
Immunol. 64(1):84-99) to produce a protein conjugated with PEG. In
addition, PEG can be added during chemical synthesis of the
protein. Modifications or sequence variations may occur at the
amino- or carboxy-terminal positions of the reference polypeptide
sequence or anywhere between those terminal positions, interspersed
either individually among the amino acids in the reference sequence
or in one or more contiguous groups within the reference sequence.
The polypeptides and peptides of this invention can be isolated,
synthetic, or recombinant. The amino acid sequences may be obtained
as individual polypeptides or peptides, or part of a complex.
[0210] Polypeptides or peptides may also be modified with a label
capable of providing a detectable signal, either directly or
indirectly, including, but not limited to, radioisotope,
fluorescent, and enzyme labels. Fluorescent labels include, for
example, Coumarin (e.g., Hydroxycoumarin, Aminocoumarin,
Methoxycoumarin), R-Phycoerythrin (PE), Fluorescein, FITC, Fluor X,
DTAF, Auramine, Alexa (e.g., Alexa Fluor.RTM. 350, -430, -488,
-532, -546, -555, -568, -594, -633, -647, -660, -680, -700, -750),
BODIPY-FL, Sulforhodamine (e.g., Texas Red), Carbocyanine,
Rhodamine, XRITC, TRITC, Lissamine Rhodamine B, Peridinin
Chlorphyll Protein (PerCP), Allophycocyanin (APC), PE-Cy5
conjugates (e.g., Cychrome, Tri-Color.RTM., Quantum Red.RTM.),
PE-Cy5.5 conjugates, PE-Cy7 conjugates, PE-Texas Red conjugates
(e.g., Red613), PC5-PE-Cy5 conjugates, PerCP-Cy5.5 conjugates
(e.g., TruRed), APC-Cy5.5 conjugates, APC-Cy7 conjugates,
ECD-PE-Texas Red conjugates, Sulfonated Pyrene (e.g., Cascade
Blue), AMCA Blue, Lucifer Yellow.
[0211] Enzymes can be conjugated by reaction with bridging
molecules such as carbodiimides, diisocyanates, glutaraldehyde, and
the like. Enzyme labels can be detected visually, or measured by
calorimetric, spectrophotometric, fluorospectrophotometric,
amperometric, or gasometric techniques. Other labeling systems,
such as avidin/biotin, colloidal gold, are known in the art, and
are commercially available.
[0212] Polypeptides, e.g. biomarker polypeptides as described
herein (peptides, and fragments, variants, and derivatives thereof)
may be produced by direct peptide synthesis using solid-phase
techniques (J. Merrifield, 1963, J. Am. Chem. Soc., 85:2149-2154;
J. Y. Roberge et al., 1995, Science, 269:202-204). Protein or
peptide synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
ABI 431A Peptide Synthesizer (PE Biosystems). Various fragments of
a biomarker polypeptide or peptide can be chemically synthesized
separately and then combined using chemical methods to produce the
full-length molecule. The newly synthesized peptide can be
substantially purified by preparative high performance liquid
chromatography (e.g., T. Creighton, 1983, Proteins, Structures and
Molecular Principles, W. H. Freeman and Co., New York, N.Y.), by
reversed-phase high performance liquid chromatography, or other
purification methods as are known in the art. The composition of
the synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). In addition, the amino acid sequence of biomarker peptide
or polypeptide or any portion thereof, may be altered during direct
synthesis and/or combined using chemical methods with sequences
from other proteins, or any part thereof, to produce a variant
peptide or polypeptide.
[0213] Polypeptides, variants, and fragments thereof may be
produced by transformation of a suitable host cell with all or part
of a polypeptide-encoding nucleic acid molecule or fragment thereof
in a suitable expression vehicle.
[0214] Those skilled in the field of molecular biology will
understand that any of a wide variety of expression systems may be
used to provide the recombinant protein. The precise host cell used
is not critical to the invention. A polypeptide of the invention
may be produced in a prokaryotic host (e.g., E. coli) or in a
eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells,
e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or
preferably COS cells). Such cells are available from a wide range
of sources (e.g., the American Type Culture Collection, Rockland,
Md.; also, see, e.g., Ausubel et al., supra). The method of
transformation or transfection and the choice of expression vehicle
will depend on the host system selected. Transformation and
transfection methods are described, e.g., in Ausubel et al.
(supra); expression vehicles may be chosen from those provided,
e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et
al., 1985, Supp. 1987).
[0215] A variety of expression systems exist for the production of
the polypeptides of the invention. Expression vectors useful for
producing such polypeptides include, without limitation,
chromosomal, episomal, and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, from bacteriophage, from
transposons, from yeast episomes, from insertion elements, from
yeast chromosomal elements, from viruses such as baculoviruses,
papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses, and vectors
derived from combinations thereof.
[0216] For example, one particular bacterial expression system for
polypeptide production is the E. coli pET expression system
(Novagen, Inc., Madison, Wis.). According to this expression
system, DNA encoding a polypeptide is inserted into a pET vector in
an orientation designed to allow expression. Since the gene
encoding such a polypeptide is under the control of the T7
regulatory signals, expression of the polypeptide is achieved by
inducing the expression of T7 RNA polymerase in the host cell. This
is typically achieved using host strains that express T7 RNA
polymerase in response to IPTG induction. Once produced,
recombinant polypeptide is then isolated according to standard
methods known in the art, for example, those described herein.
[0217] Once the recombinant polypeptide of the invention is
expressed, it is isolated, e.g., using affinity chromatography. In
one example, an antibody (e.g., produced as described herein)
raised against a polypeptide of the invention may be attached to a
column and used to isolate the recombinant polypeptide. Lysis and
fractionation of polypeptide-harboring cells prior to affinity
chromatography may be performed by standard methods (see, e.g.,
Ausubel et al., supra).
[0218] Once isolated, the recombinant protein can, if desired, be
further purified, e.g., by high performance liquid chromatography
(see, e.g., Fisher, Laboratory Techniques In Biochemistry and
Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
Polypeptides of the invention, particularly short peptide
fragments, can also be produced by chemical synthesis (e.g., by the
methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984
The Pierce Chemical Co., Rockford, Ill.). These general techniques
of polypeptide expression and purification can also be used to
produce and isolate useful peptide fragments or analogs (described
herein).
Polynucleotides
[0219] In general, the invention includes any nucleic acid sequence
encoding an ATIA polypeptide. Also included in the methods of the
invention are any nucleic acid molecule containing at least one
strand that hybridizes with such a nucleic acid sequence (e.g., an
inhibitory nucleic acid molecule, such as a dsRNA, siRNA, shRNA, or
antisense molecule). An isolated nucleic acid molecule can be
manipulated using recombinant DNA techniques well known in the art.
Thus, a nucleotide sequence contained in a vector in which 5' and
3' restriction sites are known, or for which polymerase chain
reaction (PCR) primer sequences have been disclosed, is considered
isolated, but a nucleic acid sequence existing in its native state
in its natural host is not. An isolated nucleic acid may be
substantially purified, but need not be. For example, a nucleic
acid molecule that is isolated within a cloning or expression
vector may comprise only a tiny percentage of the material in the
cell in which it resides. Such a nucleic acid is isolated, however,
as the term is used herein, because it can be manipulated using
standard techniques known to those of ordinary skill in the
art.
Screening Assays
[0220] As reported herein, the expression of ATIA polypeptide is
increased in neoplastic tissues, and in particular examples in
neoplastic tissues from patients with cancer, and in particular,
brain cancer. Accordingly, compounds that modulate the expression
or activity of ATIA polypeptide, variant, or fragment thereof are
useful in the methods of the invention for the treatment or
prevention of brain cancer, and in particular brain cancer. Any
number of methods are available for carrying out screening assays
to identify such compounds. In one approach, candidate compounds
are identified that specifically bind to and alter the activity of
a polypeptide of the invention (e.g., an ATIA activity). Methods of
assaying such biological activities are known in the art and are
described herein. The efficacy of such a candidate compound is
dependent upon its ability to interact with an ATIA polypeptide,
variant, or fragment. Such an interaction can be readily assayed
using any number of standard binding techniques and functional
assays (e.g., those described in Ausubel et al., supra). For
example, a candidate compound may be tested in vitro for
interaction and binding with a polypeptide of the invention.
Standard methods for perturbing or reducing ATIA expression include
mutating or deleting an endogenous ATIA sequence, interfering with
ATIA expression using RNAi, or microinjecting an ATIA-expressing
cell with an antibody that binds ATIA and interferes with its
function.
[0221] Potential agonists and antagonists of an ATIA polypeptide
include organic molecules, peptides, peptide mimetics,
polypeptides, nucleic acid molecules (e.g., double-stranded RNAs,
siRNAs, antisense polynucleotides), and antibodies that bind to a
nucleic acid sequence or polypeptide of the invention and thereby
inhibit or decrease its activity. Potential antagonists also
include small molecules that bind to the ATIA polypeptide thereby
preventing binding to cellular molecules with which the ATIA
polypeptide normally interacts, such that the normal biological
activity of the ATIA polypeptide is reduced or inhibited. Small
molecules of the invention preferably have a molecular weight below
2,000 daltons, more preferably between 300 and 1,000 daltons, and
most preferably between 400 and 700 daltons. It is preferred that
these small molecules are organic molecules.
[0222] For example, a recombinant polypeptide of the invention may
be purified by standard techniques from cells engineered to express
the polypeptide (e.g., those described above) and may be
immobilized on a column. A solution of candidate compounds is then
passed through the column, and a compound specific for the ATIA
polypeptide is identified on the basis of its ability to bind to
the ATIA polypeptide and be immobilized on the column. To isolate
the compound, the column is washed to remove non-specifically bound
molecules, and the compound of interest is then released from the
column and collected.
[0223] In one particular example, methods may be used to isolate a
compound bound to a polypeptide microarray. Compounds isolated by
this method (or any other appropriate method) may, if desired, be
further purified (e.g., by high performance liquid chromatography).
In addition, these candidate compounds may be tested for their
ability to alter the biological activity of an ATIA
polypeptide.
[0224] Any in vivo protein interaction detection system, for
example, any two-hybrid assay may be utilized to identify compounds
that interact with an ATIA polypeptide. Interacting compounds
isolated by this method (or any other appropriate method) may, if
desired, be further purified (e.g., by high performance liquid
chromatography). Compounds isolated by any approach described
herein may be used as therapeutics to treat cancer, e.g. brain
cancer in a human patient.
[0225] In addition, compounds that inhibit the expression of an
ATIA nucleic acid molecule whose expression is increased in a
patient having a cancer, e.g. brain cancer, are also useful in the
methods of the invention. Any number of methods are available for
carrying out screening assays to identify new candidate compounds
that alter the expression of an ATIA nucleic acid molecule. In one
working example, candidate compounds are added at varying
concentrations to the culture medium of cultured cells expressing
one of the nucleic acid sequences of the invention. Gene expression
is then measured, for example, by microarray analysis, Northern
blot analysis (Ausubel et al., supra), or RT-PCR, using any
appropriate fragment prepared from the nucleic acid molecule as a
hybridization probe. The level of gene expression in the presence
of the candidate compound is compared to the level measured in a
control culture medium lacking the candidate molecule. A compound
that promotes an alteration in the expression of an ATIA gene, or a
functional equivalent thereof, is considered useful in the
invention; such a molecule may be used, for example, as a
therapeutic to treat cancer, e.g. brain cancer in a human
patient.
[0226] In another approach, the effect of candidate compounds is
measured at the level of polypeptide production to identify those
that promote an alteration in an ATIA polypeptide level. The level
of ATIA polypeptide can be assayed using any standard method.
Standard immunological techniques include Western blotting or
immunoprecipitation with an antibody specific for an ATIA
polypeptide. For example, immunoassays may be used to detect or
monitor the expression of at least one of the polypeptides of the
invention in an organism. Polyclonal or monoclonal antibodies
(produced as described above) that are capable of binding to such a
polypeptide may be used in any standard immunoassay format (e.g.,
ELISA, Western blot, or RIA assay) to measure the level of the
polypeptide. In some embodiments, a compound that promotes a
decrease in the expression or biological activity of the
polypeptide is considered particularly useful. Again, such a
molecule may be used, for example, as a therapeutic to delay,
ameliorate, or treat cancer, e.g. brain cancer in a human
patient.
[0227] In another embodiment, a nucleic acid described herein
(e.g., an ATIA nucleic acid) is expressed as a transcriptional or
translational fusion with a detectable reporter, and expressed in
an isolated cell (e.g., mammalian or insect cell) under the control
of a heterologous promoter, such as an inducible promoter. The cell
expressing the fusion protein is then contacted with a candidate
compound, and the expression of the detectable reporter in that
cell is compared to the expression of the detectable reporter in an
untreated control cell. A candidate compound that alters the
expression of the detectable reporter is a compound that is useful
for the treatment of a brain cancer. In one embodiment, the
compound decreases the expression of the reporter.
[0228] Each of the DNA sequences listed herein may also be used in
the discovery and development of a therapeutic compound for the
treatment of brain cancer, in particular brain cancer. The encoded
protein, upon expression, can be used as a target for the screening
of drugs. Additionally, the DNA sequences encoding the amino
terminal regions of the encoded protein or Shine-Delgarno or other
translation facilitating sequences of the respective mRNA can be
used to construct sequences that promote the expression of the
coding sequence of interest. Such sequences may be isolated by
standard techniques (Ausubel et al., supra).
[0229] The invention also includes novel compounds identified by
the above-described screening assays. Optionally, such compounds
are characterized in one or more appropriate animal models to
determine the efficacy of the compound for the treatment of cancer,
e.g. brain cancer. Desirably, characterization in an animal model
can also be used to determine the toxicity, side effects, or
mechanism of action of treatment with such a compound. Furthermore,
novel compounds identified in any of the above-described screening
assays may be used for the treatment of cancer, e.g. brain cancer
in a subject. Such compounds are useful alone or in combination
with other conventional therapies known in the art.
Test Compounds and Extracts
[0230] In general, compounds capable of inhibiting the growth or
proliferation of cancer, e.g. brain cancer by altering the
expression or biological activity of an ATIA polypeptide, variant,
or fragment thereof are identified from large libraries of either
natural product or synthetic (or semi-synthetic) extracts or
chemical libraries according to methods known in the art. Numerous
methods are also available for generating random or directed
synthesis (e.g., semi-synthesis or total synthesis) of any number
of chemical compounds, including, but not limited to, saccharide-,
lipid-, peptide-, and nucleic acid-based compounds. Synthetic
compound libraries are commercially available from Brandon
Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,
Wis.). Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).
