U.S. patent application number 10/161398 was filed with the patent office on 2003-01-16 for hs2sts as modifiers of the p53 pathway and methods of use.
Invention is credited to Belvin, Marcia, Francis-Lang, Helen, Friedman, Lori, Funke, Roel P., Li, Danxi, Plowman, Gregory D..
Application Number | 20030013144 10/161398 |
Document ID | / |
Family ID | 27404397 |
Filed Date | 2003-01-16 |
United States Patent
Application |
20030013144 |
Kind Code |
A1 |
Friedman, Lori ; et
al. |
January 16, 2003 |
HS2STs as modifiers of the p53 pathway and methods of use
Abstract
Human HS2ST genes are identified as modulators of the p53
pathway, and thus are therapeutic targets for disorders associated
with defective p53 function. Methods for identifying modulators of
p53, comprising screening for agents that modulate the activity of
HS2ST are provided.
Inventors: |
Friedman, Lori; (San
Francisco, CA) ; Plowman, Gregory D.; (San Carlos,
CA) ; Belvin, Marcia; (Albany, CA) ;
Francis-Lang, Helen; (San Francisco, CA) ; Li,
Danxi; (San Francisco, CA) ; Funke, Roel P.;
(South San Francisco, CA) |
Correspondence
Address: |
JAN P. BRUNELLE
EXELIXIS, INC.
170 HARBOR WAY
P.O. BOX 511
SOUTH SAN FRANCISCO
CA
94083-0511
US
|
Family ID: |
27404397 |
Appl. No.: |
10/161398 |
Filed: |
June 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60296076 |
Jun 5, 2001 |
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60328605 |
Oct 10, 2001 |
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60357253 |
Feb 15, 2002 |
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Current U.S.
Class: |
435/7.23 ;
435/15 |
Current CPC
Class: |
G01N 33/5308 20130101;
G01N 2500/10 20130101; G01N 33/57415 20130101; G01N 2500/00
20130101; C12Q 1/485 20130101; G01N 33/5017 20130101; A61P 43/00
20180101; G01N 2333/4739 20130101; G01N 2510/00 20130101; G01N
2333/912 20130101; A61P 35/00 20180101; G01N 2333/988 20130101;
C12Q 1/6886 20130101; G01N 2333/705 20130101; C12Q 1/42 20130101;
C12Q 1/527 20130101; G01N 33/57496 20130101; G01N 33/573 20130101;
G01N 33/57484 20130101; G01N 2333/82 20130101; G01N 33/57423
20130101; G01N 33/57449 20130101; G01N 33/5011 20130101; G01N
33/574 20130101; G01N 2333/62 20130101; G01N 33/6872 20130101; G01N
2500/04 20130101; C12Q 2600/158 20130101; G01N 33/5748 20130101;
G01N 33/57419 20130101 |
Class at
Publication: |
435/7.23 ;
435/15 |
International
Class: |
G01N 033/574; C12Q
001/48 |
Claims
What is claimed is:
1. A method of identifying a candidate p53 pathway modulating
agent, said method comprising the steps of: (a) providing an assay
system comprising a purified HS2ST polypeptide or nucleic acid or a
functionally active fragment or derivative thereof; (b) contacting
the assay system with a test agent under conditions whereby, but
for the presence of the test agent, the system provides a reference
activity; and (c) detecting a test agent-biased activity of the
assay system, wherein a difference between the test agent-biased
activity and the reference activity identifies the test agent as a
candidate p53 pathway modulating agent.
2. The method of claim 1 wherein the assay system comprises
cultured cells that express the HS2ST polypeptide.
3. The method of claim 2 wherein the cultured cells additionally
have defective p53 function.
4. The method of claim 1 wherein the assay system includes a
screening assay comprising a HS2ST polypeptide, and the candidate
test agent is a small molecule modulator.
5. The method of claim 4 wherein the assay is a transferase
assay.
6. The method of claim 1 wherein the assay system is selected from
the group consisting of an apoptosis assay system, a cell
proliferation assay system, an angiogenesis assay system, and a
hypoxic induction assay system.
7. The method of claim 1 wherein the assay system includes a
binding assay comprising a HS2ST polypeptide and the candidate test
agent is an antibody.
8. The method of claim 1 wherein the assay system includes an
expression assay comprising a HS2ST nucleic acid and the candidate
test agent is a nucleic acid modulator.
9. The method of claim 8 wherein the nucleic acid modulator is an
antisense oligomer.
10. The method of claim 8 wherein the nucleic acid modulator is a
PMO.
11. The method of claim 1 additionally comprising: (d)
administering the candidate p53 pathway modulating agent identified
in (c) to a model system comprising cells defective in p53 function
and, detecting a phenotypic change in the model system that
indicates that the p53 function is restored.
12. The method of claim 11 wherein the model system is a mouse
model with defective p53 function.
13. A method for modulating a p53 pathway of a cell comprising
contacting a cell defective in p53 function with a candidate
modulator that specifically binds to a HS2ST polypeptide comprising
an amino acid sequence selected from group consisting of SEQ ID
NOs:6, 7, 8, and 9, whereby p53 function is restored.
14. The method of claim 13 wherein the candidate modulator is
administered to a vertebrate animal predetermined to have a disease
or disorder resulting from a defect in p53 function.
15. The method of claim 13 wherein the candidate modulator is
selected from the group consisting of an antibody and a small
molecule.
16. The method of claim 1, comprising the additional steps of: (d)
providing a secondary assay system comprising cultured cells or a
non-human animal expressing HS2ST, (e) contacting the secondary
assay system with the test agent of (b) or an agent derived
therefrom under conditions whereby, but for the presence of the
test agent or agent derived therefrom, the system provides a
reference activity; and (f) detecting an agent-biased activity of
the second assay system, wherein a difference between the
agent-biased activity and the reference activity of the second
assay system confirms the test agent or agent derived therefrom as
a candidate p53 pathway modulating agent, and wherein the second
assay detects an agent-biased change in the p53 pathway.
17. The method of claim 16 wherein the secondary assay system
comprises cultured cells.
18. The method of claim 16 wherein the secondary assay system
comprises a non-human animal.
19. The method of claim 18 wherein the non-human animal
mis-expresses a p53 pathway gene.
20. A method of modulating p53 pathway in a mammalian cell
comprising contacting the cell with an agent that specifically
binds a HS2ST polypeptide or nucleic acid.
21. The method of claim 20 wherein the agent is administered to a
mammalian animal predetermined to have a pathology associated with
the p53 pathway.
22. The method of claim 20 wherein the agent is a small molecule
modulator, a nucleic acid modulator, or an antibody.
23. A method for diagnosing a disease in a patient comprising: (a)
obtaining a biological sample from the patient; (b) contacting the
sample with a probe for HS2ST expression; (c) comparing results
from step (b) with a control; (d) determining whether step (c)
indicates a likelihood of disease.
24. The method of claim 23 wherein said disease is cancer.
25. The method according to claim 24, wherein said cancer is a
cancer as shown in Table 1 as having >25% expression level.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
applications Nos. 60/296,076 filed Jun. 5, 2001, 60/328,605 filed
Oct. 10, 2001, and 60/357,253 filed Feb. 15, 2002. The contents of
the prior applications are hereby incorporated in their
entirety.
BACKGROUND OF THE INVENTION
[0002] The p53 gene is mutated in over 50 different types of human
cancers, including familial and spontaneous cancers, and is
believed to be the most commonly mutated gene in human cancer
(Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al.,
Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of
mutations in the p53 gene are missense mutations that alter a
single amino acid that inactivates p53 function. Aberrant forms of
human p53 are associated with poor prognosis, more aggressive
tumors, metastasis, and short survival rates (Mitsudomi et al.,
Clin Cancer Res October 2000; 6(10):4055-63; Koshland, Science
(1993) 262:1953).
[0003] The human p53 protein normally functions as a central
integrator of signals including DNA damage, hypoxia, nucleotide
deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8).
In response to these signals, p53 protein levels are greatly
increased with the result that the accumulated p53 activates cell
cycle arrest or apoptosis depending on the nature and strength of
these signals. Indeed, multiple lines of experimental evidence have
pointed to a key role for p53 as a tumor suppressor (Levine, Cell
(1997) 88:323-331). For example, homozygous p53 "knockout" mice are
developmentally normal but exhibit nearly 100% incidence of
neoplasia in the first year of life (Donehower et al., Nature
(1992) 356:215-221).
[0004] The biochemical mechanisms and pathways through which p53
functions in normal and cancerous cells are not fully understood,
but one clearly important aspect of p53 function is its activity as
a gene-specific transcriptional activator. Among the genes with
known p53-response elements are several with well-characterized
roles in either regulation of the cell cycle or apoptosis,
including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2
(Levine, Cell (1997) 88:323-331).
[0005] Heparan sulphate (HS) proteoglycans, are ubiquitous on cell
surfaces and in the extracellular matrix, are composed of extended
polysaccharide (glycosaminoglycan) chains covalently bound to
various core proteins. HS has been associated in a variety of
biological processes, such as assembly of extracellular matrices,
control of cellular growth and differentiation, regulation of blood
coagulation, viral infection, etc. (Lindahl, U. et al. (1994)
Thromb. Res. 75, 1-32) (Lindahl, U. et al. (1998) J. Biol. Chem.
273, 24979-24982) (Salmivirta, M., et al. (1996) FASEB J. 10,
1270-1279)(Rosenberg, R, et al. (1997) J. Clin. Invest. 99,
2062-2070). The functional roles of HS appear to depend on
interactions of specific polysaccharide structures with specific
proteins. Such structures are generated in HS biosynthesis, through
the coordinated action of several enzymes. A precursor
polysaccharide composed of alternating D-glucuronic acid (GlcA) and
N-acetyl-D-glucosamine (GlcNAc; 2-deoxy-2-acetoamido-D-glucose)
units is therefore modified through a series of reactions that
include, in consecutive order, N-deacetylation deacetylation and
N-sulphation of GlcN residues, C-5 epimerization of GlcA to
L-iduronic iduronic acid (IdoA), 2-O-sulphation of uronic acid
residues, and finally 6-0- and 3-O-sulphation sulphation of GlcN
residues (Lindahl, U. et al. (1998) supra) (Salmivirta, M., et al.
(1996)supra) (Rosenberg, R, et al. (1997) supra).
[0006] Heparan sulfate 2-O-sulfotransferase (HS20ST) has been noted
as a putative sulfotransferase enzyme that may play a role in
heparan sulfate proteoglycan biosynthesis. Uronyl
2-sulfotransferase (UST or DS2ST) is a closely related enzyme that
contains sulfates iduronyl and glucuronyl that residues in
dermatan/chondroitin sulfate (Kobayashi, M. et al (1997) J. Biol.
Chem. 272, 13980-13985).
[0007] UST has ubiquitous expression of messages in a number of
human tissues and in several human cancer cell lines (Kobayashi, M.
et al. (1999) J Biol Chem 274, 10474-80).
[0008] The ability to manipulate the genomes of model organisms
such as Drosophila provides a powerful means to analyze biochemical
processes that, due to significant evolutionary conservation, has
direct relevance to more complex vertebrate organisms. Due to a
high level of gene and pathway conservation, the strong similarity
of cellular processes, and the functional conservation of genes
between these model organisms and mammals, identification of the
involvement of novel genes in particular pathways and their
functions in such model organisms can directly contribute to the
understanding of the correlative pathways and methods of modulating
them in mammals (see, for example, Mechler B M et al., 1985 EMBO J
4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K
L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M.
1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev
5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284).
For example, a genetic screen can be carried out in an invertebrate
model organism having underexpression (e.g. knockout) or
overexpression of a gene (referred to as a "genetic entry point")
that yields a visible phenotype. Additional genes are mutated in a
random or targeted manner. When a gene mutation changes the
original phenotype caused by the mutation in the genetic entry
point, the gene is identified as a "modifier" involved in the same
or overlapping pathway as the genetic entry point. When the genetic
entry point is an ortholog of a human gene implicated in a disease
pathway, such as p53, modifier genes can be identified that may be
attractive candidate targets for novel therapeutics.
[0009] All references cited herein, including sequence information
in referenced Genbank identifier numbers and website references,
are incorporated herein in their entireties.
SUMMARY OF THE INVENTION
[0010] We have discovered genes that modify the p53 pathway in
Drosophila, and identified their human orthologs, hereinafter
referred to as HS2ST. The invention provides methods for utilizing
these p53 modifier genes and polypeptides to identify candidate
therapeutic agents that can be used in the treatment of disorders
associated with defective p53 function. Preferred HS2ST-modulating
agents specifically bind to HS2ST polypeptides and restore p53
function. Other preferred HS2ST-modulating agents are nucleic acid
modulators such as antisense oligomers and RNAi that repress HS2ST
gene expression or product activity by, for example, binding to and
inhibiting the respective nucleic acid (i.e. DNA or mRNA).
[0011] HS2ST-specific modulating agents may be evaluated by any
convenient in vitro or in vivo assay for molecular interaction with
an HS2ST polypeptide or nucleic acid. In one embodiment, candidate
p53 modulating agents are tested with an assay system comprising a
HS2ST polypeptide or nucleic acid. Candidate agents that produce a
change in the activity of the assay system relative to controls are
identified as candidate p53 modulating agents. The assay system may
be cell-based or cell-free. HS2ST-modulating agents include HS2ST
related proteins (e.g. dominant negative mutants, and
biotherapeutics); HS2ST-specific antibodies; HS2ST-specific
antisense oligomers and other nucleic acid modulators; and chemical
agents that specifically bind HS2ST or compete with HS2ST binding
target. In one specific embodiment, a small molecule modulator is
identified using a transferase assay. In specific embodiments, the
screening assay system is selected from a binding assay, an
apoptosis assay, a cell proliferation assay, an angiogenesis assay,
and a hypoxic induction assay.
[0012] In another embodiment, candidate p53 pathway modulating
agents are further tested using a second assay system that detects
changes in the p53 pathway, such as angiogenic, apoptotic, or cell
proliferation changes produced by the originally identified
candidate agent or an agent derived from the original agent. The
second assay system may use cultured cells or non-human animals. In
specific embodiments, the secondary assay system uses non-human
animals, including animals predetermined to have a disease or
disorder implicating the p53 pathway, such as an angiogenic,
apoptotic, or cell proliferation disorder (e.g. cancer).
[0013] The invention further provides methods for modulating the
p53 pathway in a mammalian cell by contacting the mammalian cell
with an agent that specifically binds a HS2ST polypeptide or
nucleic acid. The agent may be a small molecule modulator, a
nucleic acid modulator, or an antibody and may be administered to a
mammalian animal predetermined to have a pathology associated the
p53 pathway.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Genetic screens were designed to identify modifiers of the
p53 pathway in Drosophila in which p53 was overexpressed in the
wing (Ollmann M, et al., Cell 2000 101: 91-101). The pipe gene was
identified as a modifier of the p53 pathway. Accordingly,
vertebrate orthologs of these modifiers, and preferably the human
orthologs, HS2ST genes (i.e., nucleic acids and polypeptides) are
attractive drug targets for the treatment of pathologies associated
with a defective p53 signaling pathway, such as cancer.
