U.S. patent application number 10/057510 was filed with the patent office on 2002-07-25 for mdm interacting protein and methods of use thereof.
This patent application is currently assigned to CuraGen Corporation. Invention is credited to Nandabalan, Krishnan, Schulz, Vincent, Yang, Meijia.
Application Number | 20020098580 10/057510 |
Document ID | / |
Family ID | 26819199 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098580 |
Kind Code |
A1 |
Nandabalan, Krishnan ; et
al. |
July 25, 2002 |
MDM interacting protein and methods of use thereof
Abstract
Disclosed are complexes of the MDM2 protein and a novel
MDM2-interacting protein (MDMIP). Also disclosed are nucleic acids
encoding the MDMIP polypeptide, as well as derivatives, fragments,
analogs and homologs of the MDMIP polypeptide and MDM2-MDMIP
complexes.
Inventors: |
Nandabalan, Krishnan;
(Guilford, CT) ; Yang, Meijia; (East Lyme, CT)
; Schulz, Vincent; (Madison, CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS,
GLOVSKY AND POPEO, P.C.
One Financial Center
Boston
MA
02111
US
|
Assignee: |
CuraGen Corporation
New Haven
CT
|
Family ID: |
26819199 |
Appl. No.: |
10/057510 |
Filed: |
January 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10057510 |
Jan 25, 2002 |
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09510252 |
Feb 22, 2000 |
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6372490 |
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60121192 |
Feb 23, 1999 |
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60122643 |
Mar 3, 1999 |
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Current U.S.
Class: |
435/325 ;
424/94.63; 435/226 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; C07K 2319/00 20130101; C07K 14/47
20130101 |
Class at
Publication: |
435/325 ;
435/226; 424/94.63 |
International
Class: |
A61K 038/48; C12N
009/64 |
Goverment Interests
[0002] STATEMENT OF GOVERNMENT SUPPORT
[0003] This invention was made with United States Government
support under award number 70NANB5H1066 awarded by the National
Institute of Standards and Technology. The United States Government
has certain rights in the invention.
Claims
What is claimed is:
1. A purified complex comprising an MDMIP binding domain of an
MDM2polypeptide and an MDM2 binding domain of an MDMIP
polypeptide.
2. The complex of claim 1, wherein the MDMIP binding domain of the
MDM2 polypeptide is present in a polypeptide comprising the amino
acid sequence of an MDMIP polypeptide.
3. The complex of claim 1, wherein the MDM2 binding domain of the
MDMIP polypeptide is present in a polypeptide comprising the amino
acid sequence of an MDMIP polypeptide.
4. The complex of claim 3, wherein the MDMIP binding domain of the
MDM2 polypeptide is present in a polypeptide comprising the amino
acid sequence of an MDMIP polypeptide.
5. The complex of claim 1, wherein the MDM2 polypeptide is a human
MDM2 polypeptide.
6. The complex of claim 5, wherein the MDMIP polypeptide is a human
MDMIP polypeptide.
7. The complex of claim 1, wherein the MDMIP polypeptide is a human
MDMIP polypeptide.
8. The complex of claim 1, wherein the MDMIP binding domain
comprises a sequence that is at least 90% identical to the amino
acid sequence of a region of an MDMIP polypeptide comprising the
amino acid sequence of SEQ ID NO: 2.
9. The complex of claim 1, wherein the MDMIP polypeptide comprises
a sequence that is at least 95% identical to the amino acid
sequence of a human MDMIP polypeptide including the amino acid
sequence of SEQ ID NO: 2.
10. The complex of claim 1, wherein the MDMIP polypeptide comprises
the amino acid sequence of SEQ ID NO: 2.
11. The complex of claim 10, wherein the MDM2 polypeptide comprises
the amino acid sequence of SEQ ID NO: 4.
12. The complex of claim 1, wherein the MDM2 polypeptide comprises
the amino acid sequence of SEQ ID NO: 4.
13. The complex of claim 1, wherein the polypeptide comprising the
MDMIP binding domain is labeled.
14. The complex of claim 1, wherein the polypeptide comprising the
MDM2 domain is labeled.
15. The complex of claim 1, wherein the MDM2-binding domain of the
MDMIP polypeptide is a fragment of an MDMIP polypeptide.
16. The complex of claim 1, wherein the MDMIP binding domain of the
MDM2 polypeptide is a fragment of an MDM2 polypeptide.
17. A chimeric polypeptide comprising six or more amino acids of an
MDM2 polypeptide covalently linked to six or more amino acids of an
MDMIP polypeptide.
18. The chimeric polypeptide of claim 17, wherein the amino acids
of the MDM2 polypeptide comprise an MDMIP-binding domain.
19. The chimeric polypeptide of claim 17, wherein the amino acids
of the MDMIP polypeptide comprise an MDM2-binding domain.
20. The chimeric polypeptide of claim 17, wherein the MDM2 binding
domain comprises an amino acid sequence at lest 90% identical to
the amino acid sequence of SEQ ID NO: 2.
21. A pharmaceutical composition comprising the complex of claim 1.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No.
09/510,252, filed Feb. 22, 2000, which claims priority from U.S.
Ser. No. 60/121,192, filed Feb. 23, 1999 and U.S. Ser. No.
60/122,643, filed Mar. 3, 1999. The contents of these applications
are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0004] The invention relates generally to polypeptides and nucleic
acids and more particularly to polypeptides that interact with the
MDM2 polypeptide, and nucleic acids encoding these MDM2-interacting
polypeptides.
BACKGROUND OF THE INVENTION
[0005] The gene MDM2 has been implicated in a variety of cellular
processes. In addition, altered expression of MDM2 has been
associated with several disease states, including cancer. For
example, the MDM2 gene has been shown to be abnormally up-regulated
in human tumors and tumor cell lines. In addition, amplification of
MDM2 genes has been reported in a variety of cancers, e.g., human
sarcoma, glioma, squamous cell carcinoma, breast cancer,
astrocytoma, leukemia and lymphoma. These results indicate that the
MDM2 protein plays a role in human carcinogenesis.
[0006] MDM2 has been reported to interact with, i.e., bind to other
proteins. Some of these proteins have themselves been associated
with tumorigenesis. For example, MDM2 has been reported to form a
complex with the p53 tumor suppressor and to block the growth
suppressive functions of p53. In addition, overexpression of MDM2
has been shown to block the transactivation, cell cycle arrest
(S/G2 phase) and apoptotic functions of p53.
[0007] MDM2 has also been shown to interact with the retinoblastoma
tumor suppressor protein pRb, and the E2F-1 and DP1 transcription
factors. Through MDM2's activation of E2F and DP1, MDM2 is thought
to stimulate a G1 to S transition in the cell cycle. In addition,
MDM2 has also been reported to interact with Numb, a protein
involved in the determination of cell fate.
SUMMARY OF THE INVENTION
[0008] The invention is based in part on the discovery of a novel
polypeptide, named MDM Interacting Protein, or "MDMIP", based on
its ability to bind to the MDM2 polypeptide.
[0009] In one aspect, the invention includes a purified complex
that includes a polypeptide which includes an MDMIP binding domain
(or region) of an MDM2 polypeptide and a polypeptide which includes
an MDM2 binding domain (or region) of an MDMIP polypeptide. The
complex can thus include, e.g., an MDM2 polypeptide and an MDMIP
polypeptide. In various embodiments, the MDM2 polypeptide, the
MDMIP polypeptide, or both polypeptides within the complex, are
human polypeptides.
[0010] In some embodiments, the MDM2 binding domain of the MDM2
polypeptide includes an amino acid sequence that is at least 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the amino
acid sequence of a region of polypeptide that includes the amino
acid sequence of SEQ ID NO: 2.
[0011] In some embodiments, the MDMIP binding domain of the MDM2
polypeptide includes an amino acid sequence that is at least 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the amino
acid sequence of a region of polypeptide that includes the amino
acid sequence encoded by the nucleic acid sequence having GenBank
accession number M92424. In some embodiments, the MDMIP binding
domain includes the amino acid sequence of SEQ ID NO: 4, e.g., it
can include the amino acid sequence of the MDM2 polypeptide encoded
by the nucleic acid sequence of M9424 (SEQ ID NO: 6). Thus, in some
embodiments, the MDMIP binding domain of the MDM2 polypeptide is
present in a polypeptide which includes the amino acid sequence of
an MDM2 polypeptide, e.g., a polypeptide including at least that
portion of the amino acid sequence of SEQ ID NO: 4 that is
sufficient to bind to an MDMIP polypeptide.
[0012] If desired, one or more of the polypeptides in the
MDM2-MDMIP complex, e.g., the MDM2-binding domain of the MDMIP
polypeptide, the MDMIP-binding domain of the MDM2 polypeptide, or
both is labeled.
[0013] In other embodiments, the complex includes a fragment of an
MDMIP polypeptide that includes an MDM2-binding domain, a fragment
of an MDM2 polypeptide that includes an MDMIP-binding domain, or
both fragments.
[0014] Another aspect of the invention includes a chimeric
polypeptide that includes a region of an MDMIP polypeptide
covalently linked, e.g., via a peptide bond, to a region of an
MDM2polypeptide. Preferably, the chimeric peptide includes 6, 8,
10, 12, 14, or 16 or more amino acids of an MDM2 polypeptide
covalently linked to 6, 8, 10, 12, 14, 16 or more amino acids of an
MDMIP polypeptide.
[0015] In some embodiments, the amino acids of the MDMIP-derived
polypeptide in the chimeric polypeptide include an MDM2 binding
domain. For example, the MDMIP-derived polypeptide can include a
polypeptide which binds to MDM2 and, optionally, includes at least
a portion of an amino acid sequence at least 80%, 85%, 90%, 95%,
98%, or 99% or more identical to the amino acid sequence of SEQ ID
NO: 2.
[0016] Similarly, in some embodiments, the amino acids of the
MDM2-derived polypeptide in the chimeric polypeptide include an
MDMIP-binding domain. Thus, the MDM2 polypeptide can include a
polypeptide which binds to MDMIP and, optionally, includes at least
a portion of an amino acid sequence at least 80%, 85%, 90%, 95%,
98%, or 99% or more identical to the amino acid sequence of SEQ ID
NO: 4, i.e., a polypeptide having the amino acid sequence encoded
by nucleic acids 312-963 of the nucleic acid sequence corresponding
to GenBank accession number M92424.
[0017] In a further aspect, the invention includes an isolated
MDMIP nucleic acid. In some embodiments, the MDMIP nucleic acid
encodes an MDMIP polypeptide, e.g., the nucleic acid may include a
nucleic acid that encodes a polypeptide at least 70%, 75%, 80%,
85%, 90%, 95%, 98% or even 99% identical to a polypeptide which
includes the amino acid sequence of SEQ ID NO: 2.
[0018] The MDMIP nucleic acid may alternatively, or in addition,
include a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%,
98% or even 99% identical to a nucleic acid which includes nucleic
acids 1-222 of FIG. 1 (SEQ ID NO: 1).
[0019] Also provided by the invention is a vector including an
MDMIP nucleic acid, as well as a cell that includes an MDMIP
nucleic acid. The MDMIP nucleic acid in the cell may be present in
a vector, i.e., the invention includes a cell that contains a
vector in which an MDMIP nucleic acid is present.
[0020] Another aspect of the invention includes a purified MDMIP
polypeptide with an amino acid sequence at least 70%, 75%, 80%,
85%, 90%, 95%, 98%, or even 99% or more identical to the amino acid
sequence of SEQ ID NO: 2. In some embodiments, the MDMIP
polypeptide binds to an MDM2 polypeptide.
[0021] In a further aspect, the invention includes an isolated
MDM2-derived nucleic acid fragment. The MDM2-derived nucleic acid
is less than the full length of a full-length MDM2 nucleic acid,
i.e., the nucleic acid includes less than the fall length reading
frame of a nucleic acid encoding a full length MDM2 polypeptide. In
some embodiments, the MDM2-derived nucleic acid encodes an
MDM2polypeptide fragment that is less than 491 amino acids, e.g.,
it is less than 450, 400, 350, 300, 250, 200, 150, 100, or 50 amino
acids, and, optionally, binds an MDMIP polypeptide, e.g., a
polypeptide having the amino acid sequence of SEQ ID NO: 2.
Alternatively, or in addition, the MDM2-derived nucleic includes a
nucleic acid that encodes a polypeptide at least 70%, 75%, 80%,
85%, 90%, 95%, 98% or even 99% identical to a polypeptide which
includes the amino acid sequence of SEQ ID NO: 4, and which is less
than 491 amino acids in length and is greater than 4 amino acids in
length. Preferably, the MDMIP binding polypeptide is greater than
5, 6, 7, 8, 9, or 10 amino acids in length and is less than 25, 50,
75, 100, 150, 200, 250, 300, 350, 400 or 450 amino acids in
length.
[0022] The MDM2-derived nucleic acid may alternatively, or in
addition, include a sequence that is at least 70%, 75%, 80%, 85%,
90%, 95%, 98% or even 99% identical to a nucleic acid which
includes the nucleic acid sequence of SEQ ID NO: 3.
[0023] Also provided by the invention is a vector including the
MDM2-derived nucleic acid, as well as a cell that includes an
MDM2-derived nucleic acid. The MDM2-derived nucleic acid in the
cell may be present in a vector, i.e., the invention includes a
cell that contains a vector in which an MDM2-derived nucleic acid
is present.
[0024] Another aspect of the invention includes a purified MDM2
polypeptide with an amino acid sequence at least 70%, 75%, 80%,
85%, 90%, 95%, 98%, or even 99% or more identical to a polypeptide
that includes the amino acid sequence of SEQ ID NO: 4.
[0025] Also provided by the invention are antibodies (e.g.,
polyclonal or monoclonal antibodies) that bind to the MDMIP-MDM2
complexes described herein. For example, the invention includes an
antibody which specifically binds to a complex that includes a
polypeptide that has an MDMIP binding domain of an MDM2 polypeptide
and a polypeptide that includes an MDM2binding domain of an MDMIP
polypeptide. In some embodiments, the antibody binds with higher
affinity to a complex that includes an MDMIP binding domain of an
MDM2 polypeptide and an MDM2 binding domain of an MDMIP polypeptide
as compared to the binding of the antibody to an isolated MDM2
polypeptide or an isolated MDMIP polypeptide.
[0026] The invention also includes antibodies (e.g., polyclonal or
monoclonal antibodies) that bind to an MDMIP polypeptide, or to an
MDMIP-binding fragment of an MDM2 polypeptide. These antibodies, in
various embodiments, may optionally bind to the herein described
MDM2-MDMIP complexes as well.
[0027] In a further aspect, the invention features a pharmaceutical
composition that includes one or more of an MDMIP polypeptide, and
MDMIP nucleic acid, a chimeric polypeptide containing sequences
derived from an MDM2 polypeptide and sequences derived from an
MDMIP polypeptide, and a complex of an MDM2-binding domain of an
MDMIP polypeptide and an MDM2-binding domain of an MDMIP
polypeptide. The pharmaceutical composition may alternatively, or
additionally, contain an antibody to any of these polypeptides,
nucleic acids or complexes.
[0028] Also included in the invention is a kit containing reagents
that detect one or more of an MDMIP polypeptide, and MDMIP nucleic
acid, a chimeric polypeptide containing sequences derived from an
MDM2 polypeptide and sequences derived from an MDMIP polypeptide,
and a complex of an MDM2-binding domain of an MDMIP polypeptide and
an MDM2-binding domain of an MDMIP polypeptide. In some
embodiments, the reagents can include one or more of an antibody to
an MDM2 polypeptide, an antibody to and MDMIP polypeptide, and/or
an antibody to a complex of an MDM2-binding domain of an MDMIP
polypeptide and an MDM2-binding domain of an MDMIP polypeptide. The
reagent may in addition include the MDMIP polypeptide for detecting
the presence of MDM2 in a sample, an MDM2 protein for detecting the
presence of MDMIP in a sample.
[0029] The invention also includes methods of identifying a
MDM2-MDMIP complex, or various components of an MDM2-MDMIP complex,
in a biological sample. For example, the invention includes a
method of identifying a MDM2 sample in a biological sample by
contacting the biological sample with an MDMIP polypeptide under
conditions allowing for the formation of MDM2-MDMIP complexes.
Levels of the MDM2-MDMIP complex, if present, are then determined,
thus allowing for determination of levels of MDM2 polypeptide in
the cell.
[0030] In some embodiments, MDM2 polypeptides are detected using a
labeled MDMIP polypeptide. Detection of labeled MDMIP-MDM2
complexes indicates the presence of a MDM2 polypeptide in the
sample.
[0031] The invention also features a method of purifying an MDM2
polypeptide from a biological sample by contacting the biological
sample with a polypeptide that includes an MDM2-binding region of
an MDMIP polypeptide.
[0032] Also included in the invention is a method of identifying an
agent which modulates MDM2 or MDMIP activity. The method includes
contacting a test agent with an MDM-MDMIP complex and measuring
binding of the agent to the complex. Binding of the agent to the
complex indicates the agent modulates MDM2 or MDMIP polypeptide
activity.
[0033] In another aspect, the invention includes a method of
identifying an agent which modulates MDMIP activity. In this method
an MDMIP polypeptide is mixed with a test agent and binding of the
agent to the complex is measured. Binding of the agent to the
complex indicates the agent modulates MDMIP polypeptide
activity.
[0034] The invention also includes a method of determining whether
a test subject has a disorder associated with aberrant expression
of MDM2 protein. The method includes providing a biological sample
from the subject, measuring the level of the complex in the
subject; and comparing the level of the complex in the sample to
the level of the complex in a reference sample whose MDM2
expression state is known. Comparing the relative levels of the
MDM2 protein in the two samples makes it possible to determine
whether the subject has a disorder associated with aberrant
expression of an MDM2 protein.
[0035] Yet another aspect of the invention is a method of treating
or preventing a disease or disorder involving aberrant levels of a
complex of a MDM2 protein and a MDMIP. The method includes
administering to a subject in which such treatment or prevention is
desired a therapeutically-effective amount of a molecule or
molecules which are capable of modulating the function of the
complex.