[0231] In one embodiment, test compounds of the invention are
present in any combinatorial library known in the art, including:
biological libraries; peptoid libraries (libraries of molecules
having the functionalities of peptides, but with a novel,
non-peptide backbone which are resistant to enzymatic degradation
but which nevertheless remain bioactive; see, e.g., Zuckermann, R.
N. et al., J. Med. Chem. 37:2678-85, 1994); spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the `one-bead one-compound`
library method; and synthetic library methods using affinity
chromatography selection. The biological library and peptoid
library approaches are limited to peptide libraries, while the
other four approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, Anticancer
Drug Des. 12:145, 1997).
[0232] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al., Proc. Natl.
Acad. Sci. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci.
USA 91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994;
Cho et al., Science 261:1303, 1993; Carrell et al., Angew. Chem.
Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed.
Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233,
1994.
[0233] Libraries of compounds may be presented in solution (e.g.,
Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature
354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria
(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No.
5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA
89:1865-1869, 1992) or on phage (Scott and Smith, Science
249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al.
Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol.
222:301-310, 1991; Ladner supra.).
[0234] Those skilled in the field of drug discovery and development
will understand that the precise source of a compound or test
extract is not critical to the screening procedure(s) of the
invention. Accordingly, virtually any number of chemical extracts
or compounds can be screened using the methods described herein.
Examples of such extracts or compounds include, but are not limited
to, plant-, fungal-, prokaryotic- or animal-based extracts,
fermentation broths, and synthetic compounds, as well as
modification of existing compounds.
[0235] When a crude extract is found to alter the biological
activity of an ATIA polypeptide, variant, or fragment thereof,
further fractionation of the positive lead extract is necessary to
isolate chemical constituents responsible for the observed effect.
Thus, the goal of the extraction, fractionation, and purification
process is the careful characterization and identification of a
chemical entity within the crude extract having anti-neoplastic
activity. Methods of fractionation and purification of such
heterogenous extracts are known in the art. If desired, compounds
shown to be useful agents for the treatment of a neoplasm, e.g.
brain cancer, are chemically modified according to methods known in
the art.
Microarrays
[0236] The methods of the invention may also be used for
microarray-based assays that provide for the high-throughput
analysis of biomarkers. The ATIA nucleic acid molecules or
polypeptides of the invention are useful as hybridizable array
elements in such a microarray. The array elements are organized in
an ordered fashion such that each element is present at a specified
location on the substrate. Useful substrate materials include
membranes, composed of paper, nylon or other materials, filters,
chips, glass slides, and other solid supports. The ordered
arrangement of the array elements allows hybridization patterns and
intensities to be interpreted as expression levels of particular
genes or proteins. Methods for making nucleic acid microarrays are
known to the skilled artisan and are described, for example, in
U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech.
14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci.
93:10614-10619, 1996), herein incorporated by reference. Methods
for making polypeptide microarrays are described, for example, by
Ge (Nucleic Acids Res. 28:e3.i-e3.vii, 2000), MacBeath et al.,
(Science 289:1760-1763, 2000), Zhu et al. (Nature Genet.
26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by
reference.
[0237] Nucleic Acid Microarrays
[0238] To produce a nucleic acid microarray oligonucleotides may be
synthesized or bound to the surface of a substrate using a chemical
coupling procedure and an ink jet application apparatus, as
described in PCT application WO95/251116 (Baldeschweiler et al.),
incorporated herein by reference. Alternatively, a gridded array
may be used to arrange and link cDNA fragments or oligonucleotides
to the surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedure.
[0239] A nucleic acid molecule (e.g. RNA or DNA) derived from a
biological sample may be used to produce a hybridization probe as
described herein. The biological samples are generally derived from
a patient, preferably as a bodily fluid (such as blood,
cerebrospinal fluid, phlegm, saliva, or urine) or tissue sample
(e.g. a tissue sample obtained by biopsy, e.g. brain tissue). For
some applications, cultured cells or other tissue preparations may
be used. The mRNA is isolated according to standard methods, and
cDNA is produced and used as a template to make complementary RNA
suitable for hybridization. Such methods are described herein. The
RNA is amplified in the presence of fluorescent nucleotides, and
the labeled probes are then incubated with the microarray to allow
the probe sequence to hybridize to complementary oligonucleotides
(e.g., ATIA nucleic acid molecules) bound to the microarray.
[0240] Incubation conditions are adjusted such that hybridization
occurs with precise complementary matches or with various degrees
of less complementarity depending on the degree of stringency
employed. For example, stringent salt concentration will ordinarily
be less than about 750 mM NaCl and 75 mM trisodium citrate,
preferably less than about 500 mM NaCl and 50 mM trisodium citrate,
and most preferably less than about 250 mM NaCl and 25 mM trisodium
citrate. Low stringency hybridization can be obtained in the
absence of organic solvent, e.g., formamide, while high stringency
hybridization can be obtained in the presence of at least about 35%
formamide, and most preferably at least about 50% formamide.
Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In
one embodiment, hybridization will occur at 30.degree. C. in 750 mM
NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment,
hybridization will occur at 37.degree. C. in 500 mM NaCl, 50 mM
trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml
denatured salmon sperm DNA (ssDNA). In yet another embodiment,
hybridization will occur at 42.degree. C. in 250 mM NaCl, 25 mM
trisodium citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA.
Useful variations on these conditions will be readily apparent to
those skilled in the art.
[0241] The removal of nonhybridized probes may be accomplished, for
example, by washing. The washing steps that follow hybridization
can also vary in stringency. Wash stringency conditions can be
defined by salt concentration and by temperature. As above, wash
stringency can be increased by decreasing salt concentration or by
increasing temperature. For example, stringent salt concentration
for the wash steps will preferably be less than about 30 mM NaCl
and 3 mM trisodium citrate, and most preferably less than about 15
mM NaCl and 1.5 mM trisodium citrate. Stringent temperature
conditions for the wash steps will ordinarily include a temperature
of at least about 25.degree. C., at least about 42.degree. C., or
at least about 68.degree. C. In one embodiment, wash steps will
occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and
0.1% SDS. In another embodiment, wash steps will occur at
42.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%
SDS. In yet another embodiment, wash steps will occur at 68.degree.
C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent
to those skilled in the art.
[0242] A detection system may be used to measure the absence,
presence, and amount of hybridization for all of the distinct
sequences simultaneously (e.g., Heller et al., Proc. Natl. Acad.
Sci. 94:2150-2155, 1997). Preferably, a scanner is used to
determine the levels and patterns of fluorescence.
[0243] Protein Microarrays
[0244] ATIA polypeptides, such as those described herein, may also
be analyzed using protein microarrays. Such arrays are useful in
high-throughput low-cost screens to identify peptide or candidate
compounds that bind a polypeptide of the invention, or fragment
thereof. Typically, protein microarrays feature a protein, or
fragment thereof, bound to a solid support. Suitable solid supports
include membranes (e.g., membranes composed of nitrocellulose,
paper, or other material), polymer-based films (e.g., polystyrene),
beads, or glass slides. For some applications, ATIA polypeptides
are spotted on a substrate using any convenient method known to the
skilled artisan (e.g., by hand or by inkjet printer). Preferably,
such methods retain the biological activity or function of the
protein bound to the substrate (e.g., ATIA antibody binding).
[0245] The protein microarray is hybridized with a detectable
probe. Such probes can be polypeptide (e.g., an ATIA antibody),
nucleic acid, or small molecules. For some applications,
polypeptide and nucleic acid probes are derived from a biological
sample taken from a patient, such as a bodily fluid (such as blood,
urine, saliva, or phlegm); a homogenized tissue sample (e.g. a
tissue sample obtained by biopsy, e.g. from the brain); or cultured
cells (e.g., lymphocytes). Probes can also include antibodies,
candidate peptides, nucleic acids, or small molecule compounds
derived from a peptide, nucleic acid, or chemical library.
Hybridization conditions (e.g., temperature, pH, protein
concentration, and ionic strength) are optimized to promote
specific interactions. Such conditions are known to the skilled
artisan and are described, for example, in Harlow, E. and Lane, D.,
Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring
Harbor Laboratories. After removal of non-specific probes,
specifically bound probes are detected, for example, by
fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric
assay), direct immunoassay, radiometric assay, or any other
suitable detectable method known to the skilled artisan.
[0246] Detection of an increase in the amount of an ATIA
polypeptide or an ATIA polynucleotide present in a patient sample
is useful as a diagnostic for the presence of brain cancer.
Optionally, ATIA detection may be combined with the detection of
other biomarkers, where the presence or level of the biomarker is
correlated with the presence of cancer, e.g. brain cancer.
ELISA
[0247] In certain embodiments, the invention provides a method for
detecting ATIA levels in biological fluids, for example in the
blood. The method is based on the finding that ATIA has a soluble
form, the extracellular portion of ATIA, and the soluble ATIA can
be detected in the culture medium of all types of ATIA-expressing
cells. Accordingly, it is possible to diagnose the disease by
assaying biological samples for the presence of the extracellular
portion of ATIA.
[0248] In preferred embodiments, soluble ATIA corresponds to SEQ ID
NOs 9 or 10, or fragments thereof.
[0249] In a preferred embodiments, ATIA levels are detected by
immunoassay, for example ELISA. Generally, immunoassays involve the
binding of ATIA and anti-ATIA antibody. The presence and amount of
binding indicate the presence and amount of ATIA present in the
sample. Examples of immunoassays include, but are not limited to,
protein arrays, ELISAs, radioimmunoassays, and immunoblots, which
are well known in the art. The antibody can be polyclonal or
monoclonal as described herein, and is preferably labeled for easy
detection. The labels can be, but are not limited to biotin,
fluorescent molecules, radioactive molecules, chromogenic
substrates, chemi-luminescence, and enzymes.
[0250] In one embodiment, ELISA, based on the capture of ATIA by
immobilized monoclonal anti-ATIA antibody followed by detection
with biotinylated polyclonal anti-ATIA antibody, is used to detect
ATIA.
[0251] Preferably, the extracellular portion of ATIA can be
detected using an ELISA assay.
[0252] The process can be automated. Automated ELISA reader devices
are well known in the art (Davis, et al. Microbiology 4th. ed., pp.
269-270 (1990)) and are commercially available (see, e.g.,
Multiskan EX and RC, Komabiotech, Seoul, Republic of Korea; HT3,
Anthos Analytical, Durham, N.C.).
Pharmaceutical Compositions
[0253] The present invention contemplates pharmaceutical
preparations comprising ATIA neutralizing antibodies, ATIA small
molecule inhibitors, or ATIA inhibitory polynucleotides, or
polypeptides, or analogs (e.g., a polynucleotide that hybridizes to
and interferes with the expression of an ATIA polynucleotide),
together with a pharmaceutically acceptable carrier.
Polynucleotides of the invention may be administered as part of a
pharmaceutical composition. The compositions should be sterile and
contain a therapeutically effective amount of the polypeptides or
nucleic acid molecules in a unit of weight or volume suitable for
administration to a subject.
[0254] These compositions ordinarily will be stored in unit or
multi-dose containers, for example, sealed ampoules or vials, as an
aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10 mL
vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous
ATIA polynucleotide solution, such as an aqueous solution of ATIA
polynucleotide or polypeptide, and the resulting mixture can then
be lyophilized. The infusion solution can be prepared by
reconstituting the lyophilized material using sterile
Water-for-Injection (WFI).
[0255] The ATIA neutralizing antibodies, ATIA small molecule
inhibitors, or ATIA inhibitory polynucleotides, or polypeptides, or
analogs may be combined, optionally, with a pharmaceutically
acceptable excipient. The term "pharmaceutically-acceptable
excipient" as used herein means one or more compatible solid or
liquid filler, diluents or encapsulating substances that are
suitable for administration into a human. The term "carrier"
denotes an organic or inorganic ingredient, natural or synthetic,
with which the active ingredient is combined to facilitate
administration. The components of the pharmaceutical compositions
also are capable of being co-mingled with the molecules of the
present invention, and with each other, in a manner such that there
is no interaction that would substantially impair the desired
pharmaceutical efficacy.
[0256] The compositions can be administered in effective amounts.
The effective amount will depend upon the mode of administration,
the particular condition being treated and the desired outcome. It
may also depend upon the stage of the condition, the age and
physical condition of the subject, the nature of concurrent
therapy, if any, and like factors well known to the medical
practitioner. For therapeutic applications, it is that amount
sufficient to achieve a medically desirable result.
[0257] With respect to a subject having cancer, and in particular a
glioblastoma or astrocytoma, an effective amount is sufficient to
stabilize, slow, or reduce the progression of the disease or
disorder.
[0258] Tumors, and in particular brain tumors, can be monitored by
imaging, for example by CAT scan, MR Spectroscopy, or MRI. Other
diagnostics include angiograms or arteriograms, which are a series
of X-rays taken after a special dye is injected into an artery,
usually in the area where the abdomen joins the top of the leg. The
dye, which flows through the blood vessels of the brain, can be
seen on X-rays. These X-rays can show the tumor and connecting
blood vessels. A brain scan may be used to reveal areas of abnormal
growth in the brain.
[0259] Generally, doses of inhibitory oligonucleotide compositions
of the present invention would be from about 0.01-1000 mg/kg per
day. Lower doses will result from certain forms of administration,
such as intravenous administration. Generally, doses of
neutralizing antibody compositions of the present invention would
be from about 0.01-1000 mg/kg per day.
[0260] In the event that a response in a subject is insufficient at
the initial doses applied, higher doses (or effectively higher
doses by a different, more localized delivery route) may be
employed to the extent that patient tolerance permits. Multiple
doses per day are contemplated to achieve appropriate systemic
levels of the ATIA polynucleotide or polypeptide compositions of
the present invention.
[0261] A variety of administration routes are available. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Other modes of administration include oral, rectal, topical,
intraocular, buccal, intravaginal, intracisternal,
intracerebroventricular, intratracheal, nasal, transdermal,
within/on implants, e.g., fibers such as collagen, osmotic pumps,
or grafts comprising appropriately transformed cells, etc., or
parenteral routes. Other useful approaches are described in Otto,
D. et al., J. Neurosci. Res. 22: 83-91 and in Otto, D. and
Unsicker, K. J. Neurosci. 10: 1912-1921.
Combination Therapies
[0262] The invention also features combination therapies.
[0263] According to certain embodiments, compositions and methods
of the invention may be used in combination with any conventional
therapy known in the art.
[0264] In particular embodiments, therapies that cross the blood
brain barrier (BBB) are particularly useful in methods of treating
or preventing brain tumors.