[0015] In vitro and in vivo methods of assessing HS2ST function are
provided herein. Modulation of the HS2ST or their respective
binding partners is useful for understanding the association of the
p53 pathway and its members in normal and disease conditions and
for developing diagnostics and therapeutic modalities for p53
related pathologies. HS2ST-modulating agents that act by inhibiting
or enhancing HS2ST expression, directly or indirectly, for example,
by affecting an HS2ST function such as enzymatic (e.g., catalytic)
or binding activity, can be identified using methods provided
herein. HS2ST modulating agents are useful in diagnosis, therapy
and pharmaceutical development.
[0016] Nucleic Acids and Polypeptides of the Invention
[0017] Sequences related to HS2ST nucleic acids and polypeptides
that can be used in the invention are disclosed in Genbank
(referenced by Genbank identifier (GI) number) as GI#s 6683563 (SEQ
ID NO:1), 12545388 (SEQ ID NO:2), and 4803734 (SEQ ID NO:5) for
nucleic acid, and GI#s 6683564 (SEQ ID NO:6), 6912420 (SEQ ID
NO:7), 4803735 (SEQ ID NO:8), and 5032219 (SEQ ID NO:9) for
polypeptides. Additionally, newly identified nucleic acid sequences
of SEQ ID NOs:3 and 4 can also be used in the invention.
[0018] HS2STs are sulfotransferase proteins with transferase
domains. The term "HS2ST polypeptide" refers to a full-length HS2ST
protein or a functionally active fragment or derivative thereof. A
"functionally active" HS2ST fragment or derivative exhibits one or
more functional activities associated with a full-length, wild-type
HS2ST protein, such as antigenic or immunogenic activity, enzymatic
activity, ability to bind natural cellular substrates, etc. The
functional activity of HS2ST proteins, derivatives and fragments
can be assayed by various methods known to one skilled in the art
(Current Protocols in Protein Science (1998) Coligan et al., eds.,
John Wiley & Sons, Inc., Somerset, N.J.) and as further
discussed below. For purposes herein, functionally active fragments
also include those fragments that comprise one or more structural
domains of an HS2ST, such as a binding domain. Protein domains can
be identified using the PFAM program (Bateman A., et al., Nucleic
Acids Res, 1999, 27:260-2; http://pfam.wustl.edu). Methods for
obtaining HS2ST polypeptides are also further described below. In
some embodiments, preferred fragments are functionally active,
domain-containing fragments comprising at least 25 contiguous amino
acids, preferably at least 50, more preferably 75, and most
preferably at least 100 contiguous amino acids of any one of SEQ ID
NOs:6, 7, 8, or 9 (an HS2ST). In further preferred embodiments, the
fragment comprises the entire transferase (functionally active)
domain.
[0019] The term "HS2ST nucleic acid" refers to a DNA or RNA
molecule that encodes a HS2ST polypeptide. Preferably, the HS2ST
polypeptide or nucleic acid or fragment thereof is from a human,
but can also be an ortholog, or derivative thereof with at least
70% sequence identity, preferably at least 80%, more preferably
85%, still more preferably 90%, and most preferably at least 95%
sequence identity with HS2ST. Normally, orthologs in different
species retain the same function, due to presence of one or more
protein motifs and/or 3-dimensional structures. Orthologs are
generally identified by sequence homology analysis, such as BLAST
analysis, usually using protein bait sequences. Sequences are
assigned as a potential ortholog if the best hit sequence from the
forward BLAST result retrieves the original query sequence in the
reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998)
95:5849-5856; Huynen M A et al., Genome Research (2000)
10:1204-1210). Programs for multiple sequence alignment, such as
CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680)
may be used to highlight conserved regions and/or residues of
orthologous proteins and to generate phylogenetic trees. In a
phylogenetic tree representing multiple homologous sequences from
diverse species (e.g., retrieved through BLAST analysis),
orthologous sequences from two species generally appear closest on
the tree with respect to all other sequences from these two
species. Structural threading or other analysis of protein folding
(e.g., using software by ProCeryon, Biosciences, Salzburg, Austria)
may also identify potential orthologs. In evolution, when a gene
duplication event follows speciation, a single gene in one species,
such as Drosophila, may correspond to multiple genes (paralogs) in
another, such as human. As used herein, the term "orthologs"
encompasses paralogs. As used herein, "percent (%) sequence
identity" with respect to a subject sequence, or a specified
portion of a subject sequence, is defined as the percentage of
nucleotides or amino acids in the candidate derivative sequence
identical with the nucleotides or amino acids in the subject
sequence (or specified portion thereof), after aligning the
sequences and introducing gaps, if necessary to achieve the maximum
percent sequence identity, as generated by the program
WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410;
http://blast.wustl.edu/blast/README.html) with all the search
parameters set to default values. The HSP S and HSP S2 parameters
are dynamic values and are established by the program itself
depending upon the composition of the particular sequence and
composition of the particular database against which the sequence
of interest is being searched. A % identity value is determined by
the number of matching identical nucleotides or amino acids divided
by the sequence length for which the percent identity is being
reported. "Percent (%) amino acid sequence similarity" is
determined by doing the same calculation as for determining % amino
acid sequence identity, but including conservative amino acid
substitutions in addition to identical amino acids in the
computation.
[0020] A conservative amino acid substitution is one in which an
amino acid is substituted for another amino acid having similar
properties such that the folding or activity of the protein is not
significantly affected. Aromatic amino acids that can be
substituted for each other are phenylalanine, tryptophan, and
tyrosine; interchangeable hydrophobic amino acids are leucine,
isoleucine, methionine, and valine; interchangeable polar amino
acids are glutamine and asparagine; interchangeable basic amino
acids are arginine, lysine and histidine; interchangeable acidic
amino acids are aspartic acid and glutamic acid; and
interchangeable small amino acids are alanine, serine, threonine,
cysteine and glycine.
[0021] Alternatively, an alignment for nucleic acid sequences is
provided by the local homology algorithm of Smith and Waterman
(Smith and Waterman, 1981, Advances in Applied Mathematics
2:482-489; database: European Bioinformatics Institute
http://www.ebi.ac.uk/MPsrch/; Smith and Waterman, 1981, J. of
Molec.Biol., 147:195-197; Nicholas et al., 1998, "A Tutorial on
Searching Sequence Databases and Sequence Scoring Methods"
(www.psc.edu) and references cited therein.; W. R. Pearson, 1991,
Genomics 11:635-650). This algorithm can be applied to amino acid
sequences by using the scoring matrix developed by Dayhoff
(Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986
Nucl. Acids Res. 14(6):6745-6763). The Smith-Waterman algorithm may
be employed where default parameters are used for scoring (for
example, gap open penalty of 12, gap extension penalty of two).
From the data generated, the "Match" value reflects "sequence
identity."
[0022] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that hybridize to the nucleic acid
sequence of any of SEQ ID NOs: 1, 2, 3, 4, or 5. The stringency of
hybridization can be controlled by temperature, ionic strength, pH,
and the presence of denaturing agents such as formamide during
hybridization and washing. Conditions routinely used are set out in
readily available procedure texts (e.g., Current Protocol in
Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons,
Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). In some embodiments, a nucleic acid molecule of the
invention is capable of hybridizing to a nucleic acid molecule
containing the nucleotide sequence of any one of SEQ ID NOs: 1, ,2
3, 4, or 5 under stringent hybridization conditions that comprise:
prehybridization of filters containing nucleic acid for 8 hours to
overnight at 65.degree. C. in a solution comprising 6.times. single
strength citrate (SSC) (1.times. SSC is 0.15 M NaCl, 0.015 M Na
citrate; pH 7.0), 5.times. Denhardt's solution, 0.05% sodium
pyrophosphate and 100 .mu.g/ml herring sperm DNA; hybridization for
18-20 hours at 65.degree. C. in a solution containing 6.times. SSC,
1.times. Denhardt's solution, 100 .mu.g/ml yeast tRNA and 0.05%
sodium pyrophosphate; and washing of filters at 65.degree. C. for 1
h in a solution containing 0.2.times. SSC and 0.1% SDS (sodium
dodecyl sulfate).
[0023] In other embodiments, moderately stringent hybridization
conditions are used that comprise: pretreatment of filters
containing nucleic acid for 6 h at 40.degree. C. in a solution
containing 35% formamide, 5.times. SSC, 50 mM Tris-HCl (pH 7.5), 5
mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA; hybridization for 18-20h at 40.degree. C. in a
solution containing 35% formamide, 5.times. SSC, 50 mM Tris-HCl (pH
7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml
salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by
washing twice for 1 hour at 55.degree. C. in a solution containing
2.times. SSC and 0.1% SDS.
[0024] Alternatively, low stringency conditions can be used that
comprise: incubation for 8 hours to overnight at 37.degree. C. in a
solution comprising 20% formamide, 5.times. SSC, 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured sheared salmon sperm DNA;
hybridization in the same buffer for 18 to 20 hours; and washing of
filters in 1.times. SSC at about 37.degree. C. for 1 hour.
[0025] Isolation, Production, Expression, and Mis-Expression of
HS2ST Nucleic Acids and Polypeptides
[0026] HS2ST nucleic acids and polypeptides, useful for identifying
and testing agents that modulate HS2ST function and for other
applications related to the involvement of HS2ST in the p53
pathway. HS2ST nucleic acids and derivatives and orthologs thereof
may be obtained using any available method. For instance,
techniques for isolating cDNA or genomic DNA sequences of interest
by screening DNA libraries or by using polymerase chain reaction
(PCR) are well known in the art. In general, the particular use for
the protein will dictate the particulars of expression, production,
and purification methods. For instance, production of proteins for
use in screening for modulating agents may require methods that
preserve specific biological activities of these proteins, whereas
production of proteins for antibody generation may require
structural integrity of particular epitopes. Expression of proteins
to be purified for screening or antibody production may require the
addition of specific tags (e.g., generation of fusion proteins).
Overexpression of an HS2ST protein for assays used to assess HS2ST
function, such as involvement in cell cycle regulation or hypoxic
response, may require expression in eukaryotic cell lines capable
of these cellular activities. Techniques for the expression,
production, and purification of proteins are well known in the art;
any suitable means therefore may be used (e.g., Higgins S J and
Hames B D (eds.) Protein Expression: A Practical Approach, Oxford
University Press Inc., New York 1999; Stanbury P F et al.,
Principles of Fermentation Technology, 2.sup.nd edition, Elsevier
Science, New York, 1995; Doonan S (ed.) Protein Purification
Protocols, Humana Press, New Jersey, 1996; Coligan J E et al,
Current Protocols in Protein Science (eds.), 1999, John Wiley &
Sons, New York). In particular embodiments, recombinant HS2ST is
expressed in a cell line known to have defective p53 function (e.g.
SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical
cancer cells, HT-29 and DLD-1 colon cancer cells, among others,
available from American Type Culture Collection (ATCC), Manassas,
Va.). The recombinant cells are used in cell-based screening assay
systems of the invention, as described further below.
[0027] The nucleotide sequence encoding an HS2ST polypeptide can be
inserted into any appropriate expression vector. The necessary
transcriptional and translational signals, including
promoter/enhancer element, can derive from the native HS2ST gene
and/or its flanking regions or can be heterologous. A variety of
host-vector expression systems may be utilized, such as mammalian
cell systems infected with virus (e.g. vaccinia virus, adenovirus,
etc.); insect cell systems infected with virus (e.g. baculovirus);
microorganisms such as yeast containing yeast vectors, or bacteria
transformed with bacteriophage, plasmid, or cosmid DNA. A host cell
strain that modulates the expression of, modifies, and/or
specifically processes the gene product may be used.
[0028] To detect expression of the HS2ST gene product, the
expression vector can comprise a promoter operably linked to an
HS2ST gene nucleic acid, one or more origins of replication, and,
one or more selectable markers (e.g. thymidine kinase activity,
resistance to antibiotics, etc.). Alternatively, recombinant
expression vectors can be identified by assaying for the expression
of the HS2ST gene product based on the physical or functional
properties of the HS2ST protein in in vitro assay systems (e.g.
immunoassays).
[0029] The HS2ST protein, fragment, or derivative may be optionally
expressed as a fusion, or chimeric protein product (i.e. it is
joined via a peptide bond to a heterologous protein sequence of a
different protein), for example to facilitate purification or
detection. A chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino acid
sequences to each other using standard methods and expressing the
chimeric product. A chimeric product may also be made by protein
synthetic techniques, e.g. by use of a peptide synthesizer
(Hunkapiller et al., Nature (1984) 310:105-111).
[0030] Once a recombinant cell that expresses the HS2ST gene
sequence is identified, the gene product can be isolated and
purified using standard methods (e.g. ion exchange, affinity, and
gel exclusion chromatography; centrifugation; differential
solubility; electrophoresis, cite purification reference).
Alternatively, native HS2ST proteins can be purified from natural
sources, by standard methods (e.g. immunoaffinity purification).
Once a protein is obtained, it may be quantified and its activity
measured by appropriate methods, such as immunoassay, bioassay, or
other measurements of physical properties, such as
crystallography.
[0031] The methods of this invention may also use cells that have
been engineered for altered expression (mis-expression) of HS2ST or
other genes associated with the p53 pathway. As used herein,
mis-expression encompasses ectopic expression, over-expression,
under-expression, and non-expression (e.g. by gene knock-out or
blocking expression that would otherwise normally occur).
[0032] Genetically Modified Animals
[0033] Animal models that have been genetically modified to alter
HS2ST expression may be used in in vivo assays to test for activity
of a candidate p53 modulating agent, or to further assess the role
of HS2ST in a p53 pathway process such as apoptosis or cell
proliferation. Preferably, the altered HS2ST expression results in
a detectable phenotype, such as decreased or increased levels of
cell proliferation, angiogenesis, or apoptosis compared to control
animals having normal HS2ST expression. The genetically modified
animal may additionally have altered p53 expression (e.g. p53
knockout). Preferred genetically modified animals are mammals such
as primates, rodents (preferably mice), cows, horses, goats, sheep,
pigs, dogs and cats. Preferred non-mammalian species include
zebrafish, C. elegans, and Drosophila. Preferred genetically
modified animals are transgenic animals having a heterologous
nucleic acid sequence present as an extrachromosomal element in a
portion of its cells, i.e. mosaic animals (see, for example,
techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763.)
or stably integrated into its germ line DNA (i.e., in the genomic
sequence of most or all of its cells). Heterologous nucleic acid is
introduced into the germ line of such transgenic animals by genetic
manipulation of, for example, embryos or embryonic stem cells of
the host animal.
[0034] Methods of making transgenic animals are well-known in the
art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci.
USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and
Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle
bombardment see U.S. Pat. No., 4,945,050, by Sandford et al.; for
transgenic Drosophila see Rubin and Spradling, Science (1982)
218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see
Berghammer A. J. et al., A Universal Marker for Transgenic Insects
(1999) Nature 402:370-371; for transgenic Zebrafish see Lin S.,
Transgenic Zebrafish, Methods Mol Biol. (2000);136:375-3830); for
microinjection procedures for fish, amphibian eggs and birds see
Houdebine and Chourrout, Experientia (1991) 47:897-905; for
transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and
for culturing of embryonic stem (ES) cells and the subsequent
production of transgenic animals by the introduction of DNA into ES
cells using methods such as electroporation, calcium phosphate/DNA
precipitation and direct injection see, e.g., Teratocarcinomas and
Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,
IRL Press (1987)). Clones of the nonhuman transgenic animals can be
produced according to available methods (see Wilmut, I. et al.
Nature 385:810-813; and PCT International Publication Nos. WO
97/07668 and WO 97/07669).
[0035] In one embodiment, the transgenic animal is a "knock-out"
animal having a heterozygous or homozygous alteration in the
sequence of an endogenous HS2ST gene that results in a decrease of
HS2ST function, preferably such that HS2ST expression is
undetectable or insignificant. Knock-out animals are typically
generated by homologous recombination with a vector comprising a
transgene having at least a portion of the gene to'be knocked out.
Typically a deletion, addition or substitution has been introduced
into the transgene to functionally disrupt it. The transgene can be
a human gene (e.g., from a human genomic clone) but more preferably
is an ortholog of the human gene derived from the transgenic host
species. For example, a mouse HS2ST gene is used to construct a
homologous recombination vector suitable for altering an endogenous
HS2ST gene in the mouse genome. Detailed methodologies for
homologous recombination in mice are available (see Capecchi,
Science (1989) 244:1288-1292; Joyner et al., Nature (1989)
338:153-156). Procedures for the production of non-rodent
transgenic mammals and other animals are also available (Houdebine
and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288;
Simms et al., Bio/Technology
[0036] 6:179-183). In a preferred embodiment, knock-out animals,
such as mice harboring a knockout of a specific gene, may be used
to produce antibodies against the human counterpart of the gene
that has been knocked out (Claesson M H et al., (1994) Scan J
Immunol 40:257-264; Declerck P J et al., (1995) J Biol Chem.
270:8397-400).
[0037] In another embodiment, the transgenic animal is a "knock-in"
animal having an alteration in its genome that results in altered
expression (e.g., increased (including ectopic) or decreased
expression) of the HS2ST gene, e.g., by introduction of additional
copies of HS2ST, or by operatively inserting a regulatory sequence
that provides for altered expression of an endogenous copy of the
HS2ST gene. Such regulatory sequences include inducible,
tissue-specific, and constitutive promoters and enhancer elements.
The knock-in can be homozygous or heterozygous.
[0038] Transgenic nonhuman animals can also be produced that
contain selected systems allowing for regulated expression of the
transgene. One example of such a system that may be produced is the
cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS
(1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase. Another example of a recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a
preferred embodiment, both Cre-LoxP and Flp-Frt are used in the
same system to regulate expression of the transgene, and for
sequential deletion of vector sequences in the same cell (Sun X et
al (2000) Nat Genet 25:83-6).
[0039] The genetically modified animals can be used in genetic
studies to further elucidate the p53 pathway, as animal models of
disease and disorders implicating defective p53 function, and for
in vivo testing of candidate therapeutic agents, such as those
identified in screens described below. The candidate therapeutic
agents are administered to a genetically modified animal having
altered HS2ST function and phenotypic changes are compared with
appropriate control animals such as genetically modified animals
that receive placebo treatment, and/or animals with unaltered HS2ST
expression that receive candidate therapeutic agent.
[0040] In addition to the above-described genetically modified
animals having altered HS2ST function, animal models having
defective p53 function (and otherwise normal HS2ST function), can
be used in the methods of the present invention. For example, a p53
knockout mouse can be used to assess, in vivo, the activity of a
candidate p53 modulating agent identified in one of the in vitro
assays described below. p53 knockout mice are described in the
literature (Jacks et al., Nature 2001;410:1111-1116, 1043-1044;
Donehower et al., supra). Preferably, the candidate p53 modulating
agent when administered to a model system with cells defective in
p53 function, produces a detectable phenotypic change in the model
system indicating that the p53 function is restored, i.e., the
cells exhibit normal cell cycle progression.
[0041] Modulating Agents
[0042] The invention provides methods to identify agents that
interact with and/or modulate the function of HS2ST and/or the p53
pathway. Such agents are useful in a variety of diagnostic and
therapeutic applications associated with the p53 pathway, as well
as in further analysis of the HS2ST protein and its contribution to
the p53 pathway. Accordingly, the invention also provides methods
for modulating the p53 pathway comprising the step of specifically
modulating HS2ST activity by administering a HS2ST-interacting or
-modulating agent.
[0043] In a preferred embodiment, HS2ST-modulating agents inhibit
or enhance HS2ST activity or otherwise affect normal HS2ST
function, including transcription, protein expression, protein
localization, and cellular or extra-cellular activity. In a further
preferred embodiment, the candidate p53 pathway- modulating agent
specifically modulates the function of the HS2ST. The phrases
"specific modulating agent", "specifically modulates", etc., are
used herein to refer to modulating agents that directly bind to the
HS2ST polypeptide or nucleic acid, and preferably inhibit, enhance,
or otherwise alter, the function of the HS2ST. The term also
encompasses modulating agents that alter the interaction of the
HS2ST with a binding partner or substrate (e.g. by binding to a
binding partner of an HS2ST, or to a protein/binding partner
complex, and inhibiting function).
[0044] Preferred HS2ST-modulating agents include small molecule
compounds; HS2ST-interacting proteins, including antibodies and
other biotherapeutics; and nucleic acid modulators such as
antisense and RNA inhibitors. The modulating agents may be
formulated in pharmaceutical compositions, for example, as
compositions that may comprise other active ingredients, as in
combination therapy, and/or suitable carriers or excipients.
Techniques for formulation and administration of the compounds may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., 19.sup.th edition.
[0045] Small Molecule Modulators
[0046] Small molecules, are often preferred to modulate function of
proteins with enzymatic function, and/or containing protein
interaction domains. Chemical agents, referred to in the art as
"small molecule" compounds are typically organic, non-peptide
molecules, having a molecular weight less than 10,000, preferably
less than 5,000, more preferably less than 1,000, and most
preferably less than 500. This class of modulators includes
chemically synthesized molecules, for instance, compounds from
combinatorial chemical libraries. Synthetic compounds may be
rationally designed or identified based on known or inferred
properties of the HS2ST protein or may be identified by screening
compound libraries. Alternative appropriate modulators of this
class are natural products, particularly secondary metabolites from
organisms such as plants or fungi, which can also be identified by
screening compound libraries for HS2ST-modulating activity. Methods
for generating and obtaining compounds are well known in the art
(Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther
J, Science (2000)151:1947-1948).
[0047] Small molecule modulators identified from screening assays,
as described below, can be used as lead compounds from which
candidate clinical compounds may be designed, optimized, and
synthesized. Such clinical compounds may have utility in treating
pathologies associated with the p53 pathway. The activity of
candidate small molecule modulating agents may be improved
several-fold through iterative secondary functional validation, as
further described below, structure determination, and candidate
modulator modification and testing. Additionally, candidate
clinical compounds are generated with specific regard to clinical
and pharmacological properties. For example, the reagents may be
derivatized and re-screened using in vitro and in vivo assays to
optimize activity and minimize toxicity for pharmaceutical
development.
[0048] Protein Modulators
[0049] Specific HS2ST-interacting proteins are useful in a variety
of diagnostic and therapeutic applications related to the p53
pathway and related disorders, as well as in validation assays for
other HS2ST-modulating agents. In a preferred embodiment,
HS2ST-interacting proteins affect normal HS2ST function, including
transcription, protein expression, protein localization, and
cellular or extra-cellular activity. In another embodiment,
HS2ST-interacting proteins are useful in detecting and providing
information about the function of HS2ST proteins, as is relevant to
p53 related disorders, such as cancer (e.g., for diagnostic
means).
[0050] An HS2ST-interacting protein may be endogenous, i.e. one
that naturally interacts genetically or biochemically with an
HS2ST, such as a member of the HS2ST pathway that modulates HS2ST
expression, localization, and/or activity. HS2ST-modulators include
dominant negative forms of HS2ST-interacting proteins and of HS2ST
proteins themselves. Yeast two-hybrid and variant screens offer
preferred methods for identifying endogenous HS2ST-interacting
proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression
Systems: A Practical Approach, eds. Glover D. & Hames B. D
(Oxford University Press, Oxford, England), pp. 169-203; Fashema SF
et al., Gene
[0051] 250:1-14; Drees B L Curr Opin Chem Biol (1999) 3:64-70;
Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S.
Pat. No. 5,928,868). Mass spectrometry is an alternative preferred
method for the elucidation of protein complexes (reviewed in, e.g.,
Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R
3.sup.rd, Trends Genet (2000) 16:5-8).
[0052] An HS2ST-interacting protein may be an exogenous protein,
such as an HS2ST-specific antibody or a T-cell antigen receptor
(see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using
antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold
Spring Harbor Laboratory Press). HS2ST antibodies are further
discussed below.
[0053] In preferred embodiments, an HS2ST-interacting protein
specifically binds an HS2ST protein. In alternative preferred
embodiments, an HS2ST-modulating agent binds an HS2ST substrate,
binding partner, or cofactor.
[0054] Antibodies
[0055] In another embodiment, the protein modulator is an HS2ST
specific antibody agonist or antagonist. The antibodies have
therapeutic and diagnostic utilities, and can be used in screening
assays to identify HS2ST modulators. The antibodies can also be
used in dissecting the portions of the HS2ST pathway responsible
for various cellular responses and in the general processing and
maturation of the HS2ST.
[0056] Antibodies that specifically bind HS2ST polypeptides can be
generated using known methods. Preferably the antibody is specific
to a mammalian ortholog of HS2ST polypeptide, and more preferably,
to human HS2ST. Antibodies may be polyclonal, monoclonal (mAbs),
humanized or chimeric antibodies, single chain antibodies, Fab
fragments, F(ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. Epitopes of HS2ST
which are particularly antigenic can be selected, for example, by
routine screening of HS2ST polypeptides for antigenicity or by
applying a theoretical method for selecting antigenic regions of a
protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A.
78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;
Sutcliffe et al., (1983) Science 219:660-66) to the amino acid
sequence shown in any of SEQ ID NOs:6, ,7 8, or 9. Monoclonal
antibodies with affinities of 10.sup.8 M.sup.-1 preferably 10.sup.9
M.sup.-1 to 10.sup.10 M.sup.-1, or stronger can be made by standard
procedures as described (Harlow and Lane, supra; Goding (1986)
Monoclonal Antibodies: Principles and Practice (2d ed) Academic
Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and
4,618,577). Antibodies may be generated against crude cell extracts
of HS2ST or substantially purified fragments thereof. If HS2ST
fragments are used, they preferably comprise at least 10, and more
preferably, at least 20 contiguous amino acids of an HS2ST protein.
In a particular embodiment, HS2ST-specific antigens and/or
immunogens are coupled to carrier proteins that stimulate the
immune response. For example, the subject polypeptides are
covalently coupled to the keyhole limpet hemocyanin (KLH) carrier,
and the conjugate is emulsified in Freund's complete adjuvant,
which enhances the immune response. An appropriate immune system
such as a laboratory rabbit or mouse is immunized according to
conventional protocols.
[0057] The presence of HS2ST-specific antibodies is assayed by an
appropriate assay such as a solid phase enzyme-linked immunosorbant
assay (ELISA) using immobilized corresponding HS2ST polypeptides.
Other assays, such as radioimmunoassays or fluorescent assays might
also be used.
[0058] Chimeric antibodies specific to HS2ST polypeptides can be
made that contain different portions from different animal species.
For instance, a human immunoglobulin constant region may be linked
to a variable region of a murine mAb, such that the antibody
derives its biological activity from the human antibody, and its
binding specificity from the murine fragment. Chimeric antibodies
are produced by splicing together genes that encode the appropriate
regions from each species (Morrison et al., Proc. Natl. Acad. Sci.
(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608;
Takeda et al., Nature (1985) 31:452-454). Humanized antibodies,
which are a form of chimeric antibodies, can be generated by
grafting complementary-determining regions (CDRs) (Carlos, T. M.,
J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a
background of human framework regions and constant regions by
recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323:
323-327). Humanized antibodies contain .about.10% murine sequences
and .about.90% human sequences, and thus further reduce or
eliminate immunogenicity, while retaining the antibody
specificities (Co M S, and Queen C. 1991 Nature 351: 501-501;
Morrison SL. 1992 Ann. Rev. Immun. 10:239-265). Humanized
antibodies and methods of their production are well-known in the
art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and
6,180,370).
[0059] HS2ST-specific single chain antibodies which are
recombinant, single chain polypeptides formed by linking the heavy
and light chain fragments of the Fv regions via an amino acid
bridge, can be produced by methods known in the art (U.S. Pat. No.
4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc.
Natl. Acad. Sci. USA
[0060] 85:5879-5883; and Ward et al., Nature (1989)
334:544-546).
[0061] Other suitable techniques for antibody production involve in
vitro exposure of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in phage or
similar vectors (Huse et al., Science (1989) 246:1275-1281). As
used herein, T-cell antigen receptors are included within the scope
of antibody modulators (Harlow and Lane, 1988, supra).
[0062] The polypeptides and antibodies of the present invention may
be used with or without modification. Frequently, antibodies will
be labeled by joining, either covalently or non-covalently, a
substance that provides for a detectable signal, or that is toxic
to cells that express the targeted protein (Menard S, et al., Int
J. Biol Markers
[0063] 4:131-134). A wide variety of labels and conjugation
techniques are known and are reported extensively in both the
scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, fluorescent emitting lanthanide metals,
chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,
recombinant immunoglobulins may be produced (U.S. Pat. No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered
and reach their targets by conjugation with membrane-penetrating
toxin proteins (U.S. Pat. No. 6,086,900).
[0064] When used therapeutically in a patient, the antibodies of
the subject invention are typically administered parenterally, when
possible at the target site, or intravenously. The therapeutically
effective dose and dosage regimen is determined by clinical
studies. Typically, the amount of antibody administered is in the
range of about 0.1 mg/kg--to about 10 mg/kg of patient weight. For
parenteral administration, the antibodies are formulated in a unit
dosage injectable form (e.g., solution, suspension, emulsion) in
association with a pharmaceutically acceptable vehicle. Such
vehicles are inherently nontoxic and non-therapeutic. Examples are
water, saline, Ringer's solution, dextrose solution, and 5% human
serum albumin. Nonaqueous vehicles such as fixed oils, ethyl
oleate, or liposome carriers may also be used. The vehicle may
contain minor amounts of additives, such as buffers and
preservatives, which enhance isotonicity and chemical stability or
otherwise enhance therapeutic potential. The antibodies'
concentrations in such vehicles are typically in the range of about
1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further
described in the literature (U.S. Pat. No. 5,859,206;
WO0073469).