[0036] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0037] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is a schematic representation of the nucleotide
sequence (SEQ ID NO: 7) of a human MDMIP polypeptide, along with an
encoded amino acid sequence (SEQ ID NO: 2). Nucleic acids 1-222
(SEQ ID NO: 1) encode the shown amino acid sequence.
[0039] FIGS. 2A and B are schematic representations of the
nucleotide sequence (SEQ ID NO: 3) (FIG. 2A) and the amino acid
sequence (SEQ ID NO: 4) of an MDMIP binding fragment of a human
MDM2 cDNA (FIG. 2B).
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention is based in part on the identification
of a novel polypeptide, named "MDMIP", which was discovered based
on its ability to bind to a fragment of a human MDM2
polypeptide.
[0041] The MDMIP polypeptide was identified in a yeast two hybrid
system designed to identify proteins ("target") based on their
ability to bind specifically to, i.e., interact with, a known
protein ("bait protein"). The bait protein used to identify
interacting fragments was a cDNA fragment containing nucleotides
312 to 963 (SEQ ID NO: 3) of the human MDM2 cDNA having GenBank
Accession Number M92424. The nucleotide sequence encoding the MDM2
bait polypeptide fragment is shown in FIG. 2 as SEQ ID NO: 3. The
amino acid sequence of the encoded bait polypeptide fragment is
shown in FIG. 2 as SEQ ID NO: 4 The full length sequence of the
human MDM2 nucleic acid is from GenBank Accession Number M92424(SEQ
ID NO: 5). The amino acid sequence of the MDM2 polypeptide encoded
by this nucleic acid is SEQ ID NO: 6.
[0042] Five polypeptides were determined to the MDM2-derived bait
polypeptide in the assay. Four of those corresponded to previously
described proteins. These polypeptides include human p53 cellular
tumor antigen (GenBank Acc. No. X54156), retinoblastoma protein
(pRB; GenBank Acc. No. M28419), E2F-1 (a pRB-binding protein with
properties of transcription factor E2F) nucleic acid sequence
(GenBank Acc. No. M96577), and ubiquitin (GenBank Acc. No.
U49869).
[0043] One interacting protein, however had not been previously
described. It was named MDMIP, for MDM Interacting Protein. The
nucleic acid sequence of a human cDNA encoding an MDMIP polypeptide
is shown in FIG. 1 as SEQ ID NO: 1. The amino acid sequence of the
polypeptide encoded by this nucleic acid sequence is shown as SEQ
ID NO: 2. Because no initiation codon is present in the reading
frame, it is possible that the sequence shown in FIG. 1 is a
partial sequence of a human MDMIP cDNA. The disclosed sequence may
correspond to the amino acid sequence at the carboxy terminal
region of a human MDMIP polypeptide.
[0044] Included within the invention are MDMIP nucleic acids and
their encoded polypeptides. Also disclosed are MDMIP-interacting
MDM2-derived polypeptides, as well as complexes that include
MDM2-binding MDMIP polypeptides and MDMIP-binding MDM2 polypeptides
and chimeric polypeptides containing MDM2 binding MDMIP
polypeptides and MDMIP binding MDM2 polypeptides. Also disclosed
are antibodies to these polypeptides and complexes, as well as
pharmaceutical compositions and methods utilizing these nucleic
acids, polypeptides, and complexes.
MDMIP Nucleic Acids
[0045] Included in the invention is an MDMIP nucleic acid. By
"MDMIP nucleic acid" is meant a nucleic acid which is at least 70%
identical to a nucleic acid including the nucleic acid sequence of
SEQ ID NO: 1. Also included in the term "MDMIP nucleic acid" is a
nucleic acid fragment that includes a portion of a sequence at
least 70% identical to a nucleic acid including the nucleic acid
sequence of SEQ ID NO: 1, provided that the fragment contains
enough sequence to specifically hybridize to a sequence at least
70% identical to a nucleic acid including the nucleic acid sequence
of SEQ ID NO: 1. Additional examples of MDMIP nucleic acids include
nucleic acids 1-442 of FIG. 1.
[0046] In some embodiments, the MDMIP nucleic acid encodes an MDMIP
polypeptide. By "MDMIP polypeptide" is meant a polypeptide at least
70% identical to the amino acid sequence of a polypeptide that
includes the amino acid sequence of SEQ ID NO: 2.
[0047] In some embodiments, the MDMIP nucleic acid encodes an MDMIP
polypeptide that includes an MDM2 binding domain. By "MDM2-binding
domain" is meant a region of amino acids sufficient to allow the
polypeptide in which the region of amino acids is present to bind
specifically to an MDM2 polypeptide. The encoded MDM2-binding
polypeptide can be derived from a full-length MDMIP polypeptide, or
from a derivative, fragment, analog, homolog or paralog of an MDMIP
polypeptide. Preferably, the derivative, fragment, analog, homolog
or paralog the has one or more of the following attributes: (i) is
functionally active (i.e., capable of exhibiting one or more
functional activities associated with full-length, wild-type MDMIP;
(ii) possesses the ability to bind the MDM2 protein; (iii) is
immunogenic or (iv) is antigenic.
[0048] In some embodiments, the fragment of an MDMIP polypeptide
includes at least 10, 20, 30, 40, or 50 amino acid residues
(preferably not larger that 35, 100 or 200 amino acid residues) of
the MDMIP polypeptide. Derivatives or analogs of the encoded MDMIP
polypeptide include, e.g., molecules which include regions which
are substantially homologous to the MDM2 protein or MDMIP in
various embodiments, of at least 50%, 60%, 70%, 80%, 90% or 95%
amino acid identity when: (i) compared to an amino acid sequence of
identical size; (ii) compared to an aligned sequence in which the
alignment is done by a computer homology program known within the
art or (iii) the encoding nucleic acid is capable of hybridizing to
a sequence encoding the MDM2 protein or MDMIP under stringent,
moderately stringent, or non-stringent conditions, as is discussed
below.
[0049] Thus, in some embodiments, the encoded MDM2-binding domain
is derived from an MDMIP polypeptide that includes a sequence that
is at least 90% identical to a polypeptide which includes the amino
acid sequence of SEQ ID NO: 2. In some embodiments, the domain is
derived from an MDMIP polypeptide which is at least 95, 98 or even
99% identical to a polypeptide including the amino acid sequence of
SEQ ID NO: 2.
[0050] In some embodiments, the encoded MDMIP polypeptide is a
polypeptide which includes the amino acid sequence of SEQ ID NO: 2.
For example, the MDMIP polypeptide may correspond to some or all of
the amino acid sequences encoded by the longest open reading frame
present in a nucleic acid that includes SEQ ID NO: 1.
Alternatively, the MDMIP polypeptide can include a region of the
amino acid sequence of SEQ ID NO: 2 that is able to bind
specifically to an MDM2 polypeptide.
[0051] Procedures for identifying regions within SEQ ID NO: 2 that
bind to MDM2 can be readily identified by one of ordinary skill in
the art. In addition, the sequence information disclosed herein for
MDMIP, e.g., the nucleic acid and amino acid sequences disclosed in
FIG. 1, may be combined with any method available within the art to
obtain longer clones encompassing additional MDMIP coding
sequences. Such sequences, for example, may encode an initiator
codon for an MDMIP polypeptide.
[0052] For example, the polymerase chain reaction (PCR) may be
utilized to amplify the sequence within a cDNA library. Similarly,
oligonucleotide primers may also be used to amplify by PCR
sequences from a nucleic acid sample (RNA or DNA), preferably a
cDNA library, from an appropriate source (e.g., the sample from
which the initial cDNA library for the modified yeast two hybrid
assay fusion population was derived).
[0053] PCR may be performed by use of, for example, a thermal
cycler and Taq polymerase. The DNA being amplified is preferably
cDNA derived from any eukaryotic species. Several different
degenerate primers may be synthesized for use in the PCR reactions.
It is also possible to vary the stringency of the hybridization
conditions used in priming the PCR reactions, to amplify nucleic
acid homologs by allowing for greater or lesser degrees of
nucleotide sequence similarity between the known nucleotide
sequence and the nucleic acid homolog being isolated. For cross
species hybridization, low stringency conditions are preferred;
whereas for same species hybridization, moderately stringent
conditions are preferred.
[0054] Any eukaryotic cell may potentially serve as the nucleic
acid source for the molecular cloning of the MDMIP sequences. The
DNA may be obtained by standard procedures known in the art from
cloned DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA
cloning, or by the cloning of genomic DNA, or fragments thereof,
purified from the desired cell. See e.g., Sambrook, et al., 1989.
Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.); and Glover,
1985. DNA Cloning: A Practical Approach (MRL Press, Ltd., Oxford,
U.K. Vol. I, II). Clones derived from genomic DNA may contain
regulatory and intronic DNA regions in addition to exonic (coding)
regions; whereas clones derived from cDNA will contain only exonic
sequences.
[0055] MDMIP nucleic acids are preferably derived from a cDNA
source. Identification of the specific cDNA containing the desired
sequence may be accomplished in a number of ways. In one method, a
portion of the MDMIP sequence (e.g., a PCR amplification product
obtained as described above), or an oligonucleotide possessing a
sequence of a portion of the known nucleotide sequence, or its
specific RNA, or a fragment thereof, may be purified, amplified,
and labeled, and the generated nucleic acid fragments may be
screened by nucleic acid hybridization utilizing a labeled probe.
See e.g., Benton & Davis, 1977. Science 196:180. In a second
method, the appropriate fragment is identified by restriction
enzyme digestion(s) and comparison of fragment sizes with those
expected from comparison to a known restriction map (if such is
available) or by DNA sequence analysis and comparison to the known
nucleotide sequence of MDMIP. In a third method, the gene of
interest may be detected utilizing assays based on the physical,
chemical or immunological properties of its expressed product. For
example, cDNA clones, or DNA clones which hybrid-select the proper
mRNAs, may be selected as a function of their production of a
protein which, for example, has similar or identical
electrophoretic migration, isoelectric focusing behavior,
proteolytic digestion maps, antigenic properties or ability to bind
the MDM2 protein. In a fourth method, should an anti-MDMIP antibody
be available, the protein of interest may be identified by the
binding of a labeled antibody to the putatively MDMIP clone in an
enzyme-linked immunosorbent assay (ELISA).
[0056] The MDMIP nucleic acid can be an isolated nucleic acid. An
"isolated" nucleic acid molecule is one that is separated from
other nucleic acid molecules which are present in the natural
source of the nucleic acid. Examples of isolated nucleic acid
molecules include, e.g., recombinant DNA molecules contained in a
vector, recombinant DNA molecules maintained in a heterologous host
cell, partially or substantially purified nucleic acid molecules,
and synthetic DNA or RNA molecules. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the nucleic
acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid) in the genomic DNA of the organism from which the nucleic
acid is derived. For example, in various embodiments, the isolated
MDMIP nucleic acid molecule can contain less than about 50 kb, 25
kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide
sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material or
culture medium when produced by recombinant techniques, or of
chemical precursors or other chemicals when chemically
synthesized.
[0057] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule including the nucleotide sequence of SEQ ID
NO: 1, and/or encoding the polypeptide including the amino acid
sequence of SEQ ID NO: 1, or a complement of any of these
nucleotide sequences, can be isolated using standard molecular
biology techniques and the sequence information provided herein.
Using all or a portion of the nucleic acid sequences of SEQ ID NO:
1 as a hybridization probe, MDMIP nucleic acid sequences can be
isolated using standard hybridization and cloning techniques (e.g.,
as described in Sambrook et al., eds., MOLECULAR CLONING: A
LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,
N.Y., 1993.).
[0058] In some embodiments, the MDMIP nucleic acid is identical in
sequence to a nucleic acid that includes SEQ ID NO: 1. In other
embodiments, the MDMIP nucleic acid differs from the nucleic acid
sequence of a nucleic acid that includes SEQ ID NO: 1. For example,
the MDMIP nucleic acid may include a sequence at least 90%, 95%,
98%, or even 99% or more identical to the nucleic acid sequence of
SEQ ID NO: 1. These sequences are referred to herein as variant
MDMIP nucleic acid sequences. An alternative way to describe
variant MDMIP nucleic acid sequences is to describe nucleic acids
that hybridize to a sequence including SEQ ID NO: 1, or to an
MDM2-binding fragment of SEQ ID NO: 1.
[0059] To determine the percent relatedness of two nucleic acid
sequences, or of two amino acid sequences (e.g., as would be done
to compare variant MDMIP polypeptides as discussed in more detail
below, or nucleic acids encoding such variant polypeptides), the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in the sequence of a first amino acid or nucleic
acid sequence for optimal alignment with a second amino or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are homologous at that
position (i.e., as used herein amino acid or nucleic acid
"homology" is equivalent to amino acid or nucleic acid
"identity").
[0060] The nucleic acid sequence homology may be determined as the
degree of identity between two sequences. The homology may be
determined using computer programs known in the art, such as GAP
software provided in the GCG program package. See Needleman and
Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the
following settings for nucleic acid sequence comparison: GAP
creation penalty of 5.0 and GAP extension penalty of 0.3, the
coding region of the analogous nucleic acid sequences referred to
above exhibits a degree of identity preferably of at least 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part
of a DNA sequence including the nucleic acid shown in SEQ ID NO: 1
or a polypeptide including the amino acid sequence shown in SEQ ID
NO: 2.
[0061] The term "sequence identity" refers to the degree to which
two polynucleotide or polypeptide sequences are identical on a
residue-by-residue basis over a particular region of comparison.
Sequence identity can be measured using sequence analysis software
(Sequence Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University
Avenue, Madison, Wis. 53705), with the default parameters
therein.
[0062] The term "percentage of sequence identity" is calculated by
comparing two optimally aligned sequences over that region of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case
of nucleic acids) occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the region of comparison (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The term "substantial identity" as
used herein denotes a characteristic of a polynucleotide sequence,
wherein the polynucleotide comprises a sequence that has at least
80 percent sequence identity, preferably at least 85 percent
identity and often 90 to 95 percent sequence identity, more usually
at least 99 percent sequence identity as compared to a reference
sequence over a comparison region.
[0063] DNA sequence polymorphisms that lead to changes in the amino
acid sequences of an MDMIP polypeptide may exist within a
population (e.g., the human population). Such genetic polymorphism
in the MDMIP gene may exist among individuals within a population
due to natural allelic variation. As used herein, the terms "gene"
and "recombinant gene" refer to nucleic acid molecules comprising
an open reading frame encoding a MDMIP polypeptide, preferably a
mammalian MDMIP polypeptide. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of the
polypeptide gene. Any and all such nucleotide variations and
resulting amino acid polymorphisms in MDMIP that are the result of
natural allelic variation and that do not alter the functional
activity of MDMIP are intended to be within the scope of the
invention.
[0064] Moreover, nucleic acid molecules encoding MDMIP proteins
from other species, and thus that have a nucleotide sequence that
differs from the human sequence of SEQ ID NO: 1, are intended to be
within the scope of the invention. Nucleic acid molecules
corresponding to natural allelic variants and homologues of the
MDMIP cDNAs of the invention can be isolated based on their
homology to the human MDMIP nucleic acids disclosed herein using
the human cDNAs, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions. For example, a soluble human MDMIP cDNA
can be isolated based on its homology to human membrane-bound
MDMIP. Likewise, a membrane-bound human MDMIP cDNA can be isolated
based on its homology to soluble human MDMIP.
[0065] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 6 nucleotides in length and
hybridizes under stringent conditions to the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO: 1. In another
embodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or
500 nucleotides in length. In another embodiment, an isolated
nucleic acid molecule of the invention hybridizes to the coding
region. As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences at least 60%
homologous to each other typically remain hybridized to each
other.
[0066] Homologs (i.e., nucleic acids encoding MDMIP proteins
derived from species other than human) or other related sequences
(e.g., paralogs) can be obtained by low, moderate or high
stringency hybridization with all or a portion of the particular
human sequence as a probe using methods well known in the art for
nucleic acid hybridization and cloning.
[0067] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a probe, primer or
oligonucleotide will hybridize to its target sequence, but to no
other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures than shorter
sequences. Generally, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion (or other salts) at pH 7.0 to 8.3 and the temperature is at
least about 30.degree. C. for short probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60.degree. C. for longer probes, primers and oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0068] Stringent conditions are known to those skilled in the art
and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the
conditions are such that sequences at least about 65%, 70%, 75%,
85%, 90%, 95%, 98%, or 99% homologous to each other typically
remain hybridized to each other. A non-limiting example of
stringent hybridization conditions is hybridization in a high salt
buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%
PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm
DNA at 65.degree. C. This hybridization is followed by one or more
washes in 0.2X SSC, 0.01% BSA at 50.degree. C. An isolated nucleic
acid molecule of the invention that hybridizes under stringent
conditions to the sequence of SEQ ID NO: 1 corresponds to a
naturally occurring nucleic acid molecule. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein).
[0069] In a second embodiment, a nucleic acid sequence that is
hybridizable to the nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO: 1, or fragments, analogs or derivatives
thereof, under conditions of moderate stringency is provided. A
non-limiting example of moderate stringency hybridization
conditions are hybridization in 6X SSC, 5X Denhardt's solution,
0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55.degree. C.,
followed by one or more washes in 1X SSC, 0.1% SDS at 37.degree. C.
Other conditions of moderate stringency that may be used are well
known in the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y., and
Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,
Stockton Press, N.Y..
[0070] In a third embodiment, a nucleic acid that is hybridizable
to the nucleic acid molecule comprising the nucleotide sequence of
SEQ ID NO: 1, or fragments, analogs or derivatives thereof, under
conditions of low stringency, is provided. A non-limiting example
of low stringency hybridization conditions are hybridization in 35%
formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP,
0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
(wt/vol) dextran sulfate at 40.degree. C., followed by one or more
washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS
at 50.degree. C. Other conditions of low stringency that may be
used are well known in the art (e.g., as employed for cross-species
hybridizations). See, e.g., Ausubel et al. (eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y., and
Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL,
Stockton Press, N.Y.; Shilo and Weinberg, 1981, Proc Natl Acad Sci
USA 78: 6789-6792.