[0265] The blood-brain barrier (BBB) is a metabolic or cellular
structure in the central nervous system that restricts the passage
of various chemical substances and microscopic objects between the
bloodstream and the neural tissue itself, while still allowing the
passage of substances essential to metabolic function (e.g.
oxygen). The BBB results from the selectivity of the tight
junctions between endothelial cells in CNS vessels that restricts
the passage of solutes.
[0266] Certain therapeutics have been designed that are able to
pass through the BBB, including, for example, etoposide and
cisplatinum.
[0267] In part, the invention is based in the finding that ATIA
expression in human glioblastoma cells (such as, but not limited
to, A172 cells), are much more sensitive to TRAIL-induced cell
death than cells which do not express ATIA. For example, U87MG
cells do not express ATIA. Accordingly, in preferred embodiments,
ATIA expression can be used as an indicator for TRAIL treatment.
TRAIL is a promising treatment of glioblastoma and is currently in
clinical trials.
[0268] The invention is based, also, on the finding that knocking
down ATIA expression in human glioblastoma cells, such as A172
cells, renders the cells more sensitive to anti-cancer drugs.
Exemplary anti-cancer drugs include, but are not limited to the
following: acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin;
[0269] altretamine; ambomycin; ametantrone acetate;
aminoglutethimide; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; interleukin II (including recombinant interleukin II,
or rIL2), interferon alfa-2a; interferon alfa-2b; interferon
alfa-n1; interferon alfa-n3; interferon beta-I a; interferon
gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide
acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol; maytansine; mechlorethamine, mechlorethamine oxide
hydrochloride rethamine hydrochloride; megestrol acetate;
melengestrol acetate; melphalan; menogaril; mercaptopurine;
methotrexate; methotrexate sodium; metoprine; meturedepa;
mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;
mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;
mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;
paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin
sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone
hydrochloride; plicamycin; plomestane; porfimer sodium;
porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;
puromycin hydrochloride; pyrazofurin; riboprine; rogletimide;
safingol; safingol hydrochloride; semustine; simtrazene; sparfosate
sodium; sparsomycin; spirogermanium hydrochloride; spiromustine;
spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;
tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate
glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine
sulfate; vindesine; vindesine sulfate; vinepidine sulfate;
vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride, improsulfan, benzodepa,
carboquone, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide, trimethylolomelamine, chlornaphazine,
novembichin, phenesterine, trofosfamide, estermustine,
chlorozotocin, gemzar, nimustine, ranimustine, dacarbazine,
mannomustine, mitobronitol, aclacinomycins, actinomycin F(1),
azaserine, bleomycin, carubicin, carzinophilin, chromomycin,
daunorubicin, daunomycin, 6-diazo-5-oxo-1-norleucine, doxorubicin,
olivomycin, plicamycin, porfiromycin, puromycin, tubercidin,
zorubicin, denopterin, pteropterin, 6-mercaptopurine, ancitabine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, enocitabine,
pulmozyme, aceglatone, aldophosphamide glycoside, bestrabucil,
defofamide, demecolcine, elfornithine, elliptinium acetate,
etoglucid, flutamide, hydroxyurea, lentinan, phenamet,
podophyllinic acid, 2-ethylhydrazide, razoxane, spirogermanium,
tamoxifen, taxotere, tenuazonic acid, triaziquone,
2,2',2''-trichlorotriethylamine, urethan, vinblastine, vincristine,
vindesine and related agents. 20-epi-1,25 dihydroxyvitamin D3;
5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;
adozelesin; aldesleukin; ALL-TK antagonists; altretamine;
ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin;
amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis
inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing
morphogenetic protein-1; antiandrogen, prostatic carcinoma;
antiestrogen; antineoplaston; antisense oligonucleotides;
aphidicolin glycinate; apoptosis gene modulators; apoptosis
regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2;
axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III
derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;
bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine; calcipotriol; calphostin C; camptothecin
derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorlns; chloroquinoxaline sulfonamide; cicaprost; cisporphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; taxel; taxel analogues; taxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pirarubicin; piritrexim; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0270] Preferred additional anti-cancer drugs are 5-fluorouracil
and leucovorin. Additional cancer therapeutics include monoclonal
antibodies such as rituximab, trastuzumab and cetuximab.
[0271] In preferred embodiments, ATIA therapy (e.g. knockdown or
inhibition) is carried out in combination with administration of
the anti-cancer drug cisplatinum.
[0272] In other preferred embodiments, ATIA therapy (e.g. knockdown
or inhibition) is carried out in combination with ionizing
radiation treatment.
[0273] The following examples are offered by way of illustration,
not by way of limitation. While specific examples have been
provided, the above description is illustrative and not
restrictive. Any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
Furthermore, many variations of the invention will become apparent
to those skilled in the art upon review of the specification. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
EXAMPLES
Example 1. Identification of ATIA as a Novel Anti-Apoptotic Protein
Against TNF-Induced Apoptosis
[0274] The proinflammatory cytokine tumor necrosis factor (TNF)
plays an important role in diverse cellular events such as septic
shock, induction of other cytokines, cell proliferation,
differentiation, necrosis and apoptosis (1, 2, 3). TNF was
originally identified as a factor that leads to rapid hemorrhagic
necrosis of transplantable tumors in mice (4). Approximately one
third of transformed cell lines were shown to be susceptible to the
cytolytic action of TNF (5). However, because of its toxicity in
animals and human, TNF did not fulfill the initial expectations
that it would be useful in the treatment of cancer (6). Studies
from many laboratories have demonstrated that the diverse
TNF-mediated biological responses are achieved through activating
multiple signaling pathways. Many of the TNF-induced cellular
responses are mediated by either one of the two TNF receptors,
TNF-R1 and TNF-R2, both of which belong to the TNF receptor
super-family (7, 8, 9). In response to TNF treatment, the
transcription factor NF-KB and MAP kinases, including ERK, p38 and
JNK, are activated in most types of cells and, in some cases,
apoptosis or necrosis could also be induced (10, 11, 12, 13, 14).
However, induction of apoptosis or necrosis is mainly achieved
through TNF-R1, which is also known as a death receptor (15, 16,
17). The initiation of these TNF-induced pathways is precisely
regulated under physiological conditions and the aberrant
commitment or failure of these pathways is often accountable for
many human diseases, such as cancer, arthritics and AIDS (1,
3).
[0275] Many molecular aspects of TNF signaling remain unknown.
Particularly, the decision of life and death in response to TNF is
still not understood. It is known now that one of the reasons for
the inefficiency of TNF killing is the activation of NF-KB in
response to TNF treatment (18). Studies from several labs have
demonstrated that NF-KB activation protects cells against
TNF-induced apoptosis (18). Inhibition of NF-kB activation renders
many types of cells TNF sensitive. Several of NF-KB's target genes,
including cIAP-1, cIAP-2 and IEX-1L, cFlip, have anti-apoptotic
properties (19, 20, 21). At the same time, results suggest that
there are also NF-KB-independent pathways that are important
components of the protective machinery against TNF-induced
apoptosis. For instance, it has been proposed that TRAF2 protects
cells against TNF-induced apoptosis independently of NF-KB (22).
The machinery of this TRAF2-dependent anti-apoptotic pathway is
largely unknown, although we found that a member of SP 1
transcription factors, LKLF, is downstream of TRAF2 and protects
cells against TNF-induced apoptosis (23). To further understand the
molecular mechanisms of TRAF2-mediated cell protection against
TNF-induced apoptosis, the experiments described herein are
directed to identifying additional proteins that function
downstream of TRAF2 and protect cells against TNF-induced
apoptosis. Through screening a retroviral cDNA expression library
for genes that protect TRAF2 null cells following TNF treatment, we
identified a novel anti-apoptotic gene, ATIA (anti-TNF-induced
apoptosis). Thehe human homolog of ATIA, Vasorin, was reported
after the present inventors cloned ATIA, and vasorin was found to
be a TGF -binding protein (24). The present inventors have
demonstrated that ATIA is a multi-functional protein that regulates
TGF-beta signaling and protects cells against TNF-induced
apoptosis. The present inventors have found that ATIA protein
localizes in the cell plasma membrane and the mitochondria and that
ATIA mutant protein targeted to mitochondria is capable of
protecting cells against TNF-induced apoptosis. The present
inventors have shown that by generating ATIA knockout mice, ATIA
protects cells against TNF-induced apoptosis in vivo.
[0276] To screen for potential anti-apoptotic genes, a retroviral
cDNA expression library was constructed with mRNA isolated from
wild-type MEFs. This library was used to infect TRAF2-/- cells. The
infected cells were induced to undergo apoptosis with TNF treatment
as TRAF2-/- MEFs are super-sensitive to TNF-induced apoptosis (25).
The surviving cells were then collected for recovering the infected
cDNA, which presumably is responsible for the increased resistance
of the infected cells to TNF-induced apoptosis. Through such a
screening approach, the gene, ATIA (anti-TNF-induced apoptosis),
which encodes a novel protein with a putative transmembrane domain
(FIG. 1A).
[0277] Initially, it was concluded that the isolated cDNA clone
encodes a full length 51 Kd protein. However, when the mouse
genomic DNA sequence became available, it was realized that only a
portion of the ATIA gene from an in-frame ATG codon had been
cloned, and that there is another 848 bps at the 5' end of the
gene. Therefore, the original clone was designated ATIAc (see FIG.
1A). The full length cDNA of ATIA encodes a protein with 673 a.a,
which has a predicted molecular weight of 72 Kd. As shown in FIG.
1B. The 673 a.a. full length protein corresponds to SEQ ID NO: 2.
As shown in FIG. 1B, ATIA is highly expressed in some tissues such
as liver, lung, kidney, and testis. Importantly, the expression of
ATIA is considerably reduced in the TRAF2-/- cells while the
ectopic-expression of TRAF2 restored the expression of ATIA (FIG.
1C). However, ATIA expression is not inducible by TNF (data not
shown). When ATIA or ATIAc was over-expressed in TRAF2-/- or human
breast carcinoma MCF7 cells, it protected cells from TNF-induced
apoptosis (FIG. 2, and data not shown). Therefore, ATIA is a
potential anti-apoptotic gene that protects cells against
TNF-induced apoptosis.
[0278] Additional information about ATIA function was provided by
studying its cellular localization. ATIA has a signal peptide at
its N-terminus and a putative transmembrane domain, suggesting that
it may localize to the plasma membrane. To test this possibility,
full length ATIA and the truncated ATIAc construct were cloned into
vectors that created c-terminal YFP-tagged ATIA proteins, ATIA-YFP
and ATIAc-YFP. These constructs were transfected into wt MEFs.
Western blot analysis with an anti-GFP antibody indicates that the
expression of the full-length ATIA-YFP gives two products, a major
one at about 130 KD and a minor one at about 110 Kd (FIG. 3A).
Since the predicted molecular weight of ATIA-YFP is about 97 Kd
without the signal peptide, the larger sizes of the products are
apparently due to additional modifications of the proteins, that it
is believed are at least partially due to glycosylation (data not
shown). The ATIAc-YFP construct gives a product at the predicted 75
Kd (FIG. 3A), indicating that the modifications are likely within
the first 231 amino acids of the ATIA protein. Confocal microscopy
demonstrated that the majority of the cells transfected with the
full-length construct had intense labeling of the plasma membrane
while some cells showed a punctate pattern in the cytoplasm (FIG.
2B). Cells transfected with the truncated ATIAc construct
demonstrated only punctuate expression and no membrane expression
(FIG. 3B). This punctate pattern resembled mitochondrial
localization. To confirm this, transfected cells were stained with
mitochondrial marker MitoTracker. Indeed, full-length ATIA
localized to the plasma membrane in the vast majority of cells with
about 30% of cells having some mitochondrial localization (FIG.
3C). ATIAc was found only in mitochondria. As ATIAc lacks the
signal peptide, this data confirmed the role of the signal peptide
in targeting ATIA to the plasma membrane. Since the expression of
ATIA-YFP plasmid yields two different sized products, these two
products may represent the ATIA protein localized to the plasma
membrane and mitochondria, respectively.
[0279] To confirm the protective ability of these two plasmids,
microinjections of TRAF2-/- MEFs with EYFP vector, ATIA-YFP or
ATIAc-YFP were performed. Cells injected with EYFP vector were
sensitive to TNF-induced apoptosis with less than 15% of the cells
surviving after 6 hours. Cells injected with either ATIA-YFP or
ATIAc-YFP plasmid were protected at a 60% or 45% survival rate,
respectively (data not shown). These results suggest that the
mitochondrially localized ATIA is capable of protecting cells
against TNF-induced apoptosis.
[0280] To further verify the membrane and mitochondrial
localizations of ATIA, cell fractionation experiments were
performed with wild-type MEF cells before and after treatment with
pronase, a proteolytic enzyme that removes exposed membrane
proteins. The presence of different proteins in total cell lysates
or in mitochondrial fractions was analyzed by Western blotting with
different antibodies. Using an anti-ATIA antibody with an epitope
to the whole intracellular domain (ICD) comprising 600 amino acids,
the endogenous ATIA protein from wild-type MEFs before pronase
treatment was detected at two different sizes in the total cell
lysate, a dominant upper band (100 Kd) and a weaker lower band (80
Kd) (FIG. 4). This is consistent with what was observed when ATIA
is overexpressed (FIG. 3). Upon pronase treatment, the upper band
of ATIA disappears while the lower band of ATIA is not affected.
However, in the enriched mitochondrial fraction, the ratio between
the upper and lower band of ATIAs is dramatically altered prior to
pronase treatment, suggesting the lower band is enriched in the
mitochondrial fraction (FIG. 4). This enrichment of the smaller
size of ATIA in the mitochondrial fraction is not affected by the
pronase treatment. Both ATIA bands are absent in the cytosolic
fraction. These results support the idea that the upper band of
ATIA is localized to the plasma membrane and the smaller form of
ATIA is in the mitochondria. Western blotting with other control
antibodies further confirmed this conclusion. Therefore, the two
forms of ATIA proteins were designated as memATIA and mitoATIA
respectively.
Example 2. ATIA Protects Cells Against TNF-Induced Apoptosis In
Vivo
[0281] To further study the physiological function of ATIA, ATIA
knockout mice were generated. As shown in FIG. 5, the region of
ATIA from a.a 449 to as 619 was deleted because this region of ATIA
is essential for protecting cells from TNF-induced apoptosis based
on in vitro study data (not shown). The presence of mutant ATIA
allele in ES cells was confirmed by PCR and DNA sequencing of the
PCR product (FIG. 5B, data not shown). The offspring were genotyped
by PCR (FIG. 5C) and confirmed by RT-PCR (Data not shown). The
ATIA+/ heterozygotic mice were backcrossed to C57BL/6 mice for 8
generations. The resulting heterozygotic mice were bred to each
other and the homozygotic wild type and knockout progeny were used
in all subsequent in vivo experiments. ATIA knockout mice appear
normal phenotypically, however, male ATIA knockout mice appear to
have impaired fertility.