[0065] Nucleic Acid Modulators
[0066] Other preferred HS2ST-modulating agents comprise nucleic
acid molecules, such as antisense oligomers or double stranded RNA
(dsRNA), which generally inhibit HS2ST activity. Preferred nucleic
acid modulators interfere with the function of the HS2ST nucleic
acid such as DNA replication, transcription, translocation of the
HS2ST RNA to the site of protein translation, translation of
protein from the HS2ST RNA, splicing of the HS2ST RNA to yield one
or more mRNA species, or catalytic activity which may be engaged in
or facilitated by the HS2ST RNA.
[0067] In one embodiment, the antisense oligomer is an
oligonucleotide that is sufficiently complementary to an HS2ST mRNA
to bind to and prevent translation, preferably by binding to the 5'
untranslated region. HS2ST-specific antisense oligonucleotides,
preferably range from at least 6 to about 200 nucleotides. In some
embodiments the oligonucleotide is preferably at least 10, 15, or
20 nucleotides in length. In other embodiments, the oligonucleotide
is preferably less than 50, 40, or 30 nucleotides in length. The
oligonucleotide can be DNA or RNA or a chimeric mixture or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may include other appending groups such as peptides, agents that
facilitate transport across the cell membrane,
hybridization-triggered cleavage agents, and intercalating
agents.
[0068] In another embodiment, the antisense oligomer is a
phosphothioate morpholino oligomer (PMO). PMOs are assembled from
four different morpholino subunits, each of which contain one of
four genetic bases (A, C, G, or T) linked to a six-membered
morpholine ring. Polymers of these subunits are joined by non-ionic
phosphodiamidate intersubunit linkages. Details of how to make and
use PMOs and other antisense oligomers are well known in the art
(e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide
and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J,
and Weller D. 1997 Antisense Nucleic Acid Drug Dev.:7:187-95; U.S.
Pat. Nos. 5,235,033; and 5,378,841).
[0069] Alternative preferred HS2ST nucleic acid modulators are
double-stranded RNA species mediating RNA interference (RNAi). RNAi
is the process of sequence-specific, post-transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene.
Methods relating to the use of RNAi to silence genes in C. elegans,
Drosophila, plants, and humans are known in the art (Fire A, et
al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363
(1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490
(2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119
(2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A.
et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature
404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);
Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M.,
et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619;
Elbashir S M, et al., 2001 Nature 411:494-498).
[0070] Nucleic acid modulators are commonly used as research
reagents, diagnostics, and therapeutics. For example, antisense
oligonucleotides, which are able to inhibit gene expression with
exquisite specificity, are often used to elucidate the function of
particular genes (see, for example, U.S. Pat. No. 6,165,790).
Nucleic acid modulators are also used, for example, to distinguish
between functions of various members of a biological pathway. For
example, antisense oligomers have been employed as therapeutic
moieties in the treatment of disease states in animals and man and
have been demonstrated in numerous clinical trials to be safe and
effective (Milligan J F, et al, Current Concepts in Antisense Drug
Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L et al.,
Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,
Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the
invention, an HS2ST-specific nucleic acid modulator is used in an
assay to further elucidate the role of the HS2ST in the p53
pathway, and/or its relationship to other members of the pathway.
In another aspect of the invention, an HS2ST-specific antisense
oligomer is used as a therapeutic agent for treatment of
p53-related disease states.
[0071] Assay Systems
[0072] The invention provides assay systems and screening methods
for identifying specific modulators of HS2ST activity. As used
herein, an "assay system" encompasses all the components required
for performing and analyzing results of an assay that detects
and/or measures a particular event. In general, primary assays are
used to identify or confirm a modulator's specific biochemical or
molecular effect with respect to the HS2ST nucleic acid or protein.
In general, secondary assays further assess the activity of a HS2ST
modulating agent identified by a primary assay and may confirm that
the modulating agent affects HS2ST in a manner relevant to the p53
pathway. In some cases, HS2ST modulators will be directly tested in
a secondary assay.
[0073] In a preferred embodiment, the screening method comprises
contacting a suitable assay system comprising an HS2ST polypeptide
with a candidate agent under conditions whereby, but for the
presence of the agent, the system provides a reference activity
(e.g. transferase activity), which is based on the particular
molecular event the screening method detects. A statistically
significant difference between the agent-biased activity and the
reference activity indicates that the candidate agent modulates
HS2ST activity, and hence the p53 pathway.
[0074] Primary Assays
[0075] The type of modulator tested generally determines the type
of primary assay.
[0076] Primary Assays for Small Molecule Modulators
[0077] For small molecule modulators, screening assays are used to
identify candidate modulators. Screening assays may be cell-based
or may use a cell-free system that recreates or retains the
relevant biochemical reaction of the target protein (reviewed in
Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and
accompanying references). As used herein the term "cell-based"
refers to assays using live cells, dead cells, or a particular
cellular fraction, such as a membrane, endoplasmic reticulum, or
mitochondrial fraction. The term "cell free" encompasses assays
using substantially purified protein (either endogenous or
recombinantly produced), partially purified or crude cellular
extracts. Screening assays may detect a variety of molecular
events, including protein-DNA interactions, protein-protein
interactions (e.g., receptor-ligand binding), transcriptional
activity (e.g., using a reporter gene), enzymatic activity (e.g.,
via a property of the substrate), activity of second messengers,
immunogenicty and changes in cellular morphology or other cellular
characteristics. Appropriate screening assays may use a wide range
of detection methods including fluorescent, radioactive,
calorimetric, spectrophotometric, and amperometric methods, to
provide a read-out for the particular molecular event detected.
[0078] Cell-based screening assays usually require systems for
recombinant expression of HS2ST and any auxiliary proteins demanded
by the particular assay. Appropriate methods for generating
recombinant proteins produce sufficient quantities of proteins that
retain their relevant biological activities and are of sufficient
purity to optimize activity and assure assay reproducibility. Yeast
two-hybrid and variant screens, and mass spectrometry provide
preferred methods for determining protein-protein interactions and
elucidation of protein complexes. In certain applications, when
HS2ST-interacting proteins are used in screens to identify small
molecule modulators, the binding specificity of the interacting
protein to the HS2ST protein may be assayed by various known
methods such as substrate processing (e.g. ability of the candidate
HS2ST-specific binding agents to function as negative effectors in
HS2ST-expressing cells), binding equilibrium constants (usually at
least about 10.sup.7 M.sup.-1, preferably at least about 10.sup.8
M.sup.-1, more preferably at least about 10.sup.9 M.sup.-1), and
immunogenicity (e.g. ability to elicit HS2ST specific antibody in a
heterologous host such as a mouse, rat, goat or rabbit). For
enzymes and receptors, binding may be assayed by, respectively,
substrate and ligand processing.
[0079] The screening assay may measure a candidate agent's ability
to specifically bind to or modulate activity of a HS2ST
polypeptide, a fusion protein thereof, or to cells or membranes
bearing the polypeptide or fusion protein. The HS2ST polypeptide
can be full length or a fragment thereof that retains functional
HS2ST activity. The HS2ST polypeptide may be fused to another
polypeptide, such as a peptide tag for detection or anchoring, or
to another tag. The HS2ST polypeptide is preferably human HS2ST, or
is an ortholog or derivative thereof as described above. In a
preferred embodiment, the screening assay detects candidate
agent-based modulation of HS2ST interaction with a binding target,
such as an endogenous or exogenous protein or other substrate that
has HS2ST--specific binding activity, and can be used to assess
normal HS2ST gene function.
[0080] Suitable assay formats that may be adapted to screen for
HS2ST modulators are known in the art. Preferred screening assays
are high throughput or ultra high throughput and thus provide
automated, cost-effective means of screening compound libraries for
lead compounds (Fernandes P B, Curr Opin Chem Biol (1998)
2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In
one preferred embodiment, screening assays uses fluorescence
technologies, including fluorescence polarization, time-resolved
fluorescence, and fluorescence resonance energy transfer. These
systems offer means to monitor protein-protein or DNA-protein
interactions in which the intensity of the signal emitted from
dye-labeled molecules depends upon their interactions with partner
molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4;
Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem
Biol (2000) 4:445-451).
[0081] A variety of suitable assay systems may be used to identify
candidate HS2ST and p53 pathway modulators (e.g. U.S. Pat. Nos.
5,550,019 and 6,133,437 (apoptosis assays); and U.S. Pat. No.
6,020,135 (p53 modulation), among others). Specific preferred
assays are described in more detail below.
[0082] Sulfotransferase Assays.
[0083] Assays for sulfotransferase activity are known in the art.
An example of a High throughput method is a continuous coupled
enzyme assay for the spectrophotometric analysis of
sulfotransferases using aryl sulfotransferase IV (Burkart M D, and
Wong C H. (1999) Anal Biochem. 274:131-7). This assay is based on
the regeneration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS)
from the desulfated 3'-phosphoadenosine-5'-phosphate (PAP) by a
recombinant aryl sulfotransferase using p-nitrophenyl sulfate as
the sulfate donor and visible spectrophotometric indicator of
enzyme turnover.
[0084] Apoptosis Assays.
[0085] Assays for apoptosis may be performed by terminal
deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end
labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear
DNA fragmentation characteristic of apoptosis (Lazebnik et al.,
1994, Nature 371, 346), by following the incorporation of
fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747).
Apoptosis may further be assayed by acridine orange staining of
tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41).
An apoptosis assay system may comprise a cell that expresses an
HS2ST, and that optionally has defective p53 function (e.g. p53 is
over-expressed or under-expressed relative to wild-type cells). A
test agent can be added to the apoptosis assay system and changes
in induction of apoptosis relative to controls where no test agent
is added, identify candidate p53 modulating agents. In some
embodiments of the invention, an apoptosis assay may be used as a
secondary assay to test a candidate p53 modulating agents that is
initially identified using a cell-free assay system. An apoptosis
assay may also be used to test whether HS2ST function plays a
direct role in apoptosis. For example, an apoptosis assay may be
performed on cells that over- or under-express HS2ST relative to
wild type cells. Differences in apoptotic response compared to wild
type cells suggests that the HS2ST plays a direct role in the
apoptotic response. Apoptosis assays are described further in U.S.
Pat. No. 6,133,437.
[0086] Cell proliferation and cell cycle assays.
[0087] Cell proliferation may be assayed via bromodeoxyuridine
(BRDU) incorporation. This assay identifies a cell population
undergoing DNA synthesis by incorporation of BRDU into
newly-synthesized DNA. Newly-synthesized DNA may then be detected
using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer
38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or by
other means.
[0088] Cell Proliferation may also be examined using
[.sup.3H]-thymidine incorporation (Chen, J., 1996, Oncogene
13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This
assay allows for quantitative characterization of S-phase DNA
syntheses. In this assay, cells synthesizing DNA will incorporate
[.sup.3H]-thymidine into newly synthesized DNA. Incorporation can
then be measured by standard techniques such as by counting of
radioisotope in a scintillation counter (e.g., Beckman LS 3800
Liquid Scintillation Counter).
[0089] Cell proliferation may also be assayed by colony formation
in soft agar (Sambrook et al., Molecular Cloning, Cold Spring
Harbor (1989)). For example, cells transformed with HS2ST are
seeded in soft agar plates, and colonies are measured and counted
after two weeks incubation.
[0090] Involvement of a gene in the cell cycle may be assayed by
flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud
Phys Chem Med 49:237-55). Cells transfected with an HS2ST may be
stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson).
[0091] Accordingly, a cell proliferation or cell cycle assay system
may comprise a cell that expresses an HS2ST, and that optionally
has defective p53 function (e.g. p53 is over-expressed or
under-expressed relative to wild-type cells). A test agent can be
added to the assay system and changes in cell proliferation or cell
cycle relative to controls where no test agent is added, identify
candidate p53 modulating agents. In some embodiments of the
invention, the cell proliferation or cell cycle assay may be used
as a secondary assay to test a candidate p53 modulating agents that
is initially identified using another assay system such as a
cell-free kinase assay system. A cell proliferation assay may also
be used to test whether HS2ST function plays a direct role in cell
proliferation or cell cycle. For example, a cell proliferation or
cell cycle assay may be performed on cells that over- or
under-express HS2ST relative to wild type cells. Differences in
proliferation or cell cycle compared to wild type cells suggests
that the HS2ST plays a direct role in cell proliferation or cell
cycle.
[0092] Angiogenesis.
[0093] Angiogenesis may be assayed using various human endothelial
cell systems, such as umbilical vein, coronary artery, or dermal
cells. Suitable assays include Alamar Blue based assays (available
from Biosource International) to measure proliferation; migration
assays using fluorescent molecules, such as the use of Becton
Dickinson Falcon HTS FluoroBlock cell culture inserts to measure
migration of cells through membranes in presence or absence of
angiogenesis enhancer or suppressors; and tubule formation assays
based on the formation of tubular structures by endothelial cells
on Matrigel.RTM. (Becton Dickinson). Accordingly, an angiogenesis
assay system may comprise a cell that expresses an HS2ST, and that
optionally has defective p53 function (e.g. p53 is over-expressed
or under-expressed relative to wild-type cells). A test agent can
be added to the angiogenesis assay system and changes in
angiogenesis relative to controls where no test agent is added,
identify candidate p53 modulating agents. In some embodiments of
the invention, the angiogenesis assay may be used as a secondary
assay to test a candidate p53 modulating agents that is initially
identified using another assay system. An angiogenesis assay may
also be used to test whether HS2ST function plays a direct role in
cell proliferation. For example, an angiogenesis assay may be
performed on cells that over- or under-express HS2ST relative to
wild type cells. Differences in angiogenesis compared to wild type
cells suggests that the HS2ST plays a direct role in
angiogenesis.
[0094] Hypoxic induction.
[0095] The alpha subunit of the transcription factor, hypoxia
inducible factor-1 (HIF-1), is upregulated in tumor cells following
exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1
stimulates the expression of genes known to be important in tumour
cell survival, such as those encoding glyolytic enzymes and VEGF.
Induction of such genes by hypoxic conditions may be assayed by
growing cells transfected with HS2ST in hypoxic conditions (such as
with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001
incubator (Precision Scientific)) and normoxic conditions, followed
by assessment of gene activity or expression by Taqman.RTM.. For
example, a hypoxic induction assay system may comprise a cell that
expresses an HS2ST, and that optionally has a mutated p53 (e.g. p53
is over-expressed or under-expressed relative to wild-type cells).