[0071] In addition to naturally-occurring allelic variants of the
MDMIP sequence that may exist in the population, an MDMIP nucleic
acid also includes nucleic acids including changes introduced by
alteration of the nucleotide sequence of SEQ ID NO: 1. In some
embodiments, these changes lead to changes in the amino acid
sequence of the encoded MDMIP protein, without altering the
functional ability of the MDMIP protein. For example, nucleotide
substitutions leading to amino acid substitutions at
"non-essential" amino acid residues can be made in the sequence of
SEQ ID NO: 1. A "non-essential" amino acid residue is a residue
that can be altered from the wild-type sequence of MDMIP without
altering the biological activity, whereas an "essential" amino acid
residue is required for binding to MDM2 or for a biological
activity mediated by an MDMIP polypeptide. For example, amino acid
residues that are conserved among the MDMIP proteins of the present
invention, are predicted to be particularly unamenable to
alteration.
[0072] In addition, amino acid residues that are conserved among
family members of the MDMIP proteins of the present invention, are
also predicted to be particularly unamenable to alteration. Other
amino acid residues, however, (e.g., those that are not conserved
or only semiconserved among members of the MDMIP proteins) may not
be essential for activity and thus are likely to be amenable to
alteration.
[0073] Another aspect of the invention pertains to nucleic acid
molecules encoding MDMIP proteins that contain changes in amino
acid residues that are not essential for activity. Such MDMIP
proteins differ in amino acid sequence from SEQ ID NO: 2, yet
retain biological activity (e.g., MDM2-binding activity). In one
embodiment, the isolated nucleic acid molecule comprises a
nucleotide sequence encoding a protein, wherein the protein
comprises an amino acid sequence at least about 45% homologous to
the amino acid sequence of SEQ ID NO: 2. Preferably, the protein
encoded by the nucleic acid molecule is at least about 60%
homologous to SEQ ID NO: 2, more preferably at least about 70%,
80%, 90%, 95%, 98%, and most preferably at least about 99%
homologous to SEQ ID NO: 2.
[0074] An isolated nucleic acid molecule encoding a MDMIP protein
homologous to the protein of SEQ ID NO: 2 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO: 1, such that
one or more amino acid substitutions, additions or deletions are
introduced into the encoded protein.
[0075] Mutations can be introduced into SEQ ID NO: 1 by standard
techniques, such as site directed mutagenesis and PCR-mediated
mutagenesis. Preferably, conservative amino acid substitutions are
made at one or more predicted non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in MDMIP is replaced with
another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced
randomly along all or part of a MDMIP coding sequence, such as by
saturation mutagenesis, and the resultant mutants can be screened
for MDMIP biological activity to identify mutants that retain
activity. Following mutagenesis of SEQ ID NO: 1 , the encoded
protein can be expressed by any recombinant technology known in the
art and the activity of the protein can be determined.
[0076] In one embodiment, a mutant MDMIP polypeptide can be assayed
for (1) the ability to form protein:protein interactions with other
MDMIP proteins, or biologically active portions thereof; (2) the
ability to form complexes with form complexes with MDM2
polypeptides, (3) the ability to form complexes with a mutant MDMIP
protein and a MDMIP ligand (including MDMIP binding domains of MDM2
polypeptides, or other MDMIP ligands); (4) the ability of a mutant
MDMIP protein to bind to an intracellular target protein or
biologically active portion thereof; (e.g., avidin proteins); (5)
the ability to bind ATP; or (6) the ability to specifically a MDMIP
protein antibody.
[0077] If desired, MDMIP nucleic acids can be derived from a CDNA
source. Identification of the specific cDNA containing the desired
sequence may be accomplished in a number of ways. In one method, a
portion of the MDMIP sequence (e.g., a PCR amplification product),
or an oligonucleotide possessing a sequence of a portion of the
known nucleotide sequence, or its specific RNA, or a fragment
thereof, may be purified, amplified, and labeled, and the generated
nucleic acid fragments may be screened by nucleic acid
hybridization utilizing a labeled probe. See e.g., Benton &
Davis, Science 196:180, 1977. In a second method, the appropriate
fragment is identified by restriction enzyme digestion(s) and
comparison of fragment sizes with those expected from comparison to
a known restriction map (if such is available) or by DNA sequence
analysis and comparison to the known nucleotide sequence of MDMIP.
In a third method, the gene of interest may be detected utilizing
assays based on the physical, chemical or immunological properties
of its expressed product. For example, cDNA clones, or DNA clones
which hybrid-select the proper mRNAs, may be selected as a function
of their production of a protein which, for example, has similar or
identical electrophoretic migration, isolectric focusing behavior,
proteolytic digestion maps, antigenic properties or ability to bind
the MDM2 protein. In a fourth method, should an anti-MDMIP antibody
be available, the protein of interest may be identified by the
binding of a labeled antibody to the putatively MDMIP clone in an
enzyme-linked immunosorbent assay (ELISA).
[0078] Also included in the invention are vectors that include
MDMIP nucleic acids, as well as cells containing MDMIP nucleic
acids or vectors containing MDMIP nucleic acids. In general, any
suitable vector known in the art can be used. For recombinant
expression of an MDMIP polypeptide, a nucleic acid containing all
or a portion of the nucleotide sequence encoding the polypeptide
may be inserted into an appropriate expression vector (i.e., a
vector which contains the necessary elements for the transcription
and translation of the inserted protein coding sequence). The
regulatory elements can be heterologous (i.e., not the native gene
promoter), or can be supplied by the native promoter for the MDMIP
polypeptide, or any MDMIP genes and/or their flanking regions.
[0079] Exemplary host-vector systems that may be used to propagate
MDMIP nucleic acids, or to express MDMIP polypeptides include,
e.g.: (i) mammalian cell systems which are infected with vaccinia
virus, adenovirus; (ii) insect cell systems infected with
baculovirus; (iii) yeast containing yeast vectors or (iv) bacteria
transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
Depending upon the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be used.
[0080] The expression of the specific proteins may be controlled by
any promoter/enhancer known in the art including, e.g.: (i) the
SV40 early promoter (see e.g. Bernoist & Chambon, Nature
290:304-310, 1981); (ii) the promoter contained within the
3'-terminus long terminal repeat of Rous Sarcoma Virus (see e.g.,
Yamamoto, et al., Cell 22:787-797, 1980); (iii) the Herpesvirus
thymidine kinase promoter (see e.g., Wagner, et al., Proc. Natl.
Acad. Sci. USA 78:1441-1445, 1981); (iv) the regulatory sequences
of the metallothionein gene (see e.g., Brinster, et al., Nature
296:39-42, 1982); (v) prokaryotic expression vectors such as the
.beta.-lactamase promoter (see e.g., Villa-Kamaroff, et al., Proc.
Natl. Acad. Sci. USA 75:3727-3731, 1978); (vi) the tac promoter
(see e.g., DeBoer, et al., Proc. Natl. Acad. Sci. USA 80:21-25,
1983).
[0081] Plant promoter/enhancer sequences within plant expression
vectors may also be utilized including, e.g.,: (i) the nopaline
synthetase promoter (see e.g., Herrar-Estrella, et al., Nature
303:209-213, 1984); (ii) the cauliflower mosaic virus 35S RNA
promoter (see e.g., Garder, et al., Nuc. Acids Res. 9:2871, 1981)
and (iii) the promoter of the photosynthetic enzyme ribulose
bisphosphate carboxylase (see e.g., Herrera-Estrella, et al.,
Nature 310:115-120, 1984).
[0082] Promoter/enhancer elements from yeast and other fungi (e.g.,
the Gal4 promoter, the alcohol dehydrogenase promoter, the
phosphoglycerol kinase promoter, the alkaline phosphatase
promoter), as well as the following animal transcriptional control
regions, which possess tissue specificity and have been used in
transgenic animals, may be utilized in the production of proteins
of the present invention.
[0083] Other animal transcriptional control sequences derived from
animals include, e.g.,: (i) the insulin gene control region active
within pancreatic .beta.-cells (see e.g., Hanahan, et al., Nature
315:115-122, 1985); (ii) the immunoglobulin gene control region
active within lymphoid cells (see e.g., Grosschedl, et al., Cell
38:647-658, 1984); (iii) the albumin gene control region active
within liver (see e.g., Pinckert, et al., Genes and Devel.
1:268-276, 1987); (iv) the myelin basic protein gene control region
active within brain oligodendrocyte cells (see e.g., Readhead, et
al., Cell 48:703-712, 1987); and (v) the gonadotrophin-releasing
hormone gene control region active within the hypothalamus (see
e.g., Mason, et al., Science 234:1372-1378, 1986).
[0084] In one embodiment, the vector includes a promoter
operably-linked to nucleic acid sequences which encode an MDMIP
polypeptide, or a fragment, derivative or homolog, thereof, one or
more origins of replication, and optionally, one or more selectable
markers (e.g., an antibiotic resistance gene). If desired, a vector
is utilized which includes a promoter operably linked to nucleic
acid sequences encoding both the MDM2 protein and MDMIP, one or
more origins of replication, and, optionally, one or more
selectable markers.
[0085] In a specific embodiment, a nucleic acid encoding an MDMIP
polypeptide, or a portion thereof, is inserted into an expression
vector. The expression vector is generated by subcloning the
sequences into the EcoRI restriction site of each of the three
available pGEX vectors (glutathione S-transferase expression
vectors; see e.g., Smith et al., Gene 7:31-40, 1988), thus allowing
the expression of products in the correct reading frame. Expression
vectors that contain the sequences of interest may be identified by
three general approaches: (i) nucleic acid hybridization, (ii)
presence or absence of "marker" gene function and/or (iii)
expression of the inserted sequences. In the first approach, MDMIP
may be detected by nucleic acid hybridization using probes
comprising sequences homologous and complementary to the inserted
sequences of interest. In the second approach, the recombinant
vector/host system may be identified and selected based upon the
presence or absence of certain "marker" functions (e.g., binding to
an antibody specific for the MDMIP polypeptide, resistance to
antibiotics, occlusion-body formation in baculovirus, and the like)
caused by the insertion of the sequences of interest into the
vector.
[0086] Expression from certain promoters may be enhanced in the
presence of certain inducer agents, thus facilitating control of
the expression of the MDMIP polypeptide.
[0087] A host cell strain may be selected which modulates the
expression of MDMIP sequences, or modifies/processes the expressed
proteins in a desired manner. Moreover, different host cells
possess characteristic and specific mechanisms for the
translational and post-translational processing and modification
(e.g., glycosylation, phosphorylation, and the like) of expressed
proteins. Appropriate cell lines or host systems may thus be chosen
to ensure the desired modification and processing of the foreign
protein is achieved. For example, protein expression within a
bacterial system can be used to produce an unglycosylated core
protein; whereas expression within mammalian cells ensures "native"
glycosylation of a heterologous protein.
[0088] A MDMIP nucleic acid can be engineered so that it encodes a
fusion polypeptide having a portion of the MDMIP polypeptide, e.g.,
linked to a second polypeptide that includes a sequence that is
derived from a polypeptide other than an MDMIP polypeptide. The
second polypeptide can include, e.g., a marker polypeptide or
fusion partner. For example, the polypeptide can be fused to a
hexa-histidine tag to facilitate purification of bacterially
expressed protein or a hemagglutinin tag to facilitate purification
of protein expressed in eukaryotic cells.
[0089] Also included in the invention is a method of making an
MDMIP polypeptide, e.g., a rat or human MDMIP polypeptide, by
providing a cell containing DNA encoding an MDMIP polypeptide and
culturing the cell under conditions permitting expression of the
MDMIP encoding DNA, i.e., production of the recombinant MDMIP by
the cell.
[0090] Various regions of the MDMIP nucleic acid can be used to
detect MDMIP nucleic acids in populations of nucleic acids. For
example, probes derived from nucleic acids 1-222 (SEQ ID NO: 1),
nucleic acids 1-486 of (SEQ ID NO: 7) or nucleic acids 223-486 (SEQ
ID NO: 8) can be used to identify MDMIP nucleic acids. The probes
can be, e.g., hybridization probes derived from these sequences, or
primers which specifically amplify these sequences in amplification
assays (such as PCR amplification assays).
MDMIP Polypeptides
[0091] Also provided in the invention is an MDMIP polypeptide, as
well as variants and fragments of an MDMIP polypeptide. Preferably,
the MDMIP polypeptide, variant or fragment binds an
MDM2-polypeptide, e.g., it includes the amino acid sequence of SEQ
ID NO: 2.
[0092] In some embodiments, the MDMIP polypeptide is purified. A
"purified" polypeptide, protein or biologically active portion
thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the MDMIP protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of MDMIP protein in which the protein is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
MDMIP protein having less than about 30% (by dry weight) of
non-MDMIP protein (also referred to herein as a "contaminating
protein"), more preferably less than about 20% of non-MDMIP
protein, still more preferably less than about 10% of non-MDMIP
protein, and most preferably less than about 5% non-MDMIP protein.
When the MDMIP protein or biologically active portion thereof is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, more preferably less than about 10%, and most preferably less
than about 5% of the volume of the protein preparation.
[0093] In some embodiments, the MDMIP polypeptide, variant, or
fragment binds an MDM2polypeptide. In some embodiments, the MDMIP
polypeptide includes an amino acid sequence at least 80% identical
to a polypeptide which includes the amino acid sequence of SEQ ID
NO: 2. More preferably, the polypeptide is at least 85, 90, 95, 98,
or even 99% or more identical. The percent relatedness of two amino
acid sequences can be determined as described above for MDMIP
nucleic acids.
[0094] The MDMIP polypeptides can be made by expressing MDMIP
nucleic acids as described above and recovering the MDMIP
polypeptide. Alternatively, the MDMIP polypeptide can be chemically
synthesized using standard techniques, e.g., by the methods
described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The
Pierce Chemical Co., Rockford, Ill.
[0095] The MDMIP polypeptide can be used to detect an MDM2
polypeptide in a biological sample by biological sample, e.g., a
cell or tissue sample from a subject, or a cell population cultured
in vitro. The sample is contacted with an MDMIP polypeptide under
conditions sufficient to allow for formation of an MDM2-MDMIP
complex (as explained below), if theMDM2 polypeptide is present in
the sample and then detecting the complex. Presence of the
MDMIP-MDM2 complex indicates the MDM2 polypeptide is present in the
sample.
[0096] The MDMIP polypeptide can also be used to remove, or purify,
an MDM2 polypeptide from a biological sample. The method includes
contacting the sample with an MDMIP polypeptide under conditions
sufficient to allow for formation of an MDM2-MDMIP complex; if the
MDM2 polypeptide is present in the sample, and removing the complex
from said sample, thereby removing said MDM2 polypeptide from said
sample.
[0097] Preferably, the MDMIP polypeptide is labeled to facilitate
detection and recovery of MDMIP-polypeptide complexes.
MDMIP Binding MDM2 Polypeptide Derivatives and Fragments
[0098] The invention also provides nucleic acids encoding
polypeptides or peptides derived from an MDM2 polypeptide or
peptide. Preferably, the nucleic acids encode polypeptides or
peptides that contain an MDMIP binding domain. Also included in the
invention are the polypeptides and peptides encoded by these
nucleic acids.
[0099] In general, an MDMIP-binding MDM2 polypeptide according to
the invention includes any region of an MDM2 polypeptide that is
less than the length of a full-length MDM2 polypeptide, but which
includes an MDMIP binding domain, e.g. is less than 491 amino acids
and is greater than 4 amino acids in length. Preferably, the
MDMIP-binding polypeptide is greater than 5, 6, 7, 8, 9, or 10
amino acids in length and is less than 25, 50, 75, 100, 150, 200,
250, 300, 350, 400 or 450 amino acids in length. The MDMIP-binding
polypeptide preferably includes SEQ ID NO: 4, or a related
sequence.
[0100] By "MDM2 polypeptide" is meant a polypeptide at least 80%
identical to the amino acid sequence of a polypeptide that includes
an amino acid sequence of a human MDM2 polypeptide, e.g., the 491
amino acid polypeptide encoded by the nucleic acid sequence
disclosed in GenBank Accession No. M92424.
[0101] By "MDMIP-binding domain" or "MDMIP-binding region" is meant
a region of amino acids sufficient to allow the polypeptide in
which the region of amino acids is present to bind specifically to
an MDMIP polypeptide.
[0102] The MDMIP binding polypeptide present in the complex will
typically include at least 6, 8, 10, 12, or 15 or more amino acids
of an MDM2 polypeptide. Preferably, the polypeptide corresponds to
a region of contiguous amino acids in a MDM2 polypeptide. In some
embodiments, the MDM2 polypeptide is a polypeptide which includes
the amino acid sequence encoded by SEQ ID NO: 3 (i.e., nucleotides
312-921 of the nucleotide sequence represented by GenBank Accession
Number M92424). The MDMIP binding polypeptide may be a full-length
MDM2 polypeptide, e.g., it may have the amino acid sequence of the
MDM2 polypeptide encoded by a human nucleic acid, which is
available as GenBank Accession No. M92424.
[0103] Alternatively, the MDMIP binding MDM2 polypeptide includes a
region of the amino acid sequence of SEQ ID NO: 4 that is able to
bind specifically to an MDMIP polypeptide. Procedures for
identifying regions within SEQ ID NO: 4 that bind to MDMIP can be
readily identified by one of ordinary skill in the art and the
procedures described herein. For example, nucleic acid sequences
containing various portions of SEQ ID NO: 3 can be tested in a
yeast two hybrid screening assay in combination with a nucleic acid
encoding the MDM2-binding region of an MDMIP polypeptide.
[0104] The invention also includes nucleic acids encoding
MDMIP-binding fragments of an MDM2 polypeptide. The nucleic acids
can include a nucleic acid sequence that is identical to a portion
of a human MDM2 polypeptide disclosed in GenBank Accession No.
M92424. Alternatively, the nucleic acid can be an MDM2 variant that
is greater than 70, 80, 85, 90, 95, 98, or even 99% identical to
the corresponding region of the human MDM2 polypeptide nucleic
acid. Alternatively, the nucleic acid encodes an MDMIP-binding MDM2
polypeptide which is greater than 70, 80, 85, 90, 95, 98, or even
99% identical to a portion of the human MDM2polypeptide disclosed
in GenBank Accession No. M92424.