[0282] To determine whether ATIA is able to protect from
TNF-induced apoptosis in vivo, a model of TNF-induced hepatitis
(26, 27) was used. ATIA-/- and ATIA.+-./.+-. litter mates from
heterozygote cross were pretreated with an inhibitor of
transcription that is specific for the liver, D-galactosamine
(GalN), which metabolically inhibits hepatocyte RNA and protein
synthesis, followed by intravenous injection of a sub-lethal dose
of TNFa (2 g/Kg). By 8 hr post-treatment, 20% of ATIA+/+ mice as
compared to more than 85% of ATIA--mice were moribund (FIG. 5D).
Histological analysis of H&E stained liver sections 4 hours
after TNFa administration demonstrated significant apoptosis in
livers of ATIA-deficient mice, while ATIA+/+ litter mates showed
little to no apoptosis (FIG. 5E). No other histological differences
were noted. TUNEL staining of liver sections also showed a dramatic
increase in TUNEL-positive cells in the ATIA-/- liver sections as
compared to the ATIA.+-./.+-. litter mates (FIG. 5E). To further
confirm this observation, we also examined TNF-induced apoptosis in
ATIA-/- MEFs. Wild-type and ATIA-/- mouse embryonic fibroblast cell
lines were established from 10.5-12.5 day old wild-type and ATIA
knockout embryos. The cell lines were verified for the loss of ATIA
by genomic PCR analysis and western blotting with the anti-ATIA
antibody (data not shown). Cells were treated with TNF and varied
doses of cyclohexamide (CHX) (FIG. 6A). At 8 hours, ATIA KO cells
show an increase in cell death compared to wild type cells as
measured by MTT assay, suggesting that the loss of ATIA increases
the sensitivity to TNF-induced apoptosis. Cell death induced by UV
radiation or staurosporine is however, only moderately affected by
the loss of ATIA (FIG. 6B). These results further support our
conclusion that ATIA protects cells against TNF-induced apoptosis.
We then examined JNK and NF-KB activation induced by TNF and found
that the loss of ATIA has no affect on TNF-induced activation of
either of these two pathways (data not shown). These results
suggested ATIA has no detectable effect on the immediate downstream
signaling by TNF, however, it protects from cell death induced by
INF.
Example 3. ATIA Inhibits TGF-Beta (TGFB) Signaling
[0283] It has been reported that the human homolog of ATIA,
Vasorin, is a TGFB-binding protein and modulates the arterial
response to injury (24). To test whether ATIA has a similar
function in regulating TGFB signaling, TGFB-induced Smad2
activation in wild-type and ATIA-/- MEFs was examined. As shown in
FIG. 7, TGF-beta-induced phosphorylation of Smad2 is dramatically
increased in ATIA-/- MEFs comparing to what in wild-type cells.
This inhibitory effect of ATIA seems to be TGFB signaling specific
since loss of ATIA does not alter BMP4-induced activation of Smad1
(FIG. 7, right panel). Therefore, ATIA attenuates TGFB-induced
activation of Smad2.
[0284] Taken together, the studies discussed above suggest that
ATIA not only regulates TGFB signalling, but also protects cells
against TNF-induced apoptosis. Since the mitochondrially localized
ATIA mutant is sufficient to protect cells against TNF-induced
apoptosis, and regulating TGFB signaling by ATIA likely occurs at
the plasma membrane, it is conceivable that there is more than one
distinct function for the ATIA protein, and that the cellular
localization of ATIA is a key factor that determines how ATIA is
functioning in the cell.
[0285] To test the hypothesis that the plasma membrane form of ATIA
is responsible for regulating TGFP signaling while the
mitochondrial ATIA protects cells against TNF-induced apoptosis,
the ATIA protein will be characterized in order to identify the
factors that determine ATIA localization, and this knowledge will
be used to show that ATIA location is an important factor in
determining its distinct functions. To further understand the
regulation of TNF-induced apoptosis, the underlying mechanism of
the ATIA protective effect against TNF-induced apoptosis will be
examined further. Since ATIA attenuates TGFB signaling, which is
known as a tumor suppressor pathway in early carcinogenesis, and
ATIA protects cell against apoptosis, ATIA is likely to be involved
in promoting tumorigenesis.
Example 4: Plasma Membrane ATIA Regulates TGFB Signaling and
Mitochondrial ATIA Protects Cells Against TNF-Induced Apoptosis
[0286] The ATIA protein will be characterized to identify the key
factors that determine ATIA cellular locations. This knowledge can
be used to address whether the localization of ATIA affects its
different functions. For instance, based on the cellular
localization of ATIA, different ATIA variant/mutant clones are
generated whose products will specifically localize to the plasma
membrane or in the mitochondria. The plasma membrane targeted ATIA
mutant can be tested for its ability to attenuate TGFB signaling,
its ability or failure to protect cells against TNF-induced
apoptosis in ATIA-/- MEFs or, in contrast, if the mitochondrial
ATIA variant only protects cells against TNF induced apoptosis, but
has no effect on TGFB signaling.
[0287] The results discussed herein suggest that the 100 Kd
endogenous ATIA membrane protein (memATIA) localizes in plasma
membrane and the 80 Kd ATIA (mitoATIA) localizes in the
mitochondria. Accordingly, finding out the differences of these two
forms of the ATIA protein will help to understand what determines
the different cellular localizations of ATIA. It has been examined
whether glycosylation causes the size difference of these two ATIA
products since the predicted molecular weight of the ATIA protein
without signal peptide is only about 70 Kd. As shown in FIG. 8,
deglycosylation decreases the molecular weights of both forms of
ATIA proteins. The results showed that the mitoATIA is only
N-glycosylated while memATIA has both N- and 0-glycosylation. After
complete deglycosylation, the memATIA protein is about 85 Kd and
the mitoATIA is about 70 Kd. Accordingly, these results suggest
that glycosylation contributes to the difference of the molecular
weights of these two ATIA proteins, but is not the sole factor.
There are two possibilities that could be accountable for the
remaining difference between memATIA and mitoATIA: 1) memATIA, but
not mitoATIA has some additional modifications; 2) an additional
proteolysis processing happens to the mitoATIA protein after its
signal peptide is removed. For the later case, it is most likely
that the additional processing of the ATIA protein happens at its
N-terminal since the two products of our c-terminal YFP tagged ATIA
plasmid have the similar difference in their molecular weights as
the endogenous counterparts do (FIGS. 3 & 4). Therefore,
N-terminal protein sequencing and mass spectrometry analysis will
be performed on these two forms of ATIA proteins.
[0288] Accordingly, the ATIA cDNA has been cloned into the V5
vector, which has a c-terminal His tag. Transient transfection
experiments have confirmed that the ATIA-His protein, as the
ATIA-YFP protein, is also present in two forms (data not shown,
FIG. 3). After establishing a stable ATIA-His overexpression cell
line with wt MEF cells, enough ATIA-His protein will be isolated,
both memATIA-His and mitoATIA-His, for N-terminal protein
sequencing and mass spectrometry analysis. Based on the information
from N-terminal protein sequencing and mass spectrometry analysis,
a variety of variant/mutant ATIA-YFP clones were created for
testing to see if any of these ATIA-YFP proteins exclusively
localizes to the mitochondria. If one of those ATIA-YFP proteins
does localize to mitochondria specifically, the ATIA-YFP clone will
be transfected into ATIA-/- cells for testing.
[0289] The results described herein have demonstrated that
mitochondrially localized ATIA mutant is capable of protecting
cells from TNF-induced apoptosis; however, the results do not
completely rule out the possibility that mitochondrial ATIA has no
effect on TGFB signaling since ATIAc does not have the LRR domain,
which is required for binding to TGFB. We anticipate that the
proposed study in the above section will address the issue whether
the mitoATIA protects cells against TNF-induced apoptosis, but does
not inhibit TGFB signaling.
[0290] To generate an ATIA mutant targeted to the plasma membrane,
the mitochondrial localization targeting signal (domain) of ATIA
will be identified and mutated. Based on previous studies with
ATIAc, it is known that the mitochondria-targeting sequence is
within 232-673 a.a of ATIA. Therefore, a serial deletion of
ATIAc-YFP protein will be performed to examine which region of ATIA
is responsible for its mitochondrial localization. Because it is
known that mitochondrial targeting sequences normally have
conserved arginine residues, different arginine residues will be
mutated within the identified region of the full length ATIA by
point mutations, and whether certain mutations on those arginine
residues will abolish the mitochondrial localization of ATIA, but
have no effect on its localization to the plasma membrane will be
tested. Based on those results, those mutant ATIA constructs will
be used to examine whether the plasma membrane localized ATIA only
inhibits TGFP signaling, but does not protect cells against
TNF-induced apoptosis in ATIA-/- MEFs. Though it is known that TGFB
can cause cells to undergo growth arrest and apoptosis in certain
types of cells, it is not expected that attenuating TGFB signaling
by memATIA will have any dramatic effect on TNF-induced apoptosis
in MEF cells in the presence of protein synthesis inhibitor, CHX,
as this blocks the de novo protein synthesis required for TGFB
functions.
Example 5: ATIA Protects Cells Against TNF-Induced Apoptosis
Through Regulating TRX2 Function
[0291] Based on the experiments described herein, it is thought
that ATIA plays a role in protecting cells against TNF-induced
apoptosis in MEF cells. To further investigate the mechanism of
ATIA mediated protection against TNF-induced apoptosis, ATIA
interacting proteins are identified. A yeast two-hybrid screen of a
mouse liver cDNA library was carried out with the full length ATIA
as the bait. After two rounds of screening, an interacting protein
was identified called Thioredoxin 2 (TRX2). Interestingly, it has
been shown that TRX2 is specifically expressed in the mitochondria
and essential for cell survival (28, 29). Deletion of TRX2 causes
massive apoptosis, accumulation of intracellular ROS and early
embryonic lethality in homozygous mice (30). Therefore, the
identification of TRX2 as a potential ATIA-interacting protein
raised the possibility that ATIA protects cells against TNF-induced
apoptosis through regulating the activity of TRX2. This possibility
is supported by the finding that the intracellular ROS level is
elevated in ATIA-/- cells comparing to wild type MEFs (FIG. 9).
[0292] After confirming the interaction between ATIA and TRX2 in
yeast under different stringencies, the ATIA and TRX2 interaction
will be examined in the mammalian systems. First the ATIA protein
will be tested for the ability to be pulled down by GST-TRX2 fusion
protein in vitro. ATIA will be overexpressed along with some of its
mutants in MEF cells and the cell lysates will be collected. GST
pull-down experiments will be carried out by mixing different cell
lysates with GST-TRX2 fusion protein while the GST protein will be
used as a control. If an interaction between ATIA and TRX2 is
detected with the in vitro pull-down experiments, the ATIA/TRX2
interaction can also be demonstrated by immuno-precipitating
endogenous proteins with anti-ATIA and/or TRX2 antibodies. In these
experiments, mitochondrial fractions will be isolated to enrich the
concentration of mitoATIA in order to limit the interference by the
memATIA. In the case that ATIA and TRX2 are loosely associated
within mitochondria and can not be detected under regular
immuno-precipitation experimental conditions, the cells will be
treated with the cross-linking reagent, DSP
[Dithiobis(succinimidylpropionate)] after isolation of mitochondria
(31). DSP covalently links the proteins in the same complex
together and allows us to detect the presence of those proteins
that are loosely associated. The cross-linker could be cleaved with
5% 13-mercaptoethanol in SDS-PAGE sample buffer at 100.degree. C.
for 5 minutes before immuno-precipitants are applied to SDS-PAGE
gel.
[0293] Next the potential regulatory effect of ATIA on TRX2
function will be examined. It is known that TRX2 is a major player
in scavenging ROS generated in mitochondria (28). Particularly, it
has been shown that TRX2 is specifically oxidized in response to
extra cellular stimuli such as TNF and H202, and the redox state of
TRX2 reflects the regulation of TRX2 function (32). Since we found
that the intracellular ROS level is elevated in ATIA-/- cells
compared to wild type MEFs (FIG. 9), it is possible that the
function of TRX2 is deregulated in ATIA-/- MEFs. It has been
confirmed that the deletion of ATIA does not affect the expression
of TRX2 in ATIA-/- cells (data not shown). Therefore, it is
important to examine the redox state of TRX2 in ATIA-/- cells
with/without TNF or H202 treatment when compared to wild-type
cells. The redox state of TRX2 will be determined by the redox
Western analysis with
4-acetamido-4'-maleimidylstibene-2,2'-disulfonic acid as described
previously (32). Meanwhile, the enzymatic activity of TRX2 isolated
from wild-type and ATIA-/- cells in vitro will be measured (33).
Based on the findings that ATIA-/- mice and MEFs are more sensitive
to TNF-induced toxicity/apoptosis and that the intracellular ROS
level is elevated in ATIA-/- MEFs, it is anticipated that the level
of the oxidized ATIA protein will be higher in ATIA-/- MEFs than in
wild-type cells. If it is found that TRX2 function is deregulated
in ATIA-/- MEFs, the role of ATIA in this process will be further
confirmed by restoring the expression of ATIA in ATIA-/- MEFs and
examining whether TRX2 function is reconstituted. Particularly, the
region of ATIA that is responsible for interacting with TRX2 will
be identified, and then subsequently experiments will be carried
out to test whether the ATIA mutant lacking the ability to interact
with TRX2 is able to restore the TRX2 function and to protect cells
against TNF-induced apoptosis. If the mutant ATIA fails to protect
cells, the possible underlying mechanism by which ATIA regulates
the function of the mitochondrial TRX system will be
investigated.
[0294] It has been reported that the TRX system in mitochondria has
three major components: the mitochondrial peroxiredoxin III, TRX2
and TRXR2 (28). Therefore, one possibility is that ATIA functions
as a scaffold protein to mediate the formation of the mitochondrial
TRX complex. To test this hypothesis, the interaction between TRX2
and TRXR2 or between TRX2 and peroxiredoxin III in wild-type and
ATIA-/- MEFs will be examined. If there is a defect in the
interaction between some of these proteins, it will then be tested
whether the affected interaction could be restored by ectopic
expression of ATIA in ATIA-/- MEFs. If the absence of ATIA does not
affect the interactions among these proteins, the role of ATIA in
this system may be to maintain the proper tertiary conformation of
the TRX complex required for its normal function. The function of
ATIA will be examined by expressing different ATIA mutants in
wild-type and ATIA-/- MEFs. It is expected that some ATIA mutants
will be identified that restore the function of TRX2 in ATIA-/-
MEFs and some mutants that disrupt the normal function of TRX2 in
wild-type cells. These studies will aid in understanding how ATIA
regulates TRX2 function.