A test agent can be added to the hypoxic induction assay system and
changes in hypoxic response relative to controls where no test
agent is added, identify candidate p53 modulating agents. In some
embodiments of the invention, the hypoxic induction assay may be
used as a secondary assay to test a candidate p53 modulating agents
that is initially identified using another assay system. A hypoxic
induction assay may also be used to test whether HS2ST function
plays a direct role in the hypoxic response. For example, a hypoxic
induction assay may be performed on cells that over- or
under-express HS2ST relative to wild type cells. Differences in
hypoxic response compared to wild type cells suggests that the
HS2ST plays a direct role in hypoxic induction.
[0096] Cell Adhesion.
[0097] Cell adhesion assays measure adhesion of cells to purified
adhesion proteins, or adhesion of cells to each other, in presence
or absence of candidate modulating agents. Cell-protein adhesion
assays measure the ability of agents to modulate the adhesion of
cells to purified proteins. For example, recombinant proteins are
produced, diluted to 2.5g/mL in PBS, and used to coat the wells of
a microtiter plate. The wells used for negative control are not
coated. Coated wells are then washed, blocked with 1% BSA, and
washed again. Compounds are diluted to 2.times. final test
concentration and added to the blocked, coated wells. Cells are
then added to the wells, and the unbound cells are washed off.
Retained cells are labeled directly on the plate by adding a
membrane-permeable fluorescent dye, such as calcein-AM, and the
signal is quantified in a fluorescent microplate reader.
[0098] Cell-cell adhesion assays measure the ability of agents to
modulate binding of cell adhesion proteins with their native
ligands. These assays use cells that naturally or recombinantly
express the adhesion protein of choice. In an exemplary assay,
cells expressing the cell adhesion protein are plated in wells of a
multiwell plate. Cells expressing the ligand are labeled with a
membrane-permeable fluorescent dye, such as BCECF, and allowed to
adhere to the monolayers in the presence of candidate agents.
Unbound cells are washed off, and bound cells are detected using a
fluorescence plate reader.
[0099] High-throughput cell adhesion assays have also been
described. In one such assay, small molecule ligands and peptides
are bound to the surface of microscope slides using a microarray
spotter, intact cells are then contacted with the slides, and
unbound cells are washed off. In this assay, not only the binding
specificity of the peptides and modulators against cell lines are
determined, but also the functional cell signaling of attached
cells using immunofluorescence techniques in situ on the microchip
is measured (Falsey J R et al., Bioconjug Chem. May-June
2001;12(3):346-53).
[0100] Primary Assays for Antibody Modulators
[0101] For antibody modulators, appropriate primary assays test is
a binding assay that tests the antibody's affinity to and
specificity for the HS2ST protein. Methods for testing antibody
affinity and specificity are well known in the art (Harlow and
Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay
(ELISA) is a preferred method for detecting HS2ST-specific
antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
[0102] Primary Assays for Nucleic Acid Modulators
[0103] For nucleic acid modulators, primary assays may test the
ability of the nucleic acid modulator to inhibit or enhance HS2ST
gene expression, preferably mRNA expression. In general, expression
analysis comprises comparing HS2ST expression in like populations
of cells (e.g., two pools of cells that endogenously or
recombinantly express HS2ST) in the presence and absence of the
nucleic acid modulator. Methods for analyzing mRNA and protein
expression are well known in the art. For instance, Northern
blotting, slot blotting, ribonuclease protection, quantitative
RT-PCR (e.g., using the TaqMan.RTM., PE Applied Biosystems), or
microarray analysis may be used to confirm that HS2ST mRNA
expression is reduced in cells treated with the nucleic acid
modulator (e.g., Current Protocols in Molecular Biology (1994)
Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4;
Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O
P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-Elie, A Curr
Opin Biotechnol 2001, 12:41-47). Protein expression may also be
monitored. Proteins are most commonly detected with specific
antibodies or antisera directed against either the HS2ST protein or
specific peptides. A variety of means including Western blotting,
ELISA, or in situ detection, are available (Harlow E and Lane D,
1988 and 1999, supra).
[0104] Secondary Assays
[0105] Secondary assays may be used to further assess the activity
of HS2ST-modulating agent identified by any of the above methods to
confirm that the modulating agent affects HS2ST in a manner
relevant to the p53 pathway. As used herein, HS2ST-modulating
agents encompass candidate clinical compounds or other agents
derived from previously identified modulating agent. Secondary
assays can also be used to test the activity of a modulating agent
on a particular genetic or biochemical pathway or to test the
specificity of the modulating agent's interaction with HS2ST.
[0106] Secondary assays generally compare like populations of cells
or animals (e.g., two pools of cells or animals that endogenously
or recombinantly express HS2ST) in the presence and absence of the
candidate modulator. In general, such assays test whether treatment
of cells or animals with a candidate HS2ST-modulating agent results
in changes in the p53 pathway in comparison to untreated (or mock-
or placebo-treated) cells or animals. Certain assays use
"sensitized genetic backgrounds", which, as used herein, describe
cells or animals engineered for altered expression of genes in the
p53 or interacting pathways.
[0107] Cell-based assays
[0108] Cell based assays may use a variety of mammalian cell lines
known to have defective p53 function (e.g. SAOS-2 osteoblasts,
H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29
and DLD-1 colon cancer cells, among others, available from American
Type Culture Collection (ATCC), Manassas, Va.). Cell based assays
may detect endogenous p53 pathway activity or may rely on
recombinant expression of p53 pathway components. Any of the
aforementioned assays may be used in this cell-based format.
Candidate modulators are typically added to the cell media but may
also be injected into cells or delivered by any other efficacious
means.
[0109] Animal Assays
[0110] A variety of non-human animal models of normal or defective
p53 pathway may be used to test candidate HS2ST modulators. Models
for defective p53 pathway typically use genetically modified
animals that have been engineered to mis-express (e.g.,
over-express or lack expression in) genes involved in the p53
pathway. Assays generally require systemic delivery of the
candidate modulators, such as by oral administration, injection,
etc.
[0111] In a preferred embodiment, p53 pathway activity is assessed
by monitoring neovascularization and angiogenesis. Animal models
with defective and normal p53 are used to test the candidate
modulator's affect on HS2ST in Matrigel.RTM. assays. Matrigel.RTM.
is an extract of basement membrane proteins, and is composed
primarily of laminin, collagen IV, and heparin sulfate
proteoglycan. It is provided as a sterile liquid at 4.degree. C.,
but rapidly forms a solid gel at 37.degree. C. Liquid Matrigel.RTM.
is mixed with various angiogenic agents, such as bFGF and VEGF, or
with human tumor cells which over-express the HS2ST. The mixture is
then injected subcutaneously(SC) into female athymic nude mice
(Taconic, Germantown, N.Y.) to support an intense vascular
response. Mice with Matrigel.RTM. pellets may be dosed via oral
(PO), intraperitoneal (IP), or intravenous (IV) routes with the
candidate modulator. Mice are euthanized 5-12 days post-injection,
and the Matrigel.RTM. pellet is harvested for hemoglobin analysis
(Sigma plasma hemoglobin kit). Hemoglobin content of the gel is
found to correlate the degree of neovascularization in the gel.
[0112] In another preferred embodiment, the effect of the candidate
modulator on HS2ST is assessed via tumorigenicity assays. In one
example, xenograft human tumors are implanted SC into female
athymic mice, 6-7 week old, as single cell suspensions either from
a pre-existing tumor or from in vitro culture. The tumors which
express the HS2ST endogenously are injected in the flank,
1.times.10.sup.5 to 1.times.10.sup.7 cells per mouse in a volume of
100 .mu.L using a 27gauge needle. Mice are then ear tagged and
tumors are measured twice weekly. Candidate modulator treatment is
initiated on the day the mean tumor weight reaches 100 mg.
Candidate modulator is delivered IV, SC, IP, or PO by bolus
administration. Depending upon the pharmacokinetics of each unique
candidate modulator, dosing can be performed multiple times per
day. The tumor weight is assessed by measuring perpendicular
diameters with a caliper and calculated by multiplying the
measurements of diameters in two dimensions. At the end of the
experiment, the excised tumors maybe utilized for biomarker
identification or further analyses. For immunohistochemistry
staining, xenograft tumors are fixed in 4% paraformaldehyde, 0. IM
phosphate, pH 7.2, for 6 hours at 4.degree. C., immersed in 30%
sucrose in PBS, and rapidly frozen in isopentane cooled with liquid
nitrogen.
[0113] Diagnostic and Therapeutic Uses
[0114] Specific HS2ST-modulating agents are useful in a variety of
diagnostic and therapeutic applications where disease or disease
prognosis is related to defects in the p53 pathway, such as
angiogenic, apoptotic, or cell proliferation disorders.
Accordingly, the invention also provides methods for modulating the
p53 pathway in a cell, preferably a cell pre-determined to have
defective p53 function, comprising the step of administering an
agent to the cell that specifically modulates HS2ST activity.
Preferably, the modulating agent produces a detectable phenotypic
change in the cell indicating that the p53 function is restored,
i.e., for example, the cell undergoes normal proliferation or
progression through the cell cycle.
[0115] The discovery that HS2ST is implicated in p53 pathway
provides for a variety of methods that can be employed for the
diagnostic and prognostic evaluation of diseases and disorders
involving defects in the p53 pathway and for the identification of
subjects having a predisposition to such diseases and
disorders.
[0116] Various expression analysis methods can be used to diagnose
whether HS2ST expression occurs in a particular sample, including
Northern blotting, slot blotting, ribonuclease protection,
quantitative RT-PCR, and microarray analysis. (e.g., Current
Protocols in Molecular Biology (1994) Ausubel F M et al., eds.,
John Wiley & Sons, Inc., chapter 4; Freeman W M et al.,
Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001,
33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001,
12:41-47). Tissues having a disease or disorder implicating
defective p53 signaling that express an HS2ST, are identified as
amenable to treatment with an HS2ST modulating agent. In a
preferred application, the p53 defective tissue overexpresses an
HS2ST relative to normal tissue. For example, a Northern blot
analysis of mRNA from tumor and normal cell lines, or from tumor
and matching normal tissue samples from the same patient, using
full or partial HS2ST cDNA sequences as probes, can determine
whether particular tumors express or overexpress HS2ST.
Alternatively, the TaqMan.RTM. is used for quantitative RT-PCR
analysis of HS2ST expression in cell lines, normal tissues and
tumor samples (PE Applied Biosystems).
[0117] Various other diagnostic methods may be performed, for
example, utilizing reagents such as the HS2ST oligonucleotides, and
antibodies directed against an HS2ST, as described above for: (1)
the detection of the presence of HS2ST gene mutations, or the
detection of either over- or under-expression of HS2ST mRNA
relative to the non-disorder state; (2) the detection of either an
over- or an under-abundance of HS2ST gene product relative to the
non-disorder state; and (3) the detection of perturbations or
abnormalities in the signal transduction pathway mediated by
HS2ST.
[0118] Thus, in a specific embodiment, the invention is drawn to a
method for diagnosing a disease in a patient, the method
comprising: a) obtaining a biological sample from the patient; b)
contacting the sample with a probe for HS2ST expression; c)
comparing results from step (b) with a control; and d) determining
whether step (c) indicates a likelihood of disease. Preferably, the
disease is cancer, most preferably a cancer as shown in TABLE 1.
The probe may be either DNA or protein, including an antibody.
EXAMPLES
[0119] The following experimental section and examples are offered
by way of illustration and not by way of limitation.
[0120] I. Drosophila p53 Screen
[0121] The Drosophila p53 gene was overexpressed specifically in
the wing using the vestigial margin quadrant enhancer. Increasing
quantities of Drosophila p53 (titrated using different strength
transgenic inserts in 1 or 2 copies) caused deterioration of normal
wing morphology from mild to strong, with phenotypes including
disruption of pattern and polarity of wing hairs, shortening and
thickening of wing veins, progressive crumpling of the wing and
appearance of dark "death" inclusions in wing blade. In a screen
designed to identify enhancers and suppressors of Drosophila p53,
homozygous females carrying two copies of p53 were crossed to 5663
males carrying random insertions of a piggyBac transposon (Fraser M
et al., Virology (1985)145:356-361). Progeny containing insertions
were compared to non-insertion-bearing sibling progeny for
enhancement or suppression of the p53 phenotypes. Sequence
information surrounding the piggyBac insertion site was used to
identify the modifier genes. Modifiers of the wing phenotype were
identified as members of the p53 pathway. Drosophila.pipe was an
enhancer of the wing phenotype. Human orthologs of the modifiers,
are referred to herein as HS2ST.
[0122] BLAST analysis (Altschul et al., supra) was employed to
identify Targets from Drosophila modifiers. For example,
representative sequences from HS2ST (GI#6912420, SEQ ID NO:7) and
HS2ST (GI#5032219, SEQ ID NO:9) share 28% and 30% amino acid
identity, respectively, with the Drosophila.pipe amino acid.
[0123] Various domains, signals, and functional subunits in
proteins were analyzed using the PSORT (Nakai K., and Horton P.,
Trends Biochem Sci, 1999, 24:34-6; Kenta Nakai, Protein sorting
signals and prediction of subcellular localization, Adv. Protein
Chem. 54, 277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids
Res, 1999, 27:260-2; http://pfam.wustl.edu), SMART (Ponting C P, et
al., SMART: identification and annotation of domains from signaling
and extracellular protein sequences. Nucleic Acids Res. 1999 Jan
1;27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne,
and Anders Krogh: A hidden Markov model for predicting
transmembrane helices in protein sequences. In Proc. of Sixth Int.
Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J.
Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen Menlo Park, Calif.: AAAI Press, 1998), and clust (Remm M,
and Sonnhammer E. Classification of transmembrane protein families
in the Caenorhabditis elegans genome and identification of human
orthologs. Genome Res. November 2000; 10(11): 1679-89)
programs.
[0124] II. High-Throughput In Vitro Fluorescence Polarization
Assay
[0125] Fluorescently-labeled HS2ST peptide/substrate are added to
each well of a 96-well microtiter plate, along with a test agent in
a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH
7.6). Changes in fluorescence polarization, determined by using a
Fluorolite FPM-2 Fluorescence Polarization Microtiter System
(Dynatech Laboratories, Inc), relative to control values indicates
the test compound is a candidate modifier of HS2ST activity.
[0126] III. High-Throughput In Vitro Binding Assay.
[0127] .sup.33P-labeled HS2ST peptide is added in an assay buffer
(100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl.sub.2, 1% glycerol, 0.5%
NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of
protease inhibitors) along with a test agent to the wells of a
Neutralite-avidin coated assay plate and incubated at 25.degree. C.
for 1 hour. Biotinylated substrate is then added to each well and
incubated for 1 hour. Reactions are stopped by washing with PBS,
and counted in a scintillation counter. Test agents that cause a
difference in activity relative to control without test agent are
identified as candidate p53 modulating agents.