[0105] Alternatively, the nucleic acid encoding an MDMIP-binding
MDM2 polypeptide may hybridize under low, medium, or high
stringency, using the parameters and conditions described above for
MDMIP nucleic acids and polypeptides.
[0106] A MDMIP-binding MDM2 nucleic acid nucleic acid can be
engineered so that it encodes a fusion polypeptide having a portion
of the MDMIP polypeptide, e.g., linked to a second polypeptide that
includes a sequence that is derived from a polypeptide other than
an MDMIP polypeptide. The second polypeptide can include, e.g., a
marker polypeptide or fusion partner. For example, the polypeptide
can be fused to a hexa-histidine tag to facilitate purification of
bacterially expressed protein or a hemagglutinin tag to facilitate
purification of protein expressed in eukaryotic cells.
[0107] A nucleic acid encoding an MDMIP binding MDM2 peptide can be
provided in vector as described above for vectors and cells
containing MDMIP polypeptides. MDM2-binding MDMIP polypeptides can
be synthesized by expressing nucleic acids encoding the MDMIP
binding polypeptides and recovering the expressed polypeptides,
e.g, using vectors and/or cells which include nucleic acid encoding
an MDMIP binding MDM2 polypeptide.
[0108] Also included in the invention is a method of making an MDM2
polypeptide fragment, e.g., a rat or human MDMIP-binding MDM2
polypeptide fragment polypeptide, by providing a cell containing
DNA encoding an MDMIP-binding MDM2 polypeptide fragment and
culturing the cell under conditions permitting expression of the
MDM2 fragment encoding DNA, i.e., production of the recombinant
MDM2 polypeptide by the cell.
Chimeric Polypeptides Including an MDM2-Binding Region of a MDMIP
Polypeptide and an MDMIP Region of an MDM2 Polypeptide
[0109] Also included in the invention is a chimeric polypeptide or
peptide which includes a region of an MDM2 polypeptide covalently
linked, e.g., via a peptide bond, to a region of an MDMIP
polypeptide. In some embodiments, the chimeric polypeptide includes
six or more amino acids of an MDM2 polypeptide covalently linked to
six or more amino acids of an MDMIP polypeptide.
[0110] Preferably, the MDM2 polypeptide in the chimeric polypeptide
includes an MDMIP binding domain. Preferably, the MDMIP polypeptide
in the chimeric polypeptide includes an MDM2-binding polypeptide.
In some embodiments, the MDM2 and MDMIP polypeptides of the
chimeric polypeptide interact to form a complex. Any MDMIP
polypeptide disclosed herein can be used in the complex, e.g., the
chimeric polypeptide can include an amino acid sequence at least
90% identical to the amino acid sequence of SEQ ID NO: 2.
Similarly, any MDM2polypeptide can be present in the chimeric
polypeptide.
[0111] Also included in the invention are nucleic acids encoding
the chimeric polypeptides or peptides, as well as vectors and cells
containing these nucleic acids.
[0112] The chimeric polypeptides can be constructed by expressing
nucleic acids encoding chimeric polypeptides using vectors and
cells as described above for MDMIP polypeptides and MDM2
polypeptides, and then recovering the chimeric polypeptides, or by
chemically synthesizing the chimeric polypeptides.
MDM2-MDMIP Complexes
[0113] In another aspect, the invention includes a purified complex
that includes the MDM2-binding domain of an MDMIP polypeptide and
an MDMIP-binding domain of a MDM2polypeptide. By purified complex
is meant a complex of polypeptide that includes an MDM2-binding
domain of an MDMIP polypeptide and a polypeptide that includes and
MDMIP binding domain of an MDM2 polypeptide.
[0114] In general, the complex can include any MDMIP polypeptide
described herein as long as it includes an MDM2 binding domain.
Similarly, any MDM2 polypeptide, or any MDM2 derived polypeptide,
can be used as long as it contains an MDMIP binding region.
[0115] Thus, the MDM2-binding polypeptide and the MDMIP-binding
polypeptide present in the complex can have the amino acid sequence
of a mammalian MDMIP polypeptide, (e.g., mouse, rat, pig, cow, dog,
monkey, frog), or of insects (e.g., fly), plants or, most
preferably, human. In some embodiments, both polypeptides have the
amino acid sequences of regions of the corresponding human
polypeptides. For example, the complex can include a human MDM2
polypeptide, or an MDMIP binding fragment of a human MDM2
polypeptide, and a human MDMIP polypeptide, or a MDM2-binding
fragment of a human MDMIP polypeptide.
[0116] In various embodiments, other components, e.g.,
polypeptides, are present in the complex in addition to the
MD-binding domain of an MDMIP polypeptide and the MDMIP binding
domain of an MDM2 polypeptide. In some embodiments, one or more
polypeptides, such as p53, Rb, E2F-1, DP1 and Numb are absent from
the complex. In additional embodiments, MDMIP binding polypeptide
and the MDM2-binding polypeptides are the only polypeptide
components present in significant levels in the complex. An example
of such a complex is a purified complex of a human MDM2 polypeptide
and a human MDMIP polypeptide.
[0117] In some embodiments, the MDM2-binding polypeptide and the
MDMIP binding polypeptide complex is a functionally active complex.
As utilized herein, the term "functionally active MDM2-binding
polypeptide and the MDMIP binding polypeptide complex" refers to
species displaying one or more known functional attributes of a
full-length MDM2 protein complexed with full-length MDMIP. These
attributes include, e.g., the control of cellular and physiological
processes, such as: (i) control of cell-cycle progression; (ii)
cellular differentiation and apoptosis; (iii) regulation of
transcription; and (iv) pathological processes including, e.g.,
hyperproliferative disorders (e.g., tumorigenesis and tumor
progression).
[0118] In other embodiments, the complex is not functionally active
i.e., the complex lacks one or more of these functional
attributes.
[0119] Either, or both, of the MDMIP or MDM2-binding polypeptides
in the complex may be labeled, i.e., attached to one or more
detectable substances. Labeling can be performed using any art
recognized method for labeling polypeptides. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerytlrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0120] The complexes can be made by expressing each polypeptide
from each nucleic acid and allowing the complex to form from the
expressed polypeptides. Any of the nucleic acids disclosed herein
that express MDM2-binding MDMIP polypeptides or EMMIP biding MDM2
polypeptides (or chimerics of these polypeptides) can be used, as
can vectors and cells expressing these polypeptides. If desired,
the complexes can then be recovered and isolated.
[0121] Once a recombinant cell expressing the MDM2 protein and/or
MDMIP, or a fragment or derivative thereof, is identified, the
individual gene product or complex may be isolated and analyzed.
This is achieved by assays that are based upon the physical and/or
functional properties of the protein or complex. The assays can
include, e.g., radioactive labeling of one or more of the
polypeptide complex components, followed by analysis by gel
electrophoresis, immunoassay, cross-linking to marker-labeled
products. The MDM2 protein-MDMIP complex may be isolated and
purified by standard methods known in the art (either from natural
sources or recombinant host cells expressing the proteins/protein
complex). These methods can include, e.g., column chromatography
(e.g., ion exchange, affinity, gel exclusion, reverse-phase, high
pressure, fast protein liquid, etc), differential centrifugation,
differential solubility, or similar methods used for the
purification of proteins.
[0122] The MDM2 protein-MDMIP complex is implicated in the
modulation of functional activities of the MDM2 protein. Such
functional activities include, e.g., (i) control of cell-cycle
progression; (ii) cellular differentiation and apoptosis; (iii)
regulation of transcription; and (iv) pathological processes
including, but not restricted to, hyperproliferative disorders
(e.g., tumorigenesis and tumor progression).
[0123] The MDM2 protein-MDMIP complex may be analyzed by
hydrophilicity analysis (see e.g., Hopp & Woods, Proc. Natl.
Acad. Sci. USA 78:3824-3828, 1981). This analysis can be used to
identify the hydrophobic and hydrophilic regions of the proteins,
thus aiding in the design of substrates for experimental
manipulation, such as in binding experiments, antibody synthesis.
Secondary structural analysis may also be performed to identify
regions of the MDM2 protein and/or MDMIP which assume specific
structural motifs. See e.g., Chou & Fasman, Biochem.
13:222-223, 1974. Manipulation, translation, secondary structure
prediction, hydrophilicity and hydrophobicity profiles, open
reading frame prediction and plotting, and determination of
sequence homologies, can be accomplished using computer software
programs available in the art.
[0124] Other methods of structural analysis, e.g., X-ray
crystallography (see e.g. Engstrom, Biochem. Exp. Biol. 11:7-13,
1974); mass spectroscopy and gas chromatography (see e.g., Methods
in Protein Science, J. Wiley and Sons, New York, N.Y., 1997) and
computer modeling (see e.g., Fletterick & Zoller, eds.,
Computer Graphics and Molecular Modeling, In: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986) may also be used to
characterize complexes.
Antibodies to MDMIP Polypeptides, MDMIP Binding Polypeptides of
MDM2 Polypeptides, and Complexes of MDMIP Polypeptides and MDM2
Polypeptides
[0125] The invention further encompasses antibodies and antibody
fragments (such as Fab or (Fab)2 fragments) that bind specifically
to any of the polypeptides or complexes described herein. By
"specifically binds" is meant an antibody that recognizes and binds
to a particular antigen, e.g., an MDMIP polypeptide of the
invention, but which does not substantially recognize or bind to
other molecules in a sample, e.g., a biological sample, which
includes an MDMIP polypeptide.
[0126] These polypeptides and complexes can include, e.g., an MDMIP
polypeptide, an MDMIP binding MDM2 polypeptide, a chimeric
MDM2-MDMIP polypeptide, or a complex of an MDMIP polypeptide and
MDM2 polypeptide. The antibodies and antibody fragments can
alternatively be raised against variants or fragments of the
complexes or polypeptides. For example, a purified MDMIP
polypeptide, or a portion, variant, or fragment thereof, can be
used as an immunogen to generate antibodies that bind MDMIP using
standard techniques for polyclonal and monoclonal antibody
preparation.
[0127] A full-length MDMIP polypeptide can be used, if desired.
Alternatively, the invention provides antigenic peptide fragments
of MDMIP polypeptides for use as immunogens. In some embodiments,
an antigenic MDMIP peptide includes at least four amino acid
residues of the amino acid sequence shown in SEQ ID NO: 2. The
antigenic peptide encompasses an epitope of MDMIP such that an
antibody raised against the peptide forms a specific immune complex
with MDMIP. In some embodiments, the antigenic peptide includes at
least 6, 8, 10, 15, 20, or 30 or more amino acid residues of an
MDMIP polypeptide. In one embodiment, epitopes encompassed by the
antigenic peptide are regions of MDMIP that bind MDM2, or are
located on the surface of the protein, e.g., hydrophilic
regions.
[0128] In some embodiments, the antibodies are raised against an
MDMIP-MDM2 complex. Preferably, the anti-complex antibodies bind
with higher affinity to the complex as compared to their affinity
for an isolated MDM2 polypeptide or an isolated MDMIP
polypeptide.
[0129] If desired, peptides containing antigenic regions can be
selected using hydropathy plots showing regions of hydrophilicity
and hydrophobicity. These plots may be generated by any method well
known in the art, including, for example, the Kyte Doolittle or the
Hopp Woods methods, either with or without Fourier transformation.
See, e.g., Hopp and Woods, Proc. Nat. Acad. Sci. USA 78:3824-3828,
1981; Kyte and Doolittle, J. Mol. Biol. 157:105-142, 1982, each
incorporated herein by reference in their entirety.
[0130] The MDMIP polypeptide sequence of SEQ ID NO: 2, or
derivatives, fragments, analogs or homologs thereof, may be
utilized as immunogens in the generation of antibodies that
specifically bind these protein components. The term "antibody" as
used herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen binding site that specifically binds
(immunoreacts with) an antigen, such as MDMIP, an MDMIP binding
polypeptide fragment of MDM2, or a complex including the two
polypeptides. Such antibodies include, e.g.,, polyclonal,
monoclonal, chimeric, single chain, Fab and F(ab,)2 fragments, and
an Fab expression library. In specific embodiments, antibodies to
human MDMIP polypeptides or complexes of human MDMIP and MDM2
polypeptides are disclosed.
[0131] Various procedures known within the art may be used for the
production of polyclonal or monoclonal antibodies. For example, for
the production of polyclonal antibodies, various suitable host
animals (e.g., rabbit, goat, mouse or other mammal) may be
immunized by injection with the native protein, or a synthetic
variant thereof, or a derivative of the foregoing. An appropriate
immunogenic preparation can contain, for example, recombinantly
expressed MDMIP, MDM2, or chimeric polypeptide including the two
polypeptides, or a complex including the two polypeptides.
Alternatively, the immunogenic polypeptide or polypeptides may be
chemically synthesized.
[0132] The preparation can further include an adjuvant. Various
adjuvants used to increase the immunological response include,
e.g., Freund's (complete and incomplete), mineral gels (e.g.,
aluminum hydroxide), surface active substances (e.g., lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions,
dinitrophenol, etc.), human adjuvants such as Bacille
Calmette-Guerin and Corynebacterium parvum, or similar
immunostimulatory agents. If desired, the antibody molecules
directed against MDMIP, MDM2, chimeras, or complexes can be
isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography
to obtain the IgG fraction.
[0133] The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope MDMIP, MDM2,
chimeras, or complex of these polypeptides. A monoclonal antibody
composition thus typically displays a single binding affinity for a
particular protein with which it immunoreacts. For preparation of
monoclonal antibodies directed towards a particular MDMIP
polypeptide, MDM2 polypeptide, chimeras, or complex, or
derivatives, fragments, analogs or homologs thereof, any technique
that provides for the production of antibody molecules by
continuous cell line culture may be utilized. Such techniques
include, e.g., the hybridoma technique (see Kohler & Milstein,
Nature 256:495-497, 1975); the trioma technique; the human B-cell
hybridoma technique (see Kozbor, et al., Immunol Today 4:72, 1983)
and the EBV hybridoma technique to produce human monoclonal
antibodies (see Cole, et al., In: Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). If desired, human
monoclonal antibodies may be prepared by using human hybridomas
(see Cote, et al., Proc. Natl. Acad. Sci. USA 80:2026-2030, 1983)
or by transforming human B-cells with Epstein Barr Virus in vitro
(see Cole, et al., In: Monoclonal Antibodies and Cancer Therapy,
supra). Each of the above citations are incorporated herein by
reference in their entirety.
[0134] Techniques can be adapted for the production of single-chain
antibodies specific to a MDMIP polypeptide, MDMIP-binding MDM2
polypeptide (or fragment), MDM2-MDMIP achimeric polypeptide, or a
complex of an MDMIP and MDM2 polypeptide (see e.g., U.S. Pat. No.
4,946,778). In addition, methods can be adapted for the
construction of F.sub.ab expression libraries (see e.g., Huse, et
al., Science 246:1275-1281, 1989) to allow rapid and effective
identification of monoclonal Fab fragments with the desired
specificity for the desired protein or derivatives, fragments,
analogs or homologs thereof. Non-human antibodies can be
"humanized" by techniques well known in the art. See e.g., U.S.
Pat. No. 5,225,539. Each of the above citations is incorporated
herein by reference. Antibody fragments that contain the idiotypes
to a MDMIP protein may be produced by techniques known in the art
including, e.g.: (i) an F.sub.(ab')2 fragment produced by pepsin
digestion of an antibody molecule; (ii) an F.sub.ab fragment
generated by reducing the disulfide bridges of an F.sub.(ab')2
fragment; (iii) an F.sub.ab fragment generated by the treatment of
the antibody molecule with papain and a reducing agent and (iv)
F.sub.v fragments.
[0135] Additionally, recombinant anti-MDMIP polypeptide, MDMIP
binding MDM2 polypeptide (or fragment), MDM2-MDMIP chimeric
polypeptide, a complex of an MDMIP and MDM2 polypeptide antibodies,
such as chimeric and humanized monoclonal antibodies, comprising
both human and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in PCT International Application No.
PCT/US86/02269; European Patent Application No. 184,187; European
Patent Application No. 171,496; European Patent Application No.
173,494; PCT International Publication No. WO 86/01533; U.S. Pat.
No. 4,816,567; European Patent Application No. 125,023; Better et
al., Science 240:1041-1043, 1988; Liu et al., Proc. Nat. Acad. Sci.
USA 84:3439-3443, 1987; Liu et al., J. Immunol. 139:3521-3526,
1987; Sun et al., Proc. Nat. Acad. Sci. USA 84:214-218, 1987;
Nishimura et al., Cancer Res. 47:999-1005, 1987; Wood et al.,
Nature 314:446-449, 1985; Shaw et al., J. Natl. Cancer Inst.
80:1553-1559, 1988; Morrison, Science 229:1202-1207, 1985; Oi et
al., BioTechniques 4:214, 1986; U.S. Pat. No. 5,225,539; Jones et
al., Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534,
1988; and Beidler et al., J. Immunol. 141:4053-4060, 1988. Each of
the above citations is incorporated herein by reference.
[0136] In one embodiment, methods for the screening of antibodies
that possess the desired specificity include, e.g., enzyme-linked
immunosorbent assay (ELISA) and other immunologically-mediated
techniques known within the art. In a specific embodiment,
selection of antibodies that are specific to a particular domain of
a MDMIP polypeptide, MDMIP binding MDM2 polypeptide (or fragment),
MDM2-MDMIP chimeric polypeptide, or a complex of an MDMIP and MDM2
polypeptide is facilitated by generation of hybridomas that bind to
the fragment of a polypeptide or complex possessing such a domain.
Antibodies that are specific for one or more domains within a given
polypeptide or complex, e.g., the domain including the amino acids
of SEQ ID NO: 2 in a MDMIP polypeptide, or derivatives, fragments,
analogs or homologs thereof, are also provided herein.