[0295] Taken together, the experiments described herein will
examine whether ATIA protects cells against TNF-induced apoptosis
through regulating TRX2 function.
Example 6: The Role of ATIA in Tumorigenesis
[0296] The studies herein suggest that that ATIA is a
multifunctional protein: it inhibits TGFB signaling and protects
cells against TNF-induced apoptosis. Since TGFB signaling is a
known tumor suppressor pathway and apoptosis plays a critical role
in preventing tumor development, ATIA may have a role in
tumorigenesis by blocking these two tumor suppressing pathways.
[0297] To explore this possibility, first the levels of ATIA
protein in several types of cancers and normal tissues were
examined using Western Blots purchased from ProSci Inc. As shown in
FIG. 10, ATIA protein level is considerably increased in brain
tumors. ATIA protein levels were examined in several human brain
tumor cell lines including A-172 (glioblastoma) and CCF-STTG 1
(astrocytoma) and found that A-172 and CCF cells have high ATIA
expression (FIG. 11).
[0298] Experiments will next examine whether ATIA plays a role in
tumorigenesis in mouse models of human tumor. There are several
existing mouse models of human brain tumor, such as the activated
Ras transgenic mouse model and the NF1 and p53 double knockout
mouse model (34, 35). Activation of Ras signaling is critical in
both models (34, 36). One of the reasons to choose these two mouse
models is that ATIA is highly expressed in the human glioblastoma
cell line, A-172 and the astrocytoma cell line, CCF-STTG1, but not
in the neuroblastoma cell line, IMR-32 (FIG. 11) and that
astrocytoma is developed in these two mouse models.
First experiments will test whether ATIA protein level is elevated
in the tumors developed in these two models. If ATIA protein is
increased in the tumors from one of these two models, for instance,
in the Ras transgenic mouse model, experiments will examine the
role of ATIA in the tumor development in this model by generating
the Ras/ATIA-/- mice through crossing the ATIA-/- mouse with the
Ras transgenic mouse. If ATIA tumor development is decreased in
Ras/ATIA-/- mice, ATIA may play an important role in tumorigenesis
in the Ras transgenic mouse model. This Ras transgenic mouse tumor
model would then be employed for a future study on the role of ATIA
in tumor promotion or growth. In particular, experiments will focus
on whether ATIA contributes to the tumor development in this mouse
model through inhibiting TNFB signaling and/or blocking
apoptosis.
[0299] If there is a failure to detect any increase of ATIA
expression in both mouse models, it suggests that the deregulation
of ATIA is not involved in the activated Ras-mediated tumorigenesis
of brain tumors. If ATIA level is increased in these models, but
deletion of ATIA has no effect on the tumor development, it implies
that ATIA function is not essential for tumorigenesis in these
models. In either case, the potential role of ATIA in tumor
development will be addressed by generating an ATIA transgenic
mouse model. With ATIA transgenic mice, experiments will address 1)
if the elevated expression of ATIA will lead to the development of
any type of tumor; 2) whether the increased expression of ATIA
protein will promote any tumor development in the presence of
carcinogens or mutations of tumor suppressors, for instance in the
p53-/- background.
[0300] The experiments described herein will provide insights about
the potential role of ATIA in tumorigenesis and establish a system
to study the involvement of ATIA in the development of certain
types of tumors, and help to identify ATIA as a therapeutic target
in treating cancer.
Example 7: Increased Expression of ATIA in Glioblastoma
[0301] Experiments were directed at the expression of ATIA in human
brain tumors. The presence of ATIA in human glioblastoma was
determined using human glioblastoma tissue stained with anti-ATIA
antibody. Samples were obtained from MD Anderson cancer center. As
shown in FIG. 12, three of four samples are positive for anti-ATIA
staining.
[0302] FIG. 13 shows that the soluble form of ATIA is present in
the cell culture medium from different types of ATIA expressing
cells including human glioblastoma A172 cells. In FIG. 13, membrane
ATIA is detected from 30 .mu.g total cell lysate and soluble ATIA
was concentrated from 1 ml A172 cell culture medium.
[0303] Next, human tissue arrays were used to examine the
expression of ATIA in glioblastoma and astrocytoma. FIGS. 14-18
show results of tissue array experiments. The tissue arrays were
purchased from Biomax US and two types of arrays have been tested
for the specific expression of ATIA in human glioblastoma and
astrocytoma. All of the experiments were repeated three times with
identical results. The glioblastoma array (BS17081) used duplicated
cores per case, with 30 cases of brain glioblastoma in three
adjacent normal tissues. The multiple brain cancer and adjacent
normal tissue array 9GL1001) used 100 cases/100 cores.
[0304] As shown in FIG. 14, 23 cases out of 30 total cases (76.7%)
are detected for high ATIA expression in both cores. FIG. 15 is a
Table showing the distribution of ATIA positive cases in the
glioblastoma array. FIG. 16 shows some examples if
immunohistochemistry with antivasorin staining from the
glioblastoma tissue arrays. Samples of glioblastoma stage IV were
compared with adjacent normal brain tissue. Specimens were examined
at 10.times. and 20.times.. As shown in FIG. 16, the glioblastoma
samples show higher staining than the controls. In FIG. 17, the
distribution of ATIA positive cases in the mixed tissue array was
examined. As shown in FIG. 17, only some astrocytoma cases are
positive by anti-vasorin staining.
[0305] FIG. 18 shows results from human tissue blots with multiple
different tissues. The human tissue blots were purchased from
ProSci Inc. The tissue blots provided by ProSci Inc. do not have
perfectly matched normal vs. tumor blots. ATIA expression (human
vasorin) can be seen in human normal tissues in the lung, and in
human tumor tissues highly expressed in the brain, and low levels
of expression in the pancreas and stomach.
Example 8: Sensitivity of ATIA Expressing Cells to TRAIL-Induced
Cell Death
[0306] Next, the sensitivity of ATIA expressing cells to
TRAIL-induced cell death was examined. FIG. 19, left panel, is a
western blot showing the expression of ATIA in various glioblastoma
cell types. The graph in the right panel of FIG. 19 shows the three
glioblastoma cell types that are treated with TRAIL (20 ng/ml) for
24 h. The results show percent viable cells after 24 h.
Example 9: Knockdown of ATIA Makes Cells More Sensitive to
Therapy
[0307] Experiments were performed that show that knocking down of
ATIA expression in human glioblastoma cells renders the cells more
sensitive to chemotherapeutic agents. FIG. 15 shows the results of
experiments where knocking down of ATIA expression in A172
glioblastoma cells renders the cells sensitive to etoposide or
cisplatinum treatment. Both etoposide and cisplatinum cross the
blood brain barrier. The top panel of FIG. 20 is a Western Blot
showing ATIA expression after siRNA knockdown. A commercial siRNA
pool (small RNAs) was used to knockdown ATIA. The bottom panel is a
graph that shows cell survival as determined by a MTT assay that
measures cell proliferation and viability. The results show that
ATIA was partially knocked down in these experiments when these
cells are treated with etoposide or cisplatinum. Next, it was
tested whether knocking down of ATIA expression in A172 cells
renders the cells sensitive to etoposide or cisplatinum treatment.
FIG. 21 is a graph that shows that cells where ATIA is knocked down
(compared with lamin control siRNA) are rendered sensitive to
cisplatinum treatment. There is a significant difference between
cisplatinum treatment and control treatment in A172 cells that have
decreased ATIA expression.
Example 10: Effect of ATIA Localization on TNF-Induced
Apoptosis
[0308] In a further set of experiments shown in FIGS. 22 and B, two
ATIA mutants were used, 1-599 ATIA and 232-671 ATIA. As shown in
FIG. 22A, the 1-599 ATIA mutant, which lost the mitochondrial
localization, but still localizes to the cell membrane, does not
protect cells against TNF-induced apoptosis. As shown in FIG. 22B,
the 232-671 mutant, which only localizes to the mitochondrial,
protects cells against apoptosis.
Example 11. ATIA Expression is Increased Under Hypoxic
Conditions
[0309] In a further set of experiments, it was shown that ATIA is a
hypoxia-inducible gene. FIG. 23 shows that ATIA is induced
following CoCl.sub.2 treatment. In FIG. 23, CoCl.sub.2 is a hypoxia
mimic, which induced hypoxia in treated cells. ATIA protein
expression, particularly in the lower band (the mitochondrial
ATIA), is considerably increased. Hypoxia-Inducible Factor (HIF)-1
is a dimeric protein complex that plays an integral role in the
body's response to hypoxia. HIF-1 is a major regulator of oxygen
homeostasis within cells. As a transcription factor, it affects and
regulates the expression of dozens of genes involved in maintaining
homeostasis as oxygen concentrations change ATIA promoter has two
HIF responsive sites. FIG. 24 is a Western blot that shows ATIA is
induced under hypoxia conditions.
Example 12. Knocking Down ATIA Sensitizes Cells to Hypoxic
Conditions
[0310] FIG. 25 is a graph that shows increased sensitivity to
CoCl.sub.2 induced Hypoxia in ATIA KO MEFs. In these experiments,
wild type (Wt) and ATIA knockout (ATIA-/-) MEFs are treated with
CoCl.sub.2 and cell death is detected at different time points as
indicated in the figure. The results shown in FIG. 25 demonstrate
that ATIA-/- MEFs are much more sensitive to CoCl.sub.2-induced
cell death. Further, FIG. 26 is a graph that shows increased
sensitivity of A172 glioblastoma cells to CoCl.sub.2 treatment when
ATIA is knocked down. Human glioblastoma A172 cells are transfected
with siRNAs of lamin or ATIA and then treated with CoCl.sub.2. ATIA
knock down renders cells more sensitive to CoCl.sub.2 induced cell
death. As shown in FIG. 27, A172 cells were transfected with siRNA
targeting lamin or ATIA for o/n, and were then subjected to 24 h
and 48 h of hypoxia (0% Oxygen). Cell death was determined using
Annexin V/PI staining method and analyzed using flow cytometry.
These results show increased sensitivity of A172 cells to hypoxia
treatment when ATIA is knocked down. Accordingly, these findings
demonstrate that ATIA is important for cancer cells to survive
through protecting hypoxia-induced cell death.
Example 13. Treatment with Small Chemical Molecules Reduces ATIA in
Cancer Cells
[0311] Experiments were performed with small chemical molecules to
reduce ATIA levels in cancer cells. Examples of small molecules
that can be used include, but are not limited to, cyclohexamide.
Cyclohexamide, at low levels (e.g. 0.1 .mu.g-1.0 .mu.g) reduces
ATIA levels in tumor cells. Preferably, cyclohexamine is
administered locally to the tumor. Results of an experiment where
tumors are treated with cyclohexamide (0.5 .mu.g) are shown in FIG.
28. FIG. 28 is a graph that shows the effect of cyclohexamide on
ATIA stability and cell apoptosis. As shown n FIG. 28, 0.5 ug of
cycloheximide causes the decrease of ATIA in A172 Cells. Hypoxia
induces A172 cells undergo apoptosis in the presence of CHX.
[0312] The present invention has been described in detail,
including the preferred embodiments thereof. However, it will be
appreciated that those skilled in the art, upon consideration of
the present disclosure, may make modifications and/or improvements
of this invention and still be within the scope and spirit of this
invention as set forth in the following claims.
[0313] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their invention.
[0314] The following specific references, also incorporated by
reference, are indicated above by corresponding reference number.
