U.S. patent application number 11/384739 was filed with the patent office on 2006-08-24 for detection and modulation of iaps and naip for the diagnosis and treatment of proliferative disease.
Invention is credited to Stephen Baird, Robert G. Korneluk, Peter Liston, Alexander E. MacKenzie, Christine Pratt, Benjamin K. Tsang.
Application Number | 20060189563 11/384739 |
Document ID | / |
Family ID | 25179735 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060189563 |
Kind Code |
A1 |
Korneluk; Robert G. ; et
al. |
August 24, 2006 |
Detection and modulation of IAPS and NAIP for the diagnosis and
treatment of proliferative disease
Abstract
Disclosed are diagnostic and prognostic kits for the detection
and treatment of proliferative diseases such as ovarian cancer,
breast cancer, and lymphoma. Also disclosed are cancer therapeutics
utilizing IAP antisense nucleic acids IAP fragments, and antibodies
which specifically bind IAP polypeptides.
Inventors: |
Korneluk; Robert G.;
(Ottawa, CA) ; MacKenzie; Alexander E.; (Ottawa,
CA) ; Liston; Peter; (Ottawa, CA) ; Baird;
Stephen; (Ottawa, CA) ; Tsang; Benjamin K.;
(Nepean, CA) ; Pratt; Christine; (Nepean,
CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
25179735 |
Appl. No.: |
11/384739 |
Filed: |
March 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09974592 |
Oct 9, 2001 |
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11384739 |
Mar 20, 2006 |
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09617053 |
Jul 14, 2000 |
6300492 |
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09974592 |
Oct 9, 2001 |
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08800929 |
Feb 13, 1997 |
6133437 |
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09617053 |
Jul 14, 2000 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A01K 2217/05 20130101;
G01N 33/5011 20130101; A61K 48/00 20130101; A61K 38/1709 20130101;
C12N 15/113 20130101; C12N 2799/022 20130101; C12Q 2600/136
20130101; G01N 33/57496 20130101; C12N 15/1135 20130101; C12Q
1/6886 20130101; A61P 35/00 20180101; C12Q 2600/106 20130101; C07K
14/4703 20130101; C12N 2310/11 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1. A method of inducing apoptosis in a cell in a mammal diagnosed
as having a proliferative disease, said method comprising
administering to said mammal a modified antisense oligonucleotide
of length sufficient to inhibit an inhibitor of apoptosis (IAP)
biological activity, wherein said antisense oligonucleotide is
complementary to a portion of human IAP-1 (HIAP-1) (SEQ ID
NO:5).
2. A method of treating a patient diagnosed as having a
proliferative disease, said method comprising administering to said
patient a modified antisense oligonucleotide of length sufficient
to inhibit an inhibitor of apoptosis (IAP) biological activity,
wherein said antisense oligonucleotide is complementary to a
portion of human IAP-1 (HIAP-1) (SEQ ID NO:5).
3. The method of claim 2, wherein said mammal is a human.
4. The method of claim 2, wherein said proliferative disease is
cancer.
5. The method of claim 4, wherein said cancer is ovarian cancer,
adenocarcinoma, lymphoma, or pancreatic cancer.
6. The method of claim 1, wherein said mammal is a human.
7. The method of claim 1, wherein said proliferative disease is
cancer.
8. The method of claim 7, wherein said cancer is ovarian cancer,
adenocarcinoma, lymphoma, or pancreatic cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a continuation application of U.S.
application Ser. No. 09/974,592, filed Oct. 9, 2001 (now allowed),
which is a continuation application of U.S. application Ser. No.
09/617,053, filed Jul. 14, 2000 (now U.S. Pat. No. 6,300,492),
which is a continuation application of U.S. application Ser. No.
08/800,929, filed Feb. 13, 1997 (now U.S. Pat. No. 6,133,437).
BACKGROUND OF THE INVENTION
[0002] The invention relates to the diagnosis and treatment of
cancer.
[0003] One way by which cells die is referred to as apoptosis, or
programmed cell death. Apoptosis often occurs as a normal part of
the development and maintenance of health tissues. The process
occurs so rapidly that it is difficult to detect. This may help to
explain why the involvement of apoptosis in a wide spectrum of
biological processes has only recently been recognized.
[0004] The apoptosis pathway is now known to play a critical role
in embryonic development, viral pathogenesis, cancer, autoimmune
disorders, and neurodegenerative disease. The failure of an
apoptotic response has been implicated in the development of
cancer, autoimmune disorders, such as lupus erythematosis and
multiple sclerosis, and in viral infections, including those
associated with herpes virus, poxvirus, and adenovirus.
[0005] Baculoviruses encode proteins that are termed inhibitors of
apoptosis proteins (IAPs) because they inhibit the apoptosis that
would otherwise occur when insect cells are infected by the virus.
These proteins are thought to work in a manner that is independent
of other viral proteins. The baculovirus IAP genes include
sequences encoding a ring zinc finger-like motif (RZF), which is
presumed to be directly involved in DNA binding, and two N-terminal
domains that consist of a 70 amino acid repeat motif termed a BIR
domain (Baculovirus IAP Repeat).
[0006] The role of apoptosis in cancer has only recently been
appreciated. The identification of growth promoting "oncogenes" in
the late 1970's gave rise to an almost universal focus on cellular
proliferation that dominated research in cancer biology for many
years. Long-standing dogma held that anti-cancer therapies
preferentially targeted rapidly dividing cancer cells relative to
"normal" cells. This explanation was not entirely satisfactory,
since some slow growing tumors are easily treated, while many
rapidly dividing tumor types are extremely resistant to anti-cancer
therapies. Progress in the cancer field has now led to a new
paradigm in cancer biology wherein neoplasia is viewed as a failure
to execute normal pathways of programmed cell death. Normal cells
receive continuous feedback from their neighbors through various
growth factors, and commit "suicide" if removed from this context.
Cancer cells somehow ignore these commands and continue
inappropriate proliferation. Cancer therapies, including radiation
and many chemotherapies, have traditionally been viewed as causing
overwhelming cellular injury. New evidence suggests that cancer
therapies actually work by triggering apoptosis.
[0007] Both normal cell types and cancer cell types display a wide
range of susceptibility to apoptotic triggers, although the
determinants of this resistance are only now under investigation.
Many normal cell types undergo temporary growth arrest in response
to a sub-lethal dose of radiation or cytotoxic chemical, while
cancer cells in the vicinity undergo apoptosis. This provides the
crucial treatment "window" of appropriate toxicity that allows
successful anti-cancer therapy. It is therefore not surprising that
resistance of tumor cells to apoptosis is emerging as a major
category of cancer treatment failure.
[0008] Compared to the numerous growth promoting oncogenes
identified to date (>100) relatively few genes have been
isolated that regulate apoptosis. The Bcl-2 gene was first
identified as an oncogene associated with the development of
follicular lymphomas. In contrast to all other oncogenes identified
to date, Bcl-2 displays no ability to promote cell proliferation,
and instead has been demonstrated to suppress apoptosis by a
variety of triggers. Elevated bcl-2 expression is associated with a
poor prognosis in neuroblastoma, prostate and colon cancer, and can
result in a multidrug resistant phenotype in vitro. Although the
study of Bcl-2 has helped revolutionize cancer paradigms, the vast
majority of human malignancies do not demonstrate aberrant Bcl-2
expression.
[0009] In contrast to the findings with bcl-2, mutation of the p53
tumor suppresser gene has been estimated to occur in up to 50% of
human cancers and is the most frequent genetic change associated
with cancer to date. The p53 protein plays a crucial role in
surveying the genome for DNA damage. The cell type and degree of
damage determines whether the cell will undergo growth arrest and
repair, or initiate apoptosis. Mutations in p53 interfere with this
activity, rendering the cell resistant to apoptosis by a wide range
of cellular insults. Some progress has been made in understanding
the molecular biology of p53, but many questions remain. p53 is
known to function as a transcription factor, with the ability to
positively or negatively regulate the expression of a variety of
genes involved in cell cycle control, DNA repair, and apoptosis
(including the anti-apoptotic Bcl-2 gene described above and the
related proapoptotic gene bax). The drug resistant phenotype
conferred by p53 alterations has been linked to Bcl-2/Bax
regulation, but this correlation does not hold for most cancer
types, leaving open the possibility that other critical genes
regulated by p53 remain to be identified.
SUMMARY OF THE INVENTION
[0010] We have discovered that IAP and NAIP overexpression are
associated with a wide range of cancer types including ovarian
cancer, adenocarcinoma, lymphoma, and pancreatic cancer. In
addition, we have found that nuclear localization fragmentation of
the IAPs, and overexpression of the IAPs in the presence of p53
mutations correlate with a cancer diagnosis, a poor prognosis, and
resistance to numerous chemotherapeutic cancer drugs. These
discoveries provide diagnostic, prognostic, and therapeutic
compounds and methods for the detection and treatment of
proliferative diseases.
[0011] In the first aspect, the invention features a method of
detecting cancer or an increased likelihood of cancer by detecting
an increase IAP gene expression or protein expression in a cell
from the mammal. In various embodiments, the detection may be
performed by contacting with IAP or NAIP nucleic acid, or a portion
thereof (which is greater than 9 nucleotides, and preferably
greater than 18 nucleotides in length), with a preparation of
nucleic acid from the cell; detecting levels of IAP or NAIP nucleic
acid using quantitative nucleic acid amplification techniques;
monitoring the levels of IAP or NAIP protein; or monitoring the
levels of IAP or NAIP biological activity. Preferably, the cell is
a cell from a mammal suspected of having a leukemia, a lymphoma,
breast cancer, pancreatic cancer, melanoma, lung cancer, or ovarian
cancer.
[0012] In one embodiment utilizing nucleic acid amplification for
detection, the invention features characterization of a cellular
IAP or NAIP nucleic acid content and levels by: (a) providing a
sample of nucleic acid; (b) providing a pair of oligonucleotides
having sequence homology to an IAP or NAIP nucleic acid; (c)
combining the pair of oligonucleotides with the cellular sample
under conditions suitable for polymerase chain reaction-mediated
nucleic acid amplification; and (d) isolating the amplified IAP
nucleic acid or fragment thereof. The isolated nucleic acid may
then be quantitated, sequenced, or otherwise characterized for the
activity it imparts on the cell or related cells in preferred
embodiments, the amplification is carried out using a
reverse-transcription polymerase chain reaction, for example, the
RACE method.
[0013] In one embodiment using nucleic acid hybridization for
detection, the invention features use of IAP or NAIP nucleic acid
isolated according to the method involving: (a) providing a
preparation of nucleic acid; (b) providing a detectably-labelled
nucleotide sequence having homology to a region of an IAP or NAIP
nucleic acid; (c) contacting the preparation of nucleic acid with
the detectably-labelled nucleic acid sequence under hybridization
conditions providing detection of nucleic acid having 50% or
greater nucleotide sequence identity; and (d) identifying IAP or
NAIP and characterizing nucleic acid by their association with the
detectable label.
[0014] In one embodiment utilizing antibodies for detection, the
invention features methods for using a purified antibody that binds
specifically to an IAP or NAIP family of proteins. Such an antibody
may be used for diagnosis and also for drug screens, prognostic
methods, and treatment methods described herein. Any standard
immunodetection method may be employed, as appropriate. Preferably,
the antibody binds specifically to XIAP, HIAP-1, HIAP-2 or NAIP. In
various embodiments, the antibody may react with other IAP
polypeptides or may be specific for one or a few IAP polypeptides.
The antibody may be a monoclonal or a polyclonal antibody.
Preferably, the antibody reacts specifically with only one of the
IAP polypeptides, for example, reacts with murine and human xiap,
but not with hiap-1 or hiap-2 from other mammalian species. In any
of the immunodetection, diagnostic and prognostic methods an
increase in IAP or NAIP polypeptide levels or an increase in the
level of certain IAP or NAIP fragments described herein (e.g.,
BIR-containing fragments or nuclear polypeptides, found to be
associated with proliferation indicate a cancer diagnosis or a poor
cancer prognosis when therapeutics which act by enhancing apoptosis
are used for treatment.
[0015] In another aspect, the invention features an IAP or NAIP
antisense oligonucleotide for use in suppressing cell
proliferation. Such nucleic acids of the invention and methods for
using them may be identified according to a method involving: (a)
providing a cell sample; (b) introducing by transformation into the
cell sample a candidate IAP or NAIP antisense oligonucleotide; (c)
expressing the candidate IAP or NAIP antisense oligonucleotide
within the cell sample; and (d) determining whether the cell sample
exhibits an altered apoptotic response, whereby increased apoptosis
identifies an anti-proliferative compound. Preferably, the cell is
a cancer cell.
[0016] In another aspect, the invention features a method of
determining the prognosis of a mammal having a proliferative
disease. The method includes detecting levels of IAP or NAIP
nucleic acids, protein levels, or biological activity, or IAP
fragments in the cell suspected to be involved in a proliferative
disease. In various embodiments, the methods of detection described
above for diagnosis may be employed. An increase in IAP or NAIP
levels indicates a proliferative disease (i.e., an increased
likelihood the cancers described herein) and, particularly if a p53
mutation is present, a poor prognosis for therapeutic approaches
which rely on enhancing apoptosis. The presence of IAP fragments of
less than 64 kD, more preferably less than 45 kD indicates in
increased likelihood that the cancer will be resistant to
chemotherapeutics which act by inducing apoptosis.
[0017] In preferred embodiments of the diagnostic and prognostic
methods, the levels being monitored are levels of IAP or NAIP
express or activity levels known to be associated with cancer
suspected or diagnosed. Most preferably, the disease is selected
from the group consisting of a breast cancer (preferably using a
hiap-2, hiap-1, HIAP-2, or HIAP-1 probe), ovarian cancer
(preferably using a hiap-2, or HIAP-2 probe), promyelocytic
leukemia, a HeLa-type carcinoma, chronic myelogenous leukemia
(preferably using a xiap, hiap-2, XIAP or HIAP-2 probe),
lymphoblastic leukemia (preferably using a xiap or XIAP probes),
Burkitt's lymphoma (preferably using a hiap-1 or HIAP-1 probe),
colorectal adenocarcinoma, lung carcinoma, and melanoma (preferably
using a xiap, or XIAP probe). Preferably, a cancer diagnosis or
poor prognosis is indicated by a 2-fold increase in expression or
activity, more preferably, at least a 10-fold increase in
expression or activity in the cell being tested.
[0018] In another aspect, the invention features a method of
identifying a compound that inhibits cancer by enhancing apoptosis.
The method includes providing a cell expressing an IAP or NAIP
polypeptide and being capable of proliferation or viability in
culture, contacting the cell with a candidate compound, and
monitoring the expression of an IAP gene, NAIP gene, a reporter
linked to IAP or NAIP regulatory sequence, levels of IAP or NAIP
polypeptides, cleavage of IAP polypeptides, and/or nuclear versus
cytoplasmic localization of IAP or NAIP polypeptides. A decrease in
the level of expression of the IAP or NAIP gene, IAP or NAIP
protein characteristics, IAP or NAIP biological activity, IAP
cleavage, or localization of protein to the nucleus, indicate the
presence of a compound which enhances apoptosis, as described
herein. In various preferred embodiments, the cell used in the
method is a fibroblast, a neuronal cell, a glial cell, a lymphocyte
(T cell or B cell), a breast cancer cell, a lymphoma cell, an
ovarian cancer cell, a leukemia cell, a pancreatic cancer cell, a
melanoma cell, or an insect cell; the preferred polypeptide
expression being monitored is XIAP, HIAP-1, HIAP-2, or NAIP (i.e.,
human or murine). In the embodiment utilizing fragment detection,
the fragment is preferably less than 64 kD, more preferably less
than 45 kD. All the detection methods described herein may be
employed, as appropriate.
[0019] In a related aspect, the invention features methods of
detecting compounds that enhance apoptosis using the interaction
trap technology and IAP or NAIP polypeptides, or fragments thereof,
as a component of the bait. In preferred embodiments, the compound
being tested as an enhancer of apoptosis is also a polypeptide.
[0020] In another aspect, the invention features a method of
treating a patient diagnosed with a proliferative disease. In the
method, apoptosis may be induced in a cell to control a
proliferative disease either alone or in combination with other
therapies by administering to the cell a negative regulator of the
IAP-dependent or NAIP anti-apoptotic pathway. The negative
regulator may be, but is not limited to, an IAP ring zinc finger,
and an IAP polypeptide that includes a ring zinc finger and lacks
at least one BIR domain. Alternatively, apoptosis may be induced in
the cell by administering a nucleic acid encoding an IAP antisense
oligonucleotide administered directly or via gene therapy (see U.S.
Pat. No. 5,576,208 for general parameters which may be applicable
in the selection of IAP or NAIP antisense oligonucleotides).
[0021] The term "oligonucleotide" refers to an oligomer or polymer
of ribonucleic acid or deoxyribonucleic acid. 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
cellular uptake and increased stability in the presence of
nucleases.
[0022] Specific examples of some preferred oligonucleotides
envisioned for this invention may contain phosphorothioates,
phosphotriesters, methyl phosphonates, short chain alkyl or
cycloalkyl intersugar linkages or short chain heteroatomic or
heterocyclic intersugar linkages. Most preferred are those with
CH.sub.2--NH--O--CH.sub.2, CH.sub.2--N(CH.sub.3)--O--CH.sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and
O--N(CH.sub.3)--CH.sub.2--CH.sub.2 backbones (where phosphodiester
is O--P--O--CH.sub.2). Also preferred are oligonucleotides having
morpholino backbone structures. Summerton, J. E. and Weller, D. D.,
U.S. Pat. No. 5,034,506. In other preferred embodiments, such as
the protein-nucleic acid (PNA) backbone, the phosphodiester
backbone of the oligonucleotide may be replaced with a polyamide
backbone, the bases being bound directly or indirectly to the aza
nitrogen atoms of the polyamide backbone. P. E. Nielsen, M. Egholm,
R. H. Berg, O Buchardt, Science 199, 254, 1497. Other preferred
oligonucleotides may contain alkyl and halogen-substituted sugar
moieties comprising one of the following at the 2' position: OH,
SH, SCH.sub.3, F, OCN, O(CH.sub.2).sub.nNH.sub.2 or
O(CH.sub.2).sub.nCH.sub.3 where n is from 1 to about 10; C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl;
Cl; Br; CN; CF.sub.3; OCF.sub.3; O--, S--, or N-alkyl; O--, S--, or
N-alkenyl; SOCH.sub.3; SO.sub.2 CH.sub.3; ONO.sub.2; NO.sub.2;
N.sub.3; NH.sub.2; heterocycloalkyl; heterocycloalkaryl;
aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving
group; a conjugate; a reporter group; an intercalator; a group for
improving the pharmacokinetic properties of an oligonucleotide; or
a group for improving the pharmacodynamic properties of an
oligonucleotide and other substituents having similar properties.
Oligonucleotides may also have sugar mimetics such as cyclobutyls
in place of the pentofuranosyl group.
[0023] Other preferred embodiments may include at least one
modified base form. Some specific examples of such modified bases
include 2-(amino)adenine, 2-(methylamino)adenine,
2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other
heterosubstituted alkyladenines. In yet another method, the
negative regulator may be a purified antibody, or a fragment
thereof, that binds specifically to an IAP polypeptide. For
example, in one preferred embodiment, the antibody may bind to an
approximately 26 kDa cleavage product of an IAP polypeptide that
includes at least one BIR domain but lacks a ring zinc finger
domain.
[0024] In two additional aspects, the invention features a
transgenic animal and methods of using the mammal for detection of
anti-cancer therapeutics. Preferably the mammal overexpresses an
IAP or NAIP polypeptide and/or expresses a NAIP or IAP antisense
oligonucleotide or IAP or NAIP fragment. In one embodiment, the
animal also has a genetic predisposition to cancer or has cancer
cells under conditions which provide for proliferation absent the
transgenic construct encoding either the antisense oligonucleotide
or fragment.
[0025] By "IAP gene" is meant a gene encoding a polypeptide having
at least one BIR domain and a ring zinc finger domain which is
capable of modulating (inhibiting or enhancing) apoptosis in a cell
or tissue when provided by other intracellular or extracellular
delivery methods (see, e.g., U.S. Ser. Nos. 08/511,485, 08/576,965,
and PCT/IB96/01022). In preferred embodiments the IAP gene is a
gene having about 50% or greater nucleotide sequence identity to at
least one of the IAP amino acid encoding sequences of FIGS. 1-4 or
portions thereof. Preferably, the region of sequence over which
identity is measured is a region encoding at least one BIR domain
and a ring zinc finger domain. Mammalian IAP genes include
nucleotide sequences isolated from any mammalian source.
Preferably, the mammal is a human. The term "IAP gene" is meant to
encompass any member of the family of genes that encode inhibitors
of apoptosis. An IAP gene may encode a polypeptide that has at
least 20%, preferably at least 30%, and most preferably at least
50% amino acid sequence identity with at least one of the conserved
regions of one of the IAP members described herein (i.e., either
the BIR or ring zinc finger domains from the human or murine xiap,
hiap-1 and hiap-2). Representative members of the IAP gene family
include, without limitation, the human and murine xiap, hiap-1, and
hiap-2 genes.
[0026] By "IAP protein" or "IAP polypeptide" is meant a
polypeptide, or fragment thereof, encoded by an IAP gene.
[0027] "NAIP gene" and "NAIP polypeptide" means the NAIP genes,
fragments thereof, and polypeptides encoded by the same described
in UK9601108.5 filed Jan. 19, 1996 and the PCT application claiming
priority from UK9601108.5 filed Jan. 17, 1997.
[0028] By "BIR domain" is meant a domain having the amino acid
sequence of the consensus sequence:
Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-
-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-X-
aa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-X-
aa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa-
1-Cys-Xaa1-Phe-Val, wherein Xaa1 is any amino acid and Xaa2 is any
amino acid or is absent (SEQ ID NO:2). Preferably, the sequence is
substantially identical to one of the BIR domain sequences provided
for xiap, hiap-1, hiap-2 herein.
[0029] By "ring zinc finger" or "RZF" is meant a domain having the
amino acid sequence of the consensus sequence:
Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-
-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa-
1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys,
wherein Xaa1 is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val
or Ile (SEQ ID NO:1).
[0030] Preferably, the sequence is substantially identical to the
RZF domains provided herein for the human or murine Xiap, Hiap-1,
or Hiap-2.
[0031] By "enhancing apoptosis" is meant increasing the number of
cells which apoptose in a given cell population. Preferably, the
cell population is selected from a group including ovarian cancer
cells, breast cancer cells, pancreatic cancer cells, T cells,
neuronal cells, fibroblasts, or any other cell line known to
proliferate in a laboratory setting. It will be appreciated that
the degree of apoptosis enhancement provided by an apoptosis
enhancing compound in a given assay will vary, but that one skilled
in the art can determine the statistically significant change in
the level of apoptosis which identifies a compound which enhances
apoptosis otherwise limited by an IAP. Preferably, "enhancing
apoptosis" means that the increase in the number of cells
undergoing apoptosis is at least 25%, more preferably the increase
is 50%, and most preferably the increase is at least one-fold.
Preferably, the sample monitored is a sample of cells which
normally undergo insufficient apoptosis (i.e., cancer cells).