[0137] An anti-MDMIP polypeptide, MDMIP binding MDM2 polypeptide
(or fragment), MDM2-MDMIP chimeric polypeptide, or a complex of an
MDMIP and MDM2 polypeptide antibodies may be used in methods known
within the art relating to the localization and/or quantitation of
a given polypeptide or complex (e.g., for use in measuring levels
of the given polypeptide complex within appropriate physiological
samples, for use in diagnostic methods, for use in imaging the
protein, and the like). In one embodiment, antibodies for the given
polypeptide or complex, or derivatives, fragments, analogs or
homologs thereof, that contain the antibody derived binding domain,
are utilized as pharmacologically-active compounds.
[0138] An anti-MDMIP polypeptide, MDMIP binding MDM2 polypeptide
(or fragment), MDM2-MDMIP chimeric polypeptide, or an
anti-MDMIP-MDM2 complex of an MDMIP and MDM2 polypeptide antibody
(e.g., monoclonal antibody) can be used to isolate MDMIP, MDM2
polypeptides, or complexes including MDMIP polypeptides and MDM2
polypeptides, by standard techniques, such as affinity
chromatography or immunoprecipitation. Thus, the anti-antibodies
disclosed herein can facilitate the purification of complexes of
MDM2 and MDMIP polypeptides from cells and of recombinantly
produced MDM2 and MDMIP polypeptides expressed in host cells.
[0139] An antibody of the invention can additionally be used to
detect MDM2, MDMIP polypeptides, or complexes of MDMIP and MDM2
polypeptides (e.g., in a cellular lysate or cell supernatant) in
order to evaluate the abundance and pattern of expression of these
polypeptides or complexes. Further, the herein disclosed antibodies
can be used diagnostically to monitor protein levels in tissue as
part of a clinical testing procedure, e.g., to, for example,
determine the efficacy of a given treatment regimen. Detection can
be facilitated by coupling (i.e., physically linking) the antibody
to a detectable substance. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
Use of MDM2 Protein-MDMIP Complexes or Their Component Polypeptides
to Identfy MDMIP and MDM2 Interacting Agents
[0140] The MDMIP, MDM2, and complexes disclosed herein can also be
used to identify compounds or other agents which modulate the
activity of MDM2 and/or MDMIP-mediated process. For example, to
identify an agent that modulates MDM2 or MDMIP activity, an
MDMIP-MDM2 complex is tested with a test agent and binding of the
agent to the complex is measured. Binding of the agent to the
complex indicates the agent modulates MDM2 or MDMIP polypeptide
activity.
[0141] Any compound or other molecule (or mixture or aggregate
thereof) can be used as a test compound. In some embodiments, the
agent can be a small peptide, or other small molecule produced by
e.g., combinatorial synthetic methods known in the art. Binding of
the compound to the complex can be determined using art recognized
methods, e.g., by immunoprecipitation using antibodies (e.g.,
antibodies against MDMIP, MDM2, or the MDM2IP-MDM2 complex). Bound
agents can be identified by comparing the relative electophoretic
mobility of complexes exposed to the test agent to the mobility of
complexes that have not been exposed to the test agent. Altered
migration of the test complexes indicates the test agent binds to
the MDMIP-MDM2 complex.
[0142] Also provided for in the invention is a method for
identifying agents which modulate MDMIP activity by contacting an
MDMIP polypeptide (or an MDMIP-binding fragment of an MDM2
polypeptide) with a test agent, and measuring binding of the agent
to the complex. Binding of the agent to the complex indicates the
agent modulates MDMIP polypeptide activity. As described above, any
art-recognized method for determining binding of a compound to a
test compound can be used.
[0143] Agents identified in the screening assays can be further
tested for their ability to alter and/or modulate cellular
functions, particularly those functions in which the MDM2 protein
has been implicated. These functions include, e.g., control of
cell-cycle progression; regulation of transcription; control of
intracellular signal transduction; and pathological processes, as
well as various other biological activities (e.g., binding to an
anti-MDM2 proteinMDMIP complex antibody).
Use of MDM2 Protein-MDMIP Complexes or Their Component Polypeptides
to Identify MDMIP and MDM2 Interacting Agents
[0144] MDM2 is implicated in multiple biological processes.
Accordingly, a variety of conditions can be or identified in
subjects by measuring the levels of MDM2-MDMIP complexes in a
subject and comparing the levels of the MDM2-MDMIP complexes to the
levels of the complexes in a reference population whose
corresponding status with respect to the compared condition is
known.
[0145] Comparable levels of the MDM2-MDMIP complex in the test
sample and the reference sample indicates the subject has the
MDM2-MDMIP complex associated disorder (or absence thereof) as that
of the reference population. In contrast, altered levels of the
complex in the test and reference populations indicates the
subject's status with respect to the MDM-MDMIP disorder is
different from that in the control population. Thus, if the
reference cell population includes cells from individuals that do
not have the MDM2-MDMIP associated disorder, a similarity in
MDMIP-MDM2 complex levels in the test and control populations
indicates the subject does not have the MDMIP-MDM2
complex-associated disorder. Conversely, a difference in the levels
of the test and reference populations indicates the subject has, or
has a predisposition to, the MDM2-MDMIP disorder.
[0146] In general, a test cell population from the subject includes
at least one cell that is capable of expressing genes encoding the
polypeptide, or polypeptides making up the measured complex (i.e.,
the cell expresses an MDMIP polypeptide, MDM2, polypeptide, or
both). By "capable of expressing" is meant that the corresponding
gene or genes is present in an intact form in the cell and can be
expressed.
[0147] In general, any reference cell population can be used, as
long as its status with respect to the measure parameter is known
(i.e., the reference cell population is known to possess or lack
the property or trait being measured in the test cell population),
and whose level of MDMIP-MDM2 complex is known. In some
embodiments, the reference cell population is made up
substantially, or preferably exclusively, of such cells whose
status is known.
[0148] Preferably, cells in the reference cell population are
derived from a tissue type as similar as possible to a test cell.
In some embodiments, the control cell is derived from the same
subject as the test cell. In other embodiments, the control cell
population is derived from a database of molecular information
derived from cells for which the assayed parameter or condition is
known. The subject is preferably a mammal. The mammal can be, e.g.,
a human, non-human primate, mouse, rat, dog, cat, horse, or
cow.
[0149] In some embodiments, the reference cell population is
derived from a plurality of cells. For example, the reference cell
population can be a database of expression patterns from previously
tested cells for which one of the herein-described parameters or
conditions is known. If desired, comparison of differentially
expressed sequences between a test cell population and a reference
cell population can be done with respect to a expression of a
control gene whose expression is independent of the parameter or
condition being measured. Expression levels of the control nucleic
acid in the test and reference nucleic acid can be used to
normalize signal levels in the compared populations.
[0150] In some embodiments, the test cell population is compared to
multiple reference cell populations. Each of the multiple reference
populations may differ in the known parameter. Thus, a test cell
population may be compared to a second reference cell population
known to contain, e.g., tumorous cells, as well as a second
reference population known to contain, e.g., non-tumorous
cells.
[0151] The test cell or cell population in any of the herein
described diagnostic or screening assays can be taken from a known
or suspected tumor containing sample or from a bodily fluid, e.g.,
biological fluid (such as blood, serum, urine, saliva, milk, ductal
fluid, or tears). For many applications, cells present in a bodily
fluid can be examined instead of a primary lesion. Thus, the need
for taking a biopsy from a known or suspected primary tumor site is
obviated.
[0152] In another aspect, disorders associated with altered levels
of MDMIP or MDM2-MDMIP complexes are identified in a subject by
measuring expression of MDMIP nucleic acids. Expression of MDMIP
sequences can be detected (if present) and measured using
techniques well known to one of ordinary skill in the art. For
example, sequences disclosed herein (e.g. SEQ ID NO: 1) can be used
to construct probes for detecting MDMIP RNA sequences in, e.g.,
northern blot hybridization analyses. As another example, the
sequences can be used to construct primers for specifically
amplifying sequences in, e.g, amplification-based detection methods
such as reverse-transcription based polymerase chain reaction.
[0153] Expression level of MDMIP sequences in the test cell
population is then compared to expression levels of the sequences
in one or more cells from a reference cell population. Expression
of the genes disclosed herein can be measured at the RNA level
using any method known in the art. For example, northern
hybridization analysis using probes which specifically recognize
one or more of these sequences can be used to determine gene
expression. Alternatively, expression can be measured using
reverse-transcription-based PCR assays, e.g., using primers
specific for the differentially expressed sequences. When
alterations in gene expression are associated with gene
amplification or deletion, sequence comparisons in test and
reference populations can be made by comparing relative amounts of
the examined DNA sequences in the test and reference cell
populations.
[0154] Levels of MDM2 protein-MDMIP complexes can be used to
determine the presence of, or predisposition to, multiple states in
a subject. For example, these complexes can serve as markers for
specific disease states that involve the disruption of
physiological processes. These processes can include e.g., control
of cell-cycle progression, cellular differentiation and apoptosis,
or regulation of transcription. In addition, a subject can be
assessed for the presence, or predisposition to, pathological
processes. These processes can include, e.g., hyperproliferative
disorders (e.g., tumorigenesis and tumor progression).
[0155] In addition to diagnostic methods, the herein described
methods can be used to assess the prognosis, or follow the course
of, a disease or condition associated with altered levels of
MDMIP-MDM2 complexes (or its components) in a subject. These
methods can additionally b use to determine the efficacy of
administered therapeutics.
[0156] To detect MDMIP-MDM2 complexes, the herein disclosed
antibodies may be used. For example, anti MDMIP-MDM2 complex
antibodies or anti-MDMIP antibodies can be used in assays (e.g.,
immunoassays) to detect, prognose, diagnose, or monitor various
conditions, diseases, and disorders characterized by aberrant
levels of MDM2 protein-MDMIP complex. They can alternatively used
to monitor the treatment of conditions associated with altered
levels of these complexes, or their components thereof.
[0157] To perform immunoassays using antibodies for MDMIP-MDM2
complexes, a sample derived from a patient is contacted with an
anti-MDM2 protein-MDMIP complex antibody under conditions such that
specific binding may occur. Specific binding by the antibody is
then detected, if present, and quantitated.
[0158] In a specific embodiment, an antibody specific for a MDM2
protein-MDMIP complex is used to analyze a tissue or serum sample
from a patient for the presence of MDM2 protein-MDMIP complex. An
aberrant level of MDM2 protein-MDMIP complex is indicative of a
diseased condition. The immunoassays which may be utilized include,
e.g., competitive and non-competitive assay systems using
techniques such as Western Blots, radio immunoassays (RIA), enzyme
linked immunosorbent assay (ELISA), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays, and protein-A immunoassays.
[0159] In some embodiments, diseases and disorders involving or
characterized by aberrant levels of MDM2 protein-MDMIP complex or a
predisposition to develop such disorders may be diagnosed by
detecting aberrant levels of MDM2 protein-MDMIP complex, or
non-complexed MDM2 protein and/or MDMIP proteins or nucleic acids
for functional activity. Suitable functional activities that can be
assayed include, e.g.,(i) binding to an interacting partner (e.g.,
the MDM2 protein, MDMIP) or (ii) by detecting mutations in MDM2
protein and/or a MDMIP RNA, DNA or protein (e.g., translocations,
truncations, changes in nucleotide or amino acid sequence relative
to wild-type MDM2 protein and/or the MDMIP) which can cause
increased or decreased expression or activity of the MDM2 protein,
a MDMIP or a MDM2 protein-MDMIP complex.
[0160] Methods which are well-known within the art (e.g.,
immunoassays, nucleic acid hybridization assays, biological
activity assays, and the like) may be used to determine whether one
or more particular MDM2 protein-MDMIP complexes are present at
either increased or decreased levels, or are absent, within samples
derived from patients suffering from a particular disease or
disorder, or possessing a predisposition to develop such a disease
or disorder, as compared to the levels in samples from subjects not
having such disease or disorder or predisposition thereto.
Additionally, these assays may be utilized to determine whether the
ratio of the MDM2 protein-MDMIP complex to the non-complexed
components (i.e. the MDM2 protein and/or the specific MDMIP) in the
complex of interest is increased or decreased in samples from
patients suffering from a particular disease or disorder or having
a predisposition to develop such a disease or disorder as compared
to the ratio in samples from subjects not having such a disease or
disorder or predisposition thereto.
[0161] Accordingly, in specific embodiments of the present
invention, diseases and disorders which involve increased/decreased
levels of one or more MDM2 protein-MDMIP complex may be diagnosed,
or their suspected presence may be screened for, or a
predisposition to develop such diseases and disorders may be
detected, by quantitatively ascertaining increased/decreased levels
of: (i) the one or more MDM2 protein-MDMIP complex; (ii) the mRNA
encoding both protein members of the complex; (iii) the complex
functional activity or (iv) mutations in the MDM2 protein or the
MDMIP (e.g., translocations in nucleic acids, truncations in the
gene or protein, changes in nucleotide or amino acid sequence
relative to wild-type MDM2 protein or the MDMIP) which
enhance/inhibit or stabilize/destabilize MDM2 protein-MDMIP complex
formation.
[0162] Antibodies directed against the MDM2 protein-MDMIP complex
can also be used to detect cells which express the protein or
protein complexes. Using such assays, specific cell types may be
quantitatively characterized in which one or more particular MDM2
protein MDMIP complex are expressed, and the presence of the
component polypeptides or protein complex may be correlated with
cell viability by techniques well-known within the art (e.g.,
florescence-activated cell sorting).
[0163] In some embodiments, expression is detected in in vitro cell
culture models which express particular MDM2 protein-MDMIP complex,
or derivatives thereof, for the purpose of characterizing and/or
isolating MDM2 protein-MDMIP complex. These detection techniques
include, e.g.,, cell-sorting of prokaryotes (see e.g., Davey &
Kell, 1996. Microbiol. Rev. 60:641-696); primary cultures and
tissue specimens from eukaryotes, including mammalian species such
as human (see e.g., Steele, et al., 1996. Clin. Obstet. Gynecol.
39:801-813) and continuous cell cultures (see e.g., Orfao &
Ruiz-Arguelles, 1996. Clin. Biochem. 29:5-9.
[0164] The observation that MDMIP interacts with MDM2 polypeptide
can also be used to detect the presence of, and, if desired, to
purify, the binding polypeptides in a biological sample. For
example, levels of MDM2 polypeptide in a biological sample can also
be measured by contacting the sample with a labeled polypeptide
including the MDM2-binding domain of an MDMIP polypeptide.
Similarly, the presence of MDMIP in a biological sample can be
measured by contacting the sample with a polypeptide that includes
an MDMIP binding region or domain of an MDM2 polypeptide.
Kits Containing Reagents for Identifying MDMIP-MDM2 Complexes and
MDM-IP Nucleic Acids
[0165] The invention additionally provides kits for diagnostic use.
The kits include one or more containers containing an anti-MDM2
protein-MDMIP complex antibody and, optionally, a labeled binding
partner to the antibody. The label incorporated into the anti-MDM2
protein-MDMIP complex antibody may include, e.g., a
chemiluminescent, enzymatic, fluorescent, calorimetric or
radioactive moiety. Alternatively, the kit may include, in one or
more containers, a pair of oligonucleotide primers (e.g., each 6-30
nucleotides in length) which are capable of acting as amplification
primers for: polymerase chain reaction (PCR; see e.g., Imis, et
al., 1990. PCR Protocols [Academic Press, Inc., San Diego,
Calif.]), ligase chain reaction, cyclic probe reaction, or other
methods known within the art. The kit may, optionally, further
comprise a predetermined amount of a purified MDM2 protein, MDMIP
or MDM2-MDMIP complex, or nucleic acids thereof, for use as a
standard or control in the aforementioned assays.
Transgenic Animals
[0166] The host cells of the invention can also be used to produce
nonhuman transgenic animals. For example, in one embodiment, a host
cell of the invention is a fertilized oocyte or an embryonic stem
cell into which MDMIP-coding sequences have been introduced. Such
host cells can then be used to create non-human transgenic animals
in which exogenous MDMIP sequences have been introduced into their
genome or homologous recombinant animals in which endogenous MDMIP
sequences have been altered. Such animals are useful for studying
the function and/or activity of MDMIP and for identifying and/or
evaluating modulators of MDMIP activity. As used herein, a
"transgenic animal" is a non-human animal, preferably a mammal,
more preferably a rodent such as a rat or mouse, in which one or
more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA that is integrated into the genome of a cell from
which a transgenic animal develops and that remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous MDMIP gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0167] A transgenic animal of the invention can be created by
introducing MDMIP-encoding nucleic acid into the male pronuclei of
a fertilized oocyte, e.g., by microinjection, retroviral infection,
and allowing the oocyte to develop in a pseudopregnant female
foster animal. The human MDMIP DNA sequence of SEQ ID NO: 1 can be
introduced as a transgene into the genome of a non-human animal.
Alternatively, a nonhuman homologue of the human MDMIP gene, such
as a mouse MDMIP gene, can be isolated based on hybridization to
the human MDMIP cDNA (described further above) and used as a
transgene. Intronic sequences and polyadenylation signals can also
be included in the transgene to increase the efficiency of
expression of the transgene. A tissue-specific regulatory
sequence(s) can be operably linked to the MDMIP transgene to direct
expression of MDMIP protein to particular cells. Methods for
generating transgenic animals via embryo manipulation and
microinjection, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan In:
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1986. Similar methods are used for
production of other transgenic animals. A transgenic founder animal
can be identified based upon the presence of the MDMIP transgene in
its genome and/or expression of MDMIP mRNA in tissues or cells of
the animals. A transgenic founder animal can then be used to breed
additional animals carrying the transgene. Moreover, transgenic
animals carrying a transgene encoding MDMIP can further be bred to
other transgenic animals carrying other transgenes.
[0168] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of a MDMIP gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the MDMIP gene. The
MDMIP gene can be a human gene (e.g., a sequence including SEQ ID
NO: 1), but more preferably, is a non-human homologue of a human
MDMIP gene. For example, a mouse homologue of human MDMIP gene of
SEQ ID NO: 1 can be used to construct a homologous recombination
vector suitable for altering an endogenous MDMIP gene in the mouse
genome. In one embodiment, the vector is designed such that, upon
homologous recombination, the endogenous MDMIP gene is functionally
disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out" vector).