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Sequence CWU 1
1
1113189DNAMus musculus 1agagaccagc ctcttacgag tcaacttcga gtctggagcc
ggagccagag accggggctg 60ggaaacccca gcccgggacg ggacgcagca gcctctggat
cccgggaccc cggacctctc 120aggaccggcc agaggtgaag gactgaggcc
ccactgaggc cttggaccgc accgcctggc 180tccttcagcc gcagtcgtct
cctgggacag aagatgcact ccaggagctg cctgccacct 240ctcctgttgt
tgcttctggt gctcctgggg tctggagtac agggttgccc atcaggctgc
300cagtgcaacc agccacagac agtcttctgc actgcccgtc agggaaccac
agtgccccga 360gacgtgccac ctgacacagt gggcctgtac atctttgaga
acggcatcac gacacttgat 420gtgggctgtt ttgctggcct tccgggcctg
cagcttctgg acttgtcaca gaaccagatc 480actagcctgc ccgggggcat
ctttcagcca cttgttaacc tcagtaacct ggacctgact 540gccaacaaac
tgcacgagat ctccaacgag accttccgtg gcctgcggcg cctggagcgc
600ctctacctgg gcaagaaccg aattcgccac atccaaccgg gtgccttcga
cgcgcttgat 660cgcctcctgg agctcaagct gccagacaat gagcttcggg
tgttgccccc attgcacttg 720ccccgcctgc tgctgcttga cctcagccac
aacagcatcc cagccctgga agccggaata 780ctggataccg ccaatgtaga
ggcattgagg ttggctggcc tagggctgcg gcagctggat 840gaggggcttt
ttggccgcct tctcaacctc catgacttgg atgtttctga caaccagttg
900gagcatatgc catctgtgat tcaaggcctg cgtggcctga cacgcctgcg
gctggctggc 960aacacccgta ttgcccagat acggcccgag gacctcgctg
gtctgactgc cctacaggaa 1020ttggatgtga gcaacctaag cctgcaggcc
ctgcccagtg acctctcgag tctctttccc 1080cgcctgcgcc tcttagcagc
tgccaggaac cccttcaact gcttgtgccc cttgagctgg 1140tttggtcctt
gggtgcgtga gaaccatgtt gtgttggcca gccctgagga gacgcgttgt
1200cactttccac ccaagaatgc tggccgactg ctcctggatc tggattatgc
agattttggc 1260tgcccagtca ccactaccac ggccacagta cctactataa
ggtctactat cagggaaccc 1320acactttcaa cttctagcca agctcccacc
tggcccagcc tcacagagcc aactacccag 1380gcctccaccg tactatcgac
tgccccacca accatgaggc cagctcctca gccccaggac 1440tgtccagcat
ccatctgcct gaatggtggt agctgccgtt tgggagcaag acaccactgg
1500gagtgcctat gccctgaggg cttcattggc ctgtactgtg agagtccagt
ggagcaaggg 1560atgaagccca gctccatacc agacactcca aggccccctc
cactgctgcc tctcagcatt 1620gagccggtga gccccacctc cttgcgtgtg
aagctgcagc gctacttgca gggtaacact 1680gtgcagctac ggagcctccg
gctcacctat cgcaacctgt ctggccctga caaacgactg 1740gtgacattac
ggctgcctgc ttcacttgca gagtatacag tcacccagct gcgacccaat
1800gccacctatt ctatctgtgt cacacccttg ggagctggac ggacacctga
aggtgaggag 1860gcctgtgggg aggccaacac ttcccaggca gtccgctcta
accatgcccc agttacccag 1920gcccgtgagg gcaacctgcc actcctcatt
gcgcctgccc tggctgctgt acttctggct 1980gtgttagccg ctgcaggggc
agcctactgt gtgcggcggg cacgggcaac ttctacagct 2040caggacaaag
ggcaggtggg gccagggact ggacccctgg aactagaggg ggtgaaagcc
2100cctttggagc caggctccaa ggcaacagag ggaggtgggg aggctttgtc
aggtggtcct 2160gaatgtgagg tgcctcttat gggctaccca gggcccagcc
ttcagggggt cctccctgct 2220aagcactaca tttagactgg tgagaaagag
cagccagggg gtcaggcttt cagtcaccac 2280cctcctgctg ccacagaagg
aagttctcag tatacaccac agtgcacgtg catgatggag 2340ctgtgggacc
ctctctgggc tgggtctcat ctgtaagctg ctacagccca gatgaactct
2400gccagccgcc agtgcatcca gtacagcgcc tgccatcttg tgcaatgtgc
aaccctggga 2460tgtgagccct gccatgtgct ggtaacatgg ctaggcatgt
tgggcttccc aaaccatgga 2520gtctggtaac cagtgaagga agcccccaga
aataatgagt ggggaaggta ctagggcact 2580ggccttggcc tcaaaagtgc
aggcacactt gaaactggaa aggaaggtgc tctgggcaca 2640tgtggatttg
cttctattgt tttgttttgt tttttctaat gtatttataa aagatctttt
2700cccatttatg ctgggaaagt gtttttcaaa ctcagtgaca aggactttgg
tttttgtaag 2760actgttgatg atatgaaggc cttttgtaag aaaataaaaa
ataaagtaaa ttgcctgtct 2820ctctggttgg gcttgagatt taaggtctgt
ggacatgcac aggattggag ggctgctgcc 2880ctgccattag aatgctctag
ccatgggtcc tgacccatgg taaggcttgc acttgggtgg 2940ggccggaaaa
tggacttgtt aggtagctta ccctaggcta ggcctcctct tctgccagca
3000ggaaccacag tgcttaatgt ataaggcaga aaggggctca tagaaaacac
agaacacaaa 3060gggaggtcac atccctcctt gggtgttctg aaagtgcagt
ccactatctt caactagaga 3120agacagcctg gagcttcctc attctagagc
ctaacagctg atcctgggac caggtggctt 3180ccagactgg 31892673PRTMus
musculus 2Met His Ser Arg Ser Cys Leu Pro Pro Leu Leu Leu Leu Leu
Leu Val 1 5 10 15 Leu Leu Gly Ser Gly Val Gln Gly Cys Pro Ser Gly
Cys Gln Cys Asn 20 25 30 Gln Pro Gln Thr Val Phe Cys Thr Ala Arg
Gln Gly Thr Thr Val Pro 35 40 45 Arg Asp Val Pro Pro Asp Thr Val
Gly Leu Tyr Ile Phe Glu Asn Gly 50 55 60 Ile Thr Thr Leu Asp Val
Gly Cys Phe Ala Gly Leu Pro Gly Leu Gln 65 70 75 80 Leu Leu Asp Leu
Ser Gln Asn Gln Ile Thr Ser Leu Pro Gly Gly Ile 85 90 95 Phe Gln
Pro Leu Val Asn Leu Ser Asn Leu Asp Leu Thr Ala Asn Lys 100 105 110
Leu His Glu Ile Ser Asn Glu Thr Phe Arg Gly Leu Arg Arg Leu Glu 115
120 125 Arg Leu Tyr Leu Gly Lys Asn Arg Ile Arg His Ile Gln Pro Gly
Ala 130 135 140 Phe Asp Ala Leu Asp Arg Leu Leu Glu Leu Lys Leu Pro
Asp Asn Glu 145 150 155 160 Leu Arg Val Leu Pro Pro Leu His Leu Pro
Arg Leu Leu Leu Leu Asp 165 170 175 Leu Ser His Asn Ser Ile Pro Ala
Leu Glu Ala Gly Ile Leu Asp Thr 180 185 190 Ala Asn Val Glu Ala Leu
Arg Leu Ala Gly Leu Gly Leu Arg Gln Leu 195 200 205 Asp Glu Gly Leu
Phe Gly Arg Leu Leu Asn Leu His Asp Leu Asp Val 210 215 220 Ser Asp
Asn Gln Leu Glu His Met Pro Ser Val Ile Gln Gly Leu Arg 225 230 235
240 Gly Leu Thr Arg Leu Arg Leu Ala Gly Asn Thr Arg Ile Ala Gln Ile
245 250 255 Arg Pro Glu Asp Leu Ala Gly Leu Thr Ala Leu Gln Glu Leu
Asp Val 260 265 270 Ser Asn Leu Ser Leu Gln Ala Leu Pro Ser Asp Leu
Ser Ser Leu Phe 275 280 285 Pro Arg Leu Arg Leu Leu Ala Ala Ala Arg
Asn Pro Phe Asn Cys Leu 290 295 300 Cys Pro Leu Ser Trp Phe Gly Pro
Trp Val Arg Glu Asn His Val Val 305 310 315 320 Leu Ala Ser Pro Glu
Glu Thr Arg Cys His Phe Pro Pro Lys Asn Ala 325 330 335 Gly Arg Leu
Leu Leu Asp Leu Asp Tyr Ala Asp Phe Gly Cys Pro Val 340 345 350 Thr
Thr Thr Thr Ala Thr Val Pro Thr Ile Arg Ser Thr Ile Arg Glu 355 360
365 Pro Thr Leu Ser Thr Ser Ser Gln Ala Pro Thr Trp Pro Ser Leu Thr
370 375 380 Glu Pro Thr Thr Gln Ala Ser Thr Val Leu Ser Thr Ala Pro
Pro Thr 385 390 395 400 Met Arg Pro Ala Pro Gln Pro Gln Asp Cys Pro
Ala Ser Ile Cys Leu 405 410 415 Asn Gly Gly Ser Cys Arg Leu Gly Ala
Arg His His Trp Glu Cys Leu 420 425 430 Cys Pro Glu Gly Phe Ile Gly
Leu Tyr Cys Glu Ser Pro Val Glu Gln 435 440 445 Gly Met Lys Pro Ser
Ser Ile Pro Asp Thr Pro Arg Pro Pro Pro Leu 450 455 460 Leu Pro Leu
Ser Ile Glu Pro Val Ser Pro Thr Ser Leu Arg Val Lys 465 470 475 480
Leu Gln Arg Tyr Leu Gln Gly Asn Thr Val Gln Leu Arg Ser Leu Arg 485
490 495 Leu Thr Tyr Arg Asn Leu Ser Gly Pro Asp Lys Arg Leu Val Thr
Leu 500 505 510 Arg Leu Pro Ala Ser Leu Ala Glu Tyr Thr Val Thr Gln
Leu Arg Pro 515 520 525 Asn Ala Thr Tyr Ser Ile Cys Val Thr Pro Leu
Gly Ala Gly Arg Thr 530 535 540 Pro Glu Gly Glu Glu Ala Cys Gly Glu
Ala Asn Thr Ser Gln Ala Val 545 550 555 560 Arg Ser Asn His Ala Pro
Val Thr Gln Ala Arg Glu Gly Asn Leu Pro 565 570 575 Leu Leu Ile Ala
Pro Ala Leu Ala Ala Val Leu Leu Ala Val Leu Ala 580 585 590 Ala Ala
Gly Ala Ala Tyr Cys Val Arg Arg Ala Arg Ala Thr Ser Thr 595 600 605
Ala Gln Asp Lys Gly Gln Val Gly Pro Gly Thr Gly Pro Leu Glu Leu 610
615 620 Glu Gly Val Lys Ala Pro Leu Glu Pro Gly Ser Lys Ala Thr Glu
Gly 625 630 635 640 Gly Gly Glu Ala Leu Ser Gly Gly Pro Glu Cys Glu
Val Pro Leu Met 645 650 655 Gly Tyr Pro Gly Pro Ser Leu Gln Gly Val
Leu Pro Ala Lys His Tyr 660 665 670 Ile 32739DNARattus norvegicus
3ggagcccggg gttgggagac ccggacgcag tagcctccgg atcccgggac cccggacctt
60tcaggaccgg ccggaggcga aggactgagg ccccattgag gccttgggcc gcaccgcccc
120gctccctcag ccacagtcgt ctcccgggac agaagatgca ctccaggagc
tgcctgccac 180ctcttctgtt gttgctcctg gtgctcctgg ggtctggagt
acagagctgc ccatcaggct 240gccagtgcaa ccaaccacag acagtcttct
gcactgcccg tcagggaacc acggtgcccc 300gagacgtgcc gcctgacaca
gtgggcctgt acatctttga gaacggcatc actacacttg 360atgtaggctg
ttttgctggc ttcccaggcc tgcagcttct ggacttgtca cagaaccaga
420tcactagcct gcccggtggc atctttcagc cacttgtgaa cctcagtaac
ctggacctga 480ctgctaacaa actgcacgag atctccaacg agaccttccg
tggcctgcgg cgcctcgaac 540gcctctacct gggcaagaac cgcattcgcc
acatccagcc tggtgccttc gatgcacttg 600accacctcct ggagctcaag
ctgccagaca atgagcttcg ggtgctgccc ccactgcact 660tgcctcgcct
gctgctgctt gacctcagcc acaacagtat cccagccctg gaagctggaa
720tactggatac tgccaatgtg gaggcactgc ggctggctgg cctcgggctg
cggcagctgg 780atgaggggct ttttggccgc cttcgcaacc tccatgacct
ggatgtttct gacaaccagt 840tggggcacat gccctccgtg attcaaggcc
tgcgtggcct gacacgcctg cggctggctg 900gcaacacccg gattgcccag
atccggcccg aggacctcgc tggcctgact gccctacagg 960aactggatgt
gagcaacctg agcctgcagg ccctgcccag tgacctctcc agtctctttc
1020cccgcctgcg cctcctagca gctgcccgaa acccctttaa ctgcttatgc
cccttgagct 1080ggtttggtcc ttgggttcgt gagagccatg ttgtgctggc
cagccctgag gagacacgtt 1140gtcacttccc acccaagaac gccggccgac
tgctcctgga gctggattat gcagattttg 1200gctgcccagt caccactacc
acagccacag ttcctactat aaggcctact gtcagggagc 1260ccacaccttc
aacttccagc caagctccca cctggcccag ccccacagag ccaactaccc
1320aggcccccat cgtactgtcc actgccccac caaccatgag gccggctcct
cagccccagg 1380actgtccagc atccatctgc ctgaatggtg gtagctgccg
tgtaggggca aaacaccacc 1440tggagtgcct gtgccccgag ggcttcattg
gcctgtactg tgagagtccc gtggaacaaa 1500ggacaaagcc cagctccata
ccggacaccc cacggccccc gcggctgctg cctctgcgca 1560ttgagccggt
gagccccacc tccctgcgtg tggagctgca gcgctacctg cagggcaaca
1620ccgtgcagct gcggagcctc cggctcacct accgcaacct gtctggccct
gacaagcggc 1680tggtgacgct gcggctgcct gcttcacttg cagagtacac
agtcacccag ctgcggccca 1740atgccaccta ttctatctgt gtcacagccc
tgggagctgg gcggacacct gaaggtgagg 1800aggcctgtgg ggaggccaac
actccccagg ccgtccgctc caaccatgcc ccagtcaccc 1860aggcccggga
gggcaacctg ccactcctca ttgcacccgc cctggctgct gtgcttctgg
1920ctgtgttggc tgcctcgggg gcagtctact gtgtgcgacg ggcgcgggca
agttccacag 1980ctcaggacaa agggcaggtg ggaccaggga ccgggcccct
ggaactagag ggggtgaaag 2040tccccttgga gccaggctcc aaggcatcag
agggaggcgg ggaggcccta tcaggtggtc 2100ctgaatgtga ggtgcccctc
atgggctacc cagggcccag tcttcagggg gtcctccctg 2160ctcagcccta
catttaagca cgtgagaagg agcagccagg aggctgggct ttcagtctcc
2220accctcctgc tgctacagaa ggaagttctc aatgcgcacc acagtgcaca
tgtgtgaccg 2280gtgctgtggg acagcagcca gtccccgacc ctctctgggc
tgggtcatct gaaagctgct 2340acagcccaaa tgaactccca gcaccagcat
ccagtacaga gcctgctgcc ttgcgcagtc 2400tgcagtcctg ggacgggaac
cctgccatgt gctggtagca tggctaggat gttgggcttc 2460ccgggccctg
gggtctggta accagtgaag gaagccccca aaaatagtgg gtagggaagg
2520cactagggcc gtggccgtgg ccccgaaagt gcaggaacac ttgaaactgg
aaaggaaggt 2580gctctgggca cacgtggatt tgcttctatt gttttgtttt
tctcctaatg tatttataaa 2640agatcttttc ccgtttatgc tgggaaaaag
tgtttttcaa actcagtgac aaggactttt 2700ggtttttgta agactattga
tgatatgaag gccttttgt 27394673PRTRattus norvegicus 4Met His Ser Arg
Ser Cys Leu Pro Pro Leu Leu Leu Leu Leu Leu Val 1 5 10 15 Leu Leu
Gly Ser Gly Val Gln Ser Cys Pro Ser Gly Cys Gln Cys Asn 20 25 30
Gln Pro Gln Thr Val Phe Cys Thr Ala Arg Gln Gly Thr Thr Val Pro 35
40 45 Arg Asp Val Pro Pro Asp Thr Val Gly Leu Tyr Ile Phe Glu Asn
Gly 50 55 60 Ile Thr Thr Leu Asp Val Gly Cys Phe Ala Gly Phe Pro
Gly Leu Gln 65 70 75 80 Leu Leu Asp Leu Ser Gln Asn Gln Ile Thr Ser
Leu Pro Gly Gly Ile 85 90 95 Phe Gln Pro Leu Val Asn Leu Ser Asn
Leu Asp Leu Thr Ala Asn Lys 100 105 110 Leu His Glu Ile Ser Asn Glu
Thr Phe Arg Gly Leu Arg Arg Leu Glu 115 120 125 Arg Leu Tyr Leu Gly
Lys Asn Arg Ile Arg His Ile Gln Pro Gly Ala 130 135 140 Phe Asp Ala
Leu Asp His Leu Leu Glu Leu Lys Leu Pro Asp Asn Glu 145 150 155 160
Leu Arg Val Leu Pro Pro Leu His Leu Pro Arg Leu Leu Leu Leu Asp 165
170 175 Leu Ser His Asn Ser Ile Pro Ala Leu Glu Ala Gly Ile Leu Asp
Thr 180 185 190 Ala Asn Val Glu Ala Leu Arg Leu Ala Gly Leu Gly Leu
Arg Gln Leu 195 200 205 Asp Glu Gly Leu Phe Gly Arg Leu Arg Asn Leu
His Asp Leu Asp Val 210 215 220 Ser Asp Asn Gln Leu Gly His Met Pro
Ser Val Ile Gln Gly Leu Arg 225 230 235 240 Gly Leu Thr Arg Leu Arg
Leu Ala Gly Asn Thr Arg Ile Ala Gln Ile 245 250 255 Arg Pro Glu Asp
Leu Ala Gly Leu Thr Ala Leu Gln Glu Leu Asp Val 260 265 270 Ser Asn
Leu Ser Leu Gln Ala Leu Pro Ser Asp Leu Ser Ser Leu Phe 275 280 285
Pro Arg Leu Arg Leu Leu Ala Ala Ala Arg Asn Pro Phe Asn Cys Leu 290
295 300 Cys Pro Leu Ser Trp Phe Gly Pro Trp Val Arg Glu Ser His Val
Val 305 310 315 320 Leu Ala Ser Pro Glu Glu Thr Arg Cys His Phe Pro
Pro Lys Asn Ala 325 330 335 Gly Arg Leu Leu Leu Glu Leu Asp Tyr Ala
Asp Phe Gly Cys Pro Val 340 345 350 Thr Thr Thr Thr Ala Thr Val Pro
Thr Ile Arg Pro Thr Val Arg Glu 355 360 365 Pro Thr Pro Ser Thr Ser
Ser Gln Ala Pro Thr Trp Pro Ser Pro Thr 370 375 380 Glu Pro Thr Thr
Gln Ala Pro Ile Val Leu Ser Thr Ala Pro Pro Thr 385 390 395 400 Met
Arg Pro Ala Pro Gln Pro Gln Asp Cys Pro Ala Ser Ile Cys Leu 405 410
415 Asn Gly Gly Ser Cys Arg Val Gly Ala Lys His His Leu Glu Cys Leu
420 425 430 Cys Pro Glu Gly Phe Ile Gly Leu Tyr Cys Glu Ser Pro Val
Glu Gln 435 440 445 Arg Thr Lys Pro Ser Ser Ile Pro Asp Thr Pro Arg
Pro Pro Arg Leu 450 455 460 Leu Pro Leu Arg Ile Glu Pro Val Ser Pro
Thr Ser Leu Arg Val Glu 465 470 475 480 Leu Gln Arg Tyr Leu Gln Gly
Asn Thr Val Gln Leu Arg Ser Leu Arg 485 490 495 Leu Thr Tyr Arg Asn
Leu Ser Gly Pro Asp Lys Arg Leu Val Thr Leu 500 505 510 Arg Leu Pro
Ala Ser Leu Ala Glu Tyr Thr Val Thr Gln Leu Arg Pro 515 520 525 Asn
Ala Thr Tyr Ser Ile Cys Val Thr Ala Leu Gly Ala Gly Arg Thr 530 535
540 Pro Glu Gly Glu Glu Ala Cys Gly Glu Ala Asn Thr Pro Gln Ala Val
545 550 555 560 Arg Ser Asn His Ala Pro Val Thr Gln Ala Arg Glu Gly
Asn Leu Pro 565 570 575 Leu Leu Ile Ala Pro Ala Leu Ala Ala Val Leu
Leu Ala Val Leu Ala 580 585 590 Ala Ser Gly Ala Val Tyr Cys Val Arg
Arg Ala Arg Ala Ser Ser Thr 595 600 605 Ala Gln Asp Lys Gly Gln Val
Gly Pro Gly Thr Gly Pro Leu Glu Leu 610 615 620 Glu Gly Val Lys Val
Pro Leu Glu Pro Gly Ser Lys Ala Ser Glu Gly 625 630 635 640 Gly Gly
Glu Ala Leu Ser Gly Gly Pro Glu Cys Glu Val Pro Leu Met 645 650 655
Gly Tyr Pro Gly Pro Ser Leu Gln Gly Val Leu Pro Ala Gln Pro Tyr 660
665 670 Ile 52864DNAHomo sapiens 5gactccggag cccgagcccg gggcgggtgg
acgcggactc gaacgcagtt gcttcgggac 60ccaggacccc ctcgggcccg acccgccagg
aaagactgag gccgcggcct gccccgcccg 120gctccctgcg ccgccgccgc
ctcccgggac agaagatgtg
ctccagggtc cctctgctgc 180tgccgctgct cctgctactg gccctggggc
ctggggtgca gggctgccca tccggctgcc 240agtgcagcca gccacagaca
gtcttctgca ctgcccgcca ggggaccacg gtgccccgag 300acgtgccacc
cgacacggtg gggctgtacg tctttgagaa cggcatcacc atgctcgacg
360caggcagctt tgccggcctg ccgggcctgc agctcctgga cctgtcacag
aaccagatcg 420ccagcctgcc cagcggggtc ttccagccac tcgccaacct
cagcaacctg gacctgacag 480ccaacaggct gcatgaaatc accaatgaga
ccttccgtgg cctgcggcgc ctcgagcgcc 540tctacctggg caagaaccgc
atccgccaca tccagcctgg tgccttcgac acgctcgacc 600gcctcctgga
gctcaagctg caggacaacg agctgcgggc actgcccccg ctgcgcctgc
660cccgcctgct gctgctggac ctcagccaca acagcctcct ggccctggag
cccggcatcc 720tggacactgc caacgtggag gcgctgcggc tggctggtct
ggggctgcag cagctggacg 780aggggctctt cagccgcttg cgcaacctcc
acgacctgga tgtgtccgac aaccagctgg 840agcgagtgcc acctgtgatc
cgaggcctcc ggggcctgac gcgcctgcgg ctggccggca 900acacccgcat
tgcccagctg cggcccgagg acctggccgg cctggctgcc ctgcaggagc
960tggatgtgag caacctaagc ctgcaggccc tgcctggcga cctctcgggc
ctcttccccc 1020gcctgcggct gctggcagct gcccgcaacc ccttcaactg
cgtgtgcccc ctgagctggt 1080ttggcccctg ggtgcgcgag agccacgtca
cactggccag ccctgaggag acgcgctgcc 1140acttcccgcc caagaacgct
ggccggctgc tcctggagct tgactacgcc gactttggct 1200gcccagccac
caccaccaca gccacagtgc ccaccacgag gcccgtggtg cgggagccca
1260cagccttgtc ttctagcttg gctcctacct ggcttagccc cacagagccg
gccactgagg 1320cccccagccc gccctccact gccccaccga ctgtagggcc
tgtcccccag ccccaggact 1380gcccaccgtc cacctgcctc aatgggggca
catgccacct ggggacacgg caccacctgg 1440cgtgcttgtg ccccgaaggc
ttcacgggcc tgtactgtga gagccagatg gggcagggga 1500cacggcccag
ccctacacca gtcacgccga ggccaccacg gtccctgacc ctgggcatcg
1560agccggtgag ccccacctcc ctgcgcgtgg ggctgcagcg ctacctccag
gggagctccg 1620tgcagctcag gagcctccgt ctcacctatc gcaacctatc
gggccctgat aagcggctgg 1680tgacgctgcg actgcctgcc tcgctcgctg
agtacacggt cacccagctg cggcccaacg 1740ccacttactc cgtctgtgtc
atgcctttgg ggcccgggcg ggtgccggag ggcgaggagg 1800cctgcgggga
ggcccataca cccccagccg tccactccaa ccacgcccca gtcacccagg
1860cccgcgaggg caacctgccg ctcctcattg cgcccgccct ggccgcggtg
ctcctggccg 1920cgctggctgc ggtgggggca gcctactgtg tgcggcgggg
gcgggccatg gcagcagcgg 1980ctcaggacaa agggcaggtg gggccagggg
ctgggcccct ggaactggag ggagtgaagg 2040tccccttgga gccaggcccg
aaggcaacag agggcggtgg agaggccctg cccagcgggt 2100ctgagtgtga
ggtgccactc atgggcttcc cagggcctgg cctccagtca cccctccacg
2160caaagcccta catctaagcc agagagagac agggcagctg gggccgggct
ctcagccagt 2220gagatggcca gccccctcct gctgccacac cacgtaagtt
ctcagtccca acctcgggga 2280tgtgtgcaga cagggctgtg tgaccacagc
tgggccctgt tccctctgga cctcggtctc 2340ctcatctgtg agatgctgtg
gcccagctga cgagccctaa cgtccccaga accgagtgcc 2400tatgaggaca
gtgtccgccc tgccctccgc aacgtgcagt ccctgggcac ggcgggccct
2460gccatgtgct ggtaacgcat gcctgggccc tgctgggctc tcccactcca
ggcggaccct 2520gggggccagt gaaggaagct cccggaaaga gcagagggag
agcgggtagg cggctgtgtg 2580actctagtct tggccccagg aagcgaagga
acaaaagaaa ctggaaagga agatgcttta 2640ggaacatgtt ttgctttttt
aaaatatata tatatttata agagatcctt tcccatttat 2700tctgggaaga
tgtttttcaa actcagagac aaggactttg gtttttgtaa gacaaacgat
2760gatatgaagg ccttttgtaa gaaaaaataa aagatgaagt gtgtttaaaa
aaaaaaaaaa 2820aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa
28646673PRTHomo sapiens 6Met Cys Ser Arg Val Pro Leu Leu Leu Pro
Leu Leu Leu Leu Leu Ala 1 5 10 15 Leu Gly Pro Gly Val Gln Gly Cys
Pro Ser Gly Cys Gln Cys Ser Gln 20 25 30 Pro Gln Thr Val Phe Cys
Thr Ala Arg Gln Gly Thr Thr Val Pro Arg 35 40 45 Asp Val Pro Pro
Asp Thr Val Gly Leu Tyr Val Phe Glu Asn Gly Ile 50 55 60 Thr Met
Leu Asp Ala Gly Ser Phe Ala Gly Leu Pro Gly Leu Gln Leu 65 70 75 80
Leu Asp Leu Ser Gln Asn Gln Ile Ala Ser Leu Pro Ser Gly Val Phe 85
90 95 Gln Pro Leu Ala Asn Leu Ser Asn Leu Asp Leu Thr Ala Asn Arg
Leu 100 105 110 His Glu Ile Thr Asn Glu Thr Phe Arg Gly Leu Arg Arg
Leu Glu Arg 115 120 125 Leu Tyr Leu Gly Lys Asn Arg Ile Arg His Ile
Gln Pro Gly Ala Phe 130 135 140 Asp Thr Leu Asp Arg Leu Leu Glu Leu
Lys Leu Gln Asp Asn Glu Leu 145 150 155 160 Arg Ala Leu Pro Pro Leu
Arg Leu Pro Arg Leu Leu Leu Leu Asp Leu 165 170 175 Ser His Asn Ser
Leu Leu Ala Leu Glu Pro Gly Ile Leu Asp Thr Ala 180 185 190 Asn Val
Glu Ala Leu Arg Leu Ala Gly Leu Gly Leu Gln Gln Leu Asp 195 200 205
Glu Gly Leu Phe Ser Arg Leu Arg Asn Leu His Asp Leu Asp Val Ser 210
215 220 Asp Asn Gln Leu Glu Arg Val Pro Pro Val Ile Arg Gly Leu Arg
Gly 225 230 235 240 Leu Thr Arg Leu Arg Leu Ala Gly Asn Thr Arg Ile
Ala Gln Leu Arg 245 250 255 Pro Glu Asp Leu Ala Gly Leu Ala Ala Leu
Gln Glu Leu Asp Val Ser 260 265 270 Asn Leu Ser Leu Gln Ala Leu Pro
Gly Asp Leu Ser Gly Leu Phe Pro 275 280 285 Arg Leu Arg Leu Leu Ala
Ala Ala Arg Asn Pro Phe Asn Cys Val Cys 290 295 300 Pro Leu Ser Trp
Phe Gly Pro Trp Val Arg Glu Ser His Val Thr Leu 305 310 315 320 Ala
Ser Pro Glu Glu Thr Arg Cys His Phe Pro Pro Lys Asn Ala Gly 325 330
335 Arg Leu Leu Leu Glu Leu Asp Tyr Ala Asp Phe Gly Cys Pro Ala Thr
340 345 350 Thr Thr Thr Ala Thr Val Pro Thr Thr Arg Pro Val Val Arg
Glu Pro 355 360 365 Thr Ala Leu Ser Ser Ser Leu Ala Pro Thr Trp Leu
Ser Pro Thr Glu 370 375 380 Pro Ala Thr Glu Ala Pro Ser Pro Pro Ser
Thr Ala Pro Pro Thr Val 385 390 395 400 Gly Pro Val Pro Gln Pro Gln
Asp Cys Pro Pro Ser Thr Cys Leu Asn 405 410 415 Gly Gly Thr Cys His
Leu Gly Thr Arg His His Leu Ala Cys Leu Cys 420 425 430 Pro Glu Gly
Phe Thr Gly Leu Tyr Cys Glu Ser Gln Met Gly Gln Gly 435 440 445 Thr
Arg Pro Ser Pro Thr Pro Val Thr Pro Arg Pro Pro Arg Ser Leu 450 455