[0128] IV. Immunoprecipitations and Immunoblotting
[0129] For coprecipitation of transfected proteins,
3.times.10.sup.6 appropriate recombinant cells containing the HS2ST
proteins are plated on 10-cm dishes and transfected on the
following day with expression constructs. The total amount of DNA
is kept constant in each transfection by adding empty vector. After
24 h, cells are collected, washed once with phosphate-buffered
saline and lysed for 20 min on ice in 1 ml of lysis buffer
containing 50 mM Hepes, pH 7.9, 250 mM NaCl, 20 mM
-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenyl
phosphate, 2 mM dithiothreitol, protease inhibitors (complete,
Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris
is removed by centrifugation twice at 15,000.times. g for 15 min.
The cell lysate is incubated with 25 .mu.l of M2 beads (Sigma) for
2 h at 4.degree. C. with gentle rocking.
[0130] After extensive washing with lysis buffer, proteins bound to
the beads are solubilized by boiling in SDS sample buffer,
fractionated by SDS-polyacrylamide gel electrophoresis, transferred
to polyvinylidene difluoride membrane and blotted with the
indicated antibodies. The reactive bands are visualized with
horseradish peroxidase coupled to the appropriate secondary
antibodies and the enhanced chemiluminescence (ECL) Western
blotting detection system (Amersham Pharmacia Biotech).
[0131] V. Expression Analysis
[0132] All cell lines used in the following experiments are NCI
(National Cancer Institute) lines, and are available from ATCC
(American Type Culture Collection, Manassas, Va. 20110-2209).
Normal and tumor tissues were obtained from Impath, UC Davis,
Clontech, Stratagene, and Ambion.
[0133] TaqMan analysis was used to assess expression levels of the
disclosed genes in various samples.
[0134] RNA was extracted from each tissue sample using Qiagen
(Valencia, Calif.) RNeasy kits, following manufacturer's protocols,
to a final concentration of 50 ng/.mu.l. Single stranded cDNA was
then synthesized by reverse transcribing the RNA samples using
random hexamers and 500 ng of total RNA per reaction, following
protocol 4304965 of Applied Biosystems (Foster City, Calif.,
http://www.appliedbiosystems.com/).
[0135] Primers for expression analysis using TaqMan assay (Applied
Biosystems, Foster City, Calif.) were prepared according to the
TaqMan protocols, and the following criteria:
[0136] a) primer pairs were designed to span introns to eliminate
genomic contamination, and
[0137] b) each primer pair produced only one product.
[0138] Taqman reactions were carried out following manufacturer's
protocols, in 25 .mu.l total volume for 96-well plates and 10 .mu.l
total volume for 384-well plates, using 300 nM primer and 250 nM
probe, and approximately 25 ng of cDNA. The standard curve for
result analysis was prepared using a universal pool of human cDNA
samples, which is a mixture of cDNAs from a wide variety of tissues
so that the chance that a target will be present in appreciable
amounts is good. The raw data were normalized using 18S rRNA
(universally expressed in all tissues and cells).
[0139] For each expression analysis, tumor tissue samples were
compared with matched normal tissues from the same patient. A gene
was considered overexpressed in a tumor when the level of
expression of the gene was 2 fold or higher in the tumor compared
with its matched normal sample. In cases where normal tissue was
not available, a universal pool of cDNA samples was used instead.
In these cases, a gene was considered overexpressed in a tumor
sample when the difference of expression levels between a tumor
sample and the average of all normal samples from the same tissue
type was greater than 2 times the standard deviation of all normal
samples (i.e., Tumor--average(all normal samples)>2.times.
STDEV(all normal samples)).
[0140] Results are shown in Table 1. Data presented in bold
indicate that greater than 50% of tested tumor samples of the
tissue type indicated in row 1 exhibited over expression of the
gene listed in column 1, relative to normal samples. Underlined
data indicates that between 25% to 49% of tested tumor samples
exhibited over expression. A modulator identified by an assay
described herein can be further validated for therapeutic effect by
administration to a tumor in which the gene is overexpressed. A
decrease in tumor growth confirms therapeutic utility of the
modulator. Prior to treating a patient with the modulator, the
likelihood that the patient will respond to treatment can be
diagnosed by obtaining a tumor sample from the patient, and
assaying for expression of the gene targeted by the modulator. The
expression data for the gene(s) can also be used as a diagnostic
marker for disease progression. The assay can be performed by
expression analysis as described above, by antibody directed to the
gene target, or by any other available detection method.
1TABLE 1 -- breast colon lung ovary HS2ST 2 12 12 30 5 14 1 7 (SEQ
ID NO: 3) GI #4803743 1 12 2 30 1 14 0 7 (SEQ ID NO: 5)
[0141]
Sequence CWU 1
1
9 1 2185 DNA Homo sapiens 1 gggaaggaag gaagagaggg aggcgggcaa
gcaggcgggc gcgggggtcg gagactgagg 60 cagtagaggg aggcgagagc
ccggcagccg cttcgcgctg tttgctggcg cgggttttgg 120 agggggcggc
cgtttagtcg gctgaggaga agcggacacc agcggcgttg gtgatagcgc 180
ctgggggagg gggactggag aggcgagaag gggggttcgc tgcggtggtt ctctcgctgt
240 cgctctctct ttgcctcgct cccggctcgg cgggctcctc ccggcgtctc
tctcgcctcc 300 ggggtcccgc tccccgcccc ccgcggtatg tcttgatccc
gagcagcggg tttcatgggg 360 ctcctcagga ttatgatgcc gcccaagttg
cagctgctgg cggtggtggc cttcgcggtg 420 gcgatgctct tcttggaaaa
ccagatccag aaactggagg agtcccgctc gaagctagaa 480 agggctattg
caagacacga agtccgagaa attgagcagc gacatacaat ggatggccct 540
cggcaagatg ccactttaga tgaggaagag gacatggtga tcatttataa cagagttccc
600 aaaacggcaa gcacttcatt taccaatatc gcctatgacc tgtgtgcaaa
gaataaatac 660 catgtccttc atatcaacac taccaaaaat aatccagtga
tgtcattgca agatcaggtg 720 cgctttgtaa agaatataac ttcctggaaa
gagatgaaac caggatttta tcatggacac 780 gtttcttact tggattttgc
aaaatttggt gtgaagaaga aaccaattta cattaatgtc 840 ataagggatc
ctattgagag gctagtttct tattattact ttctgagatt tggagatgat 900
tatagaccag ggttacggag acgaaaacaa ggagacaaaa agacctttga tgaatgtgta
960 gcagaaggtg gctcagactg tgctccagag aagctctggc ttcaaatccc
gttcttctgt 1020 ggccatagct ccgaatgctg gaatgtggga agcaggtggg
ctatggatca agccaagtat 1080 aacctaatta atgaatattt tctggtggga
gttactgaag aacttgaaga ttttatcatg 1140 ttattggagg cagcattgcc
ccggtttttc aggggtgcta ctgaactcta tcgcacagga 1200 aagaaatctc
atcttaggaa aaccacagag aagaaactcc ccactaaaca aaccattgca 1260
aaactacagc aatctgatat ttggaaaatg gagaatgagt tctatgaatt tgcactagag
1320 cagttccaat tcatcagagc ccatgccgtt cgagaaaaag atggagacct
ctacatcctc 1380 gcacaaaact ttttctatga aaagatttac cctaagtcga
actgagtata aggtgtgact 1440 attagattct tgaactaaaa tttgaccctg
tcttcacctt tgttctcagc tccacagtct 1500 ggattgctga cagtaggtgt
atatgacaat ttgtattgag ccaaattagg aaacagacag 1560 taacgtcaag
gaagtagata ctggctggca ttgtcagtgt tctaagtttc aggcattttt 1620
attttttcct ggctaaacgt tggtgaaagt tataacctcc tgcctgggag aaaatataca
1680 tcacctaaaa tgaacttatg gcaggtctaa tcaaaaggct aaatacaatt
tcagaaaagg 1740 ttctgatact cttgtttttg ataaagcatt ttttcaacta
accatgaatt aagatgagtc 1800 catttgcctc ttctgccttc actgagggtt
tgggttatac acctctactg aattgtgtta 1860 ataactgttt ggcagtgtgt
actttgtttt tgtgagtcat gtctcatgaa atttattgga 1920 atgtttaatc
atatttgcta agaaatgttt ctgctgtagt tggatttgcc catatttatg 1980
taggtggttt taatttttta aatggtgatt agtgttaaaa atcaatttaa atcatgacta
2040 atatggtaaa aagataaagc atcaaagcag tatttctcat tcctgcctcc
tcaatatcta 2100 atactgggaa gatacttcaa agaatattga gattgtctga
agttttagtt aagattttca 2160 cacattaata tcaaaaaaaa aaaaa 2185 2 6708
DNA Homo sapiens 2 agggagggaa ggaaggaaga gagggaggcg ggcaagcagg
cgggcgcggg ggtcggggac 60 tgaggcagta gagggaggcg agagcccggc
agccgcttcg cgctgtttgc tgcgcgggct 120 tttggagggg gcggccgttt
agtcggctga ggagaagcgg acaccagcgg cgttggtgat 180 agcgcctggg
ggagggggac tggagaggcg agaagggggg tcgctgcggt ggttctctcg 240
ctgtcgctct ctctttgcct cgctcccggc tcggcgggct cctcccggcg tctctctcgc
300 ctccggggtc ccgctccccg ccccccgcgg tatgtcttga tcccgagcag
cgggtttcat 360 ggggctcctc aggattatga tgccgcccaa gttgcagctg
ctggcggtgg tggccttcgc 420 ggtggcgatg ctcttcttgg aaaaccagat
ccagaaactg gaggagtccc gctcgaagct 480 agaaagggct attgcaagac
acgaagtccg agaaattgag cagcgacata caatggatgg 540 ccctcggcaa
gatgccactt tagatgagga agaggacatg gtgatcattt ataacagagt 600
tcccaaaacg gcaagcactt catttaccaa tatcgcctat gacctgtgtg caaagaataa
660 ataccatgtc cttcatatca acactaccaa aaataatcca gtgatgtcat
tgcaagatca 720 ggtgcgcttt gtaaagaata taacttcctg gaaagagatg
aaaccaggat tttatcatgg 780 acacgtttct tacttggatt ttgcaaaatt
tggtgtgaag aagaaaccaa tttacattaa 840 tgtcataagg gatcctattg
agaggctagt ttcttattat tactttctga gatttggaga 900 tgattataga
ccagggttac ggagacgaaa acaaggagac aaaaagacct ttgatgaatg 960
tgtagcagaa ggtggctcag actgtgctcc agagaagctc tggcttcaaa tcccgttctt
1020 ctgtggccat agctccgaat gctggaatgt gggaagcagg tgggctatgg
atcaagccaa 1080 gtataaccta attaatgaat attttctggt gggagttact
gaagaacttg aagattttat 1140 catgttattg gaggcagcat tgccccggtt
tttcaggggt gctactgaac tctatcgcac 1200 aggaaagaaa tctcatctta
ggaaaaccac agagaagaaa ctccccacta aacaaaccat 1260 tgcaaaacta
cagcaatctg atatttggaa aatggagaat gagttctatg aatttgcact 1320
agagcagttc caattcatca gagcccatgc cgttcgagaa aaagatggag acctctacat
1380 cctcgcacaa aactttttct atgaaaagat ttaccctaag tcgaactgag
tataaggtgt 1440 gactattaga ttcttgaact aaaatttgac cctgtcttca
cctttgttct cagctccaca 1500 gtctggattg ctgacagtag gtgtatatga
caatttgtat tgagccaaat taggaaacag 1560 acagtaacgt caaggaagta
gatactggct ggcattgtca gtgttctaag tttcaggcat 1620 ttttattttt
cctggctaaa cgttggtgaa agttataacc tcctgcctgg gagaaaatat 1680
acatcaccta aaatgaactt atggcaggtc taatcaaaag gctaaataca atttcagaaa
1740 aggttctgat actcttgttt ttgataaagc attttttcaa ctaaccatga
attaagatga 1800 gtccatttgc ctcttctgcc ttcactgagg gtttgggtta
tacacctcta ctgaattgtg 1860 ttaataactg tttggcagtg tgtactttgt
ttttgtgagt catgtctcat gaaatttatt 1920 ggaatgttta atcatatttg
ctaagaaatg tttctgctgt agttggattt gcccatattt 1980 atgtaggtgg
ttttaatttt ttaaatggtg attagtgtta aaaatcaatt taaatcatga 2040
ctaatatggt aaaaagataa agcatcaaag cagtatttct cattcctgcc tcctcaatat
2100 ctaatactgg gaagatactt caaagaatat tgagattgtc tgaagtttta
gttaagattt 2160 tcacacatta atatcaaaaa agtaagttta gtatttgttt
ctccatgggt tatttgtaaa 2220 gctgtaaact gagatatcgg tgactccgta
ttatgactcc attagtgagc tgtggtatgg 2280 gtaggatttt cctacttctt
ctgtactttt acctgtagac tatttttact aaggtgcttt 2340 ataatgtgtt
ttaaagcatt gcatttacaa aacaaggaaa atgctgtaaa tattgcatat 2400
tttatgtatt tggaccaaaa ggttacaagt aattagacaa aagtggtttt gcaccaattt
2460 tatgtcaagt aaaaccatca gacctactgt tcttgtattt ctcatttaac