[0169] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous MDMIP gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous MDMIP protein). In the homologous
recombination vector, the altered portion of the MDMIP gene is
flanked at its 5' and 3' ends by additional nucleic acid of the
MDMIP gene to allow for homologous recombination to occur between
the exogenous MDMIP gene carried by the vector and an endogenous
MDMIP gene in an embryonic stem cell. The additional flanking MDMIP
nucleic acid is of sufficient length for successful homologous
recombination with the endogenous gene. Typically, several
kilobases of flanking DNA (both at the 5' and 3' ends) are included
in the vector. See e.g., Thomas et al., Cell 51:503, 1987, for a
description of homologous recombination vectors. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced MDMIP gene has
homologously recombined with the endogenous MDMIP gene are selected
(see e.g., Li et al., Cell 69:915, 1992).
[0170] The selected cells are then injected into a blastocyst of an
animal (e.g., a mouse) to form aggregation chimeras. See e.g.,
Bradley, In: Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, Robertson, ed. IRL, Oxford, 1987, pp.113-152. A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the
homologously recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley, Curr. Opin. Biotechnol. 2:823-829, 1991; PCT International
Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO
93/04169.
[0171] In another embodiment, transgenic non-humans animals can be
produced that contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al., Proc.
Nat. Acad. Sci. USA 89:6232-6236, 1992. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al., Science 251:1351-1355, 1991. 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.
[0172] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al., Nature 385:810-813, 1997. In brief, a cell, e.g., a somatic
cell, from the transgenic animal can be isolated and induced to
exit the growth cycle and enter G.sub.0 phase. The quiescent cell
can then be fused, e.g., through the use of electrical pulses, to
an enucleated oocyte from an animal of the same species from which
the quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it develops to morula or blastocyte and then
transferred to pseudopregnant female foster animal. The offspring
borne of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0173] Alternatively, transgenic animals can be prepared as above
using nucleotide sequences encoding an MDMIP-binding fragment of an
MDM2 protein or an MDM2 protein-MDMIP chimeric polypeptide.
Therapeutic Uses of MDMIP Polypeptides, MDMIP Binding MDM2
Polypeptides, and MDM2 Protein-MDMIP Complexes
[0174] The MDM2 protein has been implicated to play a significant
role in disorders of cell-cycle progression, cell differentiation,
and transcriptional control, including cancer and
tumorigenesis.
[0175] Based in part on the discovery of the MDMIP polypeptide, the
invention provides for treatment or prevention of various diseases
and disorders by administration of a biologically-active,
therapeutic compound (hereinafter "Therapeutic"). Such Therapeutics
include, e.g.,: (i) various MDM2 proteinMDMIP complexes (e.g., the
MDM2 protein complexed with MDMIP) and derivative, fragments,
analogs and homologs thereof; (ii) antibodies directed against
these proteins and protein complexes; (iii) nucleic acids encoding
the MDM2 protein and MDMIP and derivatives, fragments, analogs and
homologs thereof; (iv) antisense nucleic acids encoding the MDM2
protein and (v) MDM2 protein IPs and MDM2 protein.MDMIP complex and
modulators (i.e., inhibitors, agonists and antagonists)
thereof.
(i) Disorders with Increased MDM2 Protein and MDM2 ProteinMDMIP
Complex Levels
[0176] Diseases and disorders which are characterized by increased
(relative to a subject not suffering from the disease or disorder)
MDM2 protein-MDMIP levels or biological activity may be treated
with Therapeutics which antagonize (i.e., reduce or inhibit) MDM2
protein-MDMIP complex formation or activity. Therapeutics which
antagonize MDM2 protein-MDMIP complex formation or activity may be
administered in a therapeutic or prophylactic manner. Therapeutics
which may be utilized include, e.g.,, the MDM2 protein or MDMIP, or
analogs, derivatives, fragments or homologs thereof, (ii) anti-MDM2
protein-MDMIP complex antibodies; (iii) nucleic acids encoding the
MDM2 protein or MDMIP; (iv) concurrent administration of a MDM2
protein and a MDMIP antisense nucleic acid and MDM2 protein and/or
MDMIP nucleic acids which are "dysfunctional" (i.e., due to a
heterologous [non-MDM2 protein and/or non-MDMIP] insertion within
the coding sequences of the MDM2 protein and MDMIP coding
sequences) are utilized to "knockout" endogenous MDM2 protein
and/or MDMIP function by homologous recombination (see e.g.,
Capecchi, Science 244:1288-1292, 1989). Alternatively, mutants or
derivatives of a first MDMIP which possess greater affinity for
MDM2 protein than the wild-type first MDMIP may be administered to
compete with a second MDMIP for binding to the MDM2 protein,
thereby reducing the levels of complex between the MDM2 protein and
the second MDMIP.
[0177] Increased levels of MDM2 protein-MDMIP complex can be
readily detected by quantifying protein and/or RNA, by obtaining a
patient tissue sample (e.g., from biopsy tissue) and assaying it in
vitro for RNA or protein levels, structure and/or activity of the
expressed MDM2 protein-MDMIP complex (or the MDM2 protein and MDMIP
mRNAs). Methods which are well-known within the art including,
e.g., immunoassays to detect MDM2 protein-MDMIP complex (e.g., by
Western blot analysis, immunoprecipitation followed by sodium
dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,
immunocytochemistry, etc.) and/or hybridization assays to detect
concurrent expression of the MDM2 protein and MDMIP mRNAs (e.g.,
Northern assays, dot blots, in situ hybridization, etc.).
(ii) Disorders with Increased MDM2 Protein and MDM2 Protein-MDMIP
Complex Levels
[0178] The invention includes methods for the reduction of MDM2
protein-MDMIP complex expression (i.e., the expression of the two
protein components of the complex and/or formation of the complex)
by targeting mRNAs which express the protein moieties. RNA
Therapeutics are differentiated into three classes: (i) antisense
species; (ii) ribozymes or (iii) RNA aptamers. See e.g., Good, et
al., 1997. Gene Therapy 4:45-54. Antisense therapy will be
discussed below. Ribozyme therapy involves the administration
(i.e., induced expression) of small RNA molecules with enzymatic
ability to cleave, bind, or otherwise inactivate specific RNAs,
thus reducing or eliminating the expression of particular proteins.
See e.g., Grassi & Marini, 1996. Ann. Med. 28:499-510. RNA
aptamers are specific RNA ligands for proteins, such as for Tat and
Rev RNA (see e.g., Good, et al., 1997. Gene Therapy 4:45-54) which
can specifically inhibit their translation.
[0179] In one embodiment, the activity or level of the MDM2 protein
may be reduced by administration of MDMIP, a nucleic acid which
encodes MDMIP or an antibody (or a derivative or fragment of the
antibody possessing the binding domain thereof) which specifically
binds to MDMIP. Similarly, the levels or activity of MDMIP may be
reduced by administration of the MDM2 protein, a nucleic acid
encoding the MDM2 protein or an antibody (or a derivative or
fragment of the antibody possessing the binding domain thereof)
which specifically binds the MDM2 protein. In another embodiment of
the present invention, diseases or disorders which are associated
with increased levels of the MDM2 protein or MDMIP, may be treated
or prevented by administration of a Therapeutic which increases
MDM2 protein-MDMIP complex formation, if the complex formation acts
to reduce or inactivate the MDM2 protein or the particular MDMIP
via MDM2 protein-MDMIP complex formation. Such diseases or
disorders may be treated or prevented by: (i) the administration of
one member of the MDM2 protein-MDMIP complex, including mutants of
one or both of the proteins which possess increased affinity for
the other member of the MDM2 protein-MDMIP complex (so as to cause
increased complex formation) or (ii) the administration of
antibodies or other molecules which serve to stabilize the MDM2
protein-MDMIP complex, or the like.
Determination of the Biological Effect of the Therapeutic
[0180] In some embodiments, suitable in vitro or in vivo assays are
utilized to determine the effect of a specific Therapeutic and
whether its administration is indicated for treatment of the
affected tissue.
[0181] For example, in vitro assays may be performed with
representative cells of the type(s) involved in the patient's
disorder, to determine if a given Therapeutic exerts the desired
effect upon the cell type(s). Compounds for use in therapy may be
tested in suitable animal model systems including, e.g. rats, mice,
chicken, cows, monkeys, rabbits, and the like, prior to testing in
human subjects. Similarly, for in vivo testing, any of the animal
model system known in the art may be used prior to administration
to human subjects.
(i) Malignancies
[0182] Components of the MDM2 protein-MDMIP complex are likely
involved in the regulation of cell proliferation. Accordingly,
Therapeutics of the present invention may be useful in the
therapeutic or prophylactic treatment of diseases or disorders
which are associated with cell hyperproliferation and/or loss of
control of cell proliferation (e.g., cancers, malignancies and
tumors). For a review of such hyperproliferation disorders, see
e.g., Fishman, et al., 1985. Medicine, 2nd ed. (J. B. Lippincott
Co., Philadelphia, Pa.).
[0183] Therapeutics of the present invention may be assayed by any
method known within the art for efficacy in treating or preventing
malignancies and related disorders. Such assays include, e.g.,, in
vitro assays utilizing transformed cells or cells derived from the
patient's tumor, as well as in vivo assays using animal models of
cancer or malignancies. Potentially effective Therapeutics, for
example, inhibit the proliferation of tumor-derived or transformed
cells in culture or cause a regression of tumors in animal models,
in comparison to the controls.
[0184] In the practice of the present invention, once a malignancy
or cancer has been shown to be amenable to treatment by modulating
(i.e., inhibiting, antagonizing or agonizing) MDM2protein-MDMIP
complex activity, that cancer or malignancy may subsequently be
treated or prevented by the administration of a Therapeutic which
serves to modulate MDM2 protein MDMIP complex formation and
function, including supplying MDM2 protein-MDMIP complex and the
individual binding partners of the protein complex (i.e., the MDM2
protein and/or MDMIP).
(ii) Pre-Malignant Conditions
[0185] The Therapeutics of the present invention which are
effective in the therapeutic or prophylactic treatment of cancer or
malignancies may also be administered for the treatment of
pre-malignant conditions and/or to prevent the progression of a
pre-malignancy to a neoplastic or malignant state. Such
prophylactic or therapeutic use is indicated in conditions known or
suspected of preceding progression to neoplasia or cancer, in
particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia or, most particularly, dysplasia has
occurred. For a review of such abnormal cell growth see e.g.,
Robbins & Angell, 1976. Basic Pathology, 2nd ed. (W. B.
Saunders Co., Philadelphia, Pa.).
[0186] Hyperplasia is a form of controlled cell proliferation
involving an increase in cell number in a tissue or organ, without
significant alteration in its structure or function. For example,
it has been demonstrated that endometrial hyperplasia often
precedes endometrial cancer. Metaplasia is a form of controlled
cell growth in which one type of mature or fully differentiated
cell substitutes for another type of mature cell. Metaplasia may
occur in epithelial or connective tissue cells. Dysplasia is
generally considered a precursor of cancer, and is found mainly in
the epithelia. Dysplasia is the most disorderly form of
non-neoplastic cell growth, and involves a loss in individual cell
uniformity and in the architectural orientation of cells. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation, and is often found in the cervix, respiratory
passages, oral cavity, and gall bladder.
[0187] Alternatively, or in addition to the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed or
malignant phenotype displayed either in vivo or in vitro within a
cell sample derived from a patient, is indicative of the
desirability of prophylactic/therapeutic administration of a
Therapeutic of the present invention which possesses the ability to
modulate MDM2 protein MDMIP complex activity. Characteristics of a
transformed phenotype include, e.g.,: (i) morphological changes;
(ii) looser substratum attachment; (iii) loss of cell-to-cell
contact inhibition; (iv) loss of anchorage dependence; (v) protease
release; (vi) increased sugar transport; (vii) decreased serum
requirement; (viii) expression of fetal antigens, (ix)
disappearance of the 250 Kdal cell-surface protein, and the like.
See e.g., Richards, et al., 1986. Molecular Pathology (W. B.
Saunders Co., Philadelphia, Pa.).
[0188] In one embodiment, a patient which exhibits one or more of
the following predisposing factors for malignancy is treated by
administration of an effective amount of a Therapeutic: (i) a
chromosomal translocation associated with a malignancy (e.g., the
Philadelphia chromosome (bcr/abl) for chronic myelogenous leukemia
and t(14;18) for follicular lymphoma, etc.); (ii) familial
polyposis or Gardner's syndrome (possible forerunners of colon
cancer); (iii) monoclonal gammopathy of undetermined significance
(a possible precursor of multiple myeloma) and (iv) a first degree
kinship with persons having a cancer or pre-cancerous disease
showing a Mendelian (genetic) inheritance pattern (e.g., familial
polyposis of the colon, Gardner's syndrome, hereditary exostosis,
polyendocrine adenomatosis, medullary thyroid carcinoma with
amyloid production and pheochromocytoma, Peutz-Jeghers syndrome,
neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid
body tumor, cutaneous melanocarcinoma, intraocular melanocarcinoma,
xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi
syndrome, albinism, Fanconi's aplastic anemia and Bloom's
syndrome).
[0189] In another embodiment, a Therapeutic of the present
invention is administered to a human patient to prevent the
progression to breast, colon, lung, pancreatic, or uterine cancer,
or melanoma or sarcoma.
(iii) Hyperproliferative and Dysproliferative Disorders
[0190] In a preferred embodiment of the present invention, a
Therapeutic is administered in the therapeutic or prophylactic
treatment of hyperproliferative or benign dysproliferative
disorders. The efficacy in treating or preventing
hyperproliferative diseases or disorders of a Therapeutic of the
present invention may be assayed by any method known within the
art. Such assays include in vitro cell proliferation assays, in
vitro or in vivo assays using animal models of hyperproliferative
diseases or disorders, or the like. Potentially effective
Therapeutics may, for example, promote cell proliferation in
culture or cause growth or cell proliferation in animal models in
comparison to controls.
[0191] Once a hyperproliferative disorder has been shown to be
amenable to treatment by modulation of MDM2 proteinMDMIP complex
activity, the hyperproliferative disease or disorder may be treated
or prevented by the administration of a Therapeutic which modulates
MDM2 proteinMDMIP complex formation (including supplying MDM2
proteinMDMIP complex and the individual binding partners of a MDM2
proteinMDMIP complex.
[0192] In some embodiments, methods are directed to the treatment
or prevention of cirrhosis of the liver (a condition in which
scarring has overtaken normal liver regeneration processes);
treatment of keloid (hypertrophic scar) formation causing
disfiguring of the skin in which the scarring process interferes
with normal renewal; psoriasis (a common skin condition
characterized by excessive proliferation of the skin and delay in
proper cell fate determination); benign tumors; fibrocystic
conditions and tissue hypertrophy (e.g., benign prostatic
hypertrophy).
Gene Therapy using MDMIP and/or MDM2 Nucleic Acids
[0193] In one embodiment, nucleic acids comprising a sequence which
encodes the MDM2 protein and/or MDMIP, or functional derivatives
thereof, are administered to modulate MDM2protein-MDMIP complex
function, using gene therapy, i. e., a nucleic acid or nucleic
acids encoding both the MDM2 protein and MDMIP, or functional
derivatives thereof, are administered to a subject. After delivery
of the nucleic acid to a subject, the nucleic acid expresses its
encoded protein(s), which then serve to exert a therapeutic effect
by modulating MDM2 proteinMDMIP complex function. Any of the
methods relating to gene therapy available within the art may be
used in the practice of the present invention. See, e.g. Goldspiel,
et al.,. Clin. Pharm. 12:488-505, 1993, and U.S. Pat. No.
5,580,859.
[0194] In one embodiment, the Therapeutic includes a nucleic acid
encoding an MDM2 protein and MDMIP nucleic acid which is part of an
expression vector expressing both proteins, or fragments or
chimeric proteins thereof, within a suitable host. In a specific
embodiment, such a nucleic acid possesses a promoter which is
operably-linked to the MDM2 protein and the MDMIP coding region(s),
or, less preferably two separate promoters linked to the MDM2
protein and the MDMIP coding regions separately; wherein the
promoter is inducible or constitutive, and, optionally,
tissue-specific. In another specific embodiment, a nucleic acid
molecule is used in which the MDM2 protein and MDMIP coding
sequences (and any other desired sequences) are flanked by regions
which promote homologous recombination at a desired site within the
genome, thus providing for intra-chromosomal expression of the MDM2
protein and the MDMIP nucleic acids. See e.g., Koller &
Smithies, 1989. Proc. Natl. Acad. Sci. USA 86:8932-8935.
[0195] Delivery of the Therapeutic nucleic acid into a patient may
be either direct (i.e., the patient is directly exposed to the
nucleic acid or nucleic acid-containing vector) or indirect (i.e.,
cells are first transformed with the nucleic acid in vitro, then
transplanted into the patient). These two approaches are known,
respectively, as in vivo or ex vivo gene therapy. For example, the
nucleic acid can be directly administered in vivo, where it is
expressed to produce the encoded product. This may be accomplished
by any of numerous methods known in the art including, e.g.: (i)
constructing it as part of an appropriate nucleic acid expression
vector and administering in a manner such that it becomes
intracellular (e.g., by infection using a defective or attenuated
retroviral or other viral vector; see U.S. Pat. No. 4,980,286) or
(ii) direct injection of naked DNA, or through the use of
microparticle bombardment (e.g., a "Gene Gun.RTM.; Biolistic,
Dupont), or by coating it with lipids, cell-surface
receptors/transfecting agents, or through encapsulation in
liposomes, microparticles, or microcapsules, or by administering it
in linkage to a peptide which is known to enter the nucleus, or by
administering it in linkage to a ligand predisposed to
receptor-mediated endocytosis (see e.g., Wu & Wu, 1987. J Biol.
Chem. 262:4429-4432), which can be used to "target" cell types
which specifically express the receptors of interest, etc.
[0196] In another specific embodiment of the present invention, a
nucleic acid-ligand complex may be produced in which the ligand
comprises a fasogenic viral peptide designed so as to disrupt
endosomes, thus allowing the nucleic acid to avoid subsequent
lysosomal degradation. In yet another specific embodiment, the
nucleic acid may be targeted in vivo for cell-specific endocytosis
and expression, by targeting a specific receptor. See e.g., PCT
Publications WO 92/06180; WO93/14188 and WO 93/20221.