[0032] By "proliferative disease" is meant a disease which is
caused by or results in inappropriately high levels of cell
division, inappropriately low levels of apoptosis, or both. For
example, cancers such as lymphoma, leukemia, melanoma, ovarian
cancer, breast cancer, pancreatic cancer, and lung cancer are all
examples of proliferative disease.
[0033] By "polypeptide" is meant any chain of more than two amino
acids, regardless of post-translational modification such as
glycosylation or phosphorylation.
[0034] By "IAP or NAIP biological activity" is meant any activity
known to be caused in vivo or in vitro by a NAIP or IAP
polypeptide.
[0035] By "substantially identical" is meant a polypeptide or
nucleic acid exhibiting at least 50%, preferably 85%, more
preferably 90%, and most preferably 95% homology to a reference
amino acid or nucleic acid sequence. For polypeptides, the length
of comparison sequences will generally be at least 16 amino acids,
preferably at least 20 amino acids, more preferably at least 25
amino acids, and most preferably 35 amino acids. For nucleic acids,
the length of comparison sequences will generally be at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides.
[0036] Sequence identity is typically measured using sequence
analysis software with the default parameters specified therein
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, and 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.
[0037] By "substantially pure polypeptide" is meant a polypeptide
that has been separated from the components that naturally
accompany it. Typically, the polypeptide is substantially pure when
it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is an IAP polypeptide that
is at least 75%, more preferably at least 90%, and most preferably
at least 99%, by weight, pure. A substantially pure IAP polypeptide
may be obtained, for example, by extraction from a natural source
(e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of
a recombinant nucleic acid encoding an IAP polypeptide, or by
chemically synthesizing the protein. Purity can be measured by any
appropriate method, e.g., by column chromatography, polyacrylamide
gel electrophoresis, or HPLC analysis.
[0038] A protein is substantially free of naturally associated
components when it is separated from those contaminants which
accompany it in its natural state. Thus, a protein which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those derived
from eukaryotic organisms but synthesized in E. coli or other
prokaryotes.
[0039] By "substantially pure DNA" is meant DNA that is free of the
genes which, in the naturally-occurring genome of the organism from
which the DNA of the invention is derived, flank the gene. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote; or which exists as a separate molecule (e.g., a cDNA or
a genomic or cDNA fragment produced by PCR or restriction
endonuclease digestion) independent of other sequences. It also
includes a recombinant DNA which is part of a hybrid gene encoding
additional polypeptide sequence.
[0040] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding (as used herein) an IAP
polypeptide.
[0041] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0042] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
mammalian (e.g., rodents such as rats or mice) and the DNA
(transgene) is inserted by artifice into the nuclear genome.
[0043] By "transformation" is meant any method for introducing
foreign molecules into a cell. Lipofection, calcium phosphate
precipitation, retroviral delivery, electroporation, and biolistic
transformation are just a few of the teachings which may be used.
For example, biolistic transformation is a method for introducing
foreign molecules into a cell using velocity driven
microprojectiles such as tungsten or gold particles. Such
velocity-driven methods originate from pressure bursts which
include, but are not limited to, helium-driven, air-driven, and
gunpowder-driven techniques. Biolistic transformation may be
applied to the transformation or transfection of a wide variety of
cell types and intact tissues including, without limitation,
intracellular organelles (e.g., and mitochondria and chloroplasts),
bacteria, yeast, fungi, algae, animal tissue, and cultured
cells.
[0044] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, e.g., an IAP polypeptide, a recombinant protein
or a RNA molecule).
[0045] By "reporter gene" is meant a gene whose expression may be
assayed; such genes include, without limitation, glucuronidase
(GUS), luciferase, chloramphenicol transacetylase (CAT), and
.beta.-galactosidase.
[0046] By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell type-specific, tissue-specific or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the native gene.
[0047] By "operably linked" is meant that a gene and one or more
regulatory sequences are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins are bound to the regulatory sequences).
[0048] By "conserved region" is meant any stretch of six or more
contiguous amino acids exhibiting at least 30%, preferably 50%, and
most preferably 70% amino acid sequence identity between two or
more of the IAP family members, (e.g., between human HIAP-1,
HIAP-2, and XIAP). Examples of preferred conserved regions are
shown (as boxed or designated sequences) in FIGS. 5-7 and Tables 1
and 2, and include, without limitation, BIR domains and ring zinc
finger domains.
[0049] By "detectably-labelled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide
probe or primer, a gene or fragment thereof, or a cDNA molecule.
Methods for detectably-labelling a molecule are well known in the
art and include, without limitation, radioactive labelling (e.g.,
with an isotope such as .sup.32P or .sup.35S) and nonradioactive
labelling (e.g., chemiluminescent labelling, e.g., fluorescein
labelling).
[0050] By "antisense," as used herein is meant an oligonucleotide,
regardless of length, that is complementary to the coding strand of
an IAP or NAIP gene. Preferably, the antisense oligonucleotide is
capable of enhancing apoptosis when present in a cell which
normally does not undergo sufficient apoptosis. Preferably, the
increase is at least 10%, relative to a control, more preferably
25%, and most preferably 1-fold or more.
[0051] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more preferably 90%, and most
preferably at least 99%, by weight, antibody, e.g., an IAP specific
antibody. A purified antibody may be obtained, for example, by
affinity chromatography using recombinantly-produced protein or
conserved motif peptides and standard techniques.
[0052] By "specifically binds" is meant an antibody that recognizes
and binds a protein but that does not substantially recognize and
bind other molecules in a sample, e.g., a biological sample, that
naturally includes protein.
[0053] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 is the human xiap cDNA sequence (SEQ ID NO:3) and the
XIAP polypeptide sequence (SEQ ID NO:4).
[0055] FIG. 2 is the human hiap-1 cDNA sequence (SEQ ID NO:5) and
the HIAP-1 polypeptide sequence (SEQ ID NO:6).
[0056] FIG. 3 is the human hiap-2 cDNA sequence (SEQ ID NO:7) and
the HIAP-2 polypeptide sequence (SEQ ID NO:8). The sequence absent
in the hiap-2-.DELTA. variant is boxed.
[0057] FIG. 4 is the murine xiap (also referred to as "miap-3")
cDNA sequence (SEQ ID NO:9) and encoded murine XIAP polypeptide
sequence (SEQ ID NO:10).
[0058] FIG. 5 is the murine hiap-1 (also referred to as "miap-1")
cDNA sequence (SEQ ID NO:11) and the encoded murine HIAP-1
polypeptide sequence (SEQ ID NO:12).
[0059] FIG. 6 is the murine hiap-2 (also referred to as "miap-2")
cDNA sequence (SEQ ID NO:13) and the encoded murine HIAP-2
polypeptide (SEQ ID NO:14).
[0060] FIG. 7 is a photograph of a Northern blot illustrating human
hiap-1 and hiap-2 mRNA expression in human tissues.
[0061] FIG. 8 is a photograph of a Northern blot illustrating human
hiap-2 mRNA expression in human tissues.
[0062] FIG. 9 is a photograph of a Northern blot illustrating human
xiap mRNA expression in human tissues.
[0063] FIG. 10A-10D are graphs depicting suppression of apoptosis
by XIAP, HIAP-1, HIAP-2, bcl-2, smn, and 6-myc.
[0064] FIG. 11 is a photograph of an agarose gel containing cDNA
fragments that were amplified, with hiap-1-specific primers, from
RNA obtained from Raji, Ramos, EB-3, Burkitt's lymphoma cells, and
Jiyoye cells, and cells from normal placenta.
[0065] FIG. 12 is a photograph of a Western blot containing protein
extracted from Jurkat and astrocytoma cells stained with an
anti-XIAP antibody. The position and size of a series of marker
proteins is indicated.
[0066] FIG. 13 is a photograph of a Western blot containing protein
extracted from Jurkat cells following treatment as described in
Example XII. The blot was stained with a rabbit polyclonal
anti-XIAP antibody. Lane 1, negative control; lane 2, anti-Fas
antibody; lane 3, anti-Fas antibody and cycloheximide; lane 4,
TNF-.alpha.; lane 5, TNF-.alpha. and cycloheximide.
[0067] FIG. 14 is a photograph of a Western blot containing protein
extracted from HeLa cells following exposure to anti-Fas
antibodies. The blot was stained with a rabbit polyclonal anti-XIAP
antibody. Lane 1, negative control; lane 2, cycloheximide; lane 3,
anti-Fas antibody; lane 4, anti-Fas antibody and cycloheximide;
lane 5, TNF-.alpha.; lane 6, TNF-.alpha. and cycloheximide.
[0068] FIGS. 15A and 15B are photographs of Western blots stained
with rabbit polyclonal anti-XIAP antibody. Protein was extracted
from HeLa cells (FIG. 21A) and Jurkat cells (FIG. 21B) immediately,
1, 2, 3, 5, 10, and 22 hours after exposure to anti-Fas
antibody.
[0069] FIGS. 16A and 16B are photographs of Western blots stained
with an anti-CPP32 antibody (FIG. 16A) or a rabbit polyclonal
anti-XIAP antibody (FIG. 16B). Protein was extracted from Jurkat
cells immediately, 3 hours, or 7 hours after exposure to an
anti-Fas antibody. In addition to total protein, cytoplasmic and
nuclear extracts are shown.
[0070] FIG. 17 is a photograph of a polyacrylamide gel following
electrophoresis of the products of an in vitro XIAP cleavage
assay.
[0071] FIGS. 18 and 19 shows the increased level of HIAP-1 and
HIAP-2 mRNA, respectively, in breast cancer cell lines having p53
mutations (lanes 5-7). The bottom portion of the figure shows the
control.
[0072] FIG. 20 shows the influence of Taxol on DNA fragmentation in
Cisplatin-sensitive (right) and resistant (left) human ovarian
epithelial cancer cells.
[0073] FIG. 21 shows the influence of Cisplatin on DNA
fragmentation in sensitive (right) and resistant (left) human
ovarian epithelial cancer cells.
[0074] FIG. 22 shows the effects of Taxol on XIAP and Hiap-2
protein levels in Cisplatin sensitive (right) and resistant (left)
human ovarian epithelial cancer cells.
[0075] FIGS. 23A and 23B show the influence of Taxol and TGF.beta.
on XIAP-2 mRNA levels in Cisplatin sensitive (right) and resistant
(left) human epithelial cancer cells.
[0076] FIGS. 24A and 24B show the effect of TGF.beta. on XIAP
protein expression (FIG. 24A) and DNA fragmentation (FIG. 24B) in
Cisplatin sensitive and resistant cells.
DETAILED DESCRIPTION
[0077] Previously, we have provided a novel family of inhibitors of
apoptosis, the IAPs, and an additional related anti-apoptotic
protein, NAIP. Here we provide identification of cancer types in
which dysregulation of the IAPs and NAIP is apparent. Our results
are of paramount importance and provide diagnostics, prognostics,
treatments, and drug screens aimed at the detection and effective
treatment of cancer.
Cancer Screening
[0078] We initially studied IAP expression levels in a variety of
normal tissues and cancer cell lines using
commercially-available-northern blots. Elevated xiap, hiap-1 and
hiap-2 mRNA was noted in a surprising number of cancer lines of
diverse lineage, including colorectal cancer, lymphoma, leukemia,
and melanoma cell lines. In contrast, Bcl-2 mRNA was elevated in
only a single cell line. Although this result reinforced the
importance of the IAPs in cancer, the question remained as to
whether the individual cancer cell lines on the blot were
representative of the cancer type. As a result, we screened panels
of cancer cell lines of particular tumor type by northern blot and
quantitative RT-PCR analysis in order to ascertain the frequency of
IAP dysregulation. The results are summarized as follows:
Burkitt's Lymphoma
[0079] We studied both the frequency and consequences of IAP
upregulation in Burkitt's lymphoma. Elevated levels of hiap-1 and
hiap-2 have been found in the vast majority of the Burkitt's cell
lines examined. Furthermore, those Burkitt's lines expressing low
levels of hiap-1 are transcriptionally activated by Epstein-Barr
virus (EBV) infection.
Breast Adenocarcinoma
[0080] A key observation was made in this survey, in which a
correlation was observed between drug resistance, p53 status and
hiap-1/2 expression. Four of the cell lines possessed wild-type
p53, while three possessed documented p53 mutations that correlated
with resistance to the anti-cancer drug adriamycin. Significantly,
the three lines which were relatively more drug resistant also
displayed elevated hiap-1 and hiap-2 mRNA levels. These results
indicate that one of the ways that p53 controls apoptosis is
through regulation of these genes.
Ovarian Carcinoma
[0081] mRNA in situ analysis suggest a role for NAIP in the
developmental biology of the ovary. Overexpression of hiap-2 and
xiap mRNA has also been documented in some ovarian cancer cell
lines.
Pancreatic Cancer
[0082] Approximately 25% of the cell lines tested to date
demonstrate hiap-1 and hiap-2 mRNA elevation.
Summary of Cancer Panels
[0083] To date, a significant fraction of cancer cell lines of each
type examined display elevated IAP levels. Our results indicate
that hiap-1 and hiap-2 tend to be the most frequently and
dramatically upregulated. The apparent coordinate regulation of
both genes was surprising given their very different normal tissue
distribution. hiap-1 and hiap-2 reside in tandem array on
chromosome 11q23, a site frequently rearranged in lymphomas and
leukemias.
Transcriptional Regulation of the IAPs in Cancer Cell Lines
[0084] Our experiments have established a correlation between p53
status and transcriptional overexpression of hiap-1 and hiap-2.
This provides an important new way in which to enhance apoptosis,
particularly in view of the fact that the mechanism by which p53
controls cell fate remains largely unknown. It has previously been
documented that wild-type p53 negatively down-regulates Bcl-2, and
positively upregulates the Bcl-2 antagonist Bax. In some cancer
cell types, mutation of p53 causes a two-fold effect; namely, the
upregulation of Bcl-2, and down regulation of Bax, both of which
contribute to the anti-apoptotic phenotype. While not wishing to
bind ourselves to a particular theory, we believe that wild-type
p53 also transcriptionally suppresses hiap-1 and hiap-2. DNA damage
that includes the increase in wild-type levels p53 levels would
therefore result in decreased hiap-1 and hiap-2 in normal cells,
resulting in apoptosis. Mutations in the p53 gene would therefore
result in a loss of transcriptional control of these iap genes. As
a result, p53 mutant cancer cells would display constitutively high
levels of hiap-1 and hiap-2, rendering the cells resistant to
anti-cancer therapies. The p53/hiap-1 and hiap-2 correlations may
be extended to the other cancer cell line panels. One may directly
demonstrate p53 regulation of the IAPs using transfection assays
and northern blot analysis.
[0085] Accordingly, we predict that cancer cells having p53
mutations (p53*) will have increased IAP levels resulting in a poor
response to chemotherapeutics. Because IAP levels may be assessed
more readily than the presence of a p53*mutation, our discovery
also provides an important improvement in cancer diagnosis and
prognosis (see below).
Transgenic Mice
[0086] We have constructed a number of IAP and NAIP transgenic
mouse expression vectors, including T-cell, B-cell, and neuronal
specific promoter constructs. Founder mice have been identified,
are viable, and for most of these constructs and we have developed
breeding colonies. These mice will likely be prone to cancers of
the tissue types in which the promoter is active. Thus the mice
provide an excellent resource for testing the efficacy of
anti-sense oligos and for screening for apoptosis enhancing cancer
therapeutics. Standard mouse drug screening models and gene
delivery protocols may be employed to utilize the mice for this
purpose.
Diagnostic/Prognostic Reagents
[0087] There is a relative lack of diagnostic and prognostic tests
which clinical oncologists may utilize in determining the
appropriate degree of intervention in the treatment of cancer.
Mutation of the p53 gene remains one of the best prognostic
indicators in cancer biology. However, the number of different
mutations identified to date is great and scattered throughout the
gene. In addition, many mutations in p53 result in an inappropriate
stabilization of the protein, which allows detection at the protein
level rather than at the mRNA level. Mutations which alter the
transactivation/repression activities of the protein are not
necessarily apparent at either the mRNA or protein levels. On the
other hand, if IAP and NAIP expression levels correlate with p53
mutation they may provide more valuable prognostic information and
assist in the determination of which patients require more
aggressive treatment. Thus the invention provides two assays for
prognosis an diagnosis. Semi-quantitative RT-PCR based assays may
be used to assay for iap and/or naip gene or protein expression
levels. Alternatively, monoclonal antibodies may be incorporated
into an ELISA (enzyme linked immunosorbent assay) type assay for
direct determination of protein levels.
Therapeutic Products
[0088] For IAP related therapies one may employ the paradigms
utilized for Bcl-2 and RAS antisense development, although
accommodation of IAP mutation is not required (in contrast to ras
antisense). Most useful are antisense constructs which enhance
apoptosis at least 10%, preferably by enhancing degradation of the
RNA in the nucleus.
[0089] In addition to antisense approached described herein the
invention features small molecule screening assays which may be
used to identify lead compounds that negatively regulate the iaps.
For example, compounds which enhance apoptosis in the presence of
IAP overexpression or which decrease the level of IAP biological
activity may be detected and are useful cancer therapeutics.
[0090] Molecules that are found, by the methods described above, to
effectively modulate IAP gene expression or polypeptide activity
may be tested further in animal models. If they continue to
function successfully in an in vivo setting, they may be used as
therapeutics to either inhibit or enhance apoptosis, as
appropriate.
Manipulation of Cancer Chemotherapeutic Drug Resistance Using an
Antisense Oligonucleotide and Fragment Approaches
[0091] We have documented that overexpression of the IAPs renders
cell lines resistant to serum growth factor withdrawal, tumor
necrosis factor alpha (TNF) and menadione exposure, all of which
are treatments that normally induce apoptosis. Herein we describe
the extension of these studies to cancer cell lines using apoptotic
triggers used in clinical situations, such as doxorubicin,
adriamycin, and methotrexate. Our findings have led up to design
antisense oligonucleotide therapeutics. Rapid screening of multiple
cell lines for apoptotic response has been made feasible through
the generation of a series of sense and antisense adenoviral IAP
and NAIP expression vectors, as well as control lacZ viruses. One
may now show enhanced drug resistance using the expression
constructs. In addition, anti-sense adenovirus constructs may be
developed and used to test reversal of the drug resistant phenotype
of appropriate cell lines. We have surveyed cancer cell lines with
the objective of identifying tumor types in which IAP
overexpression is apparent or altered and these results are
described both above and in the Examples below. Concomitant to this
research, we have designed a series of antisense oligonucleotides
to various regions of each of the iaps. These oligonucleotides may
be used to enhance drug sensitivity after testing in an assay
system, i.e., with the adenoviral vectors system. Animal modeling
of the effectiveness of antisense IAP oligos may also be employed
as a step in testing and appropriate transgenic mammals for this
are described above and also generally available in the art.
[0092] The following describes some of the testing systems which
may be employed.
Anti-Cancer Gene Therapy
[0093] Retroviral vectors, adenoviral vectors, adeno-associated
viral vectors, or other viral vectors with the appropriate tropism
for cells likely requiring enhanced apoptosis (for example, breast
cancer and ovarian cancer cells) may be used as a gene transfer
delivery system for a therapeutic gene constructs. Numerous vectors
useful for this purpose are generally known (Miller, Human Gene
Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis
and Anderson, BioTechniques 6:608-614, 1988; Tolstoshev and
Anderson, current opinion in Biotechnology 1:55-61, 1990; Sharp,
The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid
Research and Molecular Biology 36:311-322, 1987; Anderson, Science
226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et
al., BioTechniques 7:980-990, 1989; Le Gal La Salle et al., Science
259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).
[0094] Retroviral vectors are particularly well developed and have
been used in clinical settings (Rosenberg et al., N. Engl. J. Med
323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Non-viral
approaches may also be employed for the introduction of therapeutic
DNA into cells otherwise predicted to undergo apoptosis. For
example, IAP may be introduced into a neuron or a T cell by
lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413,
1987; Ono et al., Neurosci. Lett. 117:259, 1990; Brigham et al.,
Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Meth. Enz.
101:512, 1983), asialorosonucoid-polylysine conjugation (Wu et al.,
J. Biol. Chem. 263:14621, 1988; Wu et al., J. Biol. Chem.
264:16985, 1989); or, less preferably, microinjection under
surgical conditions (Wolff et al., Science 247:1465, 1990).
[0095] For any of the methods of application described above, the
therapeutic nucleic acid construct is preferably applied to the
site of the needed apoptosis event (for example, by injection).
However, it may also be applied to tissue in the vicinity of the
predicted apoptosis event or to a blood vessel supplying the cells
predicted to require enhanced apoptosis.
[0096] In the constructs described, nucleic acid expression can be
directed from any suitable promoter (e.g., the human
cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein
promoters), and regulated by any appropriate mammalian regulatory
element. For example, if desired, enhancers known to preferentially
direct gene expression in ovarian cells, breast tissue, neural
cells, T cells, or B cells may be used to direct expression. The
enhancers used could include, without limitation, those that are
characterized as tissue- or cell-specific in their expression.
Alternatively, if a clone used as a therapeutic construct,
regulation may be mediated by the cognate regulatory sequences or,
if desired, by regulatory sequences derived from a heterologous
source, including any of the promoters or regulatory elements
described above.
[0097] Less preferably, anti-cancer gene therapy is accomplished by
direct administration of the therapeutic mRNA or antisense IAP mRNA
to a cell that is expected to require enhanced apoptosis. The mRNA
may be produced and isolated by any standard technique, but is most
readily produced by in vitro transcription using an IAP related
nucleic acids under the control of a high efficiency promoter
(e.g., the T7 promoter). Administration of IAP antisense mRNA to
malignant cells can be carried out by any of the methods for direct
nucleic acid administration described above.
[0098] Ideally, the production of IAP protein by any gene therapy
approach will result in cellular levels of and/or fragments thereof
that are at least equivalent to the normal, cellular level of IAP
in an unaffected cell. Treatment by any IAP-modulating gene therapy
approach may be combined with more traditional therapies.
[0099] Another therapeutic approach within the invention involves
administration of recombinant IAP protein fragments or IAP
antibodies, either directly to the site where enhanced apoptosis is
desirable (for example, by injection) or systemically (for example,
by any conventional recombinant protein administration
technique).
[0100] The dosage of IAP, the IAP fragment, IAP mutant protein or
IAP antibody depends on a number of factors, including the size and
health of the individual patient, but, generally, between 0.1 mg
and 100 mg inclusive are administered per day to an adult in any
pharmaceutically acceptable formulation.
Administration
[0101] An IAP mutant protein or protein fragment, a gene encoding
the same, a gene encoding an IAP antisense oligonucleotide, or
modulator of an IAP may be administered within a
pharmaceutically-acceptable diluent, carrier, or excipient, in unit
dosage form. Conventional pharmaceutical practice may be employed
to provide suitable formulations or compositions to administer the
compounds to patients suffering from a disease that is caused by
excessive cell proliferation. Administration may begin before the
patient is symptomatic. Any appropriate route of administration may
be employed, for example, administration may be parenteral,
intravenous, intraarterial, subcutaneous, intramuscular,
intracranial, intraorbital, ophthalmic, intraventricular,
intracapsular, intraspinal, intracisternal, intraperitoneal,
intranasal, aerosol, suppository, or oral administration. For
example, therapeutic formulations may be in the form of liquid
solutions or suspensions; for oral administration, formulations may
be in the form of tablets or capsules; and for intranasal
formulations, in the form of powders, nasal drops, or aerosols.
[0102] Methods well known in the art for making formulations are
found, for example, in "Remington's Pharmaceutical Sciences."
Formulations for parenteral administration may, for example,
contain excipients, sterile water, or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for IAP modulatory compounds include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel.
[0103] If desired, treatment with an IAP mutant proteins or IAP
fragments, related genes, or other modulatory compounds may be
combined with more traditional therapies for the proliferative
disease such as surgery or chemotherapy.
Detection of Conditions Involving Insufficient Apoptosis
[0104] IAP polypeptides and nucleic acid sequences find diagnostic
use in the detection or monitoring of conditions involving
insufficient levels of apoptosis, i.e., proliferative disease. For
example, increased expression of IAPs, altercations in
localization, and IAP cleavage correlate with inhibition of
apoptosis and cancer in humans. Accordingly, an increase in the
level of IAP production may provide an indication of a
proliferative condition or a predisposition to such a condition.
Levels of IAP expression may be assayed by any standard technique.
For example, IAP expression in a biological sample (e.g., a biopsy
sample) may be monitored by standard Northern blot analysis or may
be aided by PCR (see, e.g., Ausubel et al., supra PCR Technology;
Principles and Applications for DNA Amplification, H. A. Ehrlich,
Ed. Stockton Press, NY; Yap et al. Nucl. Acids. Res. 19:4294,
1991).
[0105] Alternatively, a biological sample obtained from a patient
may be analyzed for one or more mutations in the IAP sequences or
p53 sequences using a mismatch detection approach. Generally, these
techniques involve PCR amplification of nucleic acid from the
patient sample, followed by identification of the mutation (i.e.,
mismatch) by either altered hybridization, aberrant electrophoretic
gel migration, binding or cleavage mediated by mismatch binding
proteins, or direct nucleic acid sequencing. Any of these
techniques may be used to facilitate mutant IAP detection, and each
is well known in the art; examples of particular techniques are
described, without limitation, in Orita et al., Proc. Natl. Acad.
Sci. USA 86:2766-2770, 1989; Sheffield et al., Proc. Natl. Acad.
Sci. USA 86:232-236, 1989).
[0106] In yet another approach, immunoassays are used to detect or
monitor IAP protein in a biological sample. IAP-specific polyclonal
or monoclonal antibodies (produced as described above) may be used
in any standard immunoassay format (e.g., ELISA, Western blot, or
RIA) to measure IAP polypeptide level or IAP levels from cancerous
control cells. These levels would be compared to wild-type IAP
levels, with a decrease in IAP production relative to a wild-type
cell indicating a condition involving increased apoptosis and a
decrease relative to a known cancer cell indicating a decreased
likelihood of an IAP related cancer. Examples of immunoassays are
described, e.g., in Ausubel et al., supra. Immunohistochemical
techniques may also be utilized for IAP detection. For example, a
tissue sample may be obtained from a patient, sectioned, and
stained for the presence of IAP using an anti-IAP antibody and any
standard detection system (e.g., one which includes a secondary
antibody conjugated to horseradish peroxidase). General guidance
regarding such techniques can be found in, e.g., Bancroft and
Stevens (Theory and Practice of Histological Techniques, Churchill
Livingstone, 1982) and Ausubel et al. (supra).
[0107] In one preferred example, a combined diagnostic method may
be employed that begins with an evaluation of IAP protein
production (for example, by immunological techniques or the protein
truncation test (Hogerrorst et al., Nature Genetics 10:208-212,
1995)) and also includes a nucleic acid-based detection technique
designed to identify more subtle IAP altercations, e.g., mutations.
As described above, a number of mismatch detection assays are
available to those skilled in the art, and any preferred technique
may be used. Mutations in IAP may be detected that either result in
enhanced IAP expression or altercations in IAP biological activity.
In a variation of this combined diagnostic method, IAP biological
activity is measured as anti-apoptotic activity using any
appropriate apoptosis assay system (for example, those described
above).
[0108] Mismatch detection assays also provide an opportunity to
diagnose an IAP-mediated predisposition to diseases caused by
insufficient apoptosis. For example, a patient heterozygous for an
IAP mutation may show no clinical symptoms and yet possess a higher
than normal probability of developing one or more types of
proliferative diseases. Given this diagnosis, a patient may take
precautions to minimize their exposure to adverse environmental
factors (for example, UV exposure or chemical mutagens) and to
carefully monitor their medical condition (for example, through
frequent physical examinations). This type of IAP diagnostic
approach may also be used to detect IAP mutations in prenatal
screens. The IAP diagnostic assays described above may be carried
out using any biological sample (for example, any biopsy sample or
bodily fluid or tissue) in which IAP is normally expressed.
Identification of a mutant IAP gene may also be assayed using these
sources for test samples.
[0109] Alternatively, an altercation in IAP activity, particularly
as part of a diagnosis for predisposition to IAP-associated
proliferative disease, may be tested using a nucleic acid sample
from any cell, for example, by mismatch detection techniques.
Preferably, the DNA sample is subjected to PCR amplification prior
to analysis.
EXAMPLE 1
Elevated IAP Levels in Cancer Cell Lines
[0110] In order to specifically demonstrate the utility of IAP gene
sequences as diagnostics and prognostics for cancer, a Human Cancer
Cell Line Multiple Tissue Northern Blot (Clontech, Palo Alto,
Calif.; #7757-1) was probed. This Northern blot contained
approximately 2 .mu.g of poly A.sup.+ RNA per lane from eight
different human cell lines: (1) promyelocytic leukemia HL-60, (2)
HeLa cell S3, (3) chronic myelogenous leukemia K-562, (4)
lymphoblastic leukemia MOLT-4, (5) Burkitt's lymphoma Raji, (6)
colorectal adenocarcinoma SW480, (7) lung carcinoma A549, and (8)
melanoma G361. As a control, a Human Multiple Tissue Northern Blot
(Clontech, Palo Alto, Calif.; #7759-1) was probed. This Northern
blot contained approximately 2 .mu.g of poly A.sup.+ RNA from eight
different human tissues: (1) spleen, (2) thymus, (3) prostate, (4)
testis, (5) ovary, (6) small intestine, (7) colon, and (8)
peripheral blood leukocytes.
[0111] The Northern blots were hybridized sequentially with: (1) a
1.6 kb probe to the xiap coding region, (2) a 375 bp hiap-2
specific probe-corresponding to the 3' untranslated region, (3) a
1.3 kb probe to the coding region of hiap-1, which cross-reacts
with hiap-2, (4) a 1.0 kb probe derived from the coding region of
bcl-2, and (5) a probe to .beta.-actin, which was provided by the
manufacturer. Hybridization was carried out at 50.degree. C.
overnight, according to the manufacturer's suggestion. The blot was
washed twice with 2.times.SSC, 0.1% SDS at room temperature for 15
minutes and then with 2.times.SSC, 0.1% SDS at 50.degree. C.
[0112] All cancer lines tested showed increased IAP expression
relative to samples from non-cancerous control tissues (Table 1).
Expression of xiap was particularly high in HeLa (S-3), chronic
myelogenous leukemia (K-562), colorectal adenocarcinoma (SW-480),
and melanoma (G-361) lines. Expression of hiap-1 was extremely high
in Burkitt's lymphoma, and was also elevated in colorectal
adenocarcinoma. Expression of hiap-2 was particularly high in
chronic myelogenous leukemia (K-562) and colorectal adenocarcinoma
(SW-480). Expression of Bcl-2 was upregulated only in HL-60
leukemia cells. TABLE-US-00001 TABLE 1 NORTHERN BLOT IAP RNA LEVELS
IN CANCER CELLS* xiap hiap1 hiap2 Promyelocytic Leukemia HL-60 + +
+ HeLa S-3 + + + Chronic Myelogenous Leukemia K-562 +++ + +++
Lymphoblastic Leukemia MOLT-4 +++ + + Burkitt's Lymphoma Raji +
+(.times.10) + Colorectal Adenocarcinoma SW-480 +++ +++ +++ Lung
Carcinoma A-549 + + + Melanoma G-361 +++ + + *Levels are indicated
by a (+) and are the approximate increase in RNA levels relative to
Northern blots of RNA from non-cancerous control cell lines. A
single plus indicates an estimated increase of at least 1-fold
[0113] These observations indicate that upregulation of the
anti-apoptotic IAP genes may be a widespread phenomenon in
proliferative diseases, perhaps occurring much more frequently than
upregulation of Bcl-2. Furthermore, upregulation may be necessary
for the establishment or maintenance of the transformed state of
cancerous cells.
[0114] In order to pursue the observation described above, i.e.,
that hiap-1 is overexpressed in the Raji Burkitt's lymphoma cell
line, RT-PCR analysis was performed in multiple Burkitt's lymphoma
cell lines. Total RNA was extracted from cells of the Raji, Ramos,
EB-3, and Jiyoye cell lines, and as a positive control, from normal
placental tissue. The RNA was reverse transcribed, and amplified by
PCR with the following set of oligonucleotide primers:
5'-AGTGCGGGTTTTTATTATGTG-3' (SEQ ID NO:15) and
5'-AGATGACCACAAGGAATAAACACTA-3' (SEQ ID NO:16), which selectively
amplify a hiap-1 cDNA fragment. RT-PCR was conducted using a Perkin
Elmer 480 Thermocycler to carry out 35 cycles of the following
program: 94.degree. C. for 1 minute, 50.degree. C. for 1.5 minutes,
and 72.degree. C. for a minute. The PCR reaction product was
electrophoresed on an agarose gel and stained with ethidium
bromide. Amplified cDNA fragments of the appropriate size were
clearly visible in all lanes containing Burkitt's lymphoma samples,
but absent in the lanes containing the normal placental tissue
sample, and absent in lanes containing negative control samples,
where template DNA was omitted from the reaction (FIG. 11).
EXAMPLE 2
IAPs in Breast Cancer
[0115] The following data relate to the regulation and role of
HIAPs in cancer cells. FIGS. 18 and 19 show data demonstrating that
HIAP-1 and HIAP-2 are both upregulated in breast cancer cell lines
that contain mutant p53. The lanes contain 20 .mu.g of total RNA
from the following lines: 1. MCF-7(clone 1, wt p53) 2. MCF-7 (clone
2, wt p53) 3. MCF-7 (American Type Culture Collection, wt p53) 4.
MCF-7 (parental line, California, wt p53) 5. MCF-7 (California,
adriamycin resistant variant, mutant p53), 6. MDA MB 231 (ATCC,
mutant p53, codon 280) 7.T47-D (ATCC, mutant p53, codon 194) 7.
ZR-75 (ATCC, wt p53). The amount of RNA loaded on each gel was
controlled for by hybridization with glycerol phosphate
dehydrogenase (GAPDH).
EXAMPLE 3
IAPs in Ovarian Cancer
Overview
[0116] Epithelial ovarian cancer is the leading cause of death from
gynecologic malignancy. Although clinical and histologic prognostic
factors such as tumor grade and surgical stage are well understood,
the biologic process that leads to uncontrolled cellular growth is
less clear. The control of cell numbers during tissue growth is
thought to be the results of a balance of cell proliferation and
cell death. An aberration in this natural homeostasis likely
contributes to malignant cellular transformation.
[0117] Recent studies on ovarian cancer cell biology have suggested
that the deregulation of apoptosis may be one of the underlying
pathologic mechanism in this disease. However, the molecular
mechanisms involved in its regulation is poorly understood and the
role and regulation of the IAP genes in ovarian cell transformation
have not been examined previously. Ovarian epithelial cancer is in
part a result of suppressed apoptosis of ovarian surface epithelial
cells. The effectiveness of certain chemotherapeutic agents rests
on their ability to induce cell death. The loss of responsiveness
of the cells to these agents is due to a desensitization of the
apoptotic process to these agents. The regulation of ovarian
epithelial cell apoptosis involves changes in the expression of IAP
genes and post-translational modification/processing of the IAP
gene products.
[0118] We have conducted experiments and now believe that IAPs play
a key role in maintaining the normal growth of ovarian surface
epithelial cells and that the overexpression of these genes leads
to cellular transformation. Furthermore, we have discovered that
the effectiveness of chemotherapeutic agents in the treatment of
this form of malignancy rests upon their ability to suppress the
expression of the IAP genes. By seeking to control the regulation
of the IAP genes in human ovarian epithelial cancer cells we have
provided a rational approach for the development of new
chemotherapeutics for patients both responsive and resistant to
current cancer drugs. Similarly, assays designed to detect
compounds which decrease IAP biological activity provide a rational
method for drug discovery.
Methods
A) Human Ovarian Epithelial Cancer Cell Culture
[0119] Cisplatin-sensitive (OV2008) and resistant (C13) human
ovarian epithelial cells were cultured in a chemically-defined
medium at 37.degree. C. for up to 48 hours in the presence or
absence of TGF.beta. (20 ng/ml), taxol (0-1.0 .mu.M) or cisplatin
(0-30 .mu.M). At the end of the culture period, cells were either
fixed for immunocytochemistry and TUNEL analyses, or snap frozen
for subsequent extraction for IAP mRNA and proteins analyses.
B) Identification of Cell Death
Nuclear Staining:
[0120] Human ovarian epithelial cancer cells were fixed (4%
formalin in PBS; 10 min. RT), washed in PBS, resuspended in Hoescht
33248 stain (0.1 .mu.g/ml PBS, 10 min) washed again and spotted
onto slides for microscopy. Nuclear staining was observed and
photographed using a Zeiss fluorescent microscope equipped with an
FITC filter. Apoptotic cells were identified by typical nuclear
morphology, and counted using randomly selected fields and numbered
photographic slides to avoid bias during counting.
Quantification of DNA Ladders:
[0121] Cellular DNA was extracted using the Qiagen Blood Amt kit.
DNA was quantified by ethidium bromide fluorescence. DNA (0.5
.mu.g) was then end labelled by incubating (20 min, RT) with Klenow
enzyme (2 U in 10 mM Tris+5 mM MgCl.sub.2) and 0.1 .mu.Ci
[.alpha.32P]dCTP. Unincorporated nucleotides were removed with the
Qiagen nucleotide removal kit and samples were resolved by
Tris-acetate-EDTA agarose (1.8%) gel electrophoresis. The gel was
then dried (2 hr, no heat) and exposed to a Bio-Rad phosphoimager
screen to densitometrically quantify low molecular weight DNA
(<15 kBp) and subsequently to x-ray film at -80.degree. C.
In Situ TUNEL Labelling of Apoptotic Cells:
[0122] To identify cell death using the in situ cell death
detection kit (Boehringer-Mannheim), slides prepared for histology
were treated (20 min. 37.degree. C.) with terminal tranferase in
the presence of FITC-conjugated dUTP.
C) Western Blot Analyses for IAPs
[0123] Protein extracts were prepared from human surface epithelial
cancer cells sonicated (8 s/cycle, 3 cycles) on ice in sucrose
buffer (0.25 M sucrose, 0.025 M NaCl, 1 mM EGTA and 15 mM Tris-HCl
pH 6.8, supplemented with 1 mM PMSF, 2 .mu.g/ml of leupeptin and 5
.mu.g/ml of aprotinin. The sonicates were centrifuged at
13,000.times.g for 10 min, the supernatants were collected and
stored at -20.degree. C. until electrophoretic analyses were
performed. Protein concentration was determined by Bio-Rad Protein
Assay. Proteins (10-30 .mu.g) were resolved by one-dimensional
SDS-PAGE, and electrophoretically transferred to nitrocellulose
membrane. Membranes were blocked with 5% non-fat milk, and
subsequently incubated with rabbit polyclonal antibody for IAP
[anti-human Hiap-2.DELTA.E(960529; 1:1000 dilution), anti-human
NAIP E1.0 (951015; 1:1000 dilution) or anti-human Xiap (1:1000
dilution) diluted in TBST (10 mM Tris-buffered saline, 0.1%
Tween-20, pH 7.5) containing 5% milk. An ECL kit was used to
visualize immunopositive protein.
D) Northern Blots for IAP mRNAs
[0124] Total RNA from ovarian surface epithelial cancer cells by
using RNeasy Kit (Qiagen). The RNA samples (10-15 .mu.g) were
quantified spectrophotometrically and size-fractioned by
electrophoresis on formaldehyde-agarose gels (1.1%) containing 1
.mu.g/ml ethidium bromide to confirm even loading of RNA samples
and adequate separation of 28S and 18S ribosomal bands. The RNAs
bands were blotted onto a nylon membrane and cross-linked by UV
light. Membranes were prehybridized in 50% formamide, saline sodium
citrate (SSC; 750 mM NaCl, 75 mM Na citrate), 1.times. Denhardt's
solution, 1% SDS, 4 mM EDTA and 100 .mu.g/ml sheared salmon sperm
DNA for 4 h at 42.degree. C. Hybridization was performed overnight
at 42.degree. C. with 20 million cpm of .sup.32P-labelled IAP cDNA
probes (rat Naip, rat Xiap or human Hiap-2) added to the
prehybridization buffer. The membranes were then washed twice with
SSC (300 mM NaCl, 30 mM Na citrate) in 0.1% SDS for 20 min at room
temperature and twice with SSC (30 mM NaCl, 3 mM sodium citrate) in
0.1% SDS for 20 min at 55.degree. C. and exposed to X-ray film at
-80.degree. C. for visualization. Densitometric analysis of various
IAPs and 28S rRNA band was performed with the Image Analysis
Systems from Bio-Rad Laboratories. Data were normalized by the
respective 28S and expressed as a percentage of the control
(defined as 100%).
Results
[0125] Cisplatin induced a concentration-dependent increase in the
incidence of apoptosis in cisplatin-sensitive (OV2008) but to a
lesser extent in--resistant (C13) human ovarian epithelial cells in
vitro (FIG. 20). Similarly, Taxol also induced apoptosis in OV2008
cells, but to a lesser extent in the C13 cells (FIG. 21).
[0126] Basal XIAP and HIAP-2 protein contents were markedly higher
in cisplatin-sensitive than resistant cells. Taxol (0.04-1.0 .mu.M)
decreased XIAP and HIAP-2 protein levels in a
concentration-dependent manner, the response being more pronounced
in sensitive than resistant cells
[0127] (FIG. 22). A lower molecular weight (approx. 45 kDa)
immunoreactive fragment of HIAP-2 was also evident in both the
sensitive and resistant cells. The content of this fragment was
increased in the C13 cells but decreased in OV2008 cells by Taxol
(FIG. 22).
[0128] Whereas Taxol (0.2 .mu.M) marked suppressed HIAP-2 mRNA
abundance in cisplatin-sensitive cells (approx. 80%), it was
ineffective in the resistant cells (FIG. 23).
[0129] TGF.beta. (20 ng/ml) induced apoptosis in OV2008 but not in
C13. Although its influence on Xiap protein content in
cisplatin-resistant cells was only marginal, it markedly suppressed
the protein level of this IAP in the cisplatin-sensitive cells
(FIG. 24A, 24B). TGF.beta. (20 ng/ml) also decreased HIAP-2 mRNA in
OV2008 but not C13 cells (FIG. 23).
Significant Observations and Possible Applications
[0130] Induction of apoptosis in human ovarian epithelial cancer
cell by taxol was accompanied by suppressed IAP gene expression.
The lost of sensitivity of the cells to the chemotherapeutic agent
may be associated with its decreased ability to express these genes
and to induce apoptosis. In drug-resistant cells, the decreased
Hiap-2 protein content (in the face of an absence of noticeable
change in Hiap-2 mRNA abundance) in the presence of Taxol was
accompanied an increase in the intensity of a 45 kDa immunoreactive
HIAP-2 protein band. These observations lead us to believe that the
45 kDa protein is a proteolytic product of HIAP-2 and plays a role
in the development of drug resistance. In addition, the sensitivity
of the IAP family in these ovarian cancer cells to Taxol suggest
possible novel sites for gene targeting in the development of new
chemotherapeutic agents for the treatment of human ovarian
epithelial cell cancer.
EXAMPLE 4
Accumulation of a 26 kDa Cleavage Protein in Astrocytoma Cells
Identification of a 26kDa Cleavage Protein
[0131] A total protein extract was prepared from Jurkat and
astrocytoma cells by sonicating them (X3 for 15 seconds at
4.degree. C.) in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 1
.mu.g/ml aprotinin, and 5 mM benzamidine. Following sonication, the
samples were centrifuged (14,000 RPM in a microfuge) for five
minutes. Twenty .mu.g of protein was loaded per well on a 10%
SDS-polyacrylamide gel, electrophoresed, and electroblotted by
standard methods to PVDF membranes. Western blot analysis,
performed as described previously, revealed that the astrocytoma
cell line (CCF-STTG1) abundantly expressed an anti-xiap reactive
band of approximately 26 kDa, despite the lack of an apoptotic
trigger event (FIG. 12). In fact, this cell line has been
previously characterized as being particularly resistant to
standard apoptotic triggers.
[0132] A 26 kDa xiap-reactive band was also observed under the
following experimental conditions. Jurkat cells (a transformed
human T cell line) were induced to undergo apoptosis by exposure to
an anti-Fas antibody (1 .mu.g/ml). Identical cultures of Jurkat
cells were exposed either to: (1) anti-Fas antibody and
cycloheximide (20 .mu.g/ml), (2) tumor necrosis factor alpha
(TNF-.alpha., at 1,000 U/ml), or (3) TNF-.alpha. and cycloheximide
(20 .mu.g/ml). All cells were harvested 6 hours after treatment
began. In addition, as a negative control, anti-Fas antibody was
added to an extract after the cells were harvested. The cells were
harvested in SDS sample buffer, electrophoresed on a 12.5% SDS
polyacrylamide gel, and electroblotted onto PVDF membranes using
standard methods. The membranes were immunostained with a rabbit
polyclonal anti-xiap antibody at 1:1000 for 1 hour at room
temperature. Following four 15 minute washes, a goat anti-rabbit
antibody conjugated to horse-radish peroxidase was applied at room
temperature for 1 hour. Unbound secondary antibody was washed away,
and chemiluminescent detection of xiap protein was performed. The
Western blot revealed the presence of the full-length, 55 kDa xiap
protein, both in untreated and treated cells. In addition, a novel,
approximately 26 kDa xiap-reactive band was also observed in
apoptotic cell extracts, but not in the control, untreated cell
extracts (FIG. 13).
[0133] Cleavage of xiap occurs in a variety of cell types,
including other cancer cell lines such as HeLa. The expression of
the 26 kDa xiap cleavage product was demonstrated in HeLa cells as
follows. HeLa cells were treated with either: (1) cyclohexamide (20
.mu.g/ml), (2) anti-Fas antibody (1 .mu.g/ml), (3) anti-Fas
antibody (1 .mu.g/ml) and cyclohexamide (20 .mu.g/ml), (4)
TNF.alpha. (1,000 U/ml), or (5) TNF.alpha. (1,000 U/ml) and
cyclohexamide (20 .mu.g/ml). All cells were harvested 18 hours
after treatment began. As above, anti-Fas antibody was added to an
extract after the cells were harvested. HeLa cells were harvested,
and the Western blot was probed under the same conditions as used
to visualize xiap-reactive bands from Jurkat cell samples. A 26 kDa
xiap band was again seen in the apoptotic cell preparations (FIG.
14). Furthermore, the degree of xiap cleavage correlated positively
with cellular exposure to apoptotic triggers. Treatment of HeLa
cells with cycloheximide or TNF.alpha. alone caused only minor
apoptosis, and little cleavage product was observed. If the cells
were treated with the anti-Fas antibody, a greater amount of
cleavage product was apparent. These data indicate that xiap is
cleaved in more than one cell type and in response to more than one
type of apoptotic trigger.
Time Course of Expression
[0134] The time course over which the 26 kDa cleavage product
accumulates was examined by treating HeLa and Jurkat cells with
anti-Fas antibody (1 .mu.g/ml) and harvesting them either
immediately, or 1, 2, 3, 5, 10, or 22 hours after treatment.
Protein extracts were prepared and Western blot analysis was
performed as described above. Both types of cells accumulated
increasing quantities of the 26 kDa cleavage product over the time
course examined (FIGS. 15A and 15B).
Subcellular Localization of the 26 kDa xiap Cleavage Product
[0135] In order to determine the subcellular location of the 26 kDa
cleavage product, Jurkat cells were induced to undergo apoptosis by
exposure to anti-Fas antibody (1 .mu.g/ml) and were then harvested
either immediately, 3 hours, or 7 hours later. Total protein
extracts were prepared, as described above, from cells harvested at
each time point. In order to prepare nuclear and cytoplasmic cell
extracts, apoptotic Jurkat cells were washed with isotonic Tris
buffered saline (pH 7.0) and lysed by freezing and thawing five
times in cell extraction buffer (50 mM PIPES, 50 mM KCl, 5 mM EGTA,
2 mM MgCl.sub.2, 1 mM DTT, and 20 .mu.M cytochalasin B). Nuclei
were pelleted by centrifugation and resuspended in isotonic Tris
(pH 7.0) and frozen at -80.degree. C. The cytoplasmic fraction of
the extract was processed further by centrifugation at 60,000 RPM
in a TA 100.3 rotor for 30 minutes. Supernatants were removed and
frozen at -80.degree. C. Samples of both nuclear and cytoplasmic
fractions were loaded on a 12.5% SDS-polyacrylamide gel, and
electroblotted onto PVDF membranes. Western blot analysis was then
performed using either an anti-CPP32 antibody (Transduction
Laboratories Lexington, Ky.; FIG. 16A) or the rabbit anti-XIAP
antibody described above (FIG. 16B).
[0136] The anti-CPP32 antibody, which recognizes the CPP32 protease
(also known as YAMA or Apopain) partitioned almost exclusively in
the cytoplasmic fraction. The 55 kDa XIAP protein localized
exclusively in the cytoplasm of apoptotic cells, in agreement with
the studies presented above, where XIAP protein in normal, healthy
COS cells was seen to localize, by immunofluoresence microscopy, to
the cytoplasm. In contrast, the 26 kDa cleavage product localized
exclusively to the nuclear fraction of apoptotic Jurkat cells.
Taken together, these observations suggest that the anti-apoptotic
component of XIAP could be the 26 kDa cleavage product, which
exerts its influence within the nucleus.
In Vitro Cleavage of xiap Protein and Characterization of the
Cleavage Product
[0137] For this series of experiments, xiap protein was labeled
with .sup.35S using the plasmid pcDNA3-6myc-XIAP, T7 RNA
polymerase, and a coupled transcription/translation kit (Promega)
according to the manufacturer's instructions. Radioactively labeled
xiap protein was separated from unincorporated methionine by column
chromatography using Sephadex G-50.TM.. In addition, extracts of
apoptotic Jurkat cells were prepared following treatment with
anti-Fas antibody (1 .mu.g/ml) for three hours. To prepare the
extracts, the cells were lysed in Triton X-100 buffer (1% Triton
X-100, 25 mM Tris HCl) on ice for two hours and then
microcentrifuged for 5 minutes. The soluble extract was retained
(and was labeled TX100). Cells were lysed in cell extraction buffer
with freeze/thawing. The soluble cytoplasmic fraction was set aside
(and labeled CEB). Nuclear pellets from the preparation of the CEB
cytoplasmic fraction were solubilized with Triton X-100 buffer,
microcentrifuged, and the soluble fractions, which contains
primarily nuclear DNA, was retained (and labeled CEB-TX100).
Soluble cell extract was prepared by lysing cells with NP-40
buffer, followed by microcentrifugation for 5 minutes (and was
labeled NP-40). In vitro cleavage was performed by incubating 16
.mu.l of each extract (CEB, TX-100, CEB-TX 100, and NP-40) with 4
.mu.l of in vitro translated XIAP protein at 37.degree. C. for 7
hours. Negative controls, containing only TX100 buffer or CEB
buffer were also included. The proteins were separated on a 10%
SDS-polyacrylamide gel, which was dried and exposed to X-ray film
overnight.
[0138] In vitro cleavage of XIAP was apparent in the CEB extract.
The observed molecular weight of the cleavage product was
approximately 36 kDa (FIG. 17). The 10 kDa shift in the size of the
cleavage product indicates that the observed product is derived
from the amino-terminus of the recombinant protein, which contains
six copies of the myc epitope (10 kDa). It thus appears that the
cleavage product possesses at least two of the BIR domains, and
that it is localized to the nucleus.
EXAMPLE 5
Characterization of IAP Activity and Intracellular Localization
Studies
[0139] The ability of IAPs to modulate apoptosis can be defined in
vitro systems in which alterations of apoptosis can be detected.
Mammalian expression constructs carrying IAP cDNAs, which are
either full-length truncated, or antisense constructs can be
introduced into cell lines such as CHO, NIH 3T3, HL60, Rat-1, or
Jurkat cells. In addition, SF21 insect cells may be used, in which
case the IAP gene is preferentially expressed using an insect heat
shock promoter. Following transfection, apoptosis can be induced by
standard methods, which include serum withdrawal, or application of
staurosporine, menadione (which induces apoptosis via free radial
formation), or anti-Fas antibodies. As a control, cells are
cultured under the same conditions as those induced to undergo
apoptosis, but either not transfected, or transfected with a vector
that lacks an IAP insert. The ability of each IAP related construct
to inhibit or enhance apoptosis upon expression can be quantified
by calculating the survival index of the cells, i.e., the ratio of
surviving transfected cells to surviving control cells. These
experiments can confirm the presence of apoptosis inhibiting
activity and, as discussed below, can also be used to determine the
functional region(s) of an IAP which may be employed to achieve
enhancement of apoptosis. These assays may also be performed in
combination with the application of additional compounds in order
to identify compounds that enhance apoptosis via IAP
expression.
EXAMPLES 6
Cell Survival Following Transfection with IAP Constructs and
Induction of Apoptosis
[0140] Specific examples of the results obtained by performing
various apoptosis suppression assays are shown in FIGS. 10A to 10D.
For example, CHO cell survival following transfection with one of
six constructs and subsequent serum withdrawal is shown in FIG.
10A. The cells were transfected using Lipofectace.TM. with 2 .mu.g
of one of the following recombinant plasmids: pCDNA3-6myc-xiap
(xiap), pCDNA3-6myc-hiap-1 (hiap-1), pCDNA3-6myc-hiap-2 (hiap-2),
pCDNA3-bcl-2 (bcl-2), pCDNA3-HA-smn (smn), and pCDNA3-6myc (6myc).
Oligonucleotide primers were synthesized to allow PCR amplification
and cloning of the xiap, hiap-1, and hiap-2 ORFs in pCDNA3
(Invitrogen). Each construct was modified to incorporate a
synthetic myc tag encoding six repeats of the peptide sequence
MEQKLISEEDL (SEQ ID NO:17), thus allowing detection of myc-IAP
fusion proteins via monoclonal anti-myc antiserum (Egan et al.,
Nature 363:45-51, 1993). Triplicate samples of cell lines in
24-well dishes were washed 5 times with serum free media and
maintained in serum free conditions during the course of the
experiment. Cells that excluded trypan blue, and that were
therefore viable, were counted with a hemocytometer immediately, 24
hours, 48 hours, and 72 hours, after serum withdrawal. Survival was
calculated as a percentage of the initial number of viable cells.
In this experiment and those presented in FIGS. 10B and 10D, the
percentage of viable cells shown represents the average of three
separate experiments performed in triplicate, +/- average
deviation.
[0141] The survival of CHO cells following transfection (with each
one of the six constructs described above) and exposure to
menadione is shown in FIG. 10B. The cells were plated in 24-well
dishes, allowed to grow overnight, and then exposed to 20 .mu.M
menadione for 1.5 hours (Sigma Chemical Co., St. Louis, Mo.).
Triplicate samples were harvested at the time of exposure to
menadione and 24 hours afterward, and survival was assessed by
trypan blue exclusion.
[0142] The survival of Rat-1 cells following transfection (with
each one of the six constructs described above) and exposure to
staurosporine is shown in FIG. 10C. Rat-1 cells were transfected
and then selected in medium containing 800 .mu.g/ml G418 for two
weeks. The cell line was assessed for resistance to
staurosporine-induced apoptosis (1 .mu.M) for 5 hours. Viable cells
were counted 24 hours after exposure to staurosporine by trypan
blue exclusion. The percentage of viable cells shown represents the
average of two experiments, .+-.average deviation.
[0143] The Rat-1 cell line was also used to test the resistance of
these cells to menadione (FIG. 10D) following transfection with
each of the six constructs described above. The cells were exposed
to 10 .mu.M menadione for 1.5 hours, and the NUMBER of viable cells
was counted 18 hours later.
EXAMPLE 7
Comparison of Cell Survival Following Transfection with Full-length
vs. Partial IAP Constructs
[0144] In order to investigate the mechanism whereby human IAPs,
including XIAP, HIAP-1, and HIAP-2, afford protection against cell
death, expression vectors were constructed that contained either:
(1) full-length IAP cDNA (as described above), (2) a portion of an
IAP gene that encodes the BIR domains, but not the RZF, or (3) a
portion of an IAP gene that encodes the RZF, but not the BIR
domains. Human and murine xiap cDNAs were tested by transient or
stable expression in HeLa, Jurkat, and CHO cell lines. Following
transfection, apoptosis was induced by serum withdrawal,
application of menadione, or application of an anti-Fas antibody.
Cell death was then assessed, as described above, by trypan blue
exclusion. As a control for transfection efficiency, the cells were
co-transfected with a .beta.-gal expression construct. Typically,
approximately 20% of the cells were successfully transfected.
[0145] When CHO cells were transiently transfected, constructs
containing full-length human or mouse xiap cDNAs conferred modest
but definite protection against cell death. In contrast, the
survival of CHO cells transfected with constructs encoding only the
BIR domains (i.e., lacking the RZF domain) was markedly enhanced 72
hours after serum deprivation. Furthermore, a large percentage of
cells expressing the BIR domains were still viable after 96 hours,
at which time no viable cells remained in the control, i.e.,
non-transfected, cell cultures, and less than 5% of the cells
transfected with the vector only, i.e., lacking a cDNA insert,
remained viable. Deletion of any of the BIR domains results in the
complete loss of apoptotic suppression, which is reflected by a
decrease in the percentage of surviving CHO cells to control levels
within 72 hours of serum withdrawal.
[0146] Stable pools of transfected CHO cells, which were maintained
for several months under G418 selection, were induced to undergo
apoptosis by exposure to 10 .mu.M menadione for 2 hours. Among the
CHO cells tested were those that were stably transfected with: (1)
full-length murine xiap cDNA (miap), (2) full-length xiap cDNA
(xiap), (3) full-length bcl-2 cDNA (Bcl-2), (4) cDNA encoding the
three BIR domains (but not the RZF) of murine xiap (BIR), and (5)
cDNA encoding the RZF (but not BIR domains) of m-xiap (RZF). Cells
that were non-transfected (CHO) or transfected with the vector only
(pcDNA3), served as controls for this experiment. Following
exposure to 10 .mu.M menadione, the transfected cells were washed
with phosphate buffered saline (PBS) and cultured for an additional
24 hours in menadione-free medium. Cell death was assessed, as
described above, by trypan blue exclusion. Less than 10% of the
non-transfected or vector-only transfected cells remained viable at
the end of the 24 hour survival period. Cells expressing the RZF
did not fare significantly better. However, expression of
full-length murine xiap, human xiap, or bcl-2, and expression of
the BIR domains, enhanced cell survival. When the concentration of
menadione was increased from 10 .mu.M to 20 .mu.M (with all other
conditions of the experiment being the same as when 10 .mu.M
menadione was applied), the percentage of viable CHO cells that
expressed the BIR domain cDNA construct was higher than the
percentage of viable cells that expressed either full-length murine
xiap or bcl-2.
EXAMPLE 8
Analysis of the Subcellular Location of Expressed RZF and BIR
Domains
[0147] The assays of cell death described above indicate that the
RZF acts as a negative regulator of the anti-apoptotic function of
IAPs. One way in which the RZF, and possibly other IAP domains, may
exert their regulatory influence is by altering the expression of
genes, whose products function in the apoptotic pathway.
[0148] In order to determine whether the subcellular locations of
expressed RZF and BIR domains are consistent with roles as nuclear
regulatory factors, COS cells were transiently transfected with the
following four constructs, and the expressed polypeptide was
localized by immunofluorescent microscopy: (1) pcDNA3-6myc-xiap,
which encodes all 497 amino acids of SEQ ID NO:4, (2)
pcDNA3-6myc-m-xiap, which encodes all 497 amino acids of mouse xiap
(SEQ ID NO:10), (3) pcDNA3-6myc-mxiap-BIR, which encodes amino
acids 1 to 341 of m-xiap (SEQ ID NO:10), and (4)
pcDNA3-6myc-mxiap-RZF, which encodes amino acids 342-497 of murine
xiap (SEQ ID NO:10). The cells were grown on multi-well tissue
culture slides for 12 hours, and then fixed and permeabilized with
methanol. The constructs used (here and in the cell death assays)
were tagged with a human Myc epitope tag at the N-terminus.
Therefore, a monoclonal anti-Myc antibody and a secondary goat
anti-mouse antibody, which was conjugated to FITC, could be used to
localize the expressed products in transiently transfected COS
cells. Full-length XIAP and MIAP were located in the cytoplasm,
with accentuated expression in the peri-nuclear zone. The same
pattern of localization was observed when the cells expressed a
construct encoding the RZF domain (but not the BIR domains).
However, cells expressing the BIR domains (without the RZF)
exhibited, primarily, nuclear staining. The protein expressed by
the BIR domain construct appeared to be in various stages of
transfer to the nucleus.
[0149] These observations are consistent with the fact that, as
described below, XIAP is cleaved within T cells that are treated
with anti-Fas antibodies (which are potent inducers of apoptosis),
and its N-terminal domain is translocated to the nucleus. As noted
in Example 2 Hiap-2 appears to undergo a similar cleavage
event.
EXAMPLE 9
Testing of Antisense Oligonucleotides
[0150] 1. Complete panel of adenovirus constructs. The panel may
consist of approximately four types of recombinant virus. A) Sense
orientation viruses for each of the IAP open reading frames. These
viruses are designed to massively overexpress the recombinant
protein in infected cells. XIAP, HIAP-1, HIAP-2, and NAIP. B)
Antisense orientation viruses in which the viral promoter drives
the synthesis of an mRNA of opposite polarity to the iap mRNA,
thereby shutting off host cell synthesis of the targeted protein
coding region. XIAP, HIAP-1, HIAP-2, and NAIP "antisense"
constructs required. C) Sub-domain expression viruses. These
constructs express only a partial IAP protein in infected cells. We
have data indicating that deletion of the zinc finger of XIAP
renders the protein more potent in protecting cell against
apoptotic triggers. This data also indicates that expression of the
zinc finger alone will indicate apoptosis by functioning as a
dominant-negative repressor of XIAP function. XIAP-.DELTA.ZF and
XIAP-.DELTA.bir viruses required. D) Control viruses. Functional
analysis of the IAPs requires suitable positive and negative
controls for comparison. Bcl-2 sense, Bcl-2 antisense, p53 sense,
and Lac Z (negative control) viruses may be utilized.
[0151] 2. Confirmation of recombinant adenovirus function.
Verification of the sense adenovirus function involves infection of
tissue culture cells and determination of protein expression
levels. We have performed western blot analysis of several of the
recombinant adenoviruses, including NAIP, XIAP and XIAP-.DELTA.ZF.
The remaining viruses may be ready readily assessed for protein
expression using the polyclonal IAP antibodies. Functional analysis
of the antisense viruses may be done at the RNA level using either
northern blots of total RNA harvested from infected tissue culture
cells or ribonuclease protection assays. Western blot analysis of
infected cells will be used to determine whether the expressed
antisense oligonucleotide interferes with IAP expression in the
host cell.
[0152] 3. Documentation that IAP overexpression results in
increased drug resistance. We have optimized cell death assays to
allow high through-put of samples with minimal sample variation.
Testing of the sense IAP adenoviruses for their ability to alter
drug sensitivity of breast and pancreatic adenocarcinoma cell lines
may be accomplished as follows. Cancer cell lines are infected with
the recombinant viruses, cultured for 5 days, then subdivided into
24 well plates. Triplicate cell receive increasing concentrations
of the anti-cancer drug under investigation. Samples are harvested
at 24, 48, and 72 hours post exposure, and assayed for the number
of viable cells in the well. The dose response curve is then
compared to uninfected and control virus (both positive and
negative) infected cells. One may document a dramatic increase in
the relative resistance of the cancer cell lines when infected with
the sense viruses, confirming our hypothesis that overexpression of
the IAP proteins contributes to the anti-apoptotic phenotype of
cancer cells. Initial experiments utilize the drugs doxorubicin,
and adriamycin.
[0153] 4. Documentation that antisense IAP overexpression results
in increased drug sensitivity. Having confirmed that IAP
overexpression renders cancer cell more resistant to
chemotherapeutic drugs, one may examine whether the antisense
adenoviruses render the same cells more sensitive. The
effectiveness of antisense IAP viruses relative to antisense. Bcl-2
virus will also be assessed as a crucial milestone.
[0154] 5. Identification of antisense oligonucleotides. Concomitant
to the adenovirus work, we have designed a series of antisense
oligonucleotides to various regions of each of the iaps. A
generally accepted model of how antisense oligonucleotides function
proposes that the formation of RNA/DNA duplexes in the nucleus
activates cellular RnaseH enzymes which then enzymatically degrade
the mRNA component of the hybrid. Virtually any region of the mRNA
can be targeted, and therefore choosing an appropriate sequence to
target is somewhat empirical. Many factors, including secondary
structure of the target mRNA and the binding affinity of the
targeted sequence determine whether a particular oligonucleotide
will be effective, necessitating several oligos for each iap. Five
oligonucleotides have been made for each iap mRNA based on the
available computer algorhythms for predicting binding affinities
and mRNA secondary structures. These and other oligos may be tested
for their ability to target their respective mRNAs for degradation
using northern blot analysis.
[0155] 6. Optimization of oligonucleotides. A secondary round of
oligonucleotides may be made when effective target regions have
been identified. These oligonucleotides target sequences in the
immediate vicinity of the most active antisense oligonucleotides
identified using methods such as those provided above. A second
round of testing by northern blot analysis may be required.
[0156] 7. Testing antisense oligonucleotides in vitro. Following
successful identification and optimization of targeting
oligonucleotides, one may test these in the tissue culture model
system using the optimal cell lines such as those described in the
cancer survey described herein. Experimental procedures may
parallel those used in the recombinant antisense adenovirus work.
Negative control oligonucleotides with miss-match sequences are
used to establish base line or non-specific effects. Assisted
transfection of the oligonucleotides using cationic lipid carriers
may be compared to unassisted transfection. Confirmation of the
effectiveness of specific antisense oligonucleotides prompts
synthesis of oligos with modified phosphodiester linkages, such as
phosphorothioate or methylimino substituted oligos. These may also
be tested in vitro.
8. Animal modeling of antisense oligonucleotide therapies.
[0157] Animal modeling of the effectiveness of the antisense IAP
approach is described here. Cell lines are routinely assessed for
their tumorigenic potential in "nude" mice, a hairless strain of
mouse that is immunocompromised, and thus extremely susceptible to
developing tumors. In the nude mouse assay, cancer cells are grown
in tissue culture and then injected under the skin at multiple
sites. The frequency with which these cells give rise to palpable
tumors within a defined period of time provides an index of the
tumorigenic potential of the cell line in the absence of
interference by a functional immune system. Preliminary assessment
of an antisense IAP therapeutic involves injection of cancer cells
infected with the recombinant adenoviruses (sense, antisense, and
control viruses) under the skin, and the tumorigenic index compared
to that of untreated cells. One may also use this model to assess
the effectiveness of systemic administration of antisense
oligonucleotides in increasing the efficacy of anti-cancer drugs in
the nude mouse model. Phosphorothioate or methylimino substituted
oligos will be assessed at this stage. This type of antisense oligo
has demonstrated enhanced cell permeability and slower clearance
rates from the body in experimental animal models.
EXAMPLE 10
Additional Apoptosis Assays
[0158] Specific examples of apoptosis assays are also provided in
the following references. Assays for apoptosis in lymphocytes are
disclosed by: Li et al., "Induction of apoptosis in uninfected
lymphocytes by HIV-1 Tat protein", Science 268:429-431, 1995;
Gibellini et al., "Tat-expressing Jurkat cells show an increased
resistance to different apoptotic stimuli, including acute human
immunodeficiency virus-type 1 (HIV-1) infection", Br. J. Haematol.
89:24-33, 1995; Martin et al., "HIV-1 infection of human CD4+ T
cells in vitro. Differential induction of apoptosis in these
cells." J. Immunol. 152:330-42, 1994; Terai et al., "Apoptosis as a
mechanism of cell death in cultured T lymphoblasts acutely infected
with HIV-1", J. Clin. Invest. 87:1710-5, 1991; Dhein et al.,
"Autocrine T-cell suicide mediated by APO-1/(Fas/CD95)11, Nature
373:438-441, 1995; Katsikis et al., "Fas antigen stimulation
induces marked apoptosis of T lymphocytes in human immunodeficiency
virus-infected individuals", J. Exp. Med. 1815:2029-2036, 1995;
Westendorp et al., Sensitization of T cells to CD95-mediated
apoptosis by HIV-1 Tat and gp12O", Nature 375:497, 1995; DeRossi et
al., Virology 198:234-44, 1994.
[0159] Assays for apoptosis in fibroblasts are disclosed by:
Vossbeck et al., "Direct transforming activity of TGF-beta on rat
fibroblasts", Int. J. Cancer 61:92-97, 1995; Goruppi et al.,
"Dissection of c-myc domains involved in S phase induction of
NIH3T3 fibroblasts", Oncogene 9:1537-44, 1994; Fernandez et al.,
"Differential sensitivity of normal and Ha-ras transformed C3H
mouse embryo fibroblasts to tumor necrosis factor: induction of
bcl-2, c-myc, and manganese superoxide dismutase in resistant
cells", Oncogene 9:2009-17, 1994; Harrington et al., "c-Myc-induced
apoptosis in fibroblasts is inhibited by specific cytokines", EMBO
J., 13:3286-3295, 1994; Itoh et al., "A novel protein domain
required for apoptosis. Mutational analysis of human Fas antigen",
J. Biol. Chem. 268:10932-7, 1993.
[0160] Assays for apoptosis in neuronal cells are disclosed by:
Melino et al., "Tissue transglutaminase and apoptosis: sense and
antisense transfection studies with human neuroblastoma cells",
Mol. Cell Biol. 14:6584-6596, 1994; Rosenbaum et al., "Evidence for
hypoxia-induced, programmed cell death of cultured neurons", Ann.
Neurol. 36:864-870, 1994; Sato et al., "Neuronal differentiation of
PC12 cells as a result of prevention of cell death by bcl-2", J.
Neurobiol. 25:1227-1234, 1994; Ferrari et al., "N-acetylcysteine D-
and L-stereoisomers prevents apoptotic death of neuronal cells", J.
Neurosci. 1516:2857-2866, 1995; Talley et al., "Tumor necrosis
factor alpha-induced apoptosis in human neuronal cells: protection
by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA",
Mol. Cell Biol. 1585:2359-2366, 1995; Talley et al., "Tumor
Necrosis Factor Alpha-Induced Apoptosis in Human Neuronal Cells:
Protection by the Antioxidant NAcetylcysteine and the Genes bcl-2
and crma", Mol. Cell. Biol. 15:2359-2366, 1995; Walkinshaw et al.,
"Induction of apoptosis in catecholaminergic PC12 cells by L-DOPA.
Implications for the treatment of Parkinson's disease.", J. Clin.
Invest. 95:2458-2464, 1995.
[0161] Assays for apoptosis in insect cells are disclosed by: Clem
et al., "Prevention of apoptosis by a baculovirus gene during
infection of insect cells", Science 254:1388-90, 1991; Crook et
al., "An apoptosis-inhibiting baculovirus gene with a zinc
finger-like motif", J. Virol. 67:2168-74, 1993; Rabizadeh et al.,
"Expression of the baculovirus p35 gene inhibits mammalian neural
cell death", J. Neurochem. 61:2318-21, 1993; Birnbaum et al., "An
apoptosis inhibiting gene from a nuclear polyhedrosis virus
encoding a polypeptide with Cys/His sequence motifs", J. Virol.
68:2521-8, 1994; Clem et al., "Control of programmed cell death by
the baculovirus genes p35 and IAP", Mol. Cell. Biol. 14:5212-5222,
1994.
EXAMPLE 11
Construction of a Transgenic Animal
[0162] Characterization of IAP genes provided information that
necessary for generation IAP transgenic animal models to be
developed by homologous recombination (for knockouts) or
transfection (for expression of IAP fragments, antisense IAP
oligonucleotides, or increased expression of wild-type or mutant
IAPs). Such models may be mammalian animal, e.g., a mouse. Such
models are useful for the identification of cancer therapeutics
alone or in combination with cancer inducing cells or agents, or
when such mice are crossed with mice genetically predisposed to
cancers.
[0163] The preferred transgenic animal overexpression in IAP and
has a predisposition to cancer. This mouse is particularly useful
for the screening of potential cancer therapeutics.
EXAMPLE 12
IAP Protein Expression
[0164] IAP genes and fragments thereof (i.e., RZF fragments) may be
expressed in both prokaryotic and eukaryotic cell types. If an IAP
fragment modulates apoptosis by exacerbating it, it may be
desirable to express that protein under control of an inducible
promoter.
[0165] In general, IAPs and fragments thereof may be produced by
transforming a suitable host cell with all or part of the
IAP-encoding cDNA fragment that has been placed into a suitable
expression vector.
[0166] Those skilled in the art of molecular biology will
understand that a wide variety of expression systems may be used to
produce the recombinant protein. The precise host cell used is not
critical to the invention, although cancer cells are preferable.
The IAP protein may be produced in a prokaryotic host (e.g., E.
coli) or in a eukaryotic host (e.g., S. cerevisiae, insect cells
such as Sf21 cells, or mammalian cells such as COS-1, NIH 3T3, or
HeLa cells, or other highly proliferative cell types). These cells
are publically available, for example, from the American Type
Culture Collection, Rockville, Md.; see also Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y., 1994). The method of transduction 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), and expression vehicles may be chosen from
those provided, e.g., in Cloning Vectors: A Laboratory Manual (P.
H. Pouwels et al., 1985, Supp. 1987).
[0167] Polypeptides of the invention, particularly short IAP
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 IAP fragments or analogs, as described
herein.
EXAMPLE 13
Anti-IAP Antibodies
[0168] In order to generate IAP-specific antibodies, an IAP coding
sequence (e.g., amino acids 180-276) can be expressed as a
C-terminal fusion with glutathione S-transferase (GST; Smith et
al., Gene 67:31-40, 1988). The fusion protein can be purified on
glutathione-Sepharose beads, eluted with glutathione, and cleaved
with thrombin (at the engineered cleavage site), and purified to
the degree required to successfully immunize rabbits. Primary
immunizations can be carried out with Freund's complete adjuvant
and subsequent immunizations performed with Freund's incomplete
adjuvant. Antibody titres are monitored by Western blot and
immunoprecipitation analyses using the thrombin-cleaved IAP
fragment of the GST-IAP fusion protein. Immune sera are affinity
purified using CNBr-Sepharose-coupled IAP protein. Antiserum
specificity is determined using a panel of unrelated GST proteins
(including GSTp53, Rb, HPV-16 E6, and E6-AP) and GST-trypsin (which
was generated by PCR using known sequences).
[0169] As an alternate or adjunct immunogen to GST fusion proteins,
peptides corresponding to relatively unique hydrophilic regions of
IAP may be generated and coupled to keyhole limpet hemocyanin (KLH)
through an introduced C-terminal lysine. Antiserum to each of these
peptides is similarly affinity purified on peptides conjugated to
BSA, and specificity is tested by ELISA and Western blotting using
peptide conjugates, and by Western blotting and immunoprecipitation
using IAP expressed as a GST fusion protein.
[0170] Alternatively, monoclonal antibodies may be prepared using
the IAP proteins described above and standard hybridoma technology
(see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al.,
Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol.
6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell
Hybridomas, Elsevier, New York, N.Y., 1981; Ausubel et al., supra).
Once produced, monoclonal antibodies are also tested for specific
IAP recognition by Western blot or immunoprecipitation analysis (by
the methods described in Ausubel et al., supra).
[0171] Antibodies that specifically recognize IAPs or fragments of
IAPs, such as those described herein containing one or more BIR
domains (but not a ring zinc finger domain), or that contain a ring
zinc finger domain (but not a BIR domain) are considered useful in
the invention. They may, for example, be used in an immunoassay to
monitor IAP expression levels or to determine the subcellular
location of an IAP or IAP fragment produced by a mammal. Antibodies
that inhibit the 26 kDa IAP cleavage product described herein
(which contains at least one BIR domain) may be especially useful
in inducing apoptosis in cells undergoing undesirable
proliferation.
[0172] Preferably, antibodies of the invention are produced using
IAP sequence that does not reside within highly conserved regions,
and that appears likely to be antigenic, as analyzed by criteria
such as those provided by the Peptide structure program (Genetics
Computer Group Sequence Analysis Package, Program Manual for the
GCG Package, Version 7, 1991) using the algorithm of Jameson and
Wolf (CABIOS 4:181, 1988). Specifically, these regions, which are
found between BIR1 and BIR2 of all IAPs, are: from amino acid 99 to
amino acid 170 of hiap-1, from amino acid 123 to amino acid 184 of
hiap-2, and from amino acid 116 to amino acid 133 of either xiap or
m-xiap. These fragments can be generated by standard techniques,
e.g., by the PCR, and cloned into the pGEX expression vector
(Ausubel et al., supra). Fusion proteins are expressed in E. coli
and purified using a glutathione agarose affinity matrix as
described in Ausubel et al. (supra). In order to minimize the
potential for obtaining antisera that is non-specific, or exhibits
low-affinity binding to IAP, two or three fusions are generated for
each protein, and each fusion is injected into at least two
rabbits. Antisera are raised by injections in series, preferably
including at least three booster injections.
EXAMPLE 14
Identification of Molecules that Modulate IAP Protein
Expression
[0173] IAP cDNAs facilitate the identification of molecules that
decrease IAP expression or otherwise enhance apoptosis normally
blocked by the IAPs. In one approach, candidate molecules are
added, in varying concentration, to the culture medium of cells
expressing IAP mRNA. IAP expression is then measured, for example,
by Northern blot analysis (Ausubel et al., supra) using an IAP
cDNA, or cDNA fragment, as a hybridization probe. The level of IAP
expression in the presence of the candidate molecule is compared to
the level of IAP expression in the absence of the candidate
molecule, all other factors (e.g., cell type and culture
conditions) being equal.
[0174] The effect of candidate molecules on IAP-mediated apoptosis
may, instead, be measured at the level of IAP protein or level of
IAP fragments using the general approach described above with
standard protein detection techniques, such as Western blotting or
immunoprecipitation with an IAP-specific antibody (for example, the
IAP antibodies described herein).
[0175] Compounds that modulate the level of IAP may be purified, or
substantially purified, or may be one component of a mixture of
compounds such as an extract or supernatant obtained from cells
(Ausubel et al., supra). In an assay of a mixture of compounds, IAP
expression is tested against progressively smaller subsets of the
compound pool (e.g., produced by standard purification techniques
such as HPLC or FPLC) until a single compound or minimal number of
effective compounds is demonstrated to modulate IAP expression.
[0176] Compounds may also be screened for their ability to enhance
IAP-mediated apoptosis. In this approach, the degree of apoptosis
in the presence of a candidate compound is compared to the degree
of apoptosis in its absence, under equivalent conditions. Again,
the screen may begin with a pool of candidate compounds, from which
one or more useful modulator compounds are isolated in a step-wise
fashion. Apoptosis activity may be measured by any standard assay,
for example, those described herein.
[0177] Another method for detecting compounds that modulate the
activity of IAPs is to screen for compounds that interact
physically with a given IAP polypeptide. These compounds may be
detected by adapting interaction trap expression systems known in
the art. These systems detect protein interactions using a
transcriptional activation assay and are generally described by
Gyuris et al. (Cell 75:791-803, 1993) and Field et al. (Nature
340:245-246, 1989), and are commercially available from Clontech
(Palo Alto, Calif.). In addition, PCT Publication WO 95/28497
describes an interaction trap assay in which proteins involved in
apoptosis, by virtue of their interaction with Bcl-2, are detected.
A similar method may be used to identify proteins and other
compounds that interact with IAPs.
[0178] Compounds or molecules that function as modulators of
IAP-mediated cell death may include peptide and non-peptide
molecules such as those present in cell extracts, mammalian serum,
or growth medium in which mammalian cells have been cultured.
TABLE-US-00002 TABLE 2 OLIGONUCLEOTIDE PRIMERS FOR THE SPECIFIC
RT-PCR AMPLIFICATION OF IAP GENES IAP Forward Primer Reverse Primer
Size of Product Gene (nucleotide position*) (nucleotide position*)
(bp) h-xiap p2415 (876-896) p2449 (1291-1311) 435 m-xiap p2566
(458-478) p2490 (994-1013) 555 h-hiap1 p2465 (827-847) p2464
(1008-1038) 211 m-hiap1 p2687 (747-767) p2684 (1177-1197) 450 hiap2
p2595 (1562-1585) p2578 (2339-2363) 801.sup.a 618.sup.b m-hiap2
p2693 (1751-1772) p2734 (2078-2100) 349 *Nucleotide position as
determined from FIGS. 1-4 for each IAP gene .sup.aPCR product size
of hiap2a .sup.bPCR product size of hiap2b
EXAMPLE 15
Assignment of xiap, hiap-1 and hiap-2 to Chromosomes Xq25 and
11q22-23 by Fluorescence in Situ Hybridization (FISH)
[0179] Fluorescence in situ hybridization (FISH) was used to
identify the chromosomal location of xiap, hiap-1 and hiap-2.
[0180] A total of 101 metaphase spreads were examined with the xiap
probe, as described above. Symmetrical fluorescent signals on
either one or both homologs of chromosome Xq25 were observed in 74%
of the cells analyzed. Following staining with hiap-1 and hiap-2
probes, 56 cells were analyzed and doublet signals in the region
11q22-23 were observed in 83% of cells examined. The xiap gene was
mapped to Xq25 while the hiap-1 and hiap-2 genes were mapped at the
border of 11q22 and 11q23 bands.
[0181] These experiments confirmed the location of the xiap gene on
chromosome Xq25. No highly consistent chromosomal abnormalities
involving band Xq25 have been reported so far in any malignancies.
However, deletions within this region are associated with a number
of immune system defects including X-linked lymphoproliferative
disease (Wu et al., Genomics 17:163-170, 1993).
[0182] Cytogenetic abnormalities of band 11q23 have been identified
in more than 50% of infant leukemias regardless of the phenotype
(Martinez-Climet et al., Leukaemia 9:1299-1304, 1995).
Rearrangements of the MLL Gene (mixed lineage leukemia or myeloid
lymphoid leukemia; Ziemin Van der Poel et al., Proc. Natl. Acad.
Sci. USA 88:10735-10739, 1991) have been detected in 80% of cases
with 11q23 translocation, however patients whose rearrangements
clearly involved regions other than the MLL gene were also reported
(Kobayashi et al., Blood 82:547-551, 1993). Thus, the IAP genes may
follow the Bcl-2 paradigm, and would therefore play an important
role in cancer transformation.
Incorporation by Reference
[0183] The following documents and all the references referred to
herein are incorporated by reference: U.S. Ser. No. 08/511,485,
filed Aug. 4, 1995; U.S. Ser. No. 08/576,956, filed Dec. 22, 1995;
PCT/IB96/01022, filed Aug. 5, 1996; U.S. Ser. No. 60/017,354, filed
Apr. 26, 1996; U.S. Ser. No. 60/030,931, filed Nov. 15, 1996
(Express Mail Labeling Number RB794124826US); U.S. Ser. No.
60/030,590, filed Nov. 14, 1996 (Express Mail Labeling Number
RB794124804US); U.S. Pat. No. 5,576,208, issued Nov. 19, 1996; and
PCT Application IB97/00142, filed Jan. 17, 1997 claiming priority
from UK 9601108.5, filed Jan. 19, 1996.
Other Embodiments
[0184] In other embodiments, the invention includes use of any
protein which is substantially identical to a mammalian IAP
polypeptides (FIGS. 1-6; SEQ ID NOs:1-14); such homologs include
other substantially pure naturally-occurring mammalian IAP proteins
as well as allelic variants; natural mutants; induced mutants; DNA
sequences which encode proteins and also hybridize to the IAP DNA
sequences of FIGS. 1-6 (SEQ ID NOS:1-14) under high stringency
conditions or, less preferably, under low stringency conditions
(e.g., washing at 2.times.SSC at 40.degree. C. with a probe length
of at least 40 nucleotides); and proteins specifically bound by
antisera directed to a IAP polypeptide. The term also includes
chimeric polypeptides that include a IAP portion.
[0185] The invention further includes use of analogs of any
naturally-occurring IAP polypeptide. Analogs can differ from the
naturally-occurring IAP protein by amino acid sequence differences,
by post-translational modifications, or by both. Analogs of the
invention will generally exhibit at least 85%, more preferably 90%,
and most preferably 95% or even 99% identity with all or part of a
naturally occurring IAP amino acid sequence. The length of sequence
comparison is at least 15 amino acid residues, preferably at least
25 amino acid residues, and more preferably more than 35 amino acid
residues. Modifications include in vivo and in vitro chemical
derivatization of polypeptides, e.g., acetylation, carboxylation,
phosphorylation, or glycosylation; such modifications may occur
during polypeptide synthesis or processing or following treatment
with isolated modifying enzymes. Analogs can also differ from the
naturally-occurring IAP polypeptide by alterations in primary
sequence. These include genetic variants, both natural and induced
(for example, resulting from random mutagenesis by irradiation or
exposure to ethanemethylsulfate or by site-specific mutagenesis as
described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A
Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al.,
supra). Also included are cyclized peptides, molecules, and analogs
which contain residues other than L-amino acids, e.g., D-amino
acids or normaturally occurring or synthetic amino acids, e.g., B
or y amino acids. In addition to full-length polypeptides, the
invention also includes IAP polypeptide fragments. As used herein,
the term "fragment," means at least 20 contiguous amino acids,
preferably at least 30 contiguous amino acids, more preferably at
least 50 contiguous amino acids, and most preferably at least 60 to
80 or more contiguous amino acids. Fragments of IAP polypeptides
can be generated by methods known to those skilled in the art or
may result from normal protein processing (e.g., removal of amino
acids from the nascent polypeptide that are not required for
biological activity or removal of amino acids by alternative mRNA
splicing or alternative protein processing events).
[0186] Preferable fragments or analogs used according to the
methods of the invention are those which facilitate specific
detection of a IAP nucleic acid or amino acid sequence in a sample
to be diagnosed. Particularly useful IAP fragments for this purpose
include, without limitation, the amino acid fragments shown in
Table 2.
[0187] The methods of the invention may use antibodies prepared by
a variety of methods. For example, the IAP or NAIP polypeptide, or
antigenic fragments thereof, can be administered to an animal in
order to induce the production of polyclonal antibodies.
Alternatively, antibodies used as described herein may be
monoclonal antibodies, which are prepared using hybridoma
technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler
et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J.
Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies
and T Cell Hybridomas, Elsevier, N.Y., 1981). The invention
features use of antibodies that specifically bind human or murine
IAP or NAIP polypeptides, or fragments thereof. In particular the
invention features "neutralizing" antibodies. By "neutralizing"
antibodies is meant antibodies that interfere with any of the
biological activities of IAP or NAIP polypeptides, particularly the
ability of IAPs to inhibit apoptosis. The neutralizing antibody may
reduce the ability of IAP polypeptides to inhibit polypeptides by,
preferably 50%, more preferably by 70%, and most preferably by 90%
or more. Any standard assay of apoptosis, including those described
herein, by those incorporated by reference and those in the art,
may be used to assess neutralizing antibodies.
[0188] In addition to intact monoclonal and polyclonal anti-IAP
antibodies, the invention features use of various genetically
engineered antibodies, humanized antibodies, and antibody
fragments, including F(ab')2, Fab', Fab, Fv and sFv fragments.
Antibodies can be humanized by methods known in the art, e.g.,
monoclonal antibodies with a desired binding specificity can be
commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo
Alto, Calif.). Fully human antibodies, such as those expressed in
transgenic animals, are also features of the invention (Green et
al., Nature Genetics 7:13-21, 1994).
[0189] Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes
methods for preparing single polypeptide chain antibodies. Ward et
al. (Nature 341:544-546, 1989) describe the preparation of heavy
chain variable domains, which they term "single domain antibodies,"
which have high antigen-binding affinities. McCafferty et al.
(Nature 348:552-554, 1990) show that complete antibody V domains
can be displayed on the surface of fd bacteriophage, that the phage
bind specifically to antigen, and that rare phage (one in a
million) can be isolated after affinity chromatography. Boss et al.
(U.S. Pat. No. 4,816,397) describe various methods for producing
immunoglobulins, and immunologically functional fragments thereof,
which include at least the variable domains of the heavy and light
chain in a single host cell. Cabilly et al. (U.S. Pat. No.
4,816,567) describe methods for preparing chimeric antibodies.
Sequence CWU 1
1
17 1 46 PRT Artificial Sequence VARIANT (1)...(46) Xaa at 2, 3, 4,
5, 6, 7, 9, 10, 11, 17, 18, 19, 20, 21, 23, 25, 30, 31, 32, 34, 35,
38, 39, 40, 41, 42, and 45 can be any amino acid; Xaa at 8 can be
Glu or Asp; Xaa at 14 and 22 can be Val or Ile. 1 Glu Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys Xaa Cys Met 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Phe Xaa Pro Cys Gly His Xaa Xaa Xaa 20 25 30
Cys Xaa Xaa Cys Ala Xaa Xaa Xaa Xaa Xaa Cys Pro Xaa Cys 35 40 45 2
68 PRT Artificial Sequence VARIANT (1)...(68) Xaa at 1, 2, 3, 6, 9,
10, 14, 15, 18, 19, 20, 21, 24, 30, 32, 33, 35, 37, 40, 42, 43, 44,
45, 46, 47, 49, 50, 51, 53, 54, 55, 56, 57, 59, 60, 61, 62, 64 and
66 can be any amino acid; Xaa at 13, 16 and 17 can be any amino
acid or absent. 2 Xaa Xaa Xaa Arg Leu Xaa Thr Phe Xaa Xaa Trp Pro
Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Leu Ala Xaa Ala Gly
Phe Tyr Tyr Xaa Gly Xaa 20 25 30 Xaa Asp Xaa Val Xaa Cys Phe Xaa
Cys Xaa Xaa Xaa Xaa Xaa Xaa Trp 35 40 45 Xaa Xaa Xaa Asp Xaa Xaa
Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Pro Xaa 50 55 60 Cys Xaa Phe Val 65
3 5232 DNA Homo sapiens variation 4623 n can be any nucleotide 3
gaaaaggtgg acaagtccta ttttcaagag aagatgactt ttaacagttt tgaaggatct
60 aaaacttgtg tacctgcaga catcaataag gaagaagaat ttgtagaaga
gtttaataga 120 ttaaaaactt ttgctaattt tccaagtggt agtcctgttt
cagcatcaac actggcacga 180 gcagggtttc tttatactgg tgaaggagat
accgtgcggt gctttagttg tcatgcagct 240 gtagatagat ggcaatatgg
agactcagca gttggaagac acaggaaagt atccccaaat 300 tgcagattta
tcaacggctt ttatcttgaa aatagtgcca cgcagtctac aaattctggt 360
atccagaatg gtcagtacaa agttgaaaac tatctgggaa gcagagatca ttttgcctta
420 gacaggccat ctgagacaca tgcagactat cttttgagaa ctgggcaggt
tgtagatata 480 tcagacacca tatacccgag gaaccctgcc atgtatagtg
aagaagctag attaaagtcc 540 tttcagaact ggccagacta tgctcaccta
accccaagag agttagcaag tgctggactc 600 tactacacag gtattggtga
ccaagtgcag tgcttttgtt gtggtggaaa actgaaaaat 660 tgggaacctt
gtgatcgtgc ctggtcagaa cacaggcgac actttcctaa ttgcttcttt 720
gttttgggcc ggaatcttaa tattcgaagt gaatctgatg ctgtgagttc tgataggaat
780 ttcccaaatt caacaaatct tccaagaaat ccatccatgg cagattatga
agcacggatc 840 tttacttttg ggacatggat atactcagtt aacaaggagc
agcttgcaag agctggattt 900 tatgctttag gtgaaggtga taaagtaaag
tgctttcact gtggaggagg gctaactgat 960 tggaagccca gtgaagaccc
ttgggaacaa catgctaaat ggtatccagg gtgcaaatat 1020 ctgttagaac
agaagggaca agaatatata aacaatattc atttaactca ttcacttgag 1080
gagtgtctgg taagaactac tgagaaaaca ccatcactaa ctagaagaat tgatgatacc
1140 atcttccaaa atcctatggt acaagaagct atacgaatgg ggttcagttt
caaggacatt 1200 aagaaaataa tggaggaaaa aattcagata tctgggagca
actataaatc acttgaggtt 1260 ctggttgcag atctagtgaa tgctcagaaa
gacagtatgc aagatgagtc aagtcagact 1320 tcattacaga aagagattag
tactgaagag cagctaaggc gcctgcaaga ggagaagctt 1380 tgcaaaatct
gtatggatag aaatattgct atcgtttttg ttccttgtgg acatctagtc 1440
acttgtaaac aatgtgctga agcagttgac aagtgtccca tgtgctacac agtcattact
1500 ttcaagcaaa aaatttttat gtcttaatct aactctatag taggcatgtt
atgttgttct 1560 tattaccctg attgaatgtg tgatgtgaac tgactttaag
taatcaggat tgaattccat 1620 tagcatttgc taccaagtag gaaaaaaaat
gtacatggca gtgttttagt tggcaatata 1680 atctttgaat ttcttgattt
ttcagggtat tagctgtatt atccattttt tttactgtta 1740 tttaattgaa
accatagact aagaataaga agcatcatac tataactgaa cacaatgtgt 1800
attcatagta tactgattta atttctaagt gtaagtgaat taatcatctg gattttttat
1860 tcttttcaga taggcttaac aaatggagct ttctgtatat aaatgtggag
attagagtta 1920 atctccccaa tcacataatt tgttttgtgt gaaaaaggaa
taaattgttc catgctggtg 1980 gaaagataga gattgttttt agaggttggt
tgttgtgttt taggattctg tccattttct 2040 tttaaagtta taaacacgta
cttgtgcgaa ttattttttt aaagtgattt gccatttttg 2100 aaagcgtatt
taatgataga atactatcga gccaacatgt actgacatgg aaagatgtca 2160
aagatatgtt aagtgtaaaa tgcaagtggc aaaacactat gtatagtctg agccagatca
2220 aagtatgtat gtttttaata tgcatagaac aaaagatttg gaaagatata
caccaaactg 2280 ttaaatgtgg tttctcttcg gggagggggg gattggggga
ggggccccag aggggtttta 2340 taggggcctt ttcactttct acttttttca
ttttgttctg ttcgaatttt ttataagtat 2400 gtattacttt tgtaatcaga
atttttagaa agtattttgc tgatttaaag gcttaggcat 2460 gttcaaacgc
ctgcaaaact acttatcact cagctttagt ttttctaatc caagaaggca 2520
gggcagttaa cctttttggt gccaatgtga aatgtaaatg attttatgtt tttcctgctt
2580 tgtggatgaa aaatatttct gagtggtagt tttttgacag gtagaccatg
tcttatcttg 2640 tttcaaaata agtatttctg attttgtaaa atgaaatata
aaatatgtct cagatcttcc 2700 aattaattag taaggattca tccttaatcc
ttgctagttt aagcctgcct aagtcacttt 2760 actaaaagat ctttgttaac
tcagtatttt aaacatctgt cagcttatgt aggtaaaagt 2820 agaagcatgt
ttgtacactg cttgtagtta tagtgacagc tttccatgtt gagattctca 2880
tatcatcttg tatcttaaag tttcatgtga gtttttaccg ttaggatgat taagatgtat
2940 ataggacaaa atgttaagtc tttcctctac ctacatttgt tttcttggct
agtaatagta 3000 gtagatactt ctgaaataaa tgttctctca agatccttaa
aacctcttgg aaattataaa 3060 aatattggca agaaaagaag aatagttgtt
taaatatttt ttaaaaaaca cttgaataag 3120 aatcagtagg gtataaacta
gaagtttaaa aatgcctcat agaacgtcca gggtttacat 3180 tacaagattc
tcacaacaaa cccattgtag aggtgagtaa ggcatgttac tacagaggaa 3240
agtttgagag taaaactgta aaaaattata tttttgttgt actttctaag agaaagagta
3300 ttgttatgtt ctcctaactt ctgttgatta ctactttaag tgatattcat
ttaaaacatt 3360 gcaaatttat tttatttatt taattttctt tttgagatgg
agtcttgctt gtcacccagg 3420 ctggagtgca gtggagtgat ctctgctcac
tgcaacctcc gccttctggg ttcaagcgat 3480 tctcgtgcct cagcttcctg
agtagctgga attacaggca ggtgccacca tgcccgacta 3540 attttttttt
atttttagta gagacggggt ttcaccatgt tggccaggct ggtatcaaac 3600
tcctgacctc aagagatcca ctcgccttgc cctcccaaag tgctgggatt acaggcttga
3660 gccaccacgc ccggctaaaa cattgcaaat ttaaatgaga gttttaaaaa
ttaaataatg 3720 actgccctgt ttctgtttta gtatgtaaat cctcagttct
tcacctttgc actgtctgcc 3780 acttagtttg gttatatagt cattaacttg
aatttggtct gtatagtcta gactttaaat 3840 ttaaagtttt ctacaagggg
agaaaagtgt taaaattttt aaaatatgtt ttccaggaca 3900 cttcacttcc
aagtcaggta ggtagttcaa tctagttgtt agccaaggac tcaaggactg 3960
aattgtttta acataaggct tttcctgttc tgggagccgc acttcattaa aattcttcta
4020 aaacttgtat gtttagagtt aagcaagact ttttttcttc ctctccatga
gttgtgaaat 4080 ttaatgcaca acgctgatgt ggctaacaag tttattttaa
gaattgttta gaaatgctgt 4140 tgcttcaggt tcttaaaatc actcagcact
ccaacttcta atcaaatttt tggagactta 4200 acagcatttg tctgtgtttg
aactataaaa agcaccggat cttttccatc taattccgca 4260 aaaattgatc
atttgcaaag tcaaaactat agccatatcc aaatcttttc cccctcccaa 4320
gagttctcag tgtctacatg tagactattc cttttctgta taaagttcac tctaggattt
4380 caagtcacca cttattttac attttagtca tgcaaagatt caagtagttt
tgcaataagt 4440 acttatcttt atttgtaata atttagtctg ctgatcaaaa
gcattgtctt aatttttgag 4500 aactggtttt agcatttaca aactaaattc
cagttaatta attaatagct ttatattgcc 4560 tttcctgcta catttggttt
tttcccctgt ccctttgatt acgggctaag gtagggtaag 4620 anngggtgta
gtgagtgtat ataatgtgat ttggccctgt gtattatgat attttgttat 4680
ttttgttgtt atattattta catttcagta gttgtttttt gtgtttccat tttaggggat
4740 aaaatttgta ttttgaacta tgaatggaga ctaccgcccc agcattagtt
tcacatgata 4800 taccctttaa acccgaatca ttgttttatt tcctgattac
acaggtgttg aatggggaaa 4860 ggggctagta tatcagtagg atatactatg
ggatgtatat atatcattgc tgttagagaa 4920 atgaaataaa atggggctgg
gctcagtggc tcacgcctgt aatcccagca ctttgggagg 4980 ctgaggcagg
tggatcacga ggtcaggaga tcgagaccat cctggctaac acggtgaaac 5040
cccgtctcta ctaaaaaaca gaaaattagc cgggcgtggt ggcgggcgcc tgtagtccca
5100 gctactcggg aggctgaggc aggagaatgg tgtgaacccg ggaggcagag
cttgcagtga 5160 gccgagatct cgccactgca ctccagcctg ggcaacagag
caagactctg tctcaaaaaa 5220 aaaaaaaaaa ag 5232 4 497 PRT Homo
sapiens 4 Met Thr Phe Asn Ser Phe Glu Gly Ser Lys Thr Cys Val Pro
Ala Asp 1 5 10 15 Ile Asn Lys Glu Glu Glu Phe Val Glu Glu Phe Asn
Arg Leu Lys Thr 20 25 30 Phe Ala Asn Phe Pro Ser Gly Ser Pro Val
Ser Ala Ser Thr Leu Ala 35 40 45 Arg Ala Gly Phe Leu Tyr Thr Gly
Glu Gly Asp Thr Val Arg Cys Phe 50 55 60 Ser Cys His Ala Ala Val
Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val 65 70 75 80 Gly Arg His Arg
Lys Val Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe 85 90 95 Tyr Leu
Glu Asn Ser Ala Thr Gln Ser Thr Asn Ser Gly Ile Gln Asn 100 105 110
Gly Gln Tyr Lys Val Glu Asn Tyr Leu Gly Ser Arg Asp His Phe Ala 115
120 125 Leu Asp Arg Pro Ser Glu Thr His Ala Asp Tyr Leu Leu Arg Thr
Gly 130 135 140 Gln Val Val Asp Ile Ser Asp Thr Ile Tyr Pro Arg Asn
Pro Ala Met 145 150 155 160 Tyr Cys Glu Glu Ala Arg Leu Lys Ser Phe
Gln Asn Trp Pro Asp Tyr 165 170 175 Ala His Leu Thr Pro Arg Glu Leu
Ala Ser Ala Gly Leu Tyr Tyr Thr 180 185 190 Gly Ile Gly Asp Gln Val
Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 195 200 205 Asn Trp Glu Pro
Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe 210 215 220 Pro Asn
Cys Phe Phe Val Leu Gly Arg Asn Leu Asn Ile Arg Ser Glu 225 230 235
240 Ser Asp Ala Val Ser Ser Asp Arg Asn Phe Pro Asn Ser Thr Asn Leu
245 250 255 Pro Arg Asn Pro Ser Met Ala Asp Tyr Glu Ala Arg Ile Phe
Thr Phe 260 265 270 Gly Thr Trp Ile Tyr Ser Val Asn Lys Glu Gln Leu
Ala Arg Ala Gly 275 280 285 Phe Tyr Ala Leu Gly Glu Gly Asp Lys Val
Lys Cys Phe His Cys Gly 290 295 300 Gly Gly Leu Thr Asp Trp Lys Pro
Ser Glu Asp Pro Trp Glu Gln His 305 310 315 320 Ala Lys Trp Tyr Pro
Gly Cys Lys Tyr Leu Leu Glu Gln Lys Gly Gln 325 330 335 Glu Tyr Ile
Asn Asn Ile His Leu Thr His Ser Leu Glu Glu Cys Leu 340 345 350 Val
Arg Thr Thr Glu Lys Thr Pro Ser Leu Thr Arg Arg Ile Asp Asp 355 360
365 Thr Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg Met Gly Phe
370 375 380 Ser Phe Lys Asp Ile Lys Lys Ile Met Glu Glu Lys Ile Gln
Ile Ser 385 390 395 400 Gly Ser Asn Tyr Lys Ser Leu Glu Val Leu Val
Ala Asp Leu Val Asn 405 410 415 Ala Gln Lys Asp Ser Met Gln Asp Glu
Ser Ser Gln Thr Ser Leu Gln 420 425 430 Lys Glu Ile Ser Thr Glu Glu
Gln Leu Arg Arg Leu Gln Glu Glu Lys 435 440 445 Leu Cys Lys Ile Cys
Met Asp Arg Asn Ile Ala Ile Val Phe Val Pro 450 455 460 Cys Gly His
Leu Val Thr Cys Lys Gln Cys Ala Glu Ala Val Asp Lys 465 470 475 480
Cys Pro Met Cys Tyr Thr Val Ile Thr Phe Lys Gln Lys Ile Phe Met 485
490 495 Ser 5 6669 DNA Homo sapiens variation (3677)...(3951) n can
be any nucleotide 5 ttgctctgtc acccagtttg gagtgcagtt atgcagtctc
acactgcaag ctctgcctca 60 tgggctcaag tgaacctcct gcctcagcct
ctcaagtagc tgggaccaca ggcaggtgcc 120 accatgtctg gctaattttt
gagtttcttt gtagagatgg tgttttgcca agtcacccag 180 tttgaggctg
gtctcaaaca cctgggctca agcaatccat ctacctcagc ctcccaaagt 240
gctgggatta caggagtgag ccatggcatg aggccttgtg gggtgtctct tttaaatgaa
300 agcatactct gtttacgtat ttgatatgaa ggaatatcct tcctttccac
aaagacaaaa 360 attatcctat ttttctcaaa acatatgtcc tttttctcta
cttttcattt ttgttacttt 420 tgatggacac atgtgttaca ttgatttcac
tttctcataa ttctgctgta agaaaaacaa 480 tagtgccagt tcaatgacaa
atagcaacag tctgttattg ctagactgtt actgttagtg 540 gagactacca
gaacagtcag tcccagtgtc agggaatcaa agagaacatg ttccctctct 600
aaagggcaca gctgctgctc agctttagct gattgctgcc ctgcaggact ataggcccag
660 tgttgctaga tcttttgatg tttcaagaga agcttggaat ctagaatgtg
atgggaagtc 720 tcttacattt aaacatgttg gcaattaatg gtaagattta
aaaatactgt ggtccaagaa 780 aaaaatggat ttggaaactg gattaaattc
aaatgaggca tgcagattaa tctacagcat 840 ggtacaatgt gaattttctg
gtttctttaa ttgcactgta attaggtaag atgttagctt 900 tggggaagct
aagtgcagag tatgcagaaa ctattatttt tgtaagtttt ctctaagtat 960
aaataaattt caaaataaaa ataaaaactt agtaaagaac tataatgcaa ttctatgtaa
1020 gccaaacata atatgtcttc cagtttgaaa cctctgggtt ttattttatt
ttattttatt 1080 tttgagacag agtcttgctg tgtcacccag gctggagtgt
agtggcacta tttcggccca 1140 ctgcaacctc cacctcccag gctcaaatga
ttctcctgcc tcagcctccg gagtagctgg 1200 gattacaggc gcgtaccacc
acacccagct aatttttgta tttttagtag agatggggtt 1260 tcaccatttt
ggccaggctg gttttgaact cctgacctca agtgatccac ttgtcttggc 1320
ctcccaaaat gctgggatta caggcgtgag ccactgcacc aggcagaggc ctctgttttt
1380 tatctctttt tggcctctac agtgcctagt aaagcacctg atacatggta
aacgatcagt 1440 aattactagt actctatttt ggagaaaatg attttttaaa
aagtcattgt gttccatcca 1500 tgagtcgttt gagttttaaa actgtctttt
tgtttgtttt tgaacaggtt tacaaaggag 1560 gaaaacgact tcttctagat
ttttttttca gtttcttcta taaatcaaaa catctcaaaa 1620 tggagaccta
aaatccttaa agggacttag tctaatctcg ggaggtagtt ttgtgcatgg 1680
gtaaacaaat taagtattaa ctggtgtttt actatccaaa gaatgctaat tttataaaca
1740 tgatcgagtt atataaggta taccataatg agtttgattt tgaatttgat
ttgtggaaat 1800 aaaggaaaag tgattctagc tggggcatat tgttaaagca
tttttttcag agttggccag 1860 gcagtctcct actggcacat tctcccatta
tgtagaatag aaatagtacc tgtgtttggg 1920 aaagatttta aaatgagtga
cagttatttg gaacaaagag ctaataatca atccactgca 1980 aattaaagaa
acatgcagat gaaagttttg acacattaaa atacttctac agtgacaaag 2040
aaaaatcaag aacaaagctt tttgatatgt gcaacaaatt tagaggaagt aaaaagataa
2100 atgtgatgat tggtcaagaa attatccagt tatttacaag gccactgata
ttttaaacgt 2160 ccaaaagttt gtttaaatgg gctgttaccg ctgagaatga
tgaggatgag aatgatggtt 2220 gaaggttaca ttttaggaaa tgaagaaact
tagaaaatta atataaagac agtgatgaat 2280 acaaagaaga tttttataac
aatgtgtaaa atttttggcc agggaaagga atattgaagt 2340 tagatacaat
tacttacctt tgagggaaat aattgttggt aatgagatgt gatgtttctc 2400
ctgccacctg gaaacaaagc attgaagtct gcagttgaaa agcccaacgt ctgtgagatc
2460 caggaaacca tgcttgcaaa ccactggtaa aaaaaaaaaa aaaaaaaaaa
aaagccacag 2520 tgacttgctt attggtcatt gctagtatta tcgactcaga
acctctttac taatggctag 2580 taaatcataa ttgagaaatt ctgaattttg
acaaggtctc tgctgttgaa atggtaaatt 2640 tattattttt tttgtcatga
taaattctgg ttcaaggtat gctatccatg aaataatttc 2700 tgaccaaaac
taaattgatg caatttgatt atccatctta gcctacagat ggcatctggt 2760
aacttttgac tgttttaaaa aataaatcca ctatcagagt agatttgatg ttggcttcag
2820 aaacatttag aaaaacaaaa gttcaaaaat gttttcagga ggtgataagt
tgaataactc 2880 tacaatgtta gttctttgag ggggacaaaa aatttaaaat
ctttgaaagg tcttatttta 2940 cagccatatc taaattatct taagaaaatt
tttaacaaag ggaatgaaat atatatcatg 3000 attctgtttt tccaaaagta
acctgaatat agcaatgaag ttcagttttg ttattggtag 3060 tttgggcaga
gtctcttttt gcagcacctg ttgtctacca taattacaga ggacatttcc 3120
atgttctagc caagtatact attagaataa aaaaacttaa cattgagttg cttcaacagc
3180 atgaaactga gtccaaaaga ccaaatgaac aaacacatta atctctgatt
atttatttta 3240 aatagaatat ttaattgtgt aagatctaat agtatcatta
tacttaagca atcatattcc 3300 tgatgatcta tgggaaataa ctattattta
attaatattg aaaccaggtt ttaagatgtg 3360 ttagccagtc ctgttactag
taaatctctt tatttggaga gaaattttag attgttttgt 3420 tctccttatt
agaaggattg tagaaagaaa aaaatgacta attggagaaa aattggggat 3480
atatcatatt tcactgaatt caaaatgtct tcagttgtaa atcttaccat tattttacgt
3540 acctctaaga aataaaagtg cttctaatta aaatatgatg tcattaatta
tgaaatactt 3600 cttgataaca gaagttttaa aatagccatc ttagaatcag
tgaaatatgg taatgtatta 3660 ttttcctcct ttgagtnagg tcttgtgctt
tttnttcctg gccactaaat ntcaccatnt 3720 ccaanaagca aantaaacct
attctgaata tttttgctgt gaaacacttg ncagcagagc 3780 tttcccncca
tgnnagaagc ttcatgagtc acacattaca tctttgggtt gattgaatgc 3840
cactgaaaca tttctagtag cctggagnag ttgacctacc tgtggagatg cctgccatta
3900 aatggcatcc tgatggctta atacacatca ctcttctgtg nagggtttta
attttcaaca 3960 cagcttactc tgtagcatca tgtttacatt gtatgtataa
agattatacn aaggtgcaat 4020 tgtgtatttc ttccttaaaa tgtatcagta
taggatttag aatctccatg ttgaaactct 4080 aaatgcatag aaataaaaat
aataaaaaat ttttcatttt ggcttttcag cctagtatta 4140 aaactgataa
aagcaaagcc atgcacaaaa ctacctccct agagaaaggc tagtcccttt 4200
tcttccccat tcatttcatt atgaacatag tagaaaacag catattctta tcaaatttga
4260 tgaaaagcgc caacacgttt gaactgaaat acgacttgtc atgtgaactg
taccgaatgt 4320 ctacgtattc cacttttcct gctggggttc ctgtctcaga
aaggagtctt gctcgtgctg 4380 gtttctatta cactggtgtg aatgacaagg
tcaaatgctt ctgttgtggc ctgatgctgg 4440 ataactggaa aagaggagac
agtcctactg aaaagcataa aaagttgtat cctagctgca 4500 gattcgttca
gagtctaaat tccgttaaca acttggaagc tacctctcag cctacttttc 4560
cttcttcagt aacacattcc acacactcat tacttccggg tacagaaaac agtggatatt
4620 tccgtggctc ttattcaaac tctccatcaa atcctgtaaa ctccagagca
aatcaagaat 4680 tttctgcctt gatgagaagt tcctacccct gtccaatgaa
taacgaaaat gccagattac 4740 ttacttttca gacatggcca ttgacttttc
tgtcgccaac agatctggca cgagcaggct 4800 tttactacat aggacctgga
gacagagtgg cttgctttgc ctgtggtgga aaattgagca 4860 attgggaacc
gaaggataat gctatgtcag aacacctgag acattttccc aaatgcccat 4920
ttatagaaaa tcagcttcaa gacacttcaa gatacacagt ttctaatctg agcatgcaga
4980 cacatgcagc ccgctttaaa acattcttta actggccctc tagtgttcta
gttaatcctg 5040 agcagcttgc aagtgcgggt ttttattatg tgggtaacag
tgatgatgtc aaatgctttt 5100 gctgtgatgg tggactcagg tgttgggaat
ctggagatga tccatgggtt caacatgcca 5160 agtggtttcc aaggtgtgag
tacttgataa gaattaaagg acaggagttc atccgtcaag 5220 ttcaagccag
ttaccctcat ctacttgaac agctgctatc cacatcagac agcccaggag 5280
atgaaaatgc agagtcatca attatccatt ttgaacctgg agaagaccat tcagaagatg
5340 caatcatgat gaatactcct gtgattaatg ctgccgtgga aatgggcttt
agtagaagcc 5400 tggtaaaaca
gacagttcag agaaaaatcc tagcaactgg agagaattat agactagtca 5460
atgatcttgt gttagactta ctcaatgcag aagatgaaat aagggaagag gagagagaaa
5520 gagcaactga ggaaaaagaa tcaaatgatt tattattaat ccggaagaat
agaatggcac 5580 tttttcaaca tttgacttgt gtaattccaa tcctggatag
tctactaact gccggaatta 5640 ttaatgaaca agaacatgat gttattaaac
agaagacaca gacgtcttta caagcaagag 5700 aactgattga tacgatttta
gtaaaaggaa atattgcagc cactgtattc agaaactctc 5760 tgcaagaagc
tgaagctgtg ttatatgagc atttatttgt gcaacaggac ataaaatata 5820
ttcccacaga agatgtttca gatctaccag tggaagaaca attgcggaga ctacaagaag
5880 aaagaacatg taaagtgtgt atggacaaag aagtgtccat agtgtttatt
ccttgtggtc 5940 atctagtagt atgcaaagat tgtgctcctt ctttaagaaa
gtgtcctatt tgtaggagta 6000 caatcaaggg tacagttcgt acatttcttt
catgaagaag aaccaaaaca tcgtctaaac 6060 tttagaatta atttattaaa
tgtattataa ctttaacttt tatcctaatt tggtttcctt 6120 aaaattttta
tttatttaca actcaaaaaa cattgttttg tgtaacatat ttatatatgt 6180
atctaaacca tatgaacata tattttttag aaactaagag aatgataggc ttttgttctt
6240 atgaacgaaa aagaggtagc actacaaaca caatattcaa tcaaaatttc
agcattattg 6300 aaattgtaag tgaagtaaaa cttaagatat ttgagttaac
ctttaagaat tttaaatatt 6360 ttggcattgt actaataccg ggaacatgaa
gccaggtgtg gtggtatgtg cctgtagtcc 6420 caggctgagg caagagaatt
acttgagccc aggagtttga atccatcctg ggcagcatac 6480 tgagaccctg
cctttaaaaa caaacagaac aaaaacaaaa caccagggac acatttctct 6540
gtcttttttg atcagtgtcc tatacatcga aggtgtgcat atatgttgaa tcacatttta
6600 gggacatggt gtttttataa agaattctgt gagaaaaaat ttaataaagc
aaccaaaaaa 6660 aaaaaaaaa 6669 6 604 PRT Homo sapiens 6 Met Asn Ile
Val Glu Asn Ser Ile Phe Leu Ser Asn Leu Met Lys Ser 1 5 10 15 Ala
Asn Thr Phe Glu Leu Lys Tyr Asp Leu Ser Cys Glu Leu Tyr Arg 20 25
30 Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser Glu Arg
35 40 45 Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn Asp
Lys Val 50 55 60 Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn Trp
Lys Arg Gly Asp 65 70 75 80 Ser Pro Thr Glu Lys His Lys Lys Leu Tyr
Pro Ser Cys Arg Phe Val 85 90 95 Gln Ser Leu Asn Ser Val Asn Asn
Leu Glu Ala Thr Ser Gln Pro Thr 100 105 110 Phe Pro Ser Ser Val Thr
His Ser Thr His Ser Leu Leu Pro Gly Thr 115 120 125 Glu Asn Ser Gly
Tyr Phe Arg Gly Ser Tyr Ser Asn Ser Pro Ser Asn 130 135 140 Pro Val
Asn Ser Arg Ala Asn Gln Glu Phe Ser Ala Leu Met Arg Ser 145 150 155
160 Ser Tyr Pro Cys Pro Met Asn Asn Glu Asn Ala Arg Leu Leu Thr Phe
165 170 175 Gln Thr Trp Pro Leu Thr Phe Leu Ser Pro Thr Asp Leu Ala
Arg Ala 180 185 190 Gly Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala
Cys Phe Ala Cys 195 200 205 Gly Gly Lys Leu Ser Asn Trp Glu Pro Lys
Asp Asn Ala Met Ser Glu 210 215 220 His Leu Arg His Phe Pro Lys Cys
Pro Phe Ile Glu Asn Gln Leu Gln 225 230 235 240 Asp Thr Ser Arg Tyr
Thr Val Ser Asn Leu Ser Met Gln Thr His Ala 245 250 255 Ala Arg Phe
Lys Thr Phe Phe Asn Trp Pro Ser Ser Val Leu Val Asn 260 265 270 Pro
Glu Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Asn Ser Asp 275 280
285 Asp Val Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser
290 295 300 Gly Asp Asp Pro Trp Val Gln His Ala Lys Trp Phe Pro Arg
Cys Glu 305 310 315 320 Tyr Leu Ile Arg Ile Lys Gly Gln Glu Phe Ile
Arg Gln Val Gln Ala 325 330 335 Ser Tyr Pro His Leu Leu Glu Gln Leu
Leu Ser Thr Ser Asp Ser Pro 340 345 350 Gly Asp Glu Asn Ala Glu Ser
Ser Ile Ile His Leu Glu Pro Gly Glu 355 360 365 Asp His Ser Glu Asp
Ala Ile Met Met Asn Thr Pro Val Ile Asn Ala 370 375 380 Ala Val Glu
Met Gly Phe Ser Arg Ser Leu Val Lys Gln Thr Val Gln 385 390 395 400
Arg Lys Ile Leu Ala Thr Gly Glu Asn Tyr Arg Leu Val Asn Asp Leu 405
410 415 Val Leu Asp Leu Leu Asn Ala Glu Asp Glu Ile Arg Glu Glu Glu
Arg 420 425 430 Glu Arg Ala Thr Glu Glu Lys Glu Ser Asn Asp Leu Leu
Leu Ile Arg 435 440 445 Lys Asn Arg Met Ala Leu Phe Gln His Leu Thr
Cys Val Ile Pro Ile 450 455 460 Leu Asp Ser Leu Leu Thr Ala Gly Ile
Ile Asn Glu Gln Glu His Asp 465 470 475 480 Val Ile Lys Gln Lys Thr
Gln Thr Ser Leu Gln Ala Arg Glu Leu Ile 485 490 495 Asp Thr Ile Leu
Val Lys Gly Asn Ile Ala Ala Thr Val Phe Arg Asn 500 505 510 Ser Leu
Gln Glu Ala Glu Ala Val Leu Tyr Glu His Leu Phe Val Gln 515 520 525
Gln Asp Ile Lys Tyr Ile Pro Thr Glu Asp Val Ser Asp Leu Pro Val 530
535 540 Glu Glu Gln Leu Arg Arg Leu Pro Glu Glu Arg Thr Cys Lys Val
Cys 545 550 555 560 Met Asp Lys Glu Val Ser Ile Val Phe Ile Pro Cys
Gly His Leu Val 565 570 575 Val Cys Lys Asp Cys Ala Pro Ser Leu Arg
Lys Cys Pro Ile Cys Arg 580 585 590 Ser Thr Ile Lys Gly Thr Val Arg
Thr Phe Leu Ser 595 600 7 3732 DNA Homo sapiens 7 gagcgcccgg
gctgatccga gccgagcggg ccgtatctcc ttgtcggcgc cgctgattcc 60
cggctctgcg gaggcctcta ggcagccgcg cagcttccgt gtttgctgcg cccgcactgc
120 gatttacaac cctgaagaat ctccctatcc ctattttgtc cccctgcagt
aataaatccc 180 attatggaga tctcgaaact ttataaaggg atatagtttg
aattctatgg agtgtaattt 240 tgtgtatgaa ttatattttt aaaacattga
agagttttca gaaagaaggc tagtagagtt 300 gattactgat actttatgct
aagcagtact tttttggtag tacaatattt tgttaggcgt 360 ttctgataac
actagaaagg acaagtttta tcttgtgata aattgattaa tgtttacaac 420
atgactgata attatagctg aatagtcctt aaatgatgaa caggttattt agtttttaaa
480 tgcagtgtaa aaagtgtgct gtggaaattt tatggctaac taagtttatg
gagaaaatac 540 cttcagttga tcaagaataa tagtggtata caaagttagg
aagaaagtca acatgatgct 600 gcaggaaatg gaaacaaata caaatgatat
ttaacaaaga tagagtttac agtttttgaa 660 ctttaagcca aattcatttg
acatcaagca ctatagcagg cacaggttca acaaagcttg 720 tgggtattga
cttcccccaa aagttgtcag ctgaagtaat ttagcccact taagtaaata 780
ctatgatgat aagctgtgtg aacttagctt ttaaatagtg tgaccatatg aaggttttaa
840 ttacttttgt ttattggaat aaaatgagat tttttgggtt gtcatgttaa
agtgcttata 900 gggaaagaag cctgcatata attttttacc ttgtggcata
atcagtaatt ggtctgttat 960 tcaggcttca tagcttgtaa ccaaatataa
ataaaaggca taatttaggt attctatagt 1020 tgcttagaat tttgttaata
taaatctctg tgaaaaatca aggagtttta atattttcag 1080 aagtgcatcc
acctttcagg gctttaagtt agtattaact caagattatg aacaaatagc 1140
acttaggtta cctgaaagag ttactacaac cccaaagagt tgtgttctaa gtagtatctt
1200 ggtaattcag agagatactc atcctacctg aatataaact gagataaatc
cagtaaagaa 1260 agtgtagtaa attctacata agagtctatc attgatttct
ttttgtggta aaaatcttag 1320 ttcatgtgaa gaaatttcat gtgaatgttt
tagctatcaa acagtactgt cacctactca 1380 tgcacaaaac tgcctcccaa
agacttttcc caggtccctc gtatcaaaac attaagagta 1440 taatggaaga
tagcacgatc ttgtcagatt ggacaaacag caacaaacaa aaaatgaagt 1500
atgacttttc ctgtgaactc tacagaatgt ctacatattc aactttcccc gccggggtgc
1560 ctgtctcaga aaggagtctt gctcgtgctg gtttttatta tactggtgtg
aatgacaagg 1620 tcaaatgctt ctgttgtggc ctgatgctgg ataactggaa
actaggagac agtcctattc 1680 aaaagcataa acagctatat cctagctgta
gctttattca gaatctggtt tcagctagtc 1740 tgggatccac ctctaagaat
acgtctccaa tgagaaacag ttttgcacat tcattatctc 1800 ccaccttgga
acatagtagc ttgttcagtg gttcttactc cagcctttct ccaaaccctc 1860
ttaattctag agcagttgaa gacatctctt catcgaggac taacccctac agttatgcaa
1920 tgagtactga agaagccaga tttcttacct accatatgtg gccattaact
tttttgtcac 1980 catcagaatt ggcaagagct ggtttttatt atataggacc
tggagatagg gtagcctgct 2040 ttgcctgtgg tgggaagctc agtaactggg
aaccaaagga tgatgctatg tcagaacacc 2100 ggaggcattt tcccaactgt
ccatttttgg aaaattctct agaaactctg aggtttagca 2160 tttcaaatct
gagcatgcag acacatgcag ctcgaatgag aacatttatg tactggccat 2220
ctagtgttcc agttcagcct gagcagcttg caagtgctgg tttttattat gtgggtcgca
2280 atgatgatgt caaatgcttt tgttgtgatg gtggcttgag gtgttgggaa
tctggagatg 2340 atccatgggt agaacatgcc aagtggtttc caaggtgtga
gttcttgata cgaatgaaag 2400 gccaagagtt tgttgatgag attcaaggta
gatatcctca tcttcttgaa cagctgttgt 2460 caacttcaga taccactgga
gaagaaaatg ctgacccacc aattattcat tttggacctg 2520 gagaaagttc
ttcagaagat gctgtcatga tgaatacacc tgtggttaaa tctgccttgg 2580
aaatgggctt taatagagac ctggtgaaac aaacagttca aagtaaaatc ctgacaactg
2640 gagagaacta taaaacagtt aatgatattg tgtcagcact tcttaatgct
gaagatgaaa 2700 aaagagaaga ggagaaggaa aaacaagctg aagaaatggc
atcagatgat ttgtcattaa 2760 ttcggaagaa cagaatggct ctctttcaac
aattgacatg tgtgcttcct atcctggata 2820 atcttttaaa ggccaatgta
attaataaac aggaacatga tattattaaa caaaaaacac 2880 agataccttt
acaagcgaga gaactgattg ataccatttt ggttaaagga aatgctgcgg 2940
ccaacatctt caaaaactgt ctaaaagaaa ttgactctac attgtataag aacttatttg
3000 tggataagaa tatgaagtat atcccaacag aagatgtttc aggtctgtca
ctggaagaac 3060 aattgaggag gttgcaagaa gaacgaactt gtaaagtgtg
tatggacaaa gaagtttctg 3120 ttgtatttat tccttgtggt catctggtag
tatgccagga atgtgcccct tctctaagaa 3180 aatgccctat ttgcaggggt
ataatcaagg gtactgttcg tacatttctc tcttaaagaa 3240 aaatagtcta
tattttaacc tgcataaaaa ggtctttaaa atattgttga acacttgaag 3300
ccatctaaag taaaaaggga attatgagtt tttcaattag taacattcat gttctagtct
3360 gctttggtac taataatctt gtttctgaaa agatggtatc atatatttaa
tcttaatctg 3420 tttatttaca agggaagatt tatgtttggt gaactatatt
agtatgtatg tgtacctaag 3480 ggagtagtgt cactgcttgt tatgcatcat
ttcaggagtt actggatttg ttgttctttc 3540 agaaagcttt gaatactaaa
ttatagtgta gaaaagaact ggaaaccagg aactctggag 3600 ttcatcagag
ttatggtgcc gaattgtctt tggtgctttt cacttgtgtt ttaaaataag 3660
gatttttctc ttatttctcc ccctagtttg tgagaaacat ctcaataaag tgctttaaaa
3720 agaaaaaaaa aa 3732 8 618 PRT Homo sapiens 8 Met His Lys Thr
Ala Ser Gln Arg Leu Phe Pro Gly Pro Ser Tyr Gln 1 5 10 15 Asn Ile
Lys Ser Ile Met Glu Asp Ser Thr Ile Leu Ser Asp Trp Thr 20 25 30
Asn Ser Asn Lys Gln Lys Met Lys Tyr Asp Phe Ser Cys Glu Leu Tyr 35
40 45 Arg Met Ser Thr Tyr Ser Thr Phe Pro Ala Gly Val Pro Val Ser
Glu 50 55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val
Asn Asp Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp
Asn Trp Lys Leu Gly 85 90 95 Asp Ser Pro Ile Gln Lys His Lys Gln
Leu Tyr Pro Ser Cys Ser Phe 100 105 110 Ile Gln Asn Leu Val Ser Ala
Ser Leu Gly Ser Thr Ser Lys Asn Thr 115 120 125 Ser Pro Met Arg Asn
Ser Phe Ala His Ser Leu Ser Pro Thr Leu Glu 130 135 140 His Ser Ser
Leu Phe Ser Gly Ser Tyr Ser Ser Leu Pro Pro Asn Pro 145 150 155 160
Leu Asn Ser Arg Ala Val Glu Asp Ile Ser Ser Ser Arg Thr Asn Pro 165
170 175 Tyr Ser Tyr Ala Met Ser Thr Glu Glu Ala Arg Phe Leu Thr Tyr
His 180 185 190 Met Trp Pro Leu Thr Phe Leu Ser Pro Ser Glu Leu Ala
Arg Ala Gly 195 200 205 Phe Tyr Tyr Ile Gly Pro Gly Asp Arg Val Ala
Cys Phe Ala Cys Gly 210 215 220 Gly Lys Leu Ser Asn Trp Glu Pro Lys
Asp Asp Ala Met Ser Glu His 225 230 235 240 Arg Arg His Phe Pro Asn
Cys Pro Phe Leu Glu Asn Ser Leu Glu Thr 245 250 255 Leu Arg Phe Ser
Ile Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg 260 265 270 Met Arg
Thr Phe Met Tyr Trp Pro Ser Ser Val Pro Val Gln Pro Glu 275 280 285
Gln Leu Ala Ser Ala Gly Phe Tyr Tyr Val Gly Arg Asn Asp Asp Val 290
295 300 Lys Cys Phe Gly Cys Asp Gly Gly Leu Arg Cys Trp Glu Ser Gly
Asp 305 310 315 320 Asp Pro Trp Val Glu His Ala Lys Trp Phe Pro Arg
Cys Glu Phe Leu 325 330 335 Ile Arg Met Lys Gly Gln Glu Phe Val Asp
Glu Ile Gln Gly Arg Tyr 340 345 350 Pro His Leu Leu Glu Gln Leu Leu
Ser Thr Ser Asp Thr Thr Gly Glu 355 360 365 Glu Asn Ala Asp Pro Pro
Ile Ile His Phe Gly Pro Gly Glu Ser Ser 370 375 380 Ser Glu Asp Ala
Val Met Met Asn Thr Pro Val Val Lys Ser Ala Leu 385 390 395 400 Glu
Met Gly Phe Asn Arg Asp Leu Val Lys Gln Thr Val Leu Ser Lys 405 410
415 Ile Leu Thr Thr Gly Glu Asn Tyr Lys Thr Val Asn Asp Ile Val Ser
420 425 430 Ala Leu Leu Asn Ala Glu Asp Glu Lys Arg Glu Glu Glu Lys
Glu Lys 435 440 445 Gln Ala Glu Glu Met Ala Ser Asp Asp Leu Ser Leu
Ile Arg Lys Asn 450 455 460 Arg Met Ala Leu Phe Gln Gln Leu Thr Cys
Val Leu Pro Ile Leu Asp 465 470 475 480 Asn Leu Leu Lys Ala Asn Val
Ile Asn Lys Gln Glu His Asp Ile Ile 485 490 495 Lys Gln Lys Thr Gln
Ile Pro Leu Gln Ala Arg Glu Leu Ile Asp Thr 500 505 510 Ile Trp Val
Lys Gly Asn Ala Ala Ala Asn Ile Phe Lys Asn Cys Leu 515 520 525 Lys
Glu Ile Asp Ser Thr Leu Tyr Lys Asn Leu Phe Val Asp Lys Asn 530 535
540 Met Lys Tyr Ile Pro Thr Glu Asp Val Ser Gly Leu Ser Leu Glu Glu
545 550 555 560 Gln Leu Arg Arg Leu Gln Glu Glu Arg Thr Cys Lys Val
Cys Met Asp 565 570 575 Lys Glu Val Ser Val Val Phe Ile Pro Cys Gly
His Leu Val Val Cys 580 585 590 Gln Glu Cys Ala Pro Ser Leu Arg Lys
Cys Pro Ile Cys Arg Gly Ile 595 600 605 Ile Lys Gly Thr Val Arg Thr
Phe Leu Ser 610 615 9 2691 DNA Mus musculus 9 attttttaaa ttgatgcatt
aacattctaa acattcatct gtttttaaat agtaaaaatt 60 gaactttgcc
ttgaatatgt aatgattcat tataacaatt atgcatagtc tttaataatc 120
tgcatatttt atgctgcttt catgtttttc ctaattaatg acttcacatg tttaatattt
180 ataatttttc tgtcatagtt tccatattta tataaaatga atacttaaga
tcagtaattc 240 tgctctgttt gtttatatac tattttccat caaaagacaa
aatgggactg aggttgaggc 300 tcgttgctaa agcactttcc taaaatgcaa
aaggccctat gatggatccc tagtacttat 360 ttaagtgaga gagaaacagg
ctgggggtgt aggtctgtta gagcatgtgt ttggcattat 420 gtgaagccca
aacactaaaa aaggagaaca aacaaaagcg cagactttaa aactcaagtg 480
gtttggtaat gtacgactct actgtttaga attaaaatgt gtcttagtta ttgtgccatt
540 atttttatgt catcactgga taatatatta gtgcttagta tcagaaatag
tccttatgct 600 ttgtgttttg aagttcctaa tgcaatgttc tctttctaga
aaaggtggac aagtcctatt 660 ttccagagaa gatgactttt aacagttttg
aaggaactag aacttttgta cttgcagaca 720 ccaataagga tgaagaattt
gtagaagagt ttaatagatt aaaaacattt gctaacttcc 780 caagtagtag
tcctgtttca gcatcaacat tggcgcgagc tgggtttctt tataccggtg 840
aaggagacac cgtgcaatgt ttcagttgtc atgcggcaat agatagatgg cagtatggag
900 actcagctgt tggaagacac aggagaatat ccccaaattg cagatttatc
aatggttttt 960 attttgaaaa tggtgctgca cagtctacaa atcctggtat
ccaaaatggc cagtacaaat 1020 ctgaaaactg tgtgggaaat agaaatcctt
ttgcccctga caggccacct gagactcatg 1080 ctgattatct cttgagaact
ggacaggttg tagatatttc agacaccata tacccgagga 1140 accctgccat
gtgtagtgaa gaagccagat tgaagtcatt tcagaactgg ccggactatg 1200
ctcatttaac ccccagagag ttagctagtg ctggcctcta ctacacaggg gctgatgatc
1260 aagtgcaatg cttttgttgt gggggaaaac tgaaaaattg ggaaccctgt
gatcgtgcct 1320 ggtcagaaca caggagacac tttcccaatt gcttttttgt
tttgggccgg aacgttaatg 1380 ttcgaagtga atctggtgtg agttctgata
ggaatttccc aaattcaaca aactctccaa 1440 gaaatccagc catggcagaa
tatgaagcac ggatcgttac ttttggaaca tggacatcct 1500 cagttaacaa
ggagcagctt gcaagagctg gattttatgc tttaggtgaa ggcgataaag 1560
tgaagtgctt ccactgtgga ggagggctca cggattggaa gccaagtgaa gacccctggg
1620 accagcatgc taagtgctac ccagggtgca aatacctatt ggatgagaag
gggcaagaat 1680 atataaataa tattcattta acccatccac ttgaggaatc
tttgggaaga actgctgaaa 1740 aaacaccacc gctaactaaa aaaatcgatg
ataccatctt ccagaatcct atggtgcaag 1800 aagctatacg aatgggattt
agcttcaagg accttaagaa aacaatggaa gaaaaaatcc 1860 aaacatccgg
gagcagctat ctatcacttg aggtcctgat tgcagatctt gtgagtgctc 1920
agaaagataa tacggaggat gagtcaagtc aaacttcatt gcagaaagac attagtactg
1980 aagagcagct aaggcgccta caagaggaga agctttccaa aatctgtatg
gatagaaata 2040 ttgctatcgt tttttttcct tgtggacatc tggccacttg
taaacagtgt gcagaagcag 2100 ttgacaaatg tcccatgtgc tacaccgtca
ttacgttcaa ccaaaaaatt tttatgtctt 2160 agtggggcac cacatgttat
gttcttcttg ctctaattga atgtgtaatg ggagcgaact 2220 ttaagtaatc
ctgcatttgc attccattag catcctgctg tttccaaatg gagaccaatg 2280
ctaacagcac tgtttccgtc taaacattca atttctggat ctttcgagtt atcagctgta
2340 tcatttagcc agtgttttac tcgattgaaa ccttagacag
agaagcattt tatagctttt 2400 cacatgtata ttggtagtac actgacttga
tttctatatg taagtgaatt catcacctgc 2460 atgtttcatg ccttttgcat
aagcttaaca aatggagtgt tctgtataag catggagatg 2520 tgatggaatc
tgcccaatga ctttaattgg cttattgtaa acacggaaag aactgcccca 2580
cgctgctggg aggataaaga ttgttttaga tgctcacttc tgtgttttag gattctgccc
2640 atttacttgg aatttattgg agttataatg tacttatatg atatttccga a 2691
10 496 PRT Mus musculus 10 Met Thr Phe Asn Ser Phe Glu Gly Thr Arg
Thr Phe Val Leu Ala Asp 1 5 10 15 Thr Asn Lys Asp Glu Glu Phe Val
Glu Glu Phe Asn Arg Leu Lys Thr 20 25 30 Phe Ala Asn Phe Pro Ser
Ser Ser Pro Val Ser Ala Ser Thr Leu Ala 35 40 45 Arg Ala Gly Phe
Leu Tyr Thr Gly Glu Gly Asp Thr Val Gln Cys Phe 50 55 60 Ser Cys
His Ala Ala Ile Asp Arg Trp Gln Tyr Gly Asp Ser Ala Val 65 70 75 80
Gly Arg His Arg Arg Ile Ser Pro Asn Cys Arg Phe Ile Asn Gly Phe 85
90 95 Tyr Phe Glu Asn Gly Ala Ala Gln Ser Thr Asn Pro Gly Ile Gln
Asn 100 105 110 Gly Gln Tyr Lys Ser Glu Asn Cys Val Gly Asn Arg Asn
Pro Phe Ala 115 120 125 Pro Asp Arg Pro Pro Glu Thr His Ala Asp Tyr
Leu Leu Arg Thr Gly 130 135 140 Gln Val Val Asp Ile Ser Asp Thr Ile
Tyr Pro Arg Asn Pro Ala Met 145 150 155 160 Cys Ser Glu Glu Ala Arg
Leu Lys Ser Phe Gln Asn Trp Pro Asp Tyr 165 170 175 Ala His Leu Thr
Pro Arg Glu Leu Ala Ser Ala Gly Leu Tyr Tyr Thr 180 185 190 Gly Ala
Asp Asp Gln Val Gln Cys Phe Cys Cys Gly Gly Lys Leu Lys 195 200 205
Asn Trp Glu Pro Cys Asp Arg Ala Trp Ser Glu His Arg Arg His Phe 210
215 220 Pro Asn Cys Phe Phe Val Leu Gly Arg Asn Val Asn Val Arg Ser
Glu 225 230 235 240 Ser Gly Val Ser Ser Asp Arg Asn Phe Pro Asn Ser
Thr Asn Ser Pro 245 250 255 Arg Asn Pro Ala Met Ala Glu Tyr Glu Ala
Arg Ile Val Thr Phe Gly 260 265 270 Thr Trp Ile Tyr Ser Val Asn Lys
Glu Gln Leu Ala Arg Ala Gly Phe 275 280 285 Tyr Ala Leu Gly Glu Gly
Asp Lys Val Lys Cys Phe His Cys Gly Gly 290 295 300 Gly Leu Thr Asp
Trp Lys Pro Ser Glu Asp Pro Trp Asp Gln His Ala 305 310 315 320 Lys
Cys Tyr Pro Gly Cys Lys Tyr Leu Leu Asp Glu Lys Gly Gln Glu 325 330
335 Tyr Ile Asn Asn Ile His Leu Thr His Pro Leu Glu Glu Ser Leu Gly
340 345 350 Arg Thr Ala Glu Lys Thr Pro Pro Leu Thr Lys Lys Ile Asp
Asp Thr 355 360 365 Ile Phe Gln Asn Pro Met Val Gln Glu Ala Ile Arg
Met Gly Phe Ser 370 375 380 Phe Lys Asp Leu Lys Lys Thr Met Glu Glu
Lys Ile Gln Thr Ser Gly 385 390 395 400 Ser Ser Tyr Leu Ser Leu Glu
Val Leu Ile Ala Asp Leu Val Ser Ala 405 410 415 Gln Lys Asp Asn Thr
Glu Asp Glu Ser Ser Gln Thr Ser Leu Gln Lys 420 425 430 Asp Ile Ser
Thr Glu Glu Gln Leu Arg Arg Leu Gln Glu Glu Lys Leu 435 440 445 Ser
Lys Ile Cys Met Asp Arg Asn Ile Ala Ile Val Phe Phe Pro Cys 450 455
460 Gly His Leu Ala Thr Cys Lys Gln Cys Ala Glu Ala Val Asp Lys Cys
465 470 475 480 Pro Met Cys Tyr Thr Val Ile Thr Phe Asn Gln Lys Ile
Phe Met Ser 485 490 495 11 2676 DNA Mus musculus 11 tgggagttcc
ccggagccct ggaggaaagc accgcaggtc tgagcagccc tgagccgggc 60
agggtggggg cagtggctaa ggcctagctg gggacgattt aaaggtatcg cgccacccag
120 ccacacccca caggccaggc gagggtgcca cccccggaga tcagaggtca
ttgctggcgt 180 tcagagccta ggaagtgggc tgcggtatca gcctagcagt
aaaaccgacc agaagccatg 240 cacaaaacta catccccaga gaaagacttg
tcccttcccc tccctgtcat ctcaccatga 300 acatggttca agacagcgcc
tttctagcca agctgatgaa gagtgctgac acctttgagt 360 tgaagtatga
cttttcctgt gagctgtacc gattgtccac gtattcagct tttcccaggg 420
gagttcctgt gtcagaaagg agtctggctc gtgctggctt ttactacact ggtgccaatg
480 acaaggtcaa gtgcttctgc tgtggcctga tgctagacaa ctggaaacaa
ggggacagtc 540 ccatggagaa gcacagaaag ttgtacccca gctgcaactt
tgtacagact ttgaatccag 600 ccaacagtct ggaagctagt cctcggcctt
ctcttccttc cacggcgatg agcaccatgc 660 ctttgagctt tgcaagttct
gagaatactg gctatttcag tggctcttac tcgagctttc 720 cctcagaccc
tgtgaacttc cgagcaaatc aagattgtcc tgctttgagc acaagtccct 780
accactttgc aatgaacaca gagaaggcca gattactcac ctatgaaaca tggccattgt
840 cttttctgtc accagcaaag ctggccaaag caggcttcta ctacatagga
cctggagata 900 gagtggcctg ctttgcgtgc gatgggaaac tgagcaactg
ggaacgtaag gatgatgcta 960 tgtcagagca ccagaggcat ttccccagct
gtccgttctt aaaagacttg ggtcagtctg 1020 cttcgagata cactgtctct
aacctgagca tgcagacaca cgcagcccgt attagaacat 1080 tctctaactg
gccttctagt gcactagttc attcccagga acttgcaagt gcgggctttt 1140
attatacagg acacagtgat gatgtcaagt gtttttgctg tgatggtggg ctgaggtgct
1200 gggaatctgg agatgacccc tgggtggaac atgccaagtg gtttccaagg
tgtgagtact 1260 tgctcagaat caaaggccaa gaatttgtca gccaagttca
agctggctat cctcatctac 1320 ttgagcagct attatctacg tcagactccc
cagaagatga gaatgcagac gcagcaatcg 1380 tgcattttgg ccctggagaa
agttcggaag atgtcgtcat gatgagcacg cctgtggtta 1440 aagcagcctt
ggaaatgggc ttcagtagga gcctggtgag acagacggtt cagcggcaga 1500
tcctggccac tggtgagaac tacaggaccg tcagtgacct cgttataggc ttactcgatg
1560 cagaagacga gatgagagag gagcagatgg agcaggcggc cgaggaggag
gagtcagatg 1620 atctagcact aatccggaag aacaaaatgg tgcttttcca
acatttgacg tgtgtgacac 1680 caatgctgta ttgcctccta agtgcaaggg
ccatcactga acaggagtgc aatgctgtga 1740 aacagaaacc acacacctta
caagcaagca cactgattga tactgtgtta gcaaaaggaa 1800 acactgcagc
aacctcattc agaaactccc ttcgggaaat tgaccctgcg ttatacagag 1860
atatatttgt gcaacaggac attaggagtc ttcccacaga tgacattgca gctctaccaa
1920 tggaagaaca gttgcggaaa ctccaggagg aaagaatgtg taaagtgtgt
atggaccgag 1980 aggtatccat cgtgttcatt ccctgtggcc atctggtcgt
gtgcaaagac tgcgctccct 2040 ctctgaggaa gtgtcccatc tgtagaggga
ccatcaaggg cacagtgcgc acatttctct 2100 cctgaacaag actaatggtc
catggctgca acttcagcca ggaggaagtt cactgtcact 2160 cccagctcca
ttcggaactt gaggccagcc tggatagcac gagacaccgc caaacacaca 2220
aatataaaca tgaaaaactt ttgtctgaag tcaagaatga atgaattact tatataataa
2280 ttttaattgg tttccttaaa agtgctattt gttcccaact cagaaaattg
ttttctgtaa 2340 acatatttac atactacctg catctaaagt attcatatat
tcatatattc agatgtcatg 2400 agagagggtt ttgttcttgt tcctgaaaag
cagggattgc ctgcactcct gaaattctca 2460 gaaagattta caatgttggc
atttatggtt cagaaactag aatcttctcc cgttgcttta 2520 agaaccggga
gcacagatgt ccatgtgttt tatgtataga aattcctgtt atttattgga 2580
tgacatttta gggatatgaa atttttataa agaatttgtg agaaaaagtt aataaagcaa
2640 cataattacc tctttttttt taaagaaaaa aaaaaa 2676 12 600 PRT Mus
musculus 12 Met Val Gln Asp Ser Ala Phe Leu Ala Lys Leu Met Lys Ser
Ala Asp 1 5 10 15 Thr Phe Glu Leu Lys Tyr Asp Phe Ser Cys Glu Leu
Tyr Arg Leu Ser 20 25 30 Thr Tyr Ser Ala Phe Pro Arg Gly Val Pro
Val Ser Glu Arg Ser Leu 35 40 45 Ala Arg Ala Gly Phe Tyr Tyr Thr
Gly Ala Asn Asp Lys Val Lys Cys 50 55 60 Phe Cys Cys Gly Leu Met
Leu Asp Asn Trp Lys Gln Gly Asp Ser Pro 65 70 75 80 Met Glu Lys His
Arg Lys Leu Tyr Pro Ser Cys Asn Phe Val Gln Thr 85 90 95 Leu Asn
Pro Ala Asn Ser Leu Glu Ala Ser Pro Arg Pro Ser Leu Pro 100 105 110
Ser Thr Ala Met Ser Thr Met Pro Leu Ser Phe Ala Ser Ser Glu Asn 115
120 125 Thr Gly Tyr Phe Ser Gly Ser Tyr Ser Ser Phe Pro Ser Asp Pro
Val 130 135 140 Asn Phe Arg Ala Asn Gln Asp Cys Pro Ala Leu Ser Thr
Ser Pro Tyr 145 150 155 160 His Phe Ala Met Asn Thr Glu Lys Ala Arg
Leu Leu Thr Tyr Glu Thr 165 170 175 Trp Pro Leu Ser Phe Leu Ser Pro
Ala Lys Leu Ala Lys Ala Gly Phe 180 185 190 Tyr Tyr Ile Gly Pro Gly
Asp Arg Val Ala Cys Phe Ala Cys Asp Gly 195 200 205 Lys Leu Ser Asn
Trp Glu Arg Lys Asp Asp Ala Met Ser Glu His Gln 210 215 220 Arg His
Phe Pro Ser Cys Pro Phe Leu Lys Asp Leu Gly Gln Ser Ala 225 230 235
240 Ser Arg Tyr Thr Val Ser Asn Leu Ser Met Gln Thr His Ala Ala Arg
245 250 255 Ile Arg Thr Phe Ser Asn Trp Pro Ser Ser Ala Leu Val His
Ser Gln 260 265 270 Glu Leu Ala Ser Ala Gly Phe Tyr Tyr Thr Gly His
Ser Asp Asp Val 275 280 285 Lys Cys Phe Cys Cys Asp Gly Gly Leu Arg
Cys Trp Glu Ser Gly Asp 290 295 300 Asp Pro Trp Val Glu His Ala Lys
Trp Phe Pro Arg Cys Glu Tyr Leu 305 310 315 320 Leu Arg Ile Lys Gly
Gln Glu Phe Val Ser Gln Val Gln Ala Gly Tyr 325 330 335 Pro His Leu
Leu Glu Gln Leu Leu Ser Thr Ser Asp Ser Pro Glu Asp 340 345 350 Glu
Asn Ala Asp Ala Ala Ile Val His Phe Gly Pro Gly Glu Ser Ser 355 360
365 Glu Asp Val Val Met Met Ser Thr Pro Val Val Lys Ala Ala Leu Glu
370 375 380 Met Gly Phe Ser Arg Ser Leu Val Arg Gln Thr Val Gln Arg
Gln Ile 385 390 395 400 Leu Ala Thr Gly Glu Asn Tyr Arg Thr Val Ser
Asp Leu Val Ile Gly 405 410 415 Leu Leu Asp Ala Glu Asp Glu Met Arg
Glu Glu Gln Met Glu Gln Ala 420 425 430 Ala Glu Glu Glu Glu Ser Asp
Asp Leu Ala Leu Ile Arg Lys Asn Lys 435 440 445 Met Val Leu Phe Gln
His Leu Thr Cys Val Thr Pro Met Leu Tyr Cys 450 455 460 Leu Leu Ser
Ala Arg Ala Ile Thr Glu Gln Glu Cys Asn Ala Val Lys 465 470 475 480
Gln Lys Pro His Thr Leu Gln Ala Ser Thr Leu Ile Asp Thr Val Leu 485
490 495 Ala Lys Gly Asn Thr Ala Ala Thr Ser Phe Arg Asn Ser Leu Arg
Glu 500 505 510 Ile Asp Pro Ala Leu Tyr Arg Asp Ile Phe Val Gln Gln
Asp Ile Arg 515 520 525 Ser Leu Pro Thr Asp Asp Ile Ala Ala Leu Pro
Met Glu Glu Gln Leu 530 535 540 Arg Lys Leu Gln Glu Glu Arg Met Cys
Lys Val Cys Met Asp Arg Glu 545 550 555 560 Val Ser Ile Val Phe Ile
Pro Cys Gly His Leu Val Val Cys Lys Asp 565 570 575 Cys Ala Pro Ser
Leu Arg Lys Cys Pro Ile Cys Arg Gly Thr Ile Lys 580 585 590 Gly Thr
Val Arg Thr Phe Leu Ser 595 600 13 3151 DNA Mus musculus 13
agttatataa aatacgaagt tttcaaaaag aaggctagtg caacagaaaa gctttgctaa
60 aacagattct tagttatttg aggtaacaaa agaaagccat gtcttgaatt
gattcgttct 120 taattataac agacttatag tggaaagggc cttaaacaca
ggcggacttt ataaaatgca 180 gtcttaggtt tatgtgcaaa atactgtctg
ttgaccagat gtattcacat gatatataca 240 gagtcaaggt ggtgatatag
aagatttaac agtgagggag ttaacagtct gtgctttaag 300 cgcagttcct
ttacagtgaa tactgtagtc ttaatagacc tgagctgact gctgcagttg 360
atgtaaccca ctttagagaa tactgtatga catcttctct aaggaaaacc agctgcagac
420 ttcactcagt tcctttcatt tcataggaaa aggagtagtt cagatgtcat
gtttaagtcc 480 ttataaggga aaagagcctg aatatatgcc ctagtaccta
ggcttcataa ctagtaataa 540 gaagttagtt atgggtaaat agatctcagg
ttacccagaa gagttcatgt gacccccaaa 600 gagtcctaac tagtgtcttg
gcaagtgaga cagatttgtc ctgtgagggt gtcaattcac 660 cagtccaagc
agaagacaat gaatctatcc agtcaggtgt ctgtggtgga gatctagtgt 720
ccaagtggtg agaaacttca tctggaagtt taagcggtca gaaatactat tactactcat
780 ggacaaaact gtctcccaga gactcggcca aggtacctta caccaaaaac
ttaaacgtat 840 aatggagaag agcacaatct tgtcaaattg gacaaaggag
agcgaagaaa aaatgaagtt 900 tgacttttcg tgtgaactct accgaatgtc
tacatattca gcttttccca ggggagttcc 960 tgtctcagag aggagtctgg
ctcgtgctgg cttttattat acaggtgtga atgacaaagt 1020 caagtgcttc
tgctgtggcc tgatgttgga taactggaaa caaggggaca gtcctgttga 1080
aaagcacaga cagttctatc ccagctgcag ctttgtacag actctgcttt cagccagtct
1140 gcagtctcca tctaagaata tgtctcctgt gaaaagtaga tttgcacatt
cgtcacctct 1200 ggaacgaggt ggcattcact ccaacctgtg ctctagccct
cttaattcta gagcagtgga 1260 agacttctca tcaaggatgg atccctgcag
ctatgccatg agtacagaag aggccagatt 1320 tcttacttac agtatgtggc
ctttaagttt tctgtcacca gcagagctgg ccagagctgg 1380 cttctattac
atagggcctg gagacagggt ggcctgtttt gcctgtggtg ggaaactgag 1440
caactgggaa ccaaaggatg atgctatgtc agagcaccgc agacattttc cccactgtcc
1500 atttctggaa aatacttcag aaacacagag gtttagtata tcaaatctaa
gtatgcagac 1560 acactctgct cgattgagga catttctgta ctggccacct
agtgttcctg ttcagcccga 1620 gcagcttgca agtgctggat tctattacgt
ggatcgcaat gatgatgtca agtgcttttg 1680 ttgtgatggt ggcttgagat
gttgggaacc tggagatgac ccctggatag aacacgccaa 1740 atggtttcca
aggtgtgagt tcttgatacg gatgaagggt caggagtttg ttgatgagat 1800
tcaagctaga tatcctcatc ttcttgagca gctgttgtcc acttcagaca ccccaggaga
1860 agaaaatgct gaccctacag agacagtggt gcattttggc cctggagaaa
gttcgaaaga 1920 tgtcgtcatg atgagcacgc ctgtggttaa agcagccttg
gaaatgggct tcagtaggag 1980 cctggtgaga cagacggttc agcggcagat
cctggccact ggtgagaact acaggaccgt 2040 caatgatatt gtctcagtac
ttttgaatgc tgaagatgag agaagagaag aggagaagga 2100 aagacagact
gaagagatgg catcaggtga cttatcactg attcggaaga atagaatggc 2160
cctctttcaa cagttgacac atgtccttcc tatcctggat aatcttcttg aggccagtgt
2220 aattacaaaa caggaacatg atattattag acagaaaaca cagataccct
tacaagcaag 2280 agagcttatt gacaccgttt tagtcaaggg aaatgctgca
gccaacatct tcaaaaactc 2340 tctgaaggaa attgactcca cgttatatga
aaacttattt gtggaaaaga atatgaagta 2400 tattccaaca gaagacgttt
caggcttgtc attggaagag cagttgcgga gattacaaga 2460 agaacgaact
tgcaaagtgt gtatggacag agaggtttct attgtgttca ttccgtgtgg 2520
tcatctagta gtctgccagg aatgtgcccc ttctctaagg aagtgcccca tctgcagggg
2580 gacaatcaag gggactgtgc gcacatttct ctcatgagtg aagaatggtc
tgaaagtatt 2640 gttggacatc agaagctgtc agaacaaaga atgaactact
gatttcagct cttcagcagg 2700 acattctact ctctttcaag attagtaatc
ttgctttatg aagggtagca ttgtatattt 2760 aagcttagtc tgttgcaagg
gaaggtctat gctgttgagc tacaggactg tgtctgttcc 2820 agagcaggag
ttgggatgct tgctgtatgt ccttcaggac ttcttggatt tggaatttgt 2880
gaaagctttg gattcaggtg atgtggagct cagaaatcct gaaaccagtg gctctggtac
2940 tcagtagtta gggtaccctg tgcttcttgg tgcttttcct ttctggaaaa
taaggatttt 3000 tctgctactg gtaaatattt tctgtttgtg agaaatatat
taaagtgttt cttttaaagg 3060 cgtgcatcat tgtagtgtgt gcagggatgt
atgcaggcaa aacactgtgt atataataaa 3120 taaatctttt taaaaagtgt
aaaaaaaaaa a 3151 14 612 PRT Mus musculus 14 Met Asp Lys Thr Val
Ser Gln Arg Leu Gly Gln Gly Thr Leu His Gln 1 5 10 15 Lys Leu Lys
Arg Ile Met Glu Lys Ser Thr Ile Leu Ser Asn Trp Thr 20 25 30 Lys
Glu Ser Glu Glu Lys Met Lys Phe Asp Phe Ser Cys Glu Leu Tyr 35 40
45 Arg Met Ser Thr Tyr Ser Ala Phe Pro Arg Gly Val Pro Val Ser Glu
50 55 60 Arg Ser Leu Ala Arg Ala Gly Phe Tyr Tyr Thr Gly Val Asn
Asp Lys 65 70 75 80 Val Lys Cys Phe Cys Cys Gly Leu Met Leu Asp Asn
Trp Lys Gln Gly 85 90 95 Asp Ser Pro Val Glu Lys His Arg Gln Phe
Tyr Pro Ser Cys Ser Phe 100 105 110 Val Gln Thr Leu Leu Ser Ala Ser
Leu Gln Ser Pro Ser Lys Asn Met 115 120 125 Ser Pro Val Lys Ser Arg
Phe Ala His Ser Ser Pro Leu Glu Arg Gly 130 135 140 Gly Ile His Ser
Asn Leu Cys Ser Ser Pro Leu Asn Ser Arg Ala Val 145 150 155 160 Glu
Asp Phe Ser Ser Arg Met Asp Pro Cys Ser Tyr Ala Met Ser Thr 165 170
175 Glu Glu Ala Arg Phe Leu Thr Tyr Ser Met Trp Pro Leu Ser Phe Leu
180 185 190 Ser Pro Ala Glu Leu Ala Arg Ala Gly Phe Tyr Tyr Ile Gly
Pro Gly 195 200 205 Asp Arg Val Ala Cys Phe Ala Cys Gly Gly Lys Leu
Ser Asn Trp Glu 210 215 220 Pro Lys Asp Asp Ala Met Ser Glu His Arg
Arg His Phe Pro His Cys 225 230 235 240 Pro Phe Leu Glu Asn Thr Ser
Glu Thr Gln Arg Phe Ser Ile Ser Asn 245 250 255 Leu Ser Met Gln Thr
His Ser Ala Arg Leu Arg Thr Phe Leu Tyr Trp 260 265 270 Pro Pro Ser
Val Pro Val Gln Pro Glu Gln Leu Ala Ser Ala Gly Phe 275 280 285 Tyr
Tyr Val Asp Arg Asn Asp Asp Val Lys Cys Phe Cys Cys Asp Gly 290 295
300 Gly Leu Arg Cys Trp Glu Pro Gly Asp Asp Pro Trp Ile Glu His Ala
305 310 315 320 Lys Trp Phe Pro Arg Cys Glu Phe Leu Ile
Arg Met Lys Gly Gln Glu 325 330 335 Phe Val Asp Glu Ile Gln Ala Arg
Tyr Pro His Leu Leu Glu Gln Leu 340 345 350 Leu Ser Thr Ser Asp Thr
Pro Gly Glu Glu Asn Ala Asp Pro Thr Glu 355 360 365 Thr Val Val His
Phe Gly Pro Gly Glu Ser Ser Lys Asp Val Val Met 370 375 380 Met Ser
Thr Pro Val Val Lys Ala Ala Leu Glu Met Gly Phe Ser Arg 385 390 395
400 Ser Leu Val Arg Gln Thr Val Gln Arg Gln Ile Leu Ala Thr Gly Glu
405 410 415 Asn Tyr Arg Thr Val Asn Asp Ile Val Ser Val Leu Leu Asn
Ala Glu 420 425 430 Asp Glu Arg Arg Glu Glu Glu Lys Glu Arg Gln Thr
Glu Glu Met Ala 435 440 445 Ser Gly Asp Leu Ser Leu Ile Arg Lys Asn
Arg Met Ala Leu Phe Gln 450 455 460 Gln Leu Thr His Val Leu Pro Ile
Leu Asp Asn Leu Leu Glu Ala Ser 465 470 475 480 Val Ile Thr Lys Gln
Glu His Asp Ile Ile Arg Gln Lys Thr Gln Ile 485 490 495 Pro Leu Gln
Ala Arg Glu Leu Ile Asp Thr Val Leu Val Lys Gly Asn 500 505 510 Ala
Ala Ala Asn Ile Phe Lys Asn Ser Leu Lys Glu Ile Asp Ser Thr 515 520
525 Leu Tyr Glu Asn Leu Phe Val Glu Lys Asn Met Lys Tyr Ile Pro Thr
530 535 540 Glu Asp Val Ser Gly Leu Ser Leu Glu Glu Gln Leu Arg Arg
Leu Gln 545 550 555 560 Glu Glu Arg Thr Cys Lys Val Cys Met Asp Arg
Glu Val Ser Ile Val 565 570 575 Phe Ile Pro Cys Gly His Leu Val Val
Cys Gln Glu Cys Ala Pro Ser 580 585 590 Leu Arg Lys Cys Pro Ile Cys
Arg Gly Thr Ile Lys Gly Thr Val Arg 595 600 605 Thr Phe Leu Ser 610
15 21 DNA Artificial Sequence Based on Homo sapiens sequence 15
agtgcgggtt tttattatgt g 21 16 25 DNA Artificial Sequence Based on
Homo sapiens sequence 16 agatgaccac aaggaataaa cacta 25 17 11 PRT
Artificial Sequence Based on Homo sapiens sequence 17 Met Glu Gln
Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10
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