tttactgtta 2520 agacatcact gaaatgaact tcagtaagct ttcaattttg
atacacagtt cattattcat 2580 aacttgaggc agtaattaca gtggaatgag
tactggacaa ggagtcaaaa aacttgattt 2640 caggtcctag ctctagcact
tacagctgtg tgatcttggg caagtcactt aacctctctt 2700 tgcctcaatt
tcctcatctt gaaatgagga taataatacc tgctgtacct acctcacagg 2760
gctgttgtga ggattaaatg agatggcatg tgaaagcact ttgaaaattg taaagcgcta
2820 tgtaaatgta aggtattata gaaacatctt taacatatag tttcatacca
ttcatttttt 2880 aacaaagaaa gggaaaagtc tgcttgtaag ctggttgaaa
aagttaatct tgatataaat 2940 ttgtgtttga taaatatcct ctcagtgttt
tatcttccat gtttcaacaa ctattgaaat 3000 atgaaatgcc tgtgaactct
taaagcttca tgagcagctg cttgagttca ggaagttcac 3060 tgttagaaat
aggctttgtt agctgactag ggtcagggaa acttttctct tcaaatttga 3120
aagctgtttc tgttttcatt ttacattatt attcagaaat ggtagctatt ctatacctat
3180 ggtttaagta aatatttctg aataaggctt caccatactg taagcatttt
aggtagattg 3240 ccttaaaggt tatgggaggg catgagggaa cacttcttat
gagaaaacat ttataaacaa 3300 aagaaacatt tataaactaa agaaaaacta
aaagaatgac agaacaatca tcttagcacc 3360 ctttcctcac aataatataa
aaatattaaa agaacatagg caggcttttt ttaaatttgg 3420 cttttttctt
tccttttttc aaattgactt ttataggtat ttcctgaaag tgtatacaaa 3480
ttatttcctc gcccaaaata aagcaccact tcaaggtgtg gtttgacatt acatgctaat
3540 gaacaaaccc agtatgcaag ttattcttgc accacatgct caaatcttct
tgaggtgcat 3600 taactctttt aggtaactag agcagtactt ggtgaactag
atcaggaggt cagtaaactt 3660 tctgtggaag ggccagagag taaatatttt
aggctttgca gcccatacgg tctctgtcac 3720 agctagtcaa ccctgccatt
ttaccacaaa agcagcaata gacattatgt aaacaaatga 3780 gcacagttat
gttccaataa aactttattt acaaaaacag atgacatccc agatgcagac 3840
catgggcaac caaccattgc actggctaaa tcattattta tggagaaatc ctctttgtgt
3900 ctctactcta gatgcctaaa agagtttata tacttctaaa agctcctaac
ttatatccaa 3960 agaattgctt tctgattcgt gtagtctctc ccacagattc
ataaactttt atgacttata 4020 ttgtttccag gtgggcatgg tttatttccc
agtttaacag ttcagaatag gggcatttat 4080 tttatcatat tttagggtgg
gttaggagta tcctttctgg agactgagaa aggggtgtat 4140 ttaattccat
caggtccagt acagtactag gagtcataat actttataat caattaaata 4200
aatagaacca ctgagacaat aatgtatttt tttaaagtgg caaatgtggt tttctttttt
4260 cagcctttgc gctttttcag tattttgacc atagggagat aattttttta
taatacaaaa 4320 gtaaccactt ggaattttaa agataatgtt atgtgtgtat
gtgaaatata tatacatata 4380 tatatatatt tcctaaaaga agaaaagata
cctttctgtt caacttgtat caactcctct 4440 tttctaattg ctgtgaaatg
gcaactgttg ataaattatt gtgattgttt taaaatctaa 4500 tgggaagtaa
aatatatttt gattttaccc agcttaatct gtaaagtagc acttaaatat 4560
atctgatagc aacacttaag atattgcatg gggattactt tcctatcatc catatgcatt
4620 tgtgcaactt caaacatatt gggtgcttct gaattcctga tgattggatt
taagctattg 4680 aaaattggat aatttaaact taatgatttt tataattttc
tgatcttaaa atttggttaa 4740 tgcctataat ctgttgcttt ttctcaatat
gtgtcctatt ggaaattcct caaatcgttg 4800 gtgccatcag tgatttacaa
acaatatttt gatattgcag atgacttgct tactgtattt 4860 gcattgttag
aaaacagttt gtagacaatg attctttttt aataaaatca aataattcta 4920
aaagtgctag agaatttaac taaaagctgg ttcccaaatg catagctggc attttaattt
4980 aaattcaaat ctacatagag aacatccgtg taaatcatct aactggattt
tcccattggt 5040 cattcccaaa cacacctatg gtcctagaat ccttaagaga
agcaccctgt aaccttttat 5100 gtggtttgcc tttaagaggc ccaggtgctt
ctcctttatg atttgagttg gcctcttcat 5160 aaattagtgc tgtttacttt
cagaggaagc agagaagttg ctgttatgtt tttgcatccg 5220 tttaccctat
gcaaagttgc tgtatgatgc caactaaact gctctttagg cagccttctg 5280
aggagaaaag caaccctgtt tcaaatccac tgccaattca gctcctctgg agtggagctt
5340 tctgatttct tggagcagga attttagaga ttgaaatgaa tgatcattta
gtcagattta 5400 tcctgtaatt tcatgcagct ttgtggcctt tgcagtacta
tttataaaat ggaccctgat 5460 ggtgatgaac tctttagaac gcattactgt
taagcctgtg ttgagacatt gatgctgtct 5520 atctcatttt ttagacagtt
tttgtagctt tctattgaga gtcaggtatg tgagcatctc 5580 tgaagcagtg
ttgaatgtaa ttttcggaaa catggattgt gtattttgac ttttatttta 5640
taaatacaca gctcaacagt gccttttttt tccctcatag tcctgttgga agatgctcac
5700 tactttctct cttctctctc cctgccctcc cccactccat tcagttgatt
catttatgca 5760 aattctgttt ccaacttgaa accattttgt cacatctgtt
ggagagataa tcactccttt 5820 tccttaacat tctgccagct ttctgatgtt
gaagtgtttc agttgactac ctgatgcaaa 5880 agctataaaa taaacagtgg
gaaggggaaa aattggtgtc ctgttttaat attttctttt 5940 gtagccttga
cactgatgga cattttccaa gctgactcag tgttcagtgt caacttaact 6000
ctcagatagt gttgccatca agaaagcatg caacatcatt ggtttctaat gattttatgg
6060 cttgtgacaa tattttatct ggactgacat gcctctgctg cttttgcttt
gtacttcatt 6120 gctggtaata aaatttcaga tggaaaactt acaaaatata
tacttaatta gaagaaaaaa 6180 atagagaaag ggctattaga attaaaaaaa
tttgaaagta acttaatcta acatttatgg 6240 cacagtttgg acatatccat
aatttttttt gggaacacac atttctgatt ttttttttcc 6300 cccttaaaga
agaaagtctc aattccattg attttcaatt cttagccact ggctcattgc 6360
tttgagcaat gcttgattga ttctatttat attatatgat attgggttga taaaatacca
6420 gttcaatgat gagttttctt aacagaattt ggtttgtact tgcagtggct
gaacaaagag 6480 catggcttga gaatcaaagg gatctgcatt tagcaatgtg
atgtcagtaa atggacataa 6540 caggattgtt gtaaaggttg ggcatgatgt
atgcaaagta ctggccaggg tagactaata 6600 actgatggca tttatatgct
gtgctggaat attgttacca agctgatgtg ccgttctcac 6660 cctgcagaat
actggttttg tcatttcata aatgatattt ttataaat 6708 3 2185 DNA Homo
sapiens 3 gggaaggaag gaagagaggg aggcgggcaa gcaggcgggc gcgggggtcg
gagactgagg 60 cagtagaggg aggcgagagc ccggcagccg cttcgcgctg
tttgctggcg cgggttttgg 120 agggggcggc cgtttagtcg gctgaggaga
agcggacacc agcggcgttg gtgatagcgc 180 ctgggggagg gggactggag
aggcgagaag gggggttcgc tgcggtggtt ctctcgctgt 240 cgctctctct
ttgcctcgct cccggctcgg cgggctcctc ccggcgtctc tctcgcctcc 300
ggggtcccgc tccccgcccc ccgcggtatg tcttgatccc gagcagcggg tttcatgggg
360 ctcctcagga ttatgatgcc gcccaagttg cagctgctgg cggtggtggc
cttcgcggtg 420 gcgatgctct tcttggaaaa ccagatccag aaactggagg
agtcccgctc gaagctagaa 480 agggctattg caagacacga agtccgagaa
attgagcagc gacatacaat ggatggccct 540 cggcaagatg ccactttaga
tgaggaagag gacatggtga tcatttataa cagagttccc 600 aaaacggcaa
gcacttcatt taccaatatc gcctatgacc tgtgtgcaaa gaataaatac 660
catgtccttc atatcaacac taccaaaaat aatccagtga tgtcattgca agatcaggtg
720 cgctttgtaa agaatataac ttcctggaaa gagatgaaac caggatttta
tcatggacac 780 gtttcttact tggattttgc aaaatttggt gtgaagaaga
aaccaattta cattaatgtc 840 ataagggatc ctattgagag gctagtttct
tattattact ttctgagatt tggagatgat 900 tatagaccag ggttacggag
acgaaaacaa ggagacaaaa agacctttga tgaatgtgta 960 gcagaaggtg
gctcagactg tgctccagag aagctctggc ttcaaatccc gttcttctgt 1020
ggccatagct ccgaatgctg gaatgtggga agcaggtggg ctatggatca agccaagtat
1080 aacctaatta atgaatattt tctggtggga gttactgaag aacttgaaga
ttttatcatg 1140 ttattggagg cagcattgcc ccggtttttc aggggtgcta
ctgaactcta tcgcacagga 1200 aagaaatctc atcttaggaa aaccacagag
aagaaactcc ccactaaaca aaccattgca 1260 aaactacagc aatctgatat
ttggaaaatg gagaatgagt tctatgaatt tgcactagag 1320 cagttccaat
tcatcagagc ccatgccgtt cgagaaaaag atggagacct ctacatcctc 1380
gcacaaaact ttttctatga aaagatttac cctaagtcga actgagtata aggtgtgact
1440 attagattct tgaactaaaa tttgaccctg tcttcacctt tgttctcagc
tccacagtct 1500 ggattgctga cagtaggtgt atatgacaat ttgtattgag
ccaaattagg aaacagacag 1560 taacgtcaag gaagtagata ctggctggca
ttgtcagtgt tctaagtttc aggcattttt 1620 attttttcct ggctaaacgt
tggtgaaagt tataacctcc tgcctgggag aaaatataca 1680 tcacctaaaa
tgaacttatg gcaggtctaa tcaaaaggct aaatacaatt tcagaaaagg 1740
ttctgatact cttgtttttg ataaagcatt ttttcaacta accatgaatt aagatgagtc
1800 catttgcctc ttctgccttc actgagggtt tgggttatac acctctactg
aattgtgtta 1860 ataactgttt ggcagtgtgt actttgtttt tgtgagtcat
gtctcatgaa atttattgga 1920 atgtttaatc atatttgcta agaaatgttt
ctgctgtagt tggatttgcc catatttatg 1980 taggtggttt taatttttta
aatggtgatt agtgttaaaa atcaatttaa atcatgacta 2040 atatggtaaa
aagataaagc atcaaagcag tatttctcat tcctgcctcc tcaatatcta 2100
atactgggaa gatacttcaa agaatattga gattgtctga agttttagtt aagattttca
2160 cacattaata tcaaaaaaaa aaaaa 2185 4 1147 DNA Homo sapiens 4
cgggtttcat ggggctcctc aggattatga tgccgcccaa gttgcagctg ctggcggtgg
60 tggccttcgc ggtggcgatg ctcttcttgg aaaaccagat ccagaaactg
gaggagtccc 120 gctcgaagct agaaagggct attgcaagac acgaagtccg
agaaattgag cagcgacata 180 caatggatgg ccctcggcaa gatgccactt
tagatgagga agaggacatg gtgatcattt 240 ataacagagt tcccaaaacg
gcaagcactt catttaccaa tatcgcctat gacctgtgtg 300 caaagaataa
ataccatgtc cttcatatca acactaccaa aaataatcca gtgatgtcat 360
tgcaagatca ggtgcgcttt gtaaagaata taacttcctg gaaagagatg aaaccaggat
420 tttatcatgg acacgtttct tacttggatt ttgcaaaatt tggtgtgaag
aagaaaccaa 480 tttacattaa tgtcataagg gatcctattg agaggctagt
ttcttattat tactttctga 540 gatttggaga tgattataga ccagggttac
ggagacgaaa acaaggagac aaaaagacct 600 ttgatgaatg tgtagcagaa
ggtggctcag actgtgctcc agagaagctc tggcttcaaa 660 tcccgttctt
ctgtggccat agctccgaat gctggaatgt gggaagcagg tgggctatgg 720
atcaagccaa gtataaccta attaatgaat attttctggt gggagttact gaagaacttg
780 aagattttat catgttattg gaggcagcat tgccccggtt tttcaggggt
gctactgaac 840 tctatcgcac aggaaagaaa tctcatctta ggaaaaccac
agagaagaaa ctccccacta 900 aacaaaccat tgcaaaacta cagcaatctg
atatttggaa aatggagaat gagttctatg 960 aatttgcact agagcagttc
caattcatca gagcccatgc cgttcgagaa aaagatggag 1020 acctctacat
cctcgcacaa aactttttct atgaaaagat ttaccctaag tcgaactgag 1080
tataaggtgt gactattaga ttcttgaact aaaatttgac cctgtcttca cctttgttct
1140 cagctcc 1147 5 4196 DNA Homo sapiens 5 cggccctccc atgtgcagcc
cggccagccg ggctctcctc ctcgcggcgg atgggtgacc 60 ttttcctggc
acgggcaggc tgtgggaggc agcggagcag gcgatgaaga agaagcagca 120
gcatcccggc ggcggcgcgg atccctggcc ccatggggcc cctatggggg gcgcccctcc
180 gggcctgggc agctggaagc gtcgggtgcc cctgctgcct ttcctgcgct
tctccctccg 240 ggactacggc ttctgcatgg ccaccctgct ggtcttctgc
ctgggctccc tcctctatca 300 gctcagcggg ggaccccctc gcttcctgct
cgacctgcgg cagtacttgg gaaattccac 360 ttacttggat gaccatggac
cacctcctag taaggtacta cctttcccaa gccaggtggt 420 gtacaacagg
gtaggcaagt gtgggagccg tactgtggtc ttgcttctga gaatcttgtc 480
ggagaagcac ggatttaatt tggtcacatc agacattcac aacaaaacca ggcttactaa
540 aaatgaacaa atggaactga ttaaaaatat aagtactgcc gaacaaccct
atttattcac 600 tcgacatgtt catttcctca acttctcaag gtttggagga
gaccagcctg tctacatcaa 660 catcattaga gaccccgtca accggttctt
atccaactat tttttccgtc gctttggaga 720 ctggagaggg gaacaaaatc
acatgatccg cacccccagc atgaggcagg aggagcgcta 780 cctggatatc
aatgagtgta ttcttgaaaa ctatcccgag tgctccaacc ccaggttatt 840
ttacatcatt ccgtactttt gtggacagca tcccagatgc agggagcctg gtgaatgggc
900 ccttgagaga gcaaagctga acgtgaatga aaacttcctg ctcgtgggga
ttcttgaaga 960 gttggaagat gtgctgctgt tactggaaag atttttacct
cattacttca agggcgtgct 1020 cagtatctac aaagacccag agcacaggaa
gcttggaaac atgactgtga cggtgaagaa 1080 gactgtcccc tctcctgagg
ctgtgcagat cctctaccag cggatgagat acgagtacga 1140 gttttaccac
tacgtcaaag agcagttcca cctgctgaag cgcaagtttg gacttaagtc 1200
tcacgtcagc aagccccccc tgaggccaca cttctttatc ccaactccac tggaaaccga
1260 ggagccaatc gacgatgaag aacaggatga tgaaaagtgg ctggaagata
tttataagag 1320 gtgatgtgac tgtgttgcct ctatggcttt atctcccttt
tccagaaagt tctttgtttg 1380 gggaagtaaa atccttaagg gactaaatta
atgcttgggt gcattaaaaa gaacaaaaca 1440 ttcccacatg ttggggtcat