Alternatively, the nucleic acid maybe introduced intracellularly
and incorporated within host cell genome for expression by
homologous recombination. See e.g., Zijlstra, et al., 1989. Nature
342:435-438.
[0197] In yet another embodiment, a viral vector which contains the
MDM2 protein and/or MDMIP nucleic acids is utilized. For example,
retroviral vectors may be employed (see e.g., Miller, et al., 1993.
Meth. Enzymol. 217:581-599) which have been modified to delete
those retroviral-specific sequences which are not required for
packaging of the viral genome and its subsequent integration into
host cell DNA. The MDM2 protein and/or MDMIP (preferably both
protein species) nucleic acids are cloned into the vector, which
facilitates delivery of the genes into a patient. See e.g., Boesen,
et al., 1994. Biotherapy 6:291-302; Kiem, et al., 1994. Blood
83:1467-1473. Additionally, adenovirus is an especially efficacious
"vehicle" for the delivery of genes to the respiratory epithelia.
Other targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
also possess the advantageous ability to infect non-dividing cells.
For a review see e.g., Kozarsky & Wilson, 1993. Curr. Opin.
Gen. Develop. 3:499-503. Adenovirus-associated virus has also been
proposed for use in gene therapy. See e.g., Walsh, et al., 1993.
Proc. Soc. Exp. Biol. Med. 204:289-300.
[0198] An additional approach to gene therapy in the practice of
the present invention involves transferring a gene into cells in in
vitro tissue culture by such methods as electroporation,
lipofection, calcium phosphate-mediated transfection, or viral
infection. Generally, the method of transfer includes the transfer
of a selectable marker to the cells. The cells are then placed
under selection pressure (e.g., antibiotic resistance) so as
facilitate the isolation of those cells which have taken up, and
are expressing the transferred gene. Those cells are then delivered
to a patient. In this specific embodiment, the nucleic acid is
introduced into a cell prior to the in vivo administration of the
resulting recombinant cell by any method known within the art
including, e.g.: transfection, electroporation, microinjection,
infection with a viral or bacteriophage vector containing the
nucleic acid sequences of interest, cell fusion,
chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, and similar methods which ensure that
the necessary developmental and physiological functions of the
recipient cells are not disrupted by the transfer. See e.g.,
Loeffler & Behr, 1993. Meth. Enzymol. 217: 599-618. The
technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its cell
progeny.
[0199] The resulting recombinant cells may be delivered to a
patient by various methods known within the art including, e.g.:
injection of epithelial cells (e.g., subcutaneously); the
application of recombinant skin cells as a skin graft onto the
patient and the intravenous injection of recombinant blood cells
(e.g., hematopoetic stem or progenitor cells). The total amount of
cells which are envisioned for use depend upon the desired effect,
patient state, etc., and may be determined by one skilled within
the art.
[0200] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include e.g., epithelial cells, endothelial cells,
keratinocytes, fibroblasts, muscle cells, hepatocytes and blood
cells. In a preferred embodiment of the present invention, the cell
utilized for gene therapy may be autologous to the patient.
[0201] In a specific embodiment in which recombinant cells are used
in gene therapy, stem or progenitor cells, which can be isolated
and maintained in vitro, may be utilized. Such stem cells include,
e.g.,, hematopoetic stem cells (HSC), stem cells of epithelial
tissues and neural stem cells (see e.g., Stemple & Anderson,
1992. Cell 71:973-985). Any technique which provides for the
isolation, propagation, and maintenance in vitro of HSC may be
used. HSCs utilized for gene therapy are, preferably, autologous to
the patient. Hence, non-autologous HSCs are, preferably, utilized
in conjunction with a method of suppressing transplantation immune
reactions of the future host/patient. See e.g., Kodo, et al., 1984.
J. Clin. Invest. 73:1377-1384. In another embodiment, HSCs may be
highly enriched (or produced in a substantially-pure form), by any
techniques known within the art, prior to administration to the
patient. See e.g., Witlock & Witte, 1982. Proc. Natl. Acad.
Sci. USA 79:3608-3612.
Anti-Sense MDMIP or MDM2 Oligonucleotides
[0202] MDM2 protein-MDMIP complex formation and function may be
inhibited by the use of anti-sense nucleic acids for the MDM2
protein and/or MDMIP. In some embodiments, nucleic acids (of at
least six nucleotides in length) which are anti-sense to a genomic
sequence (gene) or cDNA encoding the MDM2 protein and/or MDMIP, or
portions thereof, are used prophylactically or therapeutically.
Such anti-sense nucleic acids have utility as Therapeutics which
inhibit MDM2 proteinMDMIP complex formation or activity, and may be
utilized in a therapeutic or prophylactic manner.
[0203] The invention also provides methods for inhibiting
expression of the MDM2 protein and MDMIP nucleic acid sequences
within a prokaryotic or eukaryotic cell. The method includes
providing the cell with an therapeutically-effective amount of an
anti-sense nucleic acid of the MDM2 protein and MDMIP, or
derivatives thereof.
[0204] The anti-sense nucleic acids may be oligonucleotides which
may either be directly administered to a cell or which may be
produced in vivo by transcription of the exogenous, introduced
sequences. In addition, the anti-sense nucleic acid may be
complementary to either a coding (i.e., exonic) and/or non-coding
(i.e., intronic) region of the MDM2 protein or MDMIP mRNAs. The
MDM2 protein and MDMIP anti-sense nucleic acids are, at least, six
nucleotides in length and are, preferably, oligonucleotides ranging
from 6-200 nucleotides in length. In specific embodiments, the
anti-sense oligonucleotide is at least 10 nucleotides, at least 15
nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
The anti-sense oligonucleotides may be DNA or RNA (or chimeric
mixtures, derivatives or modified versions thereof), may be either
single-stranded or double-stranded and may be modified at a base,
sugar or phosphate backbone moiety.
[0205] In addition, the anti-sense oligonucleotide may include
other associated functional groups, such as peptides, moieties
which facilitate the transport of the oligonucleotide across the
cell membrane, a hybridization-triggered cross-linking agent, a
hybridization-triggered cleavageagent, and the like. See e.g.,
Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A.
86:6553-6556; PCT Publication No. WO 88/09810. In a specific
embodiment, the MDM2 protein and MDMIP antisense oligonucleotides
include catalytic RNAs or ribozymes. See, e.g., Sarver, et al.,
1990. Science 247:1222-1225.
[0206] The anti-sense oligonucleotides may be synthesized by
standard methods known within the art including, e.g.: (i)
automated phosphorothioate-mediated oligonucleotide synthesis (see
e.g., Stein, et al., 1988. Nuc. Acids Res. 16:3209) or (ii)
methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (see e.g., Sarin, et al.,
1988. Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451).
[0207] In an alternative embodiment, the MDM2 protein and MDMIP
antisense nucleic acids are produced intracellularly by
transcription of an exogenous sequence. For example, a vector may
be produced and (upon being exocytosed by the cell) transcribed in
vivo, thus producing an antisense nucleic acid (RNA) species. The
vector may either remain episomal or become
chromosomally-integrated, so long as it can be transcribed to
produce the desired antisense RNA. The vectors may be derived from
bacterial, viral, yeast or other sources known within the art,
which are utilized for replication and expression in mammalian
cells. Expression of the sequences encoding the MDM2 protein and
MDMIP antisense RNAs may be facilitated by any promoter known
within the art to function in mammalian, preferably, human cells.
Such promoters may be inducible or constitutive and include, e.g.,:
(i) the SV40 early promoter region; (ii) the promoter contained in
the 3'-terminus long terminal repeat of Rous sarcoma virus (RSV);
(iii) the Herpesvirus thymidine kinase promoter and (iv) the
regulatory sequences of the metallothionein gene.
[0208] The MDM2 protein and MDMIP antisense nucleic acids may be
utilized prophylactically or therapeutically in the treatment or
prevention of disorders of a cell type which expresses (or
over-expresses) the MDM2 protein-MDMIP complex. Cell types which
express or over-express the MDM2 protein and MDMIP RNA may be
identified by various methods known within the art including,
e.g.,, hybridization with MDM2 protein- and MDMIP-specific nucleic
acids (e.g., by Northern hybridization, dot blot hybridization, in
situ hybridization) or by observing the ability of RNA from the
specific cell type to be translated in vitro into the MDM2 protein
and the MDMIP by immunohistochemistry. If desired, primary tissue
from a patient may be assayed for the MDM2 protein and/or MDMIP
expression prior to actual treatment by, for example,
immunocytochemistry or in situ hybridization.
[0209] Pharmaceutical compositions which include an effective
amount of a MDM2 protein and MDMIP antisense nucleic acid contained
within a pharmaceutically-acceptable carrier may be administered to
a patient having a disease or disorder which is of a type that
expresses or over-expresses MDM2 protein-MDMIP complex RNA or
protein. The amount of MDM2 protein and/or MDMIP antisense nucleic
acid which will be effective in the treatment of a particular
disorder or condition will be dependant upon the nature of the
disorder or condition, and may be determined by standard clinical
techniques. Where possible, it is desirable to determine the
antisense cytotoxicity in vitro, and then in useful animal model
systems prior to testing and use in humans. In a specific
embodiment, pharmaceutical compositions comprising MDM2 protein and
MDMIP antisense nucleic acids may be administered via liposomes,
microparticles, or microcapsules. See e.g., Leonetti, et al., 1990.
Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451.
MDM2 Protein-MDMIP Complex Assays
[0210] The functional activity of MDM2 protein-MDMIP complexes (and
derivatives, fragments, analogs and homologs thereof) may be
assayed by a number of methods known within the art. For example,
putative modulators (e.g., inhibitors, agonists and antagonists) of
MDM2 protein-MDM2 protein complex activity (e.g., anti-MDM2
protein-MDMIP complex antibodies, as well as MDM2 protein or MDMIP
antisense nucleic acids) may be assayed for their ability to
modulate MDM2 protein-MDMIP complex formation and/or activity.
(i) Immunoassays
[0211] Also disclosed herein are immunoassay-based useful for
measuring the ability of an altered complex, e.g. a complex
containing derivatives, fragments, analogs and/or homologs thereof
of an MDM2 or MDMIP polypeptide to bind to, or compete with,
wild-type MDM2proteine MDMIP complex or MDMIP. Alternatively,
immunoassays be used to determine the ability of the altered
complex to bind to an anti-MDM2 protein-MDMIP complex antibody.
These immunoassays include, e.g.,, competitive and non-competitive
assay systems utilizing techniques such as radio immunoassays,
enzyme linked immunosorbent assay (ELISA), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitin reactions,
immunodiffusion assays, in situ immunoassays (e.g., using colloidal
gold, enzyme or radioisotope labels), Western blots, Northwestern
blots, precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation
assays, immunofluorescence assays, protein-A assays and
immunoelectrophoresis assays, and the like. In one specific
embodiment of the present invention, antibody binding is detected
by assaying for a label on the primary antibody. In another
specific embodiment, the binding of the primary antibody is
ascertained by the detection of the binding of a secondary antibody
(or reagent) specific for the primary antibody. In a further
embodiment, the secondary antibody is labeled.
(ii) Gene Expression Assays
[0212] The expression of the MDM2 protein or MDMIP genes (both
endogenous genes and those expressed from recombinant DNA) may be
detected using techniques known within the art including, e.g.:
Southern hybridization, Northern hybridization, restriction
endonuclease mapping, DNA sequence analysis and polymerase chain
reaction amplification (PCR) followed by Southern hybridization or
RNase protection (see e.g., Current Protocols in Molecular Biology
1997. (John Wiley and Sons, New York, N.Y.)) with probes specific
for the MDM2 protein and MDMIP genes in various cell types.
[0213] In one specific embodiment of the present invention,
Southern hybridization may be used to detect genetic linkage of the
MDM2 protein and/or MDMIP gene mutations to physiological or
pathological states. Numerous cell types, at various stages of
development, may be characterized for their expression of the MDM2
protein and MDMIP (particularly the concomitant expression of the
MDM2 protein and MDMIP within the same cells). The stringency of
the hybridization conditions for Northern or Southern blot analysis
may be manipulated to ensure detection of nucleic acids with the
desired degree of relatedness to the specific probes used.
Modification of these aforementioned methods, as well as other
methods well-known within the art, may be utilized in the practice
of the present invention.
(iii) Binding Assays
[0214] Derivatives, fragments, analogs and homologs of MDMIP may be
assayed for binding to the MDM2 protein by any method known within
the art including, e.g.: (i) the modified yeast two hybrid assay
system; (ii) immunoprecipitation with an antibody which binds to
the MDM2 protein within a complex, followed by analysis by size
fractionation of the immunoprecipitated proteins (e.g., by
denaturing or non-denaturing polyacrylamide gel electrophoresis);
(iii) Western analysis; (v) non-denaturing gel electrophoresis, and
the like.
(iv) Assays for Biological Activity
[0215] A specific embodiment of the present invention provides a
method for the screening of a derivative, fragment, analog or
homolog of the MDM2 protein for biological activity. The method
includes contacting a derivative, fragment, analog or homolog of
the MDM2 protein with MDMIP and detecting the formation of a
complex between the derivative, fragment, analog or homolog of the
MDM2 protein and MDMIP. Detection of the formation of the complex
indicates that the MDM2 protein derivative, fragment, analog or
homolog, possesses biological (e.g., binding) activity. Similarly,
an additional embodiment discloses a method for the screening a
derivative, fragment, analog or homolog of MDMIP for biological
activity. In this method, the derivative, fragment, analog or
homolog of the protein is contacted with the MDM2 protein. And
formation of a complex between the derivative, fragment, analog or
homolog of MDMIP and the MDM2 protein is detected. Detecting he
formation of the complex indicates that the MDMIP derivative,
fragment, analog, or homolog possesses biological activity.
(10) Modulation of MDM2 Polypeptide Activity
(i) Tumorigenesis
[0216] The MDM2 protein is reported to play a role in the control
of cell proliferation as well as cell-transformation and
tumorigenesis. The present invention discloses methods for
screening MDM2 protein-MDMIP complexes (and derivatives, fragments,
analogs and homologs, thereof) for its ability to alter cell
proliferation, cell transformation and/or tumorigenesis in vitro
and in vivo.
[0217] The MDM2 proteinMDMIP complex (and derivatives, fragments,
analogs and homologs, thereof) may also be screened for activity in
inducing or inhibiting cell transformation (or the progression to
malignant phenotype) in vitro. The proteins and protein complex of
the present invention may be screened by contacting either cells
with a normal phenotype (for assaying for cell transformation) or a
transformed cell phenotype (for assaying for inhibition of cell
transformation) with the protein or protein complex of the present
invention and examining the cells for acquisition or loss of
characteristics associated with a transformed phenotype (a set of
in vitro characteristics associated with a tumorigenic ability in
vivo) including, e.g.: colony formation in soft agar, a more
rounded cell morphology, looser substratum attachment, loss of
contact inhibition, loss of anchorage dependence, release of
proteases such as plasminogen activator, increased sugar transport,
decreased serum requirement, expression of fetal antigens,
disappearance of the 250 Kdal cell-surface protein, and the like.
See e.g., Luria, et al, 1978. General Virology, 3rd ed. (John Wiley
& Sons, New York, N.Y.).
[0218] The MDM2 proteinMDMIP complex (and derivatives, fragments,
analogs and homologs, thereof) may also be screened for activity to
promote or inhibit tumor formation in vivo in non-human test
animal. A vast number of animal models of hyperproliferative
disorders (e.g., tumorigenesis and metastatic spread) are known
within the art. See e.g., Lovejoy, et al, 1997. J. Pathol.
181:130-135. In a specific embodiment of the present invention, the
proteins and protein complex may be administered to a non-human
test animal (preferably a test animal predisposed to develop a type
of tumor) and the non-human test animals is subsequently examined
for an increased incidence of tumor formation in comparison with
controls animals which were not administered the proteins or
protein complex of the present invention. Alternatively, the
proteins and protein complex may be administered to non-human test
animals possessing tumors (e.g., animals in which tumors have been
induced by introduction of malignant, neoplastic, or transformed
cells or by administration of a carcinogen) and subsequently
examining the tumors within the test animals for tumor regression
in comparison to controls. Accordingly, once a hyperproliferative
disease or disorder has been shown to be amenable to treatment by
modulation of MDM2 proteine MDMIP complex activity that disease or
disorder may be treated or prevented by administration of a
Therapeutic which modulates MDM2 proteinMDMIP complex
formation.
MDM2-MDMIP Interaction Assays
[0219] The present invention discloses methods for assaying and
screening derivatives, fragments, analogs and homologs of MDMIP for
binding to MDM2 protein. The derivatives, fragments, analogs and
homologs of the MDMIP which interact with MDM2 protein may be
identified by means of a yeast two hybrid assay system (see e.g.,
Fields & Song, 1989. Nature 340:245-246) or; preferably, a
modification and improvement thereof, as described in U.S. patent
applications Ser. Nos. 08/663,824 (filed Jun. 14, 1996) and
08/874,825 (filed Jun. 13, 1997), both of which are entitled
"Identification and Comparison of Protein-Protein Interactions that
Occur in Populations and Identification of Inhibitors of These
Interactions," to Nandabalan, et al., and which are incorporated by
reference herein in their entireties.
[0220] The identification of interacting proteins by the improved
yeast two hybrid system is based upon the detection of the
expression of a reporter gene (hereinafter "Reporter Gene"), the
transcription of which is dependent upon the reconstitution of a
transcriptional regulator by the interaction of two proteins, each
fused to one half of the transcriptional regulator. The bait MDM2
protein (or derivative, fragment, analog or homolog) and prey
protein (proteins to be tested for ability to interact with the
bait protein) are expressed as fusion proteins to a DNA binding
domain, and to a transcriptional regulatory domain, respectively,
or vice versa. In a specific embodiment of the present invention,
the prey population may be one or more nucleic acids encoding
mutants of MDMIP (e.g., as generated by site-directed mutagenesis
or another method of producing mutations in a nucleotide sequence).