460 Thr Leu Gly Ile Glu Pro Val Ser Pro Thr Ser Leu Arg Val Gly Leu
465 470 475 480 Gln Arg Tyr Leu Gln Gly Ser Ser Val Gln Leu Arg Ser
Leu Arg Leu 485 490 495 Thr Tyr Arg Asn Leu Ser Gly Pro Asp Lys Arg
Leu Val Thr Leu Arg 500 505 510 Leu Pro Ala Ser Leu Ala Glu Tyr Thr
Val Thr Gln Leu Arg Pro Asn 515 520 525 Ala Thr Tyr Ser Val Cys Val
Met Pro Leu Gly Pro Gly Arg Val Pro 530 535 540 Glu Gly Glu Glu Ala
Cys Gly Glu Ala His Thr Pro Pro Ala Val His 545 550 555 560 Ser Asn
His Ala Pro Val Thr Gln Ala Arg Glu Gly Asn Leu Pro Leu 565 570 575
Leu Ile Ala Pro Ala Leu Ala Ala Val Leu Leu Ala Ala Leu Ala Ala 580
585 590 Val Gly Ala Ala Tyr Cys Val Arg Arg Gly Arg Ala Met Ala Ala
Ala 595 600 605 Ala Gln Asp Lys Gly Gln Val Gly Pro Gly Ala Gly Pro
Leu Glu Leu 610 615 620 Glu Gly Val Lys Val Pro Leu Glu Pro Gly Pro
Lys Ala Thr Glu Gly 625 630 635 640 Gly Gly Glu Ala Leu Pro Ser Gly
Ser Glu Cys Glu Val Pro Leu Met 645 650 655 Gly Phe Pro Gly Pro Gly
Leu Gln Ser Pro Leu His Ala Lys Pro Tyr 660 665 670 Ile 71501DNAMus
musculus 7tcaggctgcc agtgcaacca gccacagaca gtcttctgca ctgcccgtca
gggaaccaca 60gtgccccgag acgtgccacc tgacacagtg ggcctgtaca tctttgagaa
cggcatcacg 120acacttgatg tgggctgttt tgctggcctt ccgggcctgc
agcttctgga cttgtcacag 180aaccagatca ctagcctgcc cgggggcatc
tttcagccac ttgttaacct cagtaacctg 240gacctgactg ccaacaaact
gcacgagatc tccaacgaga ccttccgtgg cctgcggcgc 300ctggagcgcc
tctacctggg caagaaccga attcgccaca tccaaccggg tgccttcgac
360gcgcttgatc gcctcctgga gctcaagctg ccagacaatg agcttcgggt
gttgccccca 420ttgcacttgc cccgcctgct gctgcttgac ctcagccaca
acagcatccc agccctggaa 480gccggaatac tggataccgc caatgtagag
gcattgaggt tggctggcct agggctgcgg 540cagctggatg aggggctttt
tggccgcctt ctcaacctcc atgacttgga tgtttctgac 600aaccagttgg
agcatatgcc atctgtgatt caaggcctgc gtggcctgac acgcctgcgg
660ctggctggca acacccgtat tgcccagata cggcccgagg acctcgctgg
tctgactgcc 720ctacaggaat tggatgtgag caacctaagc ctgcaggccc
tgcccagtga cctctcgagt 780ctctttcccc gcctgcgcct cttagcagct
gccaggaacc ccttcaactg cttgtgcccc 840ttgagctggt ttggtccttg
ggtgcgtgag aaccatgttg tgttggccag ccctgaggag 900acgcgttgtc
actttccacc caagaatgct ggccgactgc tcctggatct ggattatgca
960gattttggct gcccagtcac cactaccacg gccacagtac ctactataag
gtctactatc 1020agggaaccca cactttcaac ttctagccaa gctcccacct
ggcccagcct cacagagcca 1080actacccagg cctccaccgt actatcgact
gccccaccaa ccatgaggcc agctcctcag 1140ccccaggact gtccagcatc
catctgcctg aatggtggta gctgccgttt gggagcaaga 1200caccactggg
agtgcctatg ccctgagggc ttcattggcc tgtactgtga gagtccagtg
1260gagcaaggga tgaagcccag ctccatacca gacactccaa ggccccctcc
actgctgcct 1320ctcagcattg agccggtgag ccccacctcc ttgcgtgtga
agctgcagcg ctacttgcag 1380ggtaacactg tgcagctacg gagcctccgg
ctcacctatc gcaacctgtc tggccctgac 1440aaacgactgg tgacattacg
gctgcctgct tcacttgcag agtatacagt cacccagctg 1500c 150181501DNAHomo
sapiens 8tccggctgcc agtgcagcca gccacagaca gtcttctgca ctgcccgcca
ggggaccacg 60gtgccccgag acgtgccacc cgacacggtg gggctgtacg tctttgagaa
cggcatcacc 120atgctcgacg caggcagctt tgccggcctg ccgggcctgc
agctcctgga cctgtcacag 180aaccagatcg ccagcctgcc cagcggggtc
ttccagccac tcgccaacct cagcaacctg 240gacctgacag ccaacaggct
gcatgaaatc accaatgaga ccttccgtgg cctgcggcgc 300ctcgagcgcc
tctacctggg caagaaccgc atccgccaca tccagcctgg tgccttcgac
360acgctcgacc gcctcctgga gctcaagctg caggacaacg agctgcgggc
actgcccccg 420ctgcgcctgc cccgcctgct gctgctggac ctcagccaca
acagcctcct ggccctggag 480cccggcatcc tggacactgc caacgtggag
gcgctgcggc tggctggtct ggggctgcag 540cagctggacg aggggctctt
cagccgcttg cgcaacctcc acgacctgga tgtgtccgac 600aaccagctgg
agcgagtgcc acctgtgatc cgaggcctcc ggggcctgac gcgcctgcgg
660ctggccggca acacccgcat tgcccagctg cggcccgagg acctggccgg
cctggctgcc 720ctgcaggagc tggatgtgag caacctaagc ctgcaggccc
tgcctggcga cctctcgggc 780ctcttccccc gcctgcggct gctggcagct
gcccgcaacc ccttcaactg cgtgtgcccc 840ctgagctggt ttggcccctg
ggtgcgcgag agccacgtca cactggccag ccctgaggag 900acgcgctgcc
acttcccgcc caagaacgct ggccggctgc tcctggagct tgactacgcc
960gactttggct gcccagccac caccaccaca gccacagtgc ccaccacgag
gcccgtggtg 1020cgggagccca cagccttgtc ttctagcttg gctcctacct
ggcttagccc cacagagccg 1080gccactgagg cccccagccc gccctccact
gccccaccga ctgtagggcc tgtcccccag 1140ccccaggact gcccaccgtc
cacctgcctc aatgggggca catgccacct ggggacacgg 1200caccacctgg
cgtgcttgtg ccccgaaggc ttcacgggcc tgtactgtga gagccagatg
1260gggcagggga cacggcccag ccctacacca gtcacgccga ggccaccacg
gtccctgacc 1320ctgggcatcg agccggtgag ccccacctcc ctgcgcgtgg
ggctgcagcg ctacctccag 1380gggagctccg tgcagctcag gagcctccgt
ctcacctatc gcaacctatc gggccctgat 1440aagcggctgg tgacgctgcg
actgcctgcc tcgctcgctg agtacacggt cacccagctg 1500c 15019501PRTMus
musculus 9Pro Ser Gly Cys Gln Cys Asn Gln Pro Gln Thr Val Phe Cys
Thr Ala 1 5 10 15 Arg Gln Gly Thr Thr Val Pro Arg Asp Val Pro Pro
Asp Thr Val Gly 20 25 30 Leu Tyr Ile Phe Glu Asn Gly Ile Thr Thr
Leu Asp Val Gly Cys Phe 35 40 45 Ala Gly Leu Pro Gly Leu Gln Leu
Leu Asp Leu Ser Gln Asn Gln Ile 50 55 60 Thr Ser Leu Pro Gly Gly
Ile Phe Gln Pro Leu Val Asn Leu Ser Asn 65 70 75 80 Leu Asp Leu Thr
Ala Asn Lys Leu His Glu Ile Ser Asn Glu Thr Phe 85 90 95 Arg Gly
Leu Arg Arg Leu Glu Arg Leu Tyr Leu Gly Lys Asn Arg Ile 100 105 110
Arg His Ile Gln Pro Gly Ala Phe Asp Ala Leu Asp Arg Leu Leu Glu 115
120 125 Leu Lys Leu Pro Asp Asn Glu Leu Arg Val Leu Pro Pro Leu His
Leu 130 135 140 Pro Arg Leu Leu Leu Leu Asp Leu Ser His Asn Ser Ile
Pro Ala Leu 145 150 155 160 Glu Ala Gly Ile Leu Asp Thr Ala Asn Val
Glu Ala Leu Arg Leu Ala 165 170 175 Gly Leu Gly Leu Arg Gln Leu Asp
Glu Gly Leu Phe Gly Arg Leu Leu 180 185 190 Asn Leu His Asp Leu Asp
Val Ser Asp Asn Gln Leu Glu His Met Pro 195 200 205 Ser Val Ile Gln
Gly Leu Arg Gly Leu Thr Arg Leu Arg Leu Ala Gly 210 215 220 Asn Thr
Arg Ile Ala Gln Ile Arg Pro Glu Asp Leu Ala Gly Leu Thr 225 230 235
240 Ala Leu Gln Glu Leu Asp Val Ser Asn Leu Ser Leu Gln Ala Leu Pro
245 250 255 Ser Asp Leu Ser Ser Leu Phe Pro Arg Leu Arg Leu Leu Ala
Ala Ala 260 265 270 Arg Asn Pro Phe Asn Cys Leu Cys Pro Leu Ser Trp
Phe Gly Pro Trp 275 280 285 Val Arg Glu Asn His Val Val Leu Ala Ser
Pro Glu Glu Thr Arg Cys 290 295 300 His Phe Pro Pro Lys Asn Ala Gly
Arg Leu Leu Leu Asp Leu Asp Tyr 305 310 315 320 Ala Asp Phe Gly Cys
Pro Val Thr Thr Thr Thr Ala Thr Val Pro Thr 325 330 335 Ile Arg Ser
Thr Ile Arg Glu Pro Thr Leu Ser Thr Ser Ser Gln Ala 340 345 350 Pro
Thr Trp Pro Ser Leu Thr Glu Pro Thr Thr Gln Ala Ser Thr Val 355 360
365 Leu Ser Thr Ala Pro Pro Thr Met Arg Pro Ala Pro Gln Pro Gln Asp
370 375 380 Cys Pro Ala Ser Ile Cys Leu Asn Gly Gly Ser Cys Arg Leu
Gly Ala 385 390 395 400 Arg His His Trp Glu Cys Leu Cys Pro Glu Gly
Phe Ile Gly Leu Tyr 405 410 415 Cys Glu Ser Pro Val Glu Gln Gly Met
Lys Pro Ser Ser Ile Pro Asp 420 425 430 Thr Pro Arg Pro Pro Pro Leu
Leu Pro Leu Ser Ile Glu Pro Val Ser 435 440 445 Pro Thr Ser Leu Arg
Val Lys Leu Gln Arg Tyr Leu Gln Gly Asn Thr 450 455 460 Val Gln Leu
Arg Ser Leu Arg Leu Thr Tyr Arg Asn Leu Ser Gly Pro 465 470 475 480
Asp Lys Arg Leu Val Thr Leu Arg Leu Pro Ala Ser Leu Ala Glu Tyr 485
490 495 Thr Val Thr Gln Leu 500 10501PRTHomo sapiens 10Pro Ser Gly
Cys Gln Cys Ser Gln Pro Gln Thr Val Phe Cys Thr Ala 1 5 10 15 Arg
Gln Gly Thr Thr Val Pro Arg Asp Val Pro Pro Asp Thr Val Gly 20 25
30 Leu Tyr Val Phe Glu Asn Gly Ile Thr Met Leu Asp Ala Gly Ser Phe
35 40 45 Ala Gly Leu Pro Gly Leu Gln Leu Leu Asp Leu Ser Gln Asn
Gln Ile 50 55 60 Ala Ser Leu Pro Ser Gly Val Phe Gln Pro Leu Ala
Asn Leu Ser Asn 65 70 75 80 Leu Asp Leu Thr Ala Asn Arg Leu His Glu
Ile Thr Asn Glu Thr Phe 85 90 95 Arg Gly Leu Arg Arg Leu Glu Arg
Leu Tyr Leu Gly Lys Asn Arg Ile 100 105 110 Arg His Ile Gln Pro Gly
Ala Phe Asp Thr Leu Asp Arg Leu Leu Glu 115 120 125 Leu Lys Leu Gln
Asp Asn Glu Leu Arg Ala Leu Pro Pro Leu Arg Leu 130 135 140 Pro Arg
Leu Leu Leu Leu Asp Leu Ser His Asn Ser Leu Leu Ala Leu 145 150 155
160 Glu Pro Gly Ile Leu Asp Thr Ala Asn Val Glu Ala Leu Arg Leu Ala
165 170 175 Gly Leu Gly Leu Gln Gln Leu Asp Glu Gly Leu Phe Ser Arg
Leu Arg 180 185 190 Asn Leu His Asp Leu Asp Val Ser Asp Asn Gln Leu
Glu Arg Val Pro 195 200 205 Pro Val Ile Arg Gly Leu Arg Gly Leu Thr
Arg Leu Arg Leu Ala Gly 210 215
220 Asn Thr Arg Ile Ala Gln Leu Arg Pro Glu Asp Leu Ala Gly Leu Ala
225 230 235 240 Ala Leu Gln Glu Leu Asp Val Ser Asn Leu Ser Leu Gln
Ala Leu Pro 245 250 255 Gly Asp Leu Ser Gly Leu Phe Pro Arg Leu Arg
Leu Leu Ala Ala Ala 260 265 270 Arg Asn Pro Phe Asn Cys Val Cys Pro
Leu Ser Trp Phe Gly Pro Trp 275 280 285 Val Arg Glu Ser His Val Thr
Leu Ala Ser Pro Glu Glu Thr Arg Cys 290 295 300 His Phe Pro Pro Lys
Asn Ala Gly Arg Leu Leu Leu Glu Leu Asp Tyr 305 310 315 320 Ala Asp
Phe Gly Cys Pro Ala Thr Thr Thr Thr Ala Thr Val Pro Thr 325 330 335
Thr Arg Pro Val Val Arg Glu Pro Thr Ala Leu Ser Ser Ser Leu Ala 340
345 350 Pro Thr Trp Leu Ser Pro Thr Glu Pro Ala Thr Glu Ala Pro Ser
Pro 355 360 365 Pro Ser Thr Ala Pro Pro Thr Val Gly Pro Val Pro Gln
Pro Gln Asp 370 375 380 Cys Pro Pro Ser Thr Cys Leu Asn Gly Gly Thr
Cys His Leu Gly Thr 385 390 395 400 Arg His His Leu Ala Cys Leu Cys
Pro Glu Gly Phe Thr Gly Leu Tyr 405 410 415 Cys Glu Ser Gln Met Gly
Gln Gly Thr Arg Pro Ser Pro Thr Pro Val 420 425 430 Thr Pro Arg Pro
Pro Arg Ser Leu Thr Leu Gly Ile Glu Pro Val Ser 435 440 445 Pro Thr
Ser Leu Arg Val Gly Leu Gln Arg Tyr Leu Gln Gly Ser Ser 450 455 460
Val Gln Leu Arg Ser Leu Arg Leu Thr Tyr Arg Asn Leu Ser Gly Pro 465
470 475 480 Asp Lys Arg Leu Val Thr Leu Arg Leu Pro Ala Ser Leu Ala
Glu Tyr 485 490 495 Thr Val Thr Gln Leu 500 116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
6xHis tag" 11His His His His His His 1 5
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