tgggagatgc ccggttttgc gggttttatt tgtttaattt 1500 tattctgtgt
tttctcttgg ctctttgggt ctttcccggg tacactagat ggctccatcc 1560
caaggcatct tgtcataaaa cagctttccc ccaccccata tcatgggaaa agggggagaa
1620 atatagcccc tagcctaata acttatcatt tgtaaaatga cttataaaaa
tattacctca 1680 atggtaggag acatccagac ttgtatattt cagtggaaat
acaaaaccac ttcagagacc 1740 agggtatctc ctctggaagg atctaagaga
aggtaagaca gattaggaca tcgaaaagga 1800 ggatggagcc aggtgccatg
gcttgagcct ataatccgag gctgaggtgg gaggatcact 1860 tgagcccagg
agtttgaggt tgcagtgagc tgtgatcaca ccactgcact ccagcctggg 1920
tgacagagtg agactctgtc tcaattaatt tttttttttt aaaggaggag gatctccatg
1980 ggtaagtggt ttctacccgc atgggtagag ttctgcctct ggtccttctc
agggggcact 2040 ttcaccaaga gcagtgtaat tatctctgaa agagcaagtc
agcttgtgcc gcatccccaa 2100 ccaatccaca gcctggagta cctttcaagg
tcaaagtgca tggccagctc cattgagaca 2160 ttccatttca aagcaccgtg
ctgacagata tcaaagtact ctagcaggga aaataatttg 2220 tttgctgtgt
aaggaagaat gtagacaaga cagataaatc tgaaggtcat gtggcatcag 2280
ggaaagggca tggctgtgtc ttttgcaccc aatatgaaac atcttctccc aacactgctt
2340 taatggaagt tctaggaacc aatttagctc aggcatttga ctcctacagc
agaagttctg 2400 agcctgacca cagatggtgt gtaatctatc aaacacaccc
ctggccaagt tgggtcctat 2460 aggacctggt actatgtact attgtaactt
ctagttccct aagaggtacc tgttttcagt 2520 aaaaaggggt cctgagttct
gtgcaggtgg aagagctacc cgagaactac ctgagttctg
2580 tgcaggtaga gtcccatttc ttatgggacc tgtgtgctcc tgagaactct
tacttgagac 2640 atcaaaaaga agcagcaaga gcttctggga cagagactgc
ttggccagct ttgtaagtaa 2700 gtggctgcct ccaatgtgat gtgagtacat
gttgggcagt ctcactgtcc taaggtatgt 2760 cttctttcca cctcccactg
cccctcccct gccacctatc aatgatgcct tggttcagtc 2820 attagaaatc
tgttgctttg agttctgaaa tattttcacc ttaaaaaaaa tgctgaaaat 2880
acacattctc ctgggaagac gataaacagc tagctaagaa gccgaggttc agtggtggca
2940 gcaggaagga cactgccaca aattttgtct atttcatatt tgtcccctag
agccagccct 3000 agcaaatgtg tgagttggga gtagttaata gtaaataaga
ctctgacttt acacaagcta 3060 cacattttat acttttcata aaccacaaag
tctctctaga attttttctg ccttcactaa 3120 aattggactg tagccaagat
ataaagcaag tcatttggaa cctgccgagt gagcactgaa 3180 gctactttat
catgagatgt gtgttaagaa ggctgcagcc cacaggagtc cagggaaggc 3240
ggggaccaca gaggcacaga gtccagcact tggccgctca tgggccttct ttctgcctca
3300 gaggacgggg gcagagaagt gatgaaggga aatgttctta gaggaggaaa
tatcctttgt 3360 cctgttcaga gagaccaggg ccctaccatt aggcatactt
tcagaagcaa cctggagaac 3420 agctatcaat catattcaaa accagtacaa
gaactgctgc ctggtaccct gtgagtcatt 3480 tctatgaaat tccatataaa
gaatgatgat aagtttacac actgtgcaat ctcacaatct 3540 gaaaataaag
ttgagttggc tgtgttttct ctgctcttgt cagaacattg ggacaattgg 3600
tcgttcaaaa acattcatcc tcttactgca agtttatctg ggtactttta cctgtgtgtt
3660 caaaggcatt tcttttcagc agtgatcatt ataacttcac aaaaaaagat
gctgacggat 3720 ttacttacag ggccttaatg ttattttgtc ccagccaaca
ccctctaggt cctaaaagtc 3780 aaggtacttc agtttatttg gcaaacatga
caacattttt ttggccctgg gcccaacagt 3840 ttgtacttca tgaaacatat
tgtacatttt acatagttta atttaaaaaa taccttttaa 3900 gctagttgat
ctttgactgt cttatttatt ataacctttc agcacattcc aaggttttag 3960
ttactcagga aggagttaat taaaatgatt ttattttggt ctgatggatg ttttttaaaa
4020 ggaaaattat tattatgaac cttcagccta ctttcttgag tgccgtaaaa
gtgcttgtaa 4080 atcttttttt ttttttaaga agaaagaaaa aaatggtgtt
tgacgttgat ggaaattcaa 4140 aaatatatat ggaactgaaa cattaactta
gctaaaataa aagcaatctg tgtttg 4196 6 356 PRT Homo sapiens 6 Met Gly
Leu Leu Arg Ile Met Met Pro Pro Lys Leu Gln Leu Leu Ala 1 5 10 15
Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln 20
25 30 Lys Leu Glu Glu Ser Arg Ser Lys Leu Glu Arg Ala Ile Ala Arg
His 35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly
Pro Arg Gln 50 55 60 Asp Ala Thr Leu Asp Glu Glu Glu Asp Met Val
Ile Ile Tyr Asn Arg 65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe
Thr Asn Ile Ala Tyr Asp Leu 85 90 95 Cys Ala Lys Asn Lys Tyr His
Val Leu His Ile Asn Thr Thr Lys Asn 100 105 110 Asn Pro Val Met Ser
Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile 115 120 125 Thr Ser Trp
Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser 130 135 140 Tyr
Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150
155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr
Phe 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg
Arg Lys Gln 180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala
Glu Gly Gly Ser Asp 195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln
Ile Pro Phe Phe Cys Gly His 210 215 220 Ser Ser Glu Cys Trp Asn Val
Gly Ser Arg Trp Ala Met Asp Gln Ala 225 230 235 240 Lys Tyr Asn Leu
Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu 245 250 255 Leu Glu
Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe 260 265 270
Arg Gly Ala Thr Glu Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg 275
280 285 Lys Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys
Leu 290 295 300 Gln Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr
Glu Phe Ala 305 310 315 320 Leu Glu Gln Phe Gln Phe Ile Arg Ala His
Ala Val Arg Glu Lys Asp 325 330 335 Gly Asp Leu Tyr Ile Leu Ala Gln
Asn Phe Phe Tyr Glu Lys Ile Tyr 340 345 350 Pro Lys Ser Asn 355 7
356 PRT Homo sapiens 7 Met Gly Leu Leu Arg Ile Met Met Pro Pro Lys
Leu Gln Leu Leu Ala 1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu
Phe Leu Glu Asn Gln Ile Gln 20 25 30 Lys Leu Glu Glu Ser Arg Ser
Lys Leu Glu Arg Ala Ile Ala Arg His 35 40 45 Glu Val Arg Glu Ile
Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln 50 55 60 Asp Ala Thr
Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg 65 70 75 80 Val
Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 90
95 Cys Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn
100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys
Asn Ile 115 120 125 Thr Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His
Gly His Val Ser 130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys
Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile
Glu Arg Leu Val Ser Tyr Tyr Tyr Phe 165 170 175 Leu Arg Phe Gly Asp
Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln 180 185 190 Gly Asp Lys
Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp 195 200 205 Cys
Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His 210 215
220 Ser Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala
225 230 235 240 Lys Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val
Thr Glu Glu 245 250 255 Leu Glu Asp Phe Ile Met Leu Leu Glu Ala Ala
Leu Pro Arg Phe Phe 260 265 270 Arg Gly Ala Thr Glu Leu Tyr Arg Thr
Gly Lys Lys Ser His Leu Arg 275 280 285 Lys Thr Thr Glu Lys Lys Leu
Pro Thr Lys Gln Thr Ile Ala Lys Leu 290 295 300 Gln Gln Ser Asp Ile
Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala 305 310 315 320 Leu Glu
Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp 325 330 335
Gly Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr 340
345 350 Pro Lys Ser Asn 355 8 406 PRT Homo sapiens 8 Met Lys Lys
Lys Gln Gln His Pro Gly Gly Gly Ala Asp Pro Trp Pro 1 5 10 15 His
Gly Ala Pro Met Gly Gly Ala Pro Pro Gly Leu Gly Ser Trp Lys 20 25
30 Arg Arg Val Pro Leu Leu Pro Phe Leu Arg Phe Ser Leu Arg Asp Tyr
35 40 45 Gly Phe Cys Met Ala Thr Leu Leu Val Phe Cys Leu Gly Ser
Leu Leu 50 55 60 Tyr Gln Leu Ser Gly Gly Pro Pro Arg Phe Leu Leu
Asp Leu Arg Gln 65 70 75 80 Tyr Leu Gly Asn Ser Thr Tyr Leu Asp Asp
His Gly Pro Pro Pro Ser 85 90 95 Lys Val Leu Pro Phe Pro Ser Gln
Val Val Tyr Asn Arg Val Gly Lys 100 105 110 Cys Gly Ser Arg Thr Val
Val Leu Leu Leu Arg Ile Leu Ser Glu Lys 115 120 125 His Gly Phe Asn
Leu Val Thr Ser Asp Ile His Asn Lys Thr Arg Leu 130 135 140 Thr Lys
Asn Glu Gln Met Glu Leu Ile Lys Asn Ile Ser Thr Ala Glu 145 150 155
160 Gln Pro Tyr Leu Phe Thr Arg His Val His Phe Leu Asn Phe Ser Arg
165 170 175 Phe Gly Gly Asp Gln Pro Val Tyr Ile Asn Ile Ile Arg Asp
Pro Val 180 185 190 Asn Arg Phe Leu Ser Asn Tyr Phe Phe Arg Arg Phe
Gly Asp Trp Arg 195 200 205 Gly Glu Gln Asn His Met Ile Arg Thr Pro
Ser Met Arg Gln Glu Glu 210 215 220 Arg Tyr Leu Asp Ile Asn Glu Cys
Ile Leu Glu Asn Tyr Pro Glu Cys 225 230 235 240 Ser Asn Pro Arg Leu
Phe Tyr Ile Ile Pro Tyr Phe Cys Gly Gln His 245 250 255 Pro Arg Cys
Arg Glu Pro Gly Glu Trp Ala Leu Glu Arg Ala Lys Leu 260 265 270 Asn
Val Asn Glu Asn Phe Leu Leu Val Gly Ile Leu Glu Glu Leu Glu 275 280
285 Asp Val Leu Leu Leu Leu Glu Arg Phe Leu Pro His Tyr Phe Lys Gly
290 295 300 Val Leu Ser Ile Tyr Lys Asp Pro Glu His Arg Lys Leu Gly
Asn Met 305 310 315 320 Thr Val Thr Val Lys Lys Thr Val Pro Ser Pro
Glu Ala Val Gln Ile 325 330 335 Leu Tyr Gln Arg Met Arg Tyr Glu Tyr
Glu Phe Tyr His Tyr Val Lys 340 345 350 Glu Gln Phe His Leu Leu Lys
Arg Lys Phe Gly Leu Lys Ser His Val 355 360 365 Ser Lys Pro Pro Leu
Arg Pro His Phe Phe Ile Pro Thr Pro Leu Glu 370 375 380 Thr Glu Glu
Pro Ile Asp Asp Glu Glu Gln Asp Asp Glu Lys Trp Leu 385 390 395 400
Glu Asp Ile Tyr Lys Arg 405 9 406 PRT Homo sapiens 9 Met Lys Lys
Lys Gln Gln His Pro Gly Gly Gly Ala Asp Pro Trp Pro 1 5 10 15 His
Gly Ala Pro Met Gly Gly Ala Pro Pro Gly Leu Gly Ser Trp Lys 20 25
30 Arg Arg Val Pro Leu Leu Pro Phe Leu Arg Phe Ser Leu Arg Asp Tyr
35 40 45 Gly Phe Cys Met Ala Thr Leu Leu Val Phe Cys Leu Gly Ser
Leu Leu 50 55 60 Tyr Gln Leu Ser Gly Gly Pro Pro Arg Phe Leu Leu
Asp Leu Arg Gln 65 70 75 80 Tyr Leu Gly Asn Ser Thr Tyr Leu Asp Asp
His Gly Pro Pro Pro Ser 85 90 95 Lys Val Leu Pro Phe Pro Ser Gln
Val Val Tyr Asn Arg Val Gly Lys 100 105 110 Cys Gly Ser Arg Thr Val
Val Leu Leu Leu Arg Ile Leu Ser Glu Lys 115 120 125 His Gly Phe Asn
Leu Val Thr Ser Asp Ile His Asn Lys Thr Arg Leu 130 135 140 Thr Lys
Asn Glu Gln Met Glu Leu Ile Lys Asn Ile Ser Thr Ala Glu 145 150 155
160 Gln Pro Tyr Leu Phe Thr Arg His Val His Phe Leu Asn Phe Ser Arg
165 170 175 Phe Gly Gly Asp Gln Pro Val Tyr Ile Asn Ile Ile Arg Asp
Pro Val 180 185 190 Asn Arg Phe Leu Ser Asn Tyr Phe Phe Arg Arg Phe
Gly Asp Trp Arg 195 200 205 Gly Glu Gln Asn His Met Ile Arg Thr Pro
Ser Met Arg Gln Glu Glu 210 215 220 Arg Tyr Leu Asp Ile Asn Glu Cys
Ile Leu Glu Asn Tyr Pro Glu Cys 225 230 235 240 Ser Asn Pro Arg Leu
Phe Tyr Ile Ile Pro Tyr Phe Cys Gly Gln His 245 250 255 Pro Arg Cys
Arg Glu Pro Gly Glu Trp Ala Leu Glu Arg Ala Lys Leu 260 265 270 Asn
Val Asn Glu Asn Phe Leu Leu Val Gly Ile Leu Glu Glu Leu Glu 275 280
285 Asp Val Leu Leu Leu Leu Glu Arg Phe Leu Pro His Tyr Phe Lys Gly
290 295 300 Val Leu Ser Ile Tyr Lys Asp Pro Glu His Arg Lys Leu Gly
Asn Met 305 310 315 320 Thr Val Thr Val Lys Lys Thr Val Pro Ser Pro
Glu Ala Val Gln Ile 325 330 335 Leu Tyr Gln Arg Met Arg Tyr Glu Tyr
Glu Phe Tyr His Tyr Val Lys 340 345 350 Glu Gln Phe His Leu Leu Lys
Arg Lys Phe Gly Leu Lys Ser His Val 355 360 365 Ser Lys Pro Pro Leu
Arg Pro His Phe Phe Ile Pro Thr Pro Leu Glu 370 375 380 Thr Glu Glu
Pro Ile Asp Asp Glu Glu Gln Asp Asp Glu Lys Trp Leu 385 390 395 400
Glu Asp Ile Tyr Lys Arg 405
* * * * *
References