Preferably, the prey populations are proteins encoded by DNA (e.g.,
cDNA, genomic DNA or synthetically generated DNA). For example, the
populations may be expressed from chimeric genes comprising cDNA
sequences derived from a non-characterized sample of a population
of cDNA from mammalian RNA. In another specific embodiment,
recombinant biological libraries expressing random peptides may be
used as the source of prey nucleic acids.
[0221] The present invention discloses methods for the screening
for inhibitors of MDMIP. In brief, the protein-protein interaction
assay may be performed as previously described herein, with the
exception that it is performed in the presence of one or more
candidate molecules. A resulting increase or decrease in Reporter
Gene activity, in relation to that which was present when the one
or more candidate molecules are absent, indicates that the
candidate molecule exerts an effect on the interacting pair. In a
preferred embodiment, inhibition of the protein interaction is
necessary for the yeast cells to survive, for example, where a
non-attenuated protein interaction causes the activation of the
URA3 gene, causing yeast to die in medium containing the chemical
5-fluoroorotic acid. See e.g. Rothstein, 1983. Meth. Enzymol.
101:167-180.
[0222] In general, the proteins comprising the bait and prey
populations are provided as fusion (chimeric) proteins, preferably
by recombinant expression of a chimeric coding sequence containing
each protein contiguous to a pre-selected sequence. For one
population, the pre-selected sequence is a DNA-binding domain that
may be any DNA-binding domain, so long as it specifically
recognizes a DNA sequence within a promoter (e.g., a
transcriptional activator or inhibitor). For the other population,
the pre-selected sequence is an activator or inhibitor domain of a
transcriptional activator or inhibitor, respectively. The
regulatory domain alone (not as a fusion to a protein sequence) and
the DNA-binding domain alone (not as a fusion to a protein
sequence) preferably, do not detectably interact, so as to avoid
false-positives in the assay. The assay system further includes a
reporter gene operably linked to a promoter that contains a binding
site for the DNA-binding domain of the transcriptional activator
(or inhibitor). Accordingly, binding of the MDM2 protein fusion
protein to a prey fusion protein leads to reconstitution of a
transcriptional activator (or inhibitor), which concomitantly
activates (or inhibits) expression of the Reporter Gene.
[0223] In a specific embodiment, the present invention discloses a
method for detecting one or more protein-protein interactions
comprising the following steps: (i) recombinantly-expressing the
MDM2 protein (or a derivative, fragment, analog or homolog thereof)
in a first population of yeast cells of a first mating type and
possessing a first fusion protein containing the MDM2 protein
sequence and a DNA-binding domain; wherein the first population of
yeast cells contains a first nucleotide sequence operably-linked to
a promoter which is "driven" by one or more DNA-binding sites
recognized by the DNA-binding domain such that an interaction of
the first fusion protein with a second fusion protein (comprising a
transcriptional activation domain) results in increased
transcription of the first nucleotide sequence; (ii) negatively
selecting to eliminate those yeast cells in the first population in
which the increased transcription of the first nucleotide sequence
occurs in the absence of the second fusion protein; (iii)
recombinantly expressing in a second population of yeast cells of a
second mating type different from the first mating type, a
plurality of the second fusion proteins; wherein the second fusion
protein is comprised of a sequence of a derivative, fragment,
analog or homolog of a MDMIP and an activation domain of a
transcriptional activator, in which the activation domain is the
same in each the second fusion protein; (iv) mating the first
population of yeast cells with the second population of yeast cells
to form a third population of diploid yeast cells, wherein the
third population of diploid yeast cells contains a second
nucleotide sequence operably linked to a promoter "driven" by a
DNA-binding site recognized by the DNA-binding domain such that an
interaction of a first fusion protein with a second fusion protein
results in increased transcription of the second nucleotide
sequence, in which the first and second nucleotide sequences can be
the same or different and (v) detecting the increased transcription
of the first and/or second nucleotide sequence, thereby detecting
an interaction between a first fusion protein and a second fusion
protein.
[0224] In a preferred embodiment, the bait (a MDM2 protein
sequence) and the prey (a library of chimeric genes) are combined
by mating the two yeast strains on solid media for a period of
approximately 6-8 hours. In a less preferred embodiment, the mating
is performed in liquid media. The resulting diploids contain both
types of chimeric genes (i.e., the DNA-binding domain fusion and
the activation domain fusion). After an interactive population is
obtained, the DNA sequences encoding the pairs of interactive
proteins are isolated by a method wherein either the DNA-binding
domain hybrids or the activation domain hybrids are amplified, in
separate reactions. Preferably, the amplification is carried out by
polymerase chain reaction (PCR; see e.g., Innis, et al., 1990. PCR
Protocols (Academic Press, Inc., San Diego, Calif.) utilizing pairs
of oligonucleotide primers specific for either the DNA-binding
domain hybrids or the activation domain hybrids. The PCR
amplification reaction may also be performed on pooled cells
expressing interacting protein pairs, preferably pooled arrays of
interactants. Other amplification methods known within the art may
also be used including, e.g., ligase chain reaction;
Q.beta.-replicase or the like. See e.g., Kricka, et al., 1995.
Molecular Probing, Blotting, and Sequencing (Academic Press, New
York, N.Y.).
[0225] In an additional embodiment of the present invention, the
plasmids encoding the DNA binding domain hybrid and the activation
domain hybrid proteins may also be isolated and cloned by any of
the methods well-known within the art. For example, but not by way
of limitation, if a shuttle (yeast to E. coli) vector is used to
express the fusion proteins, the genes may be subsequently
recovered by transforming the yeast DNA into E. coli and recovering
the plasmids from the bacteria. See e.g., Hoffman ,et al., 1987.
Gene 57:267-272.
Pharmaceutical Compositions
[0226] The invention present discloses methods of treatment and
prophylaxis by the administration to a subject of an
pharmaceutically-effective amount of a Therapeutic of the
invention. In a preferred embodiment, the Therapeutic is
substantially purified and the subject is a mammal, and most
preferably, human.
[0227] Formulations and methods of administration that can be
employed when the Therapeutic comprises a nucleic acid as described
above. Various delivery systems are known and can be used to
administer a Therapeutic of the present invention including, e.g.:
(i) encapsulation in liposomes, microparticles, microcapsules; (ii)
recombinant cells capable of expressing the Therapeutic; (iii)
receptor-mediated endocytosis (see, e.g., Wu & Wu, 1987. J.
Biol. Chem. 262:4429-4432); (iv) construction of a Therapeutic
nucleic acid as part of a retroviral or other vector, and the
like.
[0228] Methods of administration include, e.g.,, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The Therapeutics of the
present invention may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically-active agents. Administration can be systemic or
local. In addition, it may be advantageous to administer the
Therapeutic into the central nervous system by any suitable route,
including intraventricular and intrathecal injection.
Intraventricular injection may be facilitated by an
intraventricular catheter attached to a reservoir (e.g., an Ommaya
reservoir). Pulmonary administration may also be employed by use of
an inhaler or nebulizer, and formulation with an aerosolizing
agent. It may also be desirable to administer the Therapeutic
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, by injection, by means of a catheter,
by means of a suppository, or by means of an implant. In a specific
embodiment, administration may be by direct injection at the site
(or former site) of a malignant tumor or neoplastic or
pre-neoplastic tissue.
[0229] In another embodiment of the present invention, the
Therapeutic may be delivered in a vesicle, in particular a
liposome. See e.g., Langer, 1990. Science 249:1527-1533. In yet
another embodiment, the Therapeutic can be delivered in a
controlled release system including, e.g.: a delivery pump (see
e.g., Saudek, et al., 1989. New Engl. J. Med. 321:574 and a
semi-permeable polymeric material (see e.g., Howard, et al., 1989.
J. Neurosurg. 71:105). Additionally, the controlled release system
can be placed in proximity of the therapeutic target (e.g., the
brain), thus requiring only a fraction of the systemic dose. See,
e.g., Goodson, In: Medical Applications of Controlled Release 1984.
(CRC Press, Bocca Raton, Fla.).
[0230] In a specific embodiment of the present invention, where the
Therapeutic is a nucleic acid encoding a protein, the Therapeutic
nucleic acid may be administered in vivo to promote expression of
its encoded protein, by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular (e.g., by use of a retroviral vector, by
direct injection, by use of microparticle bombardment, by coating
with lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot, et al., 1991. Proc.
Natl. Acad. Sci. USA 88:1864-1868), and the like. Alternatively, a
nucleic acid Therapeutic can be introduced intracellularly and
incorporated within host cell DNA for expression, by homologous
recombination.
[0231] The present invention also provides pharmaceutical
compositions. Such compositions comprise a
therapeutically-effective amount of a Therapeutic, and a
pharmaceutically acceptable carrier. As utilized herein, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a state government or listed in the U.S.
Pharmacopoeia or other generally recognized pharmacopoeia for use
in animals and, more particularly, in humans. The term "carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered and includes, but is not limited to
such sterile liquids as water and oils.
[0232] The amount of the Therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and may be
determined by standard clinical techniques by those of average
skill within the art. In addition, in vitro assays may optionally
be employed to help identify optimal dosage ranges. The precise
dose to be employed in the formulation will also depend on the
route of administration, and the overall seriousness of the disease
or disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. However, suitable
dosage ranges for intravenous administration of the Therapeutics of
the present invention are generally about 20-500 micrograms (.mu.g)
of active compound per kilogram (Kg) body weight. Suitable dosage
ranges for intranasal administration are generally about 0.01 pg/kg
body weight to 1 mg/kg body weight. Effective doses may be
extrapolated from dose-response curves derived from in vitro or
animal model test systems. Suppositories generally contain active
ingredient in the range of 0.5% to 10% by weight; oral formulations
preferably contain 10% to 95% active ingredient.
[0233] The present invention also provides a pharmaceutical pack or
kit, comprising one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions and Therapeutics
of the present invention. Optionally associated with such
container(s) may be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0234] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1--Identification of a Complex Including an MDM2-Derived
Polypeptide and a Novel MDM2-Interacting Polypeptide
[0235] To identify proteins which bind to MDM2 proteins, expression
vectors which encode proteins for use in a yeast two hybrid
bait-prey screening assay were constructed. A cDNA encoding an
MDM2-derived polypeptide was used to produce the bait polypeptide
and libraries of human cDNA were used to produce prey polypeptides.
The MDM2 bait polypeptide was encoded by the human MDM2 nucleotide
sequence nucleotides 312-963 in the nucleic acid sequence having
GenBank Accession Number M92424; Oliner et al., Nature 358:80-83,
1992. The encoded MDM2 bait polypeptide was fused to the GAL4
activator domain.
[0236] The prey cDNAs were obtained from a human fetal brain cDNA
library of 1.5.times.10.sup.6 independent isolates. The library was
synthesized from Xho 1-digested and T15-primed fetal brain mRNA
(derived from five male/female, 19-22 week fetuses). The prey cDNAs
were directionally cloned into pBD-GAL4 (Stratagene; La Jolla,
Calif.). This plasmid is a yeast Gal4 activation domain cloning
vector and which also includes the TRP1 gene for selection of yeast
deficient in tryptophan biosynthesis.
[0237] The plasmid construct encoding the MDM2 bait polypeptide was
transformed by lithium acetate-polyethylene glycol-mediated
transformation (see e.g., Ito, et al., J. Bacteriol. 153:163-168,
1983) into the yeast strain N106r (mating type , ura3, his3, ade2,
trp1, leu2, gal4, gal80, cyh.sup.r,
Lys2::GAL1.sub.UAS-HIS3.sub.TATA-HIS3,
ura3::GAL1.sub.UAS-GAL.sub.TATA-lacZ). Plasmids containing prey
sequences were transformed into yeast strain YULH (mating type a,
ura3, his3, lys2, Ade2, trp1, leu2, gal4, gal80, GAL1-URA3,
GAL1-lacZ). The transformed yeast populations were then mated using
standard methods in the art. See e.g., Sherman, et al., Getting
Started with Yeast (Academic Press; New York, N.Y.), 1991. In
brief, the yeast cells were grown until mid- to late-log phase on
media that selected for the presence of the appropriate plasmids.
The two mating strains (.alpha. and a) were then diluted in YAPD
media, filtered onto nitrocellulose membranes and incubated at
30.degree. C. for 6-8 hours. The yeast cells were then transferred
to media selective for the desired diploids (i.e., yeast harboring
reporter genes for .beta.-galactosidase, uracil auxotrophy, and
histidine auxotrophy and expression of the vectors encoding the
bait and prey). The mating products were then plated onto synthetic
complete (SC) media (see e.g. Kaiser, et al., Methods in Yeast
Genetics (Cold Spring Harbor Laboratory Press; Cold Spring Harbor,
N.Y.) 1994.) lacking adenine and lysine (to facilitate the
selection of successful matings), leucine and tryptophan (to
facilitate the selection for expression of genes encoded by both
the bait and prey plasmids) and uracil and histidine (to facilitate
the selection for protein interactions). This medium is referred to
as SC Selective medium (hereinafter "SCS medium").
[0238] Isolates selected for successful mating, expressing both
fusion constructs, and expressing interacting proteins were
selected for further analysis.
[0239] Selected clones were first examined for expression of
.beta.-galactosidase to confirm the formation of an interaction
between the bait MDM2 protein fragment and a prey polypeptide.
Filter-lift .beta.-galactosidase assays were performed using a
protocol modified from Breeden et al., Cold Spring Harbor Quant.
Biol. 50:643-650, 1985. Colonies were patched onto SCS plates,
grown overnight and replica-plated onto Whatman No. 1 filters. The
replica filters were subsequently assayed for .beta.-galactosidase
activity. Colonies which were positive turned blue in color.
[0240] Cells from positive colonies were individually plated and
regrown as single isolates in the individual wells of 96-well
microtiter plates. Ten microliters (.mu.l) of each isolate was
lysed, the inserts contained within the pACT2 and pBD-GAL4 plasmids
were amplified by PCR using primers specific for the flanking
sequences of each vector. Approximately 200 amino-terminal
nucleotides of each insert sequence was determined using an ABI
Model 377 sequenator. Comparison to known sequences was made using
the "BLAST" computer program publicly available through the
National Center for Biotechnology Information.
[0241] Six different isolates were identified. Four of those were
determined to encode polypeptides sequences identical to already
known proteins which interact with MDM2. These polypeptides include
human p53 cellular tumor antigen (GenBank Acc. No. X54156),
retinoblastoma protein (pRB; GenBank Acc. No. M28419), E2F-1 (a
pRB-binding protein with properties of transcription factor E2F)
nucleic acid sequence (GenBank Acc. No. M96577), and ubiquitin
(GenBank Acc. No. U49869).
[0242] One isolate encoding an MDM2-interacting polypeptide include
a nucleic acid sequence that has not been previously described. The
nucleic acid sequence and its encoded polypeptide was named MDMIP,
for MDM-Interacting Polypeptide. The nucleic acid sequence and the
amino acid sequence of the encoded polypeptide are shown in FIG. 1
and SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
Example 2--Determining the Specificity of MDM2 Protein-MDIP
Interactions
[0243] To determine the overall degree of specificity for the
MDM2-MDMIP interactions, two general assays were performed. In the
first assay, N106r yeast cells were produced which expressed the
individual plasmids encoding the MDM2 proteins. These yeast cells
were plated on SCS plates, grown overnight, and examined for
growth. No growth was found for the novel interactant. This result
confirms that MDM2 is not a "self-activating" protein. Instead,
MDM2 protein requires interaction with a second protein domain for
a functional activation complex.
[0244] In the second assay, a plasmid containing the MDMIP insert
was transformed into strain YULH yeast (mating type a) and mated
with yeast strain N106r (mating type .alpha.) expressing proteins
other than the MDM2 protein. Promiscuous binders (i.e., inserts
able to bind with many other proteins in a non-specific manner)
interact in a non-specific manner with non-MDM2 protein domains,
and are subsequently discarded as non-specific interactants. MDMIP
did not show binding to proteins other than MDM2.
[0245] To further demonstrate the reproducibility and specificity
of the MDM2-MDMIP interactions, the isolated bait plasmid for the
MDM2 protein was used to transform yeast N106r (mating type
.alpha.). The interacting domain from MDMIP was transformed into
strain YULH (mating type a). The transformants were re-amplified,
and a mating was performed to recapitulate the identified MDM2
proteine MDMIP interaction. The MDM2 protein was shown to complex
in a specific manner with MDMIP. In addition, the MDM2 protein did
not show any non-specifically reaction with the CDK2 protein or
vector controls.
Example 3--Analysis of MDMIP Nucleic Acid and its Encoded
Polypeptide Sequence
[0246] The MDMIP nucleic acid sequence was examined for homology to
other nucleic acids and for the presence of open reading
frames.
[0247] The MDMIP nucleic acid sequence of 486 nucleotides (SEQ ID
NO: 1) was found to be 88% identical to soares melanocyte EST
N28611, a human cDNA clone of 440 nucleotides. However, since the
identities were found only between nucleotides 87 and 371 of MDMIP
and nucleotides 83 and 372 of EST N28611, MDMIP could not be
extended in either direction with EST N28611. Further searches did
not reveal homologies to other ESTs. Thus, the assembled sequence
could not be extended in either the 5' or 3' direction.
[0248] The MDMIP nucleotide sequence is also approximately 60%
homologous at nucleotides 86 to 418 to the cDNA sequence of the
human glutamate pyruvate transaminase (Gen Bank Accession Number
U70732; Sohocki et al., Genomics 40: 247-252, 1997).
[0249] An open reading frame (ORF) is present in the MDMIP nucleic
acid from nucleotides 1222. A BlastP search with the MDMIP amino
acid sequence showed no homology to previously describe sequences.
The translated reading frame contains no initiator methionine codon
(ATG), but has a stop codon at amino acid position 75. The protein
therefor most likely represents the carboxy terminal region of a
protein. The amino acid in position 5 (NAA) could be Glu (GAA), Gln
(CAA), Lys (AAA), or a stop codon (TAA); the amino acid in position
58 (TNT) could be Phe (TTT), Ser (TCT), Tyr (TAT), or Cys
(TGT).
OTHER EMBODIMENTS
[0250